Stimulation and inhibition of cardiac myocyte proliferation in vitro

Summary We have examined the effect of crude cardiac tissue extracts as well as that of several growth factors and triiodothyronin (T3) on DNA synthesis of cardiac myocytes in culture. Extracts from embryonic and adult cardiac tissue stimulated DNA synthesis of myocytes. Atrial myocytes exhibited overall higher degree of stimulation than their ventricular counterparts and extracts from adult atrial tissue had the highest apparent mitogenic activity for atrial myocytes. We have shown that adult heart contains basic fibroblast growth factor (bFGF), especially in the atria [1]. Transforming growth factor (TGF) and insulin-like growth factors (IGFs) are also accumulated in cardiac tissues [2, 3]. We found that bFGF and the IGFs stimulate myocyte cell proliferation and DNA synthesis. These factors also stimulate cardiac non-muscle proliferation, especially in the presence of serum. TGF inhibited proliferation and DNA synthesis and cancelled the effect of bFGF or IGFs on the myocytes. T3 also diminished the bFGF-induced mitogenic stimulation of cardiomyocytes. Our data suggest that these factors may be involved in the regulation of cardiomyocyte proliferation in vivo.Abbreviations bFGF basic Fibroblast Growth Factor - BSA Bovine Serum Albumin - DM Defined Medium - Fes Fetal calf serum - FITC Fluorescein - IGF Insulin-like Growth Factor - IgG Immunoglobulin - LI Labeling Index - PBS Phosphate Buffered Saline - T3 Triiodothyronine - TGF Transforming Growth Factor      

Creatine kinase activity of urinary bladder and skeletal muscle from control and streptozotocin-diabetic rats

The urinary bladder depends on intracellular ATP for the support of a number of essential intracellular processes including contraction. The concentration of ATP is maintained constant primarily via the rapid transfer of a phosphate from creatine phosphate (CP) to ADP catalyzed by the enzyme creatine kinase (CK). Since muscular pathologies associated with diabetes are in part related to intracellular alterations in metabolism, we have characterized the CK activity in both skeletal muscle and urinary bladder from control and streptozotocin-diabetic rats.The following is a summary of the results: 1) Bladder tissue from control rats showed linear kinetics with a V_max = 390 nmoles/mg protein/min, and a K_m = 275 µM. 2) Urinary bladder tissue isolated from diabetic rats displayed biphasic kinetics with V_max = 65 and 324 nmoles/mg protein/min, and K_m's = 10 µM and 190 µM respectively. 3) Skeletal muscle isolated from control rats showed linear kinetics with an approximate V_max of 800 nmoles/mg protein/min and a K_m of 280 µM CP. 4) Homogenates of skeletal muscle from diabetic rats showed complex kinetics not separable into distict component forms. 5) The K_m for ADP for both skeletal muscle and bladder was approximately 10 µM.These studies demonstrate that whereas bladders isolated from both control and diabetic rats possess a low-affinity isomer(s) of CK with similar maximum enzymatic activity, there is a high affinity isomer present within the urinary bladder muscle of diabetic rats that is not present in bladder tissue isolated from control rats. Skeletal muscle isolated from both diabetic and control rats exhibited a maximal activity 2 to 3 times higher than that of the bladder.     

Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.     

The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

Preparation of affinity-purified,biotinylated tetanus toxin,and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 11 to 201 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using¹²⁵I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these primed cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.Abbreviations PBS phosphate-buffered saline - STS sucrose-Tris-serum solution - NGF nerve growth factor - C collagen - PL polylysine - BBG bovine brain ganglioside mixture - GM₁ gafactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1a [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1a [N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1b galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1b [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide - NANA N-acetylneuraminic acid     

Protein phosphorylation in Streptomyces albus

The phosphorylated proteins of Streptomyces albus, radioactively labeled with [32P]orthophosphate have been analyzed by gel electrophoresis and autoradiography. More than 10 protein species were found to be phosphorylated. With [32P]ATP as substrate cell free extracts phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. From cell extract which exhibited active phosphorylated in vitro, a protein kinase has been partially purified. The kinase activity was identified in fractions corresponding to a 90 kDa protein.     

The selection of microbial communities by constant or fluctuating temperatures

Abstract The diversity of bacterial communities isolated from Antarctic lake sediment in chemostats under constant low temperature (8°C) or diurnally fluctuating temperature (1°C to 16°C) was examined. The median optimum temperature for growth of the freshwater bacteria isolated from the fluctuation chemostat was significantly lower ( P < 1%) than that for those from the constant temperature chemostat. The diversity of the enriched bacterial community isolated in the chemostat culture subjected to short-term temperature fluctuations was greater than that enriched under constant temperature. At least 4 different groups of bacteria, that occupied separate 'temperature niches', were isolated from the fluctuating chemostat compared to only one group isolated from the stable chemostat. Furthermore, a pseudomonad from the fluctuating chemostat was shown to out-compete another pseudomonad from the stable chemostat when both were subjected to the fluctuating temperature regime. However, the pseudomonad of constant (8°C) temperature origin out-competed that isolated under fluctuating conditions when subjected to a stable temperature regime.     

Solubilization and characterization of a lactogenic receptor from human placental chorion membranes

Prolactin has a wide range of actions, including osmoregulation and the control of mammary gland development and lactation. These effects are mediated through a high-affinity cell surface receptor, which has been well characterized in a number of animal tissues. The molecular characteristics of the human receptor are unknown, however. The present studies were initiated, therefore, to determine the binding and molecular characteristics of the lactogenic receptor of human placental chorion membranes. Subcellular fractionation studies showed that the bulk of the receptor sedimented in the microsomal fraction at 45,000gav. Endogenous ligand was dissociated from the receptor with 3.5 M MgCl2 or 0.05 M acetate buffer (pH 4.8) with preservation of binding activity. The microsomal receptor bound human growth hormone (hGH), human prolactin (hPRL), ovine prolactin (oPRL), and human placental lactogen (hPL) but not non-primate growth hormones, indicating a narrow specificity for lactogenic hormones. The binding was only partially reversible in agreement with the known binding kinetics of animal lactogenic receptors. The receptor was solubilized with 45% yield from the microsomes using 16 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) detergent-250 mM NaCl, and the binding activity was fully restored by a two-fold dilution in the binding reaction to reveal a KD of 0.8 nM for hGH and a binding capacity of 200 fmol of specifically bound hGH per mg of microsomal protein. Gel filtration chromatography indicated the minimum molecular weight of the ligand-receptor complex was approximately 60,000 daltons, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of covalently cross-linked 125I-hGH-receptor complexes revealed a molecular size of 58,000 daltons. When account was taken of the contribution of the ligand, a molecular weight of 36,000 for the receptor's binding domain was obtained. These data indicate that the chorion lactogenic receptor has very similar binding and molecular characteristics to the lactogenic receptors from other mammalian species. Chorion membranes are thus a convenient source of material for the further purification and characterization of the human lactogenic receptor.     

Suppression of protein kinase C and the stimulation of glucocorticoid receptor synthesis by dexamethasone in human fibroblasts derived from tumor tissue

Exposure of fibroblasts derived from keloid tissues, desmoid and dermal tissue from individuals with Gardner's syndrome (GS) to dexamethasone resulted in the suppression of protein kinase C (PKC) activity and [3H]thymidine incorporation into DNA, and a 20-fold induction of glutamine synthetase activity. Treatment of GS and keloid fibroblasts with 0.1 microM dexamethasone for 36 h increased glucocorticoid receptor (GR) synthesis, as determined by [35S]methionine labeling and immunoprecipitation with a monoclonal antibody to the human GR. The suppression of PKC activity by dexamethasone was shown to result from a loss of protein mass as determined by immunoblotting using an antibody to PKC type III. In contrast to these results, exposure of fibroblasts isolated from normal tissues to dexamethasone did not result in the suppression PKC and [3H]thymidine incorporation, there was only a sixfold induction of glutamine synthetase, and a decrease of GR synthesis. As no primary receptor binding defect could be detected, the altered response of tumor cells to steroid-occupied receptor indicates a partial post-receptor binding defect in GS and keloid cells.     

Histopathology and cell culture characteristics of liver cells from grc − and grc + rats given diethylnitrosamine

The histopathological response and cell culture characteristics of liver cells from the R16 (grc ^–) strain of rats, which carries an MHC-linked deletion, were examined one week after a single intraperitoneal injection of 200 mg/ kg body weight diethylnitrosamine (DEN) and were compared with the response of liver cells from wild type (grc+) rats. The DEN exposure induced hydropicl vacuolar changes in the parenchymal cells and a limited proliferation of oval cells in the periportal areas of the livers of both grc+ and grc rats. Primary culture of collagenase-digested livers consisted of parenchymal, bile ductular and oval-related cells as determined by cell-specific immunohistochemistry. Subpassaged cells from grc+ rats exhibited oval cell ultrastructural morphology, inducible histochemical staining for gammaglutamyl transpeptidase (GGT), and DEN-associated onset of anchorage-independent growth. Primary cultures of liver cells from R16 rats consistently failed to form cell strains upon subpassage.Abbreviations DEN diethylnitrosamine - grc growth and reproduction complex - GGT gamma-glutamyl transpeptidase - MHC major histocompatibility complex