An investigation of functional diversity of endogenous cellulase and expression of other digestive enzyme genes in freshwater crayfish (Cherax) species

The project evaluated potential of soluble cellulose as a cheap feed ingredient for major farmed Australian freshwater crayfish species testing their growth performance, digestive enzyme activity and digestive enzyme gene expression patterns. Test animals showed an innate capacity to utilise a range of carbohydrate sources including complex structural polysaccharides. Results suggest that more plant-derived ingredient can be incorporated in formulated low-cost feeds for the culture industry.

Genetic and Malt Quality Diversity of East Asian Two-Rowed Barley Accessions in Relationship to North American Malting Barley.

Because of epidemics of Fusarium head blight (FHB; caused by Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schwein.) Petch]) in the northern Great Plains of the United States and Canada in the past two decades, malting barley breeders have been forced to use nonadapted barley (Hordeum vulgare L.) accessions as sources of FHB resistance. Many of the resistant accessions are from East Asia, and limited information is available on their genetic diversity and malt quality. The objectives of this study were to determine the genetic diversity among 30 East Asian accessions and two North American cultivars. Genetic diversity was based on 49 simple-sequence repeat markers. All accessions were tested for barley grain brightness; protein content; 1,000-kernel weight; malting loss; fine-grind malt extract; content of plump kernels, free amino nitrogen, soluble protein, and wort beta-glucan; the Kolbach index (i.e., the ratio of malt soluble protein to malt total protein); a-amylase activity; diastatic power; won color; and wort viscosity. A few accessions had equal quality compared with Harrington and Conlon barley for individual traits but not for all. Qing 2, Mokkei 93-78, and Nitakia 48 could be excellent sources for increased malt extract; Nitakia 48 is a possible source for low wort viscosity; and Mokkei 93-78 and Nitakia 48 are putative sources of low beta-glucan content. The cluster analyses also implied that the malt quality of an accession cannot be predicted based on the country where it was developed.

Structural diversity and ligand specificity of lectins. The Bangalore effort

Structural studies in this laboratory encompass four of the five major classes of plant lectins, including the one discovered by us. In addition to addressing issues specific to individual lectins, the work provided insights into protein folding, quaternary association and generation of ligand specificity. Legume and beta-prism fold lectins constitute families of proteins in which small alterations in essentially the same tertiary structure lead to large variations in quaternary structure, including that involving an open structure. Strategies for generating ligand specificity include water bridges, variation in loop length, post translational modification and oligomerization. Three of the structural classes investigated have subunits with three-fold symmetry. The symmetry in the structure is reflected in the sequence to different extents in different subclasses. The evolutionary implications of this observation have been explored. The work on lectins has now been extended to those from mycobacteria.

Estudo do polimorfismo Ser49Gly do gene do receptor beta adrenérgico 1 (β-adr 1) na população do estado do Rio de Janeiro, Brasil estratificada por cor da pele e ancestralidade genômica

As doenças cardiovasculares possuem a maior taxa de óbitos no mundo, e notavelmente nos últimos anos as pesquisas genéticas sobre as mesmas estão baseadas em estudos de associação, no qual o gene suspeito que esteja em maior frequência entre os pacientes passa a ser considerado um possível fator causal. Os polimorfismos genéticos que ocorrem no receptor beta-adrenérgico podem resultar em mudanças significativas na função do receptor, podendo acarretar fisiopatologias. Neste trabalho, o objetivo foi estimar a diversidade e a frequência do polimorfismo Ser49Gly do gene do receptor beta-adrenérgico 1 a partir de uma amostra de 188 indivíduos da população do Estado do Rio de Janeiro. As frequências também foram analisadas a partir da estratificação da amostra por critério fenotípico em função do padrão de cor da pele em (negros e não negros) ou ancestralidade genética em (afrodescendente e não afrodescendente), definida através da informação dos marcadores de ancestralidade Indels e SNP de cromossomo Y, para avaliar se os padrões de ancestralidade ou cor da pele são fundamentais para a diferenciação e distanciamento genético. Fragmentos de interesse foram amplificados por PCR (reação de cadeia de polimerase) com primers específicos para o marcador Ser49Gly e as reações de genotipagem foram realizadas com enzimas de restrição Eco0109I. Os valores da heterozigosidade variaram entre 0,25-0,50 e 0,20-0,41 nos grupos estratificados por ancestralidade e cor da pele, respectivamente. No que diz respeito à análise do equilíbrio de Hardy-Weinberg, não houve um desvio significativo na distribuição do marcador nas amostras gerais do Estado do Rio de Janeiro, ou mesmo nas amostras estratificadas. A distribuição dos alelos na amostra dos 188 indivíduos da população geral do Rio de Janeiro (AC_RJ) mostrou uma frequência de 80,30% e 19,70% para o alelo selvagem e mutado Ser49Gly, respectivamente. A comparação das análises sobre a distribuição das frequências alélicas para este marcador mostrou a ocorrência de diferenças significativas na distribuição das frequências alélicas entre negros e não negros e afrodescendentes e não afrodescendentes. A diferença significativa observada entre os negros e afrodescendentes, foi em menor grau de distanciamento. A informação obtida em relação à ancestralidade foi crucial para a obtenção dos dados sobre o aumento da variável mutada do polimorfismo Ser49Gly nas populações negras e afrodescendentes do Estado Rio de Janeiro. Tal evidência, em combinação com estudos clínicos podem contribuir para uma análise pormenorizada do padrão de susceptibilidade à doença em questão, em falhas do mecanismo deste receptor.

Caracterização de beta-lactamases de espectro ampliado (ESBLs), genes de resistência aos antimicrobianos e conteúdo plasmidial em cepas de Escherichia coli e Salmonella spp. não tifóides isoladas do ambiente hospitalar e da comunidade

Enterobactérias produtoras de ESBLs são descritas tanto no ambiente hospitalar quanto na comunidade em todo o mundo. No Brasil, esses microrganismos também têm emergido como uma causa importante de infecções, sendo as enzimas CTX-M as prevalentes. O objetivo deste estudo foi analisar diferentes aspectos genotípicos relacionados à expressão da resistência aos antimicrobianos em cepas Escherichia coli e de Salmonella spp, tais como: a diversidade de ESBLs, os genes de resistência aos antimicrobianos e o conteúdo plasmidial. Os aspectos epidemiológicos das cepas produtoras de ESBLs também foram investigados. Foram estudadas 88 cepas de enterobactérias, sendo 43 E. coli e 45 cepas de Salmonella spp., de origem hospitalar e da comunidade (principalmente alimentos), isoladas na cidade do Rio de Janeiro. A expressão de ESBL foi observada em sete cepas de E. coli (7/43, 16,3%) e em uma cepa de Salmonella Typhimurium (1/45, 2,3%) e as enzimas foram identificadas como variantes de CTX-M e SHV-5, respectivamente. Entre as cepas de E. coli, a enzima CTX-M-2 foi a mais frequente (n = 4), sendo detectada em cepas isoladas de swab retal de pacientes hospitalizados, enquanto as enzimas CTX-M-59 (uma variante de CTX-M) (n = 1) e CTX-M-9 (n = 2) foram identificadas em cepas isoladas a partir de espécimes clínicos. Salmonella Typhimurium produtora de SHV-5 foi isolada do ambiente hospitalar (fórmula infantil). As cepas de E. coli produtoras das enzimas CTX-M pertenceram a grupos filogenéticos (A, B1, D) e STs (ST34, ST69, ST101) diferentes, sendo os genes blaCTX-M identificados em plasmídeos com tipo de replicon IncA/C de cerca de 150 kb (blaCTX-M-2, blaCTX-M-9, blaCTX-M-59) ou 80 kb (blaCTX-M-2). A cepa de S. Typhimurium produtora de SHV-5 pertenceu a um único clone (A-ST19) e o gene blaSHV-5 foi identificado em plasmídeo com o replicon IncL/M com aproximadamente 55Kb. Foi identificado pela primeira vez no Brasil o ST313 em um clone de S. Typhimurium (D-ST313), comumente associado com doenças invasivas severas, particulamente no continente africano. Genes que codificam para a resistência aos antimicrobianos não-beta-lactâmicos e integrons classe 1 foram identificados entre as cepas de E. coli e de Salmonella spp. multirresistentes produtoras ou não de ESBLs. Em conclusão: i) nossos resultados referentes à E. coli confirmaram a disseminação de enzimas CTX-M (principalmente variantes do grupo CTX-M-2) desde, pelo menos, o ano de 2000, em hospitais no Rio de Janeiro; demonstraram a implicação dos plasmídeos IncA/C na disseminação de genes blaCTX-M; indicaram a possível evolução intra-plasmídeo de blaCTX-M-59 a partir de blaCTX-M-2; a observação da diversidade e multiplicidade de plasmídeos poderiam fornecer plataformas genéticas para a dispersão de diferentes genes e/ou elementos de resistência aos antimicrobianos; ii) em relação à Salmonella spp. este estudo descreveu, pela primeira vez, o isolamento, a partir de fórmula infantil, de uma cepa de S. Typhimurium produtora de ESBL; foi demonstrada a associação do gene blaSHV-5 com plasmídeo do tipo IncL/M, que é considerado epidêmico; foi identificado o clone D-ST313 de S. Typhimurium, que está associado a doenças invasivas severas no continente africano, que reuniu cepas isoladas exclusivamente do ambiente hospitalar.

Diversity and abundance of ammonia-oxidizing bacteria in eutrophic and oligotrophic basins of a shallow Chinese lake (Lake Donghu)

Classical cultivation and molecular methods based on the ammonia monooxygenase gene (amoA) were used to study the abundance and diversity of beta-proteobacterial ammonia-oxidizing bacteria (AOB) in lake sediments. The eutrophic and oligotrophic basins of a Chinese shallow lake (Lake Donghu), in terms of ammonium (NH4+) concentrations, were sampled. The AOB number was significantly lower in the oligotrophic basin, but significantly higher in the eutrophic basin. In addition, using restriction fragment length polymorphism targeting the amoA, ten restriction patterns including six unique ones were found in the eutrophic basin, while five patterns were observed in the oligotrophic basin with only one unique restriction group. Phylogenetic analysis for AOB revealed that Nitrosomonas oligotropha- and Nitrosomonas ureae-related AOB and Nitrosospira-affiliated AOB were ubiquitous; the former dominated in the eutrophic basin (87.2%), while the latter dominated in the oligotrophic basin (65.5%). Furthermore, Nitrosomonas communis-related AOB was only detected in the eutrophic basin, at a small proportion (3.2%). These results indicate significant selection and adaptation of sediment AOB in lakes with differing trophic status. (C) 2009 Elsevier Masson SAS. All rights reserved.

Bacterial diversity in activated sludge from a consecutively aerated submerged membrane bioreactor treating domestic wastewater

The bacterial diversity of activated sludge from submerged membrane bioreactor (SMBR) was investigated. A 16S rDNA clone library was generated, and 150 clones were screened using restriction fragment length polymorphism (RFLP). Of the screened clones, almost full-length 16S rDNA sequences of 64 clones were sequenced. Phylogenetic tree was constructed with a database containing clone sequences from this study and bacterial rDNA sequences from NCB1 for identification purposes. The 90.6% of the clones were affiliated with the two phyla Bacteroidetes (50%) and Proteobacteria (40%), and beta-, -gamma-, and delta-Proteobacteria accounted for 7.8%, 28.1%, and 4.7%, respectively. Minor portions were affiliated with the Actinobacteria and Firmicutes (both 3.1%). Only 6 out of 64 16S rDNA sequences exhibited similarities of more than 97% to classified bacterial species, which indicated that a substantial fraction of the clone sequences were derived from unknown taxa. Rarefaction analysis of operational taxonomic units (orrUs) clusters demonstrated that 150 clones screened were still insufficient to describe the whole bacterial diversity. Measurement of water quality parameter demonstrated that performance of the SMBR maintained high level, and the SMBR system remained stable during this study.

Two thymosin-repeated molecules with structural and functional diversity coexist in Chinese mitten crab Eriocheir sinensis

Recently, beta-thymosin-like proteins with multiple thymosin domains (defined as thymosin-repeated proteins) have been identified from invertebrate. In the present study, the cDNAs of two thymosin-repeated proteins (designated EsTRP1 and EsTRP2) were cloned from Chinese mitten crab by expressed sequence tags (EST) techniques. BLAST analysis presented three and two thymosin domains in EsTRP1 and EsTRP2, respectively, with the identities amongst the five domains varying from 47% to 100%. Both EsTRP1 and EsTRP2 shared high similarities with previously identified vertebrate beta-thynnosins and invertebrate thymosin-repeated proteins (TRPs) with the identities ranging from 43% to 78%, indicating that EsTRPs were new members of the beta-thymosin family. Real-time RT-PCR assay was adopted to determine the tissue distribution of EsTRPs and their temporal expression profile in hemocytes after pathogen stimulation and injury challenge. The expression of EsTRP1 transcript was predominantly detectable in the tissues of hemocytes, hepatopancreas and gonad with the highest expression in hemocytes, while the highest expression level of EsTRP2 was found in heart. EsTRP1 mRNA expression in hemocytes significantly increased at 3 and 48 h after Listonella anguillarum challenge, but there was no significant variation in EsTRP2 temporal expression profile. The injury challenge reduced the mRNA expression of EsTRPs, with the down-regulation of EsTRP2 expression occurred earlier than that of EsTRP1. The cDNA fragments encoding their mature peptides of EsTRP1 and EsTRP2 were recombined and expressed in Escherichia coli. The activities of recombinant proteins (rEsTRP1 and rEsTRP2) were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide) and lysoplate assay. rEsTRP2 could significantly accelerate the growth of human hepatocellular carcinoma cell line, but there was no significant effect of rEsTRP1 on the tumor cell proliferation. Both rEsTRP1 and rEsTRP2 did not possess the ability of killing Micrococcus luteus and L. anguillarum. The differences in the tissue distribution of mRNA transcripts, the response to pathogen stimulation and injury challenge, and the effect of recombinant proteins on human cell proliferation, indicated that there were functional diversity between the two structurally different molecules, EsTRP1 and EsTRP2. (C) 2009 Elsevier Ltd. All rights reserved.

Incidence of diverse integrons and beta-lactamase genes in environmental Enterobacteriaceae isolates from Jiaozhou Bay, China

Environmental microbiology investigation was performed to determine the molecular diversity of beta-lactamase genes among ampicillin-resistant bacteria from Jiaozhou Bay. beta-lactamase genes were detected in 93.8% of the bacterial isolates identified as Enterobacteriaceae. The most frequently detected gene was bla(TEM), followed by bla(SHV), bla(OAX-1), bla(MOX) and bla(CMY). Most of the isolates (68.8%) were positive for the intI1 integrase gene, and two isolates were also found for the intI2 gene. The dfr and aadA gene cassettes were predominant. Anthropogenic contamination from onshore sewage processing plants might contribute predominantly to the beta-lactamase gene reservoir in the studied coastal waters. Environmental antibiotic-resistant bacteria and resistance genes may serve as bioindicators of coastal environmental quality or biotracers of the potential contamination sources. This is the first report of the prevalence and characterization of beta-lactamase genes and integrons in coastal Enterobacteriaceae from China.

Evolution of Drug Resistance: Insight on TEM -Lactamases Structure and Activity and Beta-Lactam Antibiotics

Extended-spectrum β-lactamases (ESBLs) prevalence was studied in the north of Portugal, among 193 clinical isolates belonging to citizens in a district in the boundaries between this country and Spain from a total of 7529 clinical strains. In the present study we recovered some members of Enterobacteriaceae family, producing ESBL enzymes, including Escherichia coli (67.9%), Klebsiella pneumoniae (30.6%), Klebsiella oxytoca (0.5%), Enterobacter aerogenes (0.5%), and Citrobacter freundii (0.5%). β-lactamases genes blaTEM, blaSHV, and blaCTX-M were screened by polymerase chain reaction (PCR) and sequencing approaches. TEM enzymes were among the most prevalent types (40.9%) followed by CTX-M (37.3%) and SHV (23.3%). Among our sample of 193 ESBL-producing strains 99.0% were resistant to the fourth-generation cephalosporin cefepime. Of the 193 isolates 81.3% presented transferable plasmids harboring genes. Clonal studies were performed by PCR for the enterobacterial repetitive intragenic consensus (ERIC) sequences. This study reports a high diversity of genetic patterns. Ten clusters were found for E. coli isolates and five clusters for K. pneumoniae strains by means of ERIC analysis. In conclusion, in this country, the most prevalent type is still the TEM-type, but CTX-M is growing rapidly.

Evolution of functional diversity in the cupin superfamily

The cupin superfamily of proteins is among the most functionally diverse of any described to date. It was named on the basis of the conserved beta-barrel fold ('cupa' is the Latin term for a small barrel), and comprises both enzymatic and non-enzymatic members, which have either one or two cupin domains. Within the conserved tertiary structure, the variety of biochemical function is provided by minor variation of the residues in the active site and the identity of the bound metal ion. This review discusses the advantages of this particular scaffold and provides an evolutionary analysis of 18 different subclasses within the cupin superfamily.

Phylogeny and the hierarchical organization of plant diversity

R. H. Whittaker's idea that plant diversity can be divided into a hierarchy of spatial components from alpha at the within-habitat scale through beta for the turnover of species between habitats to gamma along regional gradients implies the underlying existence of alpha, beta, and gamma niches. We explore the hypothesis that the evolution of a, (3, and gamma niches is also hierarchical, with traits that define the a niche being labile, while those defining a and 7 niches are conservative. At the a level we find support for the hypothesis in the lack of close significant phylogenetic relationship between meadow species that have similar a niches. In a second test, a niche overlap based on a variety of traits is compared between congeners and noncongeners in several communities; here, too, there is no evidence of a correlation between a niche and phylogeny. To test whether beta and gamma niches evolve conservatively, we reconstructed the evolution of relevant traits on evolutionary trees for 14 different clades. Tests against null models revealed a number of instances, including some in island radiations, in which habitat (beta niche) and elevational maximum (an aspect of the gamma niche) showed evolutionary conservatism.

Tracking phenotypically and functionally distinct T cell subsets via T cell repertoire diversity

Antigen-specific T cell receptors (TCRs) recognise complexes of immunogenic peptides (p) and major histocompatibility complex (MHC) glycoproteins. Responding T cell populations show profiles of preferred usage (or bias) toward one or few TCRβ chains. Such skewing is also observed, though less commonly, in TCRα chain usage. The extent and character of clonal diversity within individual, antigen-specific T cell sets can be established by sequence analysis of the TCRVβ and/or TCRVα CDR3 loops. The present review provides examples of such TCR repertoires in prominent responses to acute and persistent viruses. The determining role of structural constraints and antigen dose is discussed, as is the way that functionally and phenotypically distinct populations can be defined at the clonal level. In addition, clonal dissection of “high” versus “low” avidity, or “central” versus “effector” memory sets provides insights into how these antigen specific T cell responses are generated and maintained. As TCR diversity potentially influences both the protective capacity of CD8+ T cells and the subversion of immune control that leads to viral escape, analysing the spectrum of TCR selection and maintenance has implications for improving the functional efficacy of T cell responsiveness and effector function.