摘要:

AbstractIn chick embryo fibroblast cultures the 15‐ to 30‐fold enhancement of D‐glucose uptake observed when cells are starved of glucose for 24 hours is not duplicated for derivatives of glucose that compete effectively for uptake and have generally been considered to use the same carrier. 2‐deoxy‐D‐glucose, D‐mannose, D‐galactose and D‐glucosamine are derepressed progressively less sharply in that order with glucosamine uptake never more than doubled by starvation. D‐glucose at a concentration of 5.5 mM in the 24‐hour conditioning medium is a strong “repressor” resulting in low “transport” behavior for each of the five sugars cited. D‐glucosamine is equally effective at the same concentration. A 10‐fold reduction in the concentration of glucosamine (0.55 mM) allows for the escape from repression of mannose, glucose, and deoxyglucose uptake while the others remain repressed. Mannose uptake escapes as well when the glucose concentration in the “conditioning” medium is similarly reduced.Under certain conditions of starvation and cell density dramatic effects of supplemental stimulation by insulin can be achieved. Insulin withdrawal interrupts the supplemental stimulation process. Cycloheximide, actinomycin D and cordycepin block both non‐insulin and insulin‐induced derepression. Short exposure (15–30 minutes) of 24‐hour starved cells to glucose (5.5 mM) reduces glucose sharply but does not affect 3‐O‐methyl glucose uptake. If the exposure is to 2‐deoxyglucose (5.5

ISSN:0021-9541    

DOI:10.1002/jcp.1040910202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1977

数据来源: WILEY

摘要:

AbstractParenchymal cells were isolated from adult rat liver with an enzyme perfusion technique. The single‐cell suspension, representing 40–50% of the liver's hepatocytes was suspended in medium and maintained in primary culture for up to four days. The cells were found to carry out glycogen synthesis for the first eight hours in culture after which time the accumulated glycogen was gradually degraded. The ability of the liver cell cultures to accumulate glycogen was found to be dependent upon the metabolic state of the animal prior to cell isolation. Cells prepared during the feeding period from animals on the 8 + 16 feeding schedule had markedly different capacities for glycogen accumulation. Changes in glycogen metabolism were found to be due, in part, to changes in the fraction of cells involved in metabolism at any given time. High concentrations of glucose stimulated the cells to deposit glycogen but the response was reduced the longer the cells were in culture over a 3‐day period. This loss of glycogen synthesizing capacity appears to be due to a decrease in glycogen synthetase activity. The activities of pyruvate kinase, hexokinase and aldolase also decrease during the culture p

ISSN:0021-9541    

DOI:10.1002/jcp.1040910203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1977

数据来源: WILEY

摘要:

AbstractParenchymal cells from adult rat liver, isolated by a collagenase perfusion technique, have been maintained in primary culture and a detailed study on carbohydrate metabolism carried out over the initial 48‐hour culture period. The glucose concentration of the medium exerts a major influence on glycogen accumulation by the cells. Insulin, particularly at high glucose concentrations, stimulates glycogen biosynthesis, whereas glucagon prevents glycogen accumulation. Dexamethasone was without effect on glycogen metabolism. Glucose appears to stimulate glycogen accumulation by activation of glycogen synthetase enzyme. However, there is a gradual loss of synthetase activity throughout the culture period. Similar decreases in activity were noted for pyruvate kinase, aldolase and hexokinase. Glucose, insulin and dexamethasone were unable to prevent these decreases in enzyme activity. Foetal bovine serum contains fructose and this hexose appears to be the factor in serum which is responsible for the activation of glycogen accumulation in the presence of physiological glucose concentrations. The lactic acid content of the serum may also stimulate glycogen accumulation. In general, there is a gradual loss of the pattern of carbohydrate metabolism typical of differentiated hepatocytes during the culture perio

ISSN:0021-9541    

DOI:10.1002/jcp.1040910204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1977

数据来源: WILEY

摘要:

AbstractExperiments were performed to investigate the presence of colony‐forming units (CFU) in the mouse embryonic yolk sac during the developmental period in which the yolk sac is the sole hemopoietic organ. Injection of yolk sac cell suspensions from normal embryos into syngeneic, lethally irradiated adult recipients evoked a very low number of spleen colonies. However, prior cultivation of yolk sacs in vitro caused a dramatic increase in the spleen colony‐forming capacity–as high as 84‐fold–following 48 hours in culture. The yolk sac origin of the spleen colonies was confirmed by: (a) Chromosomal marker analysis; (b) dose‐response analysis; (c) demonstrating that the above colonies were not of endogenous origin induced by the mere injection of grafted cells. We conclude that the yolk sac contains many precursors of colony‐forming cells which though undetectable by immediate grafting apparently become activated in culture by an as yet unknown indu

ISSN:0021-9541    

DOI:10.1002/jcp.1040910205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1977

数据来源: WILEY

摘要:

AbstractUsing a series of cold‐sensitive variants of chemically transformed BHK‐21 cells, revertants to the normal phenotype derived from a dimethylnitrosamine transformed clone of BHK‐21 as well as revertants to the normal phenotype derived from polyoma transformed BHK‐21 cells we have demonstrated that the surface phenotype described by enhanced agglutinability with Con A and WGA can be dissociated from the transformed phenotype described by anchorage independence (growth in semisolid medium). Specifically we have demonstrated that the surface characteristic of enhanced agglutinability may be found in a variety of cell lines which fail to display an ability to grow in agar. Our work clearly shows that the two phenotypes described are not concomitantly controlled and tends to suggest that the phenotype of enhanced lectin agglutinability may be dissociated from the transformed ph

ISSN:0021-9541    

DOI:10.1002/jcp.1040910206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1977

数据来源: WILEY

摘要:

AbstractA colchicine‐resistant clone, CHRE5, has been isolated in a single step from a mutagenized culture of Chinese hamster ovary cells. Its resistance correlates with reduced colchicine permeability. At the same time, CHRE5 cells display a pattern of cross‐resistance to unrelated drugs similar to other membrane‐altered drug‐resistant mutants previously described (Ling and Thompson, 1974). However, CHRE5 cells also express a cold‐sensitivity for growth in that at 34° they do not double in number while at 38.5° they grow with a doubling time of about 22 hours. Employing synchronous cultures, the cold sensitive block in CHRE5 cells has been determined to be located prior to S in the G1 phase of the cell cycle. Mutant cells at 33.5° are not able to initiate DNA synthesis, however, cells already synthesizing DNA are able to complete the whole course of S. This cold sensitive block is reversed by shifting cells back to the higher temperature. Additonal clones with the CHRE5 phenotype have been isolated from non‐mutagenized cultures of wild‐type cells. Moreover, partial revertants of CHRE5 with increased ability to grow at 34° have been isolated and found to display increased colchicine sensitivity. These results are consistent with the hypothesis that the two phenotypes observed in CHRE5, namely, an altered plasma membrane (reduced drug permeability) and an altered ability to initiate DNA synthesis are the result of

ISSN:0021-9541    

DOI:10.1002/jcp.1040910207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1977

数据来源: WILEY

摘要:

AbstractBinding sites for tritiated cytochalasin D (3H‐CD) on the isolated plasma membrane from HEp‐2 cells were reversibly inactivated, but not dissociated from the membrane, by dialysis in 0.6 M KCl. Activity was restored by subsequent dialysis in 0.06 M KCl. Treatment with 0.2 mM ATP at low ionic strength also inactivated these sites, apparently irreversibly. Extraction of the membrane with 6% Triton X‐100 removed 75% of its protein, resulting in a two‐fold increase in specific binding activity for3H‐CD. Both high and low affinity binding sites were retained by the detergent‐extracted membrane; at least 60% of the high affinity sites were resistant to this treatment. Evidence is presented for the attachment to the HEp‐2 plasma membrane of both actin and myosin. The results support the tentative conclusion that plasma membrane binding sites for3H‐CD are peripheral proteins on the cytoplasmic face of the membrane. They are consistent with the hypothesis that myosin may be the location of the high affinity binding site and actomyosin may be the low affinity site. Comparison of these observations with those reported for the congeneric drug, cytochalasin B, suggests that CD binding sites differ from the high affinity site for

ISSN:0021-9541    

DOI:10.1002/jcp.1040910208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1977

数据来源: WILEY

摘要:

AbstractCytochalasin B (CB) was able to compete with tritiated cytochalasin D (3H‐CD) for binding sites in HEp‐2 cells. The pattern of inhibition suggested that CB associates with a low affinity class of CD binding sites. Glucose and maltose did not inhibit binding of3H‐CD to isolated HEp‐2 plasma membrane. Inhibition of hexose transport by CD was negligible, but CD did not block the potent inhibition of this transport by CB. These results indicate that CD does not bind to the high affinity CB receptor reportedly associated with the hexose transport system, and that this receptor cannot mediate the morphological effects of CD. Both CD and CB induced contraction‐zeoisis in HEp‐2 cells; CB was less potent than CD, and their effects appeared to be additive. It was concluded that the high affinity binding sites for CD and CB are different, but that these congeners share a low affinity site. Both high and low affinity sites for CD appear to mediate its morphological effects; only the low affinity class appears to be involved for CB. Possible identification of the common low affinity binding site as actomyosin (detailed in Tannenbaum et al., 1977) is furthe

ISSN:0021-9541    

DOI:10.1002/jcp.1040910209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1977

数据来源: WILEY

摘要:

AbstractA variety of metabolic and biosynthetic pathways in chick embryo fibroblasts are stimulated coordinately by many unrelated exogenous agents. Three of the best characterized components of this coordinate response are the uptake of 2‐deoxy‐D‐glucose (2‐dGlc) and of uridine and the incorporation of thymidine into DNA. Insulin stimulates and cortisol inhibits the coordinate response. In cortisol‐treated cultures, as little as 10−3units/ml of insulin may stimulate thymidine incorporation 4‐fold and 10−1units/ml may stimulate as much as 40‐fold. The higher concentrations of insulin completely override the inhibitory effect of cortisol. They also cause about a 5‐fold stimulation of the uptake of 2‐dGlc and of uridine and a 2‐fold stimulation of proline incorporation into protein. The uptake rates of 2‐dGlc and uridine double within 30 minutes after addition of insulin to cortisol‐inhibited cultures, but the incorporation of thymidine only begins to increase markedly after a 4‐hour delay. When cortisol is added to cultures in the absence of insulin, the rates of uptake of 2‐dGlc and uridine begin to decrease within two hours, but the incorporation of thymidine remains constant for two hours before beginning to decrease. Deprivation of Mg2+inhibits the accelerated coordinate response maintained by insulin, but does not further the inhibition induced by cortisol. Results with metabolic inhibitors indicate that the stimulation of 2‐dGlc and uridine uptake by insulin do not require RNA synthesis, and also suggest that they do not require protein synthesis. These and other findings can be explained by a model for coordinate control in which insulin increases and cortisol decreases the availability of Mg2+for a wide spectrum of regulatory reactions in different metabolic pathways. In this model both hormones affect only the rates of ongoing reactions and do not instruct the cell to carry out specific new reactions unless th

ISSN:0021-9541    

DOI:10.1002/jcp.1040910210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1977

数据来源: WILEY

摘要:

AbstractThe metabolism of 2′‐deoxyadenosine 5′‐phosphate (dAMP) by exponentially growing mouse fibroblasts (L‐cells) in suspension culture has been studied. Cells incubated for four hours with 0.10 μmole [3H,32P]dAMP/ml in medium containing 9.6 μmole31Pi/ml incorporate both activities at nearly linear rates into acid‐soluble and acid‐insoluble fractions. The3H/32P value increased about 30‐fold in each fraction during the incubation, indicating extensive dephosphorylation of dAMP. The DNA from treated cells was degraded enzymatically to 5′‐mono‐nucleotides, which were fractionated by ion‐exchange chromatography.3H was associated exclusively with dAMP (53%) and dGMP (47%).32P, associated with all deoxynucleotides, was at a 20‐fold higher specific activity in dAMP than in either dGMP or dCMP. The specific activity of [32P]dAMP incorporated into DNA in four hours was 24‐fold greater than that of32Piin the cellular pool. In experiments in which cells were incubated with32Piplus or minus 0.10 μmole dAMP/ml, the specific activity of dAMP was slightly less than that of dGMP or dCMP, which were equal. These results suggest that the higher specific activity of [32P]dAMP in the DNA of cells after incubation with doubly‐labeled dAMP was due to the intact penetration of some dAMP into the cells with its subsequent incorporation into DNA. Calculations, based on the amount of exogenously added [3H]thymidine incorporated into the cellular DNA in parallel cultures and the32P of dAMP isolated from this DNA, suggest that 1% of the total dAMP residues incorporated during the 4‐hour incubation were derived directly f

ISSN:0021-9541    

DOI:10.1002/jcp.1040910211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1977

数据来源: WILEY