摘要:

Abstract:The 5′‐nucleotidase activity of the purified cytoplasmic fraction preparation of bovine brain does not depend on the presence of the divalent metal ions Mg2+, Ca2+, and Cu2+in the incubation medium. The Zn2+ion (0.5 mM) causes total enzyme inhibition. Although EDTA and 8‐hydroxyquinoline inhibit the 5′‐nucleotidase from this source, it has not been possible to show the existence of metal ions in the enzyme molecule. The inhibition of 5′nucleotidase by EDTA is progressive and irreversible; when the enzyme is not preincubated with EDTA, the inhibition is overridden by metal ions. The purines (except xanthine, 0.3 mM), pyrimidines, and their nucleosides do not affect the 5′‐nucleotidase activity. The nucleoside di‐ and triphosphates are competitive enzyme inhibitors against 5′‐AMP as substrate. TheK1values of the diphosphates are lower than those determined for the corresponding triphosphates. The inhibition caused by the above nucleotides is reversed, partly or wholly, by Mg2+, depending on the molar ratio between the effectors. The inhibitory action of the ‐SH group reagents on the 5′‐nucleotidase acti

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb13558.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:Energy‐dependent internalization of125I‐labeled tetanus toxin into cultured neural cells is shown to follow an energy‐independent binding process. A three‐step model, involving receptor‐mediated binding followed by sequestration and internalization is proposed. In the first step, binding of toxin is enhanced in appearance under low ionic strength medium, at 0–4°C; it is suppressed, however, with increasing incubation temperature under physiological salt concentrations. Cell‐bound toxin is displaced by approximately 35.5% when high‐salt medium (physiological concentrations) is added to cells at 0–4°C; the effect is further amplified at 37°C. Addition of disialoganglioside GD1b(1–5 μg/ml) also lowers the amount of cell‐associated toxin. The fraction of125I‐labeled toxin retained by the cells after exposure to high‐salt medium at 0–4°C or after addition of GD1bis operationally defined as sequestered toxin. This second step, characterized by a stable association of the toxin with the neural cells, is affected by both physiological salt and by 37°C conditions. Lastly, an energy‐dependent phenomenon of firm association of tetanus toxin with neural cells, compatible with internalization, is described. The toxin residing in this fraction is bioactive and cannot be removed by salts, gangliosides, or by treatment with protease or neuraminidase. Binding, sequestration, and internalization are mutually dependent, as they are all blocked by pretreatment of cells with neuraminidase and by an enhanced energy‐independent sequestration event, which results in enhanced tetanus toxin internali

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb13559.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:The present paper describes the isolation and characterization of a clone of hybrid myelomas (3‐E7) secreting a mouse monoclonal antibody toβ‐endorphin. An examination of its specificity against a series of humanβ‐lipotropin fragments and other opioid peptides revealed that the N‐terminus portion ofβ‐endorphin is the determinant. Complete or almost complete cross‐reactivity was obtained to methionine‐ and leucine‐enkephalin,β‐lipotropin 60–65, and BAM 22; partial cross‐reactivity was seen to dynorphin1–13andα‐neo‐endorphin, whereasβ‐lipotropin,α‐N‐acetyl‐β‐endorphin, Des‐Tyr1‐β‐endorphin, in addition to a series of synthetic enkephalin derivatives, completely lacked cross‐reactivity. The use of the monoclonal antibody in radioimmunoassay (RIA) forβ‐endorphin resulted in a lower sensitivity related to respective polyclonal antibodies. An increase of 100% in tracer binding could, however, be obtained by use ofβ‐endorphin iodinated with its N‐terminal tyrosine protected by coupling to an antibody. A solid‐phase RIA was developed involving the internally3H‐labeled monoclonal antibody, which resulted in a 10‐fold increase in sensitivity as compared with the homogenous RIA. These data indicate that for the binding to this antibody a tyrosine residue in position 61 is essential, and it thus recognizes a site that is of

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb13560.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:The ontogeny of binding sites for [3H] spiperone was studied in time‐pregnant rats. Binding of [3H]spiperone to fresh homogenates of pre‐ and postnatal rat forebrain was characterized by Scatchard analysis and competition experiments with a number of dopaminergic and serotonergic agonists and antagonists and additional substances. A convenient discrimination of three high‐affinity sites, i.e., the dopaminergic D2, serotonergic S2, and spirodecanone (Sd) sites, was obtained withl‐(–)sulpiride and cis‐flupenthixol. The analgesic R5573 was found not to be specific for the Sd site but to interact with all three sites. The three binding sites became detectable in sequential order. S2and D2binding sites were first found at embryonic days 15.75 and 17.75, respectively. The Sd site did not appear before postnatal day 8. All three binding sites reached adult values at approximately postnatal day 30. During the prenatal period, the increase in the number of D2binding sites paralleled the rise in forebrain dopamine concentrations. The kinetics of D2and S2sites were the same at embryonic day 19.75 and postnatal day 30. These observations provide evidence for the presence of the receptor substrate for actions of neuroleptics on dopaminergic and serotonergic systems during

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb13561.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:Uptake and output of lactate were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14–15 days old. The chains, typically containing 30–40 μg of protein, were incubated in Eagle's minimum essential medium containing bicarbonate buffer, 6–17 mMglucose, various concentrations of lactate, and either [U‐14C]lactate, [1‐14C]glucose, or [6‐14C]glucose. The average rate of uptake of labeled lactate was measured with incubations of 5–6 h, starting with various external lactate concentrations. From these data the instantaneous relation between lactate uptake rate and concentration was deduced with a simple computerized model. The instantaneous uptake rate increased with the concentration according to a relation that fit the Michaelis‐Menten equation, with Vmax= 360 μmol/g protein/h andKm= 4.8 mM. Substantial fractions of the lactate carbon were recovered from tissue constituents and in several nonvolatile products in the medium, as well as in CO2. Glucose uptake averaged about 108 μmol/g protein/h and did not vary greatly with external lactate concentration, although the metabolic partitioning of glucose carbon was considerably affected. Regardless of initial concentration, the lactate concentration in the medium tended to change towards approximately 0.6 mM, showing that uptake equaled output at this level, with rates at about 40 μmol/g protein/h. With the steady‐state concentration of 0.6 mMlactate, about 20% of the glucose carbon was shunted out into the medium before it was reabsorbed and metabolized into various products. Lactate uptakes by neuronal and nonneuronal cultures prepared from the ganglia did not differ consistently from one another or from uptake by undissociated ganglia. The neuronal cultures tended to oxidize a greater fraction of the consumed lactate to CO2and to convert a smaller fraction of the lactate to products in the medium than did the nonneuronal cultures. Computer modeling, using known parameters for blood‐brain transport of lactate in the adult rat and data on uptake by the ganglia, suggests that lactate may supply substantial fuel to the brain, even in the presence of abundant glucose, when the lactate concentration in the blood is raised to levels commonly observed in exercising humans, such as 10–20 mM. This is in agreement with the findings of several investigators in hypoglycemic humans and in animals with intermediate blo

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb13562.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:Carbonic anhydrase from both the cytoplasmic and membrane fractions of the forebrains of rats was characterized with respect to enzymatic activity, immunoreactivity, andin vitrobiosynthesis. A procedure for the rapid purification of both membrane‐bound and soluble brain carbonic anhydrase is presented that permits retention of full enzymatic activity. Both forms of the enzyme were found to show specific activities of approximately 5500 Units/mg protein when CO2hydrating activity was determined. In addition, they exhibited similar esterase activity when assayed withp‐nitrophenyl acetate. The membrane‐bound form, although requiring detergent for extraction from membranes, was freely soluble in aqueous buffers after purification. The molecular weights of both soluble and membrane‐bound carbonic anhydrase are 30,000 daltons, and mixing experiments failed to show any significant differences with respect to size. The two forms also exhibit isoelectric points of 7.2. However, the two proteins were found to differ in two respects. Complement fixation indicated that antibodies to soluble carbonic anhydrase had a higher affinity for the soluble form than for the membrane‐bound form. The failure to observe any precursor‐product relationship between these two proteins with pulse chase studies and the establishment that carbonic anhydrase‐like proteins are synthesized on both free polysomes and the rough endoplasmic reticulum indicated that these proteins are synthesized by two separate mechanisms.In vitrosynthesis on both free and bound polysomes was determined by two independent methods using different antibodies and different analytical procedures. The basis for these findings and their physiologic importance

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb13563.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:Measurements were made of the amino acid content of a cellular clone (K55) derived from mouse cerebellar cultures transformedin vitroby simian virus‐40 (Alliot and Pessac, 1981) and that appears to be astroglial. Both the total amount of amino acids as well as the percentage of glycine in K55 cells were higher than in the mixed cultures from which they are derived. Further, glycine accumulates in the culture medium of K55 cells, but not in the medium of the parental mixed cell culture (C14), thereby suggesting that glycine is synthesized and released by K55 cell

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb13564.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:Primary astrocyte cultures from neonatal rat brains show uptake of [3H]norepinephrine ([3H]NE). This uptake has a high‐affinity component with an apparentKmof approximately 3 × 10−7M. At 10−7M[3H]NE both the initial rate of uptake and steady‐state content of [3H]NE is inhibited by up to 95% by omission of external Na+. The Na+‐dependent component of this uptake is totally inhibited by the tricyclic antidepressants desipramine (DMI) and amitryptyline with IC50values of 2 × 10−9and 4 × 10−8M, respectively. Inhibition of [3H]NE uptake by DMI shows competitive kinetics. These characteristics are essentially identical to those found for high‐affinity uptake of NE in total membrane or synaptosome fractions from rodent brains and suggests that such uptake in neural tissue is not ex

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb13565.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:Nerve growth factor treatment produces a large increase in the activity of ornithine decarboxylase and a moderate decrease in the activity ofS‐adenosylmethionine decarboxylase in PC12 cells. These changes are reflected weakly, if at all, in the levels of putrescine, spermidine, and spermine in the cells. The rates of polyamine synthesis are increased somewhat more than the overall levels, but still are not comparable in extent to the increase in the ornithine decarboxylase activity. Inhibitors of ornithine decarboxylase andS‐adenosylmethionine decarboxylase have their expected effects on the induction of ornithine decarboxylase and on the activities of both enzymes. Neither inhibitor alone, nor a combination of inhibitors, altered the rate or extent of nerve growth factor‐induced neurite outgrowth in the

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb13566.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:Brain free fatty acids (FFAs) and brain water content were measured in gerbils subjected to transient, bilateral cerebral ischemia under brief halothane anesthesia (nontreated group) and pentobarbital anesthesia (treated group). Mortality in the two groups was also evaluated. In nontreated animals, both saturated and mono‐ and polyunsaturated FFAs increased approximately 12‐fold in total at the end of a 30‐min period of ischemia; during recirculation, the level of free arachidonic acid dropped rapidly, while other FFAs gradually decreased to their preischemic levels in 90 min. In treated animals, the levels of total FFAs were lower than the nontreated group during ischemia, but higher at 90 min of reflow, and the decrease in the rate of free arachidonic acid was slower in the early period of reflow. Water content increased progressively during ischemia and recirculation with no extravasation of serum protein, but the values were consistently lower in the treated group. None of the nontreated animals survived for 2 weeks; in contrast, survival was 37.5% in the treated group. It is suggested that barbiturate protection from transient cerebral ischemia may be mediated by the attenuation of both membrane phospholipid hydrolysis during ischemia and postischemic peroxidation of accumulated free arachidonic

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb13567.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY