bluuebunny/crossref_metadata_2025 huggingface dataset:
Density dependent regulation of growth in L cell suspension cultures. III. Elevation of specific activity of acetylcholinesterase
Authors: Glinos André D. , Bartos Edwin M.
Published: February 2005

High density L cell suspension cultures were previously shown to remain viable for indefinite periods of time and to exhibit marked inhibition of DNA synthesis and mitosis while the fraction of total protein synthesis represented by collagen is increased. The present study demonstrates that regulation in this system extends to the activity of acetylcholinesterase found to be approximately 100‐fold greater in the high density populations than in low density exponentially growing cultures. Kinetic studies of the increase of the activity, its fluctuation over an extended period of time and its decrease upon resumption of exponential growth after dilution of the cultures were performed. The data obtained indicate that the enzyme does not accumulate in high density populations merely as a result of the absence of net protein synthesis and cell division but that changes of its rates of synthesis and possibly degradation are involved. The expression of regulated acetylcholinesterase activity in a cell line of connective tissue origin is considered in relation to phenotype reprogramming and to cell membrane associated growth control mechanisms.
Calcium and flagellar response during the chemotaxis of bracken spermatozoids
Authors: Brokaw C. J.
Published: February 2005

The attraction of fern spermatozoids by secretions from the female reproductive structures, and by salts of malic acid, has long been known as a classic example of precise chemotactic orientation by motile cells. Spermatozoids of the bracken fern are attracted by the partially ionized form of malic acid, bimalate ion, and also by calcium ions. Both calcium and bimalate ions must be present for chemotactic response and for response to voltage gradients, Spermatozoids swimming up a bimalate concentration gradient swim in helical paths of abnormally small radius; if they accidentally swim down the concentration gradient the radii of their path helices become abnormally large.

These observations suggest that changes in the direction of flagellar beating in response to the rate of change of bimalate ion concentration with time may be the basis for chemotactic orientation. A “coupled diffusion” hypothesis for chemo‐reception is presented, which postulates a membrane carrier which can only circulate freely in the membrane if it binds both bimalate and calcium ions. This hypothesis could explain time‐differentiation of the stimulus, the coupling of a specific stimulus — bimalate ions — to a general mediator of intracellular response — calcium ions — and the quantitative relationship between response of the spermatozoids and the chemical potential of “calcium bimalate.”
Control of normal differentiation of myeloid leukemic cells. IV. Induction of differentiation by serum from endotoxin treated mice
Authors: Fibach Eitan , Sachs Leo
Published: February 2005

Sera from different strains of mice injected with endotoxin induced clones (D+) from a cultured line of myeloid leukemic cells to undergo normal differentiation to mature granulocytes and macrophages. Other clones (D−) derived from the same cell line were not inducible by these sera to undergo normal cell differentiation. Sera from the same strains of mice that had not been injected with endotoxin, increased the cloning efficiency of D+ and D − clones but did not induce differentiation. Endotoxin serum induced differentiation in D+ cells at dilutions up to 1:64, but increased the cloning efficiency of these cells at dilutions up to 1:2048. The end point of the dilution of endotoxin serum that induced differentiation in D+ cells, was also the end point that induced the formation of colonies with differentiation from normal bone marrow cells. The results indicate that serum from endotoxin treated animals can serve as a good in vivo source to induce normal differentiation in D+ myeloid leukemic cells; that the progeny of a single leukemic cell was induced to undergo differentiation to both macrophages and granulocytes; that endotoxin serum contained two activities, one that increased cloning efficiency and the other that induced cell differentiation; and that the same material in endotoxin serum induced cell differentiation in normal and leukemic cells.
Inhibition of development of transformation by rous Sarcoma virus in cultures of high cell density
Authors: Clark Joseph M. , Bader John P.
Published: February 2005

The effect of cell density on morphological transformation of chick embryo cells by Rous Sarcoma Virus (RSV) was examined in this study, and a cell density optimum for transformation was found. Less than 10% of the transformed foci appearing at the optimum density (2.5 × 104 cells per cm2) developed at high cell densities, and the diameters of the foci (an indication of the number of cells per focus) decreased with increasing cell density. No correlation was found between the decrease in transformation at high cell densities and the effect of cell density on the initial rate of cell proliferation, although dissociation of transformation from incorporation of radioactive precursors into nucleic acids could not be established. Redistribution of cells infected at high density showed that only a small proportion of successfully infected cells developed into foci. The results indicate that transformation of cells containing the RSV genome can be suppressed by physiological factors accompanying high cell density.
Studies on the surface of chick blastoderm cells. III. Calcium binding to ionogenic sites
Authors: Harris Harriet L. , Zalik Sara E.
Published: February 2005

The effect of increasing calcium concentrations on the electrophoretic mobility of cells obtained from early chick blastoderms at successive developmental stages was studied. Cells had zero mobilities at calcium concentrations in the range of 1 to 2 × 10−2 M CaCl2, and became positively charged when calcium concentrations between 2 and 5 × 10−2 M CaCl2 were used. Results using trypsin suggest that while some calcium binding sites disappear from the surface ionogenic layer, the majority of them are not removed by this enzyme. The calcium binding capacity did not vary appreciably in the stages studied. Charge reversal induced by calcium is reversible.
Autoradiographic studies of pyridine nucleotide metabolism in human culture cells
Authors: Rechsteiner Martin , Lund Keith , Hillyard David , Olivera Baldomero
Published: February 2005

More than 80% of the intracellular pyridine metabolite pool of human culture cells is trapped by OsO4 fixation. The fixed pyridine metabolites fully exchange with nicotinamide and nicotinic acid but not with nicotinamide adenine dinucleotide. Yet, chromatography of the exchanged compounds reveals that NAD and NADP constitute more than 95%. of the fixed material. Although the mechanism of OsO4 fixation is not fully understood, such fixation has permitted the autoradiographic detection of intracellular pyridine metabolites. Cells of the human cell line, D98/AH2, synthesize pyridine nucleotides during all phases of the cell cycle at rates which do not vary by more than six‐fold. There is no difference in the apparent concentration of pyridine metabolites between nucleus and cytoplasm after ten minute or three day pulses with 3H‐nicotinic acid. The 3H‐labeled pyridine ring is lost from D98/AH2 cells upon transfer to unlabeled medium. In general, the rate of loss is uniform among cells in the population. However, in a small proportion of cells there is little or no loss. Non‐dividing cells lose the pyridine ring at approximately the same rate as dividing cells, yet the intracellular concentration of pyridine metabolites is 50% greater in non‐dividing cells.
Density dependent regulation of growth in L‐cell suspension cultures. IV. Adaptive and nonadaptive respiratory decline
Authors: Vail James M. , Glinos André D.
Published: February 2005

Respiration as an index of oxidative energy production was investigated in a L‐cell suspension culture system previously shown to exhibit density‐dependent inhibition of growth. It was found that as cultures progressed from exponential growth to high density nongrowing populations (6−10 × 106 cells/ml) over a 2‐week period, the respiratory rate determined from the total amount of oxygen consumed during the daily medium renewal cycle, declined from 5.4 to 1.8 fmoles O2/cell/min. There are two components in this decrement. The first consists of a daily recurrent decline of oxygen uptake resulting from decreased availability of medium oxygen and glutamine and is readily reversed by medium supplementation. The second component which is refractory to medium supplementation and accounts for approximately 50% of the total respiratory decline, is considered to indicate an adaptive change of the respiratory capacity of the cells. This change is reversed during the lag period which precedes resumption of exponential growth upon subculture to low cell densities. The significance of these results is discussed in relation to recent reports indicating a marked depression of respiratory activity in nongrowing dense attached cultures as well.
Cell volume regulation in ehrlich ascites tumor cells
Authors: Hendil Klavs B. , Hoffmann Else K.
Published: February 2005

Ehrlich cells subjected to anisoosmolar media show very rapid volume changes. In hypertonic media they shrink. In hypotonic media they swell but the rapid initial swelling is followed by a regulatory shrinkage lasting ca. 30 minutes.

Cells suspended in media with identical ionic concentrations but different total osmolarity (adjusted by sucrose) were compared. These studies revealed that swollen cells adjust their volume by decreasing the amount of intracellular K+ and ninhydrin positive substances. Intracellular Na+ and ATP concentrations were unchanged. Accordingly 42K+ flux analysis showed that the (passive) cell membrane permeability for K+ is increased to a minor degree and the Na+ permeability unaffected. The increased K+ permeability could not be correlated to an increase in 45Ca2+ influx.
Peritoneal exudate cells II. Kinetics of appearance of colony‐forming cells
Authors: Lin Hsiu‐San
Published: February 2005

A transient increase in the number of peritoneal colony‐forming cells in agar (PCFC) was noted in the peritoneal cavity of mice given one intraperitoneal injection of thioglycollate medium. This increase of PCFC was not accompanied by a similar degree of increase in the number of either pluripotent hemopoietic stem cells or committed hemopoietic stem cells for granulocytes and/or macrophages that are normally present in the peritoneal cavity in small numbers. Other substances, such as glycogen, starch, Freund's adjuvant, sheep red blood cells and lectins, were also capable of increasing PCFC, but to different degrees. The ability of thioglycollate medium as well as other stimulatory agents to increase PCFC appeared to be dose‐dependent.
Proliferative capacity and DNA content of aging human diploid cells in culture: A cytophotometric and autoradiographic analysis
Authors: Yanishevsky Rosalind , Mendelsohn Mortimer L. , Mayall Brian H. , Cristofalo Vincent J.
Published: February 2005

Using a combination of DNA‐cytophotometry and tritiated thymidine‐autoradiography, we have shown that the majority of nondividing cells in serially propagated human diploid cell populations have the 2C DNA content consistent with their being arrested in the G1 phase of the diploid cell cycle. Unlabeled 4C cells appear increasingly with time in culture. These may be arrested G2 diploids or they may be G1 tetraploids, since there is an associated increase in polyploidy in older cultures as evidenced by the appearance of labeled 8C cells.
Metabolic cooperation in cell culture: Studies of the mechanisms of cell interaction
Authors: Cox Rody P. , Krauss Marjorie R. , Balis M. E. , Dancis Joseph
Published: February 2005

Metabolic cooperation is a form of cell communication in which the mutant phenotype of enzyme deficient cells, as determined by incorporation of labeled substrates, is corrected in culture by contact with normal cells. Previous studies showed that metabolic cooperation between normal and hypoxanthine phosphoribosyl transferase deficient cells (HPRT−) was the result of transfer of product of the enzyme, nucleotide or nucleotide derivative, from normal to mutant cells rather than transfer of enzyme or informational macromolecules leading to the synthesis of the enzyme.

In the present study the nature and mechanisms involved in these cell interactions were investigated. Effective communication is observed within one hour of cell contact. Modifications of the extracellular environment including changes in osmolarity, concentration of sodium and divalent ions failed to interfere significantly with transfer. Changes of cell shape induced by cyclic nucleotides, hormones and urea also did not affect communication. Cytochalasin B which dissociates microfilaments and binds to cell membranes reduced metabolic cooperation while colcemide which dissociates microtubules had little effect. Enzymatic oxidation and iodination of cell surface structures abolished metabolic cooperation. The subcellular localization of label in donor cells is important in determining efficiency of transfer. Metabolic cooperation is efficient when radioactive label is primarily located in the nucleus and inefficient if the label is cytoplasmic. Cell lines previously classified as “non‐communicating” because they lack gap junctions, ionic coupling and metabolic cooperation were shown in the present study to communicate when incubated with labeled substrates for 20 hours rather than 3. Cell communication is a more generalized phenomenon among cells in contact than previously appreciated.
Cellular binding of 3H‐cytochalasin B
Authors: Mayhew E. , Poste G. , Cowden M. , Tolson N. , Maslow D.
Published: February 2005

The binding of tritium‐labelled cytochalasin B (3H‐CB) to a variety of mammalian cells was investigated. Binding studies revealed near‐equilibrium binding of 3H‐CB within 5 to 10 minutes, but the equilibrium level was influenced by 3H‐CB concentration. Binding kinetics revealed strong temperature dependence. Rapid release of up to 70% of cell‐bound 3H‐CB molecules occurred when cells were washed and returned to fresh medium without CB. The remaining 30% of cell‐bound 3H‐CB molecules dissociated more slowly. Equilibrium binding studies on a variety of diploid, heteroploid and transformed cells treated with 1 μg/ml 3H‐CB revealed between 1.7 X 107 to 5.3 X 107 3H‐CB binding sites per cell. Cellular binding of 3H‐CB was not affected by inhibition of cellular energy metabolism, RNA or protein synthesis. Modification of the cell surface by proteases, neuraminidase, hyaluronidase, ribonuclease, or occupation of cell surface saccharide residues by a variety of plant lectins did not significantly alter the pattern of 3H‐CB binding. Surface pressure measurements on CB‐treated lipid monolayers indicated that CB can interact with lipid molecules. The partition of CB in hydrophobic lipid regions of cell membrane systems as a possible mechanism of cellular binding of CB is discussed. Fractionation of 3H‐CB‐treated cells revealed binding of 3H‐CB to both the plasma membrane and by intracellular membranes.
Immunogenetic ontogeny of cellular membrane function: A review
Authors: Hubbert W. T. , Miller W. J.
Published: February 2005

Cell membrane antigens serve as recognition codes for normal cell functions (substrate transport, cell‐cell interaction, etc.). Changes in antigen‐function activity are associated with ontogeny and speciation. Some prenatal antigenic configurations are postulated to provide host protection during early development.
Expression of the neuron‐specific protein, 14‐3‐2, and steroid sulfatase in neuroblastoma cell hybrids
Authors: Arthur McMorris F. , Kolber Alan R. , Moore B. W. , Perumal A. S.
Published: February 2005

Three series of neuroblastoma X fibroblast hybrid clones were isolated from crosses between mouse or human fibroblasts and mouse or human neuroblastoma cell lines by virus‐mediated cell fusion. The expression of 14‐3‐2 protein (an acidic protein specific to neurons) and steroid sulfatase activity was studied in parental and hybrid cell lines. Steroid sulfatase was extinguished in hybrids when only one parent expressed the enzyme, but was expressed in one hybrid combination in which both parents expressed the enzyme. The neuron‐specific 14‐3‐2 protein, on the other hand, continued to be expressed in all three series of neuroblastoma x fibroblast hybrids. In most cases where these pheno‐types were expressed, they also exhibited temporal modulation; that is, specific activity is low during logarithmic growth and increases markedly during stationary phase. The glial‐specific protein S‐100 is absent from all parents and hybrids. The results are discussed in terms of mechanisms of regulation of differentiated phenotypes in mammalian cells.
Maturation of membrane function: The permeability of the rat erythroblastic leukemic cell to water and to non‐electrolytes
Authors: Hempling H. G. , Wise W. C.
Published: February 2005

The erythroblastic leukemia produced in Long‐Evans rats by the administration of 7, 8, 12 trimethylbenz (a) anthracene has been used as a model of the most immature form of the erythrocyte series. In conjunction with studies of the maturation of several other membrane functions, the permeability of this cell to water and to certain definitive non‐electrolytes was measured with osmotic methods. The hydraulic conductivity, Lp was 6.2 micro (minute)−1, (atm)−1 at 25°C, quite high and characteristic of mature erythrocytes, but different from values of 0.65 for immature myeloid cells. The effect of temperature provided an energy of activation of 4.4 kCal/mole, also typical of mature mammalian erythrocytes but again different from 13 to 18 kCal/mole for immature myeloid cells. Urea was compared to thiourea. The permeability coefficient for urea was 76.7 micra (minute)−1 ± 13.8 (S. E.); the value for thiourea was 1.55 micra (minute)−1 ± 0.18 (S. E.). Phloretin at 0.25 mM inhibited urea permeability by 90% with 50% inhibition occurring at 0.05 mM. Inhibition was reversible. Permeability to the glycols was also compatible with mature erythrocytes. We infer from these findings that the structure which underlies these basic, passive membrane functions is laid down early and persists after loss of nucleus and subsequent maturation.
The binding of cationized ferritin at the surfaces of ehrlich ascites tumor cells: The effect of pH and glutaraldehyde fixation
Authors: Subjeck J. R. , Weiss L.
Published: February 2005

The densities of cationized ferritin (CF) particles binding to the surfaces of cultured Ehrlich ascites tumor cells were determined at pH 7.4, where the ferritin stain was applied either prior to or following glutaraldehyde fixation. The densities were also determined with CF adjusted to pH 1.9 and applied after fixation. For all fixed samples there was a higher density of particles bound to microvilli than to the spaces between them. Treatment with neuraminidase removed more particles from microvilli than from the intermicrovillus spaces, but did not reduce the levels of binding to the same value.

When cationized ferritin is applied prior to fixation, an aggregation of the CF particles at the cell surface was observed, with the internalization of some clusters. This effect was independent of neuraminidase treatment.
Relationship of energy production to gluconeogenesis in renal cortical tubules
Authors: Suzuki Tadao , De Hartog Maria , Gordon Edwin E.
Published: February 2005

Isolated tubules prepared by collagenase treatment of rat renal cortex retained their ultrastructural integrity and responded to added lactate and succinate with an increase in gluconeogenesis and respiration. Inhibition of the mitochondrial respiratory chain with rotenone, or energy conservation sites with oligomycin caused a marked reduction in respiration and ATP content thereby completely inhibiting net gluconeogenesis. Dissociation of gluconeogenesis from respiration was accomplished with quinolinic acid and hydrazine, inhibitors of gluconeogenesis. At 5 × 10−3 M quinolinic acid, gluconeogenesis from succinate was inhibited approximately 50% and from lactate nearly 100%. This concentration of quinolinic acid did not affect oxygen uptake or the ATP content of tubules in the presence or absence of substrate. Hydrazine at 10−3 M resulted in approximately 75% inhibition of glucose formation from succinate and complete inhibition from lactate without interfering with respiration or ATP content. The increased mitochondrial energy generation, as manifested by accelerated respiration was independent of gluconeogenesis. The unchanging cell ATP concentration with a higher respiratory rate upon addition of exogenous substrate bespeaks increased ATP turnover. ATP utilization for the substrate‐induced enhancement of gluconeogenesis could not account for the increment in ATP hydrolysis.
Growth limitation of 3T3 mouse fibroblasts by available growth surface area and medium components
Authors: Thrash Cornelia R. , Cunningham Dennis D.
Published: February 2005

Studies with untransformed fibroblasts demonstrate that growth of these cells in culture can be limited by the availability of both growth surface and medium components. Experiments using cells grown on coverslips, in which the only variable was available growth surface, indicate that when the medium to cell ratio is high, surface area is the principal factor limiting growth. At low medium to cell ratios, however, growth of cells is predominantly limited by medium components. The final number of cells per culture is almost directly proportional to available surface area when the culture medium is changed daily.
Isolation and studies of the granules of the amebocytes of Limulus polyphemus, the horseshoe crab
Authors: Mürer Erik H. , Levin Jack , Holme Randi
Published: February 2005

Granules were isolated from the cytoplasm of the amebocytes of Limulus polyphemus, the horseshoe crab, by disruption of cells obtained from blood which had been drawn into 2 mM propranolol. The granules subsequently were purified by centrifugation through a sucrose gradient that contained heparin. Extracts of the granules were prepared by freezing and thawing the granule preparations in distilled water.

Transmission and scanning electron microscopy of the granules revealed round or ovoid particles. However, only one type of granule appeared to be present. The ultraviolet spectrum of the extract of amebocyte granules demonstrated a peak at 277 nm at pH 7.4, and a shift into two peaks of 281 nm and 290 nm at alkaline pH. Analytical ultracentrifugation revealed a pattern similar to that observed with lysates prepared from intact amebocytes. Polyacrylamide gel electrophoresis, in the presence of urea at pH 4.5, demonstrated patterns similar to those observed with amebocyte lysate. Extracts of the granules were gelled by bacterial endotoxin.

The blood of the horseshoe crab contains only one type of cell, the amebocyte. Previous studies have shown that the blood coagulation mechanism of Limulus is contained entirely within amebocytes. The current studies suggest that the granules, which pack the cytoplasm of these cells, contain all of the factors required for the coagulation of blood, including the clottable protein. The intracellularly localized coagulation system is released from amebocytes when their granules rupture during cell aggregation.
Chromosome balance and the control of malignancy
Authors: Bloch‐Shtacher Navah , Sachs Leo
Published: February 2005

The Giemsa banding pattern of the chromosomes has been analyzed in a line of transformed golden hamster cells, revertant and re‐revertant cells and their tumors. The transformed and re‐revertant cells were malignant in vivo and had gained an additional chromosome 57. Revertants with a suppression of malignancy lost this additional chromosome 57 and gained an additional chromosome 72. The tumors produced by segregants from the revertant cells were malignant, although to a lower degree than transformed and re‐revertant cells. These tumors had lost the additional chromosome 72 found in revertants and gained one or two 512 chromosomes. The results support the hypothesis that the balance between genes for expression and suppression controls malignancy. The data indicate that chromosome 72 carries genes for suppression and that chromosomes 57 and 512 carry genes for expression of malignancy. The genes on chromosome 57 seem to result in a greater degree of expression than the genes on chromosome 512. The chromosome balance that controlled malignancy in these cells, also controlled the expression and suppression of transformed properties in vitro.
Non‐histone protein synthesis during G1 phase and its relation to dna replication
Authors: Gerner Eugene W. , Meyn Raymond E. , Humphrey Ronald M.
Published: February 2005

The kinetics of non‐histone chromosomal protein (NHCP) synthesis were studied in Chinese hamster ovary (CHO) plateau phase cells stimulated to proliferate and were compared to NHCP synthesis kinetics in two populations of synchronous G1 traversing cells. In all cases, NHCP synthesis rates increase 3‐ to 5‐fold as cells traversed G1 and attained maximum values one hour before semi‐conservative DNA replication began. Similar to results in synchronous G1 cells, the molecular weight distributions of the NHCP fraction from stimulated plateau phase cells underwent only minor changes, measured by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis, as these cells moved toward S phase. Yet, during this progression after plateau phase and in the transition from early G1 to late G1 in synchronous cells, the total NHCP fraction increased significantly (1.5–2‐fold) in amount per cell. These data indicate that plateau phase cells are similar to early G1 cells, both in terms of their amounts of non‐histone per cell and in their subsequent NHCP synthesis kinetics as they move toward S phase. These results extend previous findings which suggested that NHCP synthesis was coupled to DNA replication and demonstrate that the increased NHCP synthesis and accumulation in chromatin may be a biochemical marker for G1 progression.
Degradation of abnormal proteins in heLa cells
Authors: Hendil Klavs B.
Published: February 2005

The experiments show that abnormal proteins are degraded faster than normal ones in HeLa cells. Among the fragmentary proteins made in the presence of puromycin, those with low molecular weight are least stable. Proteins made after incubation with 5‐fluorouracil or in the presence of some amino acid analogues are also unstable. Breakdown of proteins made in the presence or absence of puromycin is nearly unaffected by cycloheximide and is independent of pH between 7 and 8.
Formation of thymidine kinase and deoxycytidylate deaminase in synchronized cultures of chinese hamster cells temperature‐sensitive for DNA synthesis
Authors: Kit Saul , Jorgensen George N.
Published: February 2005

Cytosol thymidine kinase (TK) and deoxycytidylate (dCMP) deaminase formation was investigated in synchronized cultures of K12 Chinese hamster cells which have a temperature‐sensitive lesion affecting the initiation of DNA synthesis. Enzyme formation was found to be cycloheximide‐sensitive and also temperature‐dependent. Beginning at about six hours after addition of medium with 10% calf serum to serum‐depleted K12 cultures, cytosol TK and dCMP deaminase activities increased when the cultures were incubated at 36.5°, but not at 40.5°. When cultures were shifted from 36.5° to 40.5° at 4, 6, or 8 hours after serum addition, TK activity continued to increase, though not to the level observed at ten hours in cultures maintained at 36.5°. Actinomycin D addition at the time of serum reversal or four hours later blocked the TK increase normally observed at the permissive temperature at ten hours. However, when actinomycin D addition was delayed for six or eight hours after serum addition, the increase in TK measured at ten hours resembled that observed in the temperature shift‐up experiments. The results provide evidence that the mutation in K12 Chinese hamster cells most likely blocks the progression through G1 into S and suggest that transcription or post‐transcriptional processing required for TK formation is affected.
Membrane transport in synchronized ehrlich ascites tumor cells: Uptake of amino acids by the A and L system during the cell cycle
Authors: Tupper Joseph T. , Mills Barry , Zorgniotti Flavia
Published: February 2005

Using the double thymidine block technique. Ehrlich ascites tumor cells (ELD) carried in continuous spinner culture have been synchronized. Simultaneous monitoring of 3H‐thymidine incorporation, cell number and mitotic index yielded a cell cycle time of approximately 13.5 hours. This is composed of an S period of 3–4 hours, G2 of 6–8 hours and M of 1–2 hours. No appreciable G1 is present.

Ehrlich cells synchronized in this manner were used to investigate the characteristics of two neutral amino acid transport systems during progression through the cell cycle. Unidirectional influx via the Na‐dependent system A was studied using C14‐alpha‐aminoisobutyrate (AIB) as substrate. The Na‐independent system L was monitored using 3H‐leucine and 14C‐cycloleucine as substrates. Transport by the A system was minimal in M and early S. It underwent a three‐fold increase during late S and early G2. In mid G2 the transport via this system rapidly dropped and remained low again through M and early S. The intracellular/extracellular ratios of AIB indicate that the system is actively transporting AIB throughout the cell cycle. The minimum ratios of approximately 3 were achieved during early M and the maximum ratios of approximately 9 were achieved in late S, early G2. The uptake of leucine and cycloleucine by the L system was quite different during the cell cycle. Maximal unidirectional influx by this system occurred during early and mid S period. Upon progression into G2 the transport rate dropped and remained reduced throughout M. Intracellular/extracellular ratios of leucine or cycloleucine were near unity at the peak of the transport activity (early S) and dropped to values of 0.5 to 0.6 throughout the remainder of the cycle. This result indicates that inward transport by the L system is, for the most part, non‐active in growing cells.
The nature of the cells generating B‐Lymphocyte colonies in vitro
Authors: Metcalf D. , Wilson J. W. , Shortman K. , Miller J. F. A. P. , Stocker J.
Published: February 2005

B‐lymphocyte colonies were grown in semi‐solid agar from mouse spleen or lymph node cells in the presence of mercaptoethanol with or without added sheep red cells. High levels of colony‐forming cells were present in the spleen or normal mice and nu/nu (athymic) mice but colony‐forming cells were rare in the thymus and not detected in activated T‐lymphocyte populations. Colony‐forming cells were θ‐negative and most exhibited Fc receptors. Most colony‐forming cells had the sedimentation velocity of small lymphocytes, were non‐adherent and had a buoyant density similar to B‐lymphocytes. Colony‐forming cells were radiosensitive (Do 60 rads) and sensitive to cortisone. Colony formation was potentiated by the addition of adherent spleen cells or peritoneal macrophages. It is concluded that most cells forming B‐lymphocyte colonies are themselves characterisable as B‐lymphocytes.
Surface interactions between mammalian sperm and egg: Variation of spermatozoa concentration as a probe for the study of binding in vitro
Authors: Hartmann John F. , Hutchison Cameron F.
Published: February 2005

The pre‐penetration binding interactions between gametes of the golden hamster were investigated in vitro. Binding between capacitated spermatozoa and the surface of eggs, that is the zonae pellucidae with intact vitelli, as a function of the concentration of spermatozoa, followed a sigmoidal curve. This was in sharp contrast to the linear binding obtained with mechanically isolated zonae pellucidae (zonae lacking vitelli). Penetration of eggs as a function of the concentration of spermatozoa paralleled the binding curve that occurred between gametes. The binding curve obtained with uncapacitated spermatozoa and eggs was not sigmoidal but was linear after a slight lag and parallel to the curve obtained with uncapacitated spermatozoa and isolated zonae pellucidae. Taken together these results support previous work which implicated a vitelline factor in the binding reaction between the surfaces of eggs and capacitated spermatozoa. By scoring binding at one minute intervals it was possible to relate the rapid uninterrupted binding that occurs between capacitated spermatozoa and isolated zonae pellucidae with the equally rapid but transient and vitellus‐influenced binding that occurs between gametes. It was concluded that the vitelline factor acts by preventing most of the early type of binding that occurs between spermatozoa and isolated zonae pellucidae and not by terminating the early, rapid, initial binding as previously postulated. Thus, this early binding never occurs between most of the gametes that finally bind 30 to 40 minutes later and, therefore, does not play a role in the establishment of the late binding step which leads to penetration.
Increased susceptibility of murine teratocarcinoma cells to simian virus 40 and polyoma virus following treatment with 5‐bromodeoxyuridine
Authors: Speers Wendell C. , Lehman John M.
Published: February 2005

Cultures of the multipotential stem cell, embryonal carcinoma (EC), of a murine teratocarcinoma were treated with 5‐bromodeoxyuridine (BrdU). Within 2–4 days at concentrations of 1–50 m̈gm/ml of BrdU, there was a marked change in the morphology of cells observed by light and electron microscopy. A comparison of the growth potential showed that for up to four days the BrdU‐treated cultures were similar to untreated cultures. When these BrdU‐treated cells were infected with Simian virus 40 (SV40) and polyoma virus (Py), there was an increase in susceptibility of the treated cells. The untreated embryonal carcinoma cells were refractory. These results suggest that BrdU modifies the embryonal carcinoma cells to allow infection with two DNA viruses.
Degradation of abnormal proteins in HeLa cells
Authors: Prouty Walter F.
Published: February 2005

Canavanine, an arginine analog, is incorporated into HeLa cell protein when cells are incubated in the absence of arginine, and this incorporation can result in the production of nonfunctional enzymes or abnormal proteins. The cells degrade these abnormal proteins up to three times more rapidly than normal cell proteins. The capacity for selective degradation of abnormal proteins is not limited to HeLa cells since human fibroblasts also showed increased degradative rates following exposure to canavanine. In addition, enhanced degradation is not a peculiar property of canavanine incorporation since other amino acid analogs also promoted protein degradation. Thus, mammalian cells have the capacity to recognize and selectively degrade abnormal proteins.
Energy metabolism in respiration‐deficient and wild type chinese hamster fibroblasts in culture
Authors: Donnelly M. , Scheffler I. E.
Published: February 2005

This paper presents a comparison of energy metabolism in wild type and respiration‐deficient Chinese hamster cells. From previous work (DeFrancesco et al., 75) it was concluded that the mutant satisfies essentially all of its energy requirements from glycolysis and in this study we measure precisely the amount of glucose consumed and lactate produced per milligram increment of protein in exponentially growing cultures. From these measurements we calculate the amount of ATP derived from glycolysis (and hence the total energy requirement for normal proliferation) to be 105 ± 15 μmoles ATP/δ mg protein in the mutant. It is 63 ± 10 μmoles ATP/δ mg protein derived from glycolysis in wild type cells. We present evidence that the total energy requirement of wild type cells is similar to that of the mutant, suggesting that ∼40% of the energy requirement is derived from respiration. The oxidation of glutamine appears to be more significant than the complete oxidation of glucose to CO2 in these Chinese hamster fibroblasts. The amount of ATP required by the mutant cells per milligram increment of protein is relatively independent of pH.
Density regulation of amino acid transport in cultured, androgen‐responsive tumour cells
Authors: Robinson Jeffery H.
Published: February 2005

In androgen‐responsive cells, activity of a sodium‐dependent transport system for neutral amino acids is shown to be density‐dependent, whereas activity of a sodium‐independent system is not. Transport of the nonmetabolizable amino acid, α‐aminoisobutyric acid (α‐aib) was largely sodium‐dependent, whereas transport of cycloleucine (cyleu) was by both sodium‐dependent and sodium‐independent systems. The Km for cyleu transport changed with density because only the sodium‐dependent portion of this transport was density‐dependent. By contrast, the Vmax for α‐aib transport changed with density, indicating that either the amount of functional membrane carrier or the activity of the sodium pump was affected.
Reversible independent alterations in glucose transport and metabolism in cultured human cells deprived of glucose
Authors: Salter Donald W. , Cook John S.
Published: February 2005

We have measured uptake of 3H‐hexoses into diploid human cells by exposing them to brief pulses of isotopic sugar during the log‐growth, subconfluent‐growth, and confluent‐growth (contact inhibited) phases of the strain HSWP derived from human skin. 3H‐deoxyglucose appears to be taken up three times faster than 3H‐glucose. After exposure to 3H‐glucose for longer than one minute, the cells excrete ∼70% of the isotope into the medium as lactate. If lactate production (and hence excretion) is abolished by treating the cells with iodoacetic acid or dinitrofluorobenzene, neither of which inhibits transport, the uptake of 3H‐glucose is found to be in fact somewhat larger than that of 3H‐deoxyglucose. If cells are deprived of glucose for 24 hours, apparent uptake of 3H‐glucose is enhanced 10‐fold or more. This latter increase is accounted for by 2‐ to 3‐fold enhancement of true transport plus retention of > 90% of the radioactivity, since little lactate is formed or excreted in glucose‐deprived cells. Deoxyglucose, galactose, or pyruvate when present during glucose deprivation each have quantitatively different effects on the cells' capacity to produce lactate from a short pulse of glucose, but none of them prevents the enhancement of hexose transport. After restoration of 5 mM glucose to starved cells, their metabolism returns to normal (in the sense that ∼70% of the glucose taken up in a pulse is again excreted as lactate), with a half‐time of 0.5 hour; but the transport of hexoses returns to control levels much more slowly, with a half‐time of ∼6 hours. The two processes appear to be independently regulated.
Genetic control of lipopolysaccharide induced generation of serum colony stimulating factor and proliferation of splenic granulocyte/macrophage precursor cells
Authors: Apte Ron N. , Pluznik Dov H.
Published: February 2005

Administration of bacterial lipopolysaccharides (LPS) to mice causes a rise in tissue and serum colony stimulating factor (CSF) levels and in bone marrow and splenic colony forming cells (CFC). Two inbred strains of mice differing in their response to LPS were used to study the genetic control of LPS induced granulopoietic responses: a high responder strain (C3H/eB) which reacts to LPS by an elevation in serum CSF and by an increase in splenic CFC levels, and a low responder strain (C3H/HeJ) which fails to show these responses. The ability to generate serum CSF after administration of LPS is controlled by a single autosomal dominant gene, while the splenic CFC response to LPS follows the characteristic patterns of a polygenic inheritance control. The associated relationships of CSF and CFC responsiveness have been investigated in backcross (F1 x C3H/HeJ) and F2 mice. Most mice which generated high levels of CSF showed a high or intermediate CFC response and most mice which did not generate any detectable levels of serum CSF showed a low splenic CFC response. The results suggest that CSF may play a physiologic role in vivo as a granulopoietin. In addition it was shown that the genetic control mechanisms governing the CSF/CFC responses are determined by the lipid A‐KDO portion of the LPS molecule, suggesting that lipid A is the active part of the LPS molecule in stimulating granulopoiesis.
Adaptive enhancement of amino acid uptake and exodus by thymic lymphocytes: Influence of ph
Authors: Peck William A. , Rockwell Linda H. , Lichtman Marshall A.
Published: February 2005

Entry of certain free amino acids (alpha aminoisobutyric acid (AIB), alanine and proline), but not of leucine into rat thymic lymphocytes increased progressively when the cells were incubated in amino acid deficient medium. Actinomycin D, cycloheximide, or a high concentration of AIB abolished the time‐related increase in AIB accumulation, whereas exposure to a high concentration of leucine had no effect. This phenomenon could not be attributed to a progressive alteration in the nature of the incubation medium nor to reduced transinhibition of AIB uptake. The exodus of AIB also increased with time, but to a smaller degree than AIB entry.

Initial rates of AIB entry and exodus increased with increases in the pH of the incubation medium over the range 6.5–8.0. The effects of pH on entry and exodus were time‐related, increasing progressively over a 180‐minute period. Actinomycin D, cycloheximide, and 5 mM AIB nullified the magnified time related increments in AIB transport caused by prolonged incubation at pH 8.0. The influence of a given pH on transport of AIB decreased rapidly when the cells were transferred to medium of another pH, but this tendency diminished the longer the cells were exposed to the initial pH. pH influenced the entry of alanine and proline in the same fashion as that of AIB, but did not affect leucine entry.

These results indicate that thymic lymphocytes exhibit adaptive enhancement in the accumulation of free amino acids that are transported largely by the A or alanine‐preferring system, and that the adaptive process involves both entry and exodus. Moreover, alterations in pH modify entry and exodus of these same amino acids, profoundly affect the magnitude of time‐released increases, and may induce fundamental changes in the mechanism(s) serving amino acid transport.
Human erythroid colony formation in vitro: Evidence for clonal origin
Authors: Prchal Jaroslav F. , Adamson John W. , Steinmann Laura , Fialkow Philip J.
Published: February 2005

Human marrow cells, suspended in methylcellulose medium containing erythropoietin, give rise to discrete colonies of hemoglobin synthesizing cells. The presumption that such colonies originate from single progenitor cells has been tested directly in females with X‐chromosome inactivation mosaicism using glucose‐6‐phosphate dehydrogenase (G‐6‐PD) as a marker. When individual colonies were grown from marrow cells obtained from two black females heterozygous for G‐6‐PD, only one or the other isoenzyme type was observed, but not both. These results are most consistent with the interpretation that human erythroid colonies arise from single cells.
Nucleoside transport systems in Escherichia coli K12: Specificty and regulation
Authors: Munch‐Petersen Agnete , Mygind Benete
Published: February 2005

Two nucleoside transport systems have been verified and separated by mating and recombination experiments. The recipient strain was a mutant which is negative for transport of all nucleosides.

The two systems differ in specificity and in regulation. One system transports pyrimidine and adenine in specificity and in regulation. One system transports pyrimidine and adenine nucleosides, but not guanine nucleosides. It is regulated by the cytR gene. The other system transports all nucleosides and is regulated by the cytR as well as by the deoR genes.

Enzyme assays performed on whole cells of strains, able or unable to transport nucleosides, indicate that the nucleoside catabolizing enzymes are located inside the permeability barrier of the cell.
Stimulation of sugar uptake in cultured fibroblasts by epidermal growth factor (EGF) and EGF‐binding arginine esterase
Authors: Barnes David , Colowick Sidney P.
Published: February 2005

Mouse epidermal growth factor causes a rapid increase in 2‐deoxyglucose uptake in stationary phase mouse (3T3) cells or human fibroblasts. Maximum effect is approximately two fold over control levels for 3T3 cells and about 50% over controls for human fibroblasts. Maximum effect on 3T3 cells is seen about two hours after addition of 10 ng/ml EGF to the culture medium. Stimulation is easily measurable within the first fifteen minutes after addition of the hormone and may be detected at hormone concentrations as low as 0.1 ng/ml. The EGF‐binding arginine esterase found associated with EGF in the mouse submaxillary gland causes an enhancement of the EGF effect. In serum‐free medium, the EGF effect is still readily observed, but no enhancement by the esterase is seen. SV40 virus‐transformed 3T3 cells show no effect on deoxy‐glucose uptake after addition of 10 ng/ml EGF to the culture medium, but a response may be demonstrated after these cells are incubated for 12 hours or more in serumless medium. EGF stimulates transport of 3‐0‐methylglucose in stationary phase 3T3 and human fibroblasts but no EGF stimulation of α‐amino‐isobutyrate uptake in 3T3 cells is seen under conditions in which EGF clearly stimulates sugar uptake. Ammonium chloride, which is reported to inhibit intracellular degradation of human EGF by human fibroblasts, does not diminish the EGF effect on deoxyglucose uptake in human fibroblasts.
Transport of D‐allose by isolated fat‐cells: An effect of adenosine triphosphate on insulin stimulated transport
Authors: Loten E. G. , Regen D. M. , Park C. R.
Published: February 2005

D‐allose, a glucose analogue, is not metabolized by isolated fatcells and its distribution space at equilibrium in the cells is the same as that of tritiated water. Uptake of allose is inhibited by glucose and 3‐0‐methylglucose, stimulated by insulin and virtually eliminated by cytochalasin B. Counter transport of allose out of fat‐cells against a concentration gradient can be induced by exogenous glucose but not by pyruvate. It is concluded that allose is transported into fat‐cells by the same carrier mediated transport system as glucose and that it is a suitable analogue with which to study the glucose transport system. Insulin stimulated allose transport, into or out of the cell, but not basal transport, is inhibited by a brief exposure of isolated fat‐cells to exogenous ATP or ADP (but not AMP or AMP‐PNP). The antilipolytic effect of insulin is not affected. The ATP inhibition is slowly reversible.

It is suggested that ATP phosphorylates a membrane component and thereby blocks transmission of signal from the insulin receptor to the carrier system. Indirect evidence suggests that ATP does not alter the affinity of the insulin or glucose binding sites.

Insulin decreases the Km of glucose metabolism to CO2 and lipid in isolated fat‐cells and increases the Vmax. However, the hormone has no effect on the Ki of glucose as an inhibitor of allose transport. The glucose analogue, 3‐0‐methylglucose, also inhibits both glucose metabolism and allose transport. The Ki for both these processes is similar and is not affected by insulin. These results support the view that the effect of insulin on glucose transport is to raise the Vmax without a change in the Km. It appears further that sugar transport is not the major rate limiting step in metabolism at high glucose concentrations in the absence of insulin, or at most glucose concentrations in the presence of the hormone.