Molecular dynamics simulation and experimental study of the surface-display of SPA protein via Lpp-OmpA system for screening of IgG.

Staphylococcal protein A (SpA) is a major virulence factor of Staphylococcus aureus. S. aureus is able to escape detection by the immune system by the surface display of protein A. The SpA protein is broadly used to purify immunoglobulin G (IgG) antibodies. This study investigates the fusion ability of Lpp'-OmpA (46-159) to anchor and display five replicate domains of protein A with 295 residues length (SpA295) of S. aureus on the surface of Escherichia coli to develop a novel bioadsorbent. First, the binding between Lpp'-OmpA-SPA295 and IgGFc and the three-dimensional structure was investigated using molecular dynamics simulation. Then high IgG recovery from human serum by the surface-displayed system of Lpp'-OmpA-SPA295 performed experimentally. In silico analysis was demonstrated the binding potential of SPA295 to IgG after expression on LPP-OmpA surface. Surface-engineered E. coli displaying SpA protein and IgG-binding assay with SDS-PAGE analysis exhibited high potential of the expressed complex on the E. coli surface for IgG capture from human serum which is applicable to conventional immune precipitation.

Covalent immobilization of recombinant organophosphorus hydrolase on spores of Bacillus subtilis.

AIMS: Organophosphorus pesticides are widely used in agriculture. Accordingly, decontamination of these pesticides and their residual in environment is an important aim of researchers. One of the best approaches is enzymatic detoxification of these compounds with organophosphorus hydrolase (OPH). The immobilization of OPH on environmentally friendly supports is of great importance for developing stabilized enzymes for degradation of organophosphorus compounds. METHODS AND RESULTS: In this study, Bacillus subtilis spores were applied as a new matrix for immobilizing OPH for the first time; this enzyme was covalently bound to the spores by using EDC-NHS as coupling reagents and the immobilization was confirmed by enzymatic activity, Western blot, flow cytometry and fluorescence microscopic analysis. The immobilization yield was about 55% and the immobilized OPH hydrolysed paraoxon, an organophosphate substrate, without significant loss of activity was six times. The spores with immobilized OPH on their surface were successfully characterized using FT-IR analysis and SEM imaging. Thermal and pH stability was improved by immobilization of OPH on the spore surface. CONCLUSIONS: Owing to safety, environmentally friendly and low cost of spores, these spores can be employed in biosensors for monitoring and biodegradation of organophosphate contaminants in the environment and detoxification processes in bioreactors with high reusability without decrease in the activity. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe that the spore, an environmentally friendly matrix, can be used for covalent immobilization of OPH efficiently and can be applied for detoxification of organophosphorus compounds under adverse environmental conditions.

Characterization of cis-acting elements residing in the chitinase promoter of Bacillus pumilus SG2.

Bacillus pumilus SG2 is a chitinolytic bacterium that produces two chitinases, namely ChiS and ChiL. The chiS and chiL genes are consecutively expressed under a common promoter. Regulation of the chiS and chiL genes is under the control of carbon catabolite repression (CCR) in B. pumilus. This study aimed to investigate the cis-acting elements of the chitinase promoter. For this purpose, we transferred the chiS gene along with its specific promoter to Bacillus subtilis as a host. Primer extension analysis revealed two transcription start sites located 287 and 65 bp upstream of the chiS start codon. The distal promoter was highly compatible with the consensus sequence of the σ(A)-type promoters in B. subtilis, whereas the proximal promoter sequence showed less similarity to the σ(A)-type consensus sequence. A catabolite responsive element (cre), which is required for CCR in Bacillus species, was found to be 136 to 123 bp upstream of the chiS start codon. Interestingly, this cre site was located upstream of the -35 of the proximal promoter and downstream of the distal promoter. Deletion of this cre site sequence rendered the chiS expression constitutive.

Bacillus pumilus SG2 isolated from saline conditions produces and secretes two chitinases.

AIMS: Isolation and characterization of chitinases from a halotolerant Bacillus pumilus. METHODS AND RESULTS: Bacillus pumilus strain SG2 was isolated from saline conditions. It is able to produce chitinase activity at high salt concentration. SDS-PAGE analysis of the B. pumilus SG2 culture supernatant showed two major bands that were induced by chitin. The amino acid sequence of the two proteins, designated ChiS and ChiL, showed a high homology with the chitinase of B. subtilis CHU26, and chitinase A of B. licheniformis, respectively. N-terminal signal peptide of both proteins was also determined. The molecular weight and isoelectric point of the chitinases were determined to be 63 and 74 kDa, and 4.5 and 5.1, for ChiS and ChiL respectively. The genes encoding for both chitinases were isolated and their sequence determined. The regulation of the chitinase genes is under the control of the catabolite repression system. CONCLUSIONS: Secreted chitinase genes and their flanking region on the genome of B. pumilus SG2 have been identified and sequenced. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a multiple chitinases-producing B. pumilus halotolerant strain. We have identified two chitinases by using a reverse genetics approach. The chitinases show resistance to salt.

GABAA receptor-associated phosphoinositide 3-kinase is required for insulin-induced recruitment of postsynaptic GABAA receptors.

Type A gamma-aminobutyric acid (GABAA) receptors mediate most of the fast inhibitory synaptic transmission within the vertebrate brain. The regulation of this inhibition is vital in modulating neural activity. One regulator of GABAA receptor function is insulin, which can serve to enhance GABAA receptor-mediated miniature inhibitory postsynaptic currents, via an increase in the number of receptors at the plasma membrane. We set out to investigate the molecular mechanisms involved in the insulin-induced potentiation of GABAA receptor-mediated responses, by examining the role of phosphoinositide 3-kinase (PI3-K), a key mediator of the insulin response within the brain. We found that PI3-K associates with the GABAA receptor, and this interaction is increased following insulin treatment. Additionally, the beta2 subunit of the GABAA receptor appears to mediate the insulin-stimulated association with the N-terminal SH2 domain of the p85 subunit of PI3-K. Our results imply a mechanism whereby insulin can regulate changes in synaptic transmission through its downstream actions on the GABAA receptor.

Cloning and nucleotide analysis of the vp2 gene of a very virulent infectious bursal disease virus isolate from Iran.

An Iranian field isolate (IR01) of Infectious bursal disease virus (IBDV) was characterized by sequence analysis of its VP2 gene and protein. Comparison of the obtained sequences with those of IBDV isolates from other countries revealed that IR01 was similar to very virulent IBDV (vvIBDV) strains with the identities at nucleotide and amino acid levels reaching 98.198.9% and 99.199.3%, respectively. On the other hand, it was less similar to non-vvIBDV strains; with nucleotide and amino acid identities of 95.295.7% and 96.097.3%, respectively. Out of nine unique nucleotide differences found between IR01 and some other serotype 1 strains only two resulted in amino acid substitutions (Ile296Val and Thr359Lys). In phylogenetic analysis, IR01 was closely related to Asian and European vvIBDV strains. Based on these results, IR01 obviously belongs to vvIBDV strains.

Distinct molecular mechanisms and divergent endocytotic pathways of AMPA receptor internalization.

Internalization of postsynaptic AMPA receptors depresses excitatory transmission, but the underlying dynamics and mechanisms of this process are unclear. Using immunofluorescence and surface biotinylation, we characterized and quantified basal and regulated AMPA receptor endocytosis in cultured hippocampal neurons, in response to synaptic activity, AMPA and insulin. AMPA-induced AMPA receptor internalization is mediated in part by secondary activation of voltage-dependent calcium channels, and in part by ligand binding independent of receptor activation. Although both require dynamin, insulin- and AMPA-induced AMPA receptor internalization are differentially dependent on protein phosphatases and sequence determinants within the cytoplasmic tails of GluR1 and GluR2 subunits. AMPA receptors internalized in response to AMPA stimulation enter a recycling endosome system, whereas those internalized in response to insulin diverge into a distinct compartment. Thus, the molecular mechanisms and intracellular sorting of AMPA receptors are diverse, and depend on the internalizing stimulus.

Intracellular trafficking of AMPA receptors in synaptic plasticity.

Modification of ligand-gated receptor function at the postsynaptic domain is one of the most important mechanisms by which the efficacy of synaptic transmission in the nervous system is regulated. Traditionally, these types of modifications have been thought to be achieved mainly by altering the channel-gating properties or conductance of the receptors. However, recent evidence suggests that AMPA (alpha-amino-3-hydroxyl-5-methyl-4-isoxayolepropionic acid)-type ligand-gated glutamate receptors are continuously recycling between the plasma membrane and the intracellular compartments via vesicle-mediated plasma membrane insertion and clathrin-dependent endocytosis. Regulation of either receptor insertion or endocytosis results in a rapid change in the number of these receptors expressed on the plasma membrane surface and in the receptor-mediated responses, thereby playing an important role in mediating certain forms of synaptic plasticity. Thus, controlling the number of postsynaptic receptors by regulating the intracellular trafficking and plasma membrane expression of the postsynaptic receptors may be a common and important mechanism of synaptic plasticity in the mammalian central nervous system.

Expression of the ORF-2 protein of the human respiratory syncytial virus M2 gene is initiated by a ribosomal termination-dependent reinitiation mechanism.

Translation of the open reading frame 2 (ORF-2) of the human respiratory syncytial virus M2 gene initiates at one of the three initiation codons located upstream of the termination codon for the first ORF. Replacement of ORF-2 with the major ORF of the chloramphenicol acetyltransferase reporter gene followed by systematic mutagenesis of the putative initiation codons demonstrated the usage of these codons as the translational initiators for ORF-2 expression both in vitro and in vivo. While the efficiency of translation was maintained when only the first and second AUG codons were preserved in vivo, there was no apparent preference in vitro for any of the three codons when only one was present. Mutagenesis studies showed that the location of the termination codon of ORF-1 protein plays a crucial role in directing translation of ORF-2 from the upstream initiation codons in vivo. This indicates that the second ORF is accessed by the ribosomes that are departing from the first ORF and that these ribosomes reinitiate on AUG codons 5' to the point of translation termination.

Regulation of AMPA receptor-mediated synaptic transmission by clathrin-dependent receptor internalization.

Redistribution of postsynaptic AMPA- (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-) subtype glutamate receptors may regulate synaptic strength at glutamatergic synapses, but the mediation of the redistribution is poorly understood. We show that AMPA receptors underwent clathrin-dependent endocytosis, which was accelerated by insulin in a GluR2 subunit-dependent manner. Insulin-stimulated endocytosis rapidly decreased AMPA receptor numbers in the plasma membrane, resulting in long-term depression (LTD) of AMPA receptor-mediated synaptic transmission in hippocampal CA1 neurons. Moreover, insulin-induced LTD and low-frequency stimulation-(LFS-) induced homosynaptic CA1 LTD were found to be mutually occlusive and were both blocked by inhibiting postsynaptic clathrin-mediated endocytosis. Thus, controlling postsynaptic receptor numbers through endocytosis may be an important mechanism underlying synaptic plasticity in the mammalian CNS.

Detection and characterization of proteins encoded by the second ORF of the M2 gene of pneumoviruses.

The nucleotide sequence of the M2 gene of pneumonia virus of mice (PVM) was determined. The sequence showed that the gene encoded a protein of 176 amino acids with a predicted molecular mass of 20165 Da from a major ORF, which is smaller than the equivalent proteins encoded by human, bovine and ovine respiratory syncytial (RS) viruses. The PVM M2 protein is conserved, having 41% similarity to the equivalent human RS virus protein. In common with the M2 genes of the RS viruses and avian pneumovirus (APV), the PVM mRNA also contained a second ORF (ORF2) that partially overlaps the first ORF and which is capable of encoding a 98 residue polypeptide. No significant sequence identity could be detected between the putative M2 ORF2 proteins of PVM, APV and the RS viruses. The expression of the M2 ORF2 proteins of the pneumoviruses was investigated by using monospecific antisera raised against GST fusion proteins. Western blot analysis demonstrated the presence of polypeptides encoded by M2 ORF2 of PVM and RS virus corresponding with those predicted by in vitro translation studies, but this was not the case for APV. The PVM polypeptide was present as three distinct products in vivo. The PVM and RS virus polypeptides were also detected in cells by immunofluorescence, which showed that both were present in the cytoplasm with a degree of localization in inclusion bodies. No APV M2 ORF2 protein could be detected in vivo. The RS virus M2 ORF2 polypeptide was shown to accumulate during infection and the potential implications of this are discussed.