bluuebunny/crossref_metadata_2025 huggingface dataset:
Production by spleen and lymph node cells of conditioned medium with erythroid and other hemopoietic colony‐stimulating activity
Authors: Metcalf D. , Johnson G. R.
Published: February 2005

Pokeweed mitogen‐stimulated suspension cultures of mouse spleen cells produced conditioned medium able to stimulate granulocyte‐macrophage, eosinophil and megakaryocyte colony formation in agar cultures of C57BL marrow cells and granulocyte‐macrophage and erythroid colony formation in agar cultures of CBA fetal liver cells. Medium conditioned by other mouse tissues stimulated only granulocyte‐macrophage colony formation and this activity was not increased by the addition of pokeweed mitogen. Spleen cells stimulated by mixed leucocyte culture or concanavalin‐A had a weak capacity to stimulate erythroid colony formation.

Production of the factors stimulating the four types of hemopoiesis was T‐lymphocyte dependent and nu/nu spleen cells were inactive. Factor production was also dependent on adherent cells but evidence from rat‐mouse combinations suggested that the T‐lymphocytes actually produced the active factors.

Production of the four types of colony stimulating factors was radiosensitive (D0120–238 rads) and spleen cell populations of lighter buoyant density than 1.075 g/cm3 and sedimenting at 3.5–5.0 mm/hour were able to produce active conditioned media. Fractionation experiments failed to segregate spleen populations able to produce only one of the four stimulating factors.

Production of factors stimulating hemopoiesis by mitogen‐stimulated lymphoid populations could be a process contributing to the control of hemopoiesis in vivo.
Erythroid stem cells in friend‐virus infected mice
Authors: Opitz Uta , Seidel Hans‐Joachim , Bertoncello Ivan
Published: February 2005

The erythropoietic stem cell compartment was studied in Friend‐virus (polycythemic strain, FV‐P) infected DBA/2 and NMRI mice with the CFUE and BFUE technique. Early after infection there was a depression in CFUE number in bone marrow and spleen, followed by an increase of the CFUE concentration, earlier and more pronounced in the spleen than in the marrow. Three days after FV‐P infection an erythropoietin (Ep) independent CFUE population started to grow and replaced the normal Ep‐dependent population within 8 to 12 days. The shift to Ep independency was not gradual. CFUE colonies of FV‐P infected bone marrow cells were two to three times larger than control colonies after three days in vitro incubation.

BFUE colonies increased in number during the first days of infection, but were totally lost after more than ten days. After velocity sedimentation of bone marrow cells of FV‐P infected animals, however, the BFUE containing fractions showed normal BFUE colony growth and normal Ep sensitivity. In unfractionated bone marrow cell cultures BFUE colony growth could be observed later than ten days post infection when the cultures were refed with medium. It was therefore concluded that the loss of BFUE colony growth after FV‐P infection was an in vitro artefact due to inadequate culture conditions.
Satellite DNA in differentiating chick tissues
Authors: Ayres Kathleen Nelson
Published: February 2005

Embryonic chick DNA from different tissues was examined for differences which might indicate specific DNA amplification in somatic cells. The problem was approached by determining the DNA compositional heterogeneity and searching for possible variation in different tissues of the 12‐day chick. Neural retina, muscle, and whole decapitated (general) chick DNA were analyzed in CsCl and Cs2SO4 density gradients. While overloaded CsCl gradients showed a main band (ρ = 1.701 g/cm3) and a heavy shoulder (ρ = 1.716 g/cm3), overloaded Cs2SO4 gradients displayed a main band (ρ = 1.426 g/cm3) and a discrete heavy satellite (ρ = 1.447 g/cm3) This satellite, comprising approximately 1% of the whole cell DNA, appeared to be of nuclear origin and not related to mitochondrial DNA, which was found to have a density of 1.426 g/cm3 in Cs2SO4. No differences were found in the densities of the main band or the satellite DNA in the DNA samples isolated from the different tissues. However, the method of DNA isolation was found to be of crucial importance when comparing satellite DNA's among different tissues.
Factors influencing endothelial cell proliferation in vitro
Authors: Wall Robert T. , Harker Laurence A. , Quadracci Leonard J. , Striker Gary E.
Published: February 2005

The relative roles of blood cell products and plasma factors on endothelial cell proliferation were evaluated by studying the proliferative response of human umbilical vein endothelial cells to cell free plasma derived serum (CFPDS), whole blood serum (WBS), platelet released factors, fibroblast growth factor and macrophage conditioned medium in vitro. Human adult arterial smooth muscle cells were treated in a similar manner for comparison. The rate of endothelial cell proliferation was directly related to the concentrations of both WBS and CFPDS. Growth rate in WBS was marginally greater than that observed in CFPDS during early culture, however, similar confluent densities were achieved. The addition of platelet released factors to CFPDS did not further stimulate endothelial cell proliferation. In contrast smooth muscle cells were quiescent in CFPDS despite increasing serum concentrations, but proliferated actively in response to platelet released factors. Both human macrophage conditioned medium and fibroblast growth factor increased endothelial cell proliferation significantly when compared with CFPDS alone. It is concluded that endothelial cell proliferation in preconfluent cultures is dependent on plasma factors while human vascular smooth muscle cells also require cell derived mitogens such as platelet growth factor to proliferate. The release of a substance by human macrophages mitogenic for endothelial cells may be involved in endothelial cell proliferation in vivo.
Dna replication in human lymphocytes during aging
Authors: Agarwal Shyam S. , Tuffner Margaret , Loeb Lawrence A.
Published: February 2005

An analysis of the accuracy of protein and DNA synthesis in human lymphocytes with respect to aging has been carried out. The response of human peripheral lymphocytes, from young and old adults, to phytohemagglutinin was measured at varying temperatures. This should provide a sensitive test for the accumulation of altered thermolabile proteins that are rate limiting in the response to phytohemagglutinin. At 37°C the rate of thymidine incorporation as well as the induction of DNA polymerase in phytohemagglutinin‐stimulated lymphocytes from old and young adults were similar. Also at elevated temperatures, the thermosensitivity of DNA replication in lymphocytes from young and old adults was the same.

DNA polymerase was purified from PHA‐stimulated lymphocytes from young and old adults. The fidelity of DNA synthesis using poly (dC) as a template was similar with both enzymes. However, DNA polymerase‐α purified from old adults was thermolabile compared to the enzyme from young adults. Thus, while the lymphocytes from old individuals may have heat labile proteins, they do not limit their proliferative capacity.
A human serum protein system affecting muscle contractility: Characterization of the five components and their reaction sequence
Authors: Hajdu Stephen , Leonard Edward J.
Published: February 2005

A human serum protein system that affects muscle contractility, previously reported to be a calcium transport system (CTS), was fractionated into five protein components, A, B1, Bf1, B3 and C. The system was assayed on frog heart muscle in vitro by its capacity to increase contractile force and at high concentrations to cause contracture. With separated fractions, the reaction sequence was divided into three separate steps: B1 and B3 interacted with the heart in the first step, B2 and B3 in the second, and A and C in the third. Increased contractility occurred only after the third step. Partial purification, with preservation of biological activity, was achieved for B1, B2, A and C. B1 has a M.W. of about 170,000, as determined by its elution from Sephadex G‐200. B2, a labile protein of M.W. 220,000, was one of the few serum proteins precipitated by M.W. 500,000 dextran sulfate at pH 8.0. This was the basis for the preparation of highly purified B2, which was used to produce rabbit anti‐B2 antibody. Experiments with this antibody proved that B2 became bound to the heart surface. CTS‐A, the smallest protein of the system, had a M.W. of approximately 130,000. The M.W. of C was over 300,000. Purified C antigen induced rabbit antibody that inhibited the action of C without affecting the other components.
Glucose and amino acid metabolism in an established line of skeletal muscle cells
Authors: Pardridge William M. , Davidson Mayer B. , Casanello‐Ertl Delia
Published: February 2005

The suitability of an established myogenic line (L6) for the study of skeletal muscle intermediary metabolism was investigated. Myoblasts were grown in tissue culture for ten days at which time they had differentiated into multinucleated myotubes. Myotube preparations were then incubated for up to 96 hours in 10 ml of Dulbecco's modified Eagle medium containing 10% fetal calf serum. Glucose was utilized at a nearly linear rate, 3.0 nmol/min/mg protein. Intracellular glucose was detectable throughout the incubation, even when medium glucose was as low as 16 mg%. During the initial 28 hours of incubation, when net lactate production was observed, only 35% of the glucose utilized was converted to lactate. Alanine was produced in parallel to lactate at an average rate of 0.6 nmol/min/mg protein. In concert with active glutamine utilization, high rates of ammoniagenesis were observed as medium glutamine decreased from 3.3 mM to 0.49 mM and medium ammonia increased from 2.3 mM to 6.2 mM, between zero time and 96 hours of incubation, respectively. The cells maintained stable ATP and citrate levels, and physiologic intracellular lactate/pyruvate ratios (10–24) throughout 96 hours of incubation. These results suggest (1) glucose utilization by skeletal muscle in tissue culture is limited by phosphorylation, not transport; (2) as much as 50% of glucose‐derived pyruvate enters mitochondrial pathways; (3) glutamine carbon may be utilized simultaneously with glucose consumption and this process accounts for high rates of ammoniagenesis.
Amphibian cells in culture. I. Nutritional studies
Authors: Chinchar Giovina D. , Sinclair John H.
Published: February 2005

Nutritional requirements of amphibian cells in culture were studied for the purpose of modifying a minimal medium in which frog cells could proliferate and which could be used for obtaining drug‐resistant and auxotrophic variants. The serum, purine, CO2, and amino acid requirements for ICR 2A (a Rana pipiens haploid cell strain) have been investigated employing two different media: L‐15, a nonbicarbonate, amino acid‐buffered medium and Eagle's MEM, a bicarbonate‐buffered medium.

In this paper we present evidence to support the following conclusions: (1) With L‐15 as the base medium, 10% fetal calf serum (FCS) supports optimal cell growth during exponential phase. Calf serum, whole, dialyzed, or heat‐inactivated, cannot substitute for FCS and, in fact, is inhibitory. (2) Purines are required by ICR 2A cells only if grown in a nonbicarbonate‐buffered medium, since the cells under these conditions cannot produce enough endogenous CO2 to support de novo purine synthesis. (3) In addition to the amino acids considered essential for mammalian cells in culture, ICR 2A cells depend upon exogenous asparagine. Glutamine and/or aspartic acid cannot replace the asparagine requirement. However, ICR 2A cells do utilize exogenous glutamine as an oxidative substrate.
Hormonal activation of adenylate cyclase in mouse melanoma metastatic variants
Authors: Niles R. M. , Makarski J. S.
Published: February 2005

The ability of melanocyte stimulating hormone (MSH), adrenocorticotropic hormone (ACTH), and prostaglandin E1 (PGE1) to stimulate the accumulation of cyclic AMP was examined in intact mouse melanoma cells of varying metastatic potential. F1 cells (low metastatic potential) had significantly greater cyclic AMP levels in response to all three hormones than F5 (intermediate metastatic potential) and F10 (high metastatic potential) cells. The ranking of the response was as follows: MSH, F1 > F5 > F10, ACTH, F1 > F5 > F10, PGE, F1 > F10 > F5. In contrast to the above, the degree of hormonal stimulation of adenylate cyclase in broken cell preparations was virtually identical in all three melanoma cell lines. Control enzyme activity was depressed in both F5 and F10 relative to F1. The conflicting results between studies of intact vs. broken cell preparations could not be explained by increased cyclic AMP phosphodiesterase activity in F5 and F10 cells. We conclude that as the melanoma cells increase in metastatic potential, there is a significant loss in the ability of their cyclic AMP system to respond appropriately to hormonal stimuli.
Red cell aging II. Anomalous electrophoretic properties of neuraminidase treated human erythrocytes
Authors: Nordt F. J. , Knox R. J. , Seaman G. V. F.
Published: February 2005

Desialylation of human red blood cells (RBC) by Vibrio cholerae neuraminidase (VCN) was found to produce cells with electrophoretic properties which were inconsistent with the view of simple loss of N‐acetyl‐neuraminic acid (NANA) as the sole effect of VCN treatment. Modification of human RBC with 50–350 U VCN/1010 RBC for one hour at 37°C releases 90–100% of the NANA and produces a progressive decrease towards zero in their electrophoretic mobilities when measured in 0.15 M NaCl (pH 7.2) at 25°C. The appearance of positive groups on the desialylated cells was indicated by the VCN‐treated cells displaying positive mobilities below ∼ pH 5.5 and increased negative mobilities at ∼ pH 9 as well as substantial increases in their mobility at neutral pH following treatment with formaldehyde. Adsorption of about 95% of the VCN activity at 0°C to the RBC did not produce any significant change in their electrophoretic mobilities thus indicating that the observed changes in the electrophoretic properties of the RBC following VCN treatment could not be attributable to adsorption of VCN.

These studies indicate that the cationic charge groups which appear at the electrophoretic surface of the RBC after VCN treatment are probably of endogenous origin. It is suggested that this alteration rather than simple NANA release may operate to shorten the in vivo survival time of desialylated red cells.
Inhibition of basal protein degradation in rat embryo fibroblasts by cycloheximide: Correlation with activities of lysosomal proteases
Authors: Amenta J. S. , Sargus M. J. , Baccino F. M.
Published: February 2005

Rat embryo fibroblasts were grown in medium containing 14C‐leucine and 3H‐thymidine. After a 24‐hour chase in nonlabeled medium, cultures were placed in either fresh growth medium or medium containing 10–20 μg/ml cycloheximide. Cell monolayers were processed at daily intervals for three days. Four hours prior to processing, cultures were placed in fresh medium and the accumulation rate of trichloracetic acid soluble 14C in the media assayed. Cycloheximide effects a progressive decrease in the fractional degradation rate of the labeled cell protein, primarily during the first 24 hours. The specific activities of cathepsin D, cathepsin B, and neutral protease correlate closely with the fractional degradation rate. Other lysosomal hydrolases show little change during this period. The activities of the lysosomal proteases approach a new steady state which is correlated with the new steady state leve of protein synthesis. A model is proposed which relates the rate of protein break‐down in the cell to the level of protein synthesis. The data also suggests the possibility that subpopulations of high turnover and low turnover cells exist in these cultures.
Transport and phosphorylation of hexoses in normal and rous sarcoma virus‐transformed chick embryo fibroblasts
Authors: Singh Malathy , Singh Vishwa Nath , August J. T. , Horecker B. L.
Published: February 2005

Effects of transformation by Rous sarcoma virus on sugar uptake and activity and the subcellular distribution of hexokinase isozymes in chick embryo fibroblasts were examined. Transformation caused a several‐fold increase in the maximum velocity for uptake of 2‐deoxyglucose without a significant change in Km. Cytochalasin B (CB), was used to differentiate between the effects of transformation on facilitated diffusion and the nonsaturable (CB‐insensitive) mode. Transformation was found to stimulate 2‐deoxyglucose transport by both mechanisms, but the increase in transport by the CB‐insensitive mode was greater.

Transformation enhances the activity of hexokinase, the enhancement being confined to the particulate fraction of the enzyme. Heat‐inactivation and electrophoretic mobility studies showed that although hexokinase Type I is the major form in both normal and transformed fibroblasts, there is a significant increase in the proportion of the Type II isozyme in the transformed cells.
Polyene macrolide antibiotic cytotoxicity and membrane permeability alterations I. Comparative effects of four classes of polyene macrolides on mammalian cells
Authors: Fisher Paul B. , Bryson Vernon , Schaffner Carl P.
Published: February 2005

The relationship between polyene macrolide‐induced early membrane damage and cytotoxicity in B1 (hamster), B82 (mouse), and RAG (mouse) cells has been investigated. Filipin (FIL) induced the greatest immediate damage, as monitored by 51Cr release, followed by mediocidin (MED), amphotericin B‐deoxycholate (Fungizone®) (FZ) and pimaricin (PIM). For long term effect, PIM was the least toxic followed by MED, FZ, and FIL as indicated by 24‐hour survival, 72‐hour viability, and growth rate of cells. In evaluating polyene macrolide‐induced permeability alterations and cytotoxicity two types of interactions with mammalian cells were found: (1) cell toxicity at polyene macrolide levels not eliciting immediate membrane permeability changes; and (2) immediate membrane damage without long range toxicity.
The proliferative response in vitro of vascular endothelial and smooth muscle cells exposed to wound fluids and macrophages
Authors: Greenburg Gary B. , Hunt Thomas K.
Published: February 2005

The ability of wound‐derived macrophages and of fluids obtained from wounds to stimulate the proliferation of vascular endothelial cells and vascular smooth muscle cells in vitro has been investigated. Sparse cultures of endothelial and smooth muscle cells maintained in the presence of optimal concentrations of serum and wound fluid showed a 25‐fold increase and a 10‐fold increase, respectively, in final cell density when compared to cultures maintained in the presence of serum alone. The response of both endothelial and smooth muscle cells to the wound fluid was dependent on the serum or plasma‐serum concentrations in which they were maintained. The ability of the wound fluid to stimulate endothelial cell proliferation was compared to that of fibroblast growth factor (FGF), which has already been shown to be a potent mitogen for endothelial cells in vitro. The growth response of endothelial cell cultures, when exposed to either 2% wound fluid or 100 ng/ml of FGF, was found to be similar, and the normal cell morphology was maintained. To examine the possibility that the growth stimulating activity present in the wound fluid originated from macrophages, endothelial cells or smooth muscle cells were co‐cultured with macrophages obtained from the wound fluid. A significant stimulation of both endothelial and smooth muscle cells was observed with 2 × 106 macrophages, which would suggest that macrophages may be the source of the mitogenic activity of the wound fluid in vivo.
5‐fluorotryptophan resistant mutants affecting the A and L transport systems in the mouse L cell line A9
Authors: Taub Mary , Englesberg Ellis
Published: February 2005

Tryptophan transport has been examined in A9 and in mutants resistant to 5‐fluorotryptophan (5‐FT). Evidence indicates that in A9 cells two systems are present for tryptophan transport, which are analogous to the A and L systems found in Ehrlich ascites cells differing, however, in terms of amino acid specificity. Tryptophan uptake via the L system, a high affinity, low capacity system, is Na+ independent and occurs by a counter transport mechanism, while uptake via the A system, a low affinity, high capacity system, is Na+ dependent. Alanine, arginine, lysine, proline, asparagine, and aspartate (listed in order of decreasing inhibitory effect) inhibit tryptophan uptake via the A system from approximately 80‐50% while having no inhibitory effect on the L system. In addition, glutamine which inhibits tryptophan uptake by 80% via the L system only inhibits to the extent of 20% via the A system. Previous kinetic studies of 5FT resistant clone FTr37 indicated system A was altered while the analysis of the effects of the mutation on system L was inconclusive. However, in these studies Na+ independent uptake was not altered in FTr 37 indicating system L was not affected. Amino acid competition studies confirmed this observation and suggested that a change in the specificity of system A had occurred in FTr 37. The amino acid competition studies in FTr 23, indicated that the specificities of both systems differed from A9. The possibility that this change may be due to a single mutational event is discussed.
RNA synthesis by nuclei and nucleoli from ischemic liver cells
Authors: Schiaffonati Luisa , Cairo Gaetano , Bernelli‐Zazzera Aldo
Published: February 2005

Nuclei and nucleoli were isolated from rat livers subjected to an interruption of the blood supply for periods of different duration, as well as after restoration of the blood supply. They were assayed for RNA synthesis under conditions of diverse ionic strengths, and in the presence of an exogenous template, such as poly d (A–T), and actinomycin to inactivate the endogenous template; α‐amanitin was made used of to distinguish polymerase I and polymerase II dependent RNA synthesis. Nuclei and nucleoli from ischemic livers showed a severe impairment of RNA synthesis, which is likely to be due to decreased initiation frequency of the engaged polymerases, while free polymerases were essentially unchanged. Both form I and II polymerase were equally involved. After restoration of the blood supply RNA synthesis recovered with an overshooting well above normal levels of activity, lasting for at least 24 hours. Increased RNA synthesis was not followed by thymidine incorporation into DNA.
Purification and properties of human erythrocyte inosine triphosphate pyrophosphohydrolase
Authors: Vanderheiden Bernardo S.
Published: February 2005

Inosine triphosphate pyrophosphohydrolase from human erythrocytes was purified and characterized. The enzyme is highly specific for ITP and shows optimal activity in glycine buffer pH 9.6 and 50 mM MgCl2. The Km of the enzyme is 1.3 × 10−4, the Vmax = 1.2 × 10−9 and the Keq = 3.8 × 104. Human erythrocyte ITP pyrophosphohydrolase does not require SH compounds for activation. The enzyme is inhibited by Cd++, Co++, and Ca++ ions and by phydroxymercuribenzoate.
The initiation and elongation steps in protein synthesis: Relative rates in chinese hamster ovary cells during and after hyperthermic and hypothermic shocks
Authors: Oleinick Nancy L.
Published: February 2005

The relative rates of the initiation and elongation phases of protein synthesis have been determined in heat‐ and cold‐shocked CHO cells from measurements of the incorporation of 35S‐methionine into N‐terminal and internal positions of growing peptides by a modified Edman degradation. When the cells are shifted from 37°C to temperatures between 10°C and 34°C, the rate of initiation is at first reduced more extensively than that of elongation. After 20 to 30 minutes at the lower temperature, however, the cells undergo a metabolic adjustment which includes increasing the rate of initiation until it corresponds to the rate of elongation at that temperature. Calculated apparent energies of activation for initiation and elongation are in reasonable agreement with those determined in other mammalian cells. When the cooled cells are returned to 37°C, the rates of initiation and elongation recover immediately but do not exceed the control values. Exposure to elevated temperature (43°C) causes an immediate cessation of initiation and thus a delayed inhibition of elongation; upon return to 37°C, the rate of initiation is transiently elevated above the control rate, and the rate of elongation returns to the control rate after a 2‐ to 3‐minute delay. Hence, a factor which leads to supranormal rates of initiation may accumulate at high but not at low temperatures.
Dexamethasone induces irreversible G1 arrest and death of a human lymphoid cell line
Authors: Harmon Jeffrey M. , Norman Michael R. , Fowlkes Betty Jo , Thompson E. Brad
Published: February 2005

Growth of a human leukemic T‐cell line (CEM C7) in 10−6 M dexamethasone results in inhibition of growth and rapid loss of cell viability after a delay of approximately 18 to 24 hours. Analysis of dexamethasonetreated cells by flow‐microfluorometry showed that they were arrested in the G1 phase of the cell cycle. Loss of cell viability began at the same time as G1 accumulation was first detectable, and 20% of all cells were found to be blocked in G1 at this time suggesting that loss of viability and G1 arrest were coincident events. Half‐maximal and maximal effects on both viability and G1 arrest after 48 hours in steroid were nearly identical with respect to steroid concentration and corresponded to half‐maximal and full occupancy of glucocorticoid specific receptor by hormone, consistent with a glucocorticoid receptor mediated mechanism for both phenomena. Most non‐viable cells were arrested in G1, and accumulation of cells in G1 was irreversible; removal of steroid in the presence of colcemid did not result in a decreased fraction of G1 cells. Furthermore, dexamethasone treatment did not protect cells against the effects of 33258 Hoechstamplified killing of bromodeoxyuridine substituted cells exposed to light. These results show that dexamethasone arrests these leukemic cells in G1 and strongly suggest that dexamethasone‐treated cells are killed upon entry into G1.
Growth kinetics and collagen synthesis of normal skin, normal scar and keloid fibroblasts in vitro
Authors: Diegelmann Robert F. , Cohen I. Kelman , McCoy Barbara J.
Published: February 2005

Fibroblasts were isolated from keloid, normal skin, and normal scar and maintained in tissue culture for four passages. Growth kinetics were the same for all groups on days 2 through 12. However, the rate of collagen synthesis per fibroblast was greater in keloid derived cells than any controls at all growth phases. Keloid fibroblasts have an autonomous capacity to synthesize collagen at a significantly increased level in vitro, which may explain in part why these lesions are characterized by increased collagen deposition.
Correlation of the effect of Ca+2 on Na+ and K+ permeability and membrane potential of Ehrlich ascites tumor cells
Authors: Smith Thomas C. , Vernon Kim D.
Published: February 2005

The effect of Ca+2 on the transport and intracellular distribution of Na+ and K+ in Ehrlich ascites tumor cells was investigated in an effort to establish the mechanism of Ca+2‐induced hyperpolarization of the cell membrane. Inclusion of Ca+2 (2mM) in the incubation medium leads to reduced cytoplasmic concentrations of Na+, K+ and Cl− in steady state cells. In cells inhibited by ouabain, Ca+2 causes a 41% decrease in the rate of net K+ loss, but is without effect on the rate of net Na+ accumulation. Net K+ flux is reduced by 50%, while net Na+ flux is unchanged in the transport‐inhibited cells. The membrane potential of cells in Ca+2‐free medium (‐13.9 ± 0.8 mV) is unaffected by the addition of ouabain. However, the potential of cells in Ca+2‐containing medium (‐23.3 ± 1.2 mV) declines in one hour after the addition of ouabain to values comparable to those of control cells (‐15.2 ± 0.7 mV).

The results of these experiment are consistent with the postulation that Ca+2 exerts two effects on Na+ and K+ transport. First, Ca+2 reduces the membrane permeability to K+ by 25%. Second, Ca+2 alters the coupling of the Na/K active transport mechanism leding to an electrogenic hyperpolarization of the membrane.
Electrophysiological properties of human oviduct smooth muscle cells in dissociated cell culture
Authors: Sinback C. Nelson , Shain William
Published: February 2005

Intracellular recordings were made from human oviduct smooth muscle maintained in cell culture. Solitary cells isolated from one another and cells in contact with one another retained electrical properties of smooth muscle in vivo. Membrane potential of solitary cells and connected cells was −35 mV. Connected cells formed electrotonic junctions which transmitted current from one cell to another. This current spread was responsible for differences in input resistance and time constant in solitary cells, 66 MΩ and 96 msec, compared to connected cells, 26 MΩ and 56 msec. All cells expressed delayed rectification to depolarizing current pulses. Some cells generated action potentials spontaneously or in response to intracellular current puleses. Action potentials were abolished by cobalt or by EGTA. Slow wave potentials, 5‐20 mV in amplitude, occurred continuously once every 15 to 45 seconds in connected cells.
The requirement of DNA synthesis for the induction of alkaline phosphatase by bromodeoxyuridine in a derivative of the HeLa cell line
Authors: Morrow John , Stocco Douglas M. , Fralick Joe A.
Published: February 2005

Non‐lethal concentrations of bromodeoxyuridine induce a 2‐ to 5‐fold increase in the specific activity of alkaline phosphatase in HeLa subclone, S3G. Experiments employing 10‐hour pulses of BRdU showed that 48 hours were required before induction commenced, and that maximal induction was attained by 96 hours. Under conditions in which DNA synthesis was prevented with hydroxyurea induction did not occur. Upon removal of hydroxyurea both DNA synthesis and induction were rapidly reestablished. Furthermore, experiments employing radiolabelled BRdU demonstrated that the kinetics of the induction process paralleled the incorporation of the analogue into cellular DNA.

These results indicate that DNA synthesis, or some process intimately linked to DNA synthesis, is required for the induction of alkaline phosphatase, and suggest that the mode of the induction may be through the incorporation of the analogue into cellular DNA.
The importance of ornithine as a precursor for proline in mammalian cells
Authors: Smith Robert J. , Phang James M.
Published: February 2005

Ornithine aminotransferase catalyzes the reversible transamination of L‐ornithine to δ1‐pyrroline‐5‐carboxylate, the immediate precursor of proline. The direction and flux through this pathway in mammalian cells has not been established. Glutamate has generally been considered to be the most important precursor for proline biosynthesis, but recent studies in xiphoid cartilage indicate that a significant fraction of cellular proline is derived from ornithine. Using newly isolated mutant Chinese hamster ovary cells with defined defects in the proline biosynthetic pathways, we now have established that cells can grow at a maximal rate with ornithine as the sole source of proline. Furthermore, we have measured the rate of proline formation from ornithine (1.6 nmol/h/106 cells). Future studies with these mutant Chinese hamster ovary cells may offer insight into the regulatory mechanism which coordinates proline biosynthesis from ornithine and glutamate.
Ordered replication of DNA sequences: Synthesis of mouse satellite and adjacent main band sequences
Authors: Dooley Douglas C. , Ozer Harvey L.
Published: February 2005

The replication of mouse satellite DNA was delayed when synchronized 3T3 cells were exposed to low concentrations of hydroxyurea during S phase. It appears that the onset of satellite replication is not a time dependent event, but instead requires that a certain amount of main band DNA be synthesized first. Using hydroxyapatite chromatography and S1 nuclease digestion, a procedure was developed to quantitate the synthesis of both satellite and neighboring main band sequences. The replication kinetics of satellite determined by this method agree with previous estimates. Main band sequences adjacent to satellite appear to replicate in concert with satellite DNA. The results are discussed and related to the limitations of the techniques utilized.
Induction of human choriogonadotropin and follitropin in HeLa cell cultures by hyperosmolality
Authors: Fallon Robert J. , Cox Rody P. , Ghosh Nimai K.
Published: February 2005

The growth of cervical carcinoma cell (HeLa) cultures in a hyperosmolar environment stimulates increased production of the onco‐developmental peptides human choriogonadotropin (hCG) and follitropin (FSH). This effect was observed in two sublines examined in this study, HeLa65 and HeLa71. hCG and FSH were measured by radioimmunoassay using antiserum against the β‐subunit of the hormone dimer, thus insuring immunochemical specificity. The amounts of hCG and FSH produced by HeLa65 and HeLa71 cells cultured in hyperosmolar medium were 2‐ to 50‐fold higher than corresponding hormone levels in basal cultures. Synthesis of gonadotropins depended on concentration and duration of exposure to hyperosmolar medium. Levels of culture medium osmolality effective in inducing hormone production also inhibit the incorporation of 14C‐thymidine into acid‐insoluble macromolecules. Hyperosmolality thus stimulates the ectopic production of gonadotropic hormones while retarding cellular growth and nucleic acid synthesis.
The effect of hexose on chloramphenicol sensitivity and resistance in chinese hamster cells
Authors: Ziegler Michael L. , Davidson Richard L.
Published: February 2005

The toxicity and extent of growth inhibition produced by chloramphenicol (CAP) in CAPs Chinese hamster cells (line V79‐5) was found to be dependent on the type and concentration of hexose in the medium. In high levels of glucose (6.5 mM), cultures of CAPs cells underwent 7–8 population doublings in the presence of 100 μg/ml CAP and viability then dropped rapidly. In contrast, lower concentrations of glucose (1.0 mM) permitted only limited growth (2–3 doublings) in the presence of 100 μg/ml CAP, but the cells remained viable and apparently quiescent for prolonged periods of time. The growth potential of V79‐5 cells in CAP appeared specifically dependent on glucose, as mannose and galactose could not substitute for glucose. The toxicity of CAP to these cells seemed to be determined primarily by the number of cell doublings in the presence of the drug.

A CAPR derivative of V79‐5, designated 5‐3, was analyzed in order to determine whether the requirement for glucose for cell growth in the presence of CAP also occurred in cells that were isolated as resistant to the drug. In order to rigorously control the hexose in the medium, some experiments were performed with medium containing dialysed, instead of whole, fetal calf serum. It was seen that the growth of the CAPR line in the presence (but not the absence) of 100 μg/ml CAP was dependent on glucose in the medium. Thus, resistance to CAP in these cells appears to be a conditional state, dependent on glucose for expression. Furthermore, the glucose auxotrophy of these cells in the presence of CAP suggests that CAP is still affecting some activities in cells isolated as CAPR.
Tunicamycin‐mediated depletion of insulin receptors in 3T3‐L1 adipocytes
Authors: Rosen O. M. , Chia G. H. , Fung C. , Rubin C. S.
Published: February 2005

Tunicamycin, an antibiotic that inhibits protein glycosylation, elicited a rapid depletion of insulin binding activity at the surface of 3T3‐L1 adipocytes. Disappearance of insulin receptors occurred more rapidly in the presence of tunicamycin than when protein synthesis was inhibited by cycloheximide and was accompanied by a diminution in sensitivity of the adipocytes to the acute effects of insulin and anit‐insulin receptor antibody on hexose uptake and metabolism.
Transmembrane electrical and pH gradients across human erythrocytes and human peripheral lymphocytes
Authors: Deutsch C. J. , Holian A. , Holian S. K. , Daniele R. P. , Wilson D. F.
Published: February 2005

Transmembrane electrical and pH gradients have been measured across human erythrocytes and peripheral blood lymphocytes using equilibrium distributions of radioactively labelled lipophilic ions, and of weak acids and weak bases, respectively. The distributions of methylamine, trimethylamine, acetic acid and trimethylacetic acid give calculated transmembrane pH gradients (pHe‐pHi) for erythrocytes of between 0.14‐0.21 for extracellular pH values of 7.28‐7.16. The distributions of trimethylacetic acid, DMO and trimethylamine were determined for lymphocytes, establishing upper and lower limits of the calculated pH gradient over he external pH range of 6.7 to 7.7.

Tritiated triphenylmethyl phosphonium ion (TPMP) and 14C‐thiocyanate ion (SCN) equilibrium distributions were measured in order to calculate transmembrane electrical potentials, using tetraphenylboron as a catalyst to facilitate TPMP equilibrium. Transmembrane potentials of −7 to −10 mV were calculated from SCN and TPMP, respectively for red cells, and −35 to −52 mV respectively, in the case of lymphocytes. Distributions of TPMP and potassium ions were determined in the presence of valinomycin over a wide range of extracellular potassium concentrations for red cells and the calculated Nernst potentials for TPMP compared to the calculated potential using the Goldman equation for chloride and potassium ions. Distributions of TPMP, SCN and potassium ions were also determined for lymphocyte suspensions as a function of extracellular potassium and the calculated Nernst potentials for TPMP and SCN compared to the calculated potassium diffusion potential.
Two distinct mechanisms for ornithine decarboxylase regulation by polyamines in rat hepatoma cells
Authors: McCann Peter P. , Tardif Chantal , Hornsperger Jean‐Marie , Böhlen Peter
Published: February 2005

Exogenous diamines and polyamines added to rat hepatoma (HTC) cells in culture rapidly decrease ornithine decarboxylase (ODC) activity. Previous evidence has suggested that these amines act either at the level of blocking new enzyme synthesis or by the induction of a non‐competitive protein inhibitor, termed antizyme, which complexes with ODC to form an inactive complex. With the use of HMOA cells, a recently cloned rat hepatoma cell line that has a greatly stabilized ODC, it has been possible to demonstrate that 10−5 M of exogenous putrescine blocks the increase in ODC activity, but unlike in the parent HTC cell line, without induction of the antizyme or formation of any inactive ODC‐antizyme complex. However, complete blockade of ODC at 10−2 M putrescine is effected by induction of antizyme and formation of the ODC‐antizyme complex, as now evidenced by the isolation of the active enzyme and antizyme components after Sephadex column chromatography in the presence of 250 mM NaCl. These findings indicate clearly that two polyamine‐regulatory mechanisms for ODC exist and are separable in this cell line.
The effects of Clostridium perfringens enterotoxin on morphology, viability, and macromolecular synthesis in vero cells
Authors: McClane Bruce A. , McDonel James L.
Published: February 2005

Vero (African green monkey kidney) cells grown in tissue culture monolayer were sensitive to Clostridium perfringens enterotoxin. Within 30 minutes of exposure to the enterotoxin gross morphological damage was observed and within 40 minutes approximately 75% of the cells had detached. Nearly half of the cells were nonviable following 35 to 40 minutes incubation with the enterotoxin. Doses as low as 0.1 ng caused small but detectable inhibition of plating efficiency of the cells while more than 100 ng caused the inhibition to approach 100%. Total inhibition of DNA, RNA, and protein synthesis occurred within 30 minutes exposure to enterotoxin. Heat inactivated enterotoxin had no apparent effects upon cellular morphology, detachment, viability, plating efficiency, or incorporation. We propose that the enterotoxin induces structural damage to the cytoplasmic membrane which results in loss of electrolytes and other essential substances from the cells. The outcome of this process is shut down of macromolecular synthesis, gross morphological damage, and eventual death of the cell.
Sulfated mucopolysaccharides from normal and virus transformed rodent fibroblasts
Authors: Chiarugi Vincenzo P. , Dietrich Carl P.
Published: February 2005

The sulfated mucopolysaccharide composition of normal and virus transformed Balb 3T3 and BHK21 cell lines is reported. It is shown that normal 3T3 cells contain mainly chondroitin sulfate B and heparitin sulfate. Relatively higher amounts of chondroitin sulface AC were observed in polyoma virus transformed 3T3 cells, besides an absolute increase of all the three sulfated mucopolysaccharides in the polyoma and SV 40 transformed cells. It is shown also that the three sulfated mucopolysaccharides are at least in part at the cell surface. Similar differences in sulfated mucopolysaccharide composition of normal and virus transformed BHK cell lines were also observed.
Pyrimidine nucleoside, pseudouridine, and modified nucleoside excretion by growing and resting fibroblasts
Authors: Uziel Mayo , Selkirk J. K.
Published: February 2005

We are examining the relationship of RNA metabolism and de novo pyrimidine synthesis as parameters of malignant transformation. These initial experiments on normal hamster embryo fibroblasts have shown that excreted nucleosides are markers for intracellular RNA metabolism. We employed affinity chromatography to concentrate the nucleosides in the medium and sensitive column chromatographic procedures to quantitatively measure them. The excretion of pyrimidine nucleoside from hamster embryo fibroblasts in culture was found to be dependent on the growth stage of the cells, with the greatest accumulation occurring during cell quiescence. The major nucleoside excretion products, uridine and cytidine, were both normal end products of RNA metabolism and the major nucleoside excretion products from cultured cells. The modified nucleosides N‐1‐methylguanosine, N‐2‐methylguanosine, N‐2‐dimethylguanosine, N‐4‐acetylcytidine, N‐1‐methylinosine, pseudouridine, N‐1‐methyladenosine, N‐3‐methylcytidine, and 5‐methylcytidine were found, as were several unidentified nucleosides.
Relationship between histidyl‐tRNA level and protein synthesis rate in wild‐type and mutant chinese hamster ovary cells
Authors: Lofgren Don J. , Thompson Larry H.
Published: February 2005

A preliminary investigation was carried out to determine how conditional lethal mutants affected in particular aminoacyl‐tRNA synthetases may be used to study the role of tRNA charging levels in protein synthesis. The relationship between rate of protein synthesis and level of histidyl‐tRNA in wild‐type cultured Chinese hamster ovary cells was determined using the analogue histidinol to inhibit histidyl‐tRNA synthetase activity. This response was compared with that obtained using a mutant strain with a defective histidyl‐tRNA synthetase that phenotypically shows decreased rates of protein synthesis at reduced concentrations of histidine in the growth medium. The approach used was based on measuring the histidyl‐tRNA levels in live cells. The percentage charging was estimated by comparing [14C]histidine incorporated into alkali‐labile material in paired samples, one of which was treated with cycloheximide, five minutes before terminating during the incubation, to produce maximal aminoacylation. Wild‐type cells under histidinol inhibition exhibited a sensitive, sigmoidal relationship between the level of histidyl‐tRNA and the rate of protein synthesis. A decrease in the relative percentage of acylated tRNAHis from 46% to 35% elicited a large reduction in the rate of protein synthesis from 90% to 30% relative to untreated cells. An unpredicted result was that the relationship between protein synthesis and histidyl‐tRNA in the mutant was essentially linear. High acylation values for tRNAHis were associated with rates of protein synthesis that were not nearly as high as in wild‐type cells. These findings suggest that the charging levels of tRNAHis isoacceptors could play a regulatory role in determining the rate of protein synthesis under conditions of histidine starvation in normal cells. The mutant appears to be a potentially useful system for studying the pivotal role of tRNA charging in protein synthesis, assuming that the altered response in the mutant is caused by its altered synthetase.
Pyridine nucleotide synthesis in 3T3 cells
Authors: Jacobson Elaine L. , Lange Richard A. , Jacobson Myron K.
Published: February 2005

The biosynthesis of NAD has been examined in 3T3 cells. The net synthesis of pyridine nucleotides does not occur when cells are cultured in the absence of performed pyridine ring compounds; however, growth continues normally for up to four cell doublings resulting in cells with a total pyridine nucleotide content that is reduced by as much as 12‐fold. The mechanism that adjust the relative amounts of NADP and NAD are also altered such that the amount of NADP relative to NAD increases 5‐fold. Both nicotinate and nicotinamide can be used as a precursor for NAD biosynthesis, however nicotinate is utilized less efficiently than nicotinamide. The presence of functional pathways for the biosynthesis of NAD from nicotinate via nicotinate mononucleotide and nicotinate adenine dinucleotide and from nicotinamide via nicotinamide mononucleotide has been demonstrated by identification of biosynthetic intermediates following short term exposure of cells to radiolabelled precursors. When cells are grown in Dulbecco's modified Eagle's medium which contains 33 μM nicotinamide the biosynthesis of NAD proceeds by a single pathway with nicotinamide mononucleotide as the only intermediate. Nicotinamide ribonucleoside which previously has been postulated to be an intermediate in the conversion of nicotinamide to NAD is not an intermediate in NAD biosynthesis.
Properties of Na‐K pump in primary cultures of kidney cells
Authors: Becker John H. , Willis John S.
Published: February 2005

Activities related to Na‐K transport were measured in cell cultures of ground squirrel kidney cortex in order to compare these cells with those of intact kidney and of continuous cell lines. A microsomal preparation containing plasma membrane Na,K‐ATPase from fresh kidney showed twice the activity of a similar preparation from 72‐hour cultured cells. Na,K‐ATPase of homogenates of 72‐hour cells showed one‐third to one‐fourth the specific activity of that from 6‐hour cultured cells. The associated K‐dependent phosphatase activity also declined as a function of time in culture. The ouabain‐sensitive influx of K into 6‐hour cultured cells was twice as great as the K influx into 72‐hour cells. The number of sites binding 3H‐ouabain in intact cultured cells declined 81% on a cell protein basis between 6 and 72 hours in culture. This decline in ouabain binding sites was relatively greater than that of K influx, so that the K turnover number increased over this same time period.

The decline in ouabain‐sensitive K influx during culture was complementary to an increase in furosemide‐sensitive K influx. Measurements of unidirectional and net K fluxes showed that there were three components of K influx into 3‐day cultured cells: ouabain‐sensitive Na:K exchange, furosemide‐sensitive K:K exchange, and K diffusion. In the 6‐hour cultures, however, there was no furosemide‐sensitive K:K exchange.

Thus, after three days in culture ground squirrel kidney cells lose a feature characteristic of the original parent cells (high Na,K‐ATPase activity), and gain a feature common to many undifferentiated cultured cells (furosemidesensitive K:K exchange).