Design and synthesis of pyridone inhibitors of non-nucleoside reverse transcriptase

Next generation NNRTIs are sought which possess both broad spectrum antiviral activity against key mutant strains and a high genetic barrier to the selection of new mutant viral strains. Pyridones were evaluated as an acyclic conformational constraint to replace the aryl ether core of MK-4965 (1) and the more rigid indazole constraint of MK-6186 (2). The resulting pyridone compounds are potent inhibitors of HIV RT and have antiviral activity in cell culture that is superior to other next generation NNRTI’s.     

Unusual records of deepwater teleosteans trawled off the western coast of Mexico

The first records of three midwater species for the Mexican ichthyofauna (Holtbyrnia laticauda, Brama dussumieri and Cubiceps baxteri), caught off the coast of Baja California, Mexico, in mid 2014, are reported. As far as is known, they are the first verified specimens, geo‐referenced and catalogued in a reference fish collection for the west coast of Mexico. The species' known distributions were extended northward in the eastern Pacific Ocean, ranging from 3495 to 8300 km.     

Intrinsically disordered proteins: regulation and disease

Intrinsically disordered proteins (IDPs) are enriched in signaling and regulatory functions because disordered segments permit interaction with several proteins and hence the re-use of the same protein in multiple pathways. Understanding IDP regulation is important because altered expression of IDPs is associated with many diseases. Recent studies show that IDPs are tightly regulated and that dosage-sensitive genes encode proteins with disordered segments. The tight regulation of IDPs may contribute to signaling fidelity by ensuring that IDPs are available in appropriate amounts and not present longer than needed. The altered availability of IDPs may result in sequestration of proteins through non-functional interactions involving disordered segments (i.e., molecular titration), thereby causing an imbalance in signaling pathways. We discuss the regulation of IDPs, address implications for signaling, disease and drug development, and outline directions for future research.     

流行性出血热病毒R22株cDNA克隆及其特异性鉴定

用家鼠型流行性出血热病毒R22株RNA,经polyA接尾,以Oligo-dT做引物,合成cDNA。用pUC18为载体转染E.coli Mc1061,建立cDNA克隆。再经菌落杂交,选择病毒特异性的5个阳性克隆制成缺口翻译探针,与病毒RNA3个片段进行反杂交,确定RNA片段的特异性。结果表明,3个克隆为中(M)片段的cDNA,另两个分别为大(L)和小(S)片段cDNA。核苷酸序列分析证明,克隆的DNA中含病毒特异的核苷酸序列。     

High altitude tissue adaptation in Andean coots: capillarity,fibre area,fibre type and enzymatic activities of skeletal muscle

Capillarity, fibre types, fibre area and enzyme activities of different skeletal muscles (pectoralis, extensor digitorum longus), tibialis anterior, plantaris and the myocardium were compared in Andean coot (Fulica americana peruviana) native to high altitude (Junín, Perú, 4200 m) and the same species nesting at sea level. Numbers of capillaries per square millimeter were higher in all high-altitude muscles when compared with sea-level muscles (P<0.0001). Moreover, values for capillaries per fibre and capillaries in contact with each fibre were higher in digitorum and tibialis high-altitude muscles. Muscle fibres were classified as Type I, Type IIA or Type IIB on the basis of their myofibrillar ATPase pH lability. Pectoralis muscle of high-altitude and sea-level coots presented only fibres of Type IIA. In contrast, all the leg muscles studied showed a mosaic pattern of the three fibre types. Fibre areas were determined using a Leitz Texture Analysis System. Significant differences in fibre area were observed (P<0.01) between high-altitude and sea-level muscles. Mean muscle fibre diameters were also lower in the high-altitude group than in the sea-level group. The enzyme activities studied were hexokinase, lactate dehydrogenase, citrate synthase and 3-hydroxyacyl-CoA-dehydrogenase. The oxidative capacity, as reflected by citrate synthetase and hydroxyacyl-CoA-dehydrogenase activities, was greater for myocardial and pectoralis than for leg muscles. However, analysis of maximal enzyme activities showed that there were no significant differences between the glycolytic and oxidative enzyme activities of high-altitude and sea-level coots. These results suggest that in Andean coots genetically adapted to high altitude, changes in muscle capillarity and fibre size, in addition to high haemoglobin O₂ affinity and low haemoglobin concentration, are sufficient to allow adequate energy production without increases in enzymatic activities.Abbreviations BSA bovine serum albumin - C:F ratio Capillaries per fibre - CAF Capillaries in contact with each fibre - CD capillary density (mm^-2) - CS citrate synthetase - EDL muscularis digitorum longus - fra fraction reduction area - HA high altitude - HAD hydroxyacyl-CoA-dehydrogenase - HK hexokinase - LDH lactate dehydrogenase - P ₅₀ PO₂ at which hemoglobin is half saturated with O₂ - P _aO₂ arterial partial pressure of oxygen - PAS periodic acid-schiff - PEC muscularis pectoralis - PLA muscularis planaris - P _tO₂ mean tissue oxygen pressure - P _vO₂ mixed venous partial pressure of oxygen - SD standard deviation - SL sea level - TA muscularis tibialis anterior - TAS texture analysis system     

The SWISS-2DPAGE database of two-dimensional polyacrylamide gel electrophoresis, its status in 1995.

SWISS-2DPAGE is a database of proteins identified on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The current release contains 343 entries of human, yeast (Saccharomyces cerevisiae) and Escherichia coli origin, as well as virtual entries for each of the protein sequences in the SWISS-PROT database.     

Purification and properties of a coagulant thrombin-like enzyme from the venom of Bothrops leucurus

A thrombin-like enzyme from Bothrops leucurus venom, named leucurobin (leuc), was purified by gel filtration, affinity and ion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 35 kDa monomeric glycoprotein on SDS-PAGE under reducing conditions, which decreased to 29 kDa after deglycosylation with N-glycosidase F (PNGase F). The amino acid sequence of leuc was determined by automated sequencing of the intact native protein and peptides produced by digestion of the S-pyridyl-ethylated protein with trypsin. The protein sequence exhibits significant similarities with other serine proteases reported from snake venoms, and contains two potential sites of N-linked glycosylation. The proteinase split off fibrinopeptide A (FPA) rapidly from human fibrinogen; however, only negligible traces of fibrinopeptide B (FPB) were observed. In addition, the enzyme released the N-terminal peptide (Mr = 4572) containing the first 42 residues from the Bβ-chain. Leuc could neither activate factor XIII nor release kinins from heat-treated bovine plasma. Its specific clotting activity was equivalent to 198 NIH thrombin U/mg on human fibrinogen. Kinetic properties of leuc were determined using representative chromogenic substrates. The enzyme evoked the gyroxin syndrome when injected into the tail veins of mice at levels of 0.143 μg/g mouse. The inhibitory effects of PMSF and benzamidine on the amidolytic activity suggest that leuc is a serine proteinase, and inhibition by β-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Antibothropic serum, SBTI and EDTA had little or no effect on its amidolytic activity. However, the clotting effect of the enzyme was strongly inhibited by antibothropic serum. A Dixon plot showed that the hydrolysis of Bz-l-Arg-pNA by leuc was competitively inhibited by benzamidine (K_i = 1.61 ± 0.25 mM).     

Sensory, chemical and bacteriological changes during storage of iced squid (Todaropsis eblanae)

AIMS: To relate sensory shelf-life of iced whole and gutted squid to bacterial growth and chemical changes. METHODS AND RESULTS: Cooked mantles from whole and gutted individuals were rejected after 10 and 12 days of storage, respectively, due to ammoniacal off-odours. Rate of production of both ammonia and trimethylamine was highest in the whole lot. Agmatine, which was only present in trace amounts in freshly-caught squid, increased rapidly in both lots. The main microflora at the time of sensory rejection of iced whole squid included Gram-negative, motile and non-fermentative rods, which were psychrophilic and had a requirement for NaCl. 16S rDNA sequence analyses identified the strains as belonging to the genus Pseudoalteromonas. Shewanella putrefaciens, Pseudoalteromonas sp. and Pseudomonas sp. dominated in spoiled gutted squid. Identification of strains from the stomach and digestive gland of recently-captured squid showed that the main flora consisted of Photobacterium phosphoreum. CONCLUSIONS: Spoilage of iced squid is likely to result from a combination of autolytic and bacterial changes. Agmatine seems to be an excellent freshness indicator. Photobacterium phosphoreum may contribute to spoilage through activity in the digestive gland, followed by diffusion of volatile compounds and amines to the mantle. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the psychrophilic nature of P. phosphoreum and Pseudoalteromonas sp., spread-plating and low temperature incubation are recommended for bacteriological evaluation of iced squid.