Synthesis and glycosidase inhibitory activity of 5-thioglucopyranosylamines. Molecular modeling of complexes with glucoamylase

The synthesis of a series of 5-thio-D-glucopyranosylarylamines by reaction of 5-thio-D-glucopyranose pentaacetate with the corresponding arylamine and mercuric chloride catalyst is reported. The products were obtained as anomeric mixtures of the tetraacetates which can be separated and crystallized. The tetraacetates were deprotected to give alpha/beta mixtures of the parent compounds which were evaluated as inhibitors of the hydrolysis of maltose by glucoamylase G2 (GA). A transferred NOE NMR experiment with an alpha/beta mixture of 7 in the presence of GA showed that only the alpha isomer is bound by the enzyme. The Ki values, calculated on the basis of specific binding of the alpha isomers, are 0.47 mM for p-methoxy-N-phenyl-5-thio-D-glucopyranosylamine (7), 0.78 mM for N-phenyl-5-thio-D-glucopyranosylamine (8), 0.27 mM for p-nitro-N-phenyl-5-thio-D-glucopyranosylamine (9) and 0.87 mM for p-trifluoromethyl-N-phenyl-5-thio-D-glucopyranosylamine (10), and the K(m) values for the substrates maltose and p-nitrophenyl alpha-D-glucopyranoside are 1.2 and 3.7 mM, respectively. Methyl 4-amino-4-deoxy-4-N-(5'-thio-alpha-D-glucopyranosyl)-alpha-D-glucopyrano side (11) is a competitive inhibitor of GA wild-type (Ki 4 microM) and the active site mutant Trp120-->Phe GA (Ki 0.12 mM). Compounds 7, 8, and 11 are also competitive inhibitors of alpha-glucosidase from brewer's yeast, with Ki values of 1.05 mM, > 10 mM, and 0.5 mM, respectively. Molecular modeling of the inhibitors in the catalytic site of GA was used to probe the ligand-enzyme complementary interactions and to offer insight into the differences in inhibitory potencies of the ligands.     

Mapping of the human Voltage-Dependent Anion Channel isoforms 1 and 2 reconsidered

Eukaryotic porins or VDACs (Voltage-Dependent Anion-selective Channels) are integral membrane proteins forming large hydrophilic pores. Three functioning genes for VDAC isoforms have been detected in mouse and the corresponding cDNAs are known also in humans. Tissue-specific VDAC isoform 1 (HVDAC1) deficiency in human skeletal muscle is responsible of a rare mitochondrial encephalomyopathy, fatal in childhood. Since coding sequences are not affected in the patient, we focused our interest in the gene structure. HVDAC1 and 2 have been previously mapped at chromosomes Xq13-21 and 21, respectively. Screening of an human chromosome X cosmid library resulted only in the isolation of processed pseudogenes, finely mapped at Xq22 and Xp11.2. Here, we report the mapping of HVDAC1 to chromosome 5q31 and HVDAC2 to chromosome 10q22 by FISH. Exon/intron probes, designed on the basis of the mouse gene structures, were obtained by long extension PCR amplification using the whole genomic DNA as a template. The sequence of the probe extremities clearly pointed to a genuine VDAC genomic sequence. Human and mouse regions where VDAC 1 and 2 genes were mapped are known to be synthetic, thus reinforcing the mapping of the human homologues.     

Three-dimensional structure of an unusual Kunitz (STI) type trypsin inhibitor from Copaifera langsdorffii

The crystallographic structure of a novel trypsin inhibitor (CTI) from Copaifera langsdorffii is reported. The structure was solved by MIRAS procedure and refined to a crystallographic residual of 17.3% (R(free) = 20.3%) at 1.8 A resolution. Two isomorphous derivatives were obtained by quick cryo-soaking approach. CTI is the first structure of a member of Kunitz (STI) family formed by two noncovalently bound polypeptide chains and only one disulfide bridge. A standard Kunitz-type inhibitor has a single polypeptide chain and two disulfide bridges. Structural features granting CTI high inhibitory activity are discussed.     

Pulmonary aspergillosis outbreak in Rhea americana in Southern Brazil

Commercial raising of rheas is currently in expansion in the south of Brazil, and many diseases previously restricted to other avian species are currently emerging on rhea farms, especially as a result of careless management of these animals. The objective of the present article is to report a pulmonary aspergillosis outbreak that occurred in great rhea (Rhea americana) in the south of Brazil. About 50 birds aged 30 to 60 days died suddenly and one of them was submitted to autopsy which revealed the presence of white caseous nodules 0.5 mm in diameter occupying 95% of the lung area. One lung was sent to the Federal University of Santa Maria for histopathological and mycological analyses. Histopathological analysis revealed multifocal areas with necrosis and inflammatory infiltrates and the presence of fungal hyphae, giant cells and fibrous tissue proliferation at the periphery. Aspergillus fumigatus was recovered as pure culture from all culture media. This appears to be the first report of aspergillosis among great rhea in Brazil and the second in the world.     

GAA repeat instability in Friedreich ataxia YAC transgenic mice

Friedreich ataxia (FRDA) is primarily caused by an unstable GAA repeat-expansion mutation within intron 1 of the FRDA gene. However, the exact mechanisms leading to this expansion and its consequences are not fully understood. To study the dynamics of this mutation, we have generated two lines of human FRDA YAC transgenic mice that contain GAA repeat expansions within the appropriate genomic context. We have detected intergenerational instability and age-related somatic instability in both lines, with pronounced expansions found in the cerebellum. The dynamic nature of our transgenic GAA repeats is comparable with previous FRDA patient somatic tissue data. However, there is a difference between our FRDA YAC transgenic mice and other trinucleotide-repeat mouse models, which do not show pronounced repeat instability in the cerebellum. This represents the first mouse model of FRDA GAA repeat instability that will help to dissect the mechanism of this repeat.     

Enantiomeric resolution of kielcorin derivatives by HPLC on polysaccharide stationary phases using multimodal elution

Analytical HPLC methods using carbamate chiral stationary phases of polysaccharide derivatives were developed for the enantiomeric resolution of five racemic mixtures of xanthonolignoids: rac-trans-kielcorin C, rac-cis-kielcorin C, rac-trans-kielcorin D, rac-trans-isokielcorin D, and rac-trans-kielcorin E. The separations were evaluated with the stationary phases cellulose tris-3,5-dimethylphenylcarbamate, amylose tris-3,5-dimethylphenylcarbamate, amylose tris-(S)-1-phenylethylcarbamate, and amylose tris-3,5-dimethoxyphenylcarbamate under normal, reversed-phase, and polar organic elution conditions. Chiral recognition of those chiral stationary phases, the influence of mobile phases on the enantiomers separation, and the effects of structural features of the solutes on the chiral discrimination observed are discussed. The best performance was achieved on an amylose tris-3,5-dimethylphenylcarbamate phase. Polar organic conditions gave shorter retention factors and better resolutions and were a valuable alternative to the alcohol-hexane or reversed-phase conditions.     

Review of the large catfish fisheries in the upper Amazon and the stock depletion of <Emphasis Type="Italic">piraíba</Emphasis> (<Emphasis Type="Italic">Brachyplatystoma filamentosum</Emphasis>Lichtenstein)

In this paper we describe the present status of the large migratory catfish fisheries in the Upper Amazon. We present biological information about the main species and we give strong evidence that the stock of piraíba (Brachyplatystoma filamentosum),the largest catfish in the Amazon Basin is probably over-exploited. In conclusion, we raise some hypotheses about the causes and prospects for the future.     

The potential for gamete recovery from non-domestic canids and felids

Species are becoming extinct at a rate 100 times the natural background rates. Considering all mammalian orders, 24% of all Carnivora species are threatened. The goal of carnivore conservation is to reverse the decline in populations and to secure remaining populations in ways that will assure enduring public support. In this context, biotechnology is a tool with tremendous potential for assisting the conservation of endangered canid and felid species. As the first step for biotechnology development is the gamete obtainment, this review will discuss the potential of gamete recovery from non-domestic canids and felids, based on learning how to apply these procedures in the domestic carnivores. Thus, electroejaculation and obtaining both epidydimal spermatozoon and spermatogonial germ cells are indicated as techniques for male gametes recovery. In the female gametes retrieval, different methods for oocyte recovery from both antral and preantral follicles, and the possibility for ovarian tissue transplantation are discussed. Furthermore, the study discusses the responsibilities involved in the use of assisted reproduction in endangered species conservation.