bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced synthesis and extracellular accumulation of hyaluronic acid during stimulation of quiescent human fibroblasts by mouse epidermal growth factor
Authors: Lembach Kenneth J.
Published: February 2005

The effect of mouse epidermal growth factor (mEGF) on the synthesis of glycosaminoglycans and glycoproteins by human fibroblasts has been studied. The addition of physiological concentrations (10−9 M) of mEGF to quiescent cultures preincubated in the absence of serum was found to elicit an increased incorporation of 3H‐glucosamine into the glycosaminoglycans and glycoproteins of both the cellular and extracellular fractions. Although the growth response to the factor, as measured by DNA replication, was minimal under these conditions as compared with the effect of serum, the mEGF‐induced incorporation of glucosamine into these cellular constituents and into the extracellular glycoproteins was comparable to that elicited by serum shift‐up. Serum, however, caused a significantly larger incorporation of glucoasmine into extracellular, acid‐soluble glycosaminoglycans, which were shown to contain hyaluronic acid as the major component. As previously demonstrated, the growth response to mEGF can be enhanced several fold by an mEGF‐binding arginine esterase, which is normally associated with the factor in vivo, and by ascorbate. The esterase was found to increase markedly the mEGF‐induced incorporation of glucosamine into extracellular hyaluronic acid, while the addition of ascorbic acid did not significantly alter glucosamine incorporation.
Interrelationships between Water and Cellular Metabolism in Artemia cysts. V. 14CO2 incorporation
Authors: Clegg James S.
Published: February 2005

The ability of cysts of the brine shrimp, Artemia salina, to incorporate 14CO2 into organic compounds soluble in cold‐trichloroacetic acid was examined over a broad range of cellular water concentrations. Carbon dioxide was not incorporated by cysts containing less than about 0.3 g H2O/g dried cysts, the “critical hydration” for CO2‐fixation. This relationship held whether the cysts were hydrated from the liquid or the vapor phase. The incorporation of radioactivity was shown to be due exclusively to metabolic activity in the cellular component of the cyst. Above the critical hydration, the amount of 14CO2 incorporated was a function of cyst water content, but the kinds of metabolites labelled with this precursor, and their relative proportions, were found to be similar in cysts of greatly different hydration. Almost all of the radioactivity was associated with amino acids, Krebs cycle intermediates and related acids, and pyrimidine nucleotides. The fact that the pathway involved with CO2‐fixation, and subsequent metabolism of the fixation products are all initiated in cysts containing as little as 0.3 g H2O/g is particularly noteworthy since this hydration level is well within the range of the amounts of “bound water” described in the literature for a wide array of cells and tissues.
Hormonal regulation of amino acid transport and gluconeogenesis in primary cultures of adult rat liver parenchymal cells
Authors: Kletzien Rolf F. , Pariza Michael W. , Becker Joyce E. , Potter Van R.
Published: February 2005

Amino acid transport was studied in primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion technique and maintained as a monolayer in a serum‐free culture medium. These cells carried out gluconeogenesis from three carbon precursors (alanine, pyruvate, and lactate) in response to glucagon addition. Amino acid transport was assayed by measuring the uptake of the nonmetabolizable amino acid, α‐aminoisobutyric acid (AIB). Addition of insulin or glucagon to cultured rat liver parenchymal cells resulted in an increased influx of AIB which was reflected in a higher initial rate of AIB transport. The glucocorticoid, dexamethasone, when added alone to cultures did not affect AIB transport. However, prior or simultaneous addition of dexamethasone to glucagon‐treated cells caused a strong potentiation of the glucagon induction of AIB transport. Kinetic analysis of the effects of insulin and glucagon demonstrated that insulin increased the Vmax for transport without changing the Km while glucagon primarily decreased the Km for AIB transport. The effect of dexamethasone was to increase the Vmax of the low Km system.
Current status of the thiol redox model for the regulation of hexose transport by insulin
Authors: Czech Michael P.
Published: February 2005

Data obtained over the last two years pertinent to the thiol redox model for the modulation of hexose transport activity by insulin is summarized. The model proposes that activation of hexose transport in fat cells involves sulfhydryl oxidation to the disulfide form in a key protein component of the fat cell surface membrane. Theoretically, the rapid activation of transport by insulin may involve either the conversion of inactive membrane carriers to the active form as originally proposed, or the conversion of a low Vmax transport system to a high Vmax form. The present experiments showed that the percent inhibition of insulin‐activated transport rates by submaximal levels of cytochalasin B was decreased compared to its effects on basal transport. Treatment of fat cells with N‐ethylmaleimide inhibited cytochalasin B action but not transport activity. When insulin or the oxidant vitamin K5 was added to cells 5 minutes before the N‐ethylmaleimide, the elevated transport activity was also resistant to the sulfhydryl reagent, but cytochalasin B retained its potent inhibitory effect on transport. The data demonstrate that unique properties characterize basal versus insulin‐activated transport activity with respect to the sensitivity of cytochalasin B action to sulfhydryl blockade in isolated fat cells. The data are consistent with the concept that activation of transport activity reflects the conversion of a reduced (sulfhydryl) system characterized by a low Vmax to an oxidized (disulfide), high Vmax transport system.
Regulation of glucose transport in chick fibroblasts: Bicarbonate, lactate and ascorbic acid
Authors: Amos H. , Christopher C. W. , Musliner T. A.
Published: February 2005

The rate at which chick embryo fibroblasts in primary or secondary culture transport glucose or 3‐O‐methyl glucose is strongly influenced by the presence of bicarbonate ion in the culture medium. Cells growing or maintained on glucose at physiologic concentration (5.5mM) have an 8 to 10‐fold higher rate of glucose uptake than their counterparts cultivated without bicarbonate. These cells also produce more lactate as a consequence of their more rapid intake of glucose. The hydrogen acceptors, methylene blue and dehydroascorbate added to the culture medium reduce the cell capacity to transport glucose and 3‐O‐methyl glucose to levels obtaining in the bicarbonate‐free medium. There is a concomitant reduction in glucose utilized by cells during 24 hours and a further reduction in lactate formed per molecule of glucose metabolized.
Sodium: A regulator of glucose uptake in virus‐transformed and nontransformed cells
Authors: Bader John P.
Published: February 2005

Observations of cells transformed by the Bryan strain of Rous sarcoma virus (RSV‐BH) suggested that the intracellular concentrations of sodium ion (Na+) may play a critical role in cellular metabolism. In an attempt to manipulate intracellular Na+, chick embryo cells were exposed to graded concentrations of Na+ in the cellular growth medium, and the effect on capacity for glucose uptake was examined. After incubation for six hours, the incorporation rate of 2‐deoxyglucose (used as a substitute for glucose) was proportional to the external Na+ concentration over the range, 100 mM to 200 mM. Cells transformed by RSV‐BH were less responsive than nontransformed cells to differences in Na+ at low concentrations. The changes were specifically dependent upon Na+, since K+, Li+, or choline+ were ineffective as substitutes, and increasing the ionic strength above that of 120 mM Na+ was effective only when Na+ was the added cation.
Transport as a rate limiting step in glucose metabolism in virus‐transformed cells: Studies with cytochalasin B
Authors: Bissell Mina J.
Published: February 2005

Steady‐state kinetic tracer analysis and two‐dimensional chromatography and autoradiography were used to examine the relation of glucose transport to its metabolism in cultured chick embryo fibroblasts. Cytochalasin B was added to Rous sarcoma virus‐infected cells to bring the rate of glucose uptake to the level of uninfected cells. It is concluded for chick cells in culture that: (1) the transport of glucose, rather than its phosphorylation, is the rate limiting step and (2) the difference in aerobic glycolysis between normal and virus‐transformed cells at physiological glucose concentration may be a consequence of the difference in the rates of glucose uptake.
Some physicochemical properties of plasma membrane vesicles
Authors: Wallach Donald F. H.
Published: February 2005

The plasma membranes of many animal cells can be disrupted into small sealed vesicles that can be purified centrifugally and utilized for studies on membrane transport. The vesicles behave as micro‐osmometers. However, the presence of charges fixed at the internal and external surfaces of the membrane walls produce pH levels at these surfaces that deviate considerably from bulk pH. Transverse symmetry of charge distribution further leads to transverse asymmetry of surface pH. Finally, charges fixed at the internal membrane surface produce significant Donnan osmotic effects that depend upon membrane composition and ionic environment.
Cooperative binding of concanavalin a to thymocyte membrane vesicles augments uptake of α‐aminoisobutyrate
Authors: Schmidt‐Ullrich Rupert , Mann Nathan D. , Wallach Donald F. H.
Published: February 2005

(1) Membrane vesicles from rabbit thymocytes accumulate α‐aminoisobutyrate in the presence of 0.1 M NaCl. Uptake is 1/2 maximal after about 2 min and reaches a plateau value (61 pmoles/mg protein) after 30 min. (2) Up to 25 μg concanavalin A/ml, binding of the lectin describes a sigmoid curve indicative of a cooperative process. (3) At lectin concentrations up to 8 μg/ml, lectin binding enhances the uptake of α‐aminoisobutyrate (maximally 30%).
Uptake of α‐aminoisobutyric acid and phosphate by membrane vesicles derived from growing and quiescent fibroblasts
Authors: Nilsen‐Hamilton Marit , Hamilton Richard T.
Published: February 2005

Membrane vesicles derived principally from the plasma membrane and endoplasmic reticulum of mouse 3T3 cells transformed by Simian virus 40 take up α‐aminoisobutyric acid (AIB) and phosphate (Pi). When NaCl is added simultaneously with AIB or Pi, uptake rises two‐ to three‐times above the equilibrium uptake value found in the absence of NaCl.

The ability to accumulate AIB or Pi over the control value, in the presence of a Na+ gradient, is almost lost in membrane vesicles derived from benzpyrene‐transformed 3T3 cells (BP3T3) arrested in the G1 phase of the cell cycle by serum starvation.

When added to the membranes with NaCl and the uptake substrate, a combination of fibroblast growth factor (FGF) and epidermal growth factor (EGF) restores the ability of the membranes to accumulate AIB and Pi over the control value.
Large scale purification and further characterization of a rat liver cell conditioned medium multiplication stimulating activity
Authors: Dulak Norman C. , Shing Yuen W.
Published: February 2005

The application of roller‐bottle cell culture techniques and a relatively simple purification scheme has led to the isolation of milligram quantities of a polypeptide cell multiplication stimulating activity (MSA) from Buffalo rat liver cell conditioned medium. We have characterized the apparently homogeneous MSA with respect to its biological activity, its N‐terminal amino acid residue, and its amino acid composition, and have tested the MSA for growth‐promoting activity in a number of cell types.
Delayed malignancy and altered growth properties of somatic cell hybrids between rat hepatoma and mouse L‐cells
Authors: Lyons Linda B. , Thompson E. Brad
Published: February 2005

Somatic cell hybrids of mouse L‐cells with rat HTC cells were studied for their growth properties in vitro and tumorigenicity in vivo. Three hybrid clones were selected for detailed study. All were delayed in tumor formation in nude mice compared with the parent lines despite their varied growth properties in vitro.

One clone was sensitive to density dependent inhibition of growth (DDIG) and had a relatively low saturation density. A second clone was not sensitive to DDIG and had a higher saturation density. The third clone had atypical morphology, was insensitive to DDIG, and had a relatively high saturation density. All of the clones studied produced colonies suspended in agarose gel which were much smaller than those of the parents incubated for the same period of time. Only the pattern of growth in agarose gel corresponded to the delayed tumor formation in vivo of the hybrids. Sensitivity to DDIG and saturation density were not consistent with tumor growth.

The hybrid clone that was sensitive to DDIG was the only one of the three that had a nearly complete set of chromosomes derived from each of the parents. The chromosome numbers of all three clones were unchanged after growth in agarose or as tumors in the nude mice.
Surface exposure of glycosaminoglycans in resting, growing and virus transformed 3T3 cells
Authors: Vannucchi Simonetta , Chiarugi Vincenzo P.
Published: February 2005

Glycosaminoglycans (GAGs) were released by trypsin from the surface of cultured mouse cells (3T3) in two different growing states: during log‐growth phase and during resting due to serum starvation. Doubly labelled molecules from resting cells were compared with those from growing as well as from transformed cells. Reproducible differences in the elution pattern during ion exchange chromatography and in susceptibility to specific hydrolytic enzymes have been demonstrated: the GAGs pattern of growing normal cells is similar to the pattern of the cells transformed by either Polyoma or SV‐40 viruses and very different from the pattern of resting cells. Growing and transformed 3T3 show a relatively low amount of trypsin removable heparan sulphate (HS) and a relatively high amount of hyaluronic acid (HA) while resting cells exhibit an opposite ratio between the two GAGs. The lowering of HS and the increase of HA in the cell coat is therefore suspected to be more dependent upon growth than upon transformation.
Premature chromosome condensation and cell cycle analysis
Authors: Rao Potu N. , Wilson Barbara , Puck Theodore T.
Published: February 2005

The application of the phenomenon of premature chromosome condensation for cell cycle analysis in HeLa and CHO cells has been examined. Random populations of HeLa and CHO cells pulse labelled with H3‐TdR were separately fused with mitotic HeLa cells using U.V. inactivated Sendai virus. The resulting prematurely condensed chromosomes (PCC) were scored and classified into G1, S and G2‐PCC on the basis of both morphological and autoradiographic data. The results of this study indicated that the G1, S and G2 phase cells are equally susceptible to virus‐induced fusion with mitotic cells and subsequent induction into PCC. Hence the PCC method for cell cycle analysis is both practical and accurate. This study also revealed that the process of chromosome decondensation initiated during the telophase of mitosis continues throughout the G1 period reaching an ultimate state of decondensation by the end of G1, at which point the fusion of such cells with those in mitosis yield PCC with the most diffused morphology instead of the discrete single stranded structures characteristic of early G1‐PCC. Thus, the decondensation of chromatin during G1 appears to be a prerequisite for the subsequent initiation of DNA synthesis.
The effect of elevated potassium on the time course of neuron survival in cultures of dissociated dorsal root ganglia
Authors: Scott Brian S.
Published: February 2005

The effect of potassium (K+) on the time course of neuron survival has been investigated by counting neurons over a 24‐day period in live cultures of dissociated dorsal root ganglia from embryonic chick, fetal and newborn mouse, and fetal human material. In both normal K (6 mM) and in elevated K (20 mM mouse and human, 40 mM chick) there was initially a rapid exponential decrease in neuron survival. However, the magnitude of this decrease was less in the elevated K. In normal K neuron number decreased monotonically; the rate of degeneration itself decreased with time so that after 24 days neuron survival became relatively constant. In contrast, in elevated K the neuron number actually increased over a limited time interval before attaining a stable long‐term value much greater than that in normal K. Thus, elevated K enhanced long‐term survival by causing a lower rate of degeneration and also by causing an increase in neuron number during a limited period of the time in culture. From these observations and other evidence, it is argued that K can substitute to some extent for the trophic action normally exerted by the peripheral field of innervation of the DRG. It is further argued that K acts through its depolarizing effect on the membrane potential and that modification of intracellular ionic concentrations seems less likely to be involved.
Coordinate control of collagen synthesis and cell growth in chick embryo fibroblasts and the effect of viral transformation on collagen synthesis
Authors: Kamine James , Rubin Harry
Published: February 2005

Using collagenase digestion as an assay for collagen in partially synchronized secondary cultures of chick embryo fibroblasts, we find that the rate of collagen synthesis remains at a constant fraction of overall protein synthesis (5%) regardless of the growth rate of the cells even when the rate of protein synthesis is accelerated 5‐fold by adding serum and altering the pH of the culture medium. However, in cells oncogenically transformed by Rous sarcoma virus, the relative rate of collagen synthesis was decreased by 50% 24 hours after infection and was 10% of the initial rate after 5 days. This selective decrease in rate of collagen synthesis could be reversed in cells infected with an RSV temperature‐sensitive transformation‐defective mutant at the non‐permissive temperature, indicating that the decrease in the rate of collagen synthesis was not merely the result of viral infection but was a direct consequence of oncogenic transformation.
Correlated effects of external magnesium on cation content and DNA synthesis in cultured chicken embryo fibroblasts
Authors: Sanui Hisashi , Rubin Harry
Published: February 2005

Depletion of Mg2+ in the growth medium for chicken embryo fibroblasts produces a large decrease in DNA synthesis as measured by 3H‐thymidine incorporation, and concomitant decreases in cellular K+ and Mg2+ and increases in Na+ and Ca2+. In cells grown in media containing 0.2 mM Ca2+, graded reduction of Mg2+ from 0.8 mM (control) to 0.016 mM produced graded decreases in DNA synthesis to 10% of control at 0.016 mM Mg2+. Concomitantly, cell cations showed graded changes, Na+ increasing to 227%, K+ decreasing to 52.5%, Mg2+ decreasing to 57.5% and Ca2+ increasing to 153.5% of control. The effects of Mg2+ depletion on DNA synthesis and cell cation content exhibited a dependence on Ca2+ concentration, the effects being larger at low Ca2+ concentration. Use of inorganic pyrophosphate in the growth medium as a selective complexor of Mg2+ caused a marked decrease in DNA synthesis which was accompanied by changes in cellular cation content similar to those produced by direct Mg2+ depletion.

The effects of Mg2+ depletion on cell cation content are explainable in terms of changes in membrane permeability caused by rapid external surface exchange of bound divalent cations. Among the several interpretations of the data in terms of possible mechanisms by which changes in external Mg2+ concentration may affect cell metabolism, the most consistent with known properties of the system is the concept of a central role for intracellular free Mg2+ in the coordinate control of growth and metabolism in animal cells.
Effects of sugars on melanogenesis in cultured melanoma cells
Authors: Nakayasu Michie , Saeki Hisaaki , Tohda Hiroko , Oikawa Atsushi
Published: February 2005

A permanent cell line C2M of mouse melanoma B16 was highly melanized in a modified Eagle's MEM supplemented with 10% calf serum, when the medium contained 1 mM galactose and 10 mM pyruvate instead of 5.5 mM glucose. The activity of the key enzyme for melanogenesis, tyrosinase (EC 1.14.18.1), of living cells cultured in the galactose‐pyruvate medium was consistently 27 times higher than that of cells in normal MEM. This high level of tyrosinase activity was maintained in the stationary phase, in contrast to the activity of cells in normal medium, which decreased sharply in the stationary phase. It seems likely that tyrosinase activity is suppressed by the presence of glucose rather than stimulated by galactose.

This modified medium should be useful for obtaining a high level of tyrosinase activity in living cells in culture and in cell‐free extracts.
Incorporation of purine nucleosides in cultured fibroblasts from a patient with purine nucleoside phosphorylase deficiency and associated T‐cell immunodeficiency
Authors: Burke Wylie G. , Chen Shi‐Han , Scott C. Ronald , Ammann Arthur J.
Published: February 2005

Cultured skin fibroblasts from a patient with T‐cell immune deficiency and an absence of purine nucleoside phosphorylase activity in red cells were assayed for their capacity to metabolize inosine and guanosine. The cultured fibroblasts were lacking activity of nucleoside phosphorylase and, compared to normal fibroblasts, could incorporate only 2% and 4% of 14C‐inosine and 3H‐guanosine, respectively, into acid precipitable material. Autoradiography visually confirmed the failure of the NP deficient cell line to incorporate the nucleosides into nuclear material. The physiological mechanism by which the deficiency of purine nucleoside phosphorylase causes T‐cell dysfunction remains unclear.
Renal enzymes in kidney cells selected by D‐valine medium
Authors: Gilbert Scott F. , Migeon Barbara R.
Published: February 2005

The ability of D‐valine medium to inhibit fibroblasts in cultures derived from kidneys of various mammals has enabled the selective proliferation of epithelial cells in the absence of fibroblast overgrowth. Studies of these selected epithelial cells have demonstrated the presence of D‐amino acid oxidase, carbonic anhydrase, high levels of alkaline phosphatase and the renal specific pattern of lactate dehydrogenase. The presence of these renal enzymes suggests that the selected epithelial cells are of renal tubular origin and indicates that these differentiated functions are retained in cultured cells.
Nucleoside transport in mammalian cell membranes: A specific inhibitory mechanism of high affinity probes
Authors: Eilam Yael , Cabantchik Z. Ioav
Published: February 2005

The nucleoside transport of systems of hamster cells are susceptible to inhibition by S‐6‐substituted derivatives of mercaptonucleosides. The mechanism of interaction between the most potent inhibitor of the class, 6‐nitrobenzyl mercaptoinosine (NBMI) and the uridine transport system of hamster fibroblasts is studied in the present work. A kinetic description of the interaction is presented. Uridine transport is inhibited in a partially competitive fashion, leaving a substantial free fraction of the transport (20–30%) virtually insensitive to increasing concentrations of inhibitor. One interpretation compatible with the kinetic and chemical properties of the system assumes that binding of the inhibitor to carriers occurs at sites different from the substrate binding sites (allosteric binding). Such a binding induces a conformational change in the carrier as manifested in a reduced affinity to the substrate and a susceptibility to inhibition by organomercurials. The alternative interpretation postulates two parallel transport systems which display distinctly different susceptibilities to NBMI and to organomercurials. Experiments performed with non‐penetrating organomercurials show that the sulfhydryl groups related either to the alleged allosteric components or to additional carriers, are located superficially on the membrane. The binding of NBMI is reversible, the affinity is extremely high (Ki = 0.15 nMolar) and the rate of reaction could probably be diffusionally limited at low concentrations of reagent (activation energy 250 cal/mole, rate k = 1.3.108 min−1. Molar−1). The high affinity properties of the probes are used to determine the number of NBMI binding sites. A value of 47,000 and 77,000 sites/cell was obtained by two separate methods.

The possibility that allosteric properties are present in carrier systems are discussed in terms of current concepts of modulation of transport functions in biological membranes.
Induction of alkaline phosphatase in choriocarcinoma cells by 1‐β‐D‐arabinofuranosyl‐cytosine, mitomycin C, phleomycin, and cyclic nucleotides
Authors: Chou Janice Y. , Robinson J. C.
Published: February 2005

Alkaline phosphatase is induced in cultured human choriocarcinoma cells by three inhibitors of DNA synthesis which alter DNA structure: 1‐β‐D‐arabinofuranosyl‐cytosine, mitomycin C, and phleomycin. No induction is observed with the inhibitors, hydroxyurea and thymidine, which do not alter DNA structure. Cyclic AMP, analogs of cyclic nucleotides, and sodium butyrate also induce alkaline phosphatase in these cells. Among the cyclic nucleotides tested, dibutyryl cyclic AMP is the best inducer, whereas dibutyryl cyclic GMP is a poor inducer.

Induction of alkaline phosphatase by inhibitors of DNA synthesis or by exposure to dibutyryl cyclic AMP appears to utilize different mechanisms. Maximum induction is observed after simultaneous addition of both types of inducers at the concentrations found to be optimal for each inducer alone. Under these conditions, the induced activity is equal to or greater than the sum of the activities induced by each inducer. RNA synthesis and protein synthesis are required for induction.

Dibutyryl cyclic AMP added to cultures of choriocarcinoma cells is not degraded in the culture medium, but is extensively degraded in the cells. Nevertheless, significant amounts of dibutyryl and monobutyryl cyclic AMP are found intracellularly throughout the experiment. Since the cellular uptake of dibutyryl cyclic AMP is extremely slow, the amount of butyrate released by intracellular degradation cannot account for the observed induction. Neither the rate of uptake nor the stability of dibutyryl cyclic AMP are changed by the addition of 1‐β‐D‐arabinofuranosyl‐cytosine to the culture medium. Furthermore, 1‐β‐D‐arabinofuranosyl‐cytosine inhibits the induction by sodium butyrate. The results indicate that butyrate is not the major mediator of induction by dibutyryl cyclic AMP.
A cell membrane alteration specifically induced by SV40 transformation
Authors: Lago C. , Sartorius B. , Tramontano D. , Amati P.
Published: February 2005

Transformation of mouse 3T3 cells by SV40 results in a specific membrane modification which renders cells resistant to the killing action of the lipophilic antibiotic Amphotericin B. This alteration is under genetic cellular control and is specific for SV40 transformation since transformation with polyoma or mouse sarcoma viruses does not confer resistance to the antibiotic. Analogous resistance is induced by SV40 transformation of primary human fibroblast cells. The acquired resistance is not due to decreased binding of Amphotericin B and is partially reversed if cells are grown in the presence of cholesterol. The results are interpreted as a specific change of sterol structure of the membrane or the loss of a minor cholesterol fraction responsible for the killing action of the antibiotic.
Reversion in expression of hypoxanthine‐guanine phosphoribosyltransferase in 6‐thioguanine resistant neuroblastoma: Evidence for reduced enzyme levels associated with unaltered catalytic activity
Authors: Skaper Stephen D. , Spector Elaine B. , Seegmiller J. Edwin
Published: February 2005

Five clones of mouse neuroblastoma cells able to grow in hypoxanthine‐aminopterin‐thymidine containing medium were isolated from a hypoxanthine‐guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8; IMP: pyrophosphate phosphoribosyltransferase) deficient cell line. These hypoxanthine‐aminopterin‐thymidine resistant revertant clones had 45–55% of wild‐type cell HGPRT activity. Kinetic studies indicated that the HGPRT in revertant clones had a reduced maximal velocity as compared to wild type cells based on cell protein. Apparent Km values of HGPRT for hypoxanthine and 5‐phosphoribosyl‐1‐pyrophosphate were similar in wild‐type and revertant cells. Heat inactivation studies demonstrated a similar heat lability for HGPRT in revertant and wild‐type cells. An antibody fraction prepared from serum of rabbits immunized with HGPRT partially purified from mouse liver was used to measure the amount of cross‐reacting material in normal and revertant clones. The revertant clones had one‐half the amount of cross‐reacting material present in wild‐type cells, based on a given amount of cell protein. These data indicate that the revertant cells may contain fewer HGPRT molecules with unaltered catalytic activity.
Accelerated calcium ion uptake in murine thymocytes induced by concanavalin A
Authors: Ozato Keiko , Huang Leaf , Ebert James D.
Published: February 2005

The mechanism of enhancement of Ca2+ uptake by the T cell mitogen concanavalin A (Con A) was studied in murine thymocytes. Native Con A enhanced the rate of Ca2+ uptake as much as 9‐fold, an increase being observed within five minutes after Con A addition. The effect of Con A was reversed completely by α‐methyl mannopyranoside (α‐MM). Increased Ca2+ uptake was observed with increasing concentrations of Con A, between 2 and 400 μg/ml, indicating that the stimulation of Ca2+ uptake is not restricted to mitogenic lectin concentrations (0.5‐2 μg/ml). Succinyl Con A exhibits only a slight effect in the same concentration ranges as native Con A. Ca2+ uptake, both in the absence and presence of Con A, is strongly dependent on energy metabolism and is carrier mediated. The augmentation of Ca2+ uptake by native Con A is due to an enhanced Vmax. Uptake of the anion, CrO42–, by thymocytes, found to be a nonsaturable process, was also enhanced by Con A. The effect of Con A on CrO42− permeability appears to be independent of its effect on Ca2+ uptake.
Free ribosomes and growth stimulation in human peripheral lymphocytes: Activation of free ribosomes as an essential event in growth induction
Authors: Cooper Herbert L. , Braverman Richard
Published: February 2005

During the initial ten hours of growth in lymphocytes stimulated by phytohemagglutinin, the cells are converted from a state in which over 70% of all ribosomes are inactive free ribosomes, to one in which over 80% of ribosomes are in polysomes or in native ribosomal subunits. In this initial period, there was a neglible increase in total ribosomal RNA due to increased RNA synthesis, and abolition of ribosomal RNA synthesis with low concentrations of actinomycin D did not interfere with polysome formation. Therefore, the conversion is accomplished by the activation of existing free ribosomes rather than by accumulation of newly synthesized particles. The large free ribosome pool of resting lymphocytes is thus an essential source of components for accelerated protein synthesis early in lymphocyte activation, before increased synthesis can provide a sufficient number of new ribosomes. Free ribosomes accumulate once more after 24 to 48 hours of growth, when RNA and DNA synthetic activity are maximal. This reaccumulation of inactive ribosomes at the peak of growth activity may represent preparation for a return to the resting state where cells are again susceptible to stimulation.

Activation of free ribosomes to form polysomes appears to involve modification of at least two steps: (a) dissociation of free ribosomes with stabilization as native subunits, and (b) adjustment of a rate‐limiting step at initiation.
The regulation by fibroblast growth factor of early transport changes in quiescent 3T3 cells
Authors: Quinlan Dennis C. , Hochstadt Joy
Published: February 2005

This study involves the use of fibroblast growth factor (FGF) as a substitute for exogenous serum to examine the early transport changes which occur when quiescent 3T3 cells re‐initiate active growth. FGF, in nanogram amounts, together with insulin and dexamethasone, can induce mitogenesis and mitosis in 3T3 cells GO‐arrested by holding in growth medium containing 0.8% calf serum. In terms of quiescent cell transport activity enhancement, FGF is 300,000‐fold more effective than fresh serum, on a protein basis. In addition, very short exposure of serum‐depleted cells to FGF indicates that a distinct temporal or time sequence exists in the transport system activation process. For example, uptake of α‐aminoisobutyric acid (AIB) and uridine are stimulated very rapidly, whereas hypoxanthine uptake does not respond until much later. Closer analysis shows that AIB uptake is maximally enhanced within zero to two minutes after FGF addition to cells. Finally, the stimulatory effect of FGF on transport system activities is specific in terms of the proliferative state of the cells to which it is added, and in terms of the uptake systems which respond to it.
Osmotic properties of human lymphocyte
Authors: Hempling H. G. , Thompson S. , Dupre A.
Published: February 2005

The osmotic properties of human lymphocytes isolated from 15 ml of venous blood were examined. Measurements of the permeability of the membrane to water under an osmotic gradient were also made. The Boyle‐Van't Hoff relation held very well for the human lymphocyte when the cells were shrunken in hyperosmotic media to concentrations twice isosmotic. The volume of osmotically inactive material or “b” value averaged 32% of the mean corpuscular volume. These values were independent of temperature. Ponder's R ranged between 0.8 and 0.9. The average value for Lp, the hydraulic coefficient was 0.46 μ/min atm ± 0.02 (S.E.M.) at 25°C. No significant effect of age, sex, or race was noted. The effect of temperature between 10°C and 37°C was measured and heats of activation between 11.1 and 17.4 kcal/mole were calculated with a mean of 14.1 kcal/mole ± 1.6 (S.E.M.) Concanavalin A at 10 μg/ 1.5 ± 106 lymphocytes produced blastogenesis of 25% or more of the lymphocytes without clumping, agglutination, or toxicity. The mean corpuscular volume increased by 21% after 72 hours due to an increase in the “b” value which increased by 80%. The volume of free water remained constant. Histograms of the distribution of cell volumes showed that volume changes were uniform throughout the population with no evidence of agglutination or clumping. The significance of these results is discussed in the context of membrane fluidity and the state of intracellular water.
Cytotoxicity of commonly used solvents at elevated temperatures
Authors: Li Gloria C. , Hahn George M. , Shiu Esther C.
Published: February 2005

At 43°C (but not at 41°C), organic solvents used to dissolve water‐insoluble chemotherapeutic agents become themselves lethal to cells. This finding is not unique to Chinese hamster cells (HA‐1); mouse mammary sarcoma cells (EMT‐6) behave similarly. The solvent concentrations involved are in the range of those needed to make drug solutions. Hence experiments measuring drug‐cell interactions at elevated temperatures must include controls which independently measure solvent effects.
Growth stimulating activity in bovine pituitary extract specific for a rat mammary carcinoma cell line
Authors: Kano‐Sueoka Tamiko , Campbell Gerald R. , Gerber Michael
Published: February 2005

We report the isolation of a bovine pituitary growth factor (MGF) for a rat mammary carcinoma cell line, 64‐24, which was isolated from a highly hormone‐dependent mammary tumor. The MGF has been partially purified by a series of Diaflo ultrafiltration membrane sievings, isoelectric focusing and Sephadex columns. The MGF has a molecular weight of ∼︁ 1,000 to 2,000 daltons and has a U.V. absorption spectrum typical for a polypeptide. Its isoelectric point is ∼︁ pH 3.8–4.0. The factor is heat stable. The growth stimulating activity of the MGF does not stimulate other rat cell lines (22‐1, RMG or HTC lines) but is specific for the 64‐24 mammary tumor cell line. The MGF is not among previously reported pituitary hormones or growth factors.
Proliferation and differentation of normal granulopoietic cells in continuous bone marrow cultures
Authors: Williams Neil , Jackson Heather , Rabellino Enrique M.
Published: February 2005

Modified conditions are reported for successful continuous bone marrow cultures with stem cell self‐renewal and granulocyte‐macrophage differentiation. Cells cultured over several weeks were found to be identical to freshly isolated bone marrow cells. Polymorphic neutrophils derived from cultures and primary bone marrow neutrophils both showed C3 and IgG receptors and both actively phagocytosed foreign particles. Cultured and normal CFU‐c were identical, both in their dose responsiveness to CFS and in their sedimentation rate characteristics.
Studies on phosphoglucose isomerase from cultured human fibroblasts: Absence of detectable ageing effects on the enzyme
Authors: Shakespeare Valerie , Buchanan J. H.
Published: February 2005

Some properties of the enzyme phosphoglucose isomerase (PGI) from the foetal lung fibroblast strain MRC‐5 have been investigated throughout the in vitro lifespan of this cell strain.

No significant age‐related alterations in specific activity or thermostability of PGI could be detected. Titration of enzymatic activity with antibody directed against purified PGI showed no detectable differences in PGI from extracts of early passage cells compared with enzyme from senescent cells.

The effect of p‐fluorophenylalanine incorporation on PGI was examined in early passage fibroblasts. Thermostability studies showed increased heat lability of PGI from analogue treated cells when compared with enzyme from control cells at the same passage. However, no inactive PGI protein could be detected by antiserum titration in extracts from analogue‐treated cells.

The results indicate that no significant amount of altered or inactive PGI is produced in ageing fibroblasts.
Protein turnover in senescent cultured chick embryo fibroblasts
Authors: Kaftory A. , Hershko A. , Fry M.
Published: February 2005

The over‐all rates of protein synthesis, degradation and net accumulation were estimated in rapidly growing young and slowly doubling old cultures of chick fibroblasts. We find that not only the rate of protein synthesis is reduced in senescent cultures, but the average rate of protein degradation is also slowed down considerably. This decrease in the rate of protein breakdown in aging cells stands in contrast with the previously observed acceleration of this process by other conditions (such as serum deprivation or overcrowding) that lead to the cessation of cellular growth. Though the retarded protein degradation may contribute to the accumulation of abnormal proteins in senescen cells, we find that the breakdown of grossly abnormal puromycin peptides proceeds equally rapidly in young and old cultures.

The protein content of senescent cells increase by 1.8‐fold as compared to young cells, while the average cell volume is increased even more (almost 5‐fold). By contrast, consideration of the over‐all balance of protein metabolism in these cells indicaes that the average concentration of metabolically turning‐over proteins is somewhat higher in senescent than in young fibroblasts.
Age‐related and carcinogen‐induced alterations of the extracellular growth factor requirements for cell proliferation in vitro
Authors: Swierenga S. H. H. , Whitfield J. F. , Boynton A. L.
Published: February 2005

Cells from thigh muscles of fetal rats proliferate readily in vitro in medium containing homologous adult rat “plasma.” As the donor animals mature, these cells become unable to make DNA and proliferate in “plasma” medium, but retain an ability to proliferate in medium containinig fetal bovine serum (FBS). This age‐related loss of the ability to proliferate in “plasma”‐medium is due to an increasing need by the cells for an exogenous prereplicative promoter which is found in FBS and a crude preparation of bovine luteinizing hormone. Adult cells (and possibly fetal cells) also require a “cycle‐completion” factor which is found in FBS and adult rat “plasma.” The requirements for such external proliferative promoters is completely eliminated by neoplastic transformation in vivo, and neoplastic adult cells isolated from a nickel sulfide‐induced mixed rhabdomyo‐fibro‐sarcoma can make DNA and proliferate in vitro in the complete absence of exogenous growth factors.
Assay of ribonucleotide reduction in nucleotide‐permeable hamster cells
Authors: Lewis William H. , Kuzik Brian A. , Wright Jim A.
Published: February 2005

Ribonucleotide reduction was measured in Chinese hamster ovary cells made permeable to nucleotides by treatment with the detergent Tween‐80. When compared to the respective ribonucleotide reductase activity in partially purified cell extracts, CDP and GDP reductase activities in permeabilized cells responded in a similar fashion to dithiothreitol, pH, MgCl2, FeCl3, substrate concentration and the presence of positive or negative allosteric effectors. At low protein concentrations both CDP and GDP reduction with whole cells increased linearly with cell number and was greater than the activity in corresponding cell extracts.

Permeabilized cells were used to measure the level of CDP and GDP reductase in a hamster cell line resistant to the cytotoxic effects of hydroxyurea. The hydroxyurea‐resistant cell line contained four to ten times more CDP and GDP reductase activity compared to parental or revertant cell lines.

The permeabilized cell assay was also used to measure CDP and GDP reductase activities in Chinese hamster ovary cells synchronized by isoleucine starvation. CDP reductase activity was low in G1 arrested cells but increased 10‐fold by 16 hours after the readdition of isoleucine to the growth medium. GDP reductase, which is present at much higher levels, is similarly induced after isoleucine addition, but only by 2‐fold. The maximum activity of both CDP and GDP reductase occurred from 14 to 16 hours after isoleucine addition, which corresponded to the period of maximum DNA synthesis.
Phenylalanine hydroxylase in melanoma cells
Authors: Breakefield Xandra O. , Castiglione Carmela M. , Halaban Ruth , Pawelek John , Shiman Ross
Published: February 2005

A pigmented subclone of Cloudman S91 melanoma cells, PS1‐wild type, can grow in medium lacking tyrosine. This ability is conferred by phenylalanine hydroxylase activity, and not by tryptophan hydroxylase, tyrosine hydroxylase or tyrosinase activities, although the latter activity is also present in these cells. Conversion of phenylalanine to tyrosine was measured in living cells by chromatographic identification of the metabolites of [14C]phenylalanine and in cell extracts using a sensitive assay for phenylalanine hydroxylase. Phenylalanine hydroxylase activity in melanoma cell extracts was identified by its inhibition with p‐chlorophenylalanine and not with 6‐fluorotryptophan, 3‐iodotyrosine, phenylthiourea, tyrosine or tryptophan; and by adsorption with antiserum prepared against purified rat liver phenylalanine hydroxylase, and migration of immunoprecipitable activity with authentic phenylalanine hydroxylase subunits in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.
Adaptation to cycloheximide of macromolecular synthesis in Tetrahymena
Authors: Wang Ting‐Chung , Hooper Alan B.
Published: February 2005

Cycloheximide (CHI) at 10 ng/ml partially inhibited protein synthesis in exponential cultures of Tetrahymena Sp. At 20 ng/ml or greater, inhibition was complete. When protein synthesis was inhibited to any extent, cell division ceased immediately. In all instances where measured, synthesis of RNA and DNA also ceased. After a period of delay, cellular functions reinitiated in the order: (i) protein synthesis, (ii) DNA synthesis and, (iii) RNA synthesis and cell division. The delay in cell division was divided into three phases of: I, zero; II, low; and, III, fully recovered rates of exponential protein synthesis. The length of the three phases increased with increasing concentration of CHI

Prior growth of cells for one generation in the presence of 7.5 ng/ml CHI (facilitation) eliminated phase I and slightly decreased phases II and III following subsequent challenge with an inhibitory concentration of CHI. Facilitation for six generations further decreased phases II and III. Protein synthesis and cell division were not inhibited during facilitation

In the culture, succinate dehydrogenase activity did not increase during the delay but increased normally at the onset of division. In contrast, NADPH‐cytochrome c reductase activity continued to increase for an hour after inhibition of protein synthesis, was constant for a period and did not increase again until an hour after reinitiatoin of cell division and RNA synthesis

Inhibition of division of all cells was immediate and reinitiation of synthesis and cell division was non‐synchronous.
Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts
Authors: Ronald Zielke H. , Ozand Pinar T. , Tyson Tildon J. , Sevdalian David A. , Cornblath Marvin
Published: February 2005

Human diploid fibroblasts utilize both glucose and glutamine as energy sources. The utilization of glutamine by fibroblasts is regulated by glucose, and vice versa. This conclusion is supported by the following observations: (1) essentially identical growth rates were observed in Eagle's minimum essential medium (MEM)3 in which the glucose concentration was either 5.5 mM or was maintained between 25 and 40 μM, (2) the total glutamine utilization by fibroblasts increased at least 30% in medium with 25 μM to 70 μM glucose compared to medium with 5.5 mM glucose, while the rate of glutamine‐1 or 5‐14C oxidation to CO2 increased 5‐fold as the glucose concentration was decreased to zero, (3) 2 mM glutamine inhibited glucose‐6‐14C oxidation by 88% and stimulated glucose‐1‐14C by 77% in log phase cells and (4) glutamine oxidation in normal medium contributed approximately 30% of the energy requirement of human diploid fibroblasts.
Role of fatty acids in growth‐promoting effect of serum albumin on hamster cells in vitro
Authors: Nilausen Karin
Published: February 2005

Dialyzed serum albumin had considerable growth‐promoting effect on cultivated hamster cells. This effect was virtually lost on removal of the fatty acids, and it was completely restored by recombination of the fatty acid‐free albumin with the isolated and purified fatty acids.

The role of albumin itself appeared to be largely that of a carrier of fatty acids, protecting the cells against toxic effects of fatty acids in free solution. This conclusion was based on two observations: Fatty acids in the absence of albumin were growth‐inhibitory except in extremely dilute solutions, and betalactoglobulin, a protein possessing, like albumin, the ability to bind and release fatty acids, could replace albumin in the presence of fatty acids with similar growth‐promoting effect.

Examination of individual molecular types of fatty acids showed that all unsaturated acids tested were growth‐promoting, whereas the saturated acids were growth‐inhibiting, with the exception of stearic acid in low concentrations.

Although the possibility of a mitotic triggering effect was not excluded, the fatty acids presumably stimulated growth by providing substrate for cellular metabolism, since there was a direct relationship between the degree of growth stimulation and the duration of exposure of cells to the fatty acids.