The use of microarrays for the identification of the origin of genes of avian influenza viruses in wild birds

Forty-two strains of avian influenza viruses were isolated from the wild waterfowls’ feces in the city of Moscow. These viruses, as well as reference strains and some experimental reassortants, were analyzed by microarrays. The microarrays contained 176 probes to the different segments of influenza virus genome. The microarray helps to determine 1) the hemagglutinin and neuraminidase proteins subtype; 2) the primary structure of the C-terminal sequence of the viral NS1 protein, which serves as a ligand for the PDZ domain; 3) the presence of stop codons in the reading frame of PB1-F2 as well as the N66S substitution in the PB1-F2 viral protein; 4) the presence of the polybasic site for hemagglutinin cleavage. The viruses of the H3N1, H3N6, H3N8, H4N6, H1N1, H5N3, and H11N9 subtypes were identified from the group of wild birds’ isolates. All isolates contained the ESEV sequence at the C-terminus of the NS1 protein and the full-length reading frame for the PB1-F2 protein. The replacement of N66S in PB1-F2 was found in six strains. However, the presence of the ESEV sequence (ligand of PDZ domain) in the NS1 virus protein and the N66S substitution in PB1-F2 did not lead to the pathogenicity of these viruses for mice. All isolates demonstrated high yield growth in chicken embryos and were infectious and immunogenic for mice, but did not induce any clinical symptoms.Forty-two strains of avian influenza viruses were isolated from the wild waterfowls’ feces in the city of Moscow. These viruses, as well as reference strains and some experimental reassortants, were analyzed by microarrays. The microarrays contained 176 probes to the different segments of influenza virus genome. The microarray helps to determine 1) the hemagglutinin and neuraminidase proteins subtype; 2) the primary structure of the C-terminal sequence of the viral NS1 protein, which serves as a ligand for the PDZ domain; 3) the presence of stop codons in the reading frame of PB1-F2 as well as the N66S substitution in the PB1-F2 viral protein; 4) the presence of the polybasic site for hemagglutinin cleavage. The viruses of the H3N1, H3N6, H3N8, H4N6, H1N1, H5N3, and H11N9 subtypes were identified from the group of wild birds’ isolates. All isolates contained the ESEV sequence at the C-terminus of the NS1 protein and the full-length reading frame for the PB1-F2 protein. The replacement of N66S in PB1-F2 was found in six strains. However, the presence of the ESEV sequence (ligand of PDZ domain) in the NS1 virus protein and the N66S substitution in PB1-F2 did not lead to the pathogenicity of these viruses for mice. All isolates demonstrated high yield growth in chicken embryos and were infectious and immunogenic for mice, but did not induce any clinical symptoms

FAS-dependent cell death in α-synuclein transgenic oligodendrocyte models of multiple system atrophy

Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention

Применение микрочипов для идентификации происхождения генов вирусов гриппа диких птиц

Forty-two strains of avian influenza viruses were isolated from the wild waterfowl’s feces in the city of Moscow. These viruses as well as reference strains and some experimental reassortants were analyzed by microarrays. The used microarrays contained 176 probes to the different segments of influenza virus genome. The microarray allows to determine 1) the hemagglutinin and neuraminidase proteins subtype; 2) the primary structure of the C-terminal sequence of the viral NS1 protein, which serves as a ligand for the PDZ domain; 3) the presence of stop codons and substitution N66S in the reading frame of the viral protein PB1-F2; 4) the presence of the polybasic site for hemagglutinin cleavage. The viruses of H3N1, H3N6, H3N8, H4N6, H1N1, H5N3 and H11N9 subtypes were identified from the group of wild bird’s isolates. All isolates contained the ESEV sequence at the C-terminus of the NS1 protein and the full-length reading frame for the PB1-F2 protein. The replacement of N66S in PB1-F2 was found in six strains. However, the presence of ESEV sequence (ligand of PDZ domain) in the NS1 virus protein and the N66S substitution in PB1-F2 did not lead to the pathogenicity of these viruses for mice. All isolates demonstrated high yield growth in chicken embryos, were infectious and immunogenic for mice, but did not induce any clinical symptoms.В черте города Москвы из фекалий диких водоплавающих птиц изолировали 42 штамма вируса гриппа птиц и проанализировали их на микрочипах «Биогрипп», которые содержат 176 зондов к различным участкам генома вирусов гриппа. Микрочип позволяет определять: 1) субтип поверхностных белков гемагглютинина и нейраминидазы; 2) структуру С-концевой последовательности вирусного белка NS1, влияющую на степень ингибирования транскрипции клеточных хозяйских генов, в том числе ответственных за синтез интерферона; 3) наличие стоп-кодонов и мутацию N66S в рамке считывания вирусного белка PB1-F2, 4) наличие полиосновного сайта протеолитического расщепления гемагглютинина. Среди изолятов от диких птиц идентифицированы вирусы гриппа субтипов H3N1, H3N6, H3N8, H4N6, H1N1, H5N3 и H11N9. Все они содержали последовательность ESEV на С-конце белка NS1, полноразмерную рамку считывания для белка PB1-F2. Замена N66S в PB1-F2 обнаружена у шести штаммов. Однако такие маркеры патогенности, как последовательность ESEV (лиганд PDZ-домена) в вирусном белке NS1 и замена N66S PB1-F2 в контексте генома вирусов гриппа диких уток, не делали вирус патогенным для мышей. Все изоляты были высокоурожайны в куриных эмбрионах, инфекционны и иммуногенны для мышей, но не вызывали у этих животных клинических симптомов заболевания

Molecular cytogenetic organization of polytene chromosomes

The results of the works carried out in the Laboratory of Molecular Cytogenetics (Institute of Cytology and Genetics of Siberian Branch of the RAS, Novosibirsk) devoted to the molecular genetic analysis of main units of polytene chromosomes,*(1) bands, interbands, and puffs, as well as intercalary and pericentric heterochromatin,*(2) are summarized. The results are discussed in terms of the dynamic model of organization of polytene chromosomes

Regulatory Control of Human Cytosolic Branched-chain Aminotransferase by Oxidation and S-glutathionylation and its Interactions with Redox Sensitive Neuronal Proteins

Redox regulation of proteins through oxidation and S-thiolation are important regulatory processes, acting in both a protective and adaptive role in the cell. In the current study, we investigated the sensitivity of the neuronal human cytosolic branched-chain aminotransferase (hBCATc) protein to oxidation and S-thiolation, with particular attention focused on functionality and modulation of its CXXC motif. Thiol specific reagents showed significant redox cycling between the reactive thiols and the TNB anion, and using NEM, four of the six reactive thiols are critical to the functionality of hBCATc. Site-directed mutagenesis studies supported these findings where a reduced kcat (ranging from 50−70% of hBCATc) for C335S, C338S, C335/8S, and C221S, respectively, followed by a modest effect on C242S was observed. However, only the thiols of the CXXC motif (C335 and C338) were directly involved in the reversible redox regulation of hBCATc through oxidation (with a loss of 40−45% BCAT activity on air oxidation alone). Concurrent with these findings, under air oxidation, the X-ray crystallography structure of hBCATc showed a disulphide bond between C335 and C338. Further oxidation of the other four thiols was not evident until levels of hydrogen peroxide were elevated. S-thiolation experiments of hBCATc exposed to GSH provided evidence for significant recycling between GSH and the thiols of hBCATc, which implied that under reducing conditions GSH was operating as a thiol donor with minimal S-glutathionylation. Western blot analysis of WT hBCATc and mutant proteins showed that as the ratio of GSH:GSSG decreased significant S-glutathionylation occurred (with a further loss of 20% BCAT activity), preferentially at the thiols of the CXXC motif, suggesting a shift in function toward a more protective role for GSH. Furthermore, the extent of S-glutathionylation increased in response to oxidative stress induced by hydrogen peroxide potentially through a C335 sulfenic acid intermediate. Deglutathionylation of hBCATc-SSG using the GSH/glutaredoxin system provides evidence that this protein may play an important role in cellular redox regulation. Moreover, redox associations between hBCATc and several neuronal proteins were identified using targeted proteomics. Thus, our data provides strong evidence that the reactive thiol groups, in particular the thiols of the CXXC motif, play an integral role in redox regulation and that hBCATc has redox mediated associations with several neuronal proteins involved in G-protein cell signaling, indicating a novel role for hBCATc in cellular redox control

The Sun and heliosphere explorer - the Interhelioprobe mission

International audienceThe Interhelioprobe mission aims to investigate the inner heliosphere and the Sun from close distances (up to 0.3 AU) and from out of the ecliptic plane (up to 30°). In this paper we present the relevance of the mission and its main scientific objectives, describe the scientific payload, ballistic scenario and orbits of the spacecraft. Possibilities of scientific cooperation with other solar and heliospheric space missions are also mentioned