Potent inhibitory effects of the 5'-triphosphates of (E)-5-(2-bromovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil on DNA polymerase gamma

(E)-5-(2-Bromovinyl)-2'-deoxyuridine 5'-triphosphate (BrVdUTP) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil 5'-triphosphate (BrVarafUTP), which are known as specific inhibitors of herpes simplex viral (type 1 and 2) DNA polymerase, were found to be strong inhibitors of DNA polymerase gamma from human KB and murine myeloma cells. In fact BrVdUTP and BrVarafUTP were found to be stronger inhibitors of DNA polymerase gamma than of other DNA polymerases having viral (herpes simplex virus or retrovirus) origin or cellular (eukaryotic alpha and beta, or prokaryotic) origin. The mode of inhibition of DNA polymerase gamma by BrVdUTP and BrVarafUTP was competitive with respect to dTTP, the normal substrate. Whereas BrVdUTP was an efficient substrate for DNA polymerase gamma and other DNA polymerases that were examined, BrVarafUTP failed to serve as a substrate for DNA synthesis. Ki values for BrVdUTP (40 nM) and BrVarafUTP (7 nM) with DNA polymerase gamma, as determined with (rA)n.(dT) as the template.primer, were much smaller than the Km values for dTTP (0.16 microM and 0.71 microM for murine and human DNA polymerase gamma, respectively). Thus, the affinity of BrVdUTP or BrVarafUTP for DNA polymerase gamma was much stronger than that of dTTP.     

Growth hormone-releasing factor releases ACTH from an AtT-20 mouse pituitary tumor cell line but not from normal pituitary cells

Corticotropin-releasing factor (CRF) and both human pancreatic growth hormone-releasing factor (hp-GRF) and rat hypothalamic GRF (rh-GRF) stimulated ACTH release from neoplastic AtT-20 mouse pituitary tumor cells in a dose-dependent fashion, with CRF inducing a 10-fold increase and GRF a maximal increment of approximately one-half that of CRF. Neither rh-GRF nor hp-GRF induced ACTH release in normal anterior pituitary cells. Pretreatment with either dexamethasone or somatostatin prior to the addition of rh-GRF inhibited the increase in ACTH release. Both ovine CRF and rh-GRF stimulated adenosine 3,5-monophosphate production in AtT-20 cells. The weak but clearly discernible effect of GRF on ACTH release from AtT-20 cells may be due to an abnormality in the AtT-20 cell receptor.     

Molecular cloning of a cDNA coding for 70 kilodalton subunit of soluble guanylate cyclase from rat lung

A complementary DNA clone corresponding to the 70 kDa subunit of soluble guanylate cyclase (EC 4.6.1.2) of rat lung has been isolated. The primary structure of the cDNA consisted of 3063 nucleotides including a 1857-nucleotide coding region for 619 amino acids, and the calculated molecular weight was 70476. Blot hybridization of total poly(A)+RNAs from rat tissues detected a mRNA of about 3.4 kilobases. The amount of mRNA was abundant in lung, cerebrum and cerebellum, moderate in heart and kidney, and low in liver and muscle. Southern blot analysis of high molecular weight genomic DNA from rat liver indicated the presence of one gene in the rat haploid genome. The amino acid sequence of the 70 kDa subunit has partial homology with particulate guanylate cyclase from sea-urchin sperm, and protein phosphatase inhibitor I.     

Cycling of soil carbon in a Japanese red pine forest II. Changes occurring in the first year after a clear-felling

Cycling of soil carbon in the first year after a clear-felling was compared with that before the felling in a Japanese red pine forest in Hiroshima Prefecture, west Japan. The daily mean temperature at the soil surface in summer was increased after the felling in comparison to that before felling, and the water content of both the A₀ layer and the surface mineral soil was decreased due to the loss of the forest canopy. The rate of weight loss of the A₀ layer was reduced after felling. However, accumulation of the A₀ layer rapidly decreased because of the lack of litter supply to the forest floor. Low soil respiration after felling was mainly caused by the cessation of root respiration. Analysis of annual soil carbon cycling was then conducted using a compartment model. The relative decomposition rate of the A₀ layer decreased whereas that of humus and dead roots in mineral soil increased to some extent after felling. The accumulation of carbon in mineral soil, however, increased slightly due to the supply of humus from roots killed by the felling.     

ENZYME-LABELED ANTIBODIES FOR THE LIGHT AND ELECTRON MICROSCOPIC LOCALIZATION OF TISSUE ANTIGENS

Enzymes, either acid phosphatase or horseradish peroxidase, were conjugated to antibodies with bifunctional reagents. The conjugates, enzymatically and immunologically active, were employed in the immunohistochemical localization of tissue antigens utilizing the reaction product of the enzymatic reaction as the marker. Tissues reacted with acid phosphatase-labeled antibodies directed against basement membrane were stained for the enzyme with Gomori's method, and those reacted with peroxidase-labeled antibody were stained with Karnovsky's method. The reaction products of the enzymes localized in the basement membrane. Unlike the preparations of the fluorescent antibody technique, enzyme-labeled antibody preparations were permanent, could be observed with an ordinary microscope, and could be examined with the electron microscope. In the latter, specific localization of antibody occurred in the basement membrane and in the endoplasmic reticulum of cells known to synthesize basement membrane antigens. The method is sensitive because of the amplifying effect of the enzymatic activity. The ultrastructural preservation and localization were better with acid phosphatase-labeled antibody than with peroxidase-labeled antibody, but acid phosphatase conjugated antibody was unstable and difficult to prepare. Peroxidase-antibody conjugates were stable and could be stored for several months at 4°C, or indefiniely in a frozen state.     

Immunohistochemical localization and quantitative analysis of cellular glutathione peroxidase in foetal and neonatal rat tissues: Fluorescence microscopy image analysis

Summary To quantitate the developmental changes in selenium-dependent cellular glutathione peroxidase during the perinatal period, tissue sections from foetal (day 12 to day 22) and neonatal (day 6) rats were stained immunohistochemically using specific polyclonal antiserum. The intensity of the staining was quantified by fluorescence microscopy image analysis. There was a general trend of enriched glutathione peroxidase in the epithelial linings and metabolically active sites. Significant fluorescence was detected in cardiomyocytes, hepatocytes, renal tubular epithelium, bronchiolar epithelium and intestinal epithelium at day 15. The intensity increased in a stepwise manner therafter. The overall increase in the intensity of staining in the heart, liver, kidneys, lungs and intestine was 1.5-, 2.3-, 1.6-, 1.7- and 3.0-fold, respectively. The phase of most rapid increase occurred during the foetal period in the liver, intestine and heart. In the kidneys and lungs, glutathione peroxidase increased significantly during foetal life, and to a similar extent postnatally. These results suggest that the intracellular H₂O₂-scavenging system develops during the foetal period as an essential mechanism for living under atmospheric oxygen conditions. The late development observed in the kidneys and lungs is consistent with the relative biological immaturity of these organs in full-term neonates.     

Translocation of dimeric IgA through neoplastic colon cells in vitro.

We studied the translocation of dimeric IgA across epithelium, using neoplastic human colon cells in culture as a source of epithelial cells, and immunoelectronmicroscopy with peroxidase-labeled antigens and antibodies. The cells had some of the ultrastructural characteristics of normal, mature epithelial cells, i.e., polarity, desmosomal junctions, and secretory component on their basal and lateral plasma membranes. Horseradish peroxidase-labeled dimeric IgA, exposed to the cells at 0 degrees C, bound selectively to secretory component on the cell surfaces. At 37 degrees C, the bound dimeric IgA was taken into the cells by endocytosis and transported apically through the cytoplasm in vesicles. After 30 min, IgA was discharged across the apical surface. Neither colchicine (10(-4) M) nor cytochalasin B (10(-5) M) interfered with binding or endocytosis of dimeric IgA, but colchicine inhibited intracellular transport of the IgA-containing vesicles. These experiments demonstrated that dimeric IgA can be transported through living intestinal epithelial cells in vitro. The transport includes 1) specific binding of IgA dimers to secretory component on plasma membranes, 2) endocytosis of IgA in vesicles, 3) transcytoplasmic transport of the IgA-containing vesicles by a process involving microtubules, and 4) discharge of IgA at the apical surfaces.