Mosaic RNA Phage VLPs Carrying Domain III of the West Nile Virus E Protein

The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the surface of RNA phage AP205 virus-like particles (VLPs) in mosaic form. For this purpose, a 111 amino acid sequence of DIII was added via amber or opal termination codons to the C-terminus of the AP205 coat protein, and mosaic AP205-DIII VLPs were generated by cultivation in amber- or opal-suppressing Escherichia coli strains. After extensive purification to 95 % homogeneity, mosaic AP205-DIII VLPs retained up to 11–16 % monomers carrying DIII domains. The DIII domains appeared on the VLP surface because they were fully accessible to anti-DIII antibodies. Immunisation of BALB/c mice with AP205-DIII VLPs resulted in the induction of specific anti-DIII antibodies, of which the level was comparable to that of the anti-AP205 antibodies generated against the VLP carrier. The AP205-DIII-induced anti-DIII response was represented by a significant fraction of IgG2 isotype antibodies, in contrast to parallel immunisation with the DIII oligopeptide, which failed to induce IgG2 isotype antibodies. Formulation of AP-205-DIII VLPs in alum adjuvant stimulated the level of the anti-DIII response, but did not alter the fraction of IgG2 isotype antibodies. Mosaic AP205-DIII VLPs could be regarded as a promising prototype of a putative West Nile vaccine.     

The influence of exogenously supplied sucrose on glutamine synthetase and glutamate dehydrogenase levels in excisedPisum sativum roots

Glutamine synthetase (GS) level is positively influenced by exogenously supplied sucrose in isolated pea roots (similarly as nitrate reductase - NR), glutamate dehydrogenase (GDH) level negatively. Comparison with previous results shows that GS level decreases more slowly than NR level when sucrose is omitted from the medium; the rate of changes in GS level corresponds rather to that in GDH level. The increase in GDH level in the tips of isolated roots cultivated in the medium lacking sucrose stops after approx. 24 h, but continues for at least 72 h in more mature root parts. GS level decreases during the first 24 h in the tips of isolated roots (compared with roots of intact seedlings) cultivated both with sucrose and without it (without sucrose more), however it again rises in the course of further cultivation with sucrose. The differences in GS and GDH levels caused by omission of sucrose are small in isolated roots from which root tips were removed, the difference in NR level in decapitated roots is similar to that found in isolated roots with root tips left. Decapitated isolated roots cultivated without sucrose contain higher amounts of soluble sugars than corresponding roots with root tips left. These facts are dismissed with regard to sugar consumption, transport, and compartmentalisation, and with respect to production in root tips and other plant parts of unknown compounds involved in GS and GDH regulation. The results obtained suggest that GDH functions in pea roots in the deaminating direction.     

Membrane-type MMPs enable extracellular matrix permissiveness and mesenchymal cell proliferation during embryogenesis

Peri-cellular remodeling of mesenchymal extracellular matrices is considered a prerequisite for cell proliferation, motility and development. Here we demonstrate that membrane-type 3 MMP, MT3-MMP, is expressed in mesenchymal tissues of the skeleton and in peri-skeletal soft connective tissue. Consistent with this localization, MT3-MMP-deficient mice display growth inhibition tied to a decreased viability of mesenchymal cells in skeletal tissues. We document that MT3-MMP works as a major collagenolytic enzyme, enabling cartilage and bone cells to cleave high-density fibrillar collagen and modulate their resident matrix to make it permissive for proliferation and migration. Collectively, these data uncover a novel extracellular matrix remodeling mechanism required for proper function of mesenchymal cells. The physiological significance of MT3-MMP is highlighted in mice double deficient for MT1-MMP and MT3-MMP. Double deficiency transcends the combined effects of the individual single deficiencies and leads to severe embryonic defects in palatogenesis and bone formation incompatible with life. These defects are directly tied to loss of indispensable collagenolytic activities required in collagen-rich mesenchymal tissues for extracellular matrix remodeling and cell proliferation during embryogenesis.     

Associations of HLA DR and DQ molecules with Lyme borreliosis in Latvian patients

ABSTRACT: BACKGROUND: Many autoimmune diseases are associated with variants of HLA genes such as those encoding the MHC complex. This correlation is not absolute, but may help in understanding of the molecular mechanism of disease. The purpose of this study was to determine HLA-DR,-DQ alleles in Latvian patients with Lyme borreliosis and control (healthy) persons. Case patients and control subjects were similar in age, gender and ethnic heritage and differed only as regards the presence of Borrelia burgdorferi infection. The study included 20 patients with clinical stage - erythema migrans and 25 control (healthy) persons. HLA genotyping was performed by PCR with sequence-specific primers. RESULTS: The results show difference in HLA-DRB1 alleles distribution between patients and control subjects. The frequencies of HLA-DRB1 *04 (OR 8.65; p<0.022) and HLA-DRB1 *17 (03) (OR 7.00; p<0.048) were increased in the Lyme disease patients. And the frequency of allele DRB1*13 (OR 0.13; p<0.033) was lower in Borreliosis patients and higher in control group. But, significant differences in frequencies of HLA-DQ alleles we did not detect. CONCLUSIONS: HLA predisposition to Lyme borreliosis appears not to be limited to HLA molecules, but some HLA-DR alleles also have a significant influence, and, may have implications in our understanding of pathogenesis of this disease. In particular, HLA-DRB1*04 and DRB1 *17 (03) may contribute to the Lyme borreliosis development in Latvian population KEYWORDS: Lyme borreliosis, HLA alleles, PCR.     

Rhizobial nod gene-inducing activity in pea nodulation mutants: dissociation of nodulation and flavonoid response

With the characterization of the total genomes of Arabidopsis thaliana and Oryza sativa , several putative plasma membrane components have been identified. However, a lack of knowledge at the protein level, especially for hydrophobic proteins, have hampered analyses of physiological changes. To address whether protein complexes may be present in the native membrane, we subjected plasma membranes isolated from Spinacia oleracea leaves to blue-native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is well established in the separation of functional membrane protein complexes from mitochondria and chloroplasts, but a resolved protein complex pattern from PM of eukaryotic cells has previously not been reported. Using this method, protein complexes from Spinacia oleracea PM could be efficiently solubilized and separated, including the highly hydrophobic aquaporin (apparent molecular mass 230 kDa), a putative tetramer of H⁺-ATPase, and several less abundant complexes with apparent masses around or above 750 kDa. After denaturation and separation of the complexes into their subunits in a second dimension (SDS-PAGE), several of the complexes were identified as hydrophobic membrane proteins. Large amounts of protein (up to 1 mg) can be resolved in each lane, which suggests that the method could be used to study also low-abundance protein complexes, e.g. under different physiological conditions.     

Manipulation of division symmetry and developmental fate in cultures of potato microspores

The effect of media composition on microspore culture was investigated in one tetraploid and two diploid potatoes. The viability of microspores isolated from 4.5 to 5 mm buds was in the range of 33 to 52%. In media for anther culture, microspores showed no further development and lost viability within 2 days. In M1 medium containing mineral components, sucrose, uridine, cytidine, myo-inositol, glutamine and lactalbumin hydrolysate, 18 to 37% of microspores underwent mitosis within 14 days. Up to 95% of the divisions were symmetric and produced equal nuclei. Some symmetrically divided microspores eventually produced structures with 3 to 10 nuclei. The proportion of the total microspore population producing multinuclear structures reached 9% in diploid clones responsive to anther culture and 1 to 2% in recalcitrant cv. Borka. Symmetric mitoses in M1 medium were induced in the presence of glutamine and lactalbumin hydrolysate. Nucleosides and myo-inositol had no effect on microspore division. In the absence of all organic components except sucrose, most mitoses were asymmetric, formation of multinuclear structures was reduced and most pollen accumulated starch indicative of gametophytic fate. In complete M1 medium, starch accumulation was suppressed. Suppression also occurred in asymmetrically divided microspores, indicating a direct inhibition of pollen development independent of the mode of microspore division. This inhibitory effect of M1 medium might present a stress which triggers the induction of symmetric microspore division and subsequent formation of multinuclear structures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phytoextraction of trace elements by water hyacinth in contaminated area of gold mine tailing

The ability of water hyacinth (Eichhornia crassipes) to uptake Ag, Ba, Cd, Mo, and Pb from waters in gold mine tailing area was studied. All experiments were carried out in the field conditions without using of model system. Bioconcentration (BCF) and translocation factors (TF) as well as elements accumulation by plant in different points of tailings-impacted area were evaluated. It has been shown that water hyacinth demonstrates high ability to accumulate Mo, Pb, and Ba with BCF values 24,360 ± 3600, 18,800 ± 2800 and 10,040 ± 1400, respectively and is efficient in translocation of Mo and Cd. The general trend of the plant accumulation ability in relation to the studied elements corresponds to their concentration in the medium. As the distance from tailings increases, concentration of Ag, Ba and Pb in plant decreases more clearly than that of Cd, while the amount of Mo accumulated by plant doesn't drop significantly in accordance with its concentration in water. Under the conditions of the confluence of river Ur and drainage stream Ba and Ag can be considered as potential candidates for phytomining.     

Nitrile metabolism in fungi: A review of its key enzymes nitrilases with focus on their biotechnological impact

Nitriles are abundant in the plant kingdom. The ability to detoxify them is beneficial for microbes living in the plant environment. Nitrilases (NLases; EC 3.5.5.-), which hydrolyze nitriles to carboxylic acids, have been well characterized in bacteria, and classified into various substrate-specificity subtypes (aromatic NLases, aliphatic NLases, arylacetoNLases). NLases also occur in filamentous fungi, mainly in Ascomycota (subdivision Pezizomycotina), as documented by genome mining. However, the investigation of NLases in fungi has been delayed compared to bacteria. Only a few NLases (aromatic NLases) were purified from native fungal strains (mainly Fusarium), which were grown under suitable induction conditions. Over a few past years, the spectrum of known fungal NLases was broadened by expressing fungal NLase genes in Escherichia coli. Thus functional NLases were reported for the first time in fungi of genera Auricularia, Macrophomina, Nectria, Neurospora, Pichia, Talaromyces, Trichoderma and Trichophyton. Two major substrate-specificity subtypes were identified in them, i.e. aromatic NLases and arylacetoNLases, apart from a few NLases with broad substrate specificities. The biotechnological impact of fungal arylacetoNLases was explored with a focus on the enantioselective hydrolysis of (R,S)-mandelonitrile, the selective hydrolysis of one cyano group in dinitriles and the hydrolysis of nitrile precursors of the taxol sidechain. Despite recent advances, the wealth of fungal NLases whose sequences have been deposited in databases has not yet been fully exploited. Overproduction in E. coli has the potential to bring these NLases to life. This will enable to estimate the natural roles of NLases in fungi and will also provide new catalysts for biotechnological uses.