Effects of medium viscosity on kinetics of the enzymatic reaction catalyzed by bacterial RNase

Effects of medium viscosity on kinetic parameters of poly(U) hydrolysis catalyzed by RNase from Bac. intermedius 7P (binase) were studied in solutions of sucrose (4-50 wt. %) and glycerol (35-62 wt. %) in Tris--sodium acetate buffer (pH 7.5) at 25 degreesC. The rate constant of reaction kcat was practically unchanged over a wide range of viscosities (1-15 cP for sucrose and 2.5-3 cP for glycerol). In glycerol solutions, kcat slightly increased with viscosity increase from 4 to 10 cP. Addition of NaCl to the buffer medium resulted in an inhibitory effect of Na+ on kcat, prevented by 50% sucrose or 60% glycerol. It is concluded that binase-catalyzed poly(U) cleavage occurs through a "tense"-substrate mechanism, similarly to reactions catalyzed by alpha-chymotrypsin, trypsin, and laccase.     

Granule-bound starch synthase: structure, function, and phylogenetic utility

Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low- copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.      

Stereoselective disulfide formation stabilizes the local peptide conformation in nisin mimics

Nisin is a polymacrocyclic peptide antimicrobial with high activity against Gram-positive bacteria. Lanthionine and methyllanthionine bridges, closing the macrocycles, are stabilized by thioether bonds, formed between cysteines and dehydrated serine or threonine. The role of polypeptide backbone conformation in the formation of macrocycles A and B within cysteine mutants of nisin residues 1?12 is investigated here by molecular dynamics simulations. Enantiomeric combinational space of Cys3 and Cys7 and of Cys8 and Cys11 is examined for the preference of disulfide bond formation over helical turn formation within this region. A clear preference for spontaneous disulfide formation and closure of rings 3,7 and 8,11 is demonstrated for the D-Cys3, D-Cys7, L-Cys8, L-Cys11 nisin homologue, while interlinked rings A and B are obtained through disulfide bridges between L-Cys3 and D-Cys8 and between D-Cys7 and D-Cys11. This study offers a simple designer approach to solid phase synthesis of macrocyclic peptides and lantibiotic analogues.     

Resistance to Melanaphis sacchari in the sugarcane cultivar R 365

This study focuses on the resistance of sugarcane, Saccharum spec. (Poaceae), to the sugarcane aphid, Melanaphis sacchari (Zehntner) (Hemiptera: Aphididae), which vectors Sugarcane yellow leaf virus (SCYLV). Resistance was characterized in cultivar R 365, using a 3‐year field trial and laboratory experiments on potted plantlets and excised leaves. R 365 reduced aphid populations in the field by antixenosis and antibiosis. Using the electrical penetration graph technique, we detected delayed aphid salivation in phloem and inhibition of passive phloem sap uptake in R 365. The resistance factors also proved to be effective against the corn leaf aphid, Rhopalosiphum maidis (Fitch) (Hemiptera: Aphididae), another vector of SCYLV.     

Motor and psycho-cognitive clinical types in adult metachromatic leukodystrophy: genotype/phenotype relationships?

Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is biochemically characterized by an accumulation of sulfatides (sulfogalactosylceramides) mainly in oligodendrocytes and macrophages/microglia. The deficient enzyme is a lysosomal hydrolase, cerebroside sulfate sulfatase (arylsulfatase A). MLD is both a dysmyelinating and a demyelinating disease. The main clinical forms are infantile or juvenile, but some forms appear at adulthood. This disease involves also neuronal cells as sulfatides are also present in neurons in which the defect in degradation occurs also. We have studied 12 cases of adult MLD and clearly distinguished two clinical forms. One of them was characterized by mainly central nervous system motor signs (pyramidal, cerebellar, and seldom dystonia) and a peripheral neuropathy. The other form always started by behavioural abnormalities with modifications of mood, peculiar social reactions; a progressive mental deterioration occurred also. The diagnosis of schizophrenia was often mentioned. Most of these patients remained for many years without any neurological symptoms, and the diagnosis was only made when neurological signs appeared, or when Magnetic Resonance Imaging (MRI) was performed. MRI showed a diffuse demyelination, bilateral and often symmetrical, which could be temporarily limited to the periventricular areas. The diagnosis of adult MLD was biochemical, evidencing the low activity of arylsulfatase A (ASA) and sulfatide accumulation. To determine the respective participation of neurons and glial cells in the physiopathology of both the motor forms and the psycho-cognitive forms, our first approach was to search for mutations differing according to the clinical status. Motor forms involved the major adult ASA mutation P426L in a homozygote form in contrast to psycho-cognitive forms which involved as a compound heterozygote a specific I179S mutation.     

An ion-exchange based extraction method for the detection of salmonellas in soil

A method that uses a cation-exchange resin (Chelex 100) and differential centrifugation for the extraction and detection of salmonellas in soil was developed. The extraction efficiencies of a range of materials were examined and Chelex plus polyethylene glycol was identified as the best combination. Shake speeds, shake times and differential centrifugation speeds were selected to give an optimum salmonella recovery. The Chelex method accurately enumerated 1 cell per 10 g of nonsterile soil within 24 h. Addition of glycerol to soil samples enabled storage at — 70°C for 85 d without significant decreases in salmonella numbers. The Oxoid Salmonella Rapid Test (SRT) could be used to pre-screen large numbers of soil samples for the presence of salmonellas, prior to analysis by the Chelex method. The SRT method detected Salmonella typhimurium at levels as low as 2·5 cells per 10 g of nonsterile soil.     

Confidence intervals for predicted age of onset,given the size of (CAG) n repeat,in Huntington's disease

A highly significant relationship between length of trinucleotide repeat and age of onset is shown for Huntington's disease.     

Naturally occurring CCR5 extracellular and transmembrane domain variants affect HIV-1 Co-receptor and ligand binding function.

Analysis of CCR5 variants in human immunodeficiency virus, type 1 (HIV-1), high risk cohorts led to the identification of multiple single amino acid substitutions in the amino-terminal third of the HIV-1 co-receptor CCR5 suggesting the possibility of protective and permissive genotypes; unfortunately, the low frequency of these mutations did not led to correlation with function. Therefore, we used analytical methods to assess the functional and structural significance of six of these variant receptors in vitro. These studies showed three categories of effects on CCR5 function. 1) Mutations in the first extracellular domain of CCR5 severely reduce specific ligand binding and chemokine-induced chemotaxis. 2) An extracellular domain variant, A29S, when co-expressed with CD4, supported HIV-1 infection whereas the others do not. 3) The transmembrane region variants of CCR5 support monotropic HIV-1 infection that is blocked by addition of some receptor agonists. Mutations in the first and second transmembrane domains increase RANTES (regulated on activation normal T-cell expressed) binding affinity but did not affect MIP1beta binding affinity presumably based on differences in ligand-receptor interaction sites. Furthermore, the CCR5 transmembrane mutants do not respond to RANTES with the classical bell-shaped chemotactic response curve, suggesting that they are resistant to RANTES-induced desensitization. These data demonstrate that single amino acid changes in the extracellular domains of CCR5 can have profound effects on both HIV-1 co-receptor and specific ligand-induced functions, whereas mutations in the transmembrane domain only affect the response to chemokine ligands.