Cloning and Molecular Modeling of Duodenase with Respect to Evolution of Substrate Specificity within Mammalian Serine Proteases That Have Lost a Conserved Active-Site Disulfide Bond

Mammalian serine proteases such as the chromosome 14 (Homo sapiens, Mus musculus) located granzymes, chymases, cathepsin G, and related enzymes including duodenase collectively represent a special group within the chymotrypsin family which we refer to here as "granases". Enzymes of this group have lost the ancient active-site disulfide bond Cys191-Cys220 (bovine chymotrypsinogen A numbering) which is strongly conserved in classic serine proteases such as pancreatic, blood coagulation, and fibrinolysis proteases and others (granzymes A, M, K and leukocyte elastases). We sequenced the cDNA encoding bovine (Bos taurus) duodenase, a granase with unusual dual trypsin-like and chymotrypsin-like specificity. The sequence revealed a 17-residue signal peptide and two-residue (GlyLys) activation peptide typical for granases. Production of the mature enzyme is apparently accompanied by further proteolytic processing of the C-terminal pentapeptide extension of duodenase. Similar C-terminal processing is known for another dual-specific granase, human cathepsin G. Using phylogenetic analysis based on 39 granases we retraced the evolution of residues 189 and 226 crucial for serine protease primary specificity. The analysis revealed that while there is no obvious link between mutability of residue 189 and the appearance of novel catalytic properties in granases, the mutability of residue 226 evidently gives rise to different specificity subgroups within this enzyme group. The architecture of the extended substrate-binding site of granases and structural basis of duodenase dual specificity based on molecular dynamic method are discussed. We conclude that the marked selectivity of granases that is crucial to their role as regulatory proteases has evolved through the fine-tuning of specificity at three levels--primary, secondary, and conformational.     

Characterization of microsatellite DNA markers in a critically endangered species,Mekong giant catfish,Pangasianodon gigas

Microsatellite DNA markers for a critically endangered Mekong giant catfish (Pangasianodon gigas Roberts and Vidthayanon, 1991) were developed from fin clips collected from captive fish using (GT)₁₅ probe. The number of alleles per locus ranged from two to four. The expected heterozygosities ranged from 0.13 to 0.68. Also, these primers were successfully amplified in four closely related species, Pangasius bocourti, Pangasius conchophilus, Pangasius larnaudii and Pangasius sanitwongsei with the number of alleles per locus ranged from 1 to 13, 1 to 16, 1 to 12 and 1 to 4, respectively. These markers should prove to be very useful for the evaluation of genetic diversity for this species and other related Pangasius species.     

Protective and Antioxidative Effects of GM1 Ganglioside in PC12 Cells Exposed to Hydrogen Peroxide are Mediated by Trk Tyrosine Kinase

GM1 ganglioside was found to increase the survival of PC12 cells exposed to H₂O₂, its action was blocked by Trk tyrosine kinase inhibitor K-252a. Thus, the inhibition of H₂O₂ cytotoxic action by GM1 constituted 52.8 ± 4.3%, but in the presence of 1.0 μM K-252a it was only 11.7 ± 10.8%, i.e. the effect of GM1 became insignificant. Exposure to GM1 markedly reduced the increased accumulation of reactive oxygen species (ROS) and diminished the inactivation of Na⁺,K⁺-ATPase induced in PC12 cells by H₂O₂, but in the presence of K-252a GM1 did not change these metabolic parameters. The inhibitors of extracellular signal-regulated protein kinase, phosphatidyl inositol 3-kinase and protein kinase C decreased the effects of GM1. A combination of these protein kinase inhibitors reduced inhibition of H₂O₂ cytotoxic action by GM1 to the larger extent than each of the inhibitors and practically abolished the ability of GM1 to decrease H₂O₂-induced ROS accumulation. The protective and antioxidative effects of GM1 in PC12 cells exposed to H₂O₂ appear to be mediated by activation of Trk receptor tyrosine kinase and the protein kinases downstream from this enzyme.     

Use of a preparation from fungal pectin lyase in the food industry

A new enzyme preparation of fungal pectin lyase (EC 4.2.2.10) was shown to be useful for the production of cranberry juice and clarification of apple juice in the food industry. A comparative study showed that the preparation of pectin lyase is competitive with commercial pectinase products. The molecular weight of homogeneous pectin lyase was 38 kDa. Properties of the homogeneous enzyme were studied. This enzyme was most efficient in removing highly esterified pectin.     

Regulatory role of elements in the formation and accumulation of alkaloids in Papaver somniferum L. seedlings

The effects of 12 elements on the formation and accumulation of isoquinolines were studied in opium poppy seedlings (Papaver somniferum L.). Three types of concentration dependences of the effects of the elements under study were determined. The elements served as activators (Co, Mo, W, Cr, and Cu) or inhibitors of these processes (B, Fe, V, Mn, and Ca). The molecular mechanisms of action of these regulators are discussed. The optimum concentrations of Co and Mo were tested under combined treatment conditions with these elements.     

The psychomodulating effect of sub-pyrogenic doses of interleukin-1beta during chronic administration to rats

Behavioral effects of low, sub-pyrogenic doses of IL-lbeta were investigated in rats. It was shown that increasing of anxiety level started to occur as of the 5th day of the administration course. The data obtained suggest importance of pro-inflammatory cytokines in pathogenesis of psycho-emotional disorders.     

Development, ultrastructure, natural occurrence, and molecular characterization of Liebermania patagonica n. g., n. sp., a microsporidian parasite ot the grasshopper Tristira magellanica (Orthoptera: Tristiridae)

A new microsporidium, Liebermannia patagonica n. gen., n. sp., is described from midgut and gastric caecum epithelial cells of Tristira magellanica, an apterous grasshopper species of southern Patagonia, Argentina. L.patagonica is diplokaryotic, apansporoblastic, homosporous, and polysporoblastic. Transitional (from merogony to sporogony) stages and sporonts of L. patagonica were surrounded by host rough endoplasmic reticulum. The ovocylindrical spores measured 2.9 +/- 0.09 x 1.2 +/- 0.04 microm (fresh, n = 50), and they had an isofilar polar filament of only three coils and a cluster of tubules instead of a classical posterior vacuole. Prevalence was high (up to 80.6%) at the type locality for the four years sampled . Maximum likelihood , neighbor joining, maximum parismony analyses of the small submit rDNA all placed L.patagonica(Accession No. DQ 239917) in one with Orthosomella operophterae.     

Thermalkalibacillus uzonensis gen. nov. sp. nov, a novel aerobic alkali-tolerant thermophilic bacterium isolated from a hot spring in Uzon Caldera, Kamchatka

A novel thermophilic, alkali-tolerant, and CO-tolerant strain JW/WZ-YB58^T was isolated from green mat samples obtained from the Zarvarzin II hot spring in the Uzon Caldera, Kamchatka (Far East Russia). Cells were Gram-type and Gram stain-positive, strictly aerobic, 0.7–0.8 μm in width and 5.5–12 μm in length and produced terminal spherical spores of 1.2–1.6 μm in diameter with the mother cell swelling around 2 μm in diameter (drumstick-type morphology). Cells grew optimally at pH^25°C 8.2–8.4 and temperature 50–52°C and tolerated maximally 6% (w/v) NaCl. They were strict heterotrophs and could not use either CO or CO₂ (both with or without H₂) as sole carbon source, but tolerated up to 90% (v/v) CO in the headspace. The isolate grew on various complex substrates such as yeast extract, on carbohydrates, and organic acids, which included starch, d-galactose, d-mannose, glutamate, fumarate and acetate. Catalase reaction was negative. The membrane polar lipids were dominated by branched saturated fatty acids, which included iso-15:0 (24.5%), anteiso-15:0 (18.3%), iso-16:0 (9.9%), iso-17:0 (17.5%) and anteiso-17:0 (9.7%) as major constituents. The DNA G+C content of the strain is 45 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain JW/WZ-YB58^T is distantly (<93% similarity) related to members of Bacillaceae. On the basis of 16S rRNA gene sequence, physiological and phenotypic characteristics, the isolate JW/WZ-YB58^T (ATCC BAA-1258; DSM 17740) is proposed to be the type strain for the type species of the new taxa within the family Bacillaceae, Thermalkalibacillus uzoniensis gen. nov. sp. nov. The Genbank accession number for the 16S rRNA gene sequence is DQ221694.The Genbank accession number for the 16S rRNA gene sequence of strain JW/WZ-YB58^T is DQ221694.     

Molecular diagnosis and frequencies of primary hypolactasia in populations of Russia and neighboring countries

Normally, the ability to digest milk sugar (lactose) is present in every child, but not in every adult. The decrease in lactase synthesis (hypolactasia) results in the inability to digest whole milk. Recent studies of the Finnish population have associated lactase persistence in adults with allele T of the C/T_?13910 polymorphism located upstream of the lactase gene; a 100% correlation of primary hypolactasia with genotype C/C has been proved. In this study, the allele and genotype frequencies of C/T_?13910 were determined in populations of Russia. The frequencies of genotype C/C, varying from 36.6% in Russians to 88.2% in Chukchi, were close to the published medical and epidemiological data on the hypolactasia frequencies in these populations. Genotyping was carried out by three different methods to determine the optimal one. Genotype C/C proved to be the key determinant of primary hypolactasia. It was assumed that DNA diagnosis of genotype C/C provides a predictive test to detect primary hypolactasia long before its clinical manifestation.