Synthesis and antiviral activities of novel N-alkoxy-arylsulfonamide-based HIV protease inhibitors

A series of novel N-alkoxy-arylsulfonamide HIV protease inhibitors with low picomolar enzyme activity and single digit nanomolar antiviral activity is disclosed.     

Structural and functional properties of human alpha-thrombin, phosphopyridoxylated alpha-thrombin, and gamma T-thrombin. Identification of lysyl residues in alpha-thrombin that are critical for heparin and fibrin(ogen) interactions

alpha-Thrombin derivatives obtained either by site-specific modification at lysyl residues (phosphopyridoxylated) or by limited trypsinolysis (gamma T-thrombin) were compared to correlate structural modifications with the functional reactivity toward fibrin(ogen) and heparin. alpha-Thrombin phosphopyridoxylated in the absence of heparin (unprotected) showed approximately 2 mol of label incorporated/mol of thrombin, but only 1 mol of label incorporated/mol of proteinase when modified in the presence of added heparin (protected). In contrast to native alpha-thrombin, both phosphopyridoxylated alpha-thrombin derivatives failed to interact with a fibrin monomer-agarose column and had reduced fibrinogen clotting activity, which is very similar to gamma T-thrombin. Heparin accelerated the rate of antithrombin III inhibition of alpha-thrombin, heparin-protected modified-alpha-thrombin, and gamma T-thrombin in a manner consistent with a template mechanism but was without effect on unprotected modified alpha-thrombin. In a heparin-catalyzed antithrombin III inhibition assay of alpha-thrombin, we found that D-Phe-Pro-Arg chloromethyl ketone-active site-inactivated gamma T-thrombin competed for heparin binding. It has been shown that limited proteolysis/autolysis of the B-chain of alpha-thrombin in the area around Arg-B73 (in beta T/beta- and gamma T/gamma-thrombin), but not that around Lys-B154 (in gamma T/gamma-thrombin), diminishes specific interactions with fibrinogen (Hofsteenge, J., Braun, P. J., and Stone , S. R. (1988) Biochemistry 27, 2144-2151). In unprotected modified alpha-thrombin, lysyl residues B21, B65, B174, and B252 were phosphopyridoxylated. In heparin-protected modified alpha-thrombin, only lysyl residues B21 and B65 were phosphopyridoxylated. These observations suggest that lysyl residues 21/65 of the B-chain of alpha-thrombin are involved in fibrin(ogen) interactions, and lysyl residues 174/252 of the B-chain are important in heparin interactions.     

Evolution and phylogenetic utility of the period gene in Lepidoptera

Evolution and phylogenetic utility of the period gene are explored through sequence analysis of a relatively conserved 909-bp fragment in 26 lepidopteran species. Taxa range from tribes to superfamilies, primarily within the putative clade Macrolepidotera plus near outgroups, and include both strongly established and problematic groupings. Their divergence dates probably range from the late Cretaceous through much of the Tertiary. Comparisons within the same set of closely related species show that amino acid substitutions in period occur 4.9 and 44 times as frequently as they do in two other nuclear genes--dopa decarboxylase and elongation factor-1 alpha, respectively. In contrast, rates of observed synonymous substitution are within 60% of each other for these three genes. Synonymous changes in period approach saturation by the family level, whereas nonsynonymous and amino acid divergences across the Macrolepidoptera are less than half the maximal values reported for this gene. Phylogenetic analyses of period strongly supported groupings at the family level and below. In contrast to previous analyses at this level with other nuclear genes, much of the information lies in nonsynonymous change. Relationships up to the superfamily level were recovered with decreasing effectiveness, and little, if any, signal was apparent regarding relationships among superfamilies. This could reflect rapid radiation of the superfamilies, however, rather than saturation in the period locus; thus, period, in combination with other genes, remains a plausible candidate for approaching the difficult problems of lepidopteran family and superfamily relationships.      

Impact of Coal-Coking Effluent on Sediment Microbial Communities: a Multivariate Approach

The functional response to and recovery from coal-coking waste effluent was evaluated for sediment microbial communities. Twenty estimates of microbial population density, biomass, and activity were measured five times during a 15-month period. Significant effects on microbial communities were observed in response to both wastewater contamination and diversion of the wastewater. Multivariate analysis of variance and discriminant analysis indicated that accurate differentiation between uncontaminated and contaminated sediments required a minimum of nine estimates of community response. Total viable population density, ATP, alkaline phosphatase, naphthalene, and phenanthrene mineralization rates were found to be highly weighted variables in site discrimination. Lipid and glucose mineralization, nitrogen fixation, and sediment protein also contributed significantly to explaining variation among sites. Estimates of anaerobic population densities and rates of methane production contributed little to discrimination among sites in the environment examined. In general, total viable population density, ATP, and alkaline phosphatase activity were significantly depressed in contaminated sediments. However, after removal of this contamination, the previously affected sites demonstrated greater temporal variability but a closer approximation of the mean response at the control site. Naphthalene and phenanthrene mineralization did not follow the general trend and were elevated at the contaminated sites throughout the investigation. Results of the investigation supported the hypothesis that multiple functional measures of microbial community response are required to evaluate the effect of and recovery from environmental contamination. In addition, when long-term effects are evaluated, select physiological traits, i.e., polyaromatic hydrocarbon mineralization, may not reflect population and biomass estimates of community response.     

Peripheral hyaline blebs (podosomes) of macrophages

The plasmalemma and hyaline ectoplasm together constitute the sensory and motor organ of macrophages. The purpose of this study was to isolate this cell fraction in order to analyze it biochemically and functionally. Brief sonification of warmed rabbit lung macrophages caused release of heterodisperse hyaline blebs and filopodia, which were easily collected by differential centrifugation. Viewed in the electron microscope, these structures consisted of membrane-bounded sacs principally containing actin filaments. Some contained secondary lysosomes. They were enriched threefold over whole cell homogenates in specific adenylate cyclase activity and in trichloroacetic-acid-precipitable (125)I when derived from cells labeled with 125(I) by means of a lactoperoxidase-catalyzed reaction. These markers were found to have identical isopycnic densitites when macrophage homogenates were subjected to sedimentation in a focusing sucrose density gradient system, and these markers had densities distinct from those of other cytoplasmic organelles. These markers were therefore assumed to be associated with macrophage plasma membranes. The specific β- glucuronidase activity of the bleb fraction was similar to that of homogenates, but the blebs had considerably lower specific succinic dehydrogenase activity and RNA content, and DNA was undetectable. Electrophoresis of blebs solubilized in sodium dodecyl sulfate on polyacrylamide gels revealed polypeptides co-migrating with macrophage actin-binding protein, myosin, and actin; blebs also had EDTA-activated adenosine triphosphatase activity characteristic of myosin. The concentrations of actin-binding protein and myosin were higher in blebs than in cells or cytoplasmic extracts, whereas actin concentrations were similar (relative to extracts) or only slightly greater (than in cells). Blebs and intact cells had high lactate dehydrogenase activities in the presence but not the absence of Triton X-100. Blebs and cells oxidased 1-[(14)C]glucose, and the rate of glucose oxidation was increased substantially in the presence of latex beads. We conclude that intact sacs of plasmalemma encasing contractile proteins and cytoplasmic enzymes can be isolated from macrophages. They are enriched in myosin and actin-binding protein, indicating that the contractile apparatus is regulated in the cell periphery. These structures have the capacity to respond to environmental signals. We suggest the name "podosomes" for them because of their resemblance to macrophage pseudopodia. We propose that podosome formation results from rapid dissolution of the cortical gel when the membrane is in an actively extended configuration.     

Salivary alpha amylase and salivary cortisol response to fluid consumption in exercising athletes

The objective of the study was to examine salivary biomarker response to fluid consumption in exercising athletes. Exercise induces stress on the body and salivary alpha amylase (sAA) and salivary cortisol are useful biomarkers for activity in the sympathoadrenal medullary system and the hypothalamic pituitary adrenal axis which are involved in the stress response. Fifteen college students were given 150 ml and 500 ml of water on different days and blinded to fluid condition. The exercise protocol was identical for both fluid conditions using absolute exercise intensities ranging from moderate to high. Saliva was collected prior to exercise, post moderate and post high intensities and analyzed by Salimetrics assays. Exercise was significant for sAA with values different between pre-exercise (85 ± 10 U · ml⁻¹) and high intensity (284 ± 30 U · ml⁻¹) as well as between moderate intensity (204 ± 32 U · ml⁻¹) and high intensity. There was no difference in sAA values between fluid conditions at either intensity. Exercise intensity and fluid condition were each significant for cortisol. Cortisol values were different between pre-exercise (0.30 ± 0.03 ug · dL⁻¹) and high intensity (0.45 ± 0.05 ug · dL⁻¹) as well as between moderate intensity (0.33 ± 0.04 ug · dL⁻¹) and high intensity. Moderate exercise intensity cortisol was lower in the 500 ml condition (0.33 ± 0.03 ug · dL⁻¹) compared with the 150 ml condition (0.38 ± 0.03 ug · dL⁻¹). This altered physiological response due to fluid consumption could influence sport performance and should be considered. In addition, future sport and exercise studies should control for fluid consumption.     

High-level expression of the 32.5-kilodalton calelectrin in ductal epithelia as revealed by immunocytochemistry

Abstract. Calelectrins are a family of antigenically related Ca²⁺-binding proteins that have only recently been described. They have the important property of binding to membranes only in the presence of Ca²⁺. We systematically studied the tissue localization of one calelectrin, the 32.5-kilodalton species, in rats using immunocytochemistry. We found that high levels were exclusively present in the epithelial cells of bile and pancreatic ducts, renal collecting ducts, bronchial epithelia, and brain ependyma. In all of these organs, the other cells were not immunoreactive. In addition, strong immunoreactivity was found in the intercalated disks of myocardial cells, and mild immunoreactivity was observed in several endocrine tissues. In contrast, the cellular distribution of the 67-kilodalton calelectrin was more diffuse, involving most parenchymal cells in addition to the already-mentioned cells. Due to the presence of high levels of 32.5-kilodalton calelectrin in some cell types, this protein may be used as a histochemical marker for differentiated ductal epithelial cells, some specialized epithelia, myocardial cells, and Paneth cells.     

Phenanthrene Biodegradation in Freshwater Environments

Phenanthrene, a low-molecular-weight polycyclic aromatic hydrocarbon, was incubated with water samples from various reservoir systems in Tennessee to evaluate the potential for significant polycyclic aromatic hydrocarbon degradation by the indigenous microbial populations. Biodegradation was assessed by comparison of total polycyclic aromatic hydrocarbon substrate recovery in degradation flasks relative to sterile control flasks. During 1977 field studies, the mean phenanthrene biodegradation was approximately 80% after a 4-week incubation. Within a given habitat, 45% of the total variability in phenanthrene biodegradation was attributable to the physical, chemical, and microbiological site characteristics examined. Polycyclic aromatic hydrocarbon degradation was directly related to the historical environmental pollution of the sampling sites examined, the length of biodegradation assessment, temperature, and the molecular size of the polycyclic aromatic hydrocarbon substrate.