bluuebunny/crossref_metadata_2025 huggingface dataset:
The inhibition by 6‐diazo‐5‐oxo‐L‐norleucine of glutamine catabolism of the cultured human lymphoblast
Authors: Willis Randall C. , Edwin Seegmiller J.
Published: February 2005

The rapid catabolism of glutamine by the cultured human lymphoblast line WI‐L2 can be inhibited greater than 95% by incubation of cell suspensions with 6‐diazo‐5‐oxo‐L‐norleucine (DON). The inhibition persists for at least four hours after removal of DON from the cell suspension. The exposure of cells to DON inhibits over 95% of the glutaminase activity measured in lysates in the presence of either phosphate or maleate. Similarly, γ‐glutamyl transpeptidase, assayed with γ‐glutamyl‐p‐nitroanilide as substrate and glycylglycine as acceptor, is inhibited over 90%. DON‐treated and control cells accumulated radioactive material from suspensions containing [14C]‐L‐glutamine at similar initial rates; the radioactive material accumulated by the DON‐treated cells is all recoverable as glutamine while the radioactive material accumulated by untreated cells is principally recovered as glutamate.
The effect of histamine on the growth of cultured fibroblasts isolated from normal and keloid tissue
Authors: Russell James D. , Russell Shirley B. , Trupin Kathryn M.
Published: February 2005

Cultured fibroblasts derived from human keloid tissue are presented as a possible model system for studying the genetic regulation of cell growth. Histamine is shown to have a marked effect on the growth of cultured fibroblasts. A small increase in growth rate is seen during the log phase of the culture cycle and a 50% increase in cell number is observed during the plateau phase. Differences in the extent of growth stimulation are observed between strains isolated from different individuals. While most strains showed approximately 50% stimulation, a few were not stimulated and some strains gave a 100% or greater increase in cell number due to histamine. This phenotypic difference in extent of growth stimulation in response to histamine cannot be attributed to the gene or genes for keloid formation. However, elevated levels of histamine in vivo may be a contributing factor to the abnormal cell growth observed in this disorder. The extent of growth stimulation due to histamine decreases with repeated subculturing.
Effects of ph and type of sugar in the medium on tyrosinase activity in cultured melanoma cells
Authors: Saeki Hisaaki , Oikawa Atsushi
Published: February 2005

The tyrosinase (EC 1.14.18.1) activity of cultured mouse melanoma cells B16 in the stationary phase of growth, depends greatly on the pH of the medium and the kind of sugar present.

The enzyme activity of a homogenate of cells grown at pH 7.2 in Eagles's MEM supplemented with 10% new born calf serum and containing galactose in place of glucose, was about ten times that of a homogenate of cells cultured at pH 6.3 in the same medium. The tyrosinase activity changed reversibily on changing the pH of the culture medium.

When cultured at a constant pH of 7.2, cells grown with 1 mM galactose had about five times higher tyrosinase activity than cells grown with 1 mM glucose. Only a small amount of lactate accumulated in cultures with glucose and it had little effect on the enzyme activity.

These two findings explain the very low tyrosinase activity of cells cultured in medium with 5 mM glucose: the low activity is due to the presence of glucose and to the low pH resulting from conversion of glucose to lactic acid.
Regulation of protein synthesis in human diploid fibroblasts: Reduced initiation efficiency in resting cultures
Authors: Meedel Thomas H. , Levine Elliot M.
Published: February 2005

The level of poly A+ RNA in growing cultures of human diploid fibroblasts is 1.8‐fold times greater than in resting cultures. The level of functional ribosomes in growing cultures is 2.8 times that in resting cultures. Since transit times are similar in both types of cells, it can be concluded that the rate of protein synthesis in growing cultures is 2.8 times that in resting cultures. A reduced efficiency of mRNA translation at the level of initiation in resting cultures is proposed as a probable explanation for the fact that the decrease in protein synthesis rates is greater than the decrease in mRNA levels. This hypothesis is supported by the observations that: (a) poly A+ RNA is associated with smaller polysomes in resting than in growing cells, and (b) cycloheximide treatment of resting cells results in recruitment of nonpolysomal poly A+ RNA into polysomes and a shift of polysomal poly A+ RNA into larger polysomes.
Changes in membrane dynamics associated with myogenic cell fusion
Authors: Herman B. A. , Fernandez S. M.
Published: February 2005

Changes in membrane dynamic properties associated with membrane fusion are studied employing in vitro myoblast fusion as a model system. We utilize a microscopic fluorescence relaxation approach which makes feasible the study of local variations in membrane dynamics within surface subdomains of single intact cells. Studies of the average rotational mobility of the fluorescent probe‐1‐anilino‐naphthalene‐8‐sulfonate by this technique indicate that myoblast fusion activity is preceded by a generalized increased in membrane fluidity and that areas of cell contact between fusing cells exhibit higher fluidity and polarity, locally, than non‐fusion regions.
Density‐dependent effects of oxygen on the growth of mammalian fibroblasts in culture
Authors: Taylor William G. , Camalier Richard F. , Sanford Katherine K.
Published: February 2005

Various concentrations of oxygen were used to determine the optimum culture medium P for survival and proliferation of attached human and mouse fibroblasts grown from different inoculum sizes. When T−15 flasks were seeded with ⪕ 2 × 104 cells (⪕ 1.3 × 103 cells/cm2), the highest plating efficiencies and cell yields were obtained with a culture medium P of 40—60 mm Hg. At higher inoculum sizes (105 cells per T‐15) used routinely for mass cultures, no difference in cell yield or glycolytic activity was observed between cultures gassed with atmospheric, i.e., 18% O2 (growth medium P ≅ 125—135 mm Hg) and those gassed with 1% O2 (growth medium P ≅ 40—60 mm Hg). The enhanced clonal growth observed at the latter P results from an increased proliferation rate rather than more efficient attachment and survival of inoculated cells. Glucose uptake and lactic acid accumulation were increased in sparse cultures sparged with 1% O2. A slight extension of lifespan was observed in WI−38 cells serially subcultured with a gas phase of 1% O2.
The transport of L‐alanine by the hamster kidney cell line BHK21‐C13
Authors: Scott D. M. , Pateman J. A.
Published: February 2005

The uptake of L‐alanine into BHK21‐C13 cells in culture has been studied. This amino acid appears to be transported essentially via a relatively low affinity, high capacity, sodium ion dependent transport system. Inhibition studies using other amino acids or their analogues provided information about the specificity of this system. This alanine transport system was shown to exhibit a broad substrate specificity and appeared to be capable of transporting most naturally occurring neutral α‐amino acids

Kinetic studies of the inhibition of L‐alanine uptake also indicated the presence of a second neutral amino acid transport system capable of transporting this amino acid. However, it is unlikely that this second uptake system contributes greatly to L‐alanine uptake. Inhibition of the uptake of L‐leucine indicated that this transport system has a similar specificity to the "L"‐system initially described for Ehrlich ascites carcinoma cells.
Malignant mouse melanoma cells do not form tumors when mixed with cells of a non‐malignant subclone: Relationships between plasminogen activator expression by the tumor cells and the host's immune response
Authors: Newcomb Elizabeth W. , Silverstein Samuel C. , Silagi Selma
Published: February 2005

Mouse melanoma clones B559 and B78 are highly tumorigenic when injected into C57BL/6J mice. Tumor formation by these cells is suppressed when they are mixed with nonmalignant bromodeoxyuridine‐grown clone C3471 before injection. C3471 cells suppress tumor formation only in immunocompetent hosts; mixtures of B559 and C3471 cells or C3471 cells alone form tumors in antithymocyte serum (ATS)‐treated mice. Explants of C3471 tumors grown in ATS‐treated mice form tumors in immunocompetent mice, most of which regress. Inability of C3471 or mixtures of C3471 with malignant cells to grow in normal mice, as contrasted with ability to grow in immunosuppressed mice, indicates that host response is involved. Both tumorigenic clones have high plasminogen activator activity, whereas nontumorigenic clone C3471 has none. Mixture of either tumorigenic clone with C3471 cells decreases plasminogen activator in vitro. C3471 tumor explants from ATS‐treated mice initially express plasminogen activator, but lose the capacity to express this activity upon prolonged cultivation in vitro. Explants from B559 tumors retain plasminogen activator in long term culture. Close physical contact between C3471 and B559 cells appears essential both for inhibition of plasminogen activator expression by B559 cells in vitro, and for tumor suppression in vivo. These findings suggest that production of plasminogen activators by tumor cells may play an important role in suppressing the host's immune response locally to an inoculum of syngeneic tumor cells.
2‐Amino‐isobutyric acid and 3‐0‐methyl‐D‐glucose transport in 3T3, SV40‐transformed 3T3 and revertant cell lines
Authors: Dubrow Robert , Pardee Arthur B. , Pollack Robert
Published: February 2005

In order to further investigate the connection between transport and growth control, 3T3 cells, SV40 transformed 3T3 cells (SV101), and three revertant cell lines derived from SV101 which have regained certain manifestations of growth control were used. Transport rates of 2‐aminoisobutyric acid and 3‐0‐methyl‐D‐glucose were measured in sparse, confluent, serum‐starved, and serum‐stimulated cultures.

As shown before, cessation of 3T3 cell growth in G0 under conditions of confluence or serum deprivation was associated with reduced rates of transport for both compounds, whereas the density and serum dependence of growth and transport was largely eliminated in SV101. The density revertant F1SV101, which has regained density regulation of growth similar to 3T3 cells, has also regained density regulation of transport. Neither growth nor transport were serum dependent. The serum revertants AγSV7 and LsSV6 have regained both density and serum regulation of growth, but not according to the original mechanism of 3T3 cells of entry into a G0 state. Transport was high under conditions of confluence or serum deprivation. Thus for these cells rates of transport were not reduced simply as a consequence of slower cell growth nor were low transport rates responsible for growth arrest. The data are consistent with the possibility that growth arrest specifically in the G0 state could shut off a number of cellular activities, including transport.
Further observations on metabolic responses in hamster cells: Changes in udpg dehydrogenase activity
Authors: Ullrey Donna B. , Kalckar Herman M. , Christopher C. William
Published: February 2005

Administration of radioactively labeled galactose to cultured mammalian cells brings about an accumulation of metabolic products the pattern of which seems to be governed by a variety of vectors in the intracellular milieu. By manipulation of culture conditions some of these vectors appear to be a function of glycolysis. In the non‐glycolytic culture, label from a galactose probe appears as Galactose‐1‐phosphate (Gal‐1‐P) and UDPglucuronic acid (UDPGlcUA). Conversely, glycolytic culture conditions seem not to permit the formation to UDPGlcUA since the only labeled accumulation product formed was UDPHex. A suggestion is made that the difference in metabolic activity of glucose‐fed and glucose‐starved cultures may be related to the effect of NADH on the in situ activity of UDPG dehydrogenase (UDPglucose:NAD oxidoreductase, E.C. 1.1.1.22) (abbreviation, UDPG‐DH). This prompted an investigation of the effects of NAD and NADH on the activity of partially purified UDPG‐DH. The results of these experiments strongly suggest that the activity of UDPG‐DH (in situ) is negatively controlled by increased levels of NADH; the latter condition is known to exist in glycolytically active cells (Schwartz and Johnson, 1976). Added to this is a second control mechanism which is characterized by a transient inhibition of uridylyltransferase (UDP glucose:α‐D‐galactose‐1‐phosphate uridylyltransferase, E.C. 2.7.7.12). Since it is known that there is little, if any, effect on galactokinase (ATP:D‐galactose‐1‐phosphotransferase, E.C. 2.7.1.6) activity as a result of sugar starvation (Christopher et al., 1976), the low in vivo activity of uridylyltransferase contributes not only to the increased accumulation of Gal‐1‐P but also to a drastic decrease of labeled UDPhexoses, although the pre‐existing pool of UDPhexose was found to decrease only moderately under the condition of glucose starvation (30% still persisted). The benefit of this type of control is not clear.
Lack of a correlation between polyamine synthesis and DNA synthesis by cultured rat liver cells and fibroblasts
Authors: Rupniak H. T. , Paul D.
Published: February 2005

Growth stimulation of either fetal rat liver cells or rat embryo fibroblasts in culture results in considerable increases in intracellular polyamine levels as cells proceed through the cell cycle. Treatment of such cell cultures with appropriate levels of two inhibitors of polyamine synthesis, namely α‐hydrazino ornithine and methylglyoxal bis(guanylhydrazone), can essentially completely block these increases in cellular polyamine content. Under such conditions, where the elevation in intracellular polyamine content is prevented, cell cultures are nevertheless able to initiate DNA synthesis and subsequently synthesize DNA at rates comparable to untreated control cultures that have been growth‐stimulated. These two cell types therefore contain sufficient polyamines when in a resting state (G1) to enable them to enter from G1 into S phase and traverse S phase at normal rates in the absence of further polyamine synthesis. The recruitment of cells into the first cell cycle, through serum stimulation of growth, therefore appears not to be mediated or regulated by the increases in intracellular levels of polyamines that occurs under these conditions. Conversely, the arrest of growth of these cell types resulting from serum deprivation is not mediated by a limitation of intracellular polyamine content.
Membrane bound and cellular cationic changes associated with insulin stimulation of cultured cells
Authors: Sanui Hisashi , Rubin A. Harry
Published: February 2005

Insulin was employed as a stimulant in our continuing investigations of the molecular mechanisms involved in the coordinate control of cellular metabolism and growth. Incubation of chicken embryo fibroblasts for 16 hours in media containing 0–0.1 U insulin/ml resulted in a 17‐fold increase in the rate of 3H‐thymidine incorporation into DNA. Concomitantly, there were graded increases in intracellular K+ (14%) and Mg2+ (22%) and no significant change in Ca2+. These changes in cation content occurred within 10 to 30 minutes and preceded the changes in 3H‐thymidine incorporation. Insulin produced a consistent graded decrease in externally bound Mg2+ and Ca2+ and a concomitant increase in bound Na+ and K+ with no significant change in the rates of K+ and Mg2+ efflux. The results are consistent with the concept of Mg2+ as a second messenger for insulin action, as well as with the more general hypothesis that Mg2+ is the central agent in the coordinate control of metabolism and growth in animal cells.
Erythrocyte membrane antigen expression during friend cell differentiation: Analysis of two non‐inducible variants
Authors: MacDonald Marcy E. , Letarte Michelle , Bernstein Alan
Published: February 2005

Erythrocyte membrane antigens have been detected on induced Friend erythroleukemic cells with a rabbit antiserum raised against mouse erythrocyte membranes. The antibody specificities of this antiserum have been quantitatively analyzed using a cellular radioimmunoassay. After absorption with thymocytes, the rabbit anti‐erythrocyte membrane serum bound to dimethylsulfoxide (DMSO)‐induced Friend erythroleukemic cells and to mouse erythrocytes but not to uninduced Friend cells or thymocytes. Reciprocal inhibition studies demonstrated that, following complete thymocyte absorption, the antiserum detected similar antigenic specificities, termed erythrocyte membrane antigens (EMA), on both mature erythrocytes and induced Friend cells.

The expression of these erythrocyte membrane antigens was also induced on Friend cells by other agents, such as ouabain and dimethylacetamide (DMA). In contrast, exogenous hematin, which did not induce hemoglobin synthesis in the Friend cell clones used in this study, also did not induce erythrocyte membrane antigen expression.

Two independently derived variant clones which do not produce hemoglobin in reponse to DMSO were analyzed for their ability to produce erythrocyte membrane antigens in response to various inducers of Friend cell differentiation. Clone TG‐13 is not inducible by DMSO or hematin but is weakly induced by DMA for both hemoglobin production and erythrocyte membrane antigen expression. Another variant clone, M18, was also analyzed. This clone does not synthesize detectable hemoglobin when grown in either DMSO or hematin alone, but undergoes extensive hemoglobin synthesis when grown in medium containing both DMSO and hematin. M18 does, however, express erythrocyte membrane antigens when grown in DMSO alone: the presence of hematin and DMSO together in the growth medium does not enhance expression of these antigens. Thus M18 appears to be defective for hemoglobin inducibility, and this defect can be overcome by exogenous hematin; however, the expression of erythrocyte membrane antigens is not affected by this block in hemoglobin synthesis. The results with the variant clones are discussed in terms of a program for Friend cell differentiation in which the induction of hemoglobin synthesis and erythrocyte membrane antigen expression are under both co‐ordinate and separate controls.
Genetic characterization of hydroxyurea‐resistance in chinese hamster ovary cells
Authors: Lewis William H. , Wright Jim A.
Published: February 2005

Hydroxyurea is an excellent selective agent for obtaining drug‐resistant mutants. At a frequency of approximately 1 × 10−5 it was possible to select, in a single step, colonies that exhibited significant resistance to the cytotoxic effects of the drug. These hydroxyurea‐resistant cell lines maintained their resistant phenotype after extensive cultivation in the absence of the drug. Reconstruction experiments indicated that the expression of hydroxyurea‐resistance and the frequency of drug‐resistant colonies was independent of cell densities up to 5 × 105 cells per 100‐mm selection plate. Luria‐Delbrück fluctuation analyses indicated that the appearance of hydroxyurea‐resistant cells in wild type populations occurred spontaneously and at a rate of 4.8 × 10−6 per cell per generation in the presence of 0.33 mM drug. Studies with the mutagen, ethyl methane sulfonate indicated that it was capable of increasing the frequency of hydroxyurea‐resistant cells by a factor of approximately 10. Also, cell‐cell hybridization experiments showed that hydroxyurea‐resistance behaves as a dominant or codominant trait and that hydroxyurea‐resistance was a useful new genetic marker for selection of somatic cell hybrids. Furthermore, similar to many other drug‐resistant cell lines hydroxyurea‐resistant cells were found to exhibit an altered sensitivity to a number of non‐selective agents (guanazole, N‐carbamoyloxyurea, formamidoxime, and hydroxyurethane). Except for guanazole these compounds are structurally very similar to hydroxyurea and may be expected to have similar modes of action. The results presented in this paper support the view that hydroxyurea‐resistance is expressed as a normal genetic trait and is a useful genetic marker for somatic cell genetic studies.
Cell cycle dependence of the reactivation of chick erythrocyte nuclei after transplantation into mouse L929 cell cytoplasts
Authors: Lipsich Leah Ann , Lucas Joseph J. , Kates Joseph R.
Published: February 2005

Monolayer cultures of cytoplasts were formed from synchronized mouse L929 cells in the G1, S and G2 phases of the cell cycle by cytochalasin‐induced enucleation. Chick embryo erythrocytes were then fused to the cytoplasts using Sendai virus. Extensive reactivation of the dormant erythrocyte nuclei‐determined by nuclear swelling, nucleoli formation and incorporation of 3H‐uridine into RNA‐was observed upon fusion to the three types of cytoplasts. By all of these criteria, reactivation was greatest when nuclei were introduced into G1 cytoplasts.

Similar experiments in which DNA synthesis was measured showed that both G1 and G2 cytoplasts almost completely lacked the ability to induce DNA synthesis in the chick nuclei. But at least 26% of the erythrocyte nuclei within S cytoplasts exhibited significant levels of DNA synthesis. Cytoplasts prepared from cells three hours after entry into the S phase, which is six to seven hours in length in L929 cells, possessed the maximum capacity to induce DNA replication in chick nuclei. As many as 60% of the nuclei introduced into 3‐hour S phase cytoplasts synthesized significant quantities of DNA.

The effect of cycloheximide, an inhibitor of protein synthesis, on erythrocyte nuclear reactivation within G1 phase cytoplasts was also investigated. Protein synthesis was necessary to maximize the reactivation of erythrocyte nuclei. However, it appeared that protein synthesis occurring prior to enucleation was more important in the reactivation process than synthesis occurring following enucleation.
Differential effects of hydrocortisone on both growth and collagen metabolism of human fibroblasts from normal and keloid tissue
Authors: Russell James D. , Russell Shirley B. , Trupin Kathryn M.
Published: February 2005

Cultured fibroblasts isolated from normal and keloid tissue do not differ in their growth characteristics or in the rate of collagen synthesis under routine culture conditions. The addition of hydrocortisone to the culture media results in significant differences in both growth and collagen synthesis between these cell types. Collagen syntehsis is inhibited 60% in normal cultures by hydrocortisone (0.5 μg/ml) and the population size at which density‐dependent growth inhibition is achieved is increased. Keloid‐derived fibroblasts grow to a lower maximum density in the presence of hydrocortisone, while their rate of collagen syntehsis is not significantly reduced. The rate of non‐collagen protein synthesis is increased significantly by hydrocortisone in both cell types.

Comparison of normal and keloid‐derived cultures obtained from a single individual suggests that the keloid phenotype with respect to both growth and collagen synthesis is restricted to the fibroblasts isolated from the keloid nodule.
Osmotic properties of ehrlich ascites tumor cells during the cell cycle
Authors: Dupre Ann M. , Hempling H. G.
Published: February 2005

Ehrlich ascites tumor cells were grown and maintained in continuous spinner culture. The population of dividing cells was synchronized by a double thymidine block technique. Cell cycle phases were determined graphically by plotting mitotic index, cell number, and DNA synthesis against time. Changes in the osmotic properties of Ehrlich ascites tumor cells during the cell cycle are described. Permeability to water is highest at the initiation of S and progressively decreases to its lowest value just after mitosis. Heats of activation for water permeability vary during the cell cycle, ranging from 9–14 kcal/mole. Results may imply changes in the state of water in the membrane during the cycle. The volume of osmotically active cell water is highest during S and early G2 and decreases during the mitotic phase, as cells undergo division. Total water content remains stable at 82% (w/w) during the cycle. Total concentration of the three major ions (Na, K, Cl), expressed as mEq/liter total cell volume, does not change. The fraction of total cell water which is osmotically active (Ponder's R) decreased gradually from 0.75 at S to about 0.56 following mitosis. Findings suggest that a fraction of the total water within the cell exists in a “bound” form and is, therefore, incapable of being shifted under the driving force of osmotic pressure. This fraction of bound water increases during the cell cycle. Possible alterations in membrane fluidity and the state of water in the cell are discussed.
Regulation of dihydrofolate reductase gene expression in mouse fibroblasts during the transition from the resting to growing state
Authors: Johnson Lee F. , Fuhrman Carolyn L. , Wiedemann Leanne M.
Published: February 2005

The rate of accumulation of dihydrofolate reductase (DHFR) was studied in resting, growing and serum stimulated mouse 3T6 fibroblasts by first exposing the cells briefly to 10−6 M methotrexate (MTX) to inactivate specifically and irreversibly the pre‐existing enzyme, then determining the rate of recovery of reductase activity after removal of MTX. DHFR activity was quantitated by measuring the ability of a cell extract to reduce 3H‐folic acid or to bind 3H‐MTX. In all cases, recovery of enzyme activity was inhibited by cyclo‐heximide, indicating that the recovery was due to de novo synthesis of reductase.

We found that the rate of accumulation of DHFR was high in exponentially growing cells, as expected, but about 40‐fold lower in resting (G0) 3T6 cells. When resting 3T6 cells were induced to re‐enter the cell cycle following serum stimulation, we found that the rate of accumulation of DHFR increased sharply about ten hours after serum stimulation. DNA replication also began at this time. When resting cells were serum stimulated in the presence of inhibitors of DNA synthesis (hydroxyurea or cytosine arabinoside), the increase in DHFR synthesis was the same as in control stimulated cells. This indicates a lack of tight coupling between DNA synthesis and reductase gene expression. The increase in DHFR accumulation was inhibited by Actinomycin D (5 μg/ml) if the drug was added 7.5 hours after stimulation, but was not inhibited if the drug was added 15 hours after stimulation. This is consistent with the idea that DHFR gene expression is regulated at the level of transcription, and that reductase mRNA is transcribed only between 7.5 and 15 hours following stimulation.
Increased ouabain‐sensitive glycolysis of lymphocytes treated with phytohemagglutinin: Relationship to potassium transport
Authors: Segel George B. , Androphy Elliot J. , Lichtman Marshall A.
Published: February 2005

An early increase in lymphocyte plasma membrane K+ transport is essential for PHA stimulated lymphocytes to divide. Little is known about the specific source and amount of energy required to support the increased transport by activated lymphocytes. Since ouabain, a cardiac glycoside, specifically inhibits the transport ATPase, we have measured the decrement in glycolysis and tricarboxylic acid cycle activity when untreated and PHA treated lymphocytes were exposed to ouabain. This metabolic decrement represents the portion of metabolism associated with monovalent cation transport and closely related processes. Since TCA cycle activity accounted for only 0.2% of glucose consumption, aerobic glycolysis was the major source of energy, i.e., ATP, for increased transport. Approximately one‐third of the total lactate production in both control and PHA stimulated lymphocytes was ouabain‐sensitive. Ouabain sensitive lactate production in control, 105 μmol/1010 cells/hour, increased 1.8‐fold to 193 μmol/1010 cells/hour after PHA treatment. Active K+ influx in similar cell populations increased from 40 μmol/1010 cells/hour to 74 μmol/1010 cells/hour (1.9‐fold) after PHA treatment. The increment in ouabain‐sensitive energy production and K+ transport were closely correlated and, therefore, 0.38 moles of K+ are transported for each mole of ATP generated in both control and PHA treated cells. The increased requirement for transport related energy is provided by increasing the ouabain‐sensitive ATP production rather than altering the efficiency of ATP transduction.
Coordinate control of balb/c3T3 cell survival and multiplication by serum or calcium pyrophosphate complexes
Authors: Rubin A. Harry , Bowen‐Pope Daniel F.
Published: February 2005

Balb/c3T3 cells in crowded cultures detach from the dish when deprived of serum, and the survivors incorporate 3H‐thymidine at a reduced rate. The detachment becomes pronounced two hours after removal of serum, and reaches its maximum rate between two and four hours. Cells in sparse culture are not detached by serum removal, and their rate of 3H‐thymidine incorporation is only slightly reduced. As the sparse cultures grow into more crowded cultures, and the serum is depleted, increasing numbers detach. The detached cells are incapable of reattaching when placed in a new dish with ample fresh serum. The cells are leaky to cellular constituents and appear to be dead. Detachment is a consequence rather than the cause of cell death, and can be produced by agents which inhibit cellular energy metabolism. The cells on the dish which survive serum deprivation are fully viable and grow rapidly when serum is added. When they become crowded they are as sensitive to serum deprivation as was the original population. They are therefore not selected for a low serum requirement but apparently survive because they spread into the space vacated by the detaching cells and then behave as sparse cultures in response to serum variations. Insoluble complexes of Ca2+ and pyrophosphate (Ca2+‐PPi) show the same concentration dependence in promoting cell survival as in stimulating 3H‐thymidine incorporation, showing that a single substance can be responsible for both activities. It is concluded that survival and growth are part of the coordinate response of 3T3 cells to single external effectors. The results are discussed in terms of a simple model in which the coordinate response is regulated by the availability of Mg2+ for transphosphorylation reactions within the cell, and the availability depends on the binding affinity of cellular membranes for Mg2+. The difference between survival and multiplication is postulated to be in the intensity and duration rather than the kind of stimulus.
Potassium‐sodium distribution in human lymphocytes: Description by the association‐induction hypothesis
Authors: Negendank William , Shaller Calvin
Published: February 2005

Human lymphocytes were equilibrated for 48 hours over a wide range of external potassium levels, and their contents of potassium, sodium, and water determined. As external potassium rose from zero, cell potassium rose steeply in a sigmoidal fashion, reached half‐saturation at 0.4 mM external potassium, and then saturated at 129 mmoles/kg cells. The saturable cell potassium exchanged mole‐for‐mole with sodium. Analysis of the saturable components by a statistical‐mechanical adsorption model demonstrated a cooperative interaction between sites determining equilibrium potassium‐sodium distribution. Superimposed upon the saturable fraction of cell potassium was a smaller one that was non‐saturable with increasing external potassium to at least 64 mM, and that, when expressed as mmoles/liter cell water, existed in a ratio to external potassium of 0.6. The results strongly support the association‐induction hypothesis, which predicts a small non‐saturable component of ions determined by exclusion from oriented cell water and a cooperative interaction between sites throughout the cell that associate with potassium or sodium.
Binding of Salmonella minnesota R‐form glycolipid mR595 to rat fibroblasts and its effect on cell metabolism and cell behaviour
Authors: Lallier Réal , Trudel Michel , Brailovsky Carlos A. , Nigam Vijai N.
Published: February 2005

Trypsinized normal rat embryo fibroblasts and untrypsinized and trypsinized transformed rat fibroblasts have two orders of binding sites for bacterial glycolipid mR595. The high order sites fix 1–3 μg glycolipid mR595/105 cells and those of the low order fix about 6 μg glycolipid mR595/106 cells. Ca++ is required for the low order glycolipid mR595 binding to be trypsinized but not to the untrypsinized transformed rat fibroblasts. The low order binding but not to the untrypsinized transformed rat fibroblasts. The low order binding is temperature dependent with the transition temperature lying between 25 and 37°C. Exogenously added ganglioside and glycoproteins contained in the fetal calf serum do not inhibit fixation of glycolipid mR595. Only β‐lipoprotein at high concentrations is slightly inhibitory.

Glycolipid mR595 fixation to transformed fibroblast does not alter their morphology and appears to slightly improve cell attachment to substratum. Glycolipid mR595 fixation results in a lengthening of the S‐phase of the cell cycle and a reduction in 2‐deoxyglucose uptake. Uptake of inorganic phosphate is not affected. Inhibition of phospholipid synthesis is observed in mR595 fixed fibroblasts whereas synthesis of cell surface glycoproteins and the content of cellular gangliosides is not affected.
Studies on intercellular adhesion. III. Adhesion of cells to isotypic or heterotypic collecting particles and monolayers
Authors: Pessac Bernard , Girard Arlette , Alliot Françoise
Published: February 2005

Adhesion between cells of different tissues from chick embryos was studied by the modified collecting particle (Pessac et al., 1977a) and stationary monolayer assays. These assays measure the number of 3H labeled cells that adhere to the surface of cell aggregates and tissue fragments or on top of cell monolayers. The numbers of cells from a given suspension that adhere to identical or different cells can be compared. Intercellular adhesion may be considered as tissue specific if, in identical conditions, more cells adhere to identical cells than to cells from different tissues. Neural retina, cerebrum, optic tectum, liver and kidney cells from 9‐day‐old chick embryos showed no tissue specificity of adhesion, while heart cells adhered preferentially to collecting heart fragments.
Evidence for a high molecular weight factor(s) in serum which increases alkaline phosphatase specific activity in HeLa
Authors: Carlson Charles W.
Published: February 2005

HeLa (substrain Ho) grown in serum free medium showed an increase in the specific activity of alkaline phosphatase when fetal calf serum (10%) was added to the medium (9.7 nmoles/sec/mg protein to 86.8). Under the same conditions, eight intracellular enzymes showed no increase in activity. Similar results were obtained using a different serum or medium, and with a second strain of HeLa (substrain ATC).

For a given set of growth conditions, the effect of serum was dependent on its concentration and required one or more culture generations to develop. The type of isozyme expressed did not change. Neither zinc nor a total serum lipid extract would substitute for serum. The enzyme expressed by HeLaHo was not induced by prednisolone, while that in HeLaATC was. However, for cells grown in excess prednisolone without serum, the specific activity was 25% of that found for cells grown with prednisolone and serum. Cortexolone, an antagonist of prednisolone, was without effect for HeLaHo grown in A3 medium with or without serum.

The serum factor had the following characteristics. It was not lost on dialysis, treatment with DNase and RNase, or removal of lipoproteins. It was reduced after heating by 65% and after treatment with Pronase by 82%.

The data are interpreted to indicate the presence of a factor (s) in serum, probably a protein, which is involved in stimulating alkaline phosphatase specific activity.
Metabolic correction of fucosidosis fibroblasts by human α‐L‐fucosidase
Authors: Turner Bryan M. , Turner Virginia S. , Hirschhorn Kurt
Published: February 2005

Human α‐L‐fucosidase, purified from placenta, was taken up from the culture medium by skin fibroblasts from patients with fucosidosis (α‐L‐fucosidase deficiency). The rate of uptake was low (uptake coefficient = 6 × 10−4 ml.mg−1.h−1). Intracellular α‐L‐fucosidase activity was directly proportional to enzyme in the medium up to an activity of at least 40 nmoles/min/ml. No evidence for saturation of specific cell‐surface receptors was seen. However, uptake was reduced by 75% by 1 mM mannose‐6‐phosphate and by 50% by 1 mM glucose‐6‐phosphate, suggesting that uptake may be mediated by a receptor recognising a phosphorylated sugar or an analagous compound. Enzyme taken up by the cells was most active in subcellular fractions enriched with lysosomes and had an isozyme pattern, by isoelectric focusing, identical to that of the original enzyme preparation.

Fucosidosis fibroblasts were shown to accumulate low molecular‐weight, fucose‐containing compounds to a level several times greater than control cells. This stored material was eluted from Sephadex G‐25 as an asymmetrical peak with an elution volume of approximately twice the void volume of the column. Addition of placental α‐L‐fucosidase to the culture medium of fucosidosis fibroblasts prevented excessive accumulation of fucose‐containing material and accelerated the breakdown of material accumulated prior to enzyme uptake.
The accumulation of manganese from the environmental medium by the egg of Oryzias latipes
Authors: Michibata H. , Hori R.
Published: February 2005

The manganese content of the egg and embryo of the Medaka, Oryzias latipes was determined by activation analysis. A remarkable increase in the amount of manganese in the egg was observed within one hour after fertilization. The rate of increase was reduced by the gastrula stage and the concentration of manganese remained unchanged at a later stage. The accumulation of manganese by the Oryzias egg was discussed in relation to the effect of manganese on respiratory enzyme systems.
Amino acid transport in chick embryo fibroblasts: Evidence for transcriptional regulation of transport following serum addition
Authors: Borghetti Angelo F. , Tramacere Mariarosaria , Piedimonte Giuseppe , Guidotti Guido G.
Published: February 2005

Changes in amino acid transport activity by system A (a Na+‐dependent agency with affinity for a discrete group of neutral amino acids) caused by the addition of serum to serum‐deprived cultured chick embryo fibroblasts have been evaluated by measurements of 14C‐labeled L‐proline and α‐methylaminoisobutyric acid uptake under conditions approaching initial entry rates. Dialysed serum was as effective as undialysed serum in stimulating amino acid transport. This effect was inhibited by 7 μM cycloheximide, by 80 nM actinomycin D and by 40 μM cordycepin, but not by 0.3 mM cytosine arabinoside. Cultured avian fibroblasts previously incubated in a cycloheximide‐containing medium (phase of inhibited translation) in the presence of serum, subsequently exhibited a net increase of proline transport activity when transferred to a medium containing actinomycin D (phase of inhibited transcription). Omission of serum in the cycloheximide‐phase prevented the increase of transport activity during subsequent incubation in the actinomycin D‐phase; omission of serum in the actinomycin D‐phase allowed a shorter and less pronounced increase of transport activity than in the presence of serum. Additions of actinomycin D or cycloheximide slightly increased the rate of decay of amino acid transport caused by serum withdrawal. These observations suggest that in cultured avian fibroblasts, serum modulates the activity of transport system A by a mechanism acting at the transcription level.
Effects of angiotensin II and angiotensin II antagonist saralasin on cell growth and renin in 3T3 and SV3T3 cells
Authors: Schelling Pierre , Ganten Detlev , Speck Georg , Fischer Hans
Published: February 2005

Components of the renin‐angiotensin system were studied in established cell culture lines of 3T3 and SV3T3 mouse fibroblasts. The renin content in 3T3 cells was significantly higher than in virus‐transformed SV3T3 cells. With time after infection, renin decreased in Simian virus 40 transformed cells, while it increased steadily in mock‐infected 3T3 cells. In contrast to renin, angiotensinase activity was higher in SV3T3 cells.

Angiotensin II stimulated cell proliferation in 3T3 mouse fibroblasts and decreased their renin content in a dose‐related manner. In contrast, saralasin, an angiotensin receptor antagonist, inhibited cell growth in 3T3 and SV3T3 cells and caused an increase of cellular renin concentration. The angiotensin fragments angiotensin (2–8) heptapeptide and angiotensin (4–8) pentapeptide had no effect on cell growth. A significant negative correlation was found between cell proliferation and renin levels in 3T3 and SV3T3 cells irrespective of the treatment.

Our results indicate (1) that angiotensin II may be involved in cell growth regulation, (2) that a negative feedback exists between angiotensin II added and intracellular renin content, and (3) that virus infection causes a decrease in intracellular renin synthesis, while non‐specific angiotensinase activity is increased under this condition.
Evidence suggesting that a cyclic AMP‐dependent protein kinase is a positive regulator of proliferation in Cloudman S91 melanoma cells
Authors: Pawelek John M.
Published: February 2005

Wild‐type Cloudman S91 melanoma cells have a retarded rate of division when agents which raise cyclic AMP levels such as melanotropin, protaglandin E1, or cholera toxin are supplemented to the culture medium. A mutant cell line was isolated which had the opposite response, i.e., the mutant grew very slowly unless agents which raised cyclic AMP levels were present (Pawelek et al., '75a). In this report evidence is presented indicating that the molecular basis for the mutant phenotype resides in the major cyclic AMP‐dependent protein kinase found in the cells. The mutant kinase had increased thermolability and an elevated activation constant for cyclic AMP over the corresponding wild‐type kinase. It is proposed that the elevated requirement for cyclic AMP for the proliferation of cAdep cells is related to the elevated activation constant of this kinase, suggesting that the kinase is a positive regulator of proliferation in Cloudman S91 cells.
Electrophysiology of differentiating mouse spermatozoa
Authors: McGrady Angels
Published: February 2005

The morphological aspects of spermatogenesis are well described in many mammalian species, but functional changes are not completely understood. Electrophysiological parameters were investigated in primary spermatocytes and early and late spermatids isolated from the seminiferous tubules of the mouse. Substantial changes were not detected in membrane potential between different developmental stages. Membrane potential was dependent on both potassium and sodium ion concentration gradients, but not on chloride gradients. The ratio of the permeabilities PNa/PK varied according to the extracellular concentrations of sodium and potassium. Ouabain, a specific inhibitor of Na+, K+‐activated ATPase, produced a maximal reduction in membrane potential of 20% Comparisons were drawn between differentiating germ cells and previously determined properties of mature spermatozoa.
Alterations in chloride transport during differentiation of Friend virus‐transformed cells
Authors: Harper Patricia A. , Knauf Philip A.
Published: February 2005

Friend erythroleukemic cells can be induced by a variety of agents to synthesize hemoglobin and to exhibit other characteristics suggesting erythroid maturation. Upon induction of hemoglobin synthesis with dimethyl‐sulfoxide (DMSO), the chloride flux in Friend cells gradually increases, until after five days of exposure to DMSO (when the hemoglobin content of the cells approaches that of the mature erythrocyte) the flux is three times the value in non‐induced cells. A similar flux increase is observed in the presence of a different type of inducer, hypoxanthine, but no increase in flux is seen in the mutant cell line, TG‐13, which does not synthesize hemoglobin after DMSO treatment. Thus, the flux increase seems to be associated with the induction process, rather than being a direct effect of the inducing agent. After DMSO treatment, the sulphate flux decreases and the chloride/sulphate selectivity increases, as would be expected if the cells were becoming more like red cells. On the other hand, the sensitivity of the chloride flux to the inhibitor, furosemide, and to temperature is the same in the induced as in the non‐induced Friend cells, and different from that of the mature red cell. Thus, the anion transport properties of the induced Friend cell are different from those of both the non‐induced Friend cell and the mature erythrocyte. Either the system in the induced cell represents an intermediate stage in the development of the mature red cell characteristics, or else the maturation of transport function in the Friend cell differs from that in normal erythrocyte precursors.
Enhancement of chemotactic response and microtubule assembly in human leukocytes by ascorbic acid
Authors: Boxer Laurence A. , Vanderbilt Burton , Bonsib Stephen , Jersild Ralph , Yang Hsin‐Hsin , Baehner Robert L.
Published: February 2005

The incubation of human leukocytes with ascorbic acid increased chemotaxis of the cells. In addition, ascorbic acid promoted the assembly of intracellular polymorphonuclear leukocyte (PMN) microtubules as assessed by transmission electron microscopy. Prior incubation of the PMN with colchicine blocked the effect of ascorbic acid on promoting microtubule assembly. Not only did ascorbic acid promote the assembly of microtubules in vivo, but it enhanced the assembly of bovine brain tubulin into microtubules in vitro as quantitated by a glass‐fiber filtration assay and by promotion of viscosity changes. The enhancement in leukocyte mobility by ascorbate at concentrations achievable in normal tissues correlates with its ability to assemble microtubule organelles.
Plasma membrane phosphoproteins in normal and Rous sarcoma virus transformed chick embryo fibroblasts: Characterization by in vitro phosphorylation
Authors: Branton Philip E. , Landry‐Magnan Johanne
Published: February 2005

Plasma membranes isolated from normal and RSV transformed chick embryo fibroblasts were phosphorylated in vitro using endogenous protein kinase and ATP (γ32P) and the labeled phosphoproteins were analyzed by SDS‐PAGE. A number of protein phosphorylation changes were observed following transformation, however in most cases they were relatively small quantitative differences. The four major changes were in proteins of 47,000, 58,000, 75,000 and 135,000 daltons. Decreased phosphorylation of the 47,000 dalton polypeptide was found in transformed cell membranes but this alteration was shown to be due to differences in cell growth rather than transformation. Increased phosphorylation of the 75,000 dalton protein was at least partially related to virus infection. However, increased phosphorylation of the 58,000 and 135,000 dalton polypeptides were entirely transformation specific.