Morphology and diagnosis of the fourth-stage larva of <Emphasis Type=Italic>Coronocyclus labratus</Emphasis> (Looss, 1900) (Nematoda: Strongyloidea) parasitising equids

The fourth larval stage (L4) of Coronocyclus labratus (Looss, 1900) Hartwich, 1986, one of the common species of the Cyathostominae found in equids, is identified and described. The larvae found were identified as C. labratus by finding moulting forms possessing characters of both larval and adult stages. The larvae are similar to those of Cylicocyclus leptostomum (Kotlán, 1920) Foster, 1936, Cyathostomum catinatum Looss, 1900 and Cylicostephanus goldi (Boulenger, 1917) Lichtenfels, 1975. The buccal capsule (BC) wall of the L4 of Cylicocyclus leptostomum is thinner than that of Coronocyclus labratus, and the ring of the oesophageal funnel is comparatively less well developed. In C. labratus the dorsal tooth protrudes only slightly into the buccal cavity, and this larva possesses a characteristically elongate, pyriform oesophagus. In Cyathostomum catinatum, the BC walls are thicker than in Coronocyclus labratus, and the dorsal tooth is more bluntly pointed. Sublateral teeth, if present, are bluntly pointed in the former species, but usually absent or indistinct in C. labratus. Larvae of Cylicostephanus goldi differ from those of Coronocyclus labratus in the larger size of the BC.     

Complexes of gamma-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells

Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that gamma-tubulin (gamma-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, gamma-tubulin, and with anti-phosphotyrosine antibody revealed that gamma-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in gamma-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated gamma-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing gamma-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of gamma-tubulin interaction with tubulin dimers or other proteins during neurogenesis.     

Glucose-induced production of recombinant proteins in Hansenula polymorpha mutants deficient in catabolite repression

The most commonly used expression platform for production of recombinant proteins in the methylotrophic yeast Hansenula polymorpha relies on the strong and strictly regulated promoter from the gene encoding peroxisomal enzyme alcohol (or methanol) oxidase (P(MOX)). Expression from P(MOX) is induced by methanol and is partially derepressed in glycerol or xylose medium, whereas in the presence of hexoses, disaccharides or ethanol, it is repressed. The need for methanol for maximal induction of gene expression in large-scale fermentation is a significant drawback, as this compound is toxic, flammable, supports a slow growth rate and requires extensive aeration. We isolated H. polymorpha mutants deficient in glucose repression of P(MOX) due to an impaired HpGCR1 gene, and other yet unidentified secondary mutations. The mutants exhibited pronounced defects in P(MOX) regulation only by hexoses and xylose, but not by disaccharides or ethanol. With one of these mutant strains as hosts, we developed a modified two-carbon source mode expression platform that utilizes convenient sugar substrates for growth (sucrose) and induction of recombinant protein expression (glucose or xylose). We demonstrate efficient regulatable by sugar carbon sources expression of three recombinant proteins: a secreted glucose oxidase from the fungus Aspergillus niger, a secreted mini pro-insulin, and an intracellular hepatitis B virus surface antigen in these mutant hosts. The modified expression platform preserves the favorable regulatable nature of P(MOX) without methanol, making a convenient alternative to the traditional system.     

Genome sequences of the biotechnologically important Bacillus megaterium strains QM B1551 and DSM319

Bacillus megaterium is deep-rooted in the Bacillus phylogeny, making it an evolutionarily key species and of particular importance in understanding genome evolution, dynamics, and plasticity in the bacilli. B. megaterium is a commercially available, nonpathogenic host for the biotechnological production of several substances, including vitamin B(12), penicillin acylase, and amylases. Here, we report the analysis of the first complete genome sequences of two important B. megaterium strains, the plasmidless strain DSM319 and QM B1551, which harbors seven indigenous plasmids. The 5.1-Mbp chromosome carries approximately 5,300 genes, while QM B1551 plasmids represent a combined 417 kb and 523 genes, one of the largest plasmid arrays sequenced in a single bacterial strain. We have documented extensive gene transfer between the plasmids and the chromosome. Each strain carries roughly 300 strain-specific chromosomal genes that account for differences in their experimentally confirmed phenotypes. B. megaterium is able to synthesize vitamin B(12) through an oxygen-independent adenosylcobalamin pathway, which together with other key energetic and metabolic pathways has now been fully reconstructed. Other novel genes include a second ftsZ gene, which may be responsible for the large cell size of members of this species, as well as genes for gas vesicles, a second β-galactosidase gene, and most but not all of the genes needed for genetic competence. Comprehensive analyses of the global Bacillus gene pool showed that only an asymmetric region around the origin of replication was syntenic across the genus. This appears to be a characteristic feature of the Bacillus spp. genome architecture and may be key to their sporulating lifestyle.     

Nucleotide-gated KATP channels integrated with creatine and adenylate kinases: Amplification,tuning and sensing of energetic signals in the compartmentalized cellular environment

Transmission of energetic signals to membrane sensors, such as the ATP-sensitive K+ (KATP) channel, is vital for cellular adaptation to stress. Yet, cell compartmentation implies diffusional hindrances that hamper direct reception of cytosolic energetic signals. With high intracellular ATP levels, KATP channels may sense not bulk cytosolic, but rather local submembrane nucleotide concentrations set by membrane ATPases and phosphotransfer enzymes. Here, we analyzed the role of adenylate kinase and creatine kinase phosphotransfer reactions in energetic signal transmission over the strong diffusional barrier in the submembrane compartment, and translation of such signals into a nucleotide response detectable by KATP channels. Facilitated diffusion provided by creatine kinase and adenylate kinase phosphotransfer dissipated nucleotide gradients imposed by membrane ATPases, and shunted diffusional restrictions. Energetic signals, simulated as deviation of bulk ATP from its basal level, were amplified into an augmented nucleotide response in the submembrane space due to failure under stress of creatine kinase to facilitate nucleotide diffusion. Tuning of creatine kinase-dependent amplification of the nucleotide response was provided by adenylate kinase capable of adjusting the ATP/ADP ratio in the submembrane compartment securing adequate KATP channel response in accord with cellular metabolic demand. Thus, complementation between creatine kinase and adenylate kinase systems, here predicted by modeling and further supported experimentally, provides a mechanistic basis for metabolic sensor function governed by alterations in intracellular phosphotransfer fluxes.     

Molecular and genetic analysis of NS gene from high pathogenic strains of the avian influenza (H5N1) virus isolated in Kazakhstan

The high pathogenic strains of the avian influenza H5N1 virus isolated in Kazakhstan have NS of different genotypes. The influenza virus strains isolated in 2005 is of NS1E Qinghai genotype. A/swan/Mangystau/3/2006 strain is of NS2A genotype that is typical for Gs/Gd-like strains. The results of the analysis allow assuming that A/swan/Mangystau/3/2006 strain has been brought onto the territory of Kazakhstan from the European part of the continent along the Black Sea-Mediterranean flyway.     

Trypsin-induced masking of tetrodotoxin receptor of the sodium channels in mollusc neurones

At the early stage of trypsin treatment of mollusc neurones tetrodotoxin cannot block the Na⁺ current. In the course of further exposure of neurones to trypsin, tetrodotoxin-sensitivity is restored completely, so its temporal loss results from shielding rather than destruction of the tetrodotoxin-binding site. Pronase and papain do not affect the tetrodotoxin action on the Na⁺ current.     

Numerical simulation of pre- and postsurgical flow in a giant basilar aneurysm

Computational modeling of the flow in cerebral aneurysms is an evolving technique that may play an important role in surgical planning. In this study, we simulated the flow in a giant basilar aneurysm before and after surgical takedown of one vertebral artery. Patient-specific geometry and flowrates obtained from magnetic resonance (MR) angiography and velocimetry were used to simulate the flow prior to and after the surgery. Numerical solutions for steady and pulsatile flows were obtained. Highly three-dimensional flows, with strong secondary flows, were computed in the aneurysm in the presurgical and postsurgical conditions. The computational results predicted that occlusion of a vertebral artery would result in a significant increase of the slow flow region formed in the bulge of the aneurysm, where increased particle residence time and velocities lower than 2.5 cms were computed. The region of slow flow was found to have filled with thrombus following surgery. Predictions of numerical simulation methods are consistent with the observed outcome following surgical treatment of an aneurysm. The study demonstrates that computational models may provide hypotheses to test in future studies, and might offer guidance for the interventional treatment of cerebral aneurysms.     

The fully oxidized form of the cytochrome bd quinol oxidase from E. coli does not participate in the catalytic cycle: direct evidence from rapid kinetics studies

Cytochrome bd catalyzes the two-electron oxidation of either ubiquinol or menaquinol and the four-electron reduction of O(2) to H(2)O. In the current work, the rates of reduction of the fully oxidized and oxoferryl forms of the enzyme by the 2-electron donor ubiquinol-1 and single electron donor N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) have been examined by stopped-flow techniques. Reduction of the all-ferric form of the enzyme is 1000-fold slower than required for a step in the catalytic cycle, whereas the observed rates of reduction of the oxoferryl and singly-reduced forms of the cytochrome are consistent with the catalytic turnover. The data support models of the catalytic cycle which do not include the fully oxidized form of the enzyme as an intermediate.     

Cross-talk between ICAM-1 and granulocyte-macrophage colony-stimulating factor receptor signaling modulates eosinophil survival and activation

Reversal of eosinophilic inflammation has been an elusive therapeutic goal in the management of asthma pathogenesis. In this regard, GM-CSF is a primary candidate cytokine regulating eosinophil activation and survival in the lung; however, its molecular mechanism of propagation and maintenance of stimulated eosinophil activation is not well understood. In this study, we elucidate those late interactions occurring between the GM-CSF receptor and activated eosinophil signaling molecules. Using coimmunoprecipitation with GM-CSF-stimulated eosinophils, we have identified that the GM-CSF receptor beta-chain (GMRbeta) interacted with ICAM-1 and Shp2 phosphatase, as well as Slp76 and ADAP adaptor proteins. Separate experiments using affinity binding with a tyrosine-phosphorylated peptide containing an ITIM (ICAM-1 residues 480-488) showed binding to Shp2 phosphatase and GMRbeta. However, the interaction of GMRbeta with the phosphorylated ICAM-1-derived peptide was observed only with stimulated eosinophil lysates, suggesting that the interaction of GMRbeta with ICAM-1 required phosphorylated Shp2 and/or phosphorylated GMRbeta. Importantly, we found that inhibition of ICAM-1 in activated eosinophils blocked GM-CSF-induced expression of c-fos, c-myc, IL-8, and TNF-alpha. Moreover, inhibition of ICAM-1 expression with either antisense oligonucleotide or an ICAM-1-blocking Ab effectively inhibited ERK activation and eosinophil survival. We concluded that the interaction between ICAM-1 and the GM-CSF receptor was essential for GM-CSF-induced eosinophil activation and survival. Taken together, these results provide novel mechanistic insights defining the interaction between ICAM-1 and the GM-CSF receptor and highlight the importance of targeting ICAM-1 and GM-CSF/IL-5/IL-3 receptor systems as a therapeutic strategy to counter eosinophilia in asthma.