Unusual genome organization of the marine invertebrate Rapana thomasiana Grosse (Gastropoda)

The genome organization of the marine snail Rapana thomasiana Grosse (Gastropoda), genome size 2.7 pg, was studied by reassociation kinetics, S₁-nuclease assay, and restriction enzyme analysis. The slow-reassociating (single-copy) fraction represented only 21% of the genome. The average length of 80% of the single-copy sequences was less than 700 bp and the remaining 20% no longer than 1,400 bp. Longer stretches of unique DNA were not observed. The genome contained an unusually high percent-age of inverted repeats: at standard fragment length the zero-time binding fraction amounted to 25% of the genome. Foldback structures ranging from 200 bp to more than 10 kb were observed after S₁-nuclease treatment. They were randomly distributed throughout at least 85% of the genome, and the spacings between them were estimated to be about 1,600 bp on the average. The middle-repetitive DNA (45% of the genome) contained two kinetic components, repeated 430 and 65,000 times per genome, respectively. It was found that the majority of the repetitive sequences are about 300 bp long. Longer repeats (about 2,000 bp) were also observed, comprising a small portion of the genome. The inverted repeats, the middle-repetitive, and the singly-copy sequences were fully interspersed in the genome, thus indicating that R. thomasiana DNA is not organized in either the Xenopus or the Drosophila pattern type. — R. thomasiana is the only mollusc so far in which a satellite DNA has been found. It is organized in tandem repeats of 1,460 bp with a very complex organization but a low degree of divergence.     

A four-channel analysis of the tactile sensitivity of the fingertip: frequency selectivity,spatial summation,and temporal summation

Thresholds were measured for the detection of vibratory stimuli of variable frequency and duration applied to the index fingertip and thenar eminence through contactors of different sizes. The effects of stimulus frequency could be accounted for by the frequency characteristics of the Pacinian (P), non-Pacinian (NP) I, and NP III channels previously determined for the thenar eminence (Bolanowski et al., J Acoust Soc Am 84: 1680-1694, 1988; Gescheider et al., Somatosens Mot Res 18: 191-201, 2001). The effect of changing stimulus duration was also essentially identical for both sites, demonstrating the same amount of temporal summation in the P channel. Although the effect of changing stimulus frequency and changing stimulus duration did not differ for the two sites, the effect of varying the size of the stimulus was significantly greater for the thenar eminence than for the fingertip. The attenuated amount of spatial summation on the fingertip was interpreted as an indication that the mechanism of spatial summation consists of the operations of both neural integration and probability summation.     

Interaction of piroxicam with bovine serum albumin investigated by spectroscopic,calorimetric and computational molecular methods

Abstract

The binding of drugs to serum proteins is governed by weak non-covalent forces. In this study, the nature and magnitude of the interactions between piroxicam (PRX) and bovine serum albumin (BSA) was assessed using spectroscopic, calorimetric and computational molecular methods. The fluorescence data revealed an atypical behavior during PRX and BSA interaction. The quenching process of tryptophan (Trp) by PRX is a dual one (approximately equal static and dynamic quenched components). The FRET results indicate that a non-radiative transfer of energy occurred. The association constant and the number of binding sites indicate moderate PRX and BSA binding. The competitive binding study indicates that PRX is bound to site I from the hydrophobic pocket of subdomain IIA of BSA. The synchronous spectra showed that the microenvironment around the BSA fluorophores and protein conformation do not change considerably. The Trp lifetimes revealed that PRX mainly quenches the fluorescence of Trp-213 situated in the hydrophobic domain. The CD and DSC investigation show that addition of PRX stabilizes the protein structure. ITC results revealed that BSA-PRX binding involves a combination of electrostatic, hydrophobic and hydrogen interactions. The analysis of the computational data is consistent with the experimental results. This thorough investigation of the PRX-BSA binding may provide support for other studies concerning moderate affinity drugs with serum protein.

Communicated by Ramaswamy H. Sarma     

An ex-situ and in vitro approach towards the bioremediation of carcinogenic hexavalent chromium

Abstract

Chromium, ranking the second most among toxic heavy metal pollutants in the world, causing respiratory, cardiovascular and renal problems in human beings is under study herein. We examined the biological remediation of the carcinogenic Cr (VI) polluted soils by indigenous yeast isolates. The total element analysis of the treated sample was determined by Energy Dispersion X-ray Micro Analysis (EDXMA). The sample under study was observed to have a high concentration of 458.29 mgKg^?1 Cr (VI), determined by Atomic Absorption Spectroscopy (AAS) and DPC analysis. The most tolerant isolate designated as CSR was used for in vitro and ex-situ bioremediation studies of Cr (VI). The isolate achieved significant bioremediation of 86% in vitro and 75.12% in ex-situ method. The optimal conditions for in vitro bioremediation were found to be 28?°C and a pH of 6. The ITS1, 5.8S rRNA and D1, D2 domain of LSU rRNA gene characterization of the isolate CSR illustrated that it belongs to Ustilago genera. The isolate was deposited in NCBI GenBank as Ustilago sp. CSR (KY284846). Although, Ustilago is generally a pathogenic fungus, our study opens up the scope of using Ustilago spp. for bioremediation of the carcinogenic heavy metal Chromium.     

A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (> 95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.     

Manipulation of <Emphasis Type="SmallCaps">l</Emphasis>-ascorbic acid biosynthesis pathways in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>: elevated GDP-mannose pyrophosphorylase activity enhances <Emphasis Type="SmallCaps">l</Emphasis>-ascorbate levels in red fruit

Ascorbate (AsA) plays a fundamental role in redox homeostasis in plants and animals, primarily by scavenging reactive oxygen species. Three genes, representing diverse steps putatively involved in plant AsA biosynthesis pathways, were cloned and independently expressed in Solanum lycopersicum (tomato) under the control of the CaMV 35S promoter. Yeast-derived GDP-mannose pyrophosphorylase (GMPase) and arabinono-1,4-lactone oxidase (ALO), as well as myo-inositol oxygenase 2 (MIOX2) from Arabidopsis thaliana, were targeted. Increases in GMPase activity were concomitant with increased AsA levels of up to 70% in leaves, 50% in green fruit, and 35% in red fruit. Expression of ALO significantly pulled biosynthetic flux towards AsA in leaves and green fruit by up to 54 and 25%, respectively. Changes in AsA content in plants transcribing the MIOX2 gene were inconsistent in different tissue. On the other hand, MIOX activity was strongly correlated with cell wall uronic acid levels, suggesting that MIOX may be a useful tool for the manipulation of cell wall composition. In conclusion, the Smirnoff–Wheeler pathway showed great promise as a target for biotechnological manipulation of ascorbate levels in tomato.     

The Human Mast Cell Chymase Gene (CMA1): Mapping to the Cathepsin G/Granzyme Gene Cluster and Lineage-Restricted Expression

Genes encoding T-cell-receptor α/δ chains, neutrophil cathepsin G, and lymphocyte CGL/granzymes are closely linked on chromosomal band 14q11.2. The current work identifies the human mast cell chymase gene (CMA1) as the fourth protease in this cluster and maps the gene to within 150 kb of the cathepsin G gene. The gene order is centromere-T cell receptor α/δ-CGL-1/granzyme B-CGL-2/granzyme H-cathepsin G-chymase. Chymase and cathepsin G genes are shown to be cotranscribed in the human mast cell line HMC-1 and in U-937 cells. Other cells transcribe cathepsin G or CGL/granzyme genes, but not chymase genes, suggesting a capacity for independent regulation. Comparison of the 5′ flank of the chymase gene with those of cathepsin G and CGL/granzymes reveals little overall homology. Only short regions of the 5′ flanks of the human and murine chymase genes sequenced to date are similar, suggesting that they are more distantly related than human and rodent CGL-1/granzyme B, the flanks of which are highly homologous. The expression patterns and clustering of genes provide possible clues to the presence of locus control regions that orchestrate lineage-restricted expression of leukocyte and mast cell proteases.     

The rhabditid nematode Phasmarhabditis hermaphrodita as a potential biological control agent for slugs

A nematode, Phasmarhabditis hermaphrodita, known to be associated with slugs but not previously thought to be parasitic, was shown to be a parasite capable of killing the pest slug Deroceras reticulatum. The parasite infects slugs in the area beneath the mantle surrounding the shell, causing a disease with characteristic symptoms, particularly swelling of the mantle. Infection leads to death of the slug, usually between seven and 21 days afterwards. The nematode then spreads and multiplies in the cadaver. In an experiment where individual D. reticulatum were exposed to different numbers of P. hermaphrodita, a significant positive relationship was found between nematode dose and slug mortality. In two experiments on host range, the nematode was found to infect and kill all pest slug species tested: Deroceras caruanae, Arion distinctus, Arion silvaticus, Arion intermedius, Arion ater, Tandonia sowerbyi and T. budapestensis, in addition to D. reticulatum.     

The extracellular proteases produced by <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Vibrio parahaemolyticus, a Gram-negative bacterium, inhabits marine and estuarine environments and it is a major pathogen responsible globally for most cases of seafood-associated gastroenteritis in humans and acute hepatopancreatic necrosis syndrome in shrimps. There has been a dramatic worldwide increase in V. parahaemolyticus infections over the last two decades. The pathogenicity of V. parahaemolyticus has been linked to the expression of different kinds of virulence factors including extracellular proteases, such as metalloproteases and serine proteases. V. parahaemolyticus expresses the metalloproteases; PrtV, VppC, VPM and the serine proteases; VPP1/Protease A, VpSP37, PrtA. Extracellular proteases have been identified as potential virulence factors which directly digest many kinds of host proteins or indirectly are involved in the processing of other toxic protein factors. This review summarizes findings on the metalloproteases and serine proteases produced by V. parahaemolyticus and their roles in infections. Identifying the role of V. parahaemolyticus virulence-associated extracellular proteases deepens our understanding of diseases caused by this bacterium.     

Possibilities of vaccine manufacture in human diploid cell strains with a serum replacement factor.

Cell lines MDCK (canine kidney), BGM (Buffalo green monkey kidney) and human embryonic lung fibroblast will support viral growth efficiently in medium without serum. Both MRC-5 and WI-38 cell strains have been approved by the Food and Drug Administration for manufacturing viral vaccines against cytomegalovirus and varicella-zoster virus. In this study we examine these two cell lines and viruses for their ability to grow in medium containing a serum replacement. The serum substitute used is LPSR-1 (low protein serum replacement). Using LPSR-1 in defined medium, we demonstrate multipassage cell growth and viral cultivation and replication equivalent to those obtained in medium containing fetal bovine serum (FBS). Viral growth after complete elimination of FBS varies and depends on cell line and virus. Serum substitutes can eliminate FBS in the viral growth phase of vaccine production and reduce costs.