bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel mechanism for group translocation: Substrate‐product reutilization by γ‐glutamyl transpeptidase in peptide and amino acid transport
Authors: Prusiner Stanley , Doak Carola W. , Kirk Gerald
Published: February 2005

γ‐Glutamyl transpeptidase (γ‐GTP) is suggested to act as a carrier in the group translocation of oligopeptides and possibly some amino acids across cellular membranes. It is proposed that the process may involve the repetitive transfer of γ‐glutamyl groups to acceptor peptides which are being translocated from the exterior of the cell to its interior. After group translocation of the peptides has occurred with concomitant formation of γ‐glutamyl peptide products, it is suggested that the products might then be utilized as substrate for the enzyme in order to permit the translocation of other peptides from the exterior. The system is economical and requires only that it be primed with an appropriate source of γ‐glutamyl peptides, such as glutathione. In contrast to most group translocation systems previously described, substrate‐product reutilization by γ‐GTP would not be expected to accumulate peptides against a concentration gradient. Mechanisms for maintaining low intracellular concentrations of the translocated peptides are described.

Studies on acceptor substrate specificity of γ‐GTP from bovine choroid plexus and rat kidney show some glycyl peptides are much better substrates than free amino acids in accord with the proposal that γ‐GTP might be primarily involved in peptide translocation. Both kinetic and topological evidence support the suggestion that repetitive transfer of γ‐glutamyl moieties by γ‐GTP could occur during group translocation of peptides and possibly some amino acids.
The pH and temperature dependence of the interaction of steroid hormones with the transport system of glucose in human erythrocytes
Authors: Lacko L. , Wittke B. , Lacko I.
Published: February 2005

The thermodynamic parameters ΔH°, ΔF°, ΔS° and the pH dependence of the interaction between steroids and the glucose carrier in human erythrocytes have been studied. The results indicate that, according to the structure of the steroids, hydrophilic as well as hydrophobic bonds can be involved in the association. For the binding of the C‐21‐steroids to the carrier the polarity rule has been observed to be valid. On the basis of the thermodynamic parameters it has been found that in the association process of the steroids with the carrier, simultaneous randomization of ordered water molecules is important.
A serum factor requirement for the passage of cultured Vero cells through G2
Authors: Engelhard Dean L. , Jen‐Hao Mao
Published: February 2005

When Vero cells, a line derived from an African Green Monkey kidney, are grown under conditions where the saturation density is limited by serum, they deplete the growth medium of a factor necessary for cell division. The factor is a component of serum. When Vero cells are plated at low density (2 × 104/cm2) in this depleted growth medium (after dialysis against serum‐free Dulbecco's Modified Eagle's Medium) they initiate an unbalanced program of growth. Protein synthesis proceeds at the same rate as parallel cells in fresh serum, and the cells accumulate protein as a function of time. DNA synthesis is also initiated in these cells, and the amount of DNA per cell increases for the next four days after plating. However the cells quickly stop dividing. Measurements of DNA per cell using microspectrofluorometry show that the cells are accumulating in the late S and G2 period during this time. Thus we conclude that these cells cannot pass through a transition point in G2. When fresh serum is added to cells after three days in depleted growth medium, they divide before they begin to synthesize DNA. This further confirms that they are in late S and G2. Cell division is promoted in Vero cells in depleted growth medium by bovine fetuin, and to a lesser extent by bovine albumin. Cell division is not promoted by insulin, hydrocortisone, dexamethasone, linolenic acid, calcium, and trypsin inhibitor from ovomucoid. From these data we conclude that transit through G2 requires the presence of an extracellular factor.
Alterations in differentiation and pyrimidine pathway enzymes in 5‐azacytidine resistant variants of a myoblast line
Authors: Ng S. K. , Rogers Jackilynn , Sanwal B. D.
Published: February 2005

5‐azacytidine at concentrations higher than 5 μM inhibited the differentiation of a rat myoblast line in vitro. It was also somewhat cytotoxic at this level. Variants resistant to the cytotoxic effect of 5‐azacytidine were obtained which were simultaneously unable to differentiate into myotubes and exhibited altered morphology. These characteristics were retained by the variants when subcultured in the absence of the drug for over 700 generations. Several of the azacytidine resistant cells were more susceptible than the parental line to the lethal action of 5‐bromodeoxyuridine and adenosine, but not that of cytosine arabinoside, ouabain or 8‐azaguanine.

The variants were capable of transporting uridine, thymidine and 5‐azacytidine. The uridine kinase activity was one‐half to one‐third of that in the parental cells but it was not missing completely in any of the variants. Two independently isolated variants selected for detailed study showed a 2‐ to 3‐fold increase in the activity of orotidylic acid decarboxylase. This enzyme in the variants in contrast to that of the parental cells was completely insensitive to the inhibitory effect of a nucleotide generated from ATP and 5‐azacytidine in cell extracts (probably 5‐azacytidine monophosphate). These observations point to the possibility that 5‐azacytidine resistance arises in myoblasts due to an alteration of the components of two target pathways of this drug, viz., the de novo pyrimidine pathway and an undefined sequence leading to the synthesis of membrane components.
RNA synthesis and processing as a measure of phenotypic variability in cytodifferentiation and neoplasia
Authors: Bynum John W. , Regan James D. , Volkin Elliot
Published: February 2005

Neoplastic cell lines exhibit RNA synthesis and process patterns which are related to phenotypic attributes more complex than merely the rate of proliferation. Mouse neuroblastoma cells of the same genotype but different differentiated states have different ribosomal RNA precursor processing patterns, while plasmacytoma cells of different genotypes but the same differentiated state have the same pre‐ribosomal RNA processing pattern. In addition, our observations indicate that chromatin‐associated RNA is involved in cytodifferentiation and is closely related to phenotypic variability. When neuroblastoma cells are induced to differentiate, there is a 2‐ to 3‐fold increase in the labeling of chromatin‐associated RNA. Both of the differentiated cell lines, human myeloma and mouse neuroblastoma, have slow‐labeling, stable chromatin‐associated RNA while this same fraction from HeLa cells is labeled rapidly and is unstable.
Glycogen metabolism in adult rat liver parenchymal cell primary cultures
Authors: Walker P. Roy
Published: February 2005

Parenchymal 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 period.
A serum factor capable of stimulating hyaluronic acid synthesis in cultured rat fibroblasts
Authors: Tomida Mikio , Koyama Hideki , Ono Tetsuo
Published: February 2005

Calf serum as well as rat and mouse sera has a factor that stimulates hyaluronic acid synthesis in cultured rat fibroblasts. Such a factor was partially purified from calf serum and characterized. It has a molecular weight of approximately 150,000. The activity of the factor is lost by treatment with pronase and by periodate oxidation. It is suggested, therefore, that the factor is a glycoprotein. Its susceptibility to α‐mannosidase and affinity for Con A‐Sepharose may suggest that the factor contains a mannose residue(s) which is essential for the activity to induce hyaluronic acid synthesis.
Proteases stimulate proliferation of human fibroblasts
Authors: Pohjanpelto Pirkko
Published: February 2005

Incubation of primary human fibroblasts in serum‐free medium with small concentrations of thrombin, trypsin or plasmin resulted in manyfold increase in total DNA synthesis and in the number of 3H‐thymidine labelled cells. Rise in the frequency of mitoses indicates that the proteases stimulated also cell division. Because proteases induced only a fraction of cells to proliferate increase in the total number of cells remained moderate. Calf, horse and rabbit serum inhibited the growth stimulating effect of trypsin but chicken, dog and monkey serum were permissive. Specific inhibitors of proteases prevented the stimulation of cell proliferation suggesting strongly that proteases act in virtue of their enzymatic activity.
Alterations of acetylcholine enzymes in neuroblastoma cells persistently infected with lymphocytic choriomeningitis virus
Authors: Oldstone Michael B. A. , Holmstoen James , Welsh Raymond M.
Published: February 2005

Persistent infection of murine neuroblastoma cells with a relatively non‐cytopathic virus, lymphocytic choriomeningitis virus (LCMV), significantly lowered the cells' concentrations of choline acetyl transferase (CAT) and acetylcholine esterase (ACHE), enzymes which make or degrade acetylcholine. Quantities of acetylcholine enzymes remained depressed during the observation period of more than two years. This cellular luxury function was turned off without observable alterations in the cells' vital functions – growth rates, protein and RNA synthesis. Cloning experiments showed that CAT and ACHE levels were altered in the majority of LCMV infected neuroblastoma cells in culture and not limited to a specific subpopulation. Cells persistently infected with virus also contained receptors for neurotoxin A and α bungarotoxin. Six months after becoming infected, neuroblastoma cells having significant alterations in luxury functions stopped making infectious virus. Instead these cells now produced a defective interfering virus component.

Similar events to those seen in vitro with neuroblastoma cells also occurred in vivo. Mice inoculated with LCMV at birth carried high titers of LCMV in brain tissues and viral antigens in neuronal cells as adults. Some of these mice also showed significant alterations in their ability to make and degrade acetylcholine when compared to age and sex matched controls.
Rapid increase in poly(A) (+) mRNA content following inhibition of protein synthesis
Authors: Johnson Lee F. , Meister Richard
Published: February 2005

When resting 3T6 cells undergo a serum‐induced transition to the growing state, the cytoplasmic content of ribosomal, transfer and messenger RNA increase as the cells prepare for DNA synthesis. The normal linear increase in mRNA content occurs even when the production of ribosomes is blocked. In this paper we determine the effect of inhibiting protein synthesis on the increase in poly(A) (+) mRNA content. Resting cells were serum stimulated in the presence of cycloheximide or puromycin at levels which inhibit protein synthesis by greater than 95%. Cytoplasmic poly(A) (+) mRNA content was determined at various times thereafter. We found that mRNA content increased five to ten times more rapidly in drug treated cells than in control cells stimulated in the absence of inhibitors. mRNA content increased 50–70% by one hour, and 60–90% by two hours following stimulation in the presence of inhibitor, and remained more or less constant thereafter. In contrast, mRNA content increased linearly in control stimulated cultures and did not double until about 15 hours after stimulation. The rapid increase in mRNA content is most likely the result of inhibition of protein synthesis rather than a secondary effect of the drug since the same observations were made in growth stimulated cells if protein synthesis was blocked with either puromycin or cycloheximide.

A similar effect was also observed with resting 3T6, exponentially growing 3T6 and growing HeLa cells following exposure to cycloheximide, although the magnitude of the increase was less than that observed with growth stimulated cells. Puromycin had negligible effect on mRNA content in resting or exponentially growing cells.

The rapid increase in cytoplasmic poly(A) (+) mRNA content was not due to rapid unbalanced export of nuclear poly(A) (+) RNA into the cytoplasm since there was no decrease in nuclear poly(A) content following serum stimulation in the presence of cycloheximide.
Induced alteration in uptake properties of Tetrahymena and its association with the development of mating competency
Authors: Adair W. Steven , Wolfe Jason
Published: February 2005

The rate of uridine uptake in Tetrahymena declines by an order of magnitude by two hours after shiftdown to a non‐nutrient buffer. This alteration in uptake properties cannot be accounted for by an increase in the intracellular pool of uridine, an increase in apparent Km for uptake or a decline in the rate in which uridine is processed intracellularly.

It is argued that the decrease in uridine uptake is due to a reduction in numbers of functional transport molecules exposed at the cell surface and is a reflection of a developmentally related cell surface transformation.

In addition, the putative decline in functional transport molecules cannot be entirely explained by metabolic turnover of these molecules in the absence of replacement, nor does it require the synthesis of new protein. We discuss the possibility that a shift in equilibrium between accessible and inaccessible transporters is operating.

Finally, a close correlation between conditions which elicit the transport alteration and those which allow the development of mating competency suggests that the two phenomena may be coordinately regulated.
Stimulation of growth and calcium influx in cultured, bovine, aortic endothelial cells by platelets and vasoactive substances
Authors: D'amore Patricia , Shepro David
Published: February 2005

Endothelial cells (EC) line the heart and blood vessels, and they are the principal cellular components of the microvasculature. The presence of circulating platelets is believed to be necessary to maintain the integrity of the capillary endothelium. Growing EC in culture provides an opportunity to simulate the in vivo situation and to study the response of these cells to platelets and platelet secretions. The addition of platelets at a concentration of 104‐105/mm3 and substances which circulate in blood following injury (serotonin, thrombin, ADP, epinephrine, norepinephrine and histamine) stimulate endothelial proliferation from 150–1,000% of controls. That substances so diverse in form have similar effects suggests a common mode of action, such as mobilization of a second messenger. The influx of 45calcium (45Ca++) in response to these agents was found to be 5 to 24 times that of controls. The stimulation of 45Ca++ influx appears to be dose‐dependent, and it is inhibited by pre‐incubation with lanthanum chloride and specific blocking agents. Calcium as a second messenger is implicated in a variety of cellular functions including division, secretion, motility and enzyme regulation. Thus, the theorized supportive role of platelets on endothelium may be dual and operate, at least at the initial level, by a common mechanism: to mobilize calcium for stimulus‐division following injury and for stimulus‐secretion in normal metabolic activities.
Temperature sensitive mutants of BHK cells affected in cell cycle progression
Authors: Talavera Antonio , Basilico Claudio
Published: February 2005

Three different temperature sensitive mutants derived from the Syrian hamster cell line BHK 21 were found to have greatly reduced DNA synthesis at the non‐permissive temperature. These mutants are distinct by complementation analysis and behave at the non‐permissive temperature as cell cycle traverse defective mutants. Microfluorometric analysis of mutant populations arrested at the non‐permissive temperature shows an accumulation of cells with G1 DNA content. Mutants ts 13 and ts HJ4 synchronized in G1 by serum or isoleucine deprivation and shifted to the non‐permissive temperature at the time of release do not enter the S phase, while in the case of mutant ts 11 preincubation at the non‐permissive temperature before release is required to completely prevent its entry into S. Ts 13 and ts 11 are able to traverse the S phase at the non‐permissive temperature when synchronized at the boundary G1/S; in this case, preincubation of ts 11 at the non‐permissive temperature before release does not affect the ability of these cells to perform DNA synthesis. On the other hand, ts HJ4 appears to traverse S only partially when tested under similar conditions. Temperature shift experiments of mutant populations at different times after isoleucine synchronization suggest that ts 13 and ts 11 are blocked at the non‐permissive temperature in early G1, whereas ts HJ4 is probably affected near the initiation of DNA synthesis, or in some early S function.
Responses of protein synthesis and degradation in growth control of WI‐38 cells
Authors: Castor Laroy N.
Published: February 2005

The overall rates of protein synthesis and degradation in perfusion‐grown WI‐38 cells were followed in the three days after a stepdown in the serum concentration of the culture medium, from 10% to 0.3%. Within three hours after the stepdown, the rate of protein synthesis had decreased and the rate of protein degradation had increased, the combined result being the cessation of protein accumulation. The degradation rate returned over the next three days to its original value, but a zero rate of accumulation was retained because the synthesis rate continued to decline. The rate of DNA synthesis remained constant for six hours after the stepdown. It then declined steadily until reaching a minimum about eight hours later. The results show that extracellular control of protein accumulation depends on adjustments in both protein synthesis and protein degradation, and that the adjustments take place rapidly. This behavior suggests that the cell cycle is arrested after a stepdown because post‐mitotic cells are unable to accumulate additional protein. However, an alternative interpretation of the data is that at least part of the changed accumulation is the result, rather than the cause, of the cycle arrest, and that the arrest is caused by other, more specific, reactions than those of general protein metabolism.
Concanavalin A‐binding by cells of the early chick embryo
Authors: Hook S. L. , Sanders E. J.
Published: February 2005

The surfaces of cells from the early embryo of the chick were examined using electron microscope techniques for the visualization of concanavalin A‐binding sites. Horseradish peroxidase and Ferritin labelled concanavalin A were used to determine the distribution of the binding sites. All surfaces of the epiblast and hypoblast layers which were accessible to concanavalin A showed the presence of binding sites in stage 1 embryos. The ventral surface of the epiblast showed a high lectin affinity which may reflect the development of a basal lamina on this surface. The individual hypoblast cells at this stage showed a non‐uniform distribution of binding sites, having a greater affinity on the dorsal surface than the ventral. By the time of primitive streak formation (stage 4–5) the dorsal surface of the epiblast displayed increased binding sites, while the frequency of sites on the ventral surface of the endoblast was reduced. The latter may reflect a change from one cell population to another, which occurs in the lower layer of the embryo at this time. No consistent correlation could be drawn between changes in motility of cells actually invaginating through the primitive streak and changes in affinity for concanavalin A. An overall increase in affinity of the dorsal surface of the epiblast was revealed by Ferritin and may reflect the changes in surface structure occurring in readiness for the morphogenetic migrations of gastrulation.
Reversible inhibition of the hydrocortisone induction of glycerol phosphate dehydrogenase by cytochalasin B in rat glial C6 cells
Authors: Bennett Kimberly , McGinnis James F. , De Vellis Jean
Published: February 2005

The hydrocortisone (HC) induction of glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) in rat glial C6 cells was inhibited reversibly and in a dose‐dependent manner by cytochalasin B (CB). CB had no effect on basal level GPDH, total cellular RNA, DNA or protein content nor did it act as a general inhibitor of the rate of protein synthesis. CB did not appear to be acting via dissociation of microtubules since colcemid had no effect on the induction process. The addition of an alternate energy source (sodium pyruvate) did not relieve the CB inhibition of GPDH induction suggesting that CB is not exerting its effect by blocking glucose utilization. The inhibition by CB is not dependent on the temporal sequence of the induction process since it specifically inhibited GPDH induction at any time it was added. CB did not alter the rate of degradation of GPDH in these cells and direct measurements of the specific rate of synthesis of GPDH demonstrated that CB decreased the induced rate of GPDH synthesis by about 60%. The site of inhibition was more precisely defined by experiments which demonstrated a 60% decrease in specific nuclear binding of 3H‐HC even though total cellular uptake of 3H‐HC was unaffected. This effect on nuclear binding of HC is sufficient to account for the decreased accumulation of GPDH activity in CB‐treated cells.
Polyamine metabolism in enucleated mouse L‐cells
Authors: McCormick Frank
Published: February 2005

The distribution of polyamines between the nucleus and the cytoplasm, and the role of the nucleus in polyamine metabolism, have been studied using cells enucleated with cytochalasin B. Spermidine and spermine were found in the nuclear and the cytoplasmic fractions of L929 cells; their concentration was 3‐fold higher in the former fraction. Ornithine decarboxylase activity was only found in the cytoplasm, and this activity could be stimulated in enucleated cells by the addition of fresh medium. These cells synthesized putrescine actively, but the putresoine made was not converted to spermidine, and accumulated to relatively high concentrations. Similarly, methionine did not act as a precursor to spermidine in enucleated cells, in contrast to whole cells, although it was incorporated into cell protein. Spermidine synthesis, unlike putrescine synthesis, appears to be completely dependent on a nuclear component.
Development of action potential in larval muscle fibers in Drosophila melanogaster
Authors: Suzuki Nobuyuki , Kano Masaakira
Published: February 2005

In the presence of tetraethylammonium or barium ions, the larval muscle fibers of Drosophila melanogaster were found to produce an all‐or‐none action potential operated by the calcium channels. The development of this distinctive membrane property during the maturation of muscle cells was studied by measuring the maximum rate of rise of the action potential in the larval muscle fibers at different stages of development from the sixteenth to ninety‐sixth hours after hatching. The value increased significantly with age until a peak was reached at the sixty‐fourth hour, although it became lower again as puparium formation neared at about the ninety‐sixth hour. This suggests that during larval development the muscle fibers develop the ability to generate an action potential due to an inward current through the calcium channels, although the ability became lower at the later stage of larval development.
Dibutyryl cyclic AMP arrests the growth of cultivated cloudman melanoma cells in the late S and G2 phases of the cell cycle
Authors: Dipasquale Albert , McGuire Joseph
Published: February 2005

DBcAMP reversibly arrests cultivated Cloudman melanoma cells in the late S and G2 phases of the cell cycle. This is supported by the measurement of DNA synthesis by autoradiography and measurement of cellular DNA by two methods—the diphenylamine reaction and microspectrophotometry of Feulgen stained cells. We also present evidence that (1) cell division is prevented if DBcAMP is added as late in the cycle as early S phase. (2) The inhibition of cell division does not appear to be caused by products of tyrosine oxidation. (3) The increase in cell size that occurs in the presence of DBcAMP reflects continued synthesis of protein in the absence of cell division.
Role of the vacuolar apparatus in augmented protein degradation in cultured fibroblasts
Authors: Amenta J. S. , Sargus M. J. , Venkatesan S. , Shinozuka H.
Published: February 2005

Rat embryo fibroblasts, grown in Eagle's MEM with 10% serum, showed a rapid increase in autophagic vacuoles when placed in MEM with 0–1% serum. Concurrent with this response, degradation of cellular proteins showed a 2‐fold increase. We did not find any increases in cathepsin D, β‐glucuronidase, β‐galactosidase, and β‐glucosidase, or proteolytic activity of cell homogenates at pH 3.7 towards endogenous substrates. Homogenates prepared in 250 mM sucrose at pH 7.0 showed a 40% increase in protein breakdown. These data support the hypothesis that the induced increase in proteolysis, characteristic of cells placed in a nutritionally deficient medium, is effected by an activated vacuolar apparatus (lysosomes and autophagic vacuoles). We suggest, however, that this mechanism is distinct from normal protein turnover in the cell, but can be rapidly induced by appropriate alterations in the cellular environment. Finally, this induced proteolytic mechanism is not dependent upon an increase in lysosomal enzymes, but rather a structural alteration within the cell which effects a transfer of cellular proteins into the vacuolar apparatus.
Toxicity, radiation sensitivity modification, and metabolic effects of dehydroascorbate and ascorbate in mammalian cells,
Authors: Koch Cameron J. , Biaglow John E.
Published: February 2005

Dehydroascorbate, an electron affinic metabolite of vitamin C, sensitized Ehrlich ascites tumor cells, in vivo, to radiation and was selectively toxic to V79 Chinese hamster lung cells under hypoxic conditions (without radiation). The radiosensitization may involve both the electron affinic nature of dehydroascorbate as well as its ability to oxidize the intracelluar NAD(P)H and non‐protein sulfhydryl. Dehydroascorbate's oxidation of NAD(P)H required higher concentrations than other sulfhydryl oxidants such as N‐ethylmaleimide and diamide. The oxidation of NAD(P)H by dehydroascorbate could be reversed by glucose. Hypoxic cell radiosensitization of V79 cells in tissue culture by dehydroascorbate could not be easily demonstrated because of the rapid breakdown and appreciable cytotoxicity of the drug at high concentration. The cytotoxicity was found to occur with both high and low densities of V79 cells. With low cell densities small amounts of oxygen did not reduce the cytotoxicity of dehydroascorbate, but virtually eliminated the cytotoxicity of nitroaromatic electron affinic compounds (metronidazole and Ro‐07‐0582). The cytotoxicity to dense cell suspensions was found to depend upon the type of buffer included in the reaction medium. The maximum cytotoxicity was obtained in buffer free saline. The reduced form of dehydroascorbate, vitamin C, was found to be toxic only under aerobic conditions. The aerobic cytotoxicity could be prevented by the addition of catalase to the growth medium or by an increase in cell density, suggesting it was caused entirely by the production of H2O2 from the oxidation of vitamin C.
Increased membrane transport of 2‐deoxyglucose and 3‐0‐methylglucose is an early event in the transformation of chick embryo fibroblasts by rous sarcoma virus
Authors: Lang Dennis R. , Weber Michael J.
Published: February 2005

Transformation of chicken embryo fibroblasts with Rous sarcoma virus results in cells with an enhanced rate of hexose uptake. We have examined transport of the glucose analogs 2‐deoxyglucose and 3‐0‐methylglucose in cells infected with a temperature sensitive variant of the virus. In cells shifted from restrictive to permissive conditions for transformation, increased transport of the non‐phosphorylatable analog 3‐0‐methylglucose occurs at the same time as that of 2‐deoxyglucose, a phosphorylatable analog. This enhanced rate of transport can be observed within three hours of the temperature shift. There is a corresponding decrease in the transport rate of both analogs following shift to the restrictive temperature. These results suggest that increased transport is likely to be the primary event in causing transformation‐specific changes in sugar metabolism. We have also examined uptake into the internal pools of both the phosphorylated and non‐phosphorylated forms of 2‐deoxyglucose in normal cells and in cells transformed by the wild‐type virus. These data indicate a corresponding increase in the rate of accumulation of the free sugar in transformed cells and point to transport as the rate limiting step in the accumulation of 2‐deoxyglucose in both normal and transformed chicken embryo cells.
Conditions which affect initiation of animal cell division by trypsin and thrombin
Authors: Carney Darrell H. , Glenn Kevin C. , Cunningham Dennis D.
Published: February 2005

It has been well documented that trypsin or thrombin initiate proliferation of quiescent secondary chick embryo cells. However, there has been less certainty about the ability of these proteases to initiate division of quiescent mammalian cells. Accordingly, we studied the conditions under which quiescent chick embryo (CE), mouse embryo (ME), and human diploid foreskin (HF) cells respond to trypsin or thrombin

Extended culture of these cell strains in serum‐free medium increased the initiation of cell division by both proteases. Under these conditions, CE cell number was increased 90% over controls by trypsin and 70% by thrombin. In contrast, quiescent ME and HF cells both responded better to thrombin than trypsin, giving maximal increases, respectively, of 70 and 40% over controls with thrombin and 22 and 14% with trypsin. Calf serum inhibited the initiation of these cell strains, particularly the ME cells, by both trypsin and thrombin. This inhibition of initiation could be attributed to decreased proteolytic activity in the case of trypsin, but not thrombin

In contrast to the cell strains tested, quiescent cultures of the 3T3 cell line showed no detectable increase in cell number with trypsin or thrombin in the absence of serum, and only a slight increase in cell number with thrombin in the presence of serum. However, in the presence of plasma, 3T3 cell number increased up to 20% with thrombin

Initiation of cell division by proteases has been reported and confirmed mostly for early passage cell strains rather than cell lines. Our experiments with CE cells indicate that this is possibly the result of a rapid decline in protease responsiveness upon serial subculture. With these cells we found a decline in response first to trypsin, then thrombin, and finally serum

Throughout these studies, we compared the ability of trypsin and thrombin to initiate cell division under various conditions. We found several differences between the two proteases, indicating that they initiate cell division by somewhat different mechanisms.
Chloride and sulfate transport in ehrlich ascites tumor cells: Evidence for a common mechanism
Authors: Levinson Charles
Published: February 2005

The effects of phloretin, H2DIDS (4,4′‐diisothiocyano‐1,2‐diphenylethane‐2,2′‐disulfonate) and SO4−2 on anion transport in Ehrlich ascites tumor cells was studied in an effort to determine whether Cl− and SO4−2 share a common transport mechanism. Sulfate, in the presence of constant extracellular Cl− (100 mM), reduces Cl− self‐exchange by 43% (40 mM SO4−2) and Cl−−SO4−2 exchange by 36% (25 mM Cl−/O SO4−2) compared to 25 mM Cl−/50 mM SO4−2. Phloretin blocks without delay and to the same extent the self‐exchange of both Cl− and SO4−2. For example, at 10−4 M phloretin, anion transport is inhibited 28% which increases to 78% at 5 × 10−4 M. Reversibly bound H2DIDS also inhibits the self‐exchange of both Cl− and SO4−2. However, at all H2DIDS concentrations tested (0.5 − 10 × 10−5 M) SO4−2 transport was far more susceptible to inhibition than that of Cl−. H2DIDS when irreversibly bound to the cell inhibits SO4−2 but not Cl− transport

The results of these experiments are consistent with the postulation that both Cl− and SO4−2 are transported by a common mechanism possessing two reactive sites.
Thymidylate synthetase and dihydrofolic acid reductase in the stimulated human lymphocyte
Authors: Haurani Farid I. , Kardinal Carl G. , Biermann William A.
Published: February 2005

The phytohemagglutinin (PHA)‐stimulated human lymphocytes demonstrated trace or no activity of (dihydro) folate acid reductase using three methods including a radioassay, but demonstrated ample activity of thymidylate synthetase. This was true regardless of the day of harvest, (first through seventh) of the stimulated lymphocyte. The lymphocyte extracts revealed no inhibitor to the reductase enzyme when these extracts were added before the liver extracts to the assay system. When methotrexate (MTX) was added to the culture media of the lymphocytes, there was, as expected, an increase in the synthetase activity, but the expected rise in the reductase activity did not occur, it remained nil. On the other hand, MTX did influence the incorporation of nucleosides by the stimulated lymphocytes in a fashion similar to its action on the incorporation of the same nucleosides by other cells.
Heat shock dependent fluctuations of RNase activity during the cell cycle of synchronized Tetrahymena
Authors: Tarnowka M. A. , Yuyama S.
Published: February 2005

The total cellular acid RNase activity per milliliter of culture increases sharply following each heat shock in the cell cycle of Tetrahymena pyriformis ST synchronized with heat shocks spaced one generation time apart. Thus, the RNase activity per 105 cells is 24.5 units immediately after the end of the sixth heat shock, increases to 39.0 units during the following 55 minutes and decreases to 24.2 units at the start of the seventh heat shock. No change in the RNase activity occurs during the heat shock period. In logarithmically growing cells the RNase activity per 105 cells is 15.4 units. The heat shock stimulates the increase in the RNase activity, since no rapid increase occurs during the free running division cycle but a rapid increase occurs after an additional heat shock given at different times during the cell cycle

Inhibition of the increase in RNase activity by cycloheximide suggests that concurren protein synthesis is required for the stimulation of the RNase activity by the heat shock treatment.
Sodium concentrations affect metabolite uptake and cellular metabolism
Authors: Bader John P. , Sege Robert , Brown Nancy R.
Published: February 2005

Rates of uptake of glucose (measured with 3H‐2‐deoxy‐d‐glucose), galactose, and leucine increase after exposure of chick embryo cells to increasing concentrations of Na+ over the range 100 to 200 mM. Uptake of nucleosides was unaffected by [Na+] over this range. Prior exposure of cells was required for the [Na+] effect on uptake. Changes were measureable within two hours after changing [Na+], and although the capacity for deoxyglucose uptake remained constant thereafter, the capacity for leucine uptake continued to change during the next few hours. Inhibition of protein synthesis by cycloheximide, or of RNA synthesis by Actinomycin D, failed to prevent these uptake changes. Analysis of the kinetics of uptake showed that only the Km for uptake of deoxyglucose or leucine was affected by [Na+]; the maximum V for each compound remained the same. Effects of [Na+] could be distinguished from the increased capacity for glucose uptake induced by glucose starvation.

Incorporation of both radioactive uridine into RNA, and radioactive thymidine into DNA, were affected by [Na+], but the differences were not correlated with uptake of other metabolites. No differences in countable mitoses were apparent, although the growth of chick embryo cells increased slightly with increasing [Na+].

Changes in uptake due to differing [Na+] also were observed in mammalian (rat NRK) cells. However, no effects of [Na+] on rates of cell growth or saturation density were observed with these cells.
Stimulation of 86Rb+ and 32Pi movements in 3T3 cells by prostaglandins and phorbol esters
Authors: Moroney J. , Smith A. , Tomei L. D. , Wenner C. E.
Published: February 2005

The potent tumor promoter tetradecanoyl phorbol acetate (TPA) induces early changes in ion movements analogous to those induced by prostaglandins E1 and F2α. Among the earliest changes induced by TPA is a significant increase in 32Pi incorporation within 15 minutes incubation of TPA (10−8−10−6 M) with post‐confluent Swiss 3T3 mouse embryonic fibroblasts. Similarly, the active phorbol ester homolog 4‐;β‐;OH phorbol didecanoate but not the inactive stereoisomeric 4‐β‐OH phorbol didecanoate stimulated 32Pi incorporation. Also, TPA at the above concentrations stimulated 86Rb+ influx shortly after administration. Both fluxes were ouabain‐sensitive in accord with the idea that an early effect of TPA is to alter (Na+ + K+)−ATPase activity. Further, prostaglandin E1 (10−7−10−6 M) and prostaglandin F2α (3 × 10−9−10−7 M) caused a similar stimulation of 86Rb+ and 32Pi uptake. The finding that water‐soluble prostaglandin F2α also exhibited stimulatory effects indicated that those hormone‐induced responses are not mediated by solvent interactions. The similar responses of phorbol esters and prostaglandin derivatives suggests that phorbol esters and prostaglandin derivatives may act at common membrane sites

The finding that stimulatory effects were observed at discrete times in the logarithmic phase of growth suggests that the activation of membrane receptors may be cell‐cycle dependent.
Enhancement of the synthesis of specific cellular polypeptides in a temperature‐sensitive chinese hamster cell line (K12) Defective for entry into S Phase
Authors: Melero Jose A. , Fincham V.
Published: February 2005

Temperature‐sensitive Chinese hamster cells (K12) have been shown to be defective for the initiation of new rounds of DNA replication when incubated at the restrictive temperature (40.5°). By temperature shift experiments with synchronous cultures, we have marked out the step at which the mutation is expressed as the four hours preceding the initiation of DNa synthesis. The block imposed by the mutation has been shown to be irreversible. In order to approach the biochemical characterization of the temperature‐sensitive function in K12 cells, we have analyzed the cellular proteins synthesized under permissive (35°) and restrictive temperatures. The synthesis of three polypeptides is markedly enhanced when K12 cells are incubated at 40.5°. One of them (band B) has turned out to be a useful biochemical marker of the expression of K12 mutation since its synthesis is not affected in other ts‐mutants or in hybrids in which K12 mutation is complemented. In addition, the alteration in band B synthesis is irreversible and occurs during the same stage of the cell cycle at which the mutated function is expressed.
Constancy of the shift‐up point in two temperature‐sensitive mammalian cell lines that arrest in G1
Authors: Ashihara T. , Chang S. D. , Baserga R.
Published: February 2005

Two cell cycle‐specific temperature sensitive (ts) mutants of mammalian cell lines, AF8 and K12, are known to arrest in G1 when shifted to the non‐permissive temperature. We have determined the entry into S of both AF8 and K12 cells in five different growth conditions, namely: (1) quiescent sparse cultures stimulated to proliferate by serum; (2) quiescent dense cultures stimulated by serum; (3) quiescent sparse cultures stimulated by trypsinization and replating; (4) quiescent, dense cultures stimulated by trypsinization and replating; and (5) mitotic cells collected by mitotic detachment. In addition, for each cell line and for each different growth condition, we have determined the shift‐up time, i.e., the time at which a shift‐up to the nonpermissive temperature no longer prevents the entry of cells into S. In no case did K12 or AF8 enter S at the nonpermissive temperature. At the permissive temperature, the average time of entry into S varied in different growth conditions, and so did the shift‐up time. However, in both cell lines, the distance of the average shift‐up time from the average time of entry into S was remarkably constant, regardless of the growth conditions, i.e., 1.8 hours in K12 and 8.6 hours in AF8.
Relation between structure of bacterial lipopolysaccharide and the stimulation of B lymphocytes into colony formation
Authors: Sredni B. , Rozenszajn L. A.
Published: February 2005

Lipopolysaccharide (LPS) was analyzed in order to determine which component, lipid A or polysaccharide (PS), is able to stimulate B lymphocytes from ICR lymph nodes and spleen cells from nude (nu/nu) mice into forming colonies in soft agar culture. Lipid A, obtained by acid hydrolysis of LPS and solubilized by complex‐formation with bovine serum albumin, was found to be the active moiety of LPS capable of stimulating colony growth of lymphoid cells in soft agar culture. The PS portion exhibited no significant activity at the concentrations used. Glycolipids from mutant strains of S. minnesota which contain the intact lipid moiety but are deficient in PS content, were as potent as S. abortus equi LPS in stimulating B cells into colony growth. Alkaline hydrolysis of LPS which cleaves ester‐linked fatty acids, substantially decreased the number of lymphocyte colonies formed. This indicates that the intact lipid moiety is required for stimulating lymphocytes into colony formation. The synthetic glycolipid, N‐palmitoyl‐D‐glucosamine (NPG), whose structure is similar to some components of lipid A, was also able to induce B lymphocyte colony development. In summary, our data point to lipid A as the active moiety of the endotoxin which induces B lymphocytes to grow and develop into colonies in the 2‐layer soft agar culture system.
Studies on the association of the Epstein‐Barr virus genome and human chromosomes
Authors: Glaser Ronald , Nonoyama Meihan , Hampar Berge , Croce Carlo M.
Published: February 2005

We have used human‐mouse somatic cell hybrid cells to study the association between the EBV genome and the cellular genome. Attempts were made to identify a specific human chromosome(s) with which the EBV genome is associated. Our data suggest that at least in the mouse fibroblast/Burkitt lymphoma hybrid combination studied, the EBV genome is not associated with any specific human chromosome.
Diffusible factor(s) controlling density inhibition of 3T3 cell growth: A new approach
Authors: Harel L. , Jullien M. , De Monti M.
Published: February 2005

In 3T3 Swiss mouse fibroblasts, incorporation of phosphate into cells and phosphorylation of small organic compounds were increased by shaking dense cultures. This response was not obtained with SV40 transformed Swiss 3T3 cells (SV‐3T3).

It appeared likely that these results could be accounted for by an inhibitor released from 3T3 cells but not from SV‐3T3 cells.

Our new method of co‐incubation of sparse and dense cultures allowed us to demonstrate inhibition of growth and phosphate metabolism in sparse 3T3 cultures which were shaken in the presence of dense cultures. The inhibition was much less when the cultures were co‐cultivated but not shaken.

The inhibition of phosphate incorporation in acid‐soluble and acid‐insoluble fractions of sparse cultures was observed as early as 20 minutes of co‐incubation in the presence of dense cultures, so this inhibition is not the result of depletion of growth factors in the medium.

Our experiments suggest that an inhibitor(s) was released from dense cultures of 3T3 cells.
Epstein‐barr virus in somatic cell hybrids between mouse cells and human nasopharyngeal carcinoma cells
Authors: Steplewski Zenon , Koprowski Hilary , Andersson‐Anvret Maria , Klein George
Published: February 2005

Somatic cell hybrids between mouse cells and cells derived directly from NPC biopsies were produced in order to study the association of the Epstein‐Barr virus (EBV) genome and the expression of Epstein‐Barr nuclear antigen (EBNA) with the human chromosome(s). All attempts to correlate the presence of EBV‐DNA and the expression of EBNA with the presence of a particular human chromosome(s) showed that the segregation of EBV‐DNA or of EBNA and human chromosomes was dysconcordant. The data, therefore, suggest that in the hybrids studied the presence of EBA‐DNA is not determined by the presence of a specific human chromosome.
Effects of albumin on the phagocytosis of polysterene spherules by rabbit polymorphonuclear leucocytes
Authors: Beukers Harm , Deierkauf Frans A. , Blom Cornelis P. , Deierkauf Martha , Riemersma Jelle C.
Published: February 2005

Polystyrene latex spherules are rapidly phagocytized by polymorphonuclear leucocytes. Surface modifications of the latex spherules can lead to an inhibition of phagocytosis, and the purpose of this study was to analyse in particular inhibition by bovine and human serum albumin. It can be shown that albumin is bound much more strongly by the PSL particle surface than by the cell surface. Furthermore, a correlation was established between the extent to which the particle surface is covered and the extent of phagocytosis inhibition. In the binding of albumin to the particle surface ionic as well as hydrophobic interactions play a role. As a result, the particle acquires a negatively charged hydrophilic coating which effectively diminishes the number of effective cell‐particle collisions leading to engulfment.
Expression of transformation in cell hybrids. II. Nonsuppression of the transformed phenotype in hybrids between a chemically transformed and nontransformed derivatives of Balb/3T3
Authors: Jha Krishna K. , Cacciapuoti Janice , Ozer Harvey L.
Published: February 2005

Hybrid clones derived from a nitrosocarbaryl‐transformed Balb/3T3 cell line, Clone H, and a nontransformed cell line TH02 resemble the transformed parent in the clone morphology, higher saturation density, colony formation in medium with reduced serum concentration, growth in agarose and ability to form clones on Balb/3T3 monolayer. Results are discussed in the framework of genetic models which permit or require dominant mutations for the expression of transformed phenotype.