Analysis of the serotype-specific epitopes of avian infectious bronchitis virus strains Ark99 and Mass41.

The Ark and Mass serotype-specific epitopes of infectious bronchitis virus were studied by immunofluorescence and immunoprecipitation of mutant and recombinant spike glycoproteins (S protein) expressed in mouse L cells. Serotype-specific monoclonal antibodies could bind to the recombinant proteins of Ark99 and Mass41 expressed from the chimeras in which the N-terminal thirds of the S1 sequences were reciprocally exchanged. Therefore, it appears that the respective serotype-specific epitopes of both strains were localized within the C-terminal two-thirds of the S1 proteins. Deletion and insertion of a five-amino-acid fragment on the S1 proteins of Ark99 and Mass41, altered the serotype-specific epitopes. This result implies that the five-amino-acid insertion on the S1 protein of the Ark serotype was involved in determining the conformation of the protein, probably acting as a spacer. In addition, it appears that an interaction between sequences of the N-terminal third and the remaining portion of the S1 protein determines the tertiary structure of the protein as well as the conformation of the serotype-specific epitope.     

Torque generation in the flagellar motor of Escherichia coli: evidence of a direct role for FliG but not for FliM or FliN.

Among the many proteins needed for assembly and function of bacterial flagella, FliG, FliM, and FliN have attracted special attention because mutant phenotypes suggest that they are needed not only for flagellar assembly but also for torque generation and for controlling the direction of motor rotation. A role for these proteins in torque generation is suggested by the existence of mutations in each of them that produce the Mot- (or paralyzed) phenotype, in which flagella are assembled and appear normal but do not rotate. The presumption is that Mot- defects cause paralysis by specifically disrupting functions essential for torque generation, while preserving the features of a protein needed for flagellar assembly. Here, we present evidence that the reported mot mutations in fliM and fliN do not disrupt torque-generating functions specifically but, instead, affect the incorporation of proteins into the flagellum. The fliM and fliN mutants are immotile at normal expression levels but become motile when the mutant proteins and/or other, evidently interacting flagellar proteins are overexpressed. In contrast, many of the reported fliG mot mutations abolish motility at all expression levels, while permitting flagellar assembly, and thus appear to disrupt torque generation specifically. These mutations are clustered in a segment of about 100 residues at the carboxyl terminus of FliG. A slightly larger carboxyl-terminal segment of 126 residues accumulates in the cells when expressed alone and thus probably constitutes a stable, independently folded domain. We suggest that the carboxyl-terminal domain of FliG functions specifically in torque generation, forming the rotor portion of the site of energy transduction in the flagellar motor.     

Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes

The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K⁺ current and decreased the inward basal I Ca significantly. The reversal potential of both carbachol-and antibody-induced extra currents were close to –80 mV, being in proximity to the calculated E_k of –90 mV. A -adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward I_ca and outward I_k simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated I_Ca but not the isoprenaline-stimulated I_k. The antibody- or carbachol-induced outward K⁺ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated I_ca were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.     

Cloning (CAG/GTC)n STSs by an Alu-(CAG/GTC)n PCR method: an approach to human chromosome 12 and spinocerebellar ataxia 2 (SCA2).

We report here an Alu-(CAG/GTC)n PCR method for the cloning of STSs with (CAG/GTC)n sequences. We have applied this method to genomic DNA of a somatic cell hybrid containing human chromosome 12 where linkage has been found for a known familiar dominant ataxia (SCA2), which is thought to be due to a (CAG/GTC)n expansion. We have isolated several clones containing (CAG/GTC)n sequences, which include previously identified sequences that map to chromosome 12. This method could be a new PCR approach for the cloning of repeats based on their proximity to Alu sequences.     

A cholinesterase genes server (ESTHER): a database of cholinesterase-related sequences for multiple alignments, phylogenetic relationships, mutations and structural data retrieval.

We have built a database of sequences phylogenetically related to cholinesterases (ESTHER) for esterases, alpha/beta hydrolase enzymes and relatives). These sequences define a homogeneous group of enzymes (carboxylesterases, lipases and hormone-sensitive lipases) with some related proteins devoid of enzymatic activity. The purpose of ESTHER is to help comparison and alignment of any new sequence appearing in the field, to favour mutation analysis of structure-function relationships and to allow structural data recovery. ESTHER is a World Wide Web server with the URL http://www.montpellier.inra.fr:70/cholinesterase.     

Induction of glycanolytic activities by Streptomyces sp. QM-B814 growing on different glucopolymers

The actinomycete Streptomyces sp. QM-B814 is able to use polymeric glucans as sole carbon source by the induction of specific extracellular enzymatic systems. Carboxymethylcellulose and barley -glucan were the most effective inducers of endoglucanase activities towards -(1,4) and mixed -(1,3)-(1,4) linkages. Avicelase activity was induced by Avicel and carboxymethylcellulose. The higher activity towards filter paper (cellulase) was measured in cultures growing in Avicel, carboxymethylcellulose, and barley -glucan. Laminarinase activity was induced only by laminarin and lichenan failed to induce significant activity levels against any of the substrates tested.     

Inhibition of Immune Complex-Induced Inflammation by A small Molecular Weight Selectin Antagonist

The anti-inflammatory effect of a small molecular weight antagonist of P- and E-selectin-dependent cell adhesion was examined. The glycolipid sulphatide was shown to block the adherence of thrombin-activated rat platelets to HL-60 cells. This interaction is known to be dependent on P-selectin. The rat dermal reverse passive Arthus reaction was used to assess the effect of sulphatide on a neutrophil dependent inflammatory response. Sulphatide dosedependently blocked both the vascular permeability increase and cell infiltration after intraperitoneal administration. These results show that a small molecular weight compound which blocks P- and E-selectin dependent adhesion in vitro can effectively block the inflammation due to immune complex deposition. A compound with this type of profile may have therapeutic potential in the treatment of immune complex mediated diseases.     

Effect of bicarbonate and root zone temperature on uptake of Zn,Fe, Mn and Cu by different rice cultivars (Oryza sativa L.) grown in calcareous soil

Pot experiments were conducted with a calcareous soil (Inceptisol) to elucidate the effects of bicarbonate (0 and 20 mM) and root zone temperature (15° and 25°C) on the uptake of Zn, Fe, Mn and Cu by "Zn-efficient" and "Zn-inefficient" rice cultivars. Bicarbonate decreased concentrations and total uptake of Zn in shoots of "Zn-inefficient" cultivars, especially of IR 26 at 25°C, but not in Zn-efficient cultivars. Bicarbonate decreased concentrations and uptake of Fe in shoots of Zn inefficient cultivars, particularly in IR 26. Concentrations and total uptake of Mn were lower in bicarbonate treatment in the Zn-inefficient cultivars at 15°C, and in all cultivars at 25°C. However, concentration and uptake of Cu were not affected by bicarbonate in all cultivars. Compared to the 25°C root zone temperature, the concentrations and total uptake of both Zn and Cu in shoots at 15°C were lower in Zn-inefficient than in the Zn-efficient cultivars. The results indicate that Zn-efficiency in rice is causally related to high tolerance of plant to elavated bicarbonate concentrations in soil solution.