Discovery of 3-aryl-3-ethoxypropanoic acids as orally active GPR40 agonists

The G protein-coupled receptor 40 (GPR40) mediates enhancement of glucose-stimulated insulin secretion in pancreatic β cells. The GPR40 agonist has been attracting attention as a novel insulin secretagogue with glucose dependency for the treatment of type 2 diabetes. The optimization study of compound 1 led to a potent and bioavailable GPR40 agonist 24, which showed insulin secretion and glucose lowering effects in rat OGTT. Compound 24 is a potential lead compound for a novel insulin secretagogue with a low risk of hypoglycemia.     

Natural and Chernobyl-caused radioactivity in mushrooms,mosses and soil-samples of defined biotops in SW Bavaria

Summary Different mushrooms, mosses and corresponding soil samples have been collected mainly from two sites in the alpine region of southwestern Bavaria. At the end of the growthseason, September 1986, gamma spectroscopic analysis showed that the moss-, mould, and needle-layer contained considerably more ¹³⁴Cs and ¹³⁷Cs activity per unit fresh weight than eight different species of mushroom. These two isotopes were carried into the biotop mainly as a consequence of the Chernobyl accident. ¹³¹J could not be found any more in the samples ca. 5–6 months after the catastrophe. The activity of the cesium isotopes decreased with increasing soil depth. In the mushrooms the activity was relatively high in Xerocomus badius and surprisingly low in Boletus edulis; samples of the latter and of Cantharellus cibarius collected in September 1985 (before the accident) and kept deep frozen contained almost identical amounts of ¹³⁷Cs as those collected from August to October 1986. Mushrooms contained considerably more of the natural isotope ⁴⁰K than the needlelayers and the soil samples in the neighbourhood. In all mushrooms except Xerocomus badius the activity of ⁴⁰K was generally higher than the ¹³⁷Cs activity. The results indicate that except Xerocomus badius the analyzed mushrooms do not actively take up Cs from the soil, in contrast to K.     

Transformation with SV40 virus prevents retinoic acid inhibition of plasma membrane NADH diferric transferrin reductase in rat liver cells

Retinoic acid inhibits the reduction of diferric transferrin through the transplasma membrane electron transport system on fetal rat liver cells infected with a temperature-sensitive SV40 virus when the cells are in the nontransformed state cultured at 40°C. When the cells are in the transformed state (grown at the permissive 33°C temperature), retinoic acid does not inhibit the diferric transferrin reduction. Inhibition of activity of nontransformed cells is specific for retinoic acid with only slight inhibition by retinol and retinyl acetate at higher concentrations. Isolated rat liver plasma membrane NADH diferric transferrin reductase is also inhibited by retinoic acid. The effect of transformation with SV40 virus to decrease susceptibility to retinoic acid inhibition stands in contrast to much greater adriamycin inhibition of diferric transferrin reduction in the transformed cells than in nontransformed cells.     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replicationin vitro

Summary The role of DNA polymerases in the replication of SV40 DNA was studied using a T-antigen-dependent assay supplemented with a human KB cell extract. Inhibition of DNA polymerase α by addition of aphidicolin or monoclonal antibodies prevented DNA synthesis, confirming the requirement for this enzyme in replication. The replication process was unaffected by ddTTP at a concentration (5 μM) inhibitory to DNA polymerases β and γ, however, higher concentrations of ddTTP (200 μM) caused an apparent accumulation of relaxed circular plasmid with a concomitant decrease in DNA synthesis. An analysis of this replication intermediate indicated that it was formed during the replication reaction and that the replicative cycle was nearly complete. A kinetic study of ddTTP inhibition strongly suggested DNA polymerase ε (PCNA-independent DNA polymerase δ) was the target of the inhibitor and that this enzyme functions during the final stages of DNA replication.     

Idiotype network components are involved in the murine immune response to simian virus 40 large tumor antigen

Summary Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag), a monoclonal antibody specific for SV40 T-Ag (Ab-1 preparation), and a monoclonal anti-idiotypic antibody (anti-Id), designated 58D, were used to analyze the humoral immune response of Balb/c mice either immunized with recombinant SV40 T-Ag or challenged with SV40-transformed cells. Inhibition assays indicated that antibodies from mice immunized with SV40 T-Ag and from those bearing SV40 tumor inhibited the SV40 T-Ag/Ab-1 reaction. These data suggested that the antibody response in immunized or tumorchallenged mice recognized similar epitope(s) on SV40 T-Ag to that detected by the monoclonal Ab-1. These anti-(SV40 T-Ag) response antibodies also inhibited the Ab-1/anti-Id reaction and recognized the anti-Id in direct binding assays. Together, these data indicate that murine anti-(SV40 T-Ag) responses shared an idiotope with a monoclonal anti-(SV40 T-Ag) Ab-1 preparation. This idiotope, which is recognized by the monoclonal anti-Id preparation, 58D, appears to be involved in the humoral immune response to SV40 T-Ag in both SV40-T-Ag-immunized and tumor-bearing mice. The monoclonal anti-Id preparation may represent a focal point for manipulating the humoral immune response to tumors induced by SV40-transformed cells.     

Comparison of spontaneous and ultraviolet-induced mutagenesis on naked SV40 DNA and SV40 virus

Molecular aspects of mutagenesis in mammalian cells have been essentially analyzed using biological probes such as viruses and shuttle vector. Although the main data concerning the specificity of carcinogen-induced mutations are similar, the observed spontaneous mutation frequencies are significantly different when using one or the other model. This frequency is considerably higher with shuttle vectors than with viruses. We have performed an analysis of mutagenesis in order to determine if the obligatory transfection step associated with shuttle vector technology was responsible for the high mutation frequency found with these molecules. For this purpose simian virus 40 (SV40) genome used as virus or as naked DNA was introduced into permissive cells by viral infection or DNA transfection respectively. Our results show that transfection alone does not induce a higher mutation frequency on SV40 DNA the virus infection. Moreover, we have shown that the ultraviolet-light induced mutation spectrum was similar on the SV40 VP1 gene after viral infection or DNA transfection.     

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.