bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of heparin on vascular smooth muscle cells. II. Specific protein synthesis
Authors: Cochran David L. , Castellot John J. , Karnovsky Morris J.
Published: February 2005

Heparin suppresses the proliferation of vascular smooth muscle cells both in vivo and in vitro. The mechanism of action of the antiproliferative activity of heparin is not known. We have detected differences in the synthesis of specific proteins when vascular smooth muscle cells are exposed to heparin and report here that many characteristics of these protein alterations parallel the properties of the antiproliferative activity. The induction into the culture medium of a pair of proteins of approximately 35,000 dalton mw in heparintreated smooth muscle cell cultures and the antiproliferative effect of heparin share the following characteristics: 1) the effect is reversible, 2) the effect is specific for smooth muscle cells, 3) anticoagulant and non‐anticoagulant heparin are equally effective, 4) the effect is lost with time in culture and, 5) thermore, heparin causes a transient suppression of a 48,000 dalton substrateattached protein, whereas chondroitin sulfate A and C and dermatan sulfate had much less effect. Dextran sulfate was almost as effective as heparin in suppressing the synthesis of the substrate‐attached protein. These proteins appear to be noncollagenous and the induced synthesis of the 35,000 dalton proteins is inhibited by actinomycin D. Although a direct relationship between these specific protein changes and the antiproliferative effect of heparin has not been proven, these protein alterations may play a crucial role in the effect of heparin on smooth muscle cell growth.
Mitogen and co‐mitogen stimulation of lymphocytes inhibited by three Ca++ antagonists
Authors: Grier Charles E. , Mastro Andrea M.
Published: February 2005

The Ca++ requirement for in vitro lymphocyte stimulation by lectins is well known and can be demonstrated by the use of Ca++ chelators. In this study, three Ca++ antagonists were examined for their effects on lymphocyte proliferation. [3H]‐thymidine incorporation was employed to measure DNA synthesis in several systems. Stimulation and proliferation were achieved by the addition of one of the following: the mitogenic lectin concanavalin A (ConA); the combination of two co‐mitogens, the calcium ionophore A23187 and the phorbol ester, 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA), neither of which is mitogenic alone; or the non‐mitogenic lectin, wheat germ agglutinin (WGA) with TPA. These mitogenic systems were tested for their sensitivity to the Ca++ channel blockers verapamil and nicardipine and the intracellular Ca++ antagonist TMB‐8. We found that the ConA and WGA plus TPA treated cells were inhibited approximately 50% by 10 μM verapamil, nicardipine or TMB‐8. The stimulation caused by A23187 and TPA was only inhibited by TMB‐8 and nicardipine. The inhibitory effects caused by the Ca++ antagonists could not be reversed by the addition of exogenous Ca++ (0.1–1.5 mM), but were reversed by repeated washings in antagonist free media. Using TMB‐8 we saw an apparent intracellular Ca++ dependence throughout the G1 phase. Previous studies using Ca++ chelators or Ca++ antagonists suggested an endpoint at about halfway through this period.
Regulation of thymidine kinase activity in the cell cycle by a labile protein
Authors: Coppock Donald L. , Pardee Arthur B.
Published: February 2005

Previous studies have shown that the onset of DNA synthesis in Balb/c 3T3 cells appears to be regulated by a labile protein. We have found that induction of thymidine kinase (TK) activity, after quiescent cells are stimulated by the addition of serum, is similarly regulated by a labile protein. Eight hours after serum stimulation, a 6‐h pulse of cycloheximide (CHM) caused an excess delay of 2h in TK induction. A similar delay also was found in the induction of thymidylate synthase (TS). In contrast, the bonzo(a)pyrene transformed 3T3 cell line, BP‐A31, which had previously been shown to have to excess delay for the onset of DNA synthesis also had no excess delay for the induction of TK activity after a pulse of CHM. The indudction of TK was inhibited by actinomycin D and dicholoribofuranosylbenzimidizole (DRB) suggesting a requirement for new RNA synthesis. It did not appear to depend on DNA synthesis as it was not blocked by aphidicolin. In conclusion, the induction of TK activity appears to be regulated by the same labile cellular signal as the onset of DNA synthesis, and to depend on an increase in the level of TK mRNA in late G1 or early S phase.
The specific phosphorylation of a 40S ribosomal protein in growth‐arrested tetrahymena is induced by sodium
Authors: Cuny Marguerite , Sripati Conjeevaram E. , Hayes Donal H.
Published: February 2005

In the past few years, in vivo phosphorylation of ribosomal proteins has been the subject of extensive studies and the results have shown that reversible phosphorylation of small subunit ribosomal protein S6, ubiquitous in eukaryotic cells, is apparently related to regulation of protein synthesis initiation. Thus the level of protein synthesis under various conditions is correlated with the level of S6 phosphorylation. In exponentially growing Tetrahymena, however, such phosphorylation does not occur, but when these cells are transferred to starvation buffers, the rate of protein synthesis is drastically reduced and a 40S ribosomal protein analogous to S6 of higher eukaryotic cells is fully and rapidly phosphorylated in all the ribosomes. We have studied the conditions which lead to this phosphorylation in growth‐arrested Tetrahymena, in order to understand the physiological significance of this process. Our results show that there is no obvious correlation between this phosphorylation and starvation. Moreover, it is not a developmentally regulated process related to the conjugation cycle, but a modification induced by the presence of sodium ions or high concentration of Tris in the starvation buffer. The physiological significance of this process is discussed in terms of accumulation of negative charge density probably required for initiation of protein synthesis in the growth‐arrested cells starving in Na+‐containing buffers.
Amphiphilic cationic peptides mediate cell adhesion to plastic surfaces
Authors: Rideout Darryl C. , Lambert Michael , Kendall Debra A. , Moe Gregory R. , Osterman David G. , Tao H. P. , Weinstein I. Bernard , Kaiser E. T.
Published: February 2005

Four amphiphilic peptides, each with net charges of +2 or more at neutrality and molecular weights under 4 kilodaltons, were found to mediate the adhesion of normal rat kidney fibroblasts to polystyrene surfaces. Two of these peptides, a model for calcitonin (peptide 1, MCT) and melittin (peptide 2, MEL), form amphiphilic α‐helical structures at aqueous/nonpolar interfaces. The other two, a luteinizing hormone‐releasing hormone model (peptide 3, LHM) and a platelet factor model (peptide 4, MPF) form β‐strand structures in amphiphilic environments. Although it contains only 10 residues, LHM mediated adhesion to surfaces coated with solutions containing as little as 10 pmoles/ml of peptide. All four of these peptides were capable of forming monolayers at air‐buffer interfaces with collapse pressures greater than 20 dynes/cm. None of these four peptides contains the tetrapeptide sequence Arg‐Gly‐Asp‐Ser, which has been associated with fibronectin‐mediated cell adhesion. Ten polypeptides that also lacked the sequence Arg‐Gly‐Asp‐Ser but were nonamphiphilic and/or had net charges less than +2 at neutrality were all incapable of mediating cell adhesion (Pierschbacher and Ruoslahti, 1984). The morphologies of NRK cells spread on polystyrene coated with peptide LHM resemble the morphologies on fibronectin‐coated surfaces, whereas cells spread on surfaces coated with MCT or MEL exhibit strikingly different morphologies. The adhesiveness of MCT, MEL, LHM, and MPF implies that many amphiphilic cationic peptides could prove useful as well defined adhesive substrata for cell culture and for studies of the mechanism of cell adhesion.
Keratinocyte growth‐promoting activity from human placenta
Authors: O'Keefe E. J. , Payne R. E. , Russell N.
Published: February 2005

Extracts of term human placenta were tested for enhancement of proliferative growth of primary cultures of human keratinocytes. Saline extracts or supernatants from homogenates were dialyzed extensively, lyophilized, and tested in subcultures of keratinocytes in MCDB 153 medium with 0.1 mM Ca++ containing only defined supplements (insulin, hydrocortisone, transferrin, ethanolamine, phosphoethanolamine). Cells plated in the absence of EGF at moderately high densities (1000–3000 cells per cm2) formed colonies and grew in the presence of placental extract at 25–500 μg/ml. Extracts of cord serum or maternal serum were inactive, suggesting that the activity is derived from placental tissue. The activity is not EGF, since the activity in the placental extract, unlike EGF, did not promote growth at low cell density, was synergistic with EGF under some conditions, and did not produce changes in colonial morphology which occurred in the presence of EGF. Unlike keratinocyte growth‐promoting activity in bovine hypothalamic extract, the activity is nondialyzable and is destroyed at 100°C. Placental extract could not replace any of the defined components of the medium and is therefore distinct from them. The presence of activity in the placenta with distinctive properties suggests that this is a previously undescribed material with growth‐promoting properties for epithelium.
Opposing effects of dexamethasone on the clonal growth of granulocyte and macrophage progenitor cells and on the phagocytic capability of mononuclear phagocytes at different stages of differentiation
Authors: Shezen Elias , Shirman Michael , Goldman Rachel
Published: February 2005

Dexamethasone, a synthetic glucocorticosteroid, was shown to modulate the colony‐stimulating factor‐dependent clonal growth of myeloid progenitor cells in semisolid agar cultures, enhancing the formation of granulocyte colonies (50–100%) and suppressing the formation of macrophage colonies (75–97%). Modulation of the pattern of myeloid colony formation by dexamethasone (12–125 nM) was brought about when the steroid was administered to 6‐day cultures at the time of culture initiation and up to 72 hr later. Dexamethasone inhibited myeloid cell proliferation when administered to 5‐day liquid cultures at culture initiation and up to 96 hr later. Dexamethasone (12–250 nM) also enhanced the phagocytic activity of bone marrow‐derived mononuclear phagocytes toward heat‐killed (HK) yeast cells (up to 100%) and IgG‐coated sheep red blood cells (up to 60%). Enhancement of the phagocytic capability depended critically on the stage in culture at which dexamethasone was administered. Exposure to dexamethasone for 28 hr up to 96 hr of 96‐hr cultures of bone marrow cells did not lead to a modulation of phagocytic activity of the developing mononuclear phagocytes. The presence of dexamethasone during the critical period of 96 hr to 120 hr after culture initiation led to an enhanced phagocytic capability, which was statistically significant already 12 hr after the administration of the glucocorticoid. Dexamethasone induced an enhanced phagocytic activity when administered at any time after culture initiation provided that it was in culture during this critical period. When added at 120 hr of culture, dexamethasone no longer enhanced the phagocytic capability of mononuclear phagocytes and when added later than 156 hr of culture suppressed it. Dexamethasone also suppressed (up to 68%) the phagocytic capability of resident and elicited peritoneal macrophages. The results suggest that glucocorticoids shift the balance of granulocyte vs. macrophage formation at early stages of precursor cell differentiation. Reduction in mononuclear phagocyte growth and enhancement of its phagocytic capability might reflect accelerated differentiation/maturation steps. The inhibitory effect of dexamethasone on macrophage formation and on the phagocytic capability of mature mononuclear phagocytes and peritoneal macrophages might be a relevant aspect of the in vivo immune suppression encountered after glucocorticoid administration.
DNA synthesis in rat hepatocytes: Inhibition by a platelet factor and stimulation by an endogenous factor
Authors: Hayashi Izumi , Carr Brian I.
Published: February 2005

Primary monolyer cultures of adult rat hepatocytes can be induced to undergo DNA synthesis in serum‐free medium in the presence of insulin, glucagon, and epidermal growth factor (three factors). We have found that hepatocyte DNA synthesis is affected not only by an endogenous stimulant produced by the hepatocytes and released into the culture medium. Serum has a strong inhibitory effect on hepatocyte DNA synthesis. Partially purified human platelet extract (“platelet inhibitor”) inhibits the three‐factor‐induced DNA synthesis in a concenration‐dependent manner. Pure βTGF at 0.5 ng/ml as well as HPLC‐purified PDGF at 10 ng/ml completely inhibit the three‐factor‐induced DNA synthesis. Determination of the time required for the presence of the three factors and the platelet inhibitor to exert their effects indicated that the inhibition of DNA synthesis is caused not by competition of the platelet inhibitor with any of the three factors but through an independent pathway. Hepatocyte DNA synthesis is density‐dependent and is greater if medium is not changed during the course of an experiment than if medium is changed daily. Hepatocyte‐conditioned medium is also affective in stimulating DNA synthesis beyond the level induced by the three factors. These results suggest that an endogenous stimulant for hepatocyte DNA synthesis is produced by the hepatocytes themselves. Our studies demonstrate that hepatocyte DNA synthesis is subject to both stimulatory and inhibitory controls. Unlike the three factors, the endogenous stimulant can overcome the inhibition by the platelet inhibitor, suggesting the importance of these factors in the physiological control of hepatocyte DNA synthesis.
Regulation of purine biosynthesis in G1 phase‐arrested mammalian cells
Authors: Emmett Kim , Patrick Joe , Aronow Bruce , Ullman Buddy
Published: February 2005

The effects of G1 phase growth arrest on purine biosynthesis were studied in cultured S49 T lymphoma cells. Incubations of wildtype S49 cells for 18 hr with dibutyryl cyclic AMP or forskolin, two agents which induced G1 arrest, reduced the rates of purine biosynthesis by 95%. Time course and concentration dependence studies indicated that the decrease in rates of purine biosynthesis correlated with the extent of G1 phase arrest. Similar studies with somatic cell mutants deficient in some component of cyclic AMP action or metabolism indicated that the depression in purine synthetic rates required G1 arrest and did not result from cell death. Rates of RNA and DNA synthesis were also markedly diminished in the growth arrested cells. Measurements of purine rates in the presence of azaserine indicated that the block in purine biosynthesis was prior to the formation of phosphoribosylformylglycinamide. Additionally, the activities of adenylosuccinate synthetase and IMP dehydrogenase were diminished in G1 arrested cells. The levels of all controlling enzymes, substrates, and cofactors, however, were not diminished in G1 arrested cells. Despite diminished rates of purine biosynthesis, the amounts of intracellular nucleotides in G1 cells were equivalent to those in exponentially growing cells. However, the concentrations of intracellular nucleotides were 30–50% higher in the growth arrested cells. These results suggested that perturbations in the consumption of nucleotides via inhibition of nucleic acid synthesis have profound effects on the purine pathway and indicated the importance of feedback inhibition by nucleotides in the regulation of purine synthesis in situ.
Induction and/or selective retention of proteins in mammalian cells exposed to cycloheximide
Authors: Sorrentino V. , Battistini A. , Curatola A. M. , Francesco P. Di , Rossi G. B.
Published: February 2005

Exposure of a number of murine and human cell lines to low graded doses of cycloheximide (CXM) results in a pattern of protein synthesis consisting of enhanced and induced species. These can be divided into two main classes according to molecular weight (20–40 and 70–120 Kd), similar to what has been described for other agents that modify the physiological conditions of growth. In addition, the pronounced synthesis of a hitherto unreported 50‐Kd protein species has been consistently observed in all lines tested. Simultaneous exposure of cells to CXM and actinomycin D results in suppressing synthesis of some but not all protein species observed, indicating that control mechanisms at both transcriptional and posttranscriptional levels may be operative in this system.
Incorporation of [3H]thymidine by isolated fetal myoblasts and fibroblasts in response to human placental lactogen (HPL): Possible mediation of HPL action by release of immunoreactive SM‐C
Authors: Hill D. J. , Crace C. J. , Milner R. D. G.
Published: February 2005

We investigated the actions of human placental lactogen (HPL) and human growth hormone (HGH) on [3H]thymidine incorporation and the release of immunoassyable somatomedin‐C (SM‐C) by isolated myoblasts, dermal fibroblasts, and costal cartilage explants taken from human fetuses, at 11–21 weeks of gestation. The incorporation of [3H] thymidine by myoblasts and fibroblasts was significantly increased after incubation for 20 hr or 44 hr, and cell number after incubation for 7 days, in the presence of 50–250 ng/ml HPL. Incubation with HPL did not increase [3H]thymidine incorporation into cartilage explants, whereas incubation with HGH failed to enhance the uptake of this isotope by any of the tissues. Following extraction with acid‐ethanol, culture medium conditioned by exposure to myoblasts or fibroblasts for 44 hr, and to cartilage explants for 7 days, contained radioimmunoassayable SMC. Myoblast‐conditioned medium contained significantly more SM‐C [1,609 ± 953 mU/mg cell protein (mean ± SD) n = 10] than did that conditioned by fibroblasts (637 ± 323; n = 5; P < 0.02). In 1 week of culture, cartilage explants released 4.1 ± 1.1 mU/mg wet weight (n = 7). The release of immunoassayable SM‐C from cultured cells was significantly increased in the presence of 250 ng/ml HPL in five of eight experiments with myoblasts and two of four experiments with fibroblasts. Neither fibroblasts or myoblasts showed increased SM‐C release following exposure to HGH.

The results suggest that HPL, but not HGH, is growth‐promoting for some human fetal tissues in vitro and that this action is mediated, at least in part, by an increased release of somatomedins.
Serum factors that stimulate fatty acid oxidation: Properties of factors
Authors: Wice Burton M. , Stanisz Jolanta , Kennell David E.
Published: February 2005

Cultured heart muscle cells, but not HeLa cells, oxidize long‐chain fatty acids in medium containing dialyzed serum. Addition of chicken serum dialysate (or non‐dialized serum) stimulated palmitic acid oxidation by HeLa cells 10 to 20 fold. This serum activity was not eliminated by lipid extraction, ethanol or acid precipitation, alkaline phosphatase treatment, or autoclaving. About 80% was lost after any one of the following treatments: 6N HCl at 110°C for 16 hr, pepsin, Dowex cation exchange at pH 3, or 1N KOH at 100°C for 1 hr. Serum activity was separated into five or more peaks by gel filtration with Sephadex G‐10. Each of these peak fractions was further purified by HPLC using a cyanopropyl‐bonded resin. Carnitine, which is important for the transport of long‐chain fatty acids into mitochondria for oxidation, also stimulated the oxidation of palmitate. However, these serum factors are not known precursors to carnitine since its immediate precursor 4‐n‐trimethylaminobutyrate, did not stimulate palmitate oxidation. Total carnitine, including that in acylcarnitine compounds, was approxoimately 15 μM in the chicken sera to give ∼ 0.7 μM in the medium. Based on the fraction of total activity accountable by carnitine and fractional stability to acid, alkali, and pepsin, about 75% of the activity is from non‐carnitine compounds. Only one of the factors appears to be carnitine or an acylcarnitine derivative. Several lines of evidence suggest that the other factors are peptide compounds.
Cell kinetic study on GM colonies using autoradiography and collagen gel culture with special reference to unique proliferation of eosinophils
Authors: Izumi Tomoyuki , Suda Tetsuo , Shinonome Shougo , Maekawa Tadashi
Published: February 2005

Using a newly developed technique of autoradiography and collagen gel culture, a kinetic study on human GM colonies was attempted. Colonies of immature cells appeared first on day 5. The number of mixed colonies (mixture of immature cells, neutrophils, and/or monocyte‐macrophages) and neutrophil colonies attained a maximum on days 8 to 10 and a broad peak of monocyte‐macrophage colonies was observed on days 11 to 16. Eosinophil colonies appeared first on day 12, reached a maximum on day 18, and then gradually decreased. A detailed analysis of the order of appearance of the colonies suggests that mixed, neutrophil and monocyte‐macrophage colonies originate from immature cell colonies or clusters, while eosinophil colonies do not. An autoradiographic study was designed to study the proliferation characteristics of each colony. Labeling indices (LI) with 3H‐TdR of the cells in immature cell colonies were always high. LI of the cells in differentiated colonies such as neutrophil, monocyte‐macrophage, and mixed colonies were low throughout the observation period. In contrast, LI of the cells in eosinophil colonies were constantly high regardless of the size of cell aggregates and the duration of the culture period. Both mitotic indices and mean grain counts on the nuclei of eosinophils were similar to those of immature cells. These results suggest that eosinophil colonies develop from their own small clusters and that eosinophils retain a fairly good proliferative capacity even when differentiated to the level in which specific granules appear in the cytoplasm.
A direct correlation between hyperthermia‐induced membrane blebbing and survival in synchronous G1 CHO cells
Authors: Borrelli M. J. , Wong R. S. L. , Dewey W. C.
Published: February 2005

Heating synchronous G1 cells at 45.5°C for 3–20 min induced varying degrees of membrane blebbing ranging from nonblebbed cells indistinguishable from control cells to those with blebs larger than the cell itself. Both the proportion of cells exhibiting blebbing and the mean diameter of the blebs increased with heating duration. Scoring individual cells for both blebbing and colony formation demonstrated that cells with blebs larger than 50% of the cell diameter did not survive to form colonies. Electron microscopy showed that all subcellular organelles, save the ribosomes, were absent from the membrane blebs. Freeze fracture replicas revealed no changes in membrane ultra‐structure, except on some 15% of the blebs that contained bald patches devoid of membrane particles.
Substratum influence on collagen and fibronectin biosynthesis by arterial smooth muscle cells in vitro
Authors: Holderbaum Daniel , Ehrhart L. Allen
Published: February 2005

Collagen, fibronectin, and nonfibrous protein biosynthesis were examined in cultures of rabbit arterial smooth muscle cells grown on tissue culture plastic precoated either with rabbit plasma fibronectin or bovine serum albumin. Cells seeded into fibronectin‐coated wells appeared to reach confluence more quickly than counterparts grown on albumin‐coated surfaces. Measurement of 3H‐thymidine incorporation into DNA by these cultures suggested that this was probably a consequence of more rapid and efficient cell attachment rather than an increased rate of proliferation of smooth muscle cells grown on fibronectin. In preconfluent cultures, the rates of collagen and fibronectin biosynthesis were reduced to 34 and 57%, respectively, on a per‐cell basis in cultures grown on fibronectin‐coated surfaces compared with cells grown on albumin‐coated plasticware. In preconfluent cultures grown on fibronectin‐coated surfaces, a greater percentage of the total fibronectin synthesized was incorporated into the cell layer. The distribution of newly synthesized collagen between culture medium and cell layer, however, was not affected by alteration of substratum composition. There was no difference in the rate of synthesis of noncollagen proteins between the two groups of preconfluent cells. In postconfluent cultures the rates of collagen and fibronectin biosynthesis were equivalent in both albumin‐ and fibronectin‐treated cultureware. In preconfluent cultures, analyses of procollagens showed that the overall amounts of both types I and III procollagens were reduced in fibronectin‐treated wells, indicating the reduction in collagen synthesis to be general and not type‐specific. Although type V procollagen biosynthesis was not detected in either preconfluent group, it was found in postconfluent cultures. The reduction of fibronectin synthesis in cells grown in fibronectin‐coated wells was significant as early as 4 hours after plating. Together, these findings suggest that cultured arterial smooth muscle cells are capable of deriving information from their substratum and regulating the biosynthetic rates of extracellular matrix components in response to the immediate needs of the cell.
Nonhaem complexes of FeIII stimulate cell attachment and growth by a mechanism different from that of serum, 2‐oxocarboxylates, and haemproteins
Authors: Kay G. F. , Ellem K. A. O.
Published: February 2005

Most cell lines, even those producing their own growth factors, need a serum supplement when growing in several commonly used media. The requirement for serum to sustain attachment and growth in RPMI 1640 and MEM has been found to be met by a range of 2‐oxocarboxylates, by diverse coordination complexes of Felll, and by a variety of haem‐containing proteins including catalase. The latter directly implicates H2O2 in the serum shift‐down effects. H2O2 was found to accumulate in low serum media under normal laboratory lighting conditions to levels that were shown to be sufficient, when added to freshly prepared media, to explain the depressed cell performance. With the exception of some of the nonhaem Felll coordination complexes, substances found to stimulate cell attachment and growth were capable of scavenging H2O2. This suggests that an important function of serum and the 2‐oxocarboxylates (α‐keto acids) frequently used as “nonessential” medium additives is to remove H2O2 produced photodynamically during the storage and manipulation of media containing a high content of riboflavin. However, the nonhaem Felll complexes with saturated coordination shells, although capable of reducing photodynamic generation of H2O2 to a greater or lesser extent, have their prime effect by an unknown, intriguing mechanism, probably based on a common redox function.
Regulation of the A system of amino acid transport in Chinese hamster ovary cells, CHO‐K1: The difference in specificity between the apo‐repressor inactivator (apo‐ri) and the transporter and the characterization of the proposed apo‐ri
Authors: Moffett John , Englesberg Ellis
Published: February 2005

When amino acids that are generally transported through the A system are added to derepressed cultures of CHO‐K1 cells or to cultures that are undergoing starvation‐derepression, as in the co‐repressor (co‐r), co‐inactivator (co‐i), (co‐ri) assay, the A system undergoes trans‐inhibition, inactivation, and repression. The effect of inactivation and repression is not related to the ability of amino acids to bind to the A system transporter but supports a model in which these amino acids act as co‐r's/co‐i's, and by binding to a aporepressor/inactivator (apo‐ri), the product of gene R1, convert it into a repressor/inactivator (ri). For example, β‐alanine acts as a strong co‐r but does not inhibit proline transport through the A system. Hydroxyproline and histidine, although poor inhibitors of proline transport, are very effective as co‐ri's. Diaminobutyrate, phenylalanine α‐keto glutarate, pyro‐glutamate, isoleucine, and valine, compounds that inhibit A system transport, listed in decreasing order of effectiveness, are all equally poor as co‐ri's. Also the Km for the transport of 2‐(methylamino)isobutyric acid (MeAIB) through the A system is two times the concentration of MeAIB required to produce one‐half inactivation. Amino acid effectors and mutation can modify the conversion of the apo‐ri to repressor (r) and inactivator (i). The apo‐ri is converted by alanine, serine, proline, and MeAIB to ri, by β‐alanine and tryptophane to r, and by hydroxyproline to r and reduced i. The full constitutive and partial constitutive mutants alar4 and alar2, respectively, are in the same complementation group. Alar4 has no active apo‐ri while the rate of derepression of alar2 is twice and the inactivation rate is equal to that of the parent culture.
DNA‐mediated transfer of cAMP resistance in CHO cells
Authors: Abraham Irene , Brill Steven , Chapman Margaret , Hyde Jennifer , Gottesman Michael
Published: February 2005

Chinese hamster ovary (CHO) strain 10215 carries a dominant mutation which confers resistant to cAMP by virtue of an altered catalytic subunit of the cAMP‐dependent protein kinase (Evain et al., 1979). This mutation was transferred to wild‐type CHO cells by DNA‐mediated gene transfer. Based on the absence of cAMP growth inhibition, seven transformant colonies were isolated. One of these, 11586, was studied in detail. This transformant showed the same phenotype as the mutant, including resistance to the morphological changes and growth inhibitory effects of 1 mM 8‐Br‐cAMP, reduced total cAMP dependent protein kinase activity and lowered sensitivity of the kinase to cAMP activation. When the cAMP‐dependent protein kinase was fractionated on a DEAE‐cellulose column, the transformant was lacking in type II cAMP dependent protein activity, to the same degree as the mutant. The transformant and mutant, but not wild‐type cells, also failed to phosphorylate a 52,000‐dalton protein in a cAMP‐dependent manner. These characteristics support the conclusion that the gene for the mutant cAMP‐dependent protein kinase has been transferred. The ability to transfer this gene by DNA‐mediated transfer suggests that this methodology may be useful for the molecular isolation of the gene encoding the catalytic subunit of cAMP‐dependent protein kinase.
Multilineage, non‐species specific hematopoietic growth factor(s) elaborated by a feline fibroblast cell line: Enhancement by virus infection
Authors: Abkowitz Janis L. , Holly Richard D. , Segal Gerald M. , Adamson John W.
Published: February 2005

In studies designed to determine the role of feline leukemia virus (FeLV) in the pathogenesis of marrow failure in the cat, we tested medium conditioned by uninfected and FeLV‐infected feline embryonic fibroblasts (FEA) for its effect on hematopoietic colony growth in culture. As opposed to an inhibitory effect, we found that the conditioned medium (CM) from FEA or FEA/FeLV increased the in vitro growth of multiple hematopoietic progenitor cell types including erythroid burst‐forming cells (BFU‐E), granulocyte/macrophage colony‐forming cells, megakaryocytic colony‐forming cells, and mixed‐cell colony‐forming cells. Furthermore, CM enhanced the growth of progenitors in cultures of mouse or human marrow cells, as well as cat marrow cells. Stimulation of feline BFU‐E was most marked with an increment in growth of 400% over control. The human burst promoting activity (BPA) of the CM was equivalent or better than other CM available in our laboratory. The evidence suggests that the growth‐promoting activity is a constitutive product(s) released by FEA which was enhanced eightfold with virus infection. Studies with non‐adherent and T‐lymphocyte‐depleted human marrow cells and human peripheral blood cells suggest that the growth factor(s) acts directly on progenitor cells and not through readily identified accessory cells. These findings are consistent with the concept that mesenchymal cells such as fibroblasts have the capacity to release hematopoietic growth factor(s) capable of acting on primitive hematopoietic progenitors. The results provide an example of how injury of such cells, through virus infection, may enhance growth factor(s) release and influence the hematopoietic microenvironment.
Selection and characterization of mutant S49 T‐lymphoma cell lines resistant to phosphonoformic acid: Evidence for inhibition of ribonucleotide reductase
Authors: Albert Daniel A. , Gudas Lorraine J.
Published: February 2005

Phosphonoformic acid (PFA) and its congener phosphonoacetic acid (PAA) are inhibitors of viral replication whose mechanism of action appears to be the inhibition of viral DNA polymerase. These drugs inhibit mammalian DNA polymerase to a lesser extent. We sought to characterize the effects of phosphonoformic acid on mammalian cells by examining mutants of S49 cells (a mouse T‐lymphoma line), which were selected by virtue of their resistance to phosphonoformic acid. The 11 mutant lines that were resistant to growth inhibition by 3 mM PFA had a range of growth rates, cell cycle distribution abnormalities, and resistance to the inhibitory effects of thymidine, acycloguanosine (acyclovir), aphidicolin, deoxyadenosine, and novobiocin. Most mutant lines had pools of ribonucleoside triphosphates and deoxyribonucleoside triphosphates similar to those of wild‐type S49 cells. However, one line (PFA 3–9) had a greatly elevated dCTP pool. When this mutant line was further characterized, no apparent defect in DNA polymerase α activity was seen, but an increased ribonucleotide reductase activity, as assayed by CDP reduction in permeabilized cells, was observed. The CDP reductase activity in the PFA 3–9 cells decreased to wild‐type control levels, and the CDP reductase activity of wild‐type cells was also greatly reduced when PFA (2–3 mM) was added to permeabilized cells during the enzyme assay. These results demonstrate that PFA can directly inhibit ribonucleotide reductase activity in permeabilized cells. In addition, when PFA was added to exponentially growing cultures of either wild‐type or PFA 3–9 mutant cells, the drug caused an arrest in S phase of the cell cycle and a decrease in all four deoxyribonucleotide pools, with the most dramatic decrease in the dCTP pools. The reduction in the dCTP pool level could be reversed by addition of exogenous deoxycytidine, but this reversed PFA toxicity only marginally. These observations suggest that PFA is an inhibitor of mammalian ribonucleotide reductase and that partial resistance to PFA can be effected by mutation to increased CDP reductase activity resulting in a large dCTP pool. This mutation results in less than twofold resistance to PFA, suggesting that other sites of inhibition coexist.
Transient stimulation of glucose metabolism by insulin in the 1‐day chick embryo
Authors: Baroffio Anne , Raddatz Eric , Markert Michèle , Kučera Pavel
Published: February 2005

Effects of insulin upon glucose metabolism were investigated in chick embryos explanted in vitro during the first 30 h of incubation. Insulin stimulated the glucose consumption of the chick gastrula (18 h) and neurula (24 h), but had no effect on the late blastula (0 h:laying) and on the stage of six to eight somites (30 h). The increase in glucose consumption concerned both the embryonic area pellucida (AP) and extraembryonic area opaca (AO). AP responded to a greater extent (50%) and at a lower range of concentrations (0.1–1.0 ng/ml) than AO (30%; 1–100 ng/ml). Insulin had no effect on the oxygen consumption of blastoderms, whereas it stimulated the aerobic lactate production (approximately 70% of the additional glucose consumption was converted to lactate).

The nanomolar range of stimulating concentrations suggests that insulin has a specific effect in the chick embryo, and that it could modulate glucose metabolism in ovo as well. The transient sensitivity of the embryo to insulin is discussed in relation to behavior of mesodermal cells.
Retinoic acid effect on cyclic AMP‐dependent protein kinases in embryonal carcinoma cells: Studies with differentiation‐defective sublines
Authors: Plet Ariane , Gerbaud Pascale , Sherman Michael I. , Anderson Wayne B. , Brion Daniele Evain
Published: February 2005

Retinoic acid induces the differentiation of PCC4.aza 1R and Nulli‐SCC1 embryonal carcinoma (EC) cells. In response to retinoic acid treatment, the levels of cyclic AMP (cAMP)‐dependent protein kinases are enhanced in the plasma membrane within 17 hours and in the cytosol fractions of these cells within 2 to 3 days, as determined by phosphotransferase activity and by 8‐azido‐cyclic [32P]AMP binding to the RI and RII regulatory subunits. PCC4 (RA)−1 and Nulli (RA)−1 are mutant EC lines that fail to differentiate in response to retinoic acid. The former line, but not the latter, lacks cellular retinoic acid‐binding protein (cRABP). Basal levels of cAMP‐dependent protein kinase activities are elevated in PCC4 (RA)−1 cells. When these cells are treated with retinoic acid, neither cAMP‐dependent protein kinase activities nor cAMP binding activities are enhanced; rather, there is a decrease in cytosolic kinase activity and RI subunit. On the other hand, Nulli (RA)−1 cells exhibit increases both in cAMP‐dependent protein kinase activities and cAMP binding in response to retinoic acid. These results raise the possibility that cRABP mediates the enhancement of regulatory and catalytic subunits of cAMP‐dependent protein kinases in both the membrane and the cytosolic fractions of the teratocarcinoma cells. There also might be some effects of retinoic acid on the cAMP‐dependent protein kinase that are unrelated to differentiation and to the presence of cRABP.
Collagen synthesis by murine bone marrow cell culture
Authors: Waterhouse Elizabeth J. , Quesenberry Peter J. , Balian Gary
Published: February 2005

Collagen types synthesized by murine bone marrow cells were studied and the effect of lithium chloride on collagen biosynthesis in vitro was investigated. In the liquid culture system used, an adherent, mixed cell population supports hemopoiesis. Radioactive labeling of cell cultures and subsequent fractionation with ammonium sulfate, enzyme digestion, immune precipitation, and gel electrophoresis indicated that the bone marrow cells synthesized precursors to collagen types I, III, and IV, and fibronectin. A previously undescribed molecule or fragment with an apparent molecular weight of 17,000 daltons that was susceptible to bacterial collagenase and containing no interchain disulfide bonds was also identified in the culture media of both control and lithium‐treated cells. Lithium treatment did not affect the types of collagen synthesized, although the relative proportions of collagen types may differ from controls. However, lithium does have an effect on the appearance of some, as yet unidentified, non‐collagenous components in the cell culture media.
Characterization of RNA polymerase type II from human term placenta
Authors: Freund Erwin , McGuire Peter M.
Published: February 2005

RNA polymerase type II from human term placenta has been isolated and characterized with respect to its template, ammonium sulfate, divalent cation, and buffer preferences. In addition, the apparent Michaelis constants for AMP amd UMP incorporation have been determined. The enzyme was also analyzed by native and denaturing polyacrylamide gel electrophoresis, and evidence is presented that a single polypeptide is radiolabeled with azido purine nucleoside triphosphate photoprobes.
Isolation and characterization of an erythroid cell line highly inducible to form erythroid burst‐like colonies
Authors: Kimura Nobuhiro , Mak Tak W.
Published: February 2005

The study of induction of Friend erythroleukemic cell lines during the last decade has enriched our understanding of late erythroid differentiation. In comparison, little information is available on early erythroid differentiation. We describe here the isolation and characterization of a highly inducible clone from a murine erythroid cell line, which is capable of forming colonies that possess properties of the early erythroid burst progenitor. We found that a combination of erythropoietin (Epo), spleen conditioned medium (SCM), and plasma from a patient with aplastic anemia (Apa) induces over 95% of cells from this clone (clone 12) to form colonies with the properties of burst or mixed burst blast‐like colonies. Examination of the culture conditions of these cells indicated that alpha medium was more efficient for colony induction than Iscove's medium, and that the addition of two‐mercaptoethanol did not improve the induction process. These factors (EPo, SCM, and Apa) must be present for 4 days in order for induction to take place. It is hoped that the isolation of this highly inducible cell clone will enrich our understanding of early erythroid differentiation.
Permeability of human blood platelets to glycerol
Authors: Armitage W. J.
Published: February 2005

The permeability of human platelets to glycerol was determined at 37°C, 25°C, and 0°C from the rate of change of cell volume after abrupt addition of 0.5 mol/liter glycerol in phosphate‐buffered saline. Intracellular water volume was measured employing both tritiated water and a photometric method. Intracellular glycerol was measured employing tritiated glycerol. The glycerol permeability coefficient derived from the tracer cell volume data was 4.0 ± 0.7 × 10 −7 cm/s at 37°C, and 1.1 ± 0.4 × 10−7 cm/s at 25°C, and the photometric data gave a permeability coefficient of 5.4 ± 0.4 × 10−7 cm/s at 37°C. The activation energy between 23°C and 37°C for glycerol permeation was 19.8 kcal/mol. The cells were virtually impermeable to glycerol at 0°C. The minimum intracellular water volume attained after the addition of 0.5 mol/liter glycerol at 37°C determined by the photometric method was 47.8% of normal water volume, whereas the minimum water volume calculated assuming that glycerol exerted its full osmotic effect (i.e., σ = 1) was 45.6%. The reflexion coefficient was therefore assumed to be unity. Neither method of cell volume determination could be used with 1 or 2 mol/liter glycerol: adequate separation of the cells from the labeled medium could not be achieved in the tracer method; in the photometric method, it was apparent that transmittance (660 nm) was influenced by one or more variables in addition to cell volume.
A fractionation procedure of mouse bone marrow cells yielding exclusively pluripotent stem cells and committed progenitors
Authors: Bauman J. G. J. , Wagemaker G. , Visser J. W. M.
Published: February 2005

The cell surface phenotype of pluripotent hemopoietic stem cells (CFU‐S) and committed progenitors (CFU‐C1, CFU‐C2, BFU‐E) of mouse bone marrow was analyzed with respect to their binding of wheat germ agglutinin (WGA) and two monoclonal antibodies, anti‐GM‐1.2 and anti‐PGP‐1. Stained cells were fractionated on the basis of differences in fluorescence and light scatter intensity using a light‐activated cell sorter. The 6% of the cells that bound most WGA and that also had a relatively high forward light scatter (FLS) and low perpendicular light scatter (PLS) contained nearly all stem cells (CFU‐S) and progenitors. Anti‐GM‐1.2 stained only mature myeloid cells, not CFU‐S or the in vitro colony‐forming cells. Anti‐PGP‐1 stained all bone marrow cells in varying intensities: lymphoid cells were dull, CFU‐S were intermediate, CFU‐C2 were brighter, and mature myeloid cells very bright. Enrichment of progenitor cells was performed by a two‐step sorting procedure. First, the 6% most WGA‐binding cells with high FLS and low PLS were sorted out. A 10–15‐fold enrichment of progenitors and CFU‐S was obtained. Next, these cells were restained with anti‐GM‐1.2 or anti‐PGP‐1 and again fractionated on the FACS. The GM‐1.2‐negative cells were then another four‐ to sevenfold more enriched for stem cells and progenitors. Of the cells in this fraction, 95% could be assigned to a colony‐forming unit. With anti‐PGP‐1, CFU‐C2 could be partly separated from more early cells such as CFU‐S and BFU‐E.
Inhibition of DNA synthesis by an inducer of differentiation of leukemic cells, 1 alpha, 25 dihydroxy vitamin D3, precedes down regulation of the c‐myc gene
Authors: Brelvi Zamir S. , Studzinski George P.
Published: February 2005

Transient monocytic differntiation was induced in cultures of HL 60 cells by a four hour exposure to 1 alpha, 25‐dihydroxy vitamin D3 (1,25(OH)2D3). This treatment resulted in a rapid, selective, inhibition of DNA syntehsis, which was accompanied by reduced cellular levels of c‐myc mRNA, and a more gradual appearance of c‐fos mRNA. After removal of the inducer from the cultures, DNA syntehsis and c‐myc mRNA levels returned rapidly to near‐normal levels, but the expression of c‐fos gene continued to increase for 24 hr and then declined slowly. Studies with isolated nuclei showed that the inhibition of DNA synthesis can be detected earlier than the changes in transcriptional rates of the oncogenes studied, and that 1,25(OH)2D3 directly inhibits the DNA synthesis in isolated nuclei. Autoradiographic studies of [3H] thymidine incorporation showed that 1,25(OH)2D3 does not immediately block the progression of the cells into the S phase of the cell cycle, but that those cells which become differentiated as the result of a brief exposure to this inducer do have such a block. It is concluded that 1,25(OH)2D3 produces both an immediate and a delayed inhibition of DNA synthesis in HL 60 cells, that the immediate inhibition is not preceded by detectable changes in oncogene expression, and that the delayed inhibition is accompanied by an elevated expression of the c‐fos gene, and may be related to the monocytic differentiation of HL 60 cells.
Effect of alterations in the size of the vacuolar compartment on pinocytosis in J774.2 macrophages
Authors: Swanson Joel , Yirinec Brian , Burke Eugene , Bushnell Anne , Silverstein Samuel C.
Published: February 2005

J774.2 macrophages cultured in medium containing 10 mg/ml sucrose accumulate the sugar by pinocytosis and become highly vacuolated, due to the sugar's osmotic effect within the vacuolar compartment. When such cells are incubated in medium containing 0.5 mg/ml invertase, the enzyme reaches the sucrose vacuoles by pinocytosis, then cleaves the sugar to more permeant monosaccharides. Within 4 hours, the vacuoles shrink to smaller, phase‐dense organelles (Cohn and Ehrenreich, 1969, J. Exp. Med., 129:201). We have used this reversible expansion of the lysosomal compartment to address two questions: (1) Does the increased size of the lysosomal compartment affect pinocytic accumulation of solute, and (2) what is the fate of the vacuolar membrane and its soluble content during invertase‐induced vacuole shrinkage? Using lucifer yellow (LY) as a probe for pinocytic fluid influx and efflux, we found that vacuolated cells accumulated 30–50% less LY than controls and returned to higher rates of pinocytosis after invertase‐induced vacuole shrinkage. A similar reduction in LY accumulation was achieved after feeding cells latex beads to increase the size of the lysosomal compartment. Thus, treatments that increased the size of the lysosomal compartment reduced solute accumulation via pinocytosis. A dramatic shrinkage of LY‐containing sucrose vacuoles followed pinocytosis of invertase. Despite this reduction in size of the LY‐containing vacuoles, the overall rate of LY efflux did not increase significantly during invertase‐induced vacuole collapse. Electron microscopy revealed that during shrinkage, the excess vacuolar membrane was compressed into whorled membranous organelles (residual bodies), with fluid markers (colloidal gold and, by inference, LY) trapped inside. The trapping of LY inside lysosomes as J774.2 macrophages returned to their normal dimensions indicates that nearly all of the surplus membrane contents were removed from circulation as well.