BIOCHEMISTRYAT the present time biochemistry is passing through a fruitfulperiod, in fact many will say that biochemistry is now fulfilling itspromise and is helping materially to solve a few of the more complexproblems of medicine. The study of the chemistry of the vitaminsis in some cases almost complete, and the advances made duringthe past fern years can be described as little short of miraculous.Ten years or so ago it would have been extremely difficult to findanyone bold enough to suggest that at the beginning of 1938 weshould know the chemical constitution of many of the importantvitamins and have available synthetic preparations of a t leastthree of them. For practical purposes, the use of the pure naturalor synthetic vitamins is, of course, very limited (under peace-timeconditions a t least), but for the study of their physiological functionsand for the further investigation of dietetic problems, these pureproducts are of considerable value.The year 1937 has witnessed many important advances in ourknowledge about the vitamins.Of special interest might be men-tioned further syntheses of vitamin B,, the reported synthesis ofvitamin A,, the isolation from naturaJ sources of other vitamins ofthe D group, the partial clarification of the story about the vitaminB,-complex, distinct adTances in our knowledge about the chemistryof vitamin E, and the demonstration that vitamin C-deficiency isoften associated with a diminished resistance of the body to certaintypes of infection.Carbohydrate metabolism has been the subject of intensiveinvestigation in many parts of the world, and if no particularlyoutstanding advance can be mentioned, the results obtained willundoubtedly help towards a clearer understanding of this complexproblem.The views about the chemistry of muscle contractionhave certainly become more complicated during recent years, butthere is now hope that before long it will be possible to present adefinite and universally accepted general scheme to explain whathappens when a muscle fibre is stimulated and caused to contract.In chemotherapy there has been real progress, and the discoveryin 1935 of the value of the prontosil drugs has been followed by avery fruitful attack on this problem during the past two years.The less complex drug sulphanilamide, and some related compoundswhich are still more active, are proving of inestimable value for thetreatment of certain diseases, and here we can record what iWORNALL: ANIMAL.399probably the most important advance in chemotherapy for manyyears. Another noteworthy piece of work is that of H. King,E. M. Louie, and W. Yorke on the trypanocidal action of guanidinesand related compounds, an investigation which gives much promisein connection with the treatment of sleeping sickness.l n a short review of the progress of biochemistry it is naturallyimpossible to discuss more than a very small fraction of the wholefield, but in the review next year it might be possible to fill in someof the gaps. The use of deuterium and radioactive phosphorus inthe study of metabolism, and recent developments in connectionwith hormones and anti-hormones, proteins, enzymes, and thechemistry of iinmufiity have all had to be omitted from this sectionthis year.In particular it is regretted that it is not possibleto discuss here the important discoveries about the action ofprotamine-insulin, and the influence of zinc on the action of thiscompound, ordinary insulin and certain other hormones.Considerations of space have again necessitated the inclusion ofonly certain selected subjects in the Plant Biochemistry section.In view of the fact that in the last few years various aspects of thebiochemistry of the higher plants have predominated in theseR'eports, it is felt that developments in the chemistry of certain ofthe smaller organisms should be placed on record.Several yearshave elapsed since reference was made here to some of these organ-isms, and in these cases the period under review has been extendedaccordingly. The wide interest in recent inveatigations of plantvirus, and the demonstration by Stanley and others of the funda-mental chemistry concerned, demands consideration this year.1. ANIMAL BIOCHEMISTRY.The Vitamins.Vitamin A.-H. N. Holmes and R. E. Corbet have described theseparation, from certain fish liver oils, of crystalline vitamin Ain pale yellow needles, m. p. 7.5-8-0". This preparation has anextinction coefficient of 2100 and a biological assay value of 3,080,000international units per g.Analysis gives C, 83.28 ; H, 10-44%, andthe mol. wt., as determined by the freezing point depression, is294 (Karrer's formula for the vitamin requires C, 83.84 ; H, 10.56% ;mol. wt., 286).The synthesis of vitamin A has a t last been reported, this havingbeen effected from 13-iononesemicarbazormee by R. Kuhn andC. J. U. R. Morris? The identity of the synthetic product withScience, 1937, 85, 103; J. Arner. Chem. SOC., 1937, 59, 2042.Ber., 1937, 70, 853400 BIOCHEMISTRY.the natural vitamin has been shown by the mixed chromatographicadsorption test on alumina and growth tests on rats.The suggestion that a second -A exists ha’s been made by twogroups of workers. A. E. Gillam, I. M. Heilbron, E. Lederer, andV. Rosanova,3 and J. R.Edisbury, R. A. Morton, and G . W.Simpkins 4 have independently shown that in antimony trichloridesolution certain liver oils show an additional band near 6930 A.According to the latter group of investigators, this 6930 A. chromogen(designated vitamin A2) is present in halibut liver oil, but rarelyin cod liver oil and never in whale liver oil. There is apparently,as yet, no evidence as to the biological activity of this allegedsecond vitamin A.Amongst other work on this vitamin might be mentioned thatof T. Moore,5 who has determined the -A content of adult humanliver. In health the “ median ” value is found to be 220 internat.units per g. of wet tissue, whereas lower values are found in mostdiseases. The vitamin A content of the liver a t birth is very low,but it rises sharply in the earliest months and reaches a more orless static level during what would be, in normal circumstances,the later stages of lactation.6 In the latter investigations on thelivers of children under 15 years of age dying by accident or fromdisease, low liver vitamin A reserves were associated with deathfrom pneumonia, septic diseases, and heart diseases, but not acuteinfectious diseases. In general the results were in agreement withthose obtained in the study of the livers of adult^.^ The r6le of theliver in the vitamin A economy of the hen is essentially the sameas in the mammal, for administration of cod liver oil, or of avitamin A concentrate, to laying hens led to the accumulation ofthe vitamin in the liver.The vitamin A content of the eggs wasalso increased, but returned to normal when the cod liver oil treat-ment ceased, although the liver reserves were sufficient to maintainthe A content of the egg a t the raised level for about 300 ovulation^.^E. Mellanby has shown that vitamin A deficiency in young dogsproduces degenerative changes in the ganglia, nerves, and organs of’both hearing and balance inside the temporal bone. The “in-attention ” of dogs fed on these deficient diets, previously ascribedto cerebral defects, is undoubtedly due to deafness, and attemptsare being made to determine whether certain forms of deafness inman can be explained in a similar manner.Natwe, 1937, 140, 233. Ibid., p. 234.li Biochem. J . , 1937, 31, 155.6 J.B. Ellison and T. Moore, ibid., p. 165.33. M. Cruickshank and T. Moore, ibid., p. 179.Chem. and Ind., 1937, 56, 1054WORMALL ANIMAL. 401Vitamin Bl (Aneurin).-The synthesis of aneurin by R. R.Williams and J. K. Cline was mentioned last year.Q Early this yeari~ similarly successful attack on this problem, by a different method,was described by A. R. Todd and F. Bergel’lo the synthetic materialproving to be identical, as far as chemical and biological tests areconcerned, with the natural vitamin. Another synthesis has beendescribed by T. Hoshino and M. 0hta.ll The relationship betweenthe structure of aneurin and its physiological activity has beenstudied by F. Bergel and A. R. Todd.12 These authors preparedand examined the biological activity of five analogues of aneurinand were able to reach certain general conclusions as to the groupswhich are essential for vitamin B, activity.The determination of vitamin B, by chemical methods has beenstudied by several authors, for it is well recognised that a chemicalmethod would greatly facilitate many of these investigations.B.C . P. Jansen l3 described a method which involves the conversionof the vitamin into thiochrome and the determination of the latterby measuring the fluorescence with a photoelectric cell. Thismethod has been modified by W. Karrer and U. Kubli,14 J. Goudsmitand H. G . K. Westenbrink l5 and by M. A. Pyke.16 The last-named author finds that the method can be applied to a widerange of materials (milk and other foodstuffs, animal tissues andurine), although it is necessary to take special precautions in manyinstances.For the determination of the vitamin B, content ofurine it is necessary to concentrate it by adsorption on fuller’s earth.Another method which promises to be of considerable value is thatof W. H. Schopfer,17 a modification of which is described by A. P.Meiklejohn.ls The method depends on the growth-promoting effectof the vitamin on a mould, Phycomyces BZaEesZeeanus, and deter-minations can be made 011 small amounts of blood (e.g., 1-3 ml.).All workers are agreed, however, that this test is only applicable toactive concentrates and not to ordinary food materials or extracts.A fermentation test, which depends on the measurement of theaccelerating power towards the fermentation of glucose by Fleisch-mann yeast and is capable of detecting 10‘6 g.of natural or syrrtheticvitamin B,, is suggested by A. Schultz, L. Atkin, and C. N. Frey.19The “ bradycardia ” method 2o for vitamin B, determinations has9 Ann. Reports., 1936, 33, 381; cf. also J. K. Cline, R. R. Williams, and10 J., 1937, 364. l1 Proc. Imp. Acad. Tokyo, 1937,13, 101.12 J., 1937, 1504. Rec. Trav. china., 1936, 55, 1046.14 Helv. Chim. Acta, 1937, 20, 369. l5 Nature, 1937, 139, 1108.16 Biochem. J., 1937, 31, 1955. l 7 Hull. SOC. Chim. b i d . , 1935, 17, 1097.18 BiocJzein. J . , 1937, 31, 1441. l9 J . Airier. Chena. soc., 1937, 59, 948.20 T. W. Birch and L. J. Harris, Biochem. J., 1934, 28, 602.J. Finkelstein, J.Amer. Chem. Soc., 1937, 59, 1052402 BIOCHEMISTRY.been subjected to a careful reinvestigation by P. C. Leong andL. J. Harris.21 Three crystalline specimens of natural and one ofsynthetic vitamin Isl were used for the assays, and all showedapproximately the same activity (2.9 x 10-6 g. of crystallinevitamin B, hydrochloride per one I.U.). A statistical analysis ofthe results of a large number of tests shows that the accuracy ofthe method is such that the probability is 21/22 that the meanresult will be within & 22% of the true value when five " tests "are made, or within & 12% for twenty tests, each test being onedose given on one occasion to one rat.Studies on the relationship between vitamin B1 and oxidationprocesses in brain have been continued by R.A. Peters and hiscolleagues. Further investigation has shown that the action of thevitamin is related specifically to pyruvic oxidase in its aerobicreaction and the reaction can be represented as follows : 22Lactate + 0 j Pyruvate + (n)O _jr Oxidation productsZ t t Pyrophosphate (n = 4) Vitamin B,Measurements of the R.Q. and the oxygen/pyruvate ratio forpigeon brain indicate that the oxygen consumption is only aboutfour-fifths of that needed to oxidise all the pyruvate. The pro-duction of carbon dioxide is lo% in excess of that required forcomplete combustion, and it is suggested that one molecule ofpyruvic acid out of five disappears by some channel other thancomplete 0xidation.~3 The pyruvate burned does not appear toundergo conversion into succinic acid, a-ketoglutaric acid or aceto-acetic acid, and the pigeon brain " pyruvate oxidase " system, ofwhich vitamin B1 is a constituent, is not a general ~t-keto-oxidase.~~Amongst other work on vitamin BI, the discovery of K.Lohmannand P. Schuster25 that co-carboxylase is a &phosphoric ester of21 Biochem. J . , 1937, 31, 672.22 R. A. Peters, ibid., 1936, 30, 2206; Deutsche med. Woch., 1937, 63, 1144;Chem. Weelcblad, 1937, 34, 442. This theory suggesting a relationship betweenvitamin B, and pyruvate oxidation receives valuable support from the obser-vation of B. S. Platt and G. D. Lu (Pvoc. Chinese Physiol. SOC., 1936, 16;Quart. J. Med., 1936,5, 355) that in patients suffering from beri-beri there is amarked increase in the bisulphite binding substances (mainly pyruvic acid)in the blood and other body fluids. This increase appears to follow the degreeof vitamin B, deficiency.R. H. S. Thompson and R. E. Johnson (Biochem. J.,1935, 29, 694) have obtained similar results in determinations on the blood ofB,-avitaminous pigeons and rats.23 G. K. McGowan, Biochem. J., 1937,31, 1627.24 G. K. McGowan and R. A. Peters, ibid., p. 1637.Naturwiss., 1937, 25, 26; cf. also H. von Euler and R. Vestin, ibid.,p. 416. K. G. Stern and J. W. Hofer, Science, 1937, 85, 483WOR,MALL : ANIMAL. 403aneurin is of outstanding importance, and this discovery, togetherwith the above-mentioned work of the Oxford school on the partplayed by vitamin B, in brain oxidation, should throw much lighton the physiological r61e of this vitamin.The suggestion ofK. Lohmann and P. Schuster that pyruvic acid accumulation inthe experiments on avitaminous pigeon's brain is due to lack ofco-carboxylase has been tested recently by R. A. Peters,26 whofinds that, although vitamin B, may act as the diphospho-compoundin brain, the other components of the system are dissimilar fromthose of yeast.27Vitamin B, (Complex) .-There is much confusion in the literaturerelating to the vitamins of the I3 group, and it seems probable thatthis state of affairs will continue unti! we know the precise natureof all the components of this complex. Until this happens, it willprobably be wiser to group together the members of the B grocp,other than vitamin B, (aneurin), under the heading vitaminB,-complex.28Space will not allow a full review of literature on this subject,but it will probably be agreed by most authorities in this field thatthe position is a little clearer as a result of the work of the past year.The vitamin B,-complex appears to consist of at least three factors :(1) LactoAavin(2) Vitamin B, (" rat-pellagra '' or rat-antidermatitis factor)(3) Pellagra-preventing (or anti-black-tongue) factor.A considerable amount of evidence has been presented duringrecent years in support of the view that nicotinic acid (or its amide)enters somewhere into the vitamin 3,-complex. Various authorshave shown that this substance has a growth-promoting action forcertain micro-organisms29 or for pigeons or rats fed on a dietdeficient in part of the B,-complex.s C.A. Elvehjem, R. J. Madden,E'. M. Strong, and D. W. Woolley31 have shown that nicotinic acidcan cure " black-tongue " in dogs and this suggestion that the P-Pfactor and nicotinic acid are closely related, if not identical, hasreceived strong support from the recent work of L. J. Harris. Thisauthor finds that monkeys resemble human beings and dogs (and9;6 Biochem. J., 1937, 81, 2240.2 7 K. Lohmann and P. Schuster (Biochem. Z., 1937, 294, 188) have alsoreached the conclusion that the brain system differs from yeast carboxylase.28 Cf. L. J. Harris, Biochem. J., 1937, 31, 1414.29 B. C. J. G. Knight, ibid., pp. 731, 966; E. R. Holiday, ibid., p. 1299;30 C.Punk and I. C. Funk, J . Biol. Chem., 1937, 119, Proc. XXXV; D. V.s1 J. Amer. Chem. SOC., 1937, 59, 1767.J. H. Mueller, J . Biol. Chem., 1937, 120, 219.Frost and C. A. Elvehjem, ibid., 1937, 121, 265404 BIOCHEMISTRY.thus differ from rats) in requiring a factor of B, other than lacto-Aavin and vitamin B,, and this factor appears to be identical withthat which prevents pellagra in man and black-tongue in dogs.28This nutritional failure in monkeys can be cured by nicotinic acid,and this acid also produces a dramatic improvement in the con-dition of human beings suffering from pellagra.32 The suggestionis made that nicotinic acid (or its amide) may be either the P-Pvitamin itself or the less active form, or “ precursor” of a moreactive variation of the P-P vitamin, which could be formed from itin the animal body.Amongst other evidence in favour of the view that vitamin B, isof a ‘‘ tripartite ” nature, is that presented by C.E. Edgar andT. F. Macrae, who find that autoclaved yeast contains two factorswhich are required to give optimum growth of rats fed on avitamin B-deficient diet plus lactoflavin and vitamin B1.33 Thesame aut’hors 34 find that one of these factors (that present in filtratesafter exhaustive extraction of the yeast extract with fuller’s earth)appears to be distinct from vitamin B,, but is perhaps identicalwith the “filtrate factor ” from liver extracts described byS. Lepkowsky and his ~olleagues.3~ Neither nicotinic acid noriiicotinamide can, however, replace this yeast filtrate factor.36Vitamin C.-Investigations in this field have been mainly con-cerned with studies on the behaviour of ascorbic acid and relatedcompounds in the body, and the relationship between this vitaminand infection.Numerous papers z7 have been published describingthe determination of ascorbic acid in blood, urine, and tissues by amodification of the method of L. J. Harris and S. N. Ray38 or byother methods. The use of these chemical methods has undoubtedlyled to a clearer understanding of the minimum body requirementsas far as this vitamin is concerned, but its exact physiological r61eis still not clear. There is, however, a considerable amount ofevidence that ascorbic acid may have a specific function in enablingthe body to resist certain infections, and with a continuation ofthe present mass attack on this problem in various laboratories and32 Report of a lecture by L. J.Harris, Lancet, 1937, ii, 1467.33 Biochenz. J., 1937, 31, 88G.34 Ibid., p. 893.36 T. 3’. Macrae and C. E. Edgar, Biochetn. J., 1937, 31, 2225.37 P. Manceau, A. A. Polioard, and M. Ferrand, BulE. SOC. Chin~. biol.,1930, 18, 1623; W. Tschopp, Z. physiol. Chern., 1936, 244, 59; R. R. Musulinand C. G. King, J . Biol. Chem., 1936, 116, 409; F. Widenhauer, Kliiz. Woch.,1936,1& 94; R. FerrsLri rtad G. Buogo, A~ch. Pisiol., 1935,35, 125; A. Bujitanud T. Ebihara, Biockcin. z., 1937, 290, 172, 182, -192 ; P. &!kxmier, Bull. Soc.Chine. bwl., 1937, 19, 877.38 Lancet, 1935, i, 71.36 Ann.Reports, 1936, 33, 385WORMALL : ANIMAL. 405hospitals it should not be long before thc relationship betweenvitamin C-deficiency and a decreased immunity can be defined moreprecisely.There is still some disagreement as to whether the 2 : 6-dichloro-phenol-indophenol method gives a true estimate of the vitamin Ccontent of urine, blood, etc., but the same charge of non-specificitycan probably be made against the other methods.C . Mentzer and A. Vialard-Goudou39 claim that the methyleiie-blue method 40 is more specific than the 2 : 6-dichlorophenol-indophenol method, but L. J. Harris*l considers that the lattergives reliable results and that compounds containing SH groups donot interfere in practice, provided that suitable experimentalconditions are observed. A spectrophotometric determination ofascorbic acid in tissues is described by A.Chevallier and Y. Choron,4%who suggest that it, permits the detection of 0.01 mg. of the vitamin.M. Srinivasan,43 using a modification of the method of H. Tauberand I. S. Kleiner,44 has utilised an ascorbic acid oxidase for thedetermination of this vitamin in certain natural sources andconcludes that the lower values obtained are more accurate thanthose previously recorded.Some doubts have been expressed as to the exact nature of thereducing substance present in urine which is determined as vitamin C .Identscation by biological tests will be very difficult or may evenbe impossible (because of the small amount, and possibly on accountof toxic substances which may be present), but isolation andidentification by chemical means appear to offer no insuperabledifficulties.K. Hinsberg and R. Ammon45 failed, however, toisolate from normal urine the 2 : 4-dinitrophenylhydrazine derivativeof dehydroascorbic acid, and concluded that urine cannot containmore than about one-third of the amount of ascorbic acid indicatedby titration with 2 : 6-dichlorophenol-indophenol. C. P. Stewart,H. Scarborough, and P. J. Drumm 46 have more recently succeededin this quest, and have isolated from 12 litres of normal urine 20 mg.of the pure 2 : 4-dinitrophenylhydrazine derivative.T. Meuwissen and E. Noyens 47 have made similar observations,3O Bull. Xoc. Chim. biol., 1937, 19, 707.40 Cf. R. Ammon and K .Hinsberg, Klin. Woch., 1936, 15, 8 5 ; E. Trier,Ugeskr. Lceger, 1936, 98, 1238; C. Mentzer, Compt. rend. SOC. Biol., 1937,125, 330.4 1 Proc. 5th Intern. Cong. Tech. Chem. Agric. Ind., Holland, 1937, I, 112.42 Bull. SOC. Chim. biol., 1937, 19, Fill.43 Biochem. J . , 1937, 31, 1521.4 5 Biochem. Z., 1936, 288, 102.4 6 Nature, 1937, 140, 282; Biochem. .J., 1937, 31, 1874.4 7 Cited from ref. (46).44 J. Biol. Chem., 1935, 110, 559406 BIOCHEMISTRY.and there appears to be no doubt that ascorbic acid is normallypresent in ~rine.~8B. C. Guha and B. Ghosh 49 claim t o have cor&rmed their earlierobservation that rat tissues in a closed volume of air can synthesiseascorbic acid from mannose, and the negative reqults of otherauthors 50 are attributed to the fact that no synthesis can be detectedin nitrogen.J. R. Hawthorne and D. C. Harrison 51 have, however,failed to confirm this synthesis from mannose in minced rat liverincubated with Ringer-phosphate solution in the presence of oxygen ;in addition there was no increase in the vitamin C content of thelivers of rats as a result of the subcutaneous or intravenous injectionof mannose.The vitamin C requirements of man have been determined byvarious workers and the average figure usually given as about60 mg. per 70 kg. 111811.5~ P. H. O’Hara and M. N. Hauck found that2200-2800 mg. are required, at the rate of 200 mg. daily, to saturatethe tissues after feeding a deficient diet for a month, and theirresults indicated a maximum C reserve of 2500-3000 mg.53Administration by mouth may not, however, be completely satis-factory in investigations of this type, since it cannot be certainthat all the ingested vitamin is absorbed. For this reason theobservations of P.Schultzer 54 are of special interest. This authorgave a daily intravenous injection of 40 mg. of ascorbic acid to ascorbutic male adult, and found that the scurvy was cured andsaturation with ascorbic acid produced within 23 days, i.e., afteringestion of a total quantity of only 0-83 g. For satisfactory evi-dence about the reserves and the requirements of man with respectto vitamin C, it appears probable that this method of administrationwill have to be adopted, although it should not be overlooked thatfor practical purposes the information obtained from experimentswhere the vitamin is administered by mouth will be of more value.As a result of the accumulation of a large amount of evidence itseems certain that in many diseases the body is in a state of “ uii-saturation” in regard to vitamin C , although the intake may benormal or even completely adequate for healthy individuals.Whathappens to the ascorbic acid in the unhealthy is not known, but48 E. Gabbe (Klin. Woch., 1936, 15, 292) suggested that several forms ofvitamin C exist in urine, but this view has not received general acceptance.40 Nature, 1936, 138, 844. 6o Ann. Reports, 1936, 33, 387.Biochem. J . , 1937, 31, 1061.52 M. Heinemann, ibid., 1936, 30, 2299; M. Van Eekelen, ibid., p. 2291.63 J .Nutrition, 1936, 12, 413. 54 Biochem. J., 1937, 31, 1934.66 I;. J. Harris. A chapter in “ Perspectives in Biochemistry.” Editedby J. Needham and D. E. Green. Camb. Univ. Press. 1937; cf. alsorefs. 56 and 57WORMALL : ANIMAL. 407the extra consumption may be associated with the activity of theleucocytes. The early investigations arose primarily as a result ofthe observation that individuals whose diet contains a subnormalamount of vitamin C are more susceptible to certain infections, eventhough the deficiency may not be sufficient to produce scurvy.Several possible explanations of this loss of immunity can be offered,e.g., the vitamin may neutralise toxins produced by the organismsor it may play a part in the development of specific anti-bodies orof some non-specific system such as complement. This problemhas not j7et been solved, but it is not unlikely that further inves-tigation will yield results of real value in connection with immunologyand the treatment of disease.The association of a deficiency in the vitamin C intake and anincreased tendency towards infection has been observed too fre-quently to be entirely fortuitous, but it has still to be definitelyestablished that the increased usage of the vitamin in these infec-tions is not simply a result of metabolic disturbances caused by theinfection (ie., purely a secondary process).Successful results havebeen claimed, however, by many investigators following the treat-ment of certain infections with massive doses of the vitamin, and inaddition, it has been stated that periods of ill-health and epidemicshave occurred in certain countries a t a time when fruit andvegetables were not readily available.Irrespective of the questionof the exact relationship between vitamin C intake and resistanceto infection, there is thus ample justification for the advocation ofan increase in the amount of fresh fruit and vegetables to thosesuffering from certain infections. It is not possible in this reviewto quote all the work which has been carried out in this connection,but the reader will find more comprehensive surveys el~ewhere.~5As typical examples of the experiments which have been made, thework of L. J. Harris and his colleagues will be mentioned. Theseauthors have shown that osteomyelitis, in common with manyother infective conditions, causes a diminished rate of excretion ofvitamin C in the urine and a lowered response to a test dose, indi-cating an apparently increased usage of the vitamin.When thepatient is healed, he returns to normal in his usage of the vitamin.56Studies on pulmonary tuberculosis have shown that in this con-dition the deficit in vitamin C is seen in an extreme form, with agood correlation between the severity of the case and the diminutionof the urine titres.57 The suggestion is made that extra provisionof the vitamin should be made for all tuberculous patients. The56 1%. A. Abbasy, L. J. Harris, and N. Gray Hill, Lancet, 1937, 233,177.67 M. 4. Abbasy, L. J. Harris. and P. Ellman, ibid., p.181408 BIOCHEMISTRY.same authors s7 and other workers 58 have found that rheumatoidarthritis is also associated with a decreased vitamin C exmetlion anda lowered state of saturation. For the solution of this complexproblem relating to infections, a considerable amount of statisticalinformation will be required, but the task of collecting this materialwill probably be made easier by the simplified procedure for thevitamin-C urine test devised by L. J. Harris and M. A. A b b a ~ y . ~ ~It has been shown 56p 59r 59a that there is a very close parallelismbetween the dietary intake of the vitamin and its output in theurine, relatively little variation being seen between differentindividuals on the same diet. The reputed minimum intake (whichhas been determined by the amount needed to protect against anytendency to increased capillary fragility, or to cure symptoms ofincipient scurvy in mariners) is generally accepted to be about 25mg.per day. Now, this intake causes an excretion of 13 mg. perday, and it seems justifiable therefore to argue 56* 59u that, if asubject excretes less than this amount, he is “ below standard ”-not necessarily showing any clinical symptoms of deficiency, butnone the less probably not enjoying full, optimum health. Manyclinical trials have in fact shown that health is improved whenextra fruits and vegetables are included in the diet, %nd surveysbased on an analysis of urine have indicated that many workingclass subjects excreted less than the standard amount of thevitamin 59b just as an examination of national diets 5 9 ~ suggestedthat a subnormal intake was of common occurrence.The satisfactory results following administration of syntheticascorbic acid to individuals suffering from scurvy were reported bymany authors 6O very shortly after the synthesis of the vitamin wasfirst accomplished.In a very recent paper A. Elmby and E. War-burg 61 state, however, that three of their scorbutic patients failedto respond to the administration of ascorbic acid by mouth or byintravenous injection. A cure was effected, and the blood ascorbicacid was raised to a normal figure, by the administration of lemonjuice, and it is suggested that some other factor (a co-vitamin) isrequired for the absorption and retention of the vitamin.Thehypothesis that this factor is vitamin P is being investigated.Med., 1936, 35, 347; J. F. Rinehart, Ann. Intern. Med., 1936, 9, 671.6 8 J. F. Rinehart, L. D. Greenberg, and P. Baker, Proc. SOC. Exp. BioE.69 Lancet, 1937, 233, 1429.59a M. A. Abbasy, L. J. Harris, S. N. Ray, and J. R. Marrack, ibid., 1935,jg* L. J. Harris, M. A. Abbasy, and J. Yudkin (with a note by S. Kelly),59c J. B. Orr, “ Food, Health, and Iizcome,” Macmillan and Co., 1936.6o Ann. Reports, 1934, 31, 332.ii, 1399.ibid., 1936, i, 1488.61 Lancet, 1937, 233, 1363WORMALL : ANIMAL. 469Vitamin D.-Investigations carried out during the past few yearshave shown that several compounds possess antirachitic powers,and to the original vitamin 13, (calciferol) we must add several othermembers of this group.At the same time, the existence of severalprecursors of " vitamin D '' has been established. The position upto the end of last year was fully reviewed in the Annual Reports.G2Since then the search for substances having antirachitic propertiesand for substances which become active on irradia'tion or othertreatment has continued.F. Schenk 63 has isolated crystalline vitamin D, (mentioned lastyear) by hydrolysis of the m-diiiitrobenzoate ; the product hasm. p. 82-84", [%]To + 83.3' in acetone and maximum absorptiona t 2650 A. It resembles vitamin I), in giving a yellow colour withantimony trichloride and possesses an antirachitic potency equal tothat of D,.No antirachitic product other than vitamin D, couldbe isolated from halibut liver 0il.6~ A. Windaus and G. Trautmann,65starting from 22 : 23-dihydroergosterol, have obtained crystallinevitamin D, (m. p. 107-lo$', [a]:*" + 89.3" in acetone), with amaximum spectrum absorption value the same as that for vitaminAmongst the search for pro-vitamins should be mentioned theinvestigations of A. Windaus and F. Bock,66 who find that the pro-vitamin D of skin, isolated from the crude sterols of skin by adsorp-tion on alumina and fractional elution, is 7-dehydrocholesterol.Pigskin, which contains up to 5.9% of the pro-vitamin, is the richestsource encountered in this investigation. J. C. Eck, B. H. Thomas,and L. Yoder G7 have studied the chemical activation of sterols andfind that cholesterol (ordinary and purified), cholesteryl chloride,cholesterilene, dicholesteryl ether, cholestene and cholesteryl butylether are all activated by heating a t 85-90' with sulphuric acidand acetic anhydride in acetic acid, yielding it product of the sameantirachitic potency ; this product is, however, not derived from thepro-vitamin D of ordinary cholesterol, the substance which is con-verted into an antirachitic agent on irradiation.Further inves-tigations by J. C. Eck and B. H. ThomasG8 have shown thatcholesterol and cholesterilene can be rendered antirachitic by heatingwith a variety of reagents.F. W. Anderson, A. L. Bacharach, and E. L. SmithG9 havepresented revised specification values for pure calciferol (in.p. 1 16"D2.62 Ann. Repoyts, 1936, 33, 349, 390.64 H. Brockmann, 2. physiol. Chem., 1937, 245, 96.Ibid., 1937, 247, 185.6 7 J . Biol. Chem., 1937, 117, 655.I39 Analyst, 1937, 62, 430.1 3 ~ Naturwiss., 1937, 25, 159.6 6 Ibid., 1937, 245, 168.6 8 Ibid., 1937, 119, 621, 631410 BIOCHEMISTRY.(& lo), [cx]i9i1 3- 123*25-125*75” in ethyl alcohol (4% wt./vol.),E:& 2650 A. 460-500), and report that the biological activity ofeleven blends varied from 35.7 t o 45.0 internat. units per 10-6 g.The line-test assay for vitamin D, and the effect of different rachi-togenic diets on the intensity of the rickets and the response toantirachitic treatment, have been studied by A. L. Bacharach andhis c~lleagues.~OThe seasonal variation in the vitamin D content of cow’s milk hasbeen studied by J.E. Campion, K. M. Henry, S. K. Kon, andJ. Ma~kintosh,~~ who found that the direct exposure of the cow tosun- and sky-shine contributes all, and the pasture none, of theincrease in vitamin D potency of milk which takes place in thesummer.R. Nicolaysen 72 has carried out extensive investigations on themode of action of vitamin D. The results seem to indicate that theaction of this vitamin in the gut of the rat is confined to a direct actionon the absorption of calcium, and the well-known reduced absorptionof phosphorus in vitamin D deficiency is due to precipitation by theincreased amount of calcium in the bowel. The rate of absorptionof calcium from isolated loops of the small intestine increases withincreasing concentration in both normal and vitamin D-deficientrats, but the rate is lower with the latter than it is with normalrats.No difference was observed in the case of intestinal absorptionof xylose and sodium sulphate, nor was there any difference betweennormal and vitamin D-deficient rats as far as absorption of calciumfrom the abdominal cavity is concerned. The suggestion that theinfluence of vitamin D on phosphate absorption is merely a secon-dary effect is supported by the finding that isolated loops of thesmall intestine of normal and vitamin D-deficient rats are equallyeffective in absorbing potassium phosphate and sodium glycero-phospha,te.Vitamin E.-The isolation by H. 3%. Evans, 0. H. Emerson, andG .A. Emerson of substances which they named c c tocopherols ” andwhich had the formula C,9H,,0, from the unsaponifiable fraction ofwheat germ oil, and the possession of vitamin E activity by a-tocopherol, and to a lesser extent by P-tocopherol, were reportedlast ~ e a r . ~ 3 The authors have isolated the same products from cottonseed oil concentrate^,^^ and this work has undoubtedly stimulatedinterest in the relationship between the tocopherols and vitamin E.70 A. L. Bacharach, E. Allchorne, and H. E. Glynn, Biochem. J., 1936, 30,71 Biochem. J., 1937, 31, 81.73 Ann. Reports, 1936, 33, 392.74 0. H. Emerson, G. A. Emerson, and H. M. Evans, Science, 1936, 83, 421.2004; A. L. Bacharach, 2. Vitaminforschung, 1937, 6, 129.i B Ibid., pp. 107, 122, 323, 1086WORMALL : ANIMAL.41 1One significant difficulty in recognising a-tocopherol as Dhe purevitamin (or the more active of two vitamins) is the fact thai it is lesspotent in biological tests than was a similar preparation obtained byJ. C. Drummond, E. Singer, and R. J. Ma~walter,~~ and anotherobstacle is the failure of other investigators to isolate a-tocopherolin significant amounts from these oil concentrates.J. C. Drummond a,nd A. A. Hoover 76 have reinvestigated theunsaponidiable fraction of wheat germ oil and report that the resultsof attempts to separate a- and p-tocopherol as the allophanates weredisappointing. The p-allopha>nate was obtained, but in no singlecase after distillation was a-tocopherol allophanate isolated.Theiodine value of p-tocopherol suggests the presence of three ethylenelinkages, and of the two oxygen atoms, one is present in a non-phenolic reactive hydroxyl group and the other may be present inan ether linkage.77An exhaustive investigation of the unsaponifiable fractions ofrice and wheat germ oils has been carried out by A. R. Todd, F.Bergel, H. Waldmann, aEd T. S. Work.'* Three apparentlyhomogeneous alcohols, u-orysterol (m. p. 121-122"), p-orysterol(m. p. 113-114"), and y-orysterol (m. p. 119--120"), all havingthe approximate formula C,,H,,O, were isolated from rice germ oil,and wheat germ oil yielded p-amyrin, a-tritisterol (m. p. 113-114"),and a third alcohol (m. p. 174-175'). The authors suggest thatthese products, which have similar properties, may not impossiblybe mixtures of closely related compounds.All are devoid ofviitamin E activity. Compounds of the same type have beenisolated from wheat germ oil by P. Karrer and H. S a l ~ m o n , ~ ~ wh_oname their products cc- and p-tritisterols.The method of isolating tocopherols as the allophanates hasbeen improved by A. R. Todd, F. Bergel, and T. S. Work,SO whohave been able to isolate @-tocopherol allophanate in fair yieldfrom wheat germ oil. On hydrolysis this allophanate yieldsp-tocopherol, which shows full vitamin E activity in a dose of5 mg. and possibly less. The vitamin E activity of the wheatgerm oil concentrate used appears to be due almost entirely to thisalcohol. No significant amount of a-tocopherol allophanate could7 6 Biochem.J., 1935, 29, 2510.77 Cf. also E. Fernholz ( J . Arner. Chem. SOC., 1937, 59, 1154), who suggeststhat a-tocopherol is a monoether of duroquinol. F. Bergel, A. R. Todd, andT. S. Work (Chem. and Ind., 1937, 56, 1054) have also obtained duroquinolby the decomposition of active oils and in addition a quinol (m. p. 165') whichis possibly identical with #-cumoquinol. The latter authors suggest thatj3-tocopherol and " cumotocopherol" may be identical.76 Ibid., 1937, 31, 1852.7 8 Nature, 1937, 140, 361 ; Biochem. J., 1937, 31, 2247.7a Helv. Chim. Acta, 1937, 20, 424. 80 Biochem. J., 1937, 31, 3257412 BIOCHEMISTRY.be isolated, but appreciable amounts of other a,llophan' iL t es wereobtained; one of these is phytyl allophanate (m.p. 78").Further work on p-tocopherol and its isolation in larger amountswill most probably overcome the present difficulty of carrying outvitamin E assays without an accepted unit or a standard prepara-tion. This difficulty is pointed out by A. L. Bacharach, E. All-chorne, and €€. E. GlynnYs1 who discuss fully the reproductivebehaviour of female rats fed on a vitamin E-deficient diet fromweaning to maturity, and suggest that a period of vitamin E-deficiency may have an effect on the reproductive system of thematernal organism much more deep-seated than has hitherto beenbelieved. In this connection it is of interest to note that M. M. 0.Barrie 82 has found that deprivation of vitamin E leads to thyroidand anterior pituitary deficiency, and that definite pathologicalchanges in the latter can be demonstrated in vitamin E-deficientrats.Diet.The truly amazing success of investigations on vitamins duringrecent years may have tended to place in the background t,heproblem of general dietary requirements, and there was a dangerthat this more spectacular work on the chemistry of the vitaminswould completely overshadow observations on other, and probablyno less important, aspects of diet.This pitfall has, however, beenavoided, and the majority of experts on nutrition are able to takea broad view of the whole field.The present time appears opportune for a reconsideration of thedietetic requirements of man and for a general consideration of thestate of nutrition in various parts of the world.The task of makingthese surveys was undertaken by the Health Committee of theLeague of Nations, and the outcome is an excellent series ofReports 83 which may be said to contain the cream of our present-day knowledge on the subject. To the biochemist and physiologist,Volume I1 of this series will offer most interest, for it presents acompact and yet surprisingly comprehensive account of most of theessentials of nutrition. The decision of the Commission to reachagreement as to the calorie, protein, and other requirements of theaverage man will be approved by all who have had to offer "ex-81 Biochenz. J., 1937, 31, 2287.82 Nature, 1937, 139, 286; 140, 426; Lancet, 1937, 233, 251.83 League of Nations Publications. The Problem of Nutrition.Vol. I.Interim Report of the Mixed Committee on the Problem of Nutrition (Ser.L.O.N.P. 1936, 11, By 3). Vol. 11. Report on the Physiological Bases ofNutrition (1936, 11, B, 4). Vol. 111. Nutrition in Various Countries (1936,11, B, 5 ) . Vol. IV. Statistics of Food Production, Consumption and Prices(1936, 11, B, 6). Final Report (1937, IT, A, 10)WORMALL : ANIMAL. 413planations " for the widely differing accounts given in differenttext-books, and it is to be hoped that the details given in theseReports will find their way into all text-books which deal with diet.Amongst the special recommendations of the Commission are thepartial replacement of white flour by lightly milled cereals andespecially by potatoes. The last-named provide extra vitamin Cand contain more readily available calcium and phosphorus thando the cereals; they also yield more iron and B vitamins that domilled cereals.The suggestion that milk should form a conspicuouselement of the diet a t all ages should not meet with serious opposi-tion, nor will the recommendation that fresh vegetables and/orfruit should always be constituents of the normal mixed diet.Special reference is made to the high nutritive value of skimmedmilk, which contains the protein, the B and C vitamins and thecalcium and other mineral constituents of the original milk.Perusal of these reports shows that that much malnutrition un-doubtedly exists, even in the richer countries, and suggestions aremade as to the best methods of improving the general standard ofnutrition.Another report which is worthy of careful study deals with thenutritional, hygienic, and social aspects of the milk problem.8*The value of this most important foodstuff, as a source of certainof the vitamins and of protein of high biological value, is discussedin the light of modern views as to the minimum daily requirements.The diet of the cow itself has not been neglected, a.nd it is recom-mended that the animal ration should contain ample supplies ofvitamins A and D.Where necessary, vitaminised products, suchas irradiated yeast, should be supplied. After consideration of themode of collection of milk and the frequency of milk-borne diseasesin human beings, the conclusion is reached that no raw milk canbe regarded as completely safe for human consumption. Theauthors of this report suggest quite emphatically that '' all liquidmilk for human consumption should be adequately pasteurised orboiled ", and after considering all the avalilable evidence they findthat there is no real basis for the fear that pasteurisation is detri-mental to the food value of milk.The nutritive value of bread and the vexed question of white!oersus wholemeal bread have again received prominent attention,and it seems likely that the use of modern weapons will enable theprotagonists of the " wholemeal '' view to annihilate completelytheir opponents.Up to the present, the contestants have had to84 By H. C. Bendixen, U. J. Blink, J . C. Druiiimoiid, A.81. Leroy, and (2. S.JTilson, Quuvterly Bulletitz of the Health Orgunisat ion (League of Nations),1937, 6, 371414 BIOCHEMISTRY.be content with evidence which was largely indirect and oftenhypothetical, but in more ween& times it has been found possibleto define more precisely the vitamin B, requirenents of man andthe amount of this vitamin present in various types of bread. Theremoval of the germ from wheat leads to the loss of other substancesbesides B,, but it is believed that in these other instances the lossis more likely to be made good by other articles of diet. The realdeficiency of white bread as compared with wholemeal breadappears, therefore, to be concerned with the B vitamins, andparticularly B,.L. J. Harris85 finds that, whereas ordinary white bread maycontain as little as 0.12 internat.unit of vitamin B, per gram,wholemeal bread may contain as much as 0-9 unit per g., withbran bread occupying an intermediate position. These measure-ments were rendered possible by the use of the bradycardia method,for the ordinary growth tests break down when used for the assayof vitamin B, in bread. Harris concludes that the variety of“ germ-bread ” examined is not greatly superior in B, content towholemeal bread, and that the ordinary “brown bread” ascommonly sold is a reasonably good source of B,.A. Z. Baker, M. D. Wright, and J. C. Drummond 86 have reachedsimilar conclusions about the brown versus white bread problem asa result of vitamin B, determinations on various types of breadand a calculation of the B, content of present-day and past diets.A most interesting account is given of the history of milling andit is suggested that the most fundamental change, as far as thepresent problem is concerned, was the introduction of the rollermill into this country about 1870; the general adoption of thissystem led to the almost complete removal of the germ and bran.An analysis of modern diets indicates a daily vitamin B, intake offrom about 290 units at the lowest income level to 450-550 for highincome groups.From data collected from various sources it appearsthat, a’lthough 300 units of B, per day may protect man from beri-beri, the minimum daily requirement for the full maintenance ofhealth is probably about 500 units.The inadequacy of the moderndiet thus becomes apparent. The authors give the calculatedvitamin B, contents of the diet of the parish poor of 1782 (660-850units per day) and that of the Poor Law diet, City of London, 1838(1060 units per day), and they hare ample justification for statingthat the best-fed members of the population to-day consume lessvitamin B, than the parish poor of the 18th and early 19th centuries.85 Biochem. J . , 1937, 31, 799; P. C. Leong andL. J. Harris, ibid., p. 812;L. J. Harrisand P. C. Leong, J. SOC. Chena. Ind.., 1937,86, 1951..8 6 J . SOC. Chem. Ind., 1937, 56, 1 9 1 ~ WOR,MALL : ANIMAL. 415k change froin white to wholemeal bread may add to the averagediet as much as 200 units of vitamin B, per day, and cndoubtedlya strong case has been made out for a general condemnation of thewhite loaf.The last word on this question has not been said,however, and the opposing camp will probably quote the observa-tions of A. M. Copping and M. H. who conclude that theB, content of white bread is sufficiently high for this food to providea significant part of the requirements for this vitamin, bearing inmind the large amounts of bread often consumed. As against thisview must be recalled that white bread as the sole diet hasfrequently been the cause of beri-beri.Some interesting observations on the effect of overfeeding on theprotein metabolism of man are recorded by D. P. Cuthbertson,A. McCutcheon, and H. N. M u n r ~ . ~ ~ The addition of raw orboiled milk (or a mixture of beef, lactose, and butter equivalent tomilk in protein, carbohydrate, and fat content) to a diet which wasadequate for nitrogen equilibrium and for the maintenance of bodyweight, caused an increase in body weight and marked retention ofnitrogen and sulphur.The degree of retention appears to be relatedto the total increment in the energy value of the diet. In a similarmanner, the addition of glucose, or to a lesser extent fat, to anadequate diet caused nitrogen and sulphur retention.8sThe significance of the mineral constituents of the diet is in mostinstances well recognised, mainly as a result of studies on smallanimals. In this field of investigation there are not many observ-ations on man, and for this reason the experiments of R.A. McCanceand his colleaguess0 are worthy of special attention. In order toproduce a sodium chloride deficiency in normal man, McCance andcertain other volunteers lived for about 11 days on a diet which wasfree, as far as possible, from this salt. Profuse sweating was alsoinduced to increase the salt loss of the body. The symptomsobserved are fully described and it is pointed out how closely thesesymptoms resemble some of those of Addison’s disease. This salt-deficiency leads to a disordered nitrogen balance,g0 a slight inter-ference with carbohydrate rnetaboli~rn,~~ and changes in the responseof the kidney to certain tests.92 Investigations of this type areobviously most difficult to carry out and cause much inconvenienceand discomfort to the subjects concerned, but they are of inestimable8 7 Biochem,.J., 1937, 31, 1879.89 D. P. Cuthbertson and H. N. Munro, ibid., p. 691.88 Jbid., p. 681.R. A. McCmce, Proc. Roy. SOC., 1936, B, 119, 246; Lancet, 1936, i, 643,704, 765, 823.91 Idem, Biochem. J., 1937, 31, 1276.92 R. A. McCaiice and E. M. Widdowson, Proc. Roy. Soc., 1936, B, 120,228; J . Physiol., 1937, 91, 222416 RI0C)HEMlSTRY.value for the study of many diseases where salt deficiency is aprominent feature. To quote another example, the investigationsof the same authors on the absorption and excretion of irong3 havefurnished valuable information about the availability of the iron ofvarious foodstuffs and about the storage of this element in the body.Prom the results obtained it appears that the animal body has littleor no power to excrete iron and any excess which is absorbedremains in the body.Carbohydrate Oxidation.In recent times two very interesting theories have been advanced,one by Szent-Gyorgyi and the other by H.A. Krebs, to explain howsimple carbohydrates are oxidised in animal tissues. These twotheories appear to have a certain amount in common, for both areconcerned with the action of dicarboxylic acids with four carbonatoms, but fundamentally they are very different.A. Szent-Gyorgyi and his colleagues in a series of investigations 94have shown that succinic acid and related C4-dicarboxylic acidsplay it dominant rble in certain tissue oxidations. These acids forman essential link in the respiratory chain between the oxidisablefoodstuff (probably a triose) and the oxidising system cytochromeplus Warburg's atmungsferinent .Oxidation (dehydrogenation) ispictured as a, transference of hydrogen froin the foodstuff to oxalo-acetic acid, which is thereby converted into nialic acid; malic acidpasses on its hydrogen to fumaric acid, which becomes succinicacid. The succino- and nialico-dehydrogenases catalysing thesereactions should be regarded as '' hydrogen-transportases "35 Thesuccinic acid is oxidised back to fumaric acid by cytochrome, theoxidation of which is catalysed by the Warburg enzyme. Thewhole process is summarised by Szent-Gyorgyi in the followingmanner (for convenience the (&-acids are represented as free acidsand not salts) :p 2 H TO& (p2HFoodstuff 2H THz -.-+ QHz 2H SH QHz I(Triose) -4- 70 - QH*OH --+ TH C-- YH2 IOxaloacctate. Malate.Fumarate. Suecinate. IOxygen - Atmungsferment -- Cytochrome +-J93 R. A. McCance and E. M. Widdowson, Lancet, 1937, ii, 680; Biochewz. J . ,2937, 31, 2029.94 A. Szent-Gyorgyi and his colleagues, 2. physiol. Chem., 1935, 236, 1 ;1936, 244, 105; 1937, 245, 113; 247, 1, 248, 2 5 2 ; 249, 57, 61, 63, 183. 189,205, 209, 211, 217.C02H C02H COZH C02H 1 2 ~I9 5 A. Szent-Gyfirgyi, ihid., 1937, 249, 211WORMALL : ANIMAL. 41 7According to this theory the hydrogen transference is carried outby a pair of reversible reactions (oxaloacetate jt malate ; andfumarate f succinate), and since fumarase can effect the changemalate fumarate, these four compounds can be regarded as" different forms of the same substance ".Support for this theoryis given in the many publications of Szent-Gyorgyi and his col-l e a g u e ~ , ~ ~ and by the observations of other investigators who haveworked in his laboratory. F. J. Stare, for example, has shown thatliver and kidney tissues can oxidise fumarate to oxaloacetate, andthat the reverse change, the reduction of oxaloacetate to malateplus fumarate, is almost q~antitative.~6As a result of these investigations, A. KorAnyi and A. Szent-Gyorgyi were led to consider the possibility that dia,betic acidosismay be due to a destruction of the C4-dicarboxylic acid catalysissystem. To test this hypothesis, succinic acid was administered tofive diabetics, to restore the C4-acid system to normal, and satis-factory results were reported.97 Thus 10 g.of this acid per 0sper day, with a reduction later to 1 g. per day, caused the " acetonebody " excretion to fall to zero or a very small amount. The hyper-glycaemia and glycosuria remained, but with succinic acid treatmentless insulin was required to control the diabetes. Unfortunately,other authors have failed to confirm these observations. R. D.Lawrence, R. A. McCance, and N. Archer 96 treated two diabeticswith succinic acid and reported the complete failure of this treat-ment. D. M. Dunlop and W. M. Arnott 99 in a similar trial withthree patients found that succinic acid does not prevent the onsetof diabetic coma, nor does it diminish a chronic diabetic ketonuria.H.A. Krebsl has presented a somewhat different scheme toindicate the r61e of C4-dicarboxylic acids in the tissue oxidation ofcmbohydrate. Like A. Szent-Gyorgyi, Krebs considers that oxalo-acetic acid is the prime agent which is responsible for triose oxidation,but he suggests that this acid condenses with an unknown substance(a carbohydrate derivative and most probably pyruvic acid) toproduce citric acid. Citric acid is then transformed, througha-ketoghtaric, succinic, and furnaric acids, to oxaloacetic acid, thuscompleting the " citric acid cycle ". Viewed as a whole, theprocess can be regarded as the " attachment " of pyruvic acid (orits equivalent) to a C4-dicarboxylic acid, and the removal of carbondioxide and water in stages to produce once more the C4-acid. Thefollowing scheme, which is a slight modification of that given byKrebs, indicates the main features of the cycle.9 6 Biochem.J., 1936, 30, 2257.98 Brit. Med. J., 1937, ii, 214.1 Ibid., p. 736; of. also ref. 2.9 7 Deutsche med. Woch., 1937, 63, 1029.99 Lancet, 1937, 233, 738.REP .-VOL . XXXIV . 418 BIOUHEMISTRY.J.Oxaloacetic acid + carbohydrateL I VCitric acid + CO,derivative ( ? pyruvic acid)cc-Ketoglutaric acid + CO, + H,O lo Succinic acid + CO,t.1OFumaric acid + H,ONet change : CH,-CO*CO,H + 5 0 --+ 3c0, + 2H,OIt is difficult in LC short review to discuss the whole of the evidenceon which this scheme is founded, but a few of the more salientfeatures of this work might be mentioned.M:. A. Krebs andW. A. Johnson2 found that citric acid catalyses the oxidation ofcarbohydrate in muscle just as does succinic acid, and that, underanaerobic conditions, muscle can readily synthesise citric acid fromoxaloacetic acid. The same authors have also confirmed andsupplemented the observation of C. Martius and F. Knoop3 thatoxidation of citric acid by certain tissues yields a-ketoglutaric acid.This last acid is known to yield succinic acid and carbon dioxide inthe body, and the change from oxaloacetic acid (or citric acid) tosuccinic acid can be demonstrated in respiring muscle tissue whenmalonate is added to inhibit oxidation of the succinic a ~ i d . ~ * ~ Theinterconversion of succinic, fumaric and oxaloacetic acids has beenwell e~tablished.~~ This citric acid cycle appears to occur generallyin animal tissues, but not in yeast or B.coZi, and quantitativemeasurements suggest that it is the chief pathway of oxidation ofcarbohydrate in muscle (H. A. Krebs and W. A. Johnson).2The formation of ketonic substances as intermediates in carbo-hydrate metabolism has also been studied by H. A. Krebs andW. A. J~hnson,~ who suggest that acetic acid and pyruvic acidcombine to give acetopyruvic acid. Evidence is produced that thelast-named is metabolised by animal tissues (liver, muscle, etc.)to yield acetoacetic acid (aerobically) or p-hydroxybutyric acidEnzymologia, 1937, 4, 148.2. physiol. Chern., 1937, 248, 1.Biochem.J., 1937, 31, 645, 772WORMALL ANIMAL. 419plua carbon dioxide (anaerobically). The following scheme indicatesthe reactions which are most probably concerned :CH,*COeCO,H --% CH,-CO,H $- GO,CH3*CO*C0,H + CH3*C02H __P CH3*CO*CH,*CO*C0,H + H20Acetopyruvic acid.CH3*CO*CH2*C02H + CO,CH,-CO*CH,*CO*CO,H f$ Acetoacetic acid.-* CH,*CH(OH)*CH,*CO,H + CO,fl-Hydroxybutyric acid.The rate of these reactions is small compared with the citric acidcycle, but this alternative pathway of carbohydrate breakdown maybe of value in explaining certain features of diabetic acidosis. Failureof the citric acid cycle would lead to the conversion of more pyruvicacid into ‘‘ acetone bodies ”; the last-named may therefore bederived from carbohydrate as well as from fatty acids (see alsoA.Szent-Gyorgyi 94*97).These theories of carbohydrate oxidation have been describedhere at some length, but this course has been adopted because itseems likely that these views will continue to attract much attention.Criticism has been, and will be, made that the demonstration ofthe presence in tissue slices and minced tissue of systems which arecapable of effecting certain changes does not necessarily prove thatthese changes represent the main processes of metabolism in theintact animal. There is evidence, however, of a tendency to makestudies of this type more quantitative in nature, and where this isdone, it is possible to assess more accurately the significance of thevarious reactions. The investigations which have yielded evidenceagainst the theories of A.Szent-Gyorgyi and H. A. Krebs cannotbe dealt with in a short review, but the observations of J. M. Innesmight be quoted as being pertinent and typical. This author findsthat fumaric acid added to minced pigeon breast musale increasesthe oxygen uptake by an amount which is not greater than can beaccounted for by oxidation of some of the fumaric acid whichdisappears. It is suggested, therefore, that fumaric acid is utilisedas a substrate for respiration and not as a catalyst for transferenceof oxygen to other substrates in the muscle.Amongst other investigations which should have a very prominentplace in any review on oxidation are those of J. H. Quastel and hiscolleagues, who have studied the oxidation processes taking placein brain and other tissues.The inhibitory action of hydroxyrnalateon lactate, and to a lesser extent glucose, oxidation by brain6 Biochem. J., 1936, 80, 2040420 BIOCHEMlSTRY .suggests that this tissue can oxidise glucose by a mechanism whichdoes not involve lactate as an intermediary.6 Important resultshave also been obtained by studying the effect of certain narcotics(chloretone, luminal, and evipan) on brain oxidations, and it hasbeen observed that the narcotic concentrations which producenarcosis in vivo are of the same order of magnitude as those whichinhibit the in vitro respiration of cerebral cortex.’ Similar studieswith ether have shown that this substance is like the other narcoticsin some respects, but unlike in others.sThe investigations of R.A. Peters and his colleagues on therelationship between vitamin Is, and oxidations in brain have beendiscussed in the section on vitamins. For an account o€ the changeswhich are concerned with muscle contraction the reader is referredto a review by D. M. Needhamtg and to a recent paper on dis-mutations and oxidoreductions by D. E. Green, D. M. Needham,and J. G. Dewan.lo The intermediary carbohydrate metabolismin embryonic life has been the subject of extensive investigation byJ. Needham and his colleagues,11 who suggest that their results maybe interpreted upon the hypothesis that in the chick embryo thereare two separate routes of carbohydrate breakdown; (1) a veryactive non-phosphorylating glucolysis mechanism and (2) a phos-phorylating mechanism closely similar to that in muscle.Themachinery for the second system does not appear to have been laiddown fully in early embryonic development.Chemotherapy.The Prontosil Group of Drugs.-Reference was made in theseReports last year to the discovery that prontosil and prontosil Sare efficacious in the treatment of certain streptococcal infections.During the past year definite advances have been made in ourknowledge of the value and mode of action of these drugs and relatedcompounds. Most of them are given by the mouth, and, whereeffective, they usually have a marked and almost dramatic action.Unfortunately the use of these sulphonamide derivatives is riotdevoid of danger.In addition to the above-mentioned azo-compounds (prontosiland the more soluble prontosil S), sulphanilamide [p-aniinobenzene-6 M.Jowett and J. H. Quastel, Biochem. J . , 1937, 31, 275.7 Idem, ibid., p. 565. Idem, ibid., p. 1101.D “ Chemical cycles in muscle contraction.”10 Biochern. J . , 1937, 31, 2327.11 J. Needham and W. W. Nowidski, ibid., p. 1165; J. Needham, W. W.Nowiliski, K. C. Dixon, and R. P. Cook, ibid., p. 1185; J. Needham andH. Lehmann, ibid., pp. 1210, 1913.A chapter in “ Perspectivesin Biochemistry.” Cambridge Univ. Press, 1937WORMALL : ANIMAL. 42 1sulphonamide (I) or sdphonamide-PJ and many of its derivativeshave been tested. Benzylsulphanilamide [proseptasine (11)] andsodium 4 - sulphonamidophen y l- y -phenylprop ylamiI1e - ay -disulphonate[soluseptasine (IZI)] are amongst those which are available in thiscountry.(I.) N H 2 0 0 , * I V H ,(11.1 ~ H , - N H < > O , ~ N H ,(111 * ) 0p;E2*p=; C ) S 0 2 * N B ,A.T. Fuller l2 has shown that prontosil is broken down in theanimal body to yield sulphanilamide (I), an observation whichsupports the suggestion13 that the latter is the active agent inprontosil therapy. This would account for the high in vivo, a8compared with a negligible in vitro, bactericidal activity of prontosil,and for the fact that, following the administration of prontosil (orsulphanilamide), the blood of man and other animals is bactericidaltowards haemolytic streptococci.14 Various investigators haveendeavoured to find a derivative of sulphanilamide which is morepotent than this compound in the treatment of streptococcal infec-tions, but in many instances with little success.1s E.Fourneau,J. Trhfouel, F. Nitti, D. Bovet, and (Mme.) J. Trefouel lG find,however, that 4 : 4’-dinitrodiphenyl disulphide is 4-8 times andthe corresponding sulphone 10 times as active in protecting miceagainst streptococcal infections as is sulphanilamide. The samegroup of authors find a similar high activity shown by 4 :4’-di-acetamidodiphenylsulphone and 4 : 4’-diaminodiphenylsulphone.17G. A. H. Buttle, D. Stephenson, S. Smith, T. Dewing, andG. A. H. Poster 18 find that, compared with sulphanilamide, 4 : 4’-di-aminodiphenylsulphone is much more effective, and 4 : 4‘-dinitro-diphenylsulphone as effective and less toxic, when used to curestreptococcal infections in mice.Other interesting discoveries inl3 Lancet, 1937, 232, 194.13 J. and Mme. J. Trr5fouG1, F. Nitti, and 2). Bovet, Conapt. rend. Xoc. Biol.,1935, 120, 756.14 L. Colebrook, G. A. H. But’tle, and R. A. &. O’Meara, Lancet, 1936, 231,1323.16 J. and Mme. J. Trkfouel, I+’. Nitti, and D. Bovet,, Ann. I n s t . Pasteur,1937, 58, 30; W. H. Gray, G. A. H. Buttle, and D. Stephenson, Biocheni,. J . ,1937, 31, 724.16 Conzpt. rend., 1937, 204, 1763.17 E. Fourneau, (Mme.) J. Trkfoulil, J. Tri.foue1, 17. Nitti, and D. Bovet,Compt. rend., 1937, 205, 299.l8 Lancet, 1937, 232, 1331422 BIOCHEMISTRY.this field relate to the action of the “ diseptals ” A, By and C, com-pounds which have been found to be superior to some of the‘‘ prontosil ” preparations in the treatment of staphylococcal,gonococcaI, and certain other infe~ti0ns.l~ It is worthy of note thatsubstitution in the free sulphonamide group does not necessarilydestroy the bactericidal action.Diseptal C H2N<>SO2*NH<>O2*NH2E. K.Marshall, K. Emerson, and W. C. Cutting 2O have describeda method for the determination of sulphanilamide in blood andurine, based on the diazotisation of the amine and coupling of thediazo-compound with dimethyl-a-naphthylamine in acid solutionto form a purplish-red azo-dye. With the aid of this method theseauthors have found that the absorption of sulphanilamide from thealimentary tract is very rapid and is nearly complete in 4 hours;thus no advantage is gained by injecting the substance.It isexcreted in the urine mainly in the unchanged form by dogs, andboth free and conjugated by man and rabbits.21 The conjugatedform is mainly, if not entirely, p-acetamidobenzenesulphonamide.22This group of drugs has been used in the treatment of variousbacterial infections, but it is probably too early to give a dogmaticopinion as to the results of these tests. Successful results areusually obtained when the infection is due to the @-hamolyticstreptococci 23 (e.g., in puerperal sepsis, erysipelas, certain types oftonsilitis, streptococcal meningitis, etc.), and often with menin-gococcal infections Z4 and B. coli infections of the urinary tract.251 0 Cf.review by H. Hoerlein, Practitioner, 1937,139, 635.20 J . Amer. Med. ASSOC., 1937,108,953, and a, modification by E. K. Marshall,Proc. SOC. Exp. Biol. Med., 1937, 36, 422; J. Biol. Chem., 1937, 122, 263;cf. also A. T. Fuller, ref. (12).21 Cf. also A. T. Fuller, ref. (13), who found both free and conjugated formsin the urine of men and mice.82 E. K. Marshall, W. C. Cutting, andK. Emerson, Science, 1937, 85, 202.2s For the literature, see I;. Colebrook and M. Kenny, Lancet, 1936, 230,1279; 231, 1319; L. Colebrook and A. W. Purdie, ibid., 1937, 233, 1237,1292; P. H. Long and E. A. Bliss, Arch. Surg., 1937, 34, 351 ; J . Amer. Me&ASSOC., 1937, 108, 32.24 In mice:-G. A. H. Buttle, W. H. Gray, and D. Stephenson, Lancet,1936,230,1286 ; H . Proom, ibid., 1937,232,16.In man :-F. F. Schwentker,S. Gelman, and P. H. Long, J . Amer. Med. Assoc., 1937,108, 1407.26 M. Kenny, F. 13. Johnson, and T. von Haebler, Lancet, 1937, 233, 119WORMALL : ANIMAL. 423Satisfactory results are claimed by most investigators when thesedrugs are used for gonococcal infections, but others report negativeresults.Toxic effects following the use of sulphanilamide and relatedcompounds include irritation of the kidney, fever, dizziness, mildnausea, acidosis, methzmoglobinzemia, sulphmnoglobinaemia,haemolytic anzemia and agranulocytosis, of which the last two appearto be the most serious. The appearance of sulphaemoglobin in theblood of patients receiving drugs of this group has been observedby many investigators26 and is a much commoner consequence ofthis treatment than has hitherto been recognized (G.Discombe 27).H. E. Archer and G. Discombe28 conclude that sulphanilamideand related compounds catalyse in the body the reaction betweenhzmoglobin and hydrogen sulphide (which is absorbed from theintestinal tract). These authors suggest that no aperient other thanliquid paraffin should be given during treatment with these drugs,and that a low-residue diet should be given to minimise bacterialdecomposition in the colon.L. Colebrook and A. W. Purdie29 have recently reported thesatisfactory results obtained by treatment of 106 cases of puerperalfever with sulphanilamide. Some toxic effects were observed.Cyanosis, which was associated in nearly all instances with methz-moglobin or sulphzemoglobin or both in the blood, was shown byover 50% of the cases.It was concluded, however, that althoughan undesirable feature, the cyanosis had no adverse effect on theprocess of recovery. No development of agranulocytosis was.observed in these cases, but this serious sequel to sulphanilamide orprontosil treatment has been recorded by several authors.30In spite of these dangers, there appears to be no doubt thatsulphanilamide and related compounds have an extremely hightherapeutic value. Because of the possibility of toxic effects, how-ever, treatment with these drugs should be carefully controlled, andmost authorities in this field are agreed that any tendency towardsthe indiscriminate use of these drugs for every type of bacterialinfection is to be deprecated.Trypanosomiasis.-In this field of chemotherapy there have beenvery interesting developments during the past year.From the26 L. Colebrook and M. Kenny, Lancet, 1936,230, 1279; G. Discombe, ref.(27); M. A. Foulis and J. B. Barr, Brit. Med. J., 1937, i, 4-45; J. R. J. Patonand J. C. Eaton, Lancet, 1937, 232, 1159.2' Lancet, 1937, 232, 626.28 Ibid., 1937, 233, 432.29 Ibid., pp. 1237, 1292.30 J. G. G. Borst, ibid., 1937, 232, 1519; C. J. Young, Brit. Med. J., 1937,ii, 105424 BIOCHEMISTRY.scientific view point and perhaps even more important the treat-ment of trypanosomiasis and related diseases, the most promisingis that concerned with the action of guanidines and related com-pounds on trypanosomes .Over a period of many years it has been shown by many authorsthat trypanosomes metabolise large amounts of simple carbo-hydrate3I (indeed it has even been suggested that the death ofsmall animals infected with trypanosomes is due in.part to a hypo-glycaemia accompanied by the acidosis associated with the accumu-lation of lactic acid). N. and H. von Jams6 32 suggested thattrypanosomes suffering from " sugar-hunger " are more readilyphagocyted by the reticulo-endothelial cells, and that the opsoniceffect produced by Bayer 205 may depend on the toxic inhibitionby this drug of the sugar metabolism of the trypanosomes. Asa sequel to these observations the same authors studied the trypano-cidal action of synthalin (decamethylenediguanidine)? a substancewhich is known to produce hypoglycEemia in animals,33 and claimedthat this substance had a high curative effect when injected intoanimals infected with certain strains of T.bruceL3* K. Schern andR. Artagaveytia-Allende in a similar investigation 35 found that smallamounts of synthalin will cure T. equinum and T. hispanicurn butnot T. crzcxi infections. The in witro trypanocidal action ofsynthalin has been investigated fully by E. M. Lourie and W. Yorke,36who found that a concentration as low as 1 in 256,000,000 will killpractically all the trypanosomes in a suspension kept at 37" for24 hours. These authors concluded that the therapeutic action ofsynthalin is due to a direct lethal action on the trypanosomes.Very recently3' H.King, E. M. Lourie, and W. Yorke havedescribed the results of investigations which appear to open up animmense field in the search for trypanocidal agents. The in vitroand in vivo trypanocidal action of a series of hornologues of synthalinhave been tested, and a considerable therapeutic effect was observedwith those compounds containing 10 to 14 methylene groups. Otherguanidines, isothioureas, amidines, and amines, with alkyl andalkylene chains, were also prepared and tested. High trypanocidalactivity was shown by certain alkylenediamidines, compounds withtwo guanyl groups [NH:C(NH,)-] attached to the ends of a methylenechain. The most active member of two series is n-undecane-31 K. Schern, Zentr. Bakt. Orig., 1925, 96, 356, 360, 362, 440, 444, 451;Deutsche med.Woch., 1927, 53, 106; W. Yorke, A. R. D. Adams, andF. Murgatroyd, Ann. Trop. Med. Parasitol., 1927, 23, 501.32 Z . Irnmunitat., 1935, 84, 471.34 2. Immunitat., 1935, 86, 1.36 Ann. !Prop. Med. Parasitol., 1937, 31, 435.a7 Lancet, 1937, 233, 1360.33 Ann. Reports, 1927, 24, 264.36 B i d . , 1936, 89, 21, 484WORMALL : ANIMAL. 425diamidine, which is a t least as active in vitro as is synthalin andmuch less toxic. In vivo tests show quite quite definitely thatundecanediamidine can permanently cure laboratory animalsinfected with trypanosomes, and that it has a therapeutic valuemuch superior to that of synthalin. Further points of interestmentioned by these authors are that the trypanosomes do notappear to acquire very rapidly, if a t all, any resistance to the drug,and secondly that trypanosomes which have previously acquired aresistance to Bayer 205 or to arsenicals, are very sensitive toundecanediamidine.The special significance of the latter observ-ation is obvious when it is remembered that the large-scale use ofarsenicals for the treatment of sleeping sickness is accompanied bythe risk of the production of arsenic-resistant strains of trypano-somes. A further development of this work, which hasdemonstrated the high trypanocidal action of compounds with aconstitution entirely different from that of other known trypanocidalagents, will be awaited with considerable interest.Amongst other investigations on trypanosomiasis might bementioned those dealing with the action of Bayer 205 (germanin).This drug has a most powerful in vivo destructive action on trypano-somes, but in vitro it has practically no trypanocidal effect.Whenthe organisms are kept in a serum-Ringer-glucose solution a t 37”,however, a concentration of Bayer 205 of 62 mg. per 100 ml. (i.e.,62 ( ( mg.% ”) will kill all the trypanosomes present.38 N. and H.von Jancs6 39 report that as little as 1.7 mg.% is sufficient to kill theorganisms within 24 hours, but 3’. Hawking 40 finds that 125 mg.% isneeded for this purpose, although 31 mg.% will suffice to kill most ofthe trypanosomes. Much evidence is now forthcoming to show thatthe effect of this drug is indirect, and that it has an opsonin-likeaction on the trypanosomes, thus rendering them more susceptibleto the action of the phag~cytes.~~ Support for this view has beenobtained by F.Hawking,40 who finds that exposure to 10 mg.% ofBayer 205, a concentration considerably below that required tokill them, renders the trypanosomes incapable of infecting mice.A method for the determination of Bayer 205 in blood-plasma hasbeen devised by W. G. Dangerfield, W. E. Gaunt, and A. Wormall,and the presence of small but significant amounts of this drug inthe plasma of small animals three or four months after a single98 W. Yorke, F. Murgatroyd, and F. Hawking, Ann. Trop. Med. Parasitol,,1931, 25, 313.39 Zentr. Bakt. Par., 1934, 132, 257; cited from Trop. Dis. Bull., 1935, 32,22.49 In the press (personal communication to the Reporter).4 1 I,.Reiner and J. Koveskuty, Deutsche med. Woch., 1927, 53, 1988;N. and H. von Jancsb, Ann. Trop. Med. Parasitol., 1934, 28, 419426 BIOCHEMX3TRY.injection of a " normal " dose of the drug has been demonstrated.&Over this long period the amount of Bayer 205 in the plasma ismaintained at a level which may be sufficient to exert the opsonicaction noted by the above-mentioned authors.Recent developments in connection with the use of variousarsenicals for the treatment of trypanosomiasis and syphilis andthe problem of arsenic resistance were reported last year.43 Men-tion might be made, however, of a new method of approach devisedby F. Hawking, T. J. Hennelly, and J. H. Quastel44 for the studyof the efficiency of drugs used for syphilis and trypanosomiasis ofthe central nervous system.This method, which has furnishedresults of considerable interest, consists of a determination of thein vitro trypanocidal power of the cerebrospinal fluid, withdrawnafter administration of the arsenical, and comparison with the arseniccontent of the fluid.A. WORMALL.2 . PLANT BIOCHEMISTRY.Plant Viruses.Isolation and Nature of the Tobacco Mosaic Virus.--Virus diseaseswhich have presented such an enormous problem in medical andveterinary research, find a counterpart in the plant world. Foryears mosaic diseases, spotted wilt and bunchy top of tomatoes,curly top of beet, etc., have been traced to the activity of viruses,the units of which are frequently too small to be separated onbacterial filters.Although the infectivity of juices of diseasedplants suggests the virus to be an extraordinarily active livingorganism, the extreme rapidity with which its physiological effectsare translocated through plant tissue, and the speed and extent towhich the active material multiplies are perhaps more in keepingwith the properties of a biocatalyst.Controversial views as to the fundamental character of the virushave been modified very considerably by the work of W. M. Stanleyand colleagues, who have isolated from the juice of diseased tobaccoplants a crystalline substance exhibiting a high order of infectivityapproximating to 500 times that of the juice itse1f.l The methodof isolation of the active substance (fractional precipitation withammonium and magnesium sulphates), its nitrogen content ( 166y0),and its positive reaction to common protein reagents (trichloroacetic,42 Chem.and Ind., 1935, 55, 1029; Biochern. J., in the press.43 Ann. Reports, 1936, 33, 403.44 J . Pharm. Exp. Ther., 1937, 59, 167.1 Science, 1935, 81, 644; Phytopath., 1936, 26, 305; J. Biol. Chenz., 1936,81, 673POLLARD : PLANT. 427phosphotungstic, tannic acids) establish the protein character ofthe substance and confirm the earlier observations of, among others,E. Barton-Wright and A. At. McBain and also C. G. Vinson andA. W. PetreY3 who had effected a partial separation of the activematter, shown it to be nitrogenous, and speculated as to its probablechemical nature.Earlier preparations had contained phosphorusand sulphur, which, however, were separable by dialysis.The purified protein forms minute crystals (0.024-03 mm. indiameter) which are optically active ( [ a ] - 0.43') and doublyrefractive, and give characteristic X-ray patterns (R. W. G. Wyckoffand R. B. Corey4) which are identical with those observed inpreparations obtained by direct ultra-centrifuging of the juice ofdiseased plant^.^ H. P. Beale makes the interesting observationthat certain intracellular inclusions occurring in mosaic-infectedtobacco can be observed under the microscope to yield, ontreatment with dilute hydrochloric acid, crystals identical withthose obtained by Stanley. Ultracentrifugal analysis of theprotein by R.W. G. Wyckoff, J. Biscoe, and W. M. Stanley 7indicates a molecular weight of approximately 17 millions andcodrms the value obtained by I. B. Eriksson-Quensel and T.Svedberg.8 The latter workers also record the isoelectric point ofthe protein as pH 3-49.The juice of healthy plants contains proteins of molecular weightnot exceeding 30,000, and no evidence of even small amounts ofhigh-molecular proteins has been found. In diseased plants themosaic infection appears to stimulate abnormally high proteinproduction, and extracts may contain double the normal contentof total protein, of which the virus protein may constitute up toThe physical nature of the virus protein particles both withinthe plant and in vitro appears to be somewhat complex.W. N.Takahashi and T. E. Rawlins show that suspensions of the crystalsin ammonium sulphate solution contain not only visible crystalsexhibiting a stream double refraction, but also a colloidal solutionof the protein. It is suggested that the solution contains sub-microscopic rod-shaped particles. Change in the reaction of the80 yo.Nature, 1933, 132, 1003.Bot. Gaz., 1929, 87, 14; Contr. Boyce Thompson Inst., 1931, 3, 131;Science, 1934, '70, 548.4 J . Biol. Chem., 1936, 116, 51.5 R. W. G. Wyckoff and R. B. Corey, Science, 1936, 84, 513.6 Contr. Boyce Thompson Inst., 1936, 8, 333.7 J . Biol. Chem., 1937, 117, 57; also WyckoE, Compt. rend. Soc. Biol.,8 J . Amer. Chern. SOC., 1936, 58, 1863.1937,125, 5.Science, 1937, 85, 103428 BIOCHEMISTRY.suspension results in an altered proportionality between theamounts of visible crystal and colloidal solution.F. C. Bawden,N. W. Pirie, J. D. Rernal, and I. Fankuchen,lo working with anaqueous solution or suspension of the highly purified protein, havefound that this separates into two layers, the upper exhibitinganisotropy of flow, and the lower containing liquid crystals whichon drying yield a gel. The crystalline liquid and the gel giveidentical X-ray patterns. The gel stage probably consists ofhexagonally-packed particles, and the crystals of parallel, charged,rod-like molecules. The molecular weight of the protein calculatedfrom the size of the particles approximates to 17 millions (cf.Wyckoff et a?.'). In a further examination of the X-ray pattern ofthe crystals J.D. Bernal and I. Panbuchen l1 suggest that,although the long protein molecules are packed hexagonally andwith regularity a t right angles to their length, no regularity in thedirection of the packing exists. The molecules themselves aremade up of sub-molecules of definite size. Intermolecularreflections can be observed, and the authors put forward thepossibility of distinguishing and classifying viruses by means ofX-ray observations. The juice from mosaic-infected plants afterclarification by the centrifuge and storing a t a little above 0" isfound l2 to contain the virus in the form of flexible fibres, probablyconsisting of chains of protein particles weakly linked together.These fibres disintegrate or collapse when warmed to thetemperature a t which the virus loses its infectivity (70-75').The homogeneity of the crystalline virus protein has been amplydemonstrated by the constancy of the physicochemical propertiesof samples from various sources. The X-ray pattern of the crystalsis unchanged after repeated cry~tal1isation.l~ Ultracentrifugalanalysis shows the same sedimentation constant for the high-molecular protein in affected plant juice, for the crystallinecentrifugate, and for the isolated crystalline protein after extensivechemical purification. Moreover, repeated fractional crystallisationof the protein has failed to modify its infectivity.14 According toStanley,15 g.of the protein in 1 C.C. of water is almost invariablysufficient to infect the tobacco plant and in some cases 10-14 g.(i.e., approx.300 molecules) gives a positive response.Inactivation. Innumerable experiments show that whateverthe nature of the tobacco mosaic virus preparation, the presencelo Nature, 1936, 138, 1051.l2 R. J. Best, ibid., p. 628.Is R. W. G. Wyckoff and R. B. Corey, J . Bid. Chem., 1936, 116, 51.l4 H. S. Loring and W. M. Stanley, ihid., 1937, 117, 733.l1 Ibid., 1937, 139, 923.Science, 1935, 81, 644POLLARD : PLANT. 429of the high-molecular protein is always synonymous with infectiveproperties, and that treatments causing denaturation or decom-position of the protein inevitably destroy the power of inducingthe disease and of stimulating rapid formation of the characteristicprotein in tobacco-plant tissues.Thus Stanley l6 observes thatheating at 75" or transference to media of p H < 1 or > 11 denaturesthe protein and destroys virus activity. In a more detailedinvestigation R. J. Best 17 demonstrates that irreversible inactivationof the virus is initiated at pH 7.8 and is substantially complete atpH 10.2. The p=-activity curve is of the same character as theneutralisation curve of a weak acid and leads to the suggestionthat inactivation results from the neutralisation of acidic groups inthe prosthetic part of the protein molecule. Changes in the,properties of the virus are also associated with similar though notidentical ranges of pH by H. H. Thornberry.l* At pH 8.5 the viruspasses freely through a Berkefeld " W '' filter candle, but at pH 1.5it is held back and is adsorbed by the filter, from which it may beeluted by phosphate buffer solutions a t p H 8.5, a considerableconcentration of virus preparations thus being possible.Optimuminfectivity is apparent at pH 7.0-8.5. At p H 10.6 infectivity iscompletely lost in 4 hours, and at p H 11.2 inactivation is completein 5 minutes. This general range of significant pH values is inconformity with Stanley's observations.The activity of the protein appears to be eliminated fairlyrapidly by digestion with trypsin l9 and slowly by pepsin,20although it is not certain that the action in the second case is adirect proteolysis.21 Oxidising agents (hydrogen peroxide, nitrousacid) and ultra-violet light destroy the infectivity of the protein,which, however, may still exhibit certain of the initial chemicaland serological properties.22 Protein precipitants inhibit theactivity of the virus.In the case of tannic acid the effect isreversible, removal of the tannic acid restoring activity. Treatmentof plants with tannic acid before, but not after, inoculation withthe virus suppresses the infection to a degree which is proportionalto the dosage of tannic acid.23 Virus preparations are also renderednon-infective by reduced ascorbic acid, oxygen being necessary forthis action, which is catalysed by copper salts. M. Lojkin2416 Phytopath., 1935, %, 476. l7 Austral. J . Exp. Biol., 1936, 14, 323.18 Phytopath., 1935, 25, 601, 618. l8 W. M. Stanley, ibid., 1934, 24, 1055.20 Idem, ibid., p.1269.21 A. F. Ross and C. a. Vinson, Missouri Agric. Exp. Xta. Bull., 1937,22 Idem, Science, 1936, 83, 626.3 3 H, H. Thornberry, Phytopath., 1935, 25, 931.24 Contr. Boyce Thomps0.n Inst., 1936, 8, 335; 1937, 8, 446.No. 258430 BIOCHEMIS'TRY.expresses the view that this process of inactivation, which isinhibited by catalase, depends on the formation of an intermediateproduct (not dehydroascorbic acid) of a peroxide nature. It is ofinterest that neither the oxidation of reduced ascorbic acid by1 : 2 : 6-dichlorophenol-indophenol or potassium permanganatenor the autoxidation proceeding in alkaline solution in the presenceof hexoxidase provides conditions suitable for inactivation of thevirus. In this connexion A.M. Smith and W. Y. Paterson 25 recordthe significant observation that varieties of potatoes susceptible topotato mosaic virus disease show a general tendency towardslower ascorbic acid contents than do resistant varieties, althoughin individual varieties diseased stock contained larger amounts ofascorbic acid than did the healthy ones.Virus extracts lose much or all of their infectivity after treatmentwith salts of mercury, copper or silver in higher than germicidalconcentrations. In the case of copper sulphate and mercuricchloride dilution of the treated preparations (approximately I in100) results in complete reactivation of recent, but only partialreactivation of aged, preparations. Highly purified protein failedto recover after treatment with silver nitrate.Recent investigationsof these effects by J. C. Went 26 and by J. Caldwell 27 confirm theview that the action of these salts is primarily on the protein ratherthan on the cellular contents of the host plant as formerly suggestedby Stanley.It is a matter of practical interest that virus which may bewashed out from decaying plants into the soil becomes slowlyinactivated therein, aeration and drying being potent factors inthis respect. In undecayed plant tissue, however, neitherdesiccation nor freezing affects the activity of the virus.28Other Viruses.-The above considerations have concerned tobaccomosaic virus only, since this, of the many virus diseases known,has received by far the most extensive investigation on thebiochemical side.Other viruses tend to show similar properties inso far as these are as yet recorded. Much evidence indicates,however, that each virus is not necessarily a distinct entity. Thustobacco mosaic infects not only different varieties of tobacco andeven other species of the same botanical family, but in suchunrelated species as phlox and petunia, in which the normalproteins are widely different, mosaic-infected plants are found tocontain a high-molecular protein indistinguishable from that oftobacco mosaic. More extensive examination in the case of25 Biochem. J., 1937, 31, 1992.2 7 Proc. Roy. Soc., 1936, B, 119, 493.28 I. H. Hoggan and J. Johnson, J. Agrie. Res., 2936, 58, 2'91.26 Phytopath. Z., 1937, 10, 480POLLARD : PLANT.431tomato mosaic by HI. S. Loring 29 shows the indisputable identityof the protein with that of tobacco mosaic.On the other hand, Stanley 30 has isolated from Turkish tobaccothe protein associated with the yellow or aucuba-like mosaic disease.This is very closely related to the ordinary mosaic protein, butforms larger crystals and has a higher molecular weight and higherisoelectric point. It is similar to the ordinary mosaic protein inX-ray pattern31 and is inactivated by similar means. F. C. Bawdenand N. W. Pirie32 find that the protein occurring in mosaic-infected cucumber plants also possesses many of the propertiesof tobacco mosaic protein, but exhibits characteristic differences.The crystalline protein occurring in tobacco “ ring spot ” differsrather more markedly from the mosaic protein, but nevertheless isdefinitely of the same general Tobacco plants aresusceptible to at least four forms of mosaic, the ordinary variety,aucuba mosaic, “ mashed ” mosaic, and a single lesion strain, eachtending to induce the formation of a protein slightly different fromthe others.Inoculation of healthy plants with mixed viruspreparations or secondary inoculation of plants already infectedwith another virus usually results in the gradual dominance of oneof the forms 34 with a corresponding modification of proteinstructure. A form of “mutation ” among the virus proteinstherefore seems likely.Serological.-Essential differences between virus proteins demon-strated by physical or physicochemical means, in some cases maybe more definitely expressed in serological tests.Sera of animals obtained after injection of solutions of tobaccomosaic protein give a precipitin reaction with solutions of theprotein containing as little as g., with the juice from infectedplants but not with that of healthy plants.35 Chemically inactivatedprotein also produces an antiserum causing precipitation ofsolutions containing g.of active or inactivated protein.Inactivation, therefore, although modifying the chemical propertiesof the protein and its infectivity, does not necessarily destroy itsimmunological proper tie^.^^ F. C. Bawden and N. W. Pirie,37 inexamining cucumber viruses, obtained anti-sera giving specific29 H. 8. Loring and W.M. Stanley, J . Bid. Chem., 1937, 117, 733.3O Ibid., p. 325.31 R. W. G. Wyckoff and R. B. Corey, J . Biol. Chem., 1936, 116, 57.3a Nature, 1937, 139, 546.33 W. M. Stanley and R. W. (3. Wyckoff, SC~EYW, 1937,55, 181.94 H. H. McKinney, ibid., 1935, 82, 463.a5 W. M. Stanley, ibid., 1935, 81, 644.36 Idem, W., 1936,83, 626; Phytqvath., 1935, 25, 899.Nature, 1937, 139, 546432 BIOCHEMISTRY.precipitates with Q x 10-6 g. of the protein and point out that onlythose proteins serologically related to tobacco mosaic proteinexhibit anisotropy of flow and form spontaneously birefringentsolutions. Not all viruses induce precipitin reactions : those ofpeach “ yellows,” potmato leaf roll, bean mosaic, and tomato spottedwilt apparently fail in this respect.According to K. S. Chester 38serological activity in viruses is to be associated with relativestability to ageing and to temperature inactivation. On the basisof reciprocal precipitin reactions it now seems possible to classifymany viruses into groups according to their ability to producedefinite group-specific precipitins. Much remains to be done inthis connexion, but evidence already exists indicating that groupspecificity in viruses may ultimately be correlated with thestructure of the high-molecular protein.Apart from its significance in relation to virus diseases as such,current work on this subject brings out a, fundamental point ofmuch interest, vix., that physiological properties hitherto associatedwith living organisms can now be reproduced by a complex proteinmolecule. On this point Stanley 39 comments, “ It is possible thatby virtue of its size, it (the protein) is enabled to possess sufficientorganisation within the molecule to endow it with such properties(of living things).As such it would form a link between the typeof organisation within the molecule with which chemists haveconcerned themselves and the type of organisation within the cellwith which biologists have been concerned. . . , Infection may beregarded as the introduction of a few molecules of a virus proteininto a susceptible host. These few molecules appear to have theability to direct the metabolism of the host so that it produces, notnormal protein, but more of the virus protein.”Biochemistry of Certain Bacteria.The many-sidedproblem of the carbon metabolism of these organisms has beencarried forward by H.Gaffron40 in the case of the red sulphurbacteria Thiocystis. These organisms assimilate carbon dioxide indaylight but not in darkness even in the presence of hydrogen.When the cells have accumulated considerable amounts of sulphur,the evolution of carbon dioxide sometimes observed results fromsecondary metabolism of carbonaceous material. In darkness thebacteria produce hydrogen sulphide at the expense of organicreserves. This is utilised in the subsequent assimilation of carbondioxide in daylight (2C0, + H,S + 2&0 --+ 2CH,O + H,S04).Sulphur Bacteria.-Nutrition and metabolism.38 Phytopath., 1937, 27, 124.40 Biochem.Z., 1934, 269, 447; 1935,270, 1.39 Arner. J . Bot., 1937, 24, 59POLLARD : PLANT. 433Unlike the purple bacteria, Thiocystis is unable to utilise organicsubstances as hydrogen donators in the hydrogenation of carbondioxide, but can effect the reduction of sulphates to sulphides indarkness when supplied with sodium butyrate.In the case of the purple bacteria, Gaffron41 shows that underanagrobic conditions assimilation of carbon dioxide occurs in thepresence of salts of the fatty acids to extents which increase withthe molecular weight of the acid. I n an atmosphere of hydrogen,nitrates, lactates, pyruvates, and glycollates as well as carbondioxide are reduced. These organisms are apparently able toutilise the energy of infra-red light and also to assimilate oxygendirectly.According to P. 9. Roelofsen42 the purple bacteriaappear to assimilate carbon dioxide in darkness in the presence ofhydrogen, the absorption of hydrogen being proportional to theconcentration of carbon dioxide present under these conditions.Other assimilable substances are, however, necessary for thenormal growth of the organism. Oxidisable sulphur compoundssupplement the action of organic hydrogenators and increase therate of assimilation of carbon dioxide. Observed changes inoxidation-reduction potential with alteration of light intensity orconcentration of hydrogen donators are in conformity with thetheory that the bacterial suspension acts substantially as aphotoelectric half -cell. Further observations on this point byD.I. Saposhnikov43 show that the photoreduction of carbondioxide by purple sulphur bacteria is optimal a t rH 14-16 andthat one molecule of carbon dioxide is reduced for each quantumof light energy absorbed. It is noteworthy that in the case of s nentirely different organism, Streptococcus varZans, measurements ofthe photoassimilation of carbon dioxide and hydrogen by C. 8,French 44 indicate that four light quanta are required per moleculeof carbon dioxide.An interesting instance of an organism which can but does notof necessity utilise sulphur compounds in carbon assimilation isexamined by H. N a k a m ~ r a . ~ ~ The sulphur-free purple bacteriumRhodobacillus palustris develops in light or in darkness in thepresence of oxygen, but anaerobic growth is possible only in light.The assimilation process in light appears to involve the productionof an intermediate product which is subsequently decomposed inan oxidative respiration process. Since respiration does not occurin darkness, an external source of oxygen is necessary for41 Biochem.Z . , 1935, 275, 301.42 Proc. K. Akad. Wetensch. Amsterdam, 1934, 37, 660.43 Biochimia, 1937, 2, 181.45 Act4 Phytochim., 1937, 9, 189, 231.44 J . Gen. Physiol., 1937, 20, 711434 BIOCHEMISTRY.continuance of assimilation. In the presence of hydrogen sulphideand certain fatty acids, sulphur and oxidation products of the acidsare formed. It is shown that the primary reduction of carbondioxide takes place a t the expense of the hydrogen of water, theresidual hydroxyl (or hydrogen peroxide) reacting with hydrogensulphide or with hydrogen produced by dehydrogenation of thefatty acids.Specific dehydrogenases of fatty acids are present inthe organisms. The fundamental photosynthetic process, therefore,is identical with that in higher plants, in that carbon dioxide andwater are involved, and hydrogen sulphide and fatty acids areconcerned only in subsequent processes, without being essential forgrowth. In the case of Rhodospirillum giganteurn hydrogensulphide or another oxidisable sulphur compound is essential in theautotrophic culture to act as a hydrogendonator in the hydrogenationof carbon dioxide in light. In heterotrophio cultures organic acidsmay act as carbon sources, but growth is accelerated by additionof thiosulphate.The effects of different sources of sulphur on the growth of thepurple bacterium Ectothiorhodospira mobile are examined by V.A.Tschesnokov and D. I. Saposhnik~v.~~ The optimum p , forgrowth is found to be inversely related to the state of oxidation ofthe source of sulphur, e.g., NaHSO,, 7.4; Na,S,O,, 7.5; S, 8 - 5 ;Na,S, 9.0. This effect is ascribed to differences in Eh set up in themedium by the various sulphur compounds.In a series of papers R. L. Starkey records an investigation ontwo other species of sulphur bacteria, Thiobacillus thioparus andT . novellus, both of which are characterised by their ability toconvert thiosulphate into a mixture of sulphur and sulphate, othersulphur compounds being only slowly, if at all, attacked.In thecase of T. nowellus the action on thiosulphate is marked by anincreased acidity in the substrate, and is accordingly favoured byan alkaline and buffered medium. The two species are furtherdistinguished by the fact that T. thioparus can utilise nitrogen onlyin the form of ammonia, nitrite or nitrate, whereas organic formsof nitrogen are effective for 2’. n o v e l l ~ s . ~ ~ The production ofthionic acids by these organisms is now regarded as the result ofsecondary reactions, and not as an essential part of the principaltransformation .48Although the yellow substance produced by these bacteria fromthiosdphate is very generally regarded as sulphur, 0. von Deines 49had expressed the view that in the case of the larger sulphurbacteria the corresponding substance was really a polysulphide‘ti Bio&mh, 1936, 1, 63, 157.47 J. Bact., 1934, 28, 365.49 Naturwk., 1933, 21, 873. J . @en. Physiol., 1935, 18, 325POLLARD : PLANT. 435of very high sulphur content, on the ground that treatment withacid under reduced pressure yielded hydrogen sulphide. AlsoA. Monti showed that endocellular c'~uIphur droplets " producedsilver sulphidc from the nitrate, although reactions with lead andmercury salts were less definite. After a close examination of thesulphur deposits of T. thioparus Starkey 51 concludes that thesereally consist of elementary sulphur free from sulphide. He finds,however, that under certain conditions T.thioparus and T. novellusmay produce small amounts of hydrogen sulphide by hydrogenationof the initial sulphur accumulation. It would appear that thishydrogenation occurs within the bacterial cells and does not precedethe entry of sulphur into the organism.Among newly recorded sulphur transformations effected bybacteria may be mentioned the production of sulphur from 1-cystineby Achronzabacter cystinovorurn (nov. sp.) when supplied with noother source of carbon, nitrogen, or sulphur,S2 and certain activitiesof a thermal spring bacterium of the Sulphomonas thiooxidansgroup. According to 0. Baudisch, this organism, which producessulphuric acid from sulphur, is extraordinarily resistant to this acidand continues to multiply in N-solutions.I n older cultures aB~-solution of sulphuric acid may be produced in the substrate.Reduction of sulphur to hydrogen sulphide or to a thiol compoundappears to precede the oxidation process. When grown indarkness with a low oxygen tension in cultures containing thymineglycol, the bacterium produces a characteristic red pigment inthe presence of acid. It appears probable that the glycol firstloses water, yielding CO<NH.CH(~H)>C~CH~, NH- co two molecules of, ,which unite to produceoxidation of which yields the red dyePigments of purple bacteria. Purther examination of thecarotenoid pigments of the Bhodovibris by B. Karrer and U.Solmssen 53 has resulted in the isolation of five pigments, zlix.,rhodoviolascene, C42HB002, probably a, dimethyl derivative oflycopene, xhodovibrin, a polyene alcohol, rhodopurpurene, ahydrocarbon, C#&56(58), resembling but not identical withlycopene, rhodopin, an unsaturated substance having one hydroxyl50 Boll.SOC. itctl. Biol. sperirn., 1935, 10, 690.5 1 J. Bact., 1937, 33, 545.68 Helv. Clvim. Acta, 1935, 18, 306; 1936, 19, 3, 1019.52 Svensk Kern. Tidslcr., 1935, 47, 191436 BI 0 CHEMISTRY.group and probably 12 double linkings, and an amorphousflavorhodin, probably a hydrocarbon. From the sulphur-freepurple bacteria E. Schneider 54 has isolated two carotenoids, oneresembling lycopene and the other similar to photoxanthene, theratio of the former to the latter being 0.6. Comparison with theamounts of bacteriochlorophyll previously examined 55 shows theratio of chlorophyll : carotenoids in these organisms to beapproximately 2.75, a value of the same order as that obtaiiiingin the chloroplasts of higher plants.Schneider indicates thecarotenoids are concerned in carbon assimilation.From XpiriZEum rubrum C. B. Van Niel and J. H. C. Smith 56have obtained a purple pigment, spiriZEoxanthin, CP8HS603, a highlyunsaturated substance probably containing an active hydroxyl butno keto-group.An excellent survey of the activities of the sulphur bacteria byH. J. Bunke appeared in 1936.57Marine Bacteria.-A notable interest in the bacterii inhabitingsea water and sea-bottom muds has become apparent in recentyears. Much of the current work is concerned with nutritionaland environmenfal conditions which control t'he distribution ofvarious types of organisms.Sea water contains a much smallerbacterial population than does fresh water, soil, etc. S. A.Waksman and M. Hotchkiss 58 ascribe this to relative deficiency ofnutrients and energy sources, e.g., H,S, S, H,, NH,, NO,, CH,, and,perhaps more notably, to the activity of predatory protozoa, e.g.,nannoplankton. A change in biological equilibrium in sea water iseffected by alteration of the nutrient supply, of temperature, or ofthe state of aeration of the water. C. E. Renn 59 in the course ofsimilar observations emphasises the fact that the temperature of alarge proportion of the ocean is sufficiently low to be unfavournbleto bacterial multiplication and also that particulate nutrient materiallikely to encourage the formation of bacterial colonies graduallysettles to the sea bottom, carrying its bacterial population into anenvironment which in many cases is less suited to developmentthan that in which the initial colonisation occurred.Nitrifying,denitrifying, and nitrogen-fixing organisms are generally distributedin sea water, their proportions being largely influenced by theoxygen content of the water.60 I n an examination of the depth-5 6 Arch. Mikrobiol., 1935, 6, 219.54 Rev. Fac. Sci. Istanbul, 1936, 1, 74.55 2. physiol. Chem., 1934, 226, 221.57 D. 8. I. R., Chern. Res., Spec. Rept. No. 3, 48 pp.5 8 J . Bact., 1937, 33, 85; S. A. Waksman, Ecol. Monog., 1934, 4, 523.K g J . Bact., 1937, 33, 86.6o S.A. Waksman, M. Hotchkiss, and C. L. Carey, Biol. Bull., 1933, '75,137437 POLLARD : PLANT.distribution of bacterial nutrients in sea water A. Krogh 61 recordsthat the organic nitrogen and ca'rbon contents, probably the mostimportant nutrient factors likely to limit development, aresubstantially the same a t all depths. According to S. A. Waksmanand C. L. Carey G2 sea water contains sufficient dissolved organicmatter to carry an extensive bacterial population, but in manycases its utilisation is restricted by the inadequate oxygen supply.Bacterial multiplication, oxygen consumption, and the liberationof assimilable nitrogen sources are found to exhibit a directrelationship. The utilisation of nitrogen-free organic matter, e.g.,glucose, added to sea water is apparently controlled by theavailable supply of nitrogenous matter.Urea-decomposing organisms occur near the shore, especially insurface muds, and are seldom found a t depths greater than 100 m.Three groups are differentiated by C.E. Zobell and C. B. Feltham,63viz., those producing ammonia, and those which do not liberateammonia from urea, both classes developing on media containingurea as sole source of nitrogen, and a third class which multiplyonly when other sources of nitrogen (ammonium salts, amino-acids,peptone) are present in addition to urea.Luminous marine bacteria apparently utilise amino-acids (alanine,leucine, glutamic acid) and require both potassium and sodium fortheir development. Luminosity is intensified by small amounts ofcopper, zinc, iron, or manganese salts, the numbers of organismsremaining unchanged unless calcium chloride also is added.The importance of solid surfaces for the development of ma'rinebacteria is demonstrated in another paper by Z0be11.~~ Storage ofsea water causes a marked increase in bacterial numbers, the extentof the increase being great in those vessels in which the total internalsurface exposed t o the water is high.Addition of solids t o thewater produces a similar effect and microscopic observation revealsa much greater concentration of bacteria on the solid surfaces thanin the free water. Oxygen consumption, denitrification, ammoni-fication and carbohydrate decomposition increase as the area ofsolid surface increases, although these biochemical changes probablyfollow rather than accompany the enlargement of the bacterialpopulation.The effect of the solid surface may be to concentratenutrients and exo-enzymes by surface adsorption or to bring about,in the interstices between the bacterial cells and the solid surface,localised changes of environment which retard the outward diffusionof enzymes from the cells or permit the establishment of pE or ofoxidation-reduction potentials more favourable to development61 Ecol. Mofiog., 1934, 4, 421, 430.83 Science, 1935, 81, 234.e2 J. Bact., 1935, 29, 531.G4 J . Bact., 1937, 33, 86438 BIOCHEMISTRY.than those in the free water. M7. W. Smith and C. E. Zobell65find that glass slides placed in sea water rapidly attract indigenousbut not foreign types of bacteria.An interesting examination of the bacterial population of thebottom mud of the Black Sea is recorded by T.Ginsburg-Karagitscheva and K. Rodionova.66 The mud contains considerableproportiona of bituminous hydrocarbons. Organisms isolatedinclude those which decompose cellulose, proteins, and fats andalso sulphate-reducing types. The course of fat decomposition isrepresented by an increase in the degree of saturation of the acidsand in the proportion of unsaponifiable matter. The nature ofthese organisms and their chemical activities suggest a closerelationship with those occurring in various oil-bearing strata.Production of Organic Acids by Bacteria.-Propionic acid. Themechanism of the transformation of glucose into propionic acid hasbeen extensively examined by C.H. Werkman and c0lleagues,~7according to whom the general scheme involves the changes :hexose --+ hexosephosphate --+ phosphoglyceric acid -+ pyruvicacid. Further changes may take either of two courses :No dead cells are attracted.-lactic acid --+ propionic acidacetic acid + CO,Pyruvic acidsuccinic acidpropionic acid + CO,Whether the hexosephosphate and phosphoglyceric acid representessential intermediates under all conditions is not quite clear.P. Chaix and C. Fromageot 68 demonstrate that the activity ofpropionic acid bacteria is stimulated by certain sulphur compounds,e.g., cystine, methionine, glutathione (oxidised or reduced), thiolacticand thioglycollic acids, Propionibacterium I I being apparentlyunable to act on glucose in the absence of sulphur cornp0unds,6~organic thiocyanates and hydrogen sulphide being particularlyactive in this respect.'O This action of sulphur compounds is65 J .Bact., 1937, 33, 87.H. G. Wood and C. H. Werkman, Biochem. J., 1936,30,618, 624; H. G.Wood, R. W. Stone, and C. R. Werkman, ibid., 1937, 31, 349; C. H.Werkman, R. W. Stone, and H. G. Wood, Enzymologia, 1937, 4, 11, 24; J.Bact., 1937, 33, 100, 102.6e Biochem. Z., 1935, 275, 396.6 s Compt. rend., 1936, 202, 983.7O Idem, Enzymologicc, 1937,1, 321.69 P. Chaix, &id., 1935,201, 857POLLARD : PLANT. 43srelated to the pE of the medium,71 and these authors suggest thatthe enzyme system of the living organism is inactivated byoxidation, the sulphur compounds exerting a protective action.7?The lactic-fermenting system is less affected by oxygen than thatof the other stages except that concerned in the decomposition ofpyruvate, which seems uninfiuenced.Several communications show the importance of nitrogennutrition and of certain growth factors in the activity of thepropionic acid organisms.Thus C. Fromageot and P. Laroux 73record that maize contains a growth factor necessary to enablecertain of this group of organisms to utilise ammoniacal nitrogen.It is also shown 74 that, whereas several species of Propionibacteriautilise ammonia (as ammonium acetate) in the presence of palentaextract, other species fail even under these conditions through lackof an essential growth factor; in nearly all cases the efficiency ofutilisation of ammoniacal nitrogen is dependent on the nature ofthe carbon source.V. G. Lava, K. ROSS, and K. C . Blanchard 75indicate that a factor stimulating the fermentative action ofpropionic bacteria also occurs in the vitamin-B, complex. WithPropimibacterium pentoaceticum fermentation is markedly increasedby small additions of orange juice, yeast extract or potato extract,the first two named appearing to contain the necessary growthfactors. The action of the latter is due to its ammonia andasparagine contents and is operative only in the presence of growthfactors occurring in extracts of maize or liver.76 According toH. G. Wood, E. L. Tatum, and W.H. Peterson 77 the factor appearst o differ, both chemically and biologically, from other knowngrowth factors, e.g., hepatoflavin, vitamin-B,, pantothenic andindolylacetic acids, inositol, nicotinamide, and the ‘‘ X’orogenesvitamin.” In another paper 79 it is recorded that certainstrains of these bacteria producing weak growth in the absence ofamino-acids develop freely and produce more acid when suppliedwith lactoflavin.Production of lactic and other acids. The production of opticallyactive lactic acids and the transformation of one isomeride into the71 P. Chaix and C. Fromageot, Bull. Soc. Chim. biol., 1936, 18, 1436.73 C. Fromageot and P. Chaix, Enzymologia, 1937, 4, 11, 769.73 Bull. Soc. Chim. biol., 1935, 18, 797, 812.74 C.Fromageot and E. L. Piret, Arch. Mikrobiol., 1936, ’7, 551.75 Philippine J . Sci., 1936, 59, 493.76 E. L. Tatum, W. H. Peterson, and E. B. Fred, J. Bact., 1936, 32, 157;77 Ibid., 1937, 33, 227.78 A. M. Pappenheimer, jun,, Bwchem. J., 1935, 99, 2057.7@ H. G. Wood, A. A. Anderson, and C. H. Werkman, PTOC. 800. Exp.also E. L. Tatum, H. G. Wood, and W. H. Peterson, ibid., p. 167.BWZ. Med., 1937, 36, 217440 BIOCHEMISTRY,other by bacteria have long proved a source of interest. Recentwork by H. Katagiri and K. Kitahars 80 shows that S. lactis bulgarisproduces relatively more of the d- than of the l-acid even when thedl-acid is added to the fermenting glucose medium. On the otherhand, Lactobacillus pentoaceticus gives the dl-acid from glucose, andwhen either active form is added to the substrate the final productis still the inactive form.The same authors also record theracemisation of the active acids by Clostridium acetobutylicum, anaction ascribed to the presence of the enzyme racemiase. That Rand S forms of individual species may produce acids of differentoptical properties is shown by L. M. Kopeloff and N. Kopeloff.*lThe X forms of L. acidophilus and L. bulgaricus yield d-lactic acid,whereas the R forms produce the dl-acid for some time, althoughthe latter organism begins to produce d-acid in older cultures.L. G. Longsworth and D. A. McInnes,S2 in an investigation ofthe activities of L. acidophilus in media mainta.ined at constantpH, find that acid yields are highest with low pH and that for agiven pH level the production of acid reaches a minimum when theoxidation-reduction potential of the culture is high.The rates offermentation and of growth a t a constant pE tend to be inversely1-elated.8~ Conversely the growth of the culture is accompaniedby an increased Eh, which finally attains a constant andcharacteristic value.84 In a somewhat similar investigationR. W. H. Gillespie and L. P. Retger 85 show that the reversion ofEn of the culture during growth coincides with the period of mostrapid change of pE. I n buffered media these authors record thatthe final Eh reached is sufficiently specific (cf. Longsworth andMcInnes, above) to permit differentiation of different strains oforganisms, e.g., oral, intestinal, etc.Growth-promoting substances are essential t o the developmentof lactic acid-producing bacteria, streptococci included.Accordingto S. Orla-Jensen 86 the active substance is alkali-stable, is relatedto bios, and is probably pantothenic acid. In some caseslactoflavin also is required and for this purpose cannot be replacedby glutathione. In the case of L. delbriickii, E. E. Snell, E. L.Tatum, and W. H. Peterson 87 find that active growth in caseinhydrolysates containing tryptophan requires the presence of twounknown growth factors : one, possibly an acid of low molecularweight, occurs in the Neuberg filtrate of water extracts of potato,80 J . Agric. Chew. SOC. Japan, 1936, 12, 96.81 Ibid., 1935, 29, 695.84 Ibid., 1936, 32, 567.86 Nature, 1935, 135, 915; also S.Orla-Jensen and A. Snog-KjEr, Zentr.Bakt. Par., 1936, 11, 94, 434.87 J . Bact., 1937, 33, 207.J . Bact., 1937, 33, 331.83 Ibid., 1936, 31, 287.85 Ibid., 1936, 31, 14POLLARD : PLANT. 441and the other, a basic substance, is present in peptone. Bothfactors are found in liver extracts and are hydrolysable by acids.A. K. Sain 88 had previously observed that acid production by L.caucasica was stimulated by a dialysable, thermostable constituentof aqueous extracts of beans.Factors influencingthe production of gluconic acid by B. gluconicurn are examinedby S. Hermann and P. N e u s ~ h u l . ~ ~ The yield of acid variesconsiderably with the concentration of glucose supplied and withthe temperature of fermentation.In general a rise in temperaturediminishes the yield (yo) and, simultaneously, the optimum sugarconcentration. This organism also effects the oxidation of mannoseto mannonic acid, yields of 60-70y0 being obtainable. Furtheraction of B. gluconicum on calcium gluconate produces smallamounts of 2- and 5-ketogluconate and an aldehydogluconic acid,probably Z-guluronic acid. The last is also a product of the actionof B. xyZinzcrn on gluc0se.~1 T. Takahashi and T. Asai92 find,among the acid-producing bacteria of fruits, species of the gluconicacid type which convert galactose into galactonic and comenicacids, and another, Gluconoacetobacter cerinus var. ammoniacus,which oxidises glycerol to an unidentified ketogluconic acid,probably via dihydroxyacetone and succinic or glycollic acidwithout the intermediate production of glyceric and acetic acids.I n subsequent work Asai 93 shows that the optimum pH for gluconicacid production by this group of organisms is less than that formultiplication, and further that the typical biochemical changesbrought about are related to the optimum growth temperatures ofindividual species.Those growing freely a t relatively hightemperatures oxidise glucose to gluconic or glycuronic acid andacetic acid to carbon dioxide and do not act on glycerol or mannitol.Low-temperature species produce glycuronic or S-ketogluconicacid from glucose, kojic acid from niannitol (via fructose),dihydroxyacetone from glycerol, and do not ferment acetic acid.Acetone-Butanol Fermentation.-The mechanism of this type offermentation offers a problem of considerable complexity, sincethe nature and proportion of the products exhibit wide variationwith conditions of fermentation and with the nature of thecarbohydrate source.Thus K. Bernhaixer and K. Kurschner 94Bacteria producing gluconic and related acids.88 Milcrobiologiya, 1933, 2, 266.90 S. Hermann and P. Neuschul, BUZZ. SOC. Chim. bid., 1936, 18, 390.91 K. Bernhrtuer and K. Irrgang, Biochem. Z . , 1935, 280, 360; H.92 J . Agric. Chem. SOC. Japaw, 1934, 10, 604.93 Ibid., 1935, 10, 50; 11, 331, 337, 499, 610, 674.04 Biochem. Z., 1935, 280, 379.89 Biochem. Z., 1936, 287, 400.Bernhauer and B. Gorlich, ;bid., p. 367442 BIOCHEMISTRY.record that CE. butyricus in fresh cultures converts acetic acidalmost quantitatively into acetone, but in later stages yieldsincreasing amounts of ethyl alcohol.Butanol production fromstarch appears to take place via the intermediate formation ofbutyric acid. The suggestion that acetaldol or P-hydroxybutyricacid is a precursor of butyric acid in this process is negatived bythe fact that these compounds do not yield butyric acid whenadded to the actively fermenting cultures. On the other hand,addition of crotonic acid resulted in its partial transformation intobutanol with simultaneous formation of acetone and carbondioxide.In a subsequent investigation K. Bernhauer, A. Iglauer, W.Groag, and R. Kottigg5 observe that the presence of excess ofcalcium carbonate in the cultures alters the proportions if not thecharacter of the principal products, in general favouring theformation of butanol and butyric acid at the expense of acetone,acetic acid, and ethyl alcohol.Under these conditions butaldehydeis largely converted into butanol, whereas pyruvic, crotonic, andlactic acids still produce large amounts of acetic acid. Althoughthe presence in CZ. acetobutyZicum of an enzyme system capable ofreducing propionic and butyric acids to the corresponding alcoholsis fairly well established, the acceptance of butyric acid as anintermediate in the normal fermentation is by no means general.96A. Janke and V. Siedler 97 advance evidence that the formationsof butanol and of acetone by B. acetobutylicum are independentprocesses.Suspensions of washed bacilli at pE 6.0 producebutanol, butyric and acetic acids and ethyl alcohol but no acetone,but convert aldol into butyric acid without the production ofbutanol or acetone. Moreover, under these conditions acetone isnot formed from acetic acid, nor butanol from butyric. Reductionof butyric acid to butanol may occur outside the cell.Weizmann’s strain of CZ. acetobutylicum offers another exampleof the necessity of a growth-substance in association with anamino-acid for the development of bacteria. In this case,asparagine together with a constituent of yeast autolysate appearsto be essential. The active substance is neither lactoflavin norcozymase and is possibly one which is not usually included in thiscategory of biocataly~ts.~~In the commercial production of these solvents from maize mashit is found that CZ.acetobutylicum can utilise xylose, if this does not95 Biochem. Z., 1936, 287, 61.96 B. Rokusho, J . Agric. Chern. SOC. Japan, 1936, 12, 639.87 Biochem. Z., 1937, 292, 101.98 C. Weizmann and B. Rosenfeld, Biochem. J., 1937, 31, 619POLLARD : PLANT. 443exceed 40% of the mash mixture, and still produce high yields.99Bcetoin, probably formed by condensation of two molecules ofacetaldehyde, appears to be an intermediate product in theconversion of starch into acetone and butanol by B. granulobacterpectinovorunz .IBiochemistry of iioulds.Mineral Nutrition of Aspergillus niger.-The action of pota.ssiumin the nutrition of moulds appears to be directed, as in the caseof the higher plants, towards regulation of the carbohydratemetabolism.Additional evidence on this point is given by 0.Kauffmann-Cosla and R. Brull,2 who, using A . niger on Raulin'smedium, show that it exerts a catalytic effect on celluloseproduction and simultaneously inhibits the formation of lipinsfrom carbohydrates. No influence on the course of nitrogenmetabolism was apparent. A. Rippel and G. Behr3 examine theenergy relationships of the mould grown in media containingvarious levels of supply of potassium. An optimum concentrationof potassium is established, below which the energy consumptionper gram of mycelium produced falls steadily.Relationships between the development of A . niger and thesupply of magnesium form the subject of two pa'pers by J.Lavollay and F.Lab~rey.~ I n Raulin's medium growth dependson the concentration of magnesium in the medium rather than onthe absolute amount supplied. Pigmentation of the myceliumreaches a maximum when the concentration of magnesium in thesubstrate is less than the optimum for growth and is apparentlyaffected by the presence of ascorbic acid, which tends to restrictthe production of pigment. Ascorbic acid increases mycelial growthto extents which depend on the concentration of magnesiumavailable.*Secondary Nutrients.-The influence of zinc on the developmentof A . niger is directed primarily on vegetative growth, and modifiesthe course of metabolism only as a secondary effect. Therespiratory activity undergoes no change.G. M. Vassiliev showsthat with strains producing gluconic acid (alone or with citric acid)zinc restricts acid production, but ha's a stimulatory effect on otherv9 L. A. Underkofler, L. M. Christensen, and E. I. Fulmer, Id. Eng. Chem.,1036, 28, 350.1 I. Yamasaki and T. Karasima, Ernzymologia, 1937, 3, 271.2 Bull. SOC. Chim. biol., 1937, 19, 137. Arch. Milcrobiol., 1936, 7, 315.Compt. rend., 1937, 204, 1686; 205, 179.Arch. Mikrobiol., 1935, 6, 250. * H. N. Barham and B. L. Smits (p. 448) also report that pigmentationof A. flavus in xylose media is also influenced by the concentration ofmagnesium in the substrate444 BIOCHEMISTRY.strains which produce citric acid only. 0. Kauff mann-Coslal andR. Briill also observe that zinc deficiency results in n diminutionof synthesis of carbohydrates and also that of lipins and proteins.It is usually assumed that iron, unlike zinc, is concerned in thefructification rather than in the vegetative growth of A.niger.According to F. G~llmick,~ however, both zinc and iron, and alsocopper, may, under certain conditions, stimulate both inycelialgrowth and spore formation. Inter-relations between zinc andiron in respect of fungal activity are further illustrated by theobservation that iron partly counteracts the inhibitory influence oflarger dosages of zinc on fructification, but tends to increase itsaction on vegetative growth, Iron also diminishes the toxic effectsof cadmium salts.The inferior growth of A.niger on media containing suga,rpurified by alcohol (to remove bios co-enzyme R, etc.) is ascribedby R. A. Steinberg to the incidental removal of traces of zincand molybdenum salts during the purification process. It is alsosuggested that the stimulation of growth observed on the additionof yeast, malt, etc., to culture media, normally to supply accessorygrowth substances, is in reality due to the heavy-metal salts whichthese materials contain, and that growth-promoting substancesmay not be essential for the growth of the mould.Steinberg records the influence of varying proportions of zinc, iron,manganese, and copper on the growth of A . niger and indicates thatoptimum concentrations are in each case greater in media havingpH > 8. In media containing all four elements, sulphuric acid,sodium sulphate, and sodium sulphide induce a further increase ingrowth.Subsequent partial precipitation of the sulphate bybarium (itself having very little toxic action) induces deficiencysymptoms resembling those due to deficiency of nitrogen,phosphorus, magnesium, iron, or zinc. The importance of sulphurin the growth of A . niger is shown by A. Rippel and G. Bohr,lO whorecord that in potassium sulphate media the intake of sulphur ishigh and that autolysis of the mycelium results in the liberation ofcorrespondingly large proportions of organic sulphur compounds.That the intake of sulphur under these conditions is not a necessaryresult of the assimilation of potassium is shown by the fact that,when potassium is supplied as chloride instead of sulphate, nocorrespondingly large intake of chlorine occurs.A further comprehensive study l1 of the relative stimiilntory andI n other papersBull.XOC. Clziw~ b i d , 1935, 17, 1828.7 Zentr. Bakt. Par., 1936, 11, 93, 421.6, Amer. J . Bot., 1936, 23, 227; Bot. Gr'az., 1936, 9'7, 666.lo Arch. Mikrobiol., 1936, 7, 584.J . Agric. Res., 1936, 52, 439.l1 Ann. Reports, 1935, 32, 442POLLARD : PLANT. 445toxic effects of numerous metallic compounds on the growth of A .niger is reported by K. Pirschle.12 In general, metals in the anionicform of combination are more toxic than when they form thecations of salts. I n the cases examined, metals forming more thanone series of salts are more toxic in the higher state of valency.Production of Citric and Oxalic Acids by Aspergillus.-As notedin previous Reports,13 the conversion of sugars and of other organicacids into citric and oxalic acids, although examined extensivelyfrom many viewpoints, still lacks an explanation of its mechanismwhich covers anything approaching all the observed facts.Published work during the past two years has perhaps tendedmore to refute certain earlier theories than to contribute new ones,although it has in many respects clarified a number of points onwhich experimental evidence appeared contradictory.Thus T.Chrzpzcz and M. Zakomorny,14 in pursuance of thetheory that the transformation hexose + citric acid occurs byway of ethyl alcohol and acetic acid, report a further examinationof the conversion of calcium acetate into citric acid, and show thatthis change is favoured by the addition of small amounts of sugarto the medium, irrespective of the formation of oxalic acid.Enfeebled mycelium produces little citric but much oxalic acid.These authors reject the view that citric acid is derived frominycelial substance and also fail to detect the presence of aconiticacid, suggested l5 as an intermediate product in the formation ofoxalic acid.I n a further paper l6 it is shown that the addition ofsodium E-malate to calcium acetate media increases citric acidproduction, and that further increases occur if both sugar andmalate are present. The theory is advanced that both rna'lic andacetic acids are concerned in the conversion of sugar into citricacid. Chrzqszcz and Zakomorny also find l7 that prolongedpropagation of the mould leads to a form of degeneration markedby a diminished ability to produce citric acid, which is more andmore replaced by oxalic acid.Such degenerate strains regain, a tleast temporarily, their citric-producing power when propagated onglucose or sucrose media, the regeneration being favoured by thepresence of malt wort containing peptone. Guanidine and ureaappear to increase the formation of oxalic acid.R. Bonnet and J. Jacquot,l* following up earlier work,demonstrate the influence of the nitrogen nutrition of the mould12 Plccnta, 1935, 24, 649.14 Biochem. Z., 1936, 285, 340.15 K. Bernhauer and F. Slanina, Biochem.Z., 1933, 264, 109; 1934, 274, 97.16 Ibid., 1936, 285, 348.18 Compt. rend., 1935, 201, 1213.la Ann. Reports, 1935, 32, 444.Ibid., 1937, 291, 312446 BIOCHEMISTRY.on acid production, and show that with peptone or amino-acids asnitrogen source both oxalic and citric acid are formed, growth andyield of citric acid reaching a maximum after about four days andsubsequently remaining practically unchanged. Oxalic acid con-tinues to be formed over a considerable period. All commonsugars (lactose excepted) yield both acids in nitrate media, butoxalic acid was absent from all cultures in which nitrogen wassupplied as ammonium salts.The latter observations suggest that the proportions in whichcitric and oxalic acids are produced by A. niger are influenced bynutritional conditions and by the physiological state of the culture.Further evidence on this point is presented by R.Baets16,1g whoreports maximum yields of citric acid in 0.2% ammonium nitratemedia containing ZOyo of sucrose at pE 1~8-2.2. A similar resultis recorded by S. A. Barinova.20 Prolonged fermentation athigher temperatures (30") with increased concentrations ofammonium nitrate in the medium diminishes citric acid formationand results in the appearance of oxalic and gluconic acids.E. M. Johnson, E. C. Knight, and T. K. Walker 21 examine acidproduction from a different angle. Addition of small amounts ofsodium iodoacetate to mycelium of A . niger in glucose mediaincreases the rate of utilisation of sugar and the yield of acid.Larger concentrations of iodoacetate, sufficient to suppress sporeformation, also prevent the anaerobic production of ethyl alcohol,but citric acid accumulation remains unaffected.Citric acidproduction may not involve an alcoholic type of fermentation.An interesting study of the carbon balance of A . niger in relationto the glucose-+ citric acid mechanism is recorded by P. A.Wells, A. T. Moyer, and 0. E. Yields of citric acid andcarbon dioxide are found to be incompatible with any chemicalmechanism analogous to the breakdown of glucose in alcoholicfermentation, with any process involving decarboxylation ofpyruvic acid, or with Emde's mechanism.The eifect on yields of acid of neutralising the culture mediumduring fermentation is emphasised by V.A. Kirsanova,23 whofinds that with all strains examined on sucrose media acidformation ceases at pH < 3 and > 8. Addition of alkali duringfermentation increases the final yield of acid, citric, oxalic, andgluconic acids all being concerned. The efficiency of neutralisingagents was in the order CaCO, < NaOH < Na,CO,. When thereaction is thus controlled, the relative yield of oxalic acid declines19 Natuurwetensch. T;jds., 1937, 19, 5.20 Zentr. Bakt. Pm., 1936, 11, 95, 63.za J . Amer. Chem. SOC., 1936, 58, 555.21 Biochem. J., 1937, 31, 903.23 Biochimia, 1936, I, 426POLLARD : PLANT. 447with increasing time of fermentation. Frequent change of thepellicle results in increased yields of citric acid, and the interestingpractical fact emerges that with 35% sucrose solutions as much as90% of the theoretical yield of citric acid may be obtained bymaintaining the pE of the culture within the range 4-6The proposed mechanism of the production of oxalic acid viaformic acid2* is to some extent challenged by V.S. Butkevitschand L. K. Osni~kaja,~~ who observe that at 30” the yield of oxalicacid is’unaffected by the concentration of formate in the mediumand is only slightly altered by replacement of the formate bysodium carbonate. Transference of the mycelium to disodiumhydrogen phosphate buffer solutions results in a rapid accumulationof oxalic acid (up to 70% of the loss in weight of the mycelium) ina few days. The now exhausted mycelium is incapable of producingoxalic acid from formate, but when placed in sodium acetatesolution rapidly produces oxalic acid without change in its ownweight.It is suggested that oxalic acid is normally produced frommycelial substance and not from the formate, and that acetatemay be concerned in the process. This recalls Challenger’s earliertheory that the change acetate-+ oxalate takes place with theintermediate production of glycollic and glyoxylic acids. Such amechanism is now opposed by T. A. Bennet-Clarke and C. J. LaTouche26 on the grounds that glycollic acid supplied directly toactive mould cultures was rapidly utilised, but no oxalic acid wasformed. Similar results with citric acid also ruled this out as anintermediate in oxalic acid productions.In a recent paper A.Allsopp 27 throws further light on a numberof controversial points. He demonstrates a reversible equilibriumbetween oxalic acid and the reserve carbohydrate (probablyglycogen) of the mycelium. Artificially increased amounts ofoxalic acid in glucose media disappear until the same level of acidis attained as is produced by the action of the mould on glucosealone. Addition of oxalacetic, malic, succinic, pyruvic, glycollic,and other 4-, 3-, and 2-C acids to starved cultures is followed byconsumption of the acids, without corresponding production ofoxalic acid. It is unlikely, therefore, that these acids areintermediate in oxalic acid production, as has been suggested byvarious workers from time to time. The formation of oxalic acidwhen salts of the various above-mentioned acids are added toculture media is explained by a disturbance of the glycogen +glucose + oxalic equilibrium by the “trapping” of oxalic acid24 Ann.Reports, 1935, 32,44A.26 Compt. rend. Acad. Sci. U.R.S.S., 1936, 1, 361.26 New PhytoJ., 1934, 54, 211. 2 7 I b X , 1937,38, 327448 BIOCHEMISTRY.by bases liberated as the anions are assimilated by the mould. I nthe case of oxalacetate, pyruvic acid and carbon dioxide are theprincipal products, and only at a much later stage is there anyproduction of oxalic acid, which, even then, is formed in amountstotally unrelated to the proportion of oxalacetate consumed. It isconcluded, therefore, that the source of oxalic acid is the glycogenor other reserve carbohydrate of the mycelium, and that the processoccurs via sugar (glucose, fructose, galactose, arabinose, and xyloseyield oxalic acid in starved cultures} and organic acids other thanthose referred to above.Among many acids examined by additionto starved cultures, only gluconic acid yielded oxalic acid. Thetheory is advanced that oxidation of the two end carbon atoms ofa sugar molecule containing a t least 5 C produces a keto-acid andby further hydrolysis, oxalic acid ; e.g., glucose ----+ gluconicoxidationhydrolysisacid j fructuronic acid- oxalic acid + 3 or 4-C residue.It is further shown that certain organic acids, e.g., lactic acid,inhibit the accumulation of oxalic acid in mould cultures, an effectwhich is not due merely to change of p,, since hydrochloric acidin amounts to produce similar pH changes has no inhibitory action.The observation, however, affords some explanation of theapparently contradictory results obtained in cultures t o whichvarious organic acids have been added.Fermentation of Pentoses and Other Substances by VariousAspergillus Xpecies.-T. Tadokoro,28 in an investigation of sugarfermentations by A .oryxce, finds that arabinose yields principallyformic, citric, and glycollic acids with smaller proportions o€oxalic and kojic acids and glyceraldehyde. From fucose areobtained formic, glycollic, and lactic acids. I n neither case wasacetaldehyde, acetone, acetic acid, or ethyl alcohol detected. Inthe presence of a hydrogen acceptor, e.g., methylene-blue, fucoseyields maleic in place of glycollic and lactic acids.29 From xylose,H.N. Barham and B. L. Smits30 obtained considerable amountsof kojic acid (the principal acid formed) by the action of A. jlavus,utilising appropriate nutritional conditions. Optimum yields areassociated with pH 2.5-3.5 and the use of ammonium salts ratherthan nitrate as nitrogen source. The presence of zinc salts,previously observed to stimulate the activity of the mould, has noinfluence on the yield of kojic acid and iron and calcium saltsexert an inhibitory action.J. Cheymol 31 reports the fission of a glucoside, verbenaloside28 J . Agric. Chem. SOC. Japan, 1935, 11, 167, 366.29 T. Tadokoro, Bull. Chem. Xoc. Japan, 1936, 11, 239.3O Ind. Eng. Chern., 1938, 28, 667.31 Bull. SOC. Chim biol., 1937, 19, 460449 POLLARD : PLANT.into verbeiialol and glucose, by A. niger and W. €3. Deys and M. J.Dijkman32 obtained gallic acid from theotannin in the presence ofsugars with the same organism. Other carbohydrate transforxn-ations recently examined include the production of d-mannitol byA . glaucus when supplied with glycerol as sole source of carbon.33Production of Fats and Sterols by Moulds.-The formation offatty matter in moulds is usually associated with high levels ofsupply of carbon and nitrogen in the nutrient and with rapidproduction of mycelium. Under these conditions protein synthesisis considerably restri~ted.3~ A summary of data illustrating therelation between nutrient conditions and yields of fat from mouldsand bacteria is given by W.Schwartz.35According to K. Taufel, H. Thaler, and H. Schreyegg36 the fatof certain species of Citrornyces contains principally oleic andlinoleic esters and lesser proportions of stearic and palmitic esters.No acids between C, and C,, are detectable. The unsaponifiablefraction includes ergosterol.The crude fat of Rhixopus japonicus contains the same principalfatty acids, but in rather different proportions (stearic and linoleicacids low) and also yields ergosterol with a rather larger proportionof fungister01.3~ A further examination 38 of sterol production byAspergillus Jischerei is reported by P. R. Wenck, W. H. Peterson,and E. B. who indicate a general inverse relation betweenthe rate of growth of mycelium and its sterol content.The lattertends to increase with the sugar content of the medium and reachesthe maximum when the nitrogen supply is low. %'ormation ofsterols appears to be accelerated during the autolysis of mycelium.Mycelium of Aspergillus sydowi contains 0.4-0.7 yo of phospholipins,chiefly lecithin and ke~halin.~ONew Products of Mould Metabolism.-Continued investigationsof the metabolism of moulds by Raistrick and his colleagues duringthe period under review include the examination of certain productsof ,4spergiZlus terreus. On glucose media containing potassiumchloride as chlorine source this organism produces two opticallyactive chlorine-containing substances, wix., geodin, C1,H,,O,CI,,32 Proc. K . Akad. Wetensch.Amsterdam, 1937, 40, 618.33 I. Yamasaki and M. Simomura, Biochem. Z., 1937, 291, 240.3Q H. Fink, G. Haesler, and M. Schmidt, 2. Spiritusind., 1937, 60, 74, 76,35 Angew. Chem., 1937, 50, 294.37 H. Lim, J . Pac. Agric. Hoklcaid~, 1935, 3'9, 165.38 See Ann. Reports, 1935, 32, 447.39 Zentr. Bakt. Par., 1935, 11, 92, 330.413 D. W. Woolley, F. M. Strong, W. El. Peterson, and E. A. Prill, J . Amer.81.30 Fette u. Seqen, 1937, 44, 34.Chem. Xoc., 1935, 57, 2689.REP.-VOL. XXXIV. 450 BIOCHEMISTRY.m. p. 235", a dibasic acid containing two methoxy-groups, anderdin, C16Hlo07C12, m. p. 229", also a dibasic acid but containingonly one methoxy-group. Substitution of iodide or bromide forchloride in the medium failed to produce corresponding halogenatedcompound^.^^ Geodin appears to be a methyl ether of erdin.42P.W. Clutterbuck, H. Raistrick, and 3'. Reuter43 have isolatedanother metabolic product of the same organism, wix., terrein,CsHloOI, m. p. 127", probably 2-hydroxy-3 : 5-oxido-4-propenyl-cycZopen%an- 1 -one.From Penicillium paEitans 5. H. Birkinshaw and H. Raistrick 44have obtained palitantin, C14H2a04, m. p. 163", an unsa'turatedsubstance containing an aldehydo- and two hydroxyl groups.Penicillic acid, CsHl0O4, produced by P. puberulum and P. cyclopiurn,is now shown 45 to be y-keto-P-methoxy-6-methylene-Aa-hexenoicacid, which can exist in both keto and enol forms :CH,:CMe*CO*C( OMe):CE*CO*OH f CH2:CMe* (OH)*C( OMe):CH*qOThe same workers also record *6 the isolation of terrestric acid, anethylcarolic acid:' from P.terrestre.Ravenelin, a yellow colouring matter of a somewhat unusualtype among natural products, wix., a hydroxyxanthone, probably1 : 4 : S-trihydroxy:3-methylxanthone, has been obtained 48 fromthe metabolic products of Helminthosporiurn ravenelii and H .turcicum.From Fusarium culmorum four new pigments are r e p ~ r t e d , ~ ~vix., rubrofusarin, ClsH120s, rn. p. 210-211" (one methoxyl group),norrubrofusarin, m. p. > 280", aurofusarin, CsoHzoOll,HaO, m. p.> 360" (2 methoxyls), and culmorin, C1,H2,O2, m. p. 174". H.Lim 60 records the isolation, from the constituents of Rhixopusjaponicw, of a new phosphoprotein, rhizopenin, which on hydrolysisyields considerable proportions of tyrosine and tryptophan and inwhich 5-6% of the total sulphur content is in the form of cystine.A brief review of twelve years' developments in the study of thebiochemistry of moulds, more especially of the work of H.Raistrickand co-workers, is recorded by P. W. Clutterbu~k.~~41 H. Raistrick and G. Smith, Bwchem. J., 1936, 30, 1315.42 P. W. Clutterbuck, W. Koerber, and H. Raistrick, ibid., 1937, 81, 1089.43 Ibid., p. 987. 44 Ibicb., 1936, 30, 801.45 J. H. Birkinshaw, A. E. Oxford, and H. Raistrick, ibid., p. 394.46 J. H. Birkinshaw and H. Raistrick, ibid., p. 2194.4 7 See Ann. Reports, 1935, 32, 447.48 R. Raistrick, R. Robinson, and D. E. White, Biochem. J., 1936,30, 1303.49 J. N. Ashley, B. C. Hobba, and H. Raistrick, ibid., 1937, 31, 385.W J .Fac. Agric. Hokkaido, 1935, 3'7, 165.51 J . SOC. Chem. Id., 1936, 55, 5511.F,-POLLARD : PLANT. 451Biochemistry of Certain A l g aNutrition and MetaboEis,m.-Recent communications 011 thissubject are mainly concerned with the assimilation of carbon andnitrogen and with the metabolic products. Among species ofChZoreZZu examined by T. D. B e ~ k w i t h , ~ ~ considerable differencesexist in ability to utilise various forms of nitrogen. Ampply ofwhole protein is not generally essential for development and one ormore of the simpler forms (peptone, asparagine, urea, nitrate, andammonium salts) are normally effective sources of nitrogen. H.Meyer 63 examines the growth of C . Zutesviridis in media containinga wide range of organic substances.Among the simpler aliphaticalcohols, aldehydes, and acids, the lowest member is toxic in eachcase, the second member is of high nutritional value, and highermembers are utilised less and less readily as the series is ascended.I n general iso- and unsaturated acids are toxic, but p-hydroxybutyricacid is readily assimilated. Hexoses form the best sources ofcarbon, and polysaccharides have a value which is closely dependentin the ease of their hydrolysis. According to Beckwith (Zoc. cit.)only maltose and glucose, among common sugars, favourproliferation in diffuse light.W. H. Pearsall and L. Loose 54 distinguish two phases in themetabolism of Chlorella vulgaris on glucose media. I n the firststage, i.e., that of exponential proliferation, the synthesis of proteinand of protoplasmic constituents predominates.I n the secondstage the rate of increase in cell numbers slackens and is replacedby cell extension, this stage being associated with a form ofmetabolism characterised by accumulation of carbohydrates andcell-wall constituents.The interdependence of carbon and nitrogen nutrition is shownby H. L. White 55 in Lemnu. When grown with insufficiency ofpotassium, the alga accumulates large proportions of starch anddry matter per unit leaf area, but the gross rate of carbonassimilation is restricted and amylolytic activity is low. Deficiencyof nitrogen also results in carbohydrate accumulation and lowamylolytic power, the former, under these conditions, being theresult of restricted multiplication (and therefore of sugar con-sumption) and diminished respiration.The metabolism of the colourless soil-inhabiting alga Prothecaxopfli is examined by H. A. Barker.s6 Complex organic forms ofnitrogen (e.g., yeast autolysate) are apparently essential for growth,63 Publ. Univ. California Biol. Sci., 1933, 1, No. 1, 1.53 Biochem. Z., 1936, 283, 364.54 Proc. Roy. Soc., 1936, B, 121, 451.66 J . Cell. C m p . Phy.&ol., 1936, 7, 73; 1936, 8, 231,66 Ann. Bot., 1936, 60, 403452 BIOCHEMISTRY.although some ammoniacal nitrogen is also assimilated. Hexoses,certain alcohols, and fatty acids but not hydroxy-, keto-, or dibasicacids, are utilised. Under aerobic conditions glucose is convertedinto cellular matter, largely glycogen, and carbon dioxide, but inanaerobic cultures the principal product is lactic acid. 50-80~0of the carbon entering the alga is assimilated.Nitrogen fixation by the blue-green alga Nostoc rnuscorum isshown by F. E. Allison, S. R. Hoover, and H. J. Morris 57 to bemuch more intensive than that by other species of this group.Both nitrogen and carbon can be obtained from the atmosphere,and growth and nitrogen fixation proceed normally in solutions ofmineral salts. Addition of sugars to the substrate, however,markedly increases the rate of growth. Moreover, the simplerforms of combined nitrogen (nitrate, ammonia, asparagine) areoften utilised more readily than is atmospheric nitrogen. Innitrogen- free media in darkness chlorophyll is produced andgrowth and fixation of nitrogen proceed slowly, 10-12 mg. ofnitrogen being fixed per g. of glucose consumed. I n unaeratedcultures carbon may be obtained from carbonates, although thealkalinity developing in the medium may ultimately kill the cells.The nitrogen-fixation process requires the presence of calcium,although growth of the alga proceeds with no more than a trace ofthis element. The beneficial effect of humus materials on thedevelopment of the organism is ascribed to its iron content andsuggests that the mechanism of nitrogen fixation resembles thatoccurring in Axotobacter.S. Endo 58 presents evidence that glucose is the first product ofphotosynthesis in the green alga Codium Zatum, a starch-producingorganism, whereas in Chadophora Wrightiana, which contains nostarch, fructose is the first detectable sugar. The influence oforganic substances on the respiratory activities of various algae isto some extent affected by their type and molecular complexity.A. Watanabe 59 records that increased respiration in ChZoreZZaellipsoidea follows the addition to the substrate of aldehydes,polyhydric alcohols, carbohydrates, amino-acids, and somecarboxylic acids (not aliphatic). Green, brown, and red marinealga are not, in general, affected by aldehydes, alcohols, orcarbohydrates. With ChZoreZZa and the green and brown alg= thechangg in respiratory rate effected by fatty acids varies with themolecular weight and is maximal with acids containing 8-10 C.iso-Acids are less active than n-acids in this respect. Moreover,5 7 Bot. Cfax., 1937, 98, 433.8 8 Sci. Rept. Tokyo Bunrika Dnigaku, 1936, 2, 223, 291.59 Acta Phytochim., 1937, 9, 235POLLARD : PLANT. 453although unsaturated fatty acids and keto-irioiiocarboxylic acidsincrease respiration in green and brown algze, di- and tri-carboxylicacids have little or no action.Constituents of Alga?.-J. Fischer, in examining thc carotenoidpigments of certain fresh-water algae, observes that in species ofchlorophyll containing EugZena, p-carotene, zeaxanthin, lutein (asesters), and xanthophyll are commonly found. E. heliorzcbesceszsalso yields esters of euglenarhodone,60 C40H48Q4. TrentepohZia spp.also contain tc- and p-carotene (from which the alga produces smallamounts of ionone), xanthophyll, lutein, zeazanthin, and a pigmentresembling fucoxanthin.61A new sterol, pelvestrol, is isolated from Higika fusiformis byK. Shirahama,62 who also establishes the presence of fucosterol inseveral other species.Cell-wall materials in Halicystis spp. are described by G. vanIterson, j ~ n . , ~ ~ as exhibiting the properties of amyloid matter andcallose. find that X-rayexamination of the cellulose of the cell wall of Valonia ventricosaindicates that the chains of cellulose units in alternate layers fallalong meridians and along spirals terminating a t the poles. Thedirections of the chains in the alternate layers lie a t an angle ofapproximately 83" and correspond with the striations of the cellPucoidin obtained from Laminaria digitata is established byG. Lunde, E. Keen, and E. oy 65 as a carbohydrate sulphuric esterof the type RO*SO,*OR' in which R comprises 60% of fucose andR' is principally sodium with smaller amounts of potassium,ca,lcium, or magnesium.I n red, brown, and green marine algs, flavin is very generallydistributed, Irideea spp. containing notably large amounts(> 1 x I n red and brown species,from 50% upwards of the flavin present apparently occurs as aflavoprotein . 66R. D. Preston and W. T. AstburyW ~ U S .g. per g. of dry matter).A. G. POLLARD.60 2. physiol. Chem., 1936, 239, 257.62 J . Agric. Chem. Soc. Japan, 1935, 11, 980; 1936, 1% 681.63 Proc. K . A k d . Wetensch. Amsterdam, 1936, 39, 1060.64 Proc. Roy. SOC., 1937, B, 122, 76.85 2. physiol. Chem., 1937, 24'9, 189.66 A. Watanabe, Acta Phytochim., 1937, 9, 255.O1 ]bid., 1936, 243, 103