bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification of melanoma growth stimulatory activity
Authors: Richmond Ann , Thomas H. Greg 
Published: February 2005

AbstractThe Hs0294 human malignant melanoma cell line produces a monolayer mitogen that stimulates the serum free growth of low‐density cultures of Hs0294 cells. This report describes the purification of that mitogen, termed MGSA for melanoma growth stimulatory activity, from serum‐free conditioned medium from the Hs0294 cultures. MGSA has been purified from acetic acid extracts of lyophilized conditioned medium by gel filtration, reverse‐phase high‐pressure liquid chromatography (RP‐HPLC), and preparative electrophoresis, resulting in a >400,000‐fold purification. MGSA bioactivity resides in acid‐ and heat‐stable polypeptides of high and low molecular weight (24–28 kd and < 14–16 kd). However, the majority of the activity is reproducibly associated with the ˜16‐kd moiety eluting from RP‐HPLC at ∼35% acetonitrile. Reduction with dithiothreitol or B‐mercaptoethanol results in a loss of biological activity but does not convert the 24–28‐kd moieties to the<14–16‐kd forms of MGSA. 125l‐MGSA that has been purified by preparative electrophoresis (16 kd) specifically binds to Hs0294 melanoma cells and retains 100% of the growth‐stimulatory activity. The 16‐kd MGSA stimulates the proliferation of Hs0294 cells at concentrations of 0.3–30 pM. The electrophoretic mobility of MGSA is also unaltered by the preparative electrophoresis procedure, further demonstrating that this procedure does not alter the biochemical integrity of the growth factor. Purified MGSA does not enable anchorage‐independent growth of normal rat kidney (NRK) cells and is therefore different from the previously described transforming growth factors. The amino acid composition of MGSA differs from that of other previously described growth factors. These data demonstrate that MGSA represents a separate class of growth factors with biological and biochemical properties different from other growth factors.
Studies on the mechanism by which glutamine and heat shock increase lactate synthesis by l929 cells in the presence of insulin
Authors: Lanks Karl W. 
Published: February 2005

AbstractBoth glutamine and heat shock increase lactate production in the L929 cell system, Glutamine is now shown to increase hexose uptake in the presence of insulin, to inhibit pyruvate oxidation, and to provide reducing equivalents to the cytosolic compartment. The relative contribution of these processes to lactate production depends on the availability of pyruvate. When ample pyruvate is available from the culture medium, stimulation of lactate synthesis by glutamine and heat shock is transaminase dependent, suggesting that shuttling of reducing equivalents from mitochondria to cytoplasm is involved. In the absence of medium pyruvate, stimulation of glycolysis by both glutamine and heat shock is largely responsible for increased lactate synthesis. None of the observed effects of glutamine appears to be sufficient to explain the observed stimulation of glycolysis.
Effects of nutritional and hormonal factors on the metabolism of retinol‐binding protein by primary cultures of rat hepatocytes
Authors: Dixon Joseph L. , Goodman DeWitt S. 
Published: February 2005

AbstractStudies were conducted to explore hormonal and nutritional factors that might be involved in the regulation of retinol‐binding protein (RBP) synthesis and secretion by the liver. The studies employed primary cultures of hepatocytes from normal rats. When cells were cultured in Dulbecco's modified Eagle's medium alone, a high rate of RBP secretion was observed initially, which declined and became quite low by 24 hr. Supplementing the medium with amino acids maintained RBP and albumin secretion at moderate (but less than initial) rates for at least 3 days. Further addition of dexamethasone maintained the production and secretion rates of RBP, transthyretin, and albumin close to the initial rates for up to 3–5 days in culture. The effects of dexamethasone were not rapid and were not specific for RBP; half‐maximal effects were seen at 10−9 to 10−8 M levels. Hormonally treated hepatocytes produced and secreted RBP, transthyretin, and albumin at both absolute and relative rates similar to physiological values, as estimated from rates reported by others from studies in vivo (with both rats and humans) and with perfused livers. Glucagon addition partially maintained the secretion rates of these 3 proteins, but less effectively than did dexamethasone. A number of other hormones, added singly or in combination, did not affect RBP production or secretion. Addition of retinol to the cultured normal hepatocytes was without effect upon RBP secretion. These studies show that supplementing the culture medium of hepatocytes with amino acids and dexamethasone maintains RBP production and secretion for several days. In normal hepatocytes, with ample supply of retinol available within the cell, addition of exogenous retinol does not appear to influence RBP metabolism or secretion by the cells.
Calcium mobilization in permeabilized fibroblasts: Effects of inositol trisphosphate, orthovanadate, mitogens, phorbol ester, and guanosine triphosphate
Authors: Muldoon Leslie L. , Jamieson Gordon A. , Villereal Mitchel L. 
Published: February 2005

AbstractUtilizing a digitonin‐permeabilized cell system, we have studied the release of calcium from a non‐mitochondrial intracellular compartment in cultured human fibroblasts (HSWP cells). Addition of 1 mM MgATP to a monolayer of permeabilized cells in a cytosolic media buffered to 150 nM Ca with EGTA rapidly stimulates 45Ca uptake, and the subsequent addition of the putative intracellular messenger inositol trisphosphate (InsP3) induces rapid release of 85% (±6% n = 6) of the 45Ca taken up in response to ATP. Mitogenic peptides (bradykinin, vasopressin, epidermal growth factor [EGF], and insulin) and orthovanadate, which are effective in mobilizing intracellular Ca in intact cells, have little or no effect when added alone to permeabilized cells. However, in the presence of GTP these agents stimulate accumulation of inositol phosphates and release Ca from the InsP3‐sensitive pool. These data suggest that a GTP binding protein is involved in receptor mediated activation of phospholipase C, which leads to release of inositol phosphates. The GTP‐dependent release of InsP3 and the mobilization of 45Ca from the intracellular compartment are inhibited by pretreatment of cells, prior to permeabilization, with the protrein kinase C activator 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). TPA pretreatment does not affect the InsP3 stimulated Ca release. These results suggest that protein kinase C is involved in down‐regulation or inhibition of phospholipase C, or the GTP binding protein responsible for relaying the mitogenic signal from the cell surface receptor to the phospholipase C activity.
Mitogenic effect of double‐stranded RNA in human fibroblasts: Role of autogenous interferon
Authors: Vilček Jan , Kohase Masayoshi , Henriksen‐Destefano Dorothy 
Published: February 2005

AbstractThe synthetic double‐stranded RNA polyinosinate‐polycytidylate [poly‐(I).poly(C)] was mitogenic in cultures in human foreskin fibroblasts, as demonstrated by a stimulation of 3H‐thymidine incorporation and an increase in cell density. Poly(I).poly(C) is a potent inducer of interferon (IFN)‐beta in human fibroblasts. Single‐stranded poly(I) or poly(C) were not mitogenic in human fibroblasts and did not stimulate IFN production. Antiserum to interferon (IFN)‐beta, added to poly(I).poly(C)‐stimulated cultures in order to neutralize endogenously generated IFN, markedly amplified the mitogenic action. Under similar experimental conditions, antiserum to IFN‐beta did not enhance the mitogenic action of epidermal growth factor (EGF). Dexamethasone enhanced the mitogenic action of poly(I).poly(C) in a manner similar to antiserum against IFN‐beta. This effect of dexamethasone correlated with its marked inhibitory action on poly(I).poly(C)‐stimulated IFN production. Together with the results of other related studies, these findings support the notion of an evolutionary link between the generation of a mitogenic signal and IFN induction. In addition, these results support the concept that autocrine secretion of IFN‐beta can exert negative feedback control of cell proliferation.
Quantitation of protein kinase C by immunoblot—expression in different cell lines and response to phorbol esters
Authors: Stabel S. , Rodriguez‐Pena A. , Young S. , Rozengurt E. , Parker P. J. 
Published: February 2005

AbstractAntisera have been raised against human protein kinase C and also against a synthetic peptide based on the sequence of the bovine brain enzyme (LLNQEE‐GEYYNVPIPE). These antibodies react with protein kinase C from a number of species (human, murine, rat, rabbit, bovine), indicating substantial conservation of epitopes. These antisera have been used to quantitate directly protein kinase C by immunoblot analysis. We show here that there is a strict correlation between the leveis of immunoreactive polypeptide and extractable calcium‐ and phospholipid‐dependent kinase activity for various cell lines. Treatment of murine, rat, and human cells with phorbol dibutyrate was found to deplete levels of immunoreactive protein kinase C severely. A detailed study of the time course of this depletion in Swiss 3T3 cells shows that it follows precisely the loss of extractable activity. On exposure to 400 nM phorbol 12,13‐dibutyrate protein kinase C was essentially undetectable by 40 hours; the half‐life of this down‐regulation was 6.7 hours. This data thus demonstrate that the loss of immunoreactive protein kinase C and of extractable calcium‐ and phospholipid‐dependent kinase activity precisely parallels the phorbol ester induced down‐regulation of binding and responsiveness in Swiss 3T3 cells.
Retention of differentiated characteristics by cultures of defined rabbit kidney epithelia
Authors: Wilson Patricia D. , Anderson Robert J. , Breckon Ruth D. , Nathrath Walter , Schrier Robert W. 
Published: February 2005

AbstractRabbit nephron segments of proximal convoluted tubules (PCT); proximal straight tubules (PST); cortical and medullary thick ascending limbs of Henle's loop (CAL, MAL); and cortical, outer medullary, and inner medullary collecting tubules (CCT, OMCT, IMCT) were individually microdissected and grown in monolayer culture in hormone supplemented, defined media. Factors favoring a rapid onset of proliferation included young donor age, distal tubule origin, and the addition of 3% fetal calf serum to the medium. All primary cultures had polarized morphology with apical microvilli facing the medium and basement membrane‐like material adjacent to the dish. Differentiated properties characteristics of the tubular epithelium of origin retained in cultures included ultrastructural characteristics and cytochemically demonstrable marker enzyme proportions. PCT and PST were rich in alkaline phosphatase; CAL stained strongly for NaK‐ATPase; CCT contained two cell populations with regard to cytochrome oxidase reaction. A CCT‐specific antikeratin antibody (aLEA) was immunolocalized in CCT cultures, and a PST cytokeratin antibody stained PST cultures. The biochemical response of adenylate cyclase to putative stimulating agents was the same in primary cultures as in freshly isolated tubules. In PCT and PST adenylate cyclase activity was stimulated by parathyroid hormone (PTH) but not by arginine vasopressin (AVP); CAL and MAL adenylate cyclase was stimulated by neither PTH nor AVP; CCT, OMCT, and IMCT adenylate cyclase was stimulated by AVP but not by PTH. NaF stimulated adenylate cyclase activity in every cultured segment. It is concluded that primary cultures of individually microdissected rabbit PCT, PST, CAL, MAL, CCT, OMCT, and IMCT retain differentiated characteristics with regard to ultrastructure, marker enzymes, cytoskeletal proteins, and hormone response of adenylate cyclase and provide a new system for studying normal and abnormal functions of the heterogeneous tubular epithelia in the kidney.
Inhibition of fibroblast chemotaxis by recombinant human interferon γ and interferon α
Authors: Adelmann‐Grill Bernhard C. , Hein Rüdiger , Wach Franz , Krieg Thomas 
Published: February 2005

AbstractInterferons have recently been recognized as potent mediators in inflammatory processes, exerting profound effects on fibroblasts. The influence of interferons γ and α on the chemotactic movement of fibroblasts toward various attractants was, therefore, investigated. Normal human adult and embryonal dermal fibroblasts, fibrosarcoma‐derived fibroblasts and SV40‐transformed fibroblasts were tested against conditioned medium from fibroblasts, the chemotactic peptide C‐140 of fibronectin, platelet‐derived growth factor, and leukotriene B4 as attractants in the presence or absence of the interferons. Interferons γ and α inhibited chemotaxis in a dose‐dependent manner and at concentrations at least as low as 10−2 ng/ml. Inhibition was noticeable when the cells were exposed to interferon for as short a period as 60 minutes, and the effect was not readily reversible. Inhibition occurred when the cells came from sparse or dense cultures, but when platelet‐derived growth factor was the attractant and the cells had been grown at low density there was no inhibition. It is concluded that this is a specific effect, not to be wholly explained by overall increase in membrane rigidity. Inhibition of fibroblast chemotaxis by interferons may be an important regulatory mechanism during wound healing or fibrosis and metastatic spread of tumor cells.
Modulation of the nuclear antigen p105 as a function of cell‐cycle progression
Authors: Clevenger Charles V. , Epstein Alan L. , Bauer Kenneth D. 
Published: February 2005

AbstractThe characterization of the proliferation‐associated nuclear antigen designated p105 in quiescent and proliferating lymphocytes is described. Through the use of novel flow cytometric and cell‐sorting strategies the intracellular content of p105 was assessed in situ on a per cell basis. These analyses demonstrated the presence of multiple cellular subpopulations within the cell cycle differing significantly in p105 content. The data revealed that the flow cytometric quantitation of p105 levels may effectively discriminate cycling from noncycling cells. Immunogold electron microscopy revealed that the modulation of this interchromatin‐associated antigen was correlated with a significant degree of nuclear restructuring. In conjunction with cell sorting, immunogold electron microscopy and immunoblot controls demonstrated that the cell‐cycle‐related modulation in p105 cannot be accounted for by increased cellular mass or antigen sequestration. The significance of these controls and of the potential role of p105 in cellular proliferation is discussed.
Growth‐promoting activity in serum‐free medium of kallikreinlike arginylesteropeptidases from rat submaxillary gland
Authors: Catalioto R. M. , Négrel R. , Gaillard D. , Ailhaud G. 
Published: February 2005

AbstractThe characterization and purification of the growth‐promoting activity present in rat submaxillary gland extracts, known to be required for the proliferation of adipose precursor cells in serum‐free medium, have been undertaken. Fractionation of the extracts by ion‐exchange chromatography, gel filtration, and affinity chromatography on immobilized benzamidine allowed the copurification of the mitogenic activity with two distinct arginylesteropeptidases of apparent molecular weight 25,000; one of these enzymes has been purified to homogeneity and shown to be immunologically related to tonin, a well‐characterized kallikreinlike protease from submaxillary gland. The specificity of both enzymes was similar to that of plasma and glandular kallikreins, as indicated by the relative rates of hydrolysis of peptide p‐nitroanilide substrates. Prior treatment of the kallikreinlike proteases with phenylmethylsulfonylfluoride or aprotinin abolished completely both mitogenic and arginylesteropeptidase activities, indicating that enzymatic activity was essential for the manifestation of their growth‐promoting ability. The kallikreinlike proteases from rat submaxillary gland were able to replace thrombin to support the proliferation of Chinese hamster lung fibroblasts in serum‐free medium. These results underline the role of proteases in controlling cell growth and are discussed in light of adipose tissue development.
Effect of potassium depletion of Hep 2 cells on intracellular pH and on chloride uptake by anion antiport
Authors: Madshus Inger Helene , Tønnessen Tor Inge , Olsnes Sjur , Sandvig Kirsten 
Published: February 2005

AbstractThe effect of K+ depletion of Hep 2 cells on ion fluxes, internal pH, cell volume, and membrane potential was studied. The cells were depleted of K+ by incubation in K+‐free buffer with or without a preceding exposure to hypotonic medium. Efflux of K+ in cells not exposed to hypotonic medium was inhibited by furosemide or by incubation in Na+‐free medium, indicating that in this case at least part of the K+ efflux occurs by Na+/K+/Cl− cotransport. After exposure to hypotonic medium, K+ efflux was not inhibited by furosemide, whereas it was partly inhibited by 4,4′‐diisothiocyano‐2,2′‐stilbene‐disulfonic acid (DIDS). Exposure to hypotonic medium induced acidification of the cytosol, apparently because of efflux of protons from intracellular acidic vesicles. When isotonicity was restored, a rebound alkalinization of the cytosol was induced, because of activation of the Na+/H+ antiporter. While hypotonic shock and a subsequent incubation in K+‐free buffer rapidly depolarized the cells, depolarization occurred much more slowly when the K+ depletion was carried out by incubation in K+‐free buffer alone. The cell volume was reduced in both cases. K+ depletion by either method strongly reduced the ability of the cells to accumulate 36Cl− by anion antiport, and K+‐depleted cells were unable to increase the rate of 36Cl− uptake in response to alkalinization of the cytoscl.
Effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of Hep 2 cells by picornaviruses
Authors: Madshus Inger Helene , Sandvig Kirsten , Olsnes Sjur , van Deurs Bo 
Published: February 2005

AbstractPotassium depletion after a brief exposure of the cells to hypotonic medium was used to inhibit endocytosis from coated pits in Hep 2 cells. After such treatment the endocytic uptake of transferrin was arrested, and electron microscopy revealed that virtually no coated pits were present at the cell surface, while smooth (uncoated) pits were abundant. Under the same conditions the cells were strongly protected against poliovirus, while the cytopathogenic effect of human rhinovirus type 2, HRV 2, was increased. The cytopathogenic effect of encephalomyocarditis (EMC) virus was only slightly affected.Potassium depletion without hypotonic shock reduced the endocytic uptake of transferrin 2–3‐fold and the number of coated pits at the cell surface about 3‐fold. Furthermore, the cells were not protected against poliovirus after such treatment. The data indicate that the productive uptake of poliovirus occurs by receptor‐mediated endocytosis from coated pits, while the productive uptake of the other two picornaviruses may occur by another endocytic pathway. In order to efficiently arrest endocytosis from coated pits in these cells, hypotonic shock seems to be a critical component of the potassium depletion protocol.
Establishment and some mutational characteristics of 3T3‐like near‐diploid mouse cell line
Authors: Sasaki M. S. , Kodama S. 
Published: February 2005

AbstractA stable and nonmalignant near‐diploid cell line, designated m5S, has been established by serial in vitro transfers of embryonic skin cells of ICR mouse. The cells are stable in karyotype and growth, display high sensitivity to contact inhibition of cell division, are nontumorigenic in nude mice, and the transformation and mutation may be reproducibly tested. Unlike most of the mouse cell lines, the m5S underwent a unique karyotypic change during establishment and showed high stability in a diploid range, making amenable to the study of mutagenesis. The m5S is nearly unique; it represents the second near‐diploid cell line sensitive to postconfluence inhibition of cell division derived from mouse embryo cells to be reported and characterized. These cells are being successfully used for qualitative and quantitative studies of oncogenic transformation as well as mutation to drug resistance by radiation and chemicals.
Antiproliferative effects of heparin on vascular smooth muscle cells are reversed by epidermal growth factor
Authors: Reilly Christopher F. , Fritze Linda M. S. , Rosenberg Robert D. 
Published: February 2005

AbstractHeparin and related glycosaminoglycans are potent inhibitors of both in vivo and in vitro smooth muscle cell (SMC) proliferation. We have found that epidermal growth factor (EGF) reverses the antiproliferative effects of heparin. Other known SMC mitogens, including platelet‐derived growth factor (PDGF), insulin‐like growth factor‐1 (IGF‐1), and thrombin, were unable to prevent heparin action. The EGF specificity was further demonstrated by developing a biological growth assay in which EGF or PDGF, at concentrations as low as 1 ng/ml, stimulated SMC growth in the absence of other serum components. Under these conditions, EGF, but not PDGF, suppressed heparin inhibition as well. The ability of EGF to reverse heparin inhibition was only observed when mitogen and glycosaminoglycan were added to SMC at similar times. If SMC were pretreated with heparin for 48 hours prior to EGF addition, the protective effects of EGF were lost. Heparin did not directly prevent 125I‐EGF or platelet‐derived EGF‐like peptides from binding to the EGF receptor on SMC. However, cultures that were pretreated with heparin for 48 hours bound 49% less 125I‐EGF than cultures that had been pretreated with the mucopolysac‐charide for only 2 hours or that had not been preexposed to heparin. In previous studies, we have established that heparin exerts its maximal inhibitory activity after a 48‐hour treatment of SMC (Reilly et al. 1986). Taken together, these data suggest that heparin may exert its antiproliferative potential by slowly and specifically altering SMC response to EGF‐like mitogens of platelet origin.