DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).     

Flavones are inhibitors of HIV-1 proteinase.

Substituted gamma-chromones were found to weakly inhibit HIV-1 proteinase, an important enzyme in the replication and processing of the AIDS virus. Chromones bearing hydroxyl substituents and a phenolic group at the 2-position (flavones) were the most active compounds and structure-activity relationships for a limited series of flavone inhibitors are presented. Dixon plots are reported and a possible mechanism for flavone-induced inhibition is proposed. The results are also compared with those for some structurally related non-peptidic inhibitors of HIV-1 proteinase. Since some flavonoid compounds have already been shown to have antiviral activity against AIDS, the present observations of anti-HIV-1 proteinase activity may be particularly significant.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced nicotinamide-adenine dinucleotide by affinity chromatography.

Rabbit muscle lactate dehydrogenase was subjected to frontal affinity chromatography on Sepharose-oxamate in the presence of various concentrations of NADH and sodium phosphate buffer (0.05 M, pH 6.8) containing 0.5 M-NaCl. Quantitative interpretation of the results yields an intrinsic association constant of 9.0 x 10 (4)M-1 for the interaction of enzyme with NADH at 5 degrees C, a value that is confirmed by equilibrium-binding measurements. In a second series of experiments, zonal affinity chromatography of a mouse tissue extract under the same conditions was used to evaluate assoication constants of the order 2 x 10(5)M-1, 3 x 10(5)M-1, 4 x 10(5)M-1, 7 x 10(5)M-1 and 2 x 10(6)M-1 for the interaction of NADH with the M4, M3H, M2H2, MH3 and H4 isoenzymes respectively of lactate dehydrogenase.     

Delimitation of the maritime boundary in the gulf of Maine area

The compulsory dispute settlement regime included in the 1982 Law of the Sea Convention is recognized as one of the most comprehensive in a modern international convention. Yet, in the recent application of this regime, the question has arisen as to whether the procedural prerequisites associated with the LOS Convention's compulsory dispute settlement mechanism are so arduous as to avoid binding and compulsory jurisdiction in most instances. This article addresses that question by examining, in particular, the reasoning of the Southern Bluefin Tuna arbitration tribunal, which found Article 281 of Section 1 of the LOS Convention to bar jurisdiction to the compulsory dispute settlement mechanism prescribed by the Convention, and offers suggestions as to how states might distinguish or overcome the barriers imposed by the Southern Bluefin Tuna tribunal in future cases.     

Widespread Shortening of 3’ Untranslated Regions and Increased Exon Inclusion Are Evolutionarily Conserved Features of Innate Immune Responses to Infection

The contribution of pre-mRNA processing mechanisms to the regulation of immune responses remains poorly studied despite emerging examples of their role as regulators of immune defenses. We sought to investigate the role of mRNA processing in the cellular responses of human macrophages to live bacterial infections. Here, we used mRNA sequencing to quantify gene expression and isoform abundances in primary macrophages from 60 individuals, before and after infection with Listeria monocytogenes and Salmonella typhimurium. In response to both bacteria we identified thousands of genes that significantly change isoform usage in response to infection, characterized by an overall increase in isoform diversity after infection. In response to both bacteria, we found global shifts towards (i) the inclusion of cassette exons and (ii) shorter 3’ UTRs, with near-universal shifts towards usage of more upstream polyadenylation sites. Using complementary data collected in non-human primates, we show that these features are evolutionarily conserved among primates. Following infection, we identify candidate RNA processing factors whose expression is associated with individual-specific variation in isoform abundance. Finally, by profiling microRNA levels, we show that 3’ UTRs with reduced abundance after infection are significantly enriched for target sites for particular miRNAs. These results suggest that the pervasive usage of shorter 3’ UTRs is a mechanism for particular genes to evade repression by immune-activated miRNAs. Collectively, our results suggest that dynamic changes in RNA processing may play key roles in the regulation of innate immune responses.     

Inhibition of HIV-1 proteinase by metal ions.

1. Certain metal ions have been identified as inhibitors (IC50 1-20 microM) of the aspartic proteinase of Human Immunodeficiency Virus Type 1 (HIV-PR). 2. By contrast most simple metal ions do not inhibit this enzyme. 3. Those that did inhibit have in common a high charge/size ratio or "hard" acidic nature, preferring to combine covalently with oxygen donor ligands. 4. Some evidence from independent X-ray crystal structure determinations suggests that the metalloinhibitors identified here may bind in the active site of the enzyme via coordination to the carboxylate side chains of the essential active site residues Asp 25 and 125. 5. Although the measured inhibition is only microM, very few enzyme-inhibitor interactions can be taking place and so more complex metalloinhibitors with ligands that can also bind to peptide side chains of the enzyme might be significantly more potent inhibitors of HIV-PR and of viral replication.     

Hemoglobin-degrading, aspartic proteases of blood-feeding parasites: substrate specificity revealed by homology models

Blood-feeding parasites, including schistosomes, hookworms, and malaria parasites, employ aspartic proteases to make initial or early cleavages in ingested host hemoglobin. To better understand the substrate affinity of these aspartic proteases, sequences were aligned with and/or three-dimensional, molecular models were constructed of the cathepsin D-like aspartic proteases of schistosomes and hookworms and of plasmepsins of Plasmodium falciparum and Plasmodium vivax, using the structure of human cathepsin D bound to the inhibitor pepstatin as the template. The catalytic subsites S5 through S4' were determined for the modeled parasite proteases. Subsequently, the crystal structure of mouse renin complexed with the nonapeptidyl inhibitor t-butyl-CO-His-Pro-Phe-His-Leu [CHOHCH(2)]Leu-Tyr-Tyr-Ser- NH(2) (CH-66) was used to build homology models of the hemoglobin-degrading peptidases docked with a series of octapeptide substrates. The modeled octapeptides included representative sites in hemoglobin known to be cleaved by both Schistosoma japonicum cathepsin D and human cathepsin D, as well as sites cleaved by one but not the other of these enzymes. The peptidase-octapeptide substrate models revealed that differences in cleavage sites were generally attributable to the influence of a single amino acid change among the P5 to P4' residues that would either enhance or diminish the enzymatic affinity. The difference in cleavage sites appeared to be more profound than might be expected from sequence differences in the enzymes and hemoglobins. The findings support the notion that selective inhibitors of the hemoglobin-degrading peptidases of blood-feeding parasites at large could be developed as novel anti-parasitic agents.     

Examination of ras oncoproteins in human plasma from healthy controls and workers exposed to petroleum emissions, including benzene-related compounds

Ras oncoproteins in blood plasma from workers exposed to petroleum emissions and unexposed controls were examined from Polish and Estonian samples. Twenty-four workers and 35 unexposed controls were examined from Poland and 97 exposed and 40 unexposed controls from Estonia. Of the Estonian workers, 50 were exposed to benzene in a benzene production plant and 47 to polyaromatic hydrocarbons and benzene in a cokery. Blood plasma proteins were separated by gel electrophoresis, transferred to a nitrocellulose membrane by Western blotting and detected by chemiluminescence using a monoclonal antibody as the primary antibody. There were no statistically significant differences between the exposed and the control groups in either the Polish or the Estonian samples.