bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of glutathione in the inhibition of sea urchin egg mitosis by phenyl glyoxal
Authors: Amy Christopher M. , Rebhun Lionel I.
Published: February 2005

Cell division in fertilized sea urchin eggs was reversibly inhibited when the ketoaldehyde phenyl glyoxal (PG) at a concentration of 0.1 mM was added to eggs for ten minutes prior to the formation of the mitotic spindle. We investigated whether inhibition of mitosis was due to PG binding to the cell surface (as previously suggested by Stein and Berestecky, '74) or to some intracellular effect. When 14C‐PG was added to eggs, label was readily taken up into the egg cytoplasm; very little label was associated with the egg surface. In the cytoplasm PG combined with equimolar amounts of reduced glutathione (GSH), decreasing the levels of cellular GSH to less than 15% of normal and accounting for at least 50% of the PG taken up by eggs. The concentrations of oxidized and protein‐bound glutathione were unaffected by PG treatment. We showed that glyoxalase enzymes were present in sea urchin eggs and were capable of metabolizing the PG‐GSH complex, thereby restoring GSH to normal levels after PG was removed from the sea water. Though some other effect of PG cannot be ruled out, the major fate of PG in eggs was to combine with GSH, and the transient decrease in GSH which resulted could lead to inhibition of mitosis. While other reports (Nath and Rebhun, '76; Oliver et al., '76) have shown that reagents which oxidize GSH disrupt microtubule‐related events, our results showed that such inhibition could be caused by decreased GSH levels alone.
Effect of serum on prostaglandin production during the co‐culture of human thyroid cells and peripheral blood mononuclear cells
Authors: Herman E. A. , Yamamoto M. , Rapoport B.
Published: February 2005

Prostaglandin generation by human peripheral blood mononuclear cells is enhanced during co‐culture with human thyroid cells. The objective of the present study was to determine the influence of various sera on this process.

Human thyroid adenoma cell monolayers were cultured with normal human peripheral blood mononuclear cells for three days in the presence of a variety of sera, or serum fractions. Prostaglandin E (PGE) in the medium was measured by bioassay or by radioimmunoassay. Significantly more PGE was generated in cultures containing fetal calf serum than in those containing human serum. This difference was not abolished by dialysis of the human serum. When the 50% (NH4) 2SO4 precipitate of the serum was used, PGE generation was similar to that in fetal calf serum, indicating the presence of an inhibitory factor in human serum. The degree of this inhibitory activity was similar in autologous and heterologous human serum, as well as in normal subjects and patients with Graves' disease. Gel filtration and ion‐exchange chomatography of human serum showed the inhibitor to co‐migrate with albumin. Evidence presented suggests that the inhibitor is not albumin itself but is, instead, a factor tightly bound to albumin. Inhibitory activity was also found in rabbit, goat, rat and cow serum.

Prostaglandins are potent modulators of immune‐cell function. These data indicate that this process may be modulated by a factor in mammalian serum. The relative absence of this factor in fetal serum may have important implications in regard to the profound changes which occur in the immune system after birth.
Studies on the mechanism of Ca2+ stimulation of plasminogen activator synthesis/release by swiss 3T3 cells
Authors: Chou Iih‐Nan (George) , Cox Robert , Black Paul H.
Published: February 2005

Stimulation of postconfluent Swiss 3T3 cells in serum‐free medium with 4.3 mM Ca2+ results in marked increases in both released and cell‐associated plasminogen activator (PA). Increased release of PA commenced approximately 10 to 12 hours post‐stimulation and continued to increase steadily until 48 hours at which time the stimulated cells (4.3 mM Ca2+) released approximately 14 times more PA than control cells (1.8 mM Ca2+). Sr2+, like Ca2+, also stimulates PA synthesis/release either in the presence or in the absence of 1.8 mM Ca2+ whereas an excess of Mg2+ inhibits Ca2+ stimulation. Supranormal [Pi] in the medium stimulates PA synthesis/release in the presence of 1.8 mM Ca2+. Further, optimal stimulation by 4.3 mM Ca2+ requires a normal level of Pi (1.0 mM). Elevation of medium [Ca2+] or [Pi] results in an enhanced uptake of Ca2+. The facts that cycloheximide treatment completely abolishes the Ca2+ stimulatory effect and that an increase in cell associated PA precedes release indicate that PA release is coupled to synthesis of new PA. Ca2+ stimulation of PA synthesis/release also requires continuous energy production and RNA as well as protein synthesis. A hypothesis is proposed to explain the relationship between stimulation of PA production and its enhanced release from cells stimulated by elevated [Ca2+] or [Pi] in the media. The possibility that PA release may be an example of the phenomenon of membrane shedding as opposed to secretion is discussed.
Effects of low electrolyte media on salt loss and hemolysis of mammalian red blood cells
Authors: Zeidler R. B. , Kim H. D.
Published: February 2005

Cation loss and hemolysis of various mammalian red cells suspended in isotonic non‐electrolyte media were investigated. Sucrose buffered with 10 mM Tris‐Hepes, pH 7.4 was used as the non‐permeable non‐electrolyte. Mammals from which the red cells were derived include the human, guinea pig, rat, rabbit, newborn calf, newborn piglet and pig, all of which contain K as the predominant cation species (HK type) and the dog, cat, sheep and cow, all of which possess Na as the predominant cation species (LK type). Of HK cells, a rapid efflux of K takes place from humans, rats and guinea pigs. Of LK type cells, the dog and cat exhibit an augmented membrane permeability to Na. The governing factors which influence cation permeability are the change in pH, temperature, and ionic strength. In response to increase in pH, the red cells of humans, dogs and cats become more permeable to cations, whereas the red cells of rat and rabbit are unaffected. In response to increase in temperature, HK type cells exhibit augmented K efflux, while the Na loss from the dog and cat cells manifest a well‐defined maximum at near 37°C. In all cases, a small substitution of sucrose by an equal number of osmoles of salts results in a dramatic decrease in cation loss. By contrast, the red cells of the rabbit, newborn calf, adult cow, newborn piglet, adult pig and sheep display no discernible increase in ion‐permeability under the conditions alluded to above. In some species including the newborn calf, dog and cat, an extensive hemolysis occurs usually within an hour in isotonic buffered sucrose solution. The osmolarity of sucrose solution affects these cells differently in that as the osmolarity increases from 200‐500 mM, hemolytic rates of the calf and dog reach a saturation near 300 mM sucrose, whereas the hemolytic rate of the cat decreases progressively. Common features pertaining to this hemolysis are (1) the intracellular alkalinization process; and (2) the diminution of the cell volume which take place prior to and onset of hemolysis. SITS, a potent anion transport inhibitor, completely protects the cells from hemolysis by inhibiting chloride flux and the concomitant rise in intracellular pH.
Somatic cell hybrids between totipotent mouse teratocarcinoma and rat hepatoma cells
Authors: Litwack Gerald , Croce Carlo M.
Published: February 2005

We have produced somatic cell hybrids between totipotent mouse teratocarcinoma cells and rat hepatoma cells. These hybrids were tested for the expression of liver specific functions expressed in the hepatoma cell parent and for their ability to differentiate when injected into nude mice. The results of this study indicate that hybrid cell clones do not resemble either of the parental cells, since they do not produce albumin and tyrosine aminotransferase that are expressed in the rat hepatoma parent, and are incapable of forming either teratocarcinoma or hepatomas when injected in experimental animals.
Potassium fluxes and ouabain binding in growing, density‐inhibited and rous sarcoma virus‐transformed chicken embryo cells
Authors: Johnson Marcia A. , Weber Michael J.
Published: February 2005

Potassium fluxes, ouabain binding, and Na+ and K+ intracellular concentrations were determined for cultures of growing normal, density‐inhibited and Rous sarcoma virus‐transformed chicken embryo fibroblasts. No significant differences in K+ influx or ouabain binding were detected between growing normal cells and Rous sarcoma virus‐transformed cells; however, ouabain binding and ouabain‐sensitive K+ influx were 1.5‐ to 1.8‐fold lower in density‐inhibited cells. Thus, potassium influx in this system can be classified as a growth‐related, but not transformation‐specific change. As determined by both flame photometry and radioisotopic (42K) equilibration, growing normal and density‐inhibited cells had similar potassium contents, whereas transformed cells exhibited 1.4‐fold higher potassium levels. Sodium ion levels, as measured by flame photometry, were also 2‐ to 4.5‐fold higher in transformed than normal or density‐inhibited cells. Complementary studies of potassium efflux showed a 1.3‐ to 1.5‐fold higher rate (based on the percentage of pool exiting the cell) in growing normal versus density‐inhibited or transformed fibroblasts. Because of the larger potassium pool in transformed cells, efflux based on absolute number of potassium ions is similar in normal and transformed chicken embryo fibroblasts.
Studies on the penetration of mammalian cells by deoxyribonucleoside‐5′‐phosphates
Authors: Waqar M. Anwar , Taber Robert L. , Huberman Joel A.
Published: February 2005

We have tested the ability of [5′‐32P]‐deoxyribonucleoside monophosphates (dNMPs) to penetrate living mouse fibroblast L cells and human HeLa cells. Under the conditions of our experiments, small numbers of apparently intact dNMP molecules appeared to penetrate into the interior of L cells and be incorporated into DNA. This incorporation was not due to mycoplasma contamination nor to extracellular hydrolysis of the dNMPs followed by resynthesis inside the cell. Under these same conditions, penetration of HeLa cells by intact dNMPs did not occur to a significant extent. However, HeLa cells were capable of hydrolyzing extracellular dNMPs to Pi and deoxyribonucleosides at a much faster rate than L cells.

These experiments provide a starting point for attempts to specifically label the DNA in intact, living eukaryotic cells with [32P]‐dNMPs.
Reversible inactivation of autosomal alleles in chinese hamster cells
Authors: Bradley W. E. C.
Published: February 2005

Evidence is presented which suggests that a CHO‐derived thymidine kinase (Tk) heterozygote, ts201, can reversibly inactivate the wild type Tk allele, and that this event may result in simultaneous inactivation of a linked allele, galactokinase (Glk). The clone ts201 was isolated as a revertant of a stable Tk− 5‐bromodeoxyuridine (BrdU)‐resistant mutant of CHO. The reacquired Tk activity differed from that of the wild type with respect to Km and heat resistance, supporting the contention that ts201 was genetically heterozygous (Tk+/−). At frequencies varying from 0.02–0.5, segregants of ts201 could be isolated by cloning in BrdU at 39°. These derivatives were phenotypically Tk− (by enzymology and by autoradiography of 3H‐dT labeled cells), but after removal of BrdU, reacquired the Tk+ phenotype at frequencies varying randomly from clone to clone. From mutagenized populations of Ts201 two variants were isolated, 71t and 72c, by selection at 39° in 2‐deoxygalactose (2‐dgal). Resistance to 2‐dgal has been correlated with a mutation in the gene for Glk, which is syntenic with that for Tk and evidence is presented suggesting that 71t and 72c are Glk+/−. Cloning efficiencies in doubly selective media and autoradiographic data showed that at 39° a coordination existed between the levels of Tk and Glk, which in 71t was inverse, and in 72c, direct. These data led to the hypothesis that pairs of linked alleles can be inactivated or re‐expressed simultaneously. The inactivating effect was temperature‐sensitive, since at 33°, the cloning efficiency in BrdU and in 2‐dgal was very low.
DNA synthesis: Evaluation of a hydrodynamic method for measuring the rate of replication fork movement
Authors: Richter Andrea , Hand Roger
Published: February 2005

When replicating DNA is labeled sequentially with radioactive and density tracers and analyzed by equilibrium centrifugation, the fraction banding at heavier than normal density is inversely proportional to the rate of replication fork movement if there is a sharp transition from one tracer to the other on the newly synthesized chains (Painter and Schaefer, '69). Primate CV‐1 DNA labeled for 5 to 30 minutes with 3H‐dThd and then for three hours with BrdUrd in the presence of FdUrd bands in a bimodal distribution in alkaline CsCl, rather than in a continuous distribution with a skew toward heavier density seen when FdUrd is omitted and centrifugation is in neutral CsCl. The heavy density peak represents interspersion of both tracers in the DNA and is caused by slow transition from dThd to BrdUrd incorporation when the tracers are switched in the labeling medium. This may result from preferential uptake and incorporation of dThd over BrdUrd. Because of the interspersion, calculation of the rate of replication fork movement is inaccurate. Reversal of the labeling sequence with administration of the long density pulse before the radioactive pulse reduces the problem of interspersion. Using this sequence of labeling, estimates of the rate of fork movement of 0.36–0.38 μm/min are obtained when the 3H pulse time is long enough to allow accurate measurement of the fraction of heavy DNA. Analysis by fiber autoradiography yields a rate of 0.56 μm/min in the same cell line. If appropriate precautions are taken to minimize mixing of the two tracers in the precursor pool and to ensure that the fraction of heavy DNA is measured accurately, the hydrodynamic technique provides an objective method of measuring rate of fork movement that gives values only slightly lower than those obtained by autoradiography.
Phenotypic characterization of ouabain‐resistant Aedes albopictus cells
Authors: Mento Steven J. , Malinoski Frank , Stollar Victor
Published: February 2005

The phenotype of a ouabain‐resistant Aedes albopictus cell line has been partially characterized. Treatment of ouabain‐sensitive cells with 0.005–1.0 mM ouabain resulted in an 80% reduction in the uptake of 86rubidium (86Rb+), an ion with an affinity for the K+ pump binding site; ouabain‐resistant cells showed only a 40% reduction with 1.0 mM ouabain. When ouabain‐sensitive cells were incubated in the presence of ouabain (0.1 mM) for one and one‐half to three hours, the molar ratio of intracellular Na+/K+ rose from 0.2 to 4.2. In ouabain‐resistant cells, a similar treatment had very little effect. Based on [3H] ouabain‐binding studies, ouabain‐resistant cells were estimated to have 60% fewer binding sites per cell than ouabain‐sensitive cells. The spontaneous mutation rate from ouabain sensitivity to ouabain resistance was calculated to be 1–6 X 10−8 mutations/cell/generation, a value similar to that reported for mammalian cells at the analogous locus.
Serum of lipopolysacharide‐treated mice contains two types of colony‐stimulating factor, separable by affinity chromatography
Authors: Staber F. G. , Burgess A. W.
Published: February 2005

Serum from mice traated with bacterial lipopolysaccharide (LPS) was fractionated by Con A‐Sepharose affinity chromatography, and assayed in vitro for colony‐stimulating factor (CSF) using mouse bone marrow cells. The CSF failing to bind to concanavalin A‐Sepharose (pool A) had similar biological properties to the unfractionated serum, i.e., it stimulated the formation of about equal numbers of granulocytic, mixed granulocyte‐macrophage and macrophage colonies. The fraction eluted from the Con A‐Sepharose column with α‐methyl‐D‐glucopyranoside (pool B) had a steeper dose‐response curve than either the unfractionated serum or the pool A CSF and most of the colonies were composed of macrophages. A mixture of the pool A and pool B CSFs stimulated colonies in a similar way as unfractionated serum and pool A. The apparent molecular weights of the two types of CSF were determined by two different gel‐filtration procedures. Sephacryl S‐200 gel‐filtration suggested an apparent molecular weight of 85,000 for pool A CSF and 180,000 for pool B CSF. Gel‐filtration on Sepharose CL‐6B in the presence of guanidine hydrochloride (6M) yielded an apparent molecular weight of approximately 23,000 for pool A CSF and 33,000 for pool B CSF. The colony‐forming cells (CFC) responding to pool B CSF were found to have a relatively high sedimentation velocity (peak sedimentation velocity 5.6–6.2 mm/hr) compared to the CFC responding to mouse‐lung conditioned medium (MLCM) whose peak sedimentation velocity was between 4.0–4.5 mm/hour. The CFC responding to pool A CSF had an intermediate sedimentation velocity (peak 4.6–5.2 mm/hour). A time‐course analysis of the morphology of clones or colonies in cultures stimulated with either MLCM or pool B CSF showed that the proporation of different colony types depends significantly on the incubation period and suggested that pool B CSF induced an early commitment of CFC towards macrophage differentiation.
Transcriptional activity of nuclei from WI‐38 cells at various passages
Authors: Nose Kiyoshi , Okamoto Hiroshi
Published: February 2005

Transcriptional activity of nuclei from human diploid fibroblast (WI‐38) cells at different passage levels was studied with endogenous RNA polymerase. The rate and extent of the RNA synthesis decreased when the nuclei were were prepared from senescent cells. The decreased activity of RNA synthesis in senescent cell nuclei was demonstrated from both the transcriptional assay and the estimation of the processing activity, i.e., polyadenylation of the transcripts.
Effect of phagocytosis on guinea pig granulocyte membrane markers
Authors: Smolen James E. , Karnovsky Manfred L.
Published: February 2005

The internalization of membrane markers during phagocytosis was followed in guinea pig granulocytes as a function of the extent of particle ingestion. The plasmalemma was carefully labeled with diazotized 35S‐sulfanilate, or by treatment with periodate and sodium 3H‐borohydride. These treatments provided general membrane markers. More specific markers used were 5′‐nucleotidase, neuraminidase‐releasable membrane sialate, and concanavalin A binding sites. In all cases except the last, internalization of membrane was directly determined on isolated phagosomes; disappearance of binding sites from the cell surface was followed in the last instance. Both phagosomal levels and disappearance from the surface were measured in the case of 5′‐nucleotidase, permitting balance studies. Phagocytosis was determined as uptake of paraffin emulsions labeled with Oil‐Red 0. “Marker/particle” ratios were determined as the percent external marker internalized per mg paraffin ingested.

The “marker/particle” ratios for cells with chemically labeled membranes were considered to reflect random internalization of membrane entities. Colchicine had no effect on those ratios. Internalization of 5′‐nucleotidase and neuraminidase‐releasable sialate gave “marker/particle” ratios similar to those of the random markers, and these increased in the presence of colchicine. Concanavalin A binding sites did not appear to be removed from the cell surface in the absence of colchicine, but disappearance was observed in its presence. Control experiments indicated that these changes due to colchicine must be very cautiously interpreted. Our results differ from those obtained by others, and the reasons due to species, cell type, experimental design, etc. are discussed. Maximal particle uptake was computed to require internalization of one‐fifth to one‐third of the total external membrane of the cells—based on the assumption of random internalization of markers.
Glucose and amino acid metabolism in neonatal rat skeletal muscle in tissue culture
Authors: Pardridge William M. , Duducgian‐Vartavarian Luiza , Casanello‐Ertl Delia , Jones Michael R. , Kopple Joel D.
Published: February 2005

The characteristics of glucose and amino acid metabolism over a 98‐hour incubation period were studied in a primary culture of neonatal rat skeletal muscle cells. The cells formed large myotubes in culture, were spontaneously highly contractile, and had cell phosphocreatine levels exceeding ATP concentrations. Medium glucose fell from 7.2±0.2 to 1.5±0.1 mM between 0 and 98 hours; intracellular glucose was readily detectable, indicating glycolysis was limited by phosphorylation, not glucose transport. Alanine levels in the medium increased from 0.06±0.01 to 0.82±0.04 mM between 0 and 48 hours and decreased to 0.72±0.04 mM by 98 hours. The period of net alanine production correlated with the rise in the cell mass action ratio of the alanine aminotransferase reaction. Cell aspartate, glutamate, and calculated oxalacetate levels were inversely related to the cell NADH/NAD+ ratio, as represented by the intracellular lactate/pyruvate ratio (r=0.78–0.88). The branched chain amino acids (leucine, isoleucine, valine) were actively utilized, e.g., medium leucine fell from 0.70±0.01 to 0.30±0.06 mM between 0 and 98 hours. In addition, arginine and serine consumption was observed in conjunction with ornithine, proline, and glycine production. Conclusions: (1) A major driving force for the high rates of alanine production by skeletal muscle cells in tissue culture is the active utilization of branched chain amino acids. (2) Intracellular aspartate and glutamate pools are linked, probably via the malate‐aspartate shuttle, to the cell NADH/NAD+ redox state. (3) Muscle cells in tissue culture metabolize significant amounts of arginine and serine in association with the production of ornithine and proline, and these pathways may possibly be related to creatine production.
Density‐dependent and adaptive regulation of glucose transport in primary cell cultures of the R3230AC rat mammary adenocarcinoma
Authors: Gay Roger J. , Hilf Russell
Published: February 2005

Transport of 3‐O‐methyl‐(1‐3H)‐D‐glucose (3‐OMG) was studied in primary cell cultures of the R3230AC rat mammary adenocarcinoma. Fastest rates of carrier‐mediated 3‐OMG transport (vc) were temporally associated with fastest cell growth, as were the rates of 3H‐labeled thymidine, uridine, and leucine incorporation into macromolecules. The decrease in vc for 3‐OMG observed as cultured cells approached quiescence was due to a 4‐fold decrease in Vmax with the Km remaining relatively constant (4–9mM). Provided adequate time was allowed for cells to adapt, (6–12hr), the vc for 3‐OMG transport was found to be inversely related to the concentration of glucose in the medium. Within 6 hours after switching cells from standard growth medium (5mM glucose) to medium containing no glucose (5 mM fructose), a 2‐fold increase in vc for 3‐OMG transport was observed in both fast and slow growing cells. The glucose‐starvation induced increase in 3‐OMG transport was due to an increase in Vmax; the Km remained constant, and was not significantly influenced by the presence of serum (10%) or insulin (5 m̈g/ml) or by [5 mM] galactose, mannose, fructose, 2‐deoxy‐D‐glucose, 3‐OMG, or mannitol. Readdition of glucose (5 mM) to transport‐activated cells (deprived of glucose for 9–11 hr) resulted in a rapid return of 3‐OMG transport to basal levels. In the presence of cycloheximide (10 m̈g/ml) or actinomycin D (10 m̈g/ml), this glucose‐induced decline in carrier function was largely blocked. In fast‐growing cells, the addition of either of these inhibitors, in the presence or absence of glucose, resulted in an initial rise in the rates of 3‐OMG transport, followed by a linear decrease. Compared to fast‐growing cells, the cycloheximide‐induced increase in 3‐OMG transport was greater and longer sustained in slow‐growing cells. Regardless of their growth rate, cell cultures preincubated in medium containing glucose and cycloheximide exhibited decreases in 3‐OMG transport when transferred to medium containing fructose, with or without actinomycin D. Slow‐growing cells preincubated in medium containing fructose and cycloheximide exhibited an increase in 3‐OMG transport when switched to medium containing fructose, whereas similarly treated fast growing cell cultures displayed a slight decrease. These experiments suggest that the adaptive regulation of glucose transport in these mammary tumor cell cultures occurred at the transcriptional level and was influenced by the rates of cellular growth. A model in which a metabolite of glucose acts to enhance the synthesis or stabilization of a mRNA species specific for a putative carrier inactivator protein is proposed.
The effect of mouse lung granulocyte‐macrophage colony‐stimulating factor and other colony‐stimulating activities on the proliferation and differentiation of murine bone marrow cells in long‐term cultures
Authors: Williams Neil , Burgess Antony W.
Published: February 2005

The roles of colony‐stimulating factors in long‐term bone marrow cultures were studied and compared. After single additions of high concentrations of unpurified colony‐stimulating activities to the cultures, rapid deterioration of the cultures was observed. This appears to result from contaminants in the stimulatory preparations. Cultures to which one purified colony‐stimulating factor (CSF) from endotoxin mouse lung‐conditioned medium was added did not run down ten weeks after addition and were found to be the same as the controls. The deterioration of the cultures to which unpurified stimulators were added could not be accounted for by accelerated granulopoiesis leading to subsequent exhaustion of the cultures. The inability of purified CSF to affect the cellularity of the suspension cells did not result from instability or masking of the activity in the cultures, nor did CSF preferentially stimulate the cells within the adherent layer. The suspension cells responded to purified CSF after separation from the adherent cells. The data suggest that if CSFs are marrow stimulators, their effects in turn may be stringently regulated within the marrow.
Inhibitors of RNA synthesis and passage of chick embryo fibroblasts through the G1 period
Authors: Chadwick David E. , Ignotz George G. , Ignotz Ronald A. , Lieberman Irving
Published: February 2005

Events that are essential for progression through the G1 period begin immediately or shortly after resting chick embryo cells are given fresh medium with serum. The following observations support the contention that the critical events include the production of non‐ribosomal RNAs: (1) Addition to the “shift‐up” medium of either of two inhibitors of RNA formation, comptothecin or 5, 6‐dichloro−1‐β‐D‐ribofuranosylbenzimidazole, delays the onset of DNA replication by about the length of time the cells are exposed to the drugs. (2) Although entry into the S phase is delayed by the inhibitors, the slopes of the DNA response curves are identical to that of control cultures. (3) Neither drug reduces significantly the rate of overall protein synthesis. Observations (2) and (3) are taken to mean that expansion of the G1 period is not due to cell damage. (4) A third inhibitor of RNA synthesis, cordycepin, also delays passage of stimulated cells throgh the G1 phase, but, in this case, the length of the delay period is greater than that of the exposure period. (5) A low dose of actinomycin D does not impede movement towards the S phase, even though the synthesis of preribosomal RNA is considerably reduced.

The possibility is considered that the essential G1 molecules are mRNAs.
The permeability of the human red cell to deuterium oxide (heavy water)
Authors: Karan Daniel M. , Macey Robert I.
Published: February 2005

Using a stopped‐flow device, the osmotic water permeability of human red cells to D2O and H2O was studied as a function of temperature and under the influence of the sulfhydryl reagent paracholoromercuribenzene sulfonic acid (PCMBS), an inhibitor of water transport. The ratio, permeability (D2O)/permeability (H2O) at each temperature can be predicted simply by assuming that permeability varies inversely with macroscopic viscosity. When water permeability is inhibited with PCMBS, this dependency on viscosity vanishes; the inhibited permeabilities in D2O and H2O are indistinguishable.
Patterns of chromosome segregation in chinese hamster × mouse cell hybrids between permanent cell lines and thymus cells
Authors: Lasserre Chantal , Jami Jacques , Aviles Dominique
Published: February 2005

Hybrids between a tumorigenic Chinese hamster cell line (DC3F‐aza) and normal mouse thymus cells very rapidly lost most of their mouse chromosomes, whereas hybrids between tumorigenic mouse cell lines (either Cl.1D of L cell line origin, or PCC4‐aza1 teratocarcinoma cells) and normal Chinese hamster thymus cells lost most of their hamster chromosomes. From three such fusion experiments, 20 cell lines were developed which all followed the same evolution, namely, the elimination of the majority of the chromosomes contributed by the normal thymus cell. In some hybrids, the elimination process resulted in the total absence of intact chromosomes contributed by the thymus cell parent. Such hybrids were distinguished from revertant parental cells growing in the selective hybrids were distinguished from revertant parental cells growing in the selective medium by the presence of at least one enzyme in their cell extracts which displayed the electrophoretic mobility of the enzyme of the thymus cell parent. These observations, together with data from other reports, suggest that, as a rule, interspecific cell hybrids which develop upon fusion between normal diploid cells and tumorigenic cell lines maintain the chromosomes of the latter and eliminate preferentially many or most of the chromosomes contributed by the normal cell parents, independent of the respective species of the parental cells.
The responses of hemopoietic precursor cells in mice to bacterial cell‐wall components
Authors: Staber Fritz G. , Johnson Gregory R.
Published: February 2005

The influence upon differnt cellular and humoral parameters of hemopoiesis of three structurally unrelated, highly purified bacterial cell‐wall components (BCWC) was investigated. The spleens of C57BL/6 mice assayed 6 days after the injection of either lipid A or outer‐membrane lipoportein, but not murein, showed a marked increase in granulocyte‐macrophage, eosinophil, and megakaryocyte progenitor cell levels. The number of pluripotent hemopoietic stem cells (CFU‐S) also increased in the spleens of mice treated with either lipid A or lipoprotein. Similar results were obtained following the injection of lipoprotein or lipid A into CBA or C57BL/6.nu mice. Genetically anemic Wf/Wf mice were found to have spontaneously elevated numbers of splenic progenitor cells, which increased further after the injection of lipid A. The proportions of the different splenic progenitor cell types were similar in both untreated and lipid A treated Wf/Wf mice, and in normal littermate controls.

When tested in vitro, unfractionated or partially purified post‐lipid A serum was found to stimulate the growth of granulocyte‐macrophage progenitor cells (GM‐CFC), but no detectable stimulation of eosinohphil, megakryocyte, or erythroid progenitor cells was observed. The data suggest that the rise in splenic levels of the different progenitor cells is not mediated by the corresponding types of CSF, but more likely by proliferation and differentiation of CFU‐S.
Tumor promoter modulation of epidermal growth factor‐ and nerve growth factor‐induced adhesion and growth factor binding of PC‐12 pheochromocytoma cells
Authors: Chandler Charles E. , Herschman Harvey R.
Published: February 2005

Nerve growth factor (NGF) has previously been shown to increase the rate of adhesion of PC‐12 pheochromocytoma cells to cell culture dishes. This increase in the rate of adhesion was postulated to be important in NGF‐mediated neurite outgrowth. We now report that epidermal growth factor (EGF) is also able to increase the rate of adhesion of PC‐12 cells to cell culture dishes, but does not elicit neurite outgrowth. The dose‐response curve for EGF is bell‐shaped, in contrast to the more classically shaped dose‐response curve obtained with NGF.

Tetradecanoyl‐phorbol‐acetate (TPA), a potent tumor promoter, blocks the EGF‐induced increase in adhesion rate of PC‐12 cells, but does not alter the NGF‐induced increase in adhesion rate. TPA shifts the EGF binding curve to the right for PC‐12 cells, but does not alter maximal EGF binding at saturating concentrations of EGF. The binding of NGF to PC‐12 cells is not affected by TPA. NGF‐induced neurite formation by PC‐12 cells is unaffected by TPA, in contrast to the previously reported delay of neurite outgrowth of serum‐deprived neuroblastoma cells and NGF‐exposed chick embryonic ganglia cells.

NGF and EGF both cause a decrease in the number of short microvilli and an increase in the number of long microvilli on PC‐12 cells. TPA blocks the decrease in the number of short microvilli in EGF‐treated cells, but not in NGF‐treated cells. Long microvilli formation is blocked by TPA in both conditions, suggesting the latter are not involved in the increased adhesion rates.
Phorbol myristate acetate induced neutrophil autotoxicity
Authors: Tsan Min‐Fu
Published: February 2005

The viability of neutrophils in the condition under which they kill neoplastic cells was studied. In the presence of phorbol myristate acetate (PMA) the 51Cr‐release by human neutrophils was markedly stimulated. The PMA‐induced 51Cr‐release by neutrophils correlated well with the number of nonviable neutrophils as determined by the uptake of trypan blue. Phorbol myristate acetate had no effect on the 51Cr‐release by lymphocytes, LPC‐1 myeloma cells, ovarian ascites tumor cells, or neutrophils from a patient with chronic granulomatous disease. This suggests that the effect of PMA is not due to its nonspecific toxic effect; instead, it is dependent on the reactive oxygen species produced by the normal neutrophils. Catalase, cytochrome C, histidine, and methionine inhibited the PMA‐induced 51Cr‐release by human neutrophils, whereas superoxide dismutase, myeloperoxidase inhibitors, and some hydroxyl radical scavengers or singlet oxygen quenchers had no effect. The clumping of neutrophils induced by PMA was also important in the PMA‐induced 51Cr‐release by human neutrophils.
Effect of arachidonic acid and the chemotactic factor F‐Met‐Leu‐Phe on cation transport in rabbit neutrophils
Authors: Sha'Afi R. I. , Naccache P. H. , Alobaidi T. , Molski T. F. P. , Volpi M.
Published: February 2005

A detailed examination of the effects of exogenous arachidonate on cation metabolism in rabbit neutrophils was undertaken. Arachidonic acid stimulates the movement of 45Ca into and out of the neutrophils with a net result, in the presence of extracellular calcium, of increasing the steady‐state level of 45Ca. Arachidonate also increases the uptake of 22Na. These effects of arachidonate are specific to these cations, concentration‐dependent, and sensitive to lipoxygenase inhibitors. At the concentrations used in this study arachidonate does not influence the permeability of human erythrocytes to 45Ca. Furthermore, both arachidonic acid and F‐Met‐Leu‐Phe release calcium from a previously unexchangeable intracellular pool and the effect of the two stimuli are not additive. Arachidonic acid‐dependent, but not F‐Met‐Leu‐Phe‐dependent, calcium release is sensitive to lipoxygenase inhibitors. These two stimuli thus appear to release is sensitive to lipoxygenase inhibitors. These two stimuli thus appear to release calcium from the same pool(s) by separate mechanisms. The results summarized above are consistent with the hypothesis that one or more arachidonate metabolites are involved in the mechanism underlying the chemotactic factor induced permeability changes in rabbit neutrophils.
N‐Carbamoyloxyurea‐resistant Chinese hamster ovary cells with elevated levels of ribonucleotide reductase activity
Authors: Hards Robert G. , Wright Jim A.
Published: February 2005

We describe the isolation and characterization of a Chinese hamster ovary cell line selected for resistance to N‐carbamoyloxyurea. Using the mammalian cell permeabilization assay developed in our laboratory, a detailed analysis of the target enzyme, ribonucleotide reductase (EC 1.17.4.1), was carried out. Both drug‐resistant and parental wild‐type cells required the same optimum conditions for enzyme activity. The Ki values for N‐carbamoyloxyurea inhibition of CDP reduction were 2.0 mM for NCR‐30A cells and 2.3 mM for wild‐type cells, while the Ki value for ADP reduction was 2.3 mM for both cell lines. Although the Ki values remained essentially unchanged, the Vmax values for NCR‐30A cells were 1.01 nmoles dCDP formed/5 × 106 cells/hour and 1.83 nmoles dADP/5 × 106 cells/hour, while those for the wild‐type cells were 0.49 nmoles dCDP produced/5 × 106 cells/hour and 1.00 nmoles dADP/5 × 106 cells/hour. This approximate twofold increase in reductase activity at least partially accounts for a 2.6‐fold increase in D10 value for cellular resistance to N‐carbamoyloxyurea exhibited by NCR‐30A cells. The NCR‐30A cell line was also cross‐resistant to the antitumor agents, hydroxyurea and guanazole. No differences in Ki values for inhibition of CDP and ADP reduction by these two drugs were detected and cellular resistance could be entirely accounted for by the elevation in activity of the reductase in the NCR‐30A cell line. The properties of N‐carbamoyloxyurea‐resistance cells indicate they should be useful for further investigations into the regulation of mammalian enzyme activity.
Methionine utilization in long‐term human lymphoid cell lines
Authors: Guarini L. , Sturman J. A. , Gaull G. E. , Beratis N. G.
Published: February 2005

Long‐term lymphoid cell lines (LTL) cultured under normal conditions use methionine primarily for protein synthesis, although a significant proportion is converted to S‐adenosylmethionine (SAM) and used for synthesis of the polyamines, spermidine, and spermine. When LTL are cultured under conditions of high cell density, there is an initial phase of rapid protein synthesis and accumulation of SAM as found under normal culture conditions, but this soon ceases. Polyamine synthesis is small under these conditions, despite the presence of relatively large amounts of SAM.
The production and localization of laminin in cultured vascular and corneal endothelial cells
Authors: Gospodarowicz D. , Greenburg G. , Foidart J. M. , Savion N.
Published: February 2005

The production and localization of laminin, as a function of cell density (sparse versus confluent cultures) and growth stage (actively growing versus resting cultures), has been compared on the cell surfaces of cultured vascular and corneal endothelial cells. Comparison of the abilities of the two types of cells to secrete laminin and fibronectin into their incubation medium reveals that vascular endothelial cells can secrete 20‐fold as much laminin as can corneal endothelial cells. In contrast, both cell types produce comparable amounts of fibronectin. Furthermore, if one compares the secretion of laminin and fibronectin as a function of cell growth, it appears that the laminin released into the medium by either vascular or corneal endothelial cells, is a function of cell density and cell growth, since this release is most pronounced when the cells are sparse and actively growing, and decreases by 10‐ and 30‐fold, respectively, when either vascular or corneal endothelial cell cultures become confluent. With regard to fibronectin secretion, no such variation can be seen with vascular endothelial cell cultures, regardless of whether they are sparse and actively growing or confluent and resting. Corneal endothelial cell cultures, demonstrated a twofold increase in fibronectin production when they were confluent and resting as compared to when they were sparse and actively growing. When the distribution of laminin versus fibronectin within the apical and basal cell surfaces of cultured corneal and vascular endothelial cells is compared, one can observe that unlike fibronectin, which in sparse and subconfluent cultures can be seen to be associated with both the apical cell surface. In confluent cultures, laminin can be found associated primarily with the extracellular matrix beneath the cell monolayer, where it codistributes with type IV collagen.
Effect of glucose on aspartate and glutamate synthesis by human diploid fibroblasts
Authors: Zielke H. Ronald , Sumbilla Carlota M. , Ozand Pinar T.
Published: February 2005

The quantity of aspartate, but not of glutamate, synthesized by human diploid fibroblasts (HDF) was inversely proportional to the glucose concentration. Forty hours after confluent cells were refed with media containing 1.5 mM or 55 μM glucose, the aspartate concentration was 8 μM in medium high in glucose and 28 μM in medium low in glucose; glutamate was 350 μM at both glucose levels. The label incorporation from [U‐14C] glutamine (0.8 C/ml) into aspartate after 40 hours in 1.5 mM glucose medium was less than 1000 DPM/ml and into glutamate 540,000 DPM/ml. The respective label incorporation into aspartate in 55 μM glucose medium was 118,000 DPM/ml and into glutamate 450,000 DPM/ml. In media with 1.5 mM glucose, label incorporation into both aspartate and glutamate was observed from [6‐14C] glucose, [3‐14C] pyruvate, and [1‐14C] pyruvate. Since [1‐14C] pyruvate labeled aspartate in 1.5 mM glucose medium, whereas [U‐14C] glutamine did not, the observations support a cystolic pathway of aspartate synthesis from glycolytic intermediates in the presence of glucose. However, when the glucose concentration is decreased, glutamine appears to be the primary carbon source for aspartate synthesis. Therefore, both the quantity and the pathway of aspartate synthesis in HDF are functions of the glucose concentration. It is postulated that increased accumulation of aspartate in the media of HDF at confluency may be explained by decreased availability of acetyl CoA or of NADH.
Gonadotropin stimulation of pregnenolone metabolism in Chinese Hamster Ovary cells in culture
Authors: Evain Daniele , Anderson Wayne B. , Saez Jose M.
Published: February 2005

To determine if Chinese Hamster Ovary (CHO) cells in culture are able to metabolize steroids, CHO cells were incubated in defined medium with [14C]pregnenolone. As shown, [14C]pregnenolone is metabolized to progesterone and other δ53β steroids; this steroidogenic response is appreciably enhanced upon exposure of the cells to 50 nM gonadotropins (human chorionic gonadotropin and follicle‐stimulating hormone). The primary metabolites that accumulate in the medium upon treatment with gonadotropins are 16α‐17β‐dihydroxydehydroepiandrosterone. Exposure of the CHO cells to gonadotropins induces significant increases in the activities of 16α‐hydroxylase, 17α‐hydroxylase, and 17–20 lyase. Similar results are obtained when the CHO cells are treated with 0.1 mM 8‐bromocyclic AMP, indicating that the gonadotropin enhancement of steroid metabolism is a cyclic AMP‐mediated process. CHO cells apparently lack the cholesterol desmolase complex since 14C‐cholesterol is not utilized by these cells to produce other steroid metabolites. These results indicate that CHO cells offer an in vitro system for the study of certain aspects of gonadotropin stimulation of steroidogenesis.
Cation fluxes and volume regulation by human lymphocytes
Authors: Bui A. H. , Wiley J. S.
Published: February 2005

The ionic basis of volume regulation by human peripheral blood lymphocytes in hypotonic Tyrode's medium has been studied. The intracellular water space of lymphocytes increased to a maximum after 1 min in 0.68 × isotonic Tyrode's but returned to the isotonic value by 20 min at 37°C. During this phase of volume regulation (1–20 min) both 42K+ efflux and 42K+ influx were stimulated severalfold, but the increase in 42K+ efflux exceeded the influx, resulting in a net loss of 20% of the lymphocyte K+. The increase in 42K+ efflux during the phase of cell shrinkage was unaffected by ouabain or by quinidine. Hypotonicity increased both the ouabain‐sensitive (active) and ouabain‐insensitive components of 42K+ influx by 76% and 123% respectively. Hypotonic shock stimulated 22Na+ influx by only 25%, but cell Na+ content was unchanged at 1 min and even decreased after 20 min. Thus active K+ influx and Na+ extrusion is increased by hypotonicity, but greater pumping cannot explain the net decrease in cell cations that leads to volume regulation. The 45Ca2+ uptake was not significantly changed by hypotonicity. Although volume regulation was abolished in a hypotonic high K medium, 42K+ efflux was still stimulated 2‐fold by the reduction in tonicity. These findings support the hypothesis that volume regulation in hypotonic media occurs largely by a passive loss of cell K+, which results from a selective increase in membrane permeability to this ion. The increase in K+ permeability in hypotonic media is observed even in the absence of volume regulation by the cell.
The hexose transport system in the human K‐562 chronic myelogenous leukemia‐derived cell
Authors: Dozier J. C. , Diedrich D. F. , Turco S. J.
Published: February 2005

Kinetics of glucose transport in K‐562 cells was studied using 3‐0‐methylglucose, a nonmetabolizable analog of glucose. A Km of 3.7 mM and Vmax of 32.0 nmoles/minute/106 cells was found for the process. D‐Glucose, phloretin, and phlorizin competitively inhibit the transport of 3‐0‐methylglucose with Ki values of 4.1 mM, 4.1 μM and 225 μM, respectively, whereas L‐glucose did not inhibit transport at all. The results indicate that K‐562 cells, which are known to have erythropoietic characteristics, possess a glucose carrier system similar to the one in adult human erythrocytes. However, the Vmax data suggest that more copies of the carrier are present in the malignant cell, presumably to support the high rate of anaerobic glycolysis.
Control of cellular proliferation in HeLa‐S3 suspension cultures. Characterization of cultures utilizing acridine orange staining procedures
Authors: Bauer Kenneth D. , Dethlefsen Lyle A.
Published: February 2005

Growth control is investigated in detail in fed and unfed HeLa‐S3 suspension cultures. Two‐step acridine orange staining and flow cytometric analysis indicate declines in cellular red fluorescence (proportional to RNA content) of 40–50% between exponential and plateau phase in both culture types. Cellular green fluorescence (DNA content) assessed simultaneously indicates an increment of cells with G1‐DNA content in plateau phase in the unfed cultures, while fed cultures show a brief increment in G1‐phase cells in the transition phase followed by a recovery in plateau phase to a value similar to that of exponential cultures. Temporal declines in the 3H‐thymidine pulse‐labeling index are observed in both culture systems. These data along with the flow cytometry data indicate a distinct G1‐arrest in the unfed plateau cultures and suggest a random arrest of cells about the cell cycle in fed plateau cultures. Acidic acridine orange staining and flow cytometric analysis furthermore indicate the occurrence of a quiescent population comprising approximately 34% of the total cells and consisting of both dead and viable cells in plateau phase unfed cultures. In contrast, fed plateau cultures show approximately 14% quiescent, mostly dead cells. Also, both culture systems show temporal declines in the clonogenic index and a longer cell‐cycle transit time in plateau phase relative to exponential phase. These findings confirm earlier work which indicates that the environment has a profound influence on the mode of growth control for mammalian cells in vitro.