Insulin suppresses hepatitis B surface antigen expression in human hepatoma cells

The human hepatoma Hep3B cells contain integrated hepatitis B viral genome and continually secret hepatitis B surface antigen (HBsAg). The production of HBsAg (but not alpha-fetoprotein) was suppressed by addition of low concentrations (0.1-1 nM) of insulin into serum-free medium. In addition, the suppression of HBsAg production by insulin was paralleled with the decrease in HBsAg mRNA abundance. Insulin also cause a rapid rate of disappearance of HBsAg mRNA (t 1/2, 2 h) in Hep3B cells. The Hep3B cells carry specific receptor with high affinity for insulin (Kd = 1.8 nM). The receptor showed an insulin-dependent protein tyrosine kinase activity. The half-maximal insulin concentration for the activation of the receptor kinase was about 5 nM. Only very high concentrations of insulin-like growth factor I and human proinsulin can compete for the insulin receptor binding and suppress HBsAg production, this suggests that insulin may act through its receptor binding to suppress HBsAg expression in human hepatoma Hep3B cells.     

Certain aspects of so-called post cholecystectomy syndrome

The study involved 126 patients with cholelithiasis. Set of biochemical, radiological, endoscopic, and ultrasound examinations was carried out in these patients. Potential coincidence of clinical symptoms and causes of so-called postcholecystectomy syndrome was the purpose of a 1-year follow-up studies. The significance of the results are discussed.     

Reductive biotransformations of organic compounds by cells or enzymes of yeast.

Saccharomyces cerevisiae catalyses the asymmetric reductive biotransformation of a variety of compounds containing a carbonyl group or carbon-carbon double bond. Oxidoreductases participating in these reactions which have commercial potential in biotransformation processes are likely to have relatively broad substrate specificity. Important carbonyl reductases falling into this category include YADH- and yeast NADP-dependent beta-ketoester reductases. The enoyl reductase component of the FAS complex may have a role in asymmetric yeast reduction of carbon-carbon double bonds of unnatural substrates. Other nicotinamide-requiring oxidoreductases of yeast are also surveyed to rationalize observed biotransformations of whole yeast cells in terms of specific enzymes. Genetic and protein engineering may enable enzymes to be tailored to accept new substrates. A greater understanding of the enzymes and reactions involved will facilitate further optimization and exploitation of these catalytic systems in industrial processes.     

3H]L-657,743 (MK-912): a new, high affinity, selective radioligand for brain alpha 2-adrenoceptors

L-657,743 (MK-912), a highly potent and selective alpha 2-adrenoceptor antagonist was tritiated to a high specific activity and its binding characteristics to brain tissue were determined. The specific binding of [3H]L-657,743 to rat cerebrocortex was saturable, reversible, and dependent on tissue concentration. In saturation studies, [3H]L-657,743 binding was resolved into two high affinity components exhibiting Kd values of 86 pM and 830 pM with densities of 82 fmol/mg protein and 660 fmol/mg protein, respectively. Based on the binding potencies of a variety of compounds with differing receptor selectivities, the sites labeled by [3H]L-657,743 were characteristic of alpha 2-adrenoceptors. In contrast to alpha 2-antagonists, alpha 2-agonists displayed shallow competition curves. In the presence of 100 microM GTP, Gpp(NH)p or 150 mM NaCl, the competition curve for epinephrine was shifted to the right, whereas that for yohimbine was unaffected. In studies utilizing human cerebrocortical tissue, [3H]L-657,743 also bound with high affinity to sites characteristic of alpha 2-adrenoceptors.     

Chromogenic redox assay for beta-lactamases yielding water-insoluble products. II. Heterogeneous sandwich assay for hCG.

A very sensitive and rapid heterogeneous sandwich enzyme immunoassay for human chorionic gonadotropin (hCG) is described. The assay is based on the application of the novel chromogenic redox substrate system for beta-lactamase which is used as label. The chromogen system consists of a thioacetylcephalosporin beta-lactamase substrate, which upon turnover by the enzyme label releases the thiolate with the concomitant reduction of the tetrazolium salt to a colored formazan. The concentration of the formazan is directly related to the amount of the hormone in the sample and is read spectrophotometrically. The enzyme-antibody conjugates, produced through use of heterobifunctional maleimide crosslinker, maintain 90% of the enzyme activity after 30 days at 25 degrees C. Concentrations of the hormone as low as 5 mIU/ml, equivalent to 25 fmol/ml, are detectable in 3 h.     

Structure of a ricin mutant showing rescue of activity by a noncatalytic residue.

Ricin A chain is an N-glycosidase which removes a single adenine base from a conservative loop of 28S rRNA, thereby inactivating eukaryotic ribosomes. The mechanism of action has been proposed to include transition-state stabilization of an oxycarbonium ion on the substrate ribose by interaction with Glu 177. Conversion of Glu 177 to Gln reduces activity nearly 200-fold [Ready, M. P., Kim, Y., & Robertus, J. D. (1991) Proteins: Struct., Funct., Genet. 10, 270-278] while conversion to Ala (E177A) reduces activity only 20-fold [Schlossman, D., Withers, D., Welsh, P., Alexander, A., Robertus, J., & Frankel, A. (1989) Mol. Cell. Biol. 9, 5012-5021]. X-ray analysis of the latter mutant protein shows that a residue at the edge of the active site, Glu 208, rotates into the space left vacant by the mutation. Its rearranged carboxylate partially substitutes for that of Glu 177. This is equivalent to the rescue of enzyme activity by a second-site reversion. Kinetic analysis shows the E177A mutation affects kcat and not Km, consistent with the notion that the carboxylate serves in transition-state stabilization.     

Control of the Amiloride-Sensitive Na+ Current in Salivary Duct Cells by Extracellular Sodium

We have previously reported that intralobular salivary duct cells contain an amiloride-sensitive Na⁺ conductance (probably located in the apical membranes). Since the amiloride-sensitive Na⁺ conductances in other tight epithelia have been reported to be controlled by extracellular (luminal) Na⁺, we decided to use whole-cell patch clamp techniques to investigate whether the Na⁺ conductance in salivary duct cells is also regulated by extracellular Na⁺. Using Na⁺-free pipette solutions, we observed that the whole-cell Na⁺ conductance increased when the extracellular Na⁺ was increased, whereas the whole-cell Na⁺ permeability, as defined in the Goldman equation, decreased. The dependency of the whole-cell Na⁺ conductance on extracellular Na⁺ could be described by the Michaelis-Menten equation with a K _m of 47.3 mmol/1 and a maximum conductance (G _max) of 2.18 nS. To investigate whether this saturation of the Na⁺ conductance with increasing extracellular Na⁺ was due to a reduction in channel activity or to saturation of the single-channel current, we used fluctuation analysis of the noise generated during the onset of blockade of the Na⁺ current with 200 μmol/l 6-chloro-3,5-diaminopyrazine-2-carboxamide. Using this technique, we estimated the single channel conductance to be 4 pS when the channel was bathed symmetrically in 150 mmol/l Na⁺ solutions. We found that Na⁺ channel activity, defined as the open probability multiplied by the number of available channels, did not alter with increasing extracellular Na⁺. On the other hand, the single-channel current saturated with increasing extracellular Na⁺ and, consequently, whole-cell Na⁺ permeability declined. In other words, the decline in Na⁺ permeability in salivary duct cells with increasing extracellular Na⁺ concentration is due simply to saturation of the single-channel Na⁺ conductance rather than to inactivation of channel activity. Received: 27 July 1995/Revised: 7 December 1995     

Floristic diversity and co-occurrences in a subtropical broad-leaved forest and two contrasting regrowth stands in central-west Yunnan Province,China

Many of the natural forested ecosystems that still remain in mainland China are being cleared with potentially detrimental effects on woody plant species diversity on both local and regional scales. The most extensive stand of subtropical broad-leaved forest remaining in China is located in Yunnan Province. In an effort to document the influence of human-induced disturbance on Yunnan's woody flora, floristic inventories were conducted in a stand of primary forest and in regrowth stands located in its interior and along its outer margin in the Xujiaba Nature Sanctuary in the Ailao Mountain Range. Of particular interest was the location of the disturbance relative to the primary forest source area. A total of 134 woody plant species representing 74 genera and 43 families were recorded. The floristics of the two regrowth stands were significantly different from each other, with < 10% of their respective floras comprised of co-occurring species. The interior regrowth stand had a higher number of co-occurring species with the primary forest; however, > 40% were still non-co-occurring.The principal families represented in the primary forest and the interior regrowth stand were Aquifoliaceae, Berberidaceae, Fagaceae, Lauraceae, Rosaceae, Smilacaceae, Symplocaceae, Theaceae, and Vacciniaceae. The three dominant species with relative importance values ranging from > 5% to 18% in both the primary forest and the interior regrowth stand were Castanopsis wattii, Lithocarpus jingdongensis, and Symplocos sumuntia. The edge regrowth stands had the lowest species diversity and were dominated by the native pine Pinus yunnanensis, with a relative importance of 24%. The principal families represented in the edge regrowth stand were Betulaceae, Ericaceae, Fagaceae, Myricaceae, Pinaceae, and Theaceae. Only the Fagaceae and Theaceae were well-represented in all three stands. The results of the study document the low species diversity in post-cutting regrowth on the margins of the primary forest as compared with post-cutting regrowth in the forest interior.