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111.
Miriam YH Ueda Paulo G Alvarenga Juliana M Real Eloisa de Sá Moreira Aripuan? Watanabe Ana Maria Passos-Castilho Matheus Vescovi Yana Novis Vanderson Rocha Adriana Seber Jose SR Oliveira Celso A Rodrigues Celso FH Granato 《Memórias do Instituto Oswaldo Cruz》2015,110(4):461-467
Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem
cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and
early diagnosis of related clinical diseases, constitute essential measures to
improve outcomes. A prospective survey on the incidence and clinical features of
HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the
impact of this infection on HSCT outcome remains unclear. A rapid test based on
real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen
and quantify clinical samples for HHV-6. The detection step was based on reaction
with TaqMan® hydrolysis probes. A set of previously described primers and
probes have been tested to evaluate efficiency, sensitivity and reproducibility. The
target efficiency range was 91.4% with linearity ranging from 10-106
copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of
plasma. The qPCR assay developed in the present study was simple, rapid and
sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this
test may be useful as a practical tool to help elucidate the clinical relevance of
HHV-6 infection and reactivation in different scenarios and to determine the need for
surveillance. 相似文献
112.
Ernesto Nicolás Jordi Bacardit Tina Ferrer Ernest Giralt 《Letters in Peptide Science》1996,2(6):353-362
Summary Short peptides spanning the helicoidal sequences of the uteroglobin monomer (crystal forms P21 and C2221) were synthesized and studied by circular dichroism spectroscopy. None of them showed any secondary structure in the absence of HFIP. However, most peptides achieved a helical conformation when this structuring agent was used, with the exception of the analogue corresponding to the helicoidal fragment 19–24 (helix II, crystal P21). These results indicate that other factors, such as interchain interactions, have to contribute to helix stabilization in the molecule. On the other hand, while peptides corresponding to N- and C-terminal fragments that contain the first and fourth helices of the monomer, respectively (1–14 and 48–70) achieved a -like structure when 10–15% of HFIP was used, this behaviour was not observed when TFE was used. Moreover, substitution of cysteine by -aminobutyric acid at position 3 increased both the helicity of fragment 1–14 and its ability to adopt a -like structure, but the opposite effect was observed for fragment 48–70 when -aminobutyric acid was introduced at position 69. These results indicate that this part of the protein might be sensitive to the chemical environment it is exposed to and that the two cysteine residues at positions 3 and 69 of the monomer could play a different role in the folding process. 相似文献
113.
Complexes of Co(II), Ni(II), and Cu(II) with dideprotonated Acm are synthesized and characterized. Acm acts as bidentate ligand through the N-sulfonamido atom and the N-thiadiazole atom except for K6CoAcm4.6H2O in which Acm behaves as monodentate through the N-sulfonamido atom. 相似文献
114.
Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated) versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of “Methylation-Insensitive Primers” (MIPs), having degenerated bases (G/A) to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies. 相似文献
115.
116.
Vercheval L Bauvois C di Paolo A Borel F Ferrer JL Sauvage E Matagne A Frère JM Charlier P Galleni M Kerff F 《The Biochemical journal》2010,432(3):495-504
The activity of class D β-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate-limiting step for the wild-type OXA-10 β-lactamase. 相似文献
117.
118.
Fernández-Arrojo L Guazzaroni ME López-Cortés N Beloqui A Ferrer M 《Current opinion in biotechnology》2010,21(6):725-733
Microbial enzymes have many known applications as biocatalysts. However, only a few of them are currently employed for biocatalysis even though an annotated collection of more than 190 billion bases is available in metagenome sequence databases from uncultured and highly diverse microbial populations. This review aims at providing conceptual and technical bases for the translation of metagenome data into both experimental and computational frameworks that facilitates a comprehensive analysis of the biocatalysts diversity space. We will also briefly present the status of the current capabilities that assess and predict catalytic potential of environmental sites and track its diversity and evolution in large-scale biocatalysis process resulting from studies applying metagenomics in association with gene fingerprinting, catabolic arrays and complementary '-omics'. 相似文献
119.
Violetta La Cono Francesco Smedile Manuel Ferrer Peter N. Golyshin Laura Giuliano Michail M. Yakimov 《Microbial biotechnology》2010,3(5):595-606
Marine Crenarchaeota, ubiquitous and abundant organisms in the oceans worldwide, remain metabolically uncharacterized, largely due to their low cultivability. Identification of candidate genes for bicarbonate fixation pathway in the Cenarchaeum symbiosum A was an initial step in understanding the physiology and ecology of marine Crenarchaeota. Recent cultivation and genome sequencing of obligate chemoautotrophic Nitrosopumilus maritimus SCM1 were a major breakthrough towards understanding of their functioning and provide a valuable model for experimental validation of genomic data. Here we present the identification of multiple key components of 3-hydroxipropionate/4-hydroxybutyrate cycle, the fifth pathway in carbon fixation, found in data sets of environmental sequences representing uncultivated superficial and bathypelagic Crenarchaeota from Sargasso sea (GOS data set) and KM3 (Mediterranean Sea) and ALOHA (Atlantic ocean) stations. These organisms are likely to use acetyl-CoA/propionyl-CoA carboxylase(s) as CO2-fixing enzyme(s) to form succinyl-CoA, from which one molecule of acetyl-CoA is regenerated via 4-hydroxybutyrate cleavage and another acetyl-CoA to be the pathway product. The genetic distinctiveness and matching sympatric abundance imply that marine crenarchaeal genotypes from the three different geographic sites share similar ecophysiological properties, and therefore may represent fundamental units of marine ecosystem functioning. To couple results of sequence comparison with the dark ocean primary production, dissolved inorganic carbon fixation rates were measured at KM3 Station (3000 m depth, Eastern Mediterranean Sea), i.e. at the same site and depth used for metagenomic library construction. 相似文献
120.
José María Vieites Azam Ghazi Ana Beloqui Julio Polaina José M. Andreu Olga V. Golyshina Taras Y. Nechitaylo Agnes Waliczek Michail M. Yakimov Peter N. Golyshin Manuel Ferrer 《Microbial biotechnology》2010,3(1):48-58
Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(kcat/Km)]CE/[(kcat/Km)]FAE factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys281, Asp282, Asn316 and Lys317) situated close to the catalytic core (Ser143/Asp273/His305) and a deletion of a 34-AA–long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in kcat/Km values) and enzymes with inverted specificity ((kcat/Km)CE/(kcat/Km)FAE ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to −5.6 J mol−1), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to −13.7 J mol−1) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of ‘hot spot’ mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction. 相似文献