bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of a brewer's yeast extract on growth and glucose metabolism of cells in culture
Authors: Høstmark Arne T. , Alexander Jan , Grønnerød Ole , Askevold Rolf , Von Kraemer Bryn Monica
Published: February 2005

Brewer's yeast preparations influence glucose metabolism in vivo and in isolated tissues. We have studied the effect of a brewer's yeast extract on glucose metabolism and grwoth of rat hepatoma and human embryonic cells. Growth of the rat hepatoma cells was very much stimulated by the extract in a concentration‐dependent manner. Glucose uptake was, on the other hand, appreciably inhibited, and lactate uptake completely abolished by the extract. Insulin stimulated cell growth and inhibited lactate uptake but did not affect the glucose level. Insulin and the extract had additive effects on growth and lactate uptake of the hepatoma cells. The inhibition by the brewer's yeast extract of glucose uptake was, however, antagonized by insulin. Niacin or Cr3+, which are suggested to be components of a “glucose tolerance factor” of brewer's yeast, did not affect growth or glucose and lactate uptake. The glucose uptake of the human embryonic cells was strongly inhibited by the brewer's yeast extract. Cell growth and lactate production were not influenced by the extract or by insulin; however, when both insulin and extract were present simultaneously, a slight stimulation of growth and inhibition of lactate production was observed. The results indicate that brewer's yeast can have appreciable direct effects on cells and that not all of these effects are “insulin‐like”.
The effect of membrane‐fluidizing agents on the adhesion of CHO cells
Authors: Juliano R. L. , Gagalang E.
Published: February 2005

Treatment of CHO cells with drugs which are known to increase membrane lipid fluidity reduced the cells' ability to adhere to protein coated substrates. The concentrations of local anesthetics, nonionic detergents or aliphatic alcohols required to reduce CHO cell adhesion by 50% were similar to those reported to block nerve conduction, indicating that these drugs can affect the membrane at physiologically significant concentrations. Nonionic detergents and aliphatic alcohols, but not local anesthetics, caused increases in the fluidity of CHO plasma membranes (measured by fluorescence polarization) at concentrations which inhibited cell adhesion. The adhesion versus temperature profile had a sigmoidal shape, suggesting that a temperature dependent cooperative process such as a lipid phase transition, might be involved. However, the temperature profile for CHO membrane fluidity manifested no discontinuities, indicating the absence of any discrete phase transitions of the lipid matrix. This observation, coupled with the result that the inhibition of CHO cell adhesion produced by low temperatures was not relieved by drugs which can increase membrane fluidity, suggests that the reduced adhesion seen at low temperature is probably not due to reduced lipid fluidity.
Serum‐dependent regulation of α‐aminoisobutyric acid uptake in bovine granulosa cells
Authors: Allen W. Ross , Nilsen‐Hamilton Marit , Hamilton Richard T. , Gospodarowicz Denis
Published: February 2005

The removal of serum from the medium of ovarian granulosa cells in exponential or confluent stages of growth results in a rapid and pronounced decrease in the rate of transport of the non‐metabolizable amino acid, α‐aminoisobutyric acid. This decrease is rapidly and completely reversed by the addition of serum. The decrease and its reversal are insensitive to inhibitors of RNA and protein synthesis and are unaffected by a number of other metabolic inhibitors. The serum requirement cannot be replaced by peptide hormones known to stimulate cell division and secretion by these cells. These data are consistent with a model of post‐translational control of AIB transport by a high‐molecular‐weight component of serum.
A comparative study of glycosaminoglycans in cultures of human, normal and malignant glial cells
Authors: Glimelius B. , Norling B. , Westermark B. , Wasteson Å.
Published: February 2005

The glycosaminoglycans (GAG) of human cultured normal glial and malignant glioma cell lines were studied using 35S‐sulphate or 3H‐glucosamine as markers. 35S‐labelled GAG were assayed by precipitation with cetylpyridinium chloride; 3H‐labelled sulphated GAG and 3H‐labelled hyaluronic acid were quantitated after separation on a DEAE‐cellulose column. The net production of GAG and the distribution, composition and turnover of GAG were similar in all of the normal cell lines tested, but showed a great variability in the malignant cell lines. Most of the glioma cell lines produced more hyaluronic acid and less sulphated GAG than the normal cell lines, but exceptions were noted. The GAG of the trypsin susceptible (pericellular) pool of normal glial cells consisted mainly of heparan sulphate with only minor amounts of other GAG. The analogous material of most glioma cells showed hyaluronic acid as the major GAG. Material liberated by trypsin from EDTA‐detached cells (membrane fraction) was enriched in heparan sulphate as compared to the entire pericellular pool. Substrate attached material (SAM) left with the plastic dish after EDTA treatment of normal cultures was rich in heparan sulphate, whereas SAM of glioma cells lacked heparan sulphate or showed greatly reduced amounts of this component. Release of newly synthesized GAG to the extracellular medium was a rapid process in the normal cells but was more or less delayed in the glioma cells. The extracellular medium of the malignant glioma cultures was consistently poor in dermatan sulphate, as compared to that of normal cultures.
Gonadotropin stimulation of cyclic AMP levels in Chinese Hamster Ovary cells in culture
Authors: Evain Daniele , Anderson Wayne B.
Published: February 2005

When Chinese Hamster Ovary (CHO) cells, incubated in serum‐free medium, are exposed to gonadotropins a transient increase in the intracellular concentration of cyclic AMP is observed. Maximum accumulation of cyclic AMP is noted 30 minutes after addition of either human chorionic gonadotropin (hCG) or follicle stimulating hormone (FSH). Within one to two hours after hormone addition, the intracellular concentrations of cyclic AMP have returned to basal levels. The enhancement of intracellular cyclic AMP levels by hCG is hormone concentration dependent, with maximal stimulation observed at 10 μg/ml hCG.

The exogenous addition of gonadotropins also slows the growth rate of CHO cells. This effect on growth seems to be mediated through cyclic AMP since the growth rate of a mutant of CHO cells defective in the catalytic subunit of cyclic AMP dependent protein kinase is only slightly decreased.
Active cell aggregation by immature rat Sertoli cells in primary culture: A role for cell surface glycoproteins
Authors: Bordy Michael J. , Berger Sheldon , Desjardins Claude , Davis John C.
Published: February 2005

Follicle stimulating hormone (FSH) stimulates “colony formation” by immature rat Sertoli cells in primary culture. “Colony formation” involves cell aggregation. Consequently, the involvement of cell surface glycoproteins in cell aggregation was investigated by treatment of dissociated 10‐day rat testis cells with sodium metaperiodate, glucosamine, various lectins, tunicamycin, and puromycin. Treatment of control cultures with 5 μM glucosamine stimulated cell aggregation; however, glucosamine did not affect FSH‐stimulated cultures. Treatment of dissociated testis cells with 5 μM sodium metaperiodate, 10 μg/ml castor bean agglutinin (ricin), or 2.5 μg/ml horseshoe crab agglutinin inhibited FSH stimulation of cell aggregation. A similar inhibition of cell aggregation was observed following addition of 10 μg/ml puromycin or tunicamycin to culture media from 0‐ to 18‐hours incubation. Treatment with soybean agglutinin, concanavalin A, or wheat germ agglutinin had no effect. The galactose‐specific lectins, Ricin, Ricinus communis agglutinin I, and Bendeirea simplicifolia agglutinin, inhibit the FSH stimulation of 3H‐aminoacid incorporation as well as cell aggregation in 24‐hour cultres. The inhibition of cell aggregation by sodium metaperiodate treatment was reversed with 5 μM sodium borohydride reduction. Sodium metaperiodate treatment did not alter cell viability (as assayed with trypan blue dye exclusion), did not alter cell attachment, nor significantly decrease 125I‐FSH binding by cultured testis cells. The results suggest that FSH stimulation of cell aggregation by immature rat Sertoli cells requires cell surface glycoprotein interactions. Furthermore, the specificity of lectin inhibition suggests that glycoproteins with terminal galactose and sialic acid residues are required for the FSH induction of cell aggregation.
Selection and characterization of a variant of murine L5178Y lymphoma resistant to local anesthetics
Authors: Yau Tom M. , Kim S. C. , Crissman H. A.
Published: February 2005

A variant of murine L5178Y lymphoma resistant to procaine hydrochloride (PH) was selected by exposing the cells to gradual increments of PH in the growth medium until the cell grew exponentially in the presence of 1.5 mM PH. Using cinephotomicrography, it was observed that the majority of cells that initially succumbed to PH failed to undergo successful mitosis. With respect to chromosomal, cell size distribution and flow microfluorometric analyses, the PH‐resistant cells are very similar to a spontaneous tetraploid cell line (R1T) previously cloned. The isolated cells, designated R1/P, were also found to be cross‐resistant to analogues of PH, namely, lidocaine, tetracaine and dibucaine. The naturally‐occurring tetraploid cell line (R1T) was also found to be more resistant to local anesthetics, although not to the same extent as R1/P cells. Since the enzyme that hydrolyzes procaine appears to be absent in all these lymphoid cell lines, the difference in resistance does not appear to depend on differences in the ability of these cells to remove the agent. It is suggested that an alteration in the structure and/or function of the plasma membrane in R1/P cells have rendered them either less sensitive to the membrane‐perturbing effects of the local anesthetics or less permeable to local anesthetics molecules. The ability of local anesthetics to affect membranes and cytoskeleton structures may play a role in the genesis and/or selection of these cell variants.
Expression of malignancy in hybrids between normal and malignant cells
Authors: Croce Carlo M. , Barrick Joan , Linnenbach Alban , Koprowski Hilary
Published: February 2005

Somatic cell hybrids between either normal human fibroblasts, phenotypically normal mouse fibroblasts or mouse peritoneal macrophages and HT1080 human diploid fibrosarcoma cells were studied for their ability to form tumors in nude mice. The results of this study indicate that tumorigenic behavior is expressed as a dominant trait in both human‐human and mouse‐human hybrid cells.
Isolation of rat hepatoma cell variants selectively resistant to dexamethasone inhibition of plasminogen activator
Authors: Seifert Sarah Carlson , Gelehrter Thomas D.
Published: February 2005

Glucocorticoids induce several phenotypic changes in rat hepatoma cells in tissue culture, including the inhibition of plasminogen activator activity. Variant cell lines resistant to dexamethasone inhibtion of plasminogen activator activity have been isolated using an agar‐fibrin overlay technique to identify colonies with fibrinolytic (plasminogen activator) activity. The variants are resistant to concentrations of dexamethasone 1,000 times that necessary to completely inhibit plasminogen activator activity in wild‐type cells. The variant phenotype has been inherited in a stable manner for more than 300 generations in continuous culture in the absence of dexamethasone. These variants are unique in that the resistance is not secondary to defective or absent glucocorticoid receptors but is due to a lesion specific for regulation of plasminogen activator. Fluctuation analyses support the hypothesis that resistance to dexamethasone arises randomly and is not induced by dexamethasone. Because HTC cells are heteroploid and karyotypically highly variable, variants are thought to arise primarily by chromosomal segregation events. These variants provide a valuable tool for studying the mechanism of hormonal regulation of plasminogen activator as well as the role of proteases in hormonal regulation of membrane functions.
Regulation of protein synthesis in mitotic HeLa cells
Authors: Tarnowka Miroslaw A. , Baglioni Corrado
Published: February 2005

Mitotic HeLa cells (M cells) synthesize protein at about 25% of the rate of S phase cells. This decrease in protein synthesis is due to a reduction in the rate of initiation. However, extracts prepared from M cells are almost as active in protein synthesis as S cell extracts. Both cell extracts are quite active in in vitro initiation of protein synthesis. Moreover, two steps in initiation, binding of Met‐tRNAf to 40S ribosomal subunits and binding of mRNA to ribosomes, show similar activity in both extracts. The difference in protein synthesizing activity observed in vivo is largely eliminated in the preparation of cell‐free systems. The ribosomes of M cells contain small mot wt RNA, which inhibits protein synthesis in vitro. This RNA, which has possibly a nuclear origin, may be a cause of the reduction in the rate of protein synthesis in M cells.
Regulation of surface antigen expression in Paramecium primaurelia. II. Role of the surface antigen itself
Authors: Capdeville Yvonne
Published: February 2005

In the wild‐type strains, 156 and 168, of Paramecium primaurelia, the alleles G156 and G168 expressed at medium temperature specify two immunologically distinguishable surface antigens 156G and 168G, whose phenotypic expression shows allelic exclusion, the majority of heterozygotes being phenotypically [156G] while a small minority is phenotypically [156G–168G]. At high temperature, the antigens coded by another locus, generally the D locus, are expressed. This system, displaying both intergenic and interallelic exclusion, provides favourable material to analyze the respective roles of the genome, of the antigens expressed and of the environmental conditions, in particular temperature, on the regulation of the expression of surface antigens.

This analysis was carried out by studying the variations of the expression of surface antigens as a function of temperature, culture medium and previously expressed antigens in different genetic situations (a) in homozygotes: the wild‐type strains 156 and 168, and the isogenized strains “G156 isogenic 168” carrying the G156 allele in a 168 genetic background; (b) in heterozygotes of the two phenotypic classes of heterozygotes, [156G] and [156G–168G]. The results show that (1) the thermal stability of the expression of a given surface antigen and its rate of re‐appearance at the cell surface depend on its own specificity: (2) in heterozygotes [156G–168G], the stability of the expression of the antigen 156G is modified and “adjusted” to that of the less stable surface antigen 168G, and (3) the surface antigen itself exerts a positive control on the maintenance of its own expression.

An interpretative model of “transmembranous control” is proposed to account for the regulation of the expression of surface antigens in Paramecium.
Control of the Balb/c‐3T3 cell cycle by nutrients and serum factors: Analysis using platelet‐derived growth factor and platelet‐poor plasma
Authors: Stiles C. D. , Isberg R. R. , Pledger W. J. , Antoniades H. N. , Scher C. D.
Published: February 2005

Much controversy regarding the relationship between nutrients and serum in regulation of cell growth can be reconciled by recognizing that serum contains multiple factors which regulate different events in the cell cycle. Serum was fractioned into a platelet‐derived growth factor (PDGF), which induces cells to become competent to synthesize DNA, and plasma which allows competent cells to traverse G0/G1 and enter the S phase. Nutrients are not required for the cellular response to PDGF; however amino acids are required for plasma to promote the entry of PDGF‐treated, competent cells into S phase. The nutrient independent, PDGF‐modulated, growth regulatory event (competence) is located 12 hours prior to the G1/S phase boundary in quiescent, density‐arrested Balb/c‐3T3 cells. The nutrient dependent, plasma‐modulated event is located six hours prior to the G1/S phase boundary and corresponds in time to a plasma dependent growth arrest point. Moreover, plasma controls the concentration of amino acids required for DNA synthesis. Infection of density‐arrested Balb/c‐3T3 cells with SV40 overrides both the nutrient independent and the nutrient dependent growth regulatory events.
Stimulation of endothelial cell proliferation by precursors of thymidylate
Authors: McAuslan B. R. , Reilly W. , Hannan G. N.
Published: February 2005

Bovine corneal and aortal endothelial cell cultures were established from primary explants and subcultured for at least 40 passages. With both cell, exogenous thymidine, folate or folinate markedly increased the proliferation of these cells and decreased their serum requirement in Medium 199.

Medium 199 supplemented with thymidine was particularly useful for cell survival at low densities; clones were readily produced when single cells were plated as low as 0.07 cells. cm−2. In contrast to the results of others, neither fibroblast growth factor nor epidermal growth factor were necessary for cell proliferation or survival at low densities.
Effect of temperature on protein and immunoglobulin synthesis and secretion in two mouse myeloma cell lines
Authors: Craig Nessly
Published: February 2005

Protein synthesis in differentiated MOPC‐21 and MPC‐11 mouse myeloma cells was studied to determine the basis for the differences in the temperature and actinomycin D sensitivity of translation between non‐differentiated mouse L‐cells and differentiated rabbit reticulocytes. The temperature dependence of total protein synthesis was similar to that of L‐cells and reticulocytes, being biphasic in Arrhenius plots with apparent activation energies of approximately 25 and 42 kcal/mol, above and below 25°C. The dependence of the secretion process was different since it was not biphasic, having a single activation energy of about 22 kcal/mol. Myeloma polysomes were like L‐cell polysomes in their response to lower temperature and reached a minimum level of 50% at 15°C. This response was also found for the specific polysomes synthesizing the IgG H‐ and L‐chains. In the presence of actinomycin D, myeloma polysomes declined exponentially with a half‐life of ∼6 hours. These two L‐cell‐like responses were not found in reticulocytes. Translation of both the IgG mRNAs and the non‐IgG mRNAs was reduced by lower temperatures and actinomycin D, even though the L‐chain mRNA was slightly more resistant, suggesting that this mRNA is slightly more efficient. The results of these experiments suggest that the translational differences between L‐cells and reticulocytes are not mRNA dependent, but are cell type differences.
A new cell surface marker of aging in human diploid fibroblasts
Authors: Aizawa Shinichi , Mitsui Youji
Published: February 2005

The relationship of cell surface changes to proliferative decline of human diploid fibroblasts was investigated using the concanavalin A‐mediated red blood cell adsorption assay. The amount of the red blood cells adsorbed to human diploid fibroblasts via concanavalin A increased continously from the early phases of cell passage up through cell senescence, while the amount of 3H‐concanavalin A binding did not change to a significant extent. The red blood cell adsorption is not a function of cell cycle phase and time spent in culture. Cocultivation of young cells with old cells also did not affect the adsorption capacity of respective cells. Thus, the concanavalin A‐mediated red blood cell adsorption can be expected to serve as a new cell surface marker for aging in vitro. Using this marker, it was revealed that transient cell size or 3H‐thymidine incorporating capacity do not have a direct relationship with the division age of a cell. Small rapidly dividing cells in old populations resemble large slowly dividing or nondividing cells of the same populations and differ from small rapidly dividing cells in young populations, in terms of cell surface properties.
The inhibition by xanthine phosphodiesterase inhibitors of the induction of alkaline phosphatase activity in HeLa cells: Relationship of enzyme activity to cyclic AMP concentrations
Authors: Wharton Walker , Goz Barry
Published: February 2005

The three xanthine derivatives, caffeine, theophylline and 3‐isobutyl‐1‐methyl‐xanthine (IBMX) produced dose‐dependent increases in cyclic AMP concentrations in HeLa cells after long term treatment. Only IBMX produced increases over the first 60 minutes, with a peak of approximately 5‐fold control values five to 10 minutes after the addition of the drug. About four hours after the addition of either 0.67 or 1.0 mM IBMX there was a second peak in the concentration of cyclic AMP which was at least as large and usually larger than the peak observed at five to ten minutes. Neither caffeine nor theophylline increased cyclic AMP concentrations above control values until one hour after addition of the compounds, and there was no indication of a peak in the concentration at four hours. Between 24 and 72 hours, all three compounds produced elevations in cyclic AMP levels that were steadily maintained. At any given concentration, the order of potency was IBMX > theophylline > caffeine. If the xanthine derivatives were removed from the medium after 24 hours of treatment, the cyclic AMP concentrations fell to control levels within one hour. Treatment with 5‐iodo‐2′‐deoxyuridine (IdUrd) or hydrocortisone alone did not change the levels of cyclic AMP, nor did the presence of these inducers of alkaline phosphatase activity alter the effects of the xanthine derivations on cyclic AMP concentrations. The data showed a significant correlation between the magnitude of the increase in cyclic AMP concentrations over the period from 24 to 72 hours and the degree of inhibition by the xanthine derivatives of the induction of alkaline phosphatase activity.
High affinity concanavalin a binding to sterol‐depleted L cells
Authors: Marshall Jan D. , Heiniger Hans‐Jörg , Waymouth Charity
Published: February 2005

Binding of Concanavalin A to mouse L cells which were grown in serum free, chemically defined medium and depleted of their membrane sterol by blocking their de novo sterol synthesis was investigated. Kinetic analysis of binding data implied positive cooperativity, with two different affinities for Con A, in both experimental and control cultures. The amount of Con A bound to the cell surface at saturation was approximately 0.5 picomoles per mg cellular protein in controls and approximately 1.0 picomoles per mg cellular protein in 25‐hydroxycholesterol treated cultures (which had a reduced sterol concentration of up to 50% in their plasma membranes). This phenomenon was reversed when cholesterol or mevalonate was added to the inhibited cultures to compensate for their inability to synthesize sterol. Our findings indicate that lectin binding to specific glycoprotein receptors is influenced by membrane lipid composition.
Mechanochemical proteins, cell motility and cell‐cell contacts: The localization of mechanochemical proteins inside cultured cells at the edge of an in vitro “wound”
Authors: Gotlieb Avrum I. , Heggeness M. H. , Ash J. F. , Singer S. J.
Published: February 2005

We have examined the distribution of several mechanochemical proteins inside rat A10 cells in monolayer culture, both in sparse cultures and at the edges of in vitro “wounds” in confluent cultures. The proteins examined were actin, myosin, tropomyosin, α‐actinin, filamin, and tubulin. In each experiment, a pair of these proteins (one of which was usually actin) were examined simultaneously by double fluorescence staining methods. Actin was specifically stained by a method based on heavy meromyosin binding, whille the other proteins were specifically stained by indirect immunofluorescence procedures. The most important of the various results described was obtained with cells moving out from the edge of an in vitro wound. Within the flat leading lamella of such a cell, there was an extended region in which myosin was severely depleted or absent compared to the proximal regions of the same cells. By contrast, the other proteins were abundantly present throughout the leading lamella, except for tropomyosin, which was somewhat depleted but not as extensively as myosin. In Nomarski optics, there was no detectable morphological differentiation between the region depleted of myosin and the more proximal portion of the same lamella. While the depletion of myosin from the motile regions of cells does not rule out the involvement of some form of an actomyosin sliding filament mechanism, it suggests that other molecular mechanisms for generating motility be seriously considered.
Role of hydrogen bonding in red cell aggregation
Authors: Jan K. K.
Published: February 2005

The role of hydrogen bonding in red cell aggregation induced by dextran was studied with the use of urea, an inhibitor for hydrogen bonding. In order to avoid hemolysis of red cells by the high concentration of urea, the studies were performed on human red cells hardened in glutaraldehyde. The degree of red cell aggregation at Hct = 45% was estimated by the use of a coaxial cylinder viscometer. The viscometric aggregation index (VAI) was calculated from viscosity values at shear rates of 52 sec−1 (ηH) and 0.05 sec−1 (ηL); VAI = (ηL–ηH)/ηH. Red cells with surface charge intact and with charge removal by neuraminidase treatment were studied. Urea at high concentrations, e.g., 6 M, significantly inhibited red cell aggregation induced by dextran. These findings indicate that hydrogen bonding plays an important role in dextran‐induced red cell aggregation. An understanding of the nature of the forces involved in red cell aggregation serves to establish the physicochemical principles of cell‐to‐cell interactions induced by macromolecules.
The growth response of cells in medium made hyperosmolal with electrolytes or mannitol
Authors: Swanson T. Leonard
Published: February 2005

A comparison of the growth rates of established human lymphoid and tumor cell lines was performed in nutrient medium made hyperosmolal with mannitol, NaCl, or mixtures of NaCl and KCl at a constant Na/K ratio. It was found that considerably higher osmolalities were attained with mannitol than electrolytes before a reduction in the growth rate of the culture was observed. This suggests that mannitol and electrolytes affected the growth rate through different mechanisms. Mannitol uptake was studied with two of the cell lines and both cell lines were found to be permeable to mannitol. This eventually would have eliminated the osmolality gradient between the interior of the cell and the medium, and could explain why higher osmolalities were obtained with mannitol before the growth rate was effected. In addition, initial experiments showed that these cell lines may also be able to metabolize mannitol.
Effect of cell density on energy‐dependent calcium uptake by Balb/c 3T3 membranes is independent of protein synthesis and attachment to substratum
Authors: Moore Leon , Pastan Ira
Published: February 2005

Membranes isolated from subconfluent cultures of Balb/c 3T3 cells have low energy‐dependent calcium uptake activity. Replating confluent cells at low density results in a prompt fall of energy‐dependent calcium uptake by membrane fractions. The level to which uptake activity falls is a function of the density at which the cells are plated (Moore and Pastan, '77b). To determine if regulation of energy‐dependent uptake of calcium by membrane fractions is dependent upon attachment to a substrate and to further characterize conditions that regulate the process, we examined calcium uptake activity of membranes isolated from cells in suspension. With cells in suspension energy‐dependent calcium uptake activity of isolated membranes falls promptly if cells are diluted to a low density (<105 cells/ml) and is a function of cell density. When cells in suspension at low cell densities are concentrated to high cell densities (>2 X 106 cells/ml), calcium uptake activity of the isolated membrane fraction is increased as a function of cell density. These changes of membrane calcium uptake activity occur promptly and do not require protein synthesis.
Insulin stimulation of glucose entry in cultured human fibroblasts
Authors: Howard Barbara V. , Mott David M. , Fields Rose M. , Bennett Peter H.
Published: February 2005

The effect of insulin on glucose entry has been studied in monolayer cultures of human diploid fibroblastic cells. Influence of insulin on total cell glucose incorporation was evaluated using [14C] glucose. Glucose incorporation was increased up to two‐fold in the presence of insulin. Insulin action occurred within 30 minutes and could be observed with insulin concentrations as low as 10−10 M (10 μU/ml). The action of insulin was enhanced by preincubation in glucose‐free medium. After glucose starvation the cells converted glucose primarily to glycogen and nucleotides, and the stimulation by insulin was observed equally in both fractions.

Influence of insulin on the kinetics of hexose transport was studied using 2‐deoxyglucose and 3‐0‐methyl glucose. A large diffusion component was corrected using ρ‐chloromercuribenzoic acid or phloridzin. Km for facilitated diffusion averaged 1.9 mM for 2‐deoxyglucose and 5.3 mM for 3‐0‐methyl glucose, and Vmax ranged from 10–24 nmoles/min/mg cell protein. Insulin resulted in a 150% increase in Vmax with no significant change in Km. The data suggest that human diploid fibroblasts can be a useful system for the study of insulin's glucoregulatory action.
Adenosine transport and metabolism in mouse leukemia cells and in canine thymocytes and peripheral blood leukocytes
Authors: Lum Caliann T. , Marz Richard , Plagemann Peter G. W. , Wohlhueter Robert M.
Published: February 2005

The intracellular accumulation of free [3H] adenosine was measured by rapid kinetic techniques in P388 murine leukemia cells in which adenosine metabolism (phosphorylation and deamination) was completely prevented by depletion of cellular ATP and by treatment with deoxycoformycin. Nonlinear regression of integrated rate equations on the data demonstrate that the time courses of labeled adenosine accumulation at various extracellular adenosine concentrations in zero‐trans and equilibrium exchange protocols are well described by a simple, completely symmetrical, transport model with a carrier:substrate affinity constant of about 150 μM. Adenosine transport was not affected by 1 mM deoxycoformycin indicating that this analog has a low affinity for the nucleoside transport system. The transport capacity of dog thymocytes and peripheral leukocytes was similar to that of P388 cells. Transport was not inhibited by deoxycoformycin and remained constant during the first two hours after mitogenic stimulation with concanavalin A.

In untreated, metabolizing P388 cells transport was found to be the major determinant of the rate of intracellular metabolism, regardless of the extracellular adenosine concentration (up to at least 160 μM), but the long‐term accumulation (longer than 30–60 seconds) of radioactivity from extracellular adenosine strictly reflected the rate of formation of nucleotides (mainly ATP). The metabolism of adenosine by whole cells was entirely consistent with the kinetic properties of the transport system and those of the metabolic enzymes. At low exogenous adenosine concentrations (1 μM and below) transport was slow enough to allow direct phosphorylation of most of the entering adenosine. The remainder was deaminated and rapidly converted to nucleotides via inosine, hypoxanthine, and IMP. At concentrations of 100 μM or higher, on the other hand, influx exceeded the maximum velocity of adenosine kinase about 100 times so that most of the entering adenosine was deaminated. But since the maximum velocity of adenosine deaminase exceeded those of nucleoside phosphorylase and hypoxanthine/guanine phosphoribosyltransferase about 5 and 100 times, respectively, hypoxanthine and inosine rapidly exited from the cells and accumulated in the medium. A 98% reduction of adenosine transport (at 100 μM), caused by the transport inhibitor Persantin, inhibited adenosine deamination by whole cells to about the same extent as transport, whereas adenosine phosphorylation was relatively little affected; thus in the presence of Persantin, transport and metabolism resembled that occurring at the low adenosine concentration. These and other results indicate that adenosine deamination is an event distinct from transport, which occurs only subsequent to adenosine's transport into the cell.
Differential effects of cytochalasin B and caffeine on control of DNA synthesis in normal and transformed cells
Authors: O'Neill Frank J.
Published: February 2005

Evidence is presented which suggests the existence of at least two growth control points in cultured mammalian cells. These controls are in “parallel” rather than in “series.” It is suggested that in normal and some transformed cells the controls are coupled while in SV40 transformed cells and tumor cells they are uncoupled. One control is revealed by cytochalasin B (CB) treatment since in normal and some kinds of transformed cells, CB treatment results in the accumulation or the arrest of cells in G1. In this case, the control is normal or coupled. In DNA tumor virus transformed cells and many cultured tumor cells (excluding human glioblastomas) CB does not arrest cells although it prevents cytoplasmic division. Such cells continue DNA synthesis and nuclear division and become highly multinucleated. The effects of CB are not concentration dependent. Here the control is defective or uncoupled. Another control is revealed by caffeine treatment. Again, normal and some types of transformed cells are accumulated in G1 following caffeine treatment, while SV40 transformed cells and tumor cells continue DNA synthesis unabated. The effects of caffeine in arresting cells are concentration dependent. Similarly the control point revealed by caffeine is coupled in normal cells and altered or uncoupled in some SV40 transformed and tumor cells. Although CB and caffeine do not inhibit DNA synthesis in tumor and SV40 transformed cells when administered separately, they cause G1 accumulation when administered in combination. This observation can be explained by the possibility that some transformed cells can utilize alternate or parallel mechanisms to progress through G1 when one mechanism is blocked (CB or caffeine). When both are blocked the cells become arrested in G1. The human glioblastomas are unique among the cultured human tumors thus far examined since they are partially arrested by CB. However they are like other human tumors in that caffeine does not significantly affect DNA synthesis.
Ribosome biosynthesis in Tetrahymena thermophila. III. Regulation of ribosomal RNA degradation in growing and growth arrested cells
Authors: Sutton Claudia A. , Hallberg Richard L.
Published: February 2005

We have measured the turnover rate of ribosomal RNA in exponentially growing Tetrahymena thermophila cells, cells entering the plateau phase of growth, and nutrient‐deprived (starved) cells. Ribosomal RNA is stable in cells in early log phase growth but it begins to turnover as the cells begin a deceleratory growth phase prior to entering a plateau state. Likewise, rRNA in cells transferred from early log phase growth to a starvation medium begins to be degraded immediately upon starvation. In both cases the degradation of rRNA exhibits biphasic kinetics. A rapid initial exponential degradation with a half time of nine and one‐half hours lasting for six hours is followed by a slower exponential degradation with a half‐life of 35 hours. When starved cells are transferred to fresh growth medium turnover of rRNA ceases. The evidence presented suggests that the alteration in degradation rate is a regulated process which is most likely independent of the cell cycle.
Regulation of phosphodiesterase and ornithine decarboxylase by cAMP is cell cycle independent
Authors: Kaiser Nurit , Bourne Henry R. , Insel Paul A. , Coffino Philip
Published: February 2005

Cyclic AMP (cAMP) causes growth arrest in G1 and induction of cAMP phosphodiesterase and decrease of ornithine decarboxylase in S49 mouse lymphoma cells. Dibutyryl cAMP treatment of partially synchronized cells causes similar changes in activities of both enzymes, regardless of position in the cell cycle. This suggests that cAMP regulation of these enzymes is not mediated by growth perturbation.
Potentiation by p‐fluorophenylalanine of the division‐delaying effects of high hydrostatic pressure in synchronized Tetrahymena pyriformis
Authors: Walker Eric , Wheatley Denys N.
Published: February 2005

Cultures of heat‐synchronized Tetrahymena pyriformis, growing in a proteose peptone medium, were subjected to short pulses of the amino acid analogue, p‐flurophenylalanine, and high hydrostatic pressure. The pulses of these agents were chosen so that, when applied individually, they did not appreciably delay cell division. However, combined treatments, analogue pulse followed by pressure pulse, produced a pronounced synergism. The results are interpreted as further evidence to support the inclusion of analogue division proteins in pressure labile assemblies during the progression of Tetrahymena into division.
Rapid alterations in cellular morphology and plasma membrane structure induced in rat thymocytes by mitogenic stimuli
Authors: Hume David A. , Thornton Mary R. , Weidemann Maurice J. , Speth Volker
Published: February 2005

Under conditions where a maximum stimulation of 3‐O‐methyl‐glucose transport is observed, three thymocyte mitogens (concanavalin A, ionophore A23187 and hydrogen peroxide) cause cell rounding and a decrease in the density of intra‐membrane particles on the plasma membrane. The early effects of mitogens on the thymocyte plasma membrane are similar to those of osmotic shock.
Colchicine resistant friend cells: Application to the study of actinomycin D induced erythroid differentiation
Authors: Crichley Valerie , Mager Dixie , Bernstein Alan
Published: February 2005

Colchicine resistant (CchR) mutants have been isolated from Friend erythroeleukemic cells by successive single‐step selections. Measurements of the rate of uptake of [3H]‐colchicine into whole cells, and the binding of [3H]‐colchicine to cytoplasmic extracts, suggest that these mutants are colchicine‐resistant due to a reduced membrane permeability to colchicine, rather than an altered intracellular colchicine‐binding target. Consistent with this conclusion is the observation that non‐toxic concentrations of Tween–80, a non‐ionic detergent, potentiated colchicine uptake into mutant cells. In addition, these Friend cell mutants, like CchR mutants of other cell types, are cross‐resistant to a variety of unrelated drugs, including daunomycin, puromycin, emetine, and actinomycin D.

A comparison of the dose‐response curves for the induction of Friend cell differentiation by actinomycin D of both wild‐type and two CchR cells suggests that actinomycin D permeation is required for its effects on Friend cell differentiation. Potentiation of actinomycin D uptake by Tween–80 significantly lowered the concentration of drug required to induce hemoglobin synthesis in the CchR cells, but had no significant effect on either actinomycin D induction of CchS cells or DMSO induction of both CchS and CchR cells. In common with other chemical inducers of Friend cell differentiation, the addition of actinomycin D results in an early decrease in 86 RbCl uptake, although this effect on transport occurred 14 hours later than that observed with DMSO.
Synthesis of proteins and RNA of the 60S ribosomal subunit in HeLa cells recovering from valine deprivation
Authors: Raikow Radmila B. , Vaughan Maurice H.
Published: February 2005

The synthesis of ribosomes in HeLa cells was studied during recovery from a 20‐hour deprivation for valine. The rates of incorporation of labeled precursors into ribosomal pre‐RNA, processed rRNA, total cellular proteins, and proteins of the 60S ribosomal subunit returned to normal or nearly normal levels immediately after restoration of valine to the medium. Specific proteins of the 60S ribosomal subunit, whose apparent net synthesis is reduced more than that of the other proteins of the 60S ribosomal subunit during valine deprivation, were no longer undersynthesized after valine was restored. This rapid recovery suggests that the apparent decrease in the net rate of synthesis of these ribosomal proteins during valine deprivation is effected at the translational or post‐translational level. No evidence of significant synchrony in any particular stage of the cell cycle was observed after a 20‐hr valine deprivation. Key words: 60S ribosomal subunit; HeLa, cells; valine deprivation.
Growth‐modulating tripeptide (glycylhistidyllysine): Associaton with copper and iron in plasma, and stimulation of adhesiveness and growth of hepatoma cells in culture by tripeptide‐metal ion complexes
Authors: Pickart Loren , Thaler M. Michael
Published: February 2005

The tripeptide H‐Gly‐His‐Lys‐OH (GHL) is a human plasma constituent which has been previously shown to modulate the growth and viability of a variety of cell types and organisms. Experimental observations presented herein indicate that GHL is complexed with the transition metal ions Cu++ and Fe++ in vivo and may exert its biological effects as a peptide‐metal chelate. At physiological pH in vitro, GHL associates with ionic copper, cobalt, iron, molybdenum, manganese, nickel, and zinc, but has no affinity for calcium, manganese, potassium, and sodium. GHL acts synergistically with copper, iron, cobalt, and zinc to alter patterns of cell growth in monolayer cultures of a tumorigenic hepatoma cell line (HTC4). These transition metals induce cellular flattening and adhesion to support surfaces, and inhibit DNA synthesis and lactic acid production when growth is limited by reduction of serum concentrations in medium. These inhibitory effects are neutralized, and intercellular adhesion and growth are stimulated by GHL in medium at nanomolar concentrations. Cu and Fe are the most active metals when combined with GHL. The results suggest that the inability of HTC4 cultures to replicate without adequate concentrations of serum in medium may reflect deficiency of GHL and transition metals, which appear to form complexes prior to interaction with cells. Chelation of transition metals with GHL and, potentially, with other growth‐modulating peptide factors in plasma or medium, may provide a mechanism for expression and regulation of biological activities influenced by transition metals and polypeptide growth factors. The observed effects of GHL‐metal complexes, including stimulation of cellular adhesiveness to substratum (flattening) and intercellular attachment (monolayer formation), appear to satisfy requirements for growth of hepatoma cells in monolayer culture.
Vital DNA staining and cell sorting by flow microfluorometry
Authors: Lydon M. J. , Keeler K. D. , Thomas D. B.
Published: February 2005

A procedure has been investigated for sorting viable cells according to their DNA content. Cells are stained with the U.V. activated fluorochromes 4′6‐diamidino‐2‐pheylindole (DAPI), Hoechst 33258 or Hoechst 33342, and sorted with a Fluorescence Activated Cell Sorter. Hoechst 33342 is a suitable vital stain for a varietyof cell types. Hoechst 33258 and DAPI, however, are quantitative vital stains for CHO cells only. Cloning efficiency is unaffected by the sorting procedure, and these stains are not mutagenic at concentrations suitable for vital staining. Potential applications of this procedure to cell biology are discussed.
The cellular basis of self renewal in culture by human acute myeloblastic leukemia blast cell progenitors
Authors: Chang Lawrence J. A. , Till James E. , McCulloch Ernest A.
Published: February 2005

Blast cells from patients with Acute Myeloblastic Leukemia (AML) were separated according to cell size using velocity sedimentation under unit gravity. Fractions obtained in this way were plated in methyl cellulose with a growth stimulator present in media conditioned by leukocytes in the presence of phytohemagglutinin (PHA‐LCM). Colonies of blast cells form under these conditions. Pooled cell suspensions from such colonies were plated in microwells; the plating efficiency of such suspensions is a measure of blast progenitor self‐renewal occurring in the original blast colonies. Self‐renewal assays on each fraction indicated that self renewal among blast progenitors is heterogeneously distributed with subpopulations differing in renewal capacities. The results are consistent with the view that blast cell subpopulations in AML undergo a series of transitions associated with decreasing self renewal capacity, analogous to that observed in normal hemopoiesis, where proliferative capacity decreases with increasing differentiation.
Role of lysosomes in protein turnover: Catch‐up proteolysis after release from NH4Cl inhibition
Authors: Amenta J. S. , Brocher S. C.
Published: February 2005

Cultured rat embryo fibroblasts, when placed in media with 10% serum containing 20 mM NH4Cl, show an inhibition of protein degradation and, concurrently, an accumulation of numerous, large vacuoles, partially filled with cellular debris. Cells placed in a serum‐free media exhibit an enhanced degradation of cell protein, which is also inhibited by NH4Cl. When these cells are removed from media containing NH4Cl and placed in fresh media, the material accumulated in these vacuoles is rapidly and quantitatively released to the media in both an acid‐soluble and acid‐insoluble form. NH4Cl inhibits rapidly and specifically the lysosomal proteolytic mechanism, and is without effect on the basal turnover mechanism. The lysosomal proteolytic mechanism accounts for approximately 25% of protein turnover, and, at least in low density cultures, can be stimulated to levels which account for more than half of the protein turnover in the cell. The major pathway for the degradation of fast turnover proteins appears to be separate from lysosomal mechanism.
Lectin‐dependent neutrophil cytotoxicity: Enhanced susceptibility of desialylated red cells
Authors: Tsan Min‐Fu , Scheffel Ursula , Denison Rebecca C.
Published: February 2005

Lectin‐dependent neutrophil cytotoxicity against autologous human red cells was studied using an 111In(indium)‐release assay. Human red cells were not readily killed by neutrophils in the presence of phytohemagglutinin (PHA). However, removal of red cell membrane sialic acids (desialylation) markedly enhanced their susceptibility to PHA‐dependent neutrophil cytotoxicity. This neutrophil cytotoxicity was dependent on the energy supplied by anaerobic glycolysis, but it was independent of erythrophagocytosis. Catalase, superoxide dismutase, KCN, and Na azide did not inhibit PHA‐dependent neutrophil cytotoxicity. Neutrophils from a patient with chronic granulomatous disease, in the presence of PHA, also killed desialylated red cells normally. On the other hand, desialylation of neutrophils had no effect on the expression of their cytotoxic effect. The results suggest that desialylated red cells are much more susceptible to lectin‐dependent neutrophil cytotoxicity than normal red cells, and that lectin‐dependent neutrophil cytotoxicity is independent of reactive oxygen species.