Cloning and expression of a cDNA encoding a catalytically active fragment of calf thymus DNA polymerase alpha

A calf thymus cDNA expression library was constructed in the EcoRI site of lambda gt11 and probed with an antibody raised against calf thymus DNA polymerase alpha. Three classes of antibody-reactive clones were isolated. The largest class carried a 1.9 kilobase calf cDNA insert and expressed a 165-175 kilodalton beta-galactosidase:calf fusion protein which displayed DNA polymerase activity. The characteristic responses of the polymerase activity to alpha-specific inhibitors and antibodies identified the 1.9 kilobase cDNA as a sequence specifically derived from the structural gene encoding the pol alpha catalytic core.     

Mathematical model for human myeloma relating growth kinetics and drug resistance

We present a computer-based mathematical model that can simulate characteristic features of the clinical time course of human myeloma. It asserts that therapy resistance in myeloma cells is an inherited trait associated with the longer inter-mitotic times of some cells and that the strength of this trait affects tumour growth characteristics. These kinetic differences within the malignant cell clone may also influence therapeutic efficacy. In the model, the same total therapy, administered in different time-dose fractions, could be 'curative' or 'minimally effective' depending on kinetic properties. For example, as others have shown, in myeloma pulsed intermittent therapy is often more effective than low dose continuous therapy. According to our model this finding is compatible with a high coefficient of inheritability of resistance from one cell generation to the next. The model also suggests that if there are subclones of varying resistance, a therapy must have some effect on each of them if it is to be employed in a curative fashion. While many aspects of the model are not yet clinically testable, exploration of its concepts might increase knowledge about fundamental neoplastic mechanisms.     

Growth hormone regulates the abundance of insulin-like growth factor I RNA in adult rat liver

Insulin-like growth factor I (IGF-I) is a mitogenic polypeptide present in the plasma of man and rat that is thought to mediate the actions of pituitary growth hormone on cartilage to promote skeletal elongation. In the rat, plasma levels of IGF-I show both developmental and hormonal regulation: levels are low at birth, increase with age, and are decreased in growth hormone-deficient adult animals. The present study demonstrates that these changes in plasma IGF-I reflect the abundance of IGF-I RNA in rat liver. A human IGF-I cDNA probe hybridized to multiple RNA species in adult rat liver with sizes 8.6, 4.6, 3.2, 2.1, and 1.0-1.4 kilobases. These RNA species were decreased by greater than 80% in neonatal (2- and 12-day-old) rat liver and by greater than 90% in liver from adult rats made growth hormone-deficient by hypophysectomy. Treatment of hypophysectomized rats with growth hormone increased the abundance of all species of IGF-I RNA. These results suggest that growth hormone regulates the expression of its physiological mediator by altering the synthesis, stability, or both of IGF-I RNA in rat liver.     

Isolation and primary structure of the califins, three biologically active egg-laying hormone-like peptides from the atrial gland of Aplysia californica

The atrial gland of the marine mollusk Aplysia californica contains several biologically active peptides that are thought to be important in reproductive function. In the present study, three novel peptides, which we named califin A, B, and C, were purified from extracts of atrial glands by high performance liquid chromatography, and their primary structures were determined. Each consists of a 36-residue subunit bound by a single disulfide bond to an 18-residue subunit. The large subunits differ from each other by one or two residues, whereas the small subunits are identical. The large subunits are 78-83% homologous to egg-laying hormone (ELH), a 36-residue peptide synthesized by the neuroendocrine bag cells of Aplysia. Like ELH, the califins excite LB and LC cells of the abdominal ganglion and cause egg laying when injected into sexually mature animals. Based on previously described DNA sequence data, each califin is likely to be derived from one of several precursor proteins that are encoded by members of the ELH gene family. Califin A is encoded on the peptide A precursor, and califin B may be encoded on the peptide B precursor. No gene encoding califin C has been sequenced. Because peptides A and B are also biologically active, the precursors encoding them and califins A and B are polyproteins. The possible role of atrial gland peptides as pheromones is discussed.     

Site-specific maturation of enveloped viruses in L cells treated with cytochalasin B

Treatment of infected L cells with 10 micrograms/ml cytochalasin B (CB) was found to promote a rapid relocalization of viral glycoproteins on the cell surface. Whereas the vesicular stomatitis virus G protein and the influenza virus hemagglutinin were uniformly distributed on the surface of untreated cells, in CB-treated cells, they were strikingly concentrated at cell extremities in the regions of clustered blebs. Glycoprotein concentration at cell extremities was accompanied by preferential maturation of virus particles from the same sites; both vesicular stomatitis and influenza viruses budded predominantly from the vicinity of clustered blebs. This effect of CB was completely reversible. Removal of CB from the cell growth medium resulted in a return of viral glycoproteins to the uniform distribution characteristic of untreated cells and to uniform virus budding. The results of this study are interpreted in terms of a model that suggests that preferential budding of viruses from the regions of bleb clusters is due to the concentration of viral glycoproteins at these sites.     

Detection of immunologically cross-reacting capsid protein of alphaviruses on the surfaces of infected L929 cells.

Hyperimmune, but not normal immune, monospecific antiserum made to capsid protein of Sindbis virus (SIN) was found to cause cytolysis equally well of both SIN- and Semliki Forest virus-infected L929 cells in antibody-dependent, complement-mediated cytotoxicity assays. The cell surface reactivity of the hyperimmune antiserum was also demonstrated by solid-phase radioimmune assays with unfixed infected cells or infected cells fixed with low concentrations of glutaraldehyde (0.025%) before reactivity with antisera. Higher concentrations of glutaraldehyde lowered the sensitivity of detection. Purified SIN capsid protein specifically inhibited antibody-dependent, complement-mediated cytotoxicity by the monospecific anti-capsid protein serum on SIN- and Semliki Forest virus-infected target cells. That hyperimmune anti-SIN serum also cross-reacts with capsid protein on the surface of Semliki Forest virus-infected cells was suggested by the fact that capsid protein inhibited cross-cytolysis in the antibody-dependent, complement-mediated cytotoxicity assay. The latter antiserum was collected after repeated injections of purified virions over a 9-month period. The results suggest that hyperimmune monospecific antisera made to SIN capsid protein or hyperimmune antisera to SIN or Semliki Forest virions detect homologous and cross-reacting capsid protein determinants on the surface of infected cells.     

Histamine H2 receptors on chondrocytes derived from human, canine and bovine articular cartilage.

Histamine (1-100 microM) induced a concentration-dependent increase in intracellular cyclic AMP in monolayer cultures of human, canine and foetal-bovine articular chondrocytes. The dose-response curve for histamine in each culture was progressively displaced to the right with increasing concentrations of cimetidine, an H2-receptor antagonist. The histamine-induced cyclic AMP elevation in human articular chondrocytes was also significantly decreased by ranitidine, another H2 antagonist, but not by the H1 antagonists mepyramine and chlorpheniramine. These findings indicate that histamine activates chondrocyte adenylate cyclase through an H2 receptor. The cyclic AMP response of human chondrocytes to histamine was many times greater than that measured for synovial fibroblasts under similar conditions. Such findings suggest that mast-cell-chondrocyte interactions in vivo may contribute to changed chondrocyte metabolism in joint disease.     

Dexamethasone and indomethacin modify endotoxin-induced respiratory failure in pigs

We studied the porcine pulmonary response to endotoxemia before and after administration of nonsteroidal antiinflammatory drugs (NSAID, i.e., indomethacin or flunixin meglumine) or dexamethasone (DEX). Escherichia coli endotoxin was infused intravenously into anesthetized 10- to 12-wk old pigs for 4.5 h. In endotoxemic pigs, the phase 1 (i.e., 0-2 h) increases in pulmonary arterial pressure, pulmonary vascular resistance (PVR), and alveolar-arterial O2 gradient and the decreases in cardiac index (CI) and lung dynamic compliance (Cdyn) were blocked by NSAID. Thus phase 1 changes were cyclooxygenase dependent. Furthermore, these effects were blocked or greatly attenuated by DEX. During phase 2 of endotoxemia (i.e., 2-4.5 h), the increased PVR and decreased CI and Cdyn were not blocked by NSAID but were attenuated by DEX, suggesting the presence of cyclooxygenase-independent metabolites. Both NSAID and DEX blocked the endotoxin-induced increases in lung water, bronchoalveolar lavage (BAL) neutrophil, and BAL albumin content. The fall in plasma proteins persisted in NSAID but not DEX-treated pigs. We conclude that endotoxemia in the pig causes severe acute respiratory failure largely mediated by cyclooxygenase and possibly lipoxygenase products of arachidonic acid metabolism.     

Relationship between ion requirements for respiration and membrane transport in a marine bacterium.

Intact cells of the marine bacterium Alteromonas haloplanktis 214 oxidized NADH, added to the suspending medium, by a process which was stimulated by Na+ or Li+ but not K+. Toluene-treated cells oxidized NADH at three times the rate of untreated cells by a mechanism activated by Na+ but not by Li+ or K+. In the latter reaction, K+ spared the requirement for Na+. Intact cells of A. haloplanktis oxidized ethanol by a mechanism stimulated by either Na+ or Li+. The uptake of alpha-aminoisobutyric acid by intact cells of A. haloplanktis in the presence of either NADH or ethanol as an oxidizable substrate required Na+, and neither Li+ nor K+ could replace it. The results indicate that exogenous and endogenous NADH and ethanol are oxidized by A. haloplanktis by processes distinguishable from one another by their requirements for alkali metal ions and from the ion requirements for membrane transport. Intact cells of Vibrio natriegens and Photobacterium phosphoreum oxidized NADH, added externally, by an Na+-activated process, and intact cells of Vibrio fischeri oxidized NADH, added externally, by a K+-activated process. Toluene treatment caused the cells of all three organisms to oxidize NADH at much faster rates than untreated cells by mechanisms which were activated by Na+ and spared by K+.