New solvent-producing Clostridium sp. strains, hydrolyzing a wide range of polysaccharides, are closely related to Clostridium butyricum

Thirteen new Clostridium strains, previously isolated from soil and found to produce high amounts of solvents from glucose, hydrolyzed a great variety of α- and β-glycans, including raw starch, xylan, pectin, inulin and cellulose. The sequences of the PCR-amplified DNA fragments containing the variable 3′ part of one of the 16S rRNA genes were 99.5% identical. The macrorestriction pattern of two endonucleolytic digests of chromosomal DNA in the pulsed-field gel electrophoresis (PFGE) confirmed their high homogeneity on the DNA level. The complete 16S rRNA gene sequence of three selected strains was 99.8% identical to the 16S rRNA gene sequence from Clostridium butyricum and separates them from C. acetobutylicum. To the closely related four species of solventogenic clostridia a new group of strains has to be added, which has a great potential for the direct fermentation of biomass. Journal of Industrial Microbiology & Biotechnology (2001) 27, 329–335. Received 12 September 2000/ Accepted in revised form 25 July 2001     

msmsEval: tandem mass spectral quality assignment for high-throughput proteomics

Background  

In proteomics experiments, database-search programs are the method of choice for protein identification from tandem mass spectra. As amino acid sequence databases grow however, computing resources required for these programs have become prohibitive, particularly in searches for modified proteins. Recently, methods to limit the number of spectra to be searched based on spectral quality have been proposed by different research groups, but rankings of spectral quality have thus far been based on arbitrary cut-off values. In this work, we develop a more readily interpretable spectral quality statistic by providing probability values for the likelihood that spectra will be identifiable.     

Limited genetic diversity in <Emphasis Type="Italic">Salmonella enterica</Emphasis> Serovar Enteritidis PT13

Background  

Salmonella enterica serovar Enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. In Canada phage types (PT) 4, 8 and 13 predominate and in 2005 a large foodborne PT13 outbreak occurred in the province of Ontario. The ability to link strains during this outbreak was difficult due to the apparent clonality of PT13 isolates in Canada, as there was a single dominant pulsed-field gel electrophoresis (PFGE) profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. The aim of this study was to perform comparative genomic hybridization (CGH), DNA sequence-based typing (SBT) genomic analyses, plasmid analyses, and automated repetitive sequence-based PCR (rep-PCR) to identify epidemiologically significant traits capable of subtyping S. Enteritidis PT13.     

Involvement of a calpain-like protease in the processing of the murine interleukin 1 alpha precursor

Interleukin (IL) 1 alpha is synthesized as a 33-kDa precursor that is enzymatically cleaved to the 15-17-kDa forms that are found in the culture supernatants of activated macrophages. We have explored the possibility that calcium might enhance IL-1 processing and secretion via the stimulation of a calcium-dependent protease. We have found that lysates prepared from human peripheral blood monocytes, the human histiocytic lymphoma cell line U937, and the murine macrophage cell line P388D1 contain a calcium-dependent IL-1 alpha processing activity that cleaves the IL-1 alpha precursor to its mature form. Although NIH 3T3 mouse fibroblast cell lysates also contain IL-1 processing activity, lysates from the murine thymoma EL-4, the human epidermoid cell line HEp-2, and the human foreskin fibroblast line FS-4 lack this activity. IL-1 processing activity is inhibited by leupeptin and exhibits a molecular mass of 80-110 kDa. The processing activity is also inhibited by a monoclonal antibody directed against calpain type I. These results indicate that the processing of the IL-1 alpha precursor is mediated, at least in part, by a member of the calpain family of proteases. Mixing experiments revealed that lysates from EL-4 or HEp-2 cells contain an inhibitor(s) of the calpain-like protease in macrophage extracts. It is, therefore, likely that many non-macrophage cell types are unable to process the IL-1 alpha precursor because the calpain present in these cells is only weakly active due to the presence of a specific inhibitor(s) such as calpastatin.     

Loss of alveolar membrane diffusing capacity and pulmonary capillary blood volume in pulmonary arterial hypertension

Background

Reduced gas transfer in patients with pulmonary arterial hypertension (PAH) is traditionally attributed to remodeling and progressive loss of pulmonary arterial vasculature that results in decreased capillary blood volume available for gas exchange.

Methods

We tested this hypothesis by determination of lung diffusing capacity (DL) and its components, the alveolar capillary membrane diffusing capacity (D_m) and lung capillary blood volume (V_c) in 28 individuals with PAH in comparison to 41 healthy individuals, and in 19 PAH patients over time. Using single breath simultaneous measure of diffusion of carbon monoxide (DL_CO) and nitric oxide (DL_NO), DL and D_m were respectively determined, and V_c calculated. D_m and V_c were evaluated over time in relation to standard clinical indicators of disease severity, including brain natriuretic peptide (BNP), 6-minute walk distance (6MWD) and right ventricular systolic pressure (RVSP) by echocardiography.

Results

Both DL_CO and DL_NO were reduced in PAH as compared to controls and the lower DL in PAH was due to loss of both D_m and V_c (all p < 0.01). While DL_CO of PAH patients did not change over time, DL_NO decreased by 24 ml/min/mmHg/year (p = 0.01). Consequently, D_m decreased and V_c tended to increase over time, which led to deterioration of the D_m/V_c ratio, a measure of alveolar-capillary membrane functional efficiency without changes in clinical markers.

Conclusions

The findings indicate that lower than normal gas transfer in PAH is due to loss of both D_m and V_c, but that deterioration of D_m/V_c over time is related to worsening membrane diffusion.     

Regulation of DNA replication within the immunoglobulin heavy-chain locus during B cell commitment

The temporal order of replication of mammalian chromosomes appears to be linked to their functional organization, but the process that establishes and modifies this order during cell differentiation remains largely unknown. Here, we studied how the replication of the Igh locus initiates, progresses, and terminates in bone marrow pro-B cells undergoing B cell commitment. We show that many aspects of DNA replication can be quantitatively explained by a mechanism involving the stochastic firing of origins (across the S phase and the Igh locus) and extensive variations in their firing rate (along the locus). The firing rate of origins shows a high degree of coordination across Igh domains that span tens to hundreds of kilobases, a phenomenon not observed in simple eukaryotes. Differences in domain sizes and firing rates determine the temporal order of replication. During B cell commitment, the expression of the B-cell-specific factor Pax5 sharply alters the temporal order of replication by modifying the rate of origin firing within various Igh domains (particularly those containing Pax5 binding sites). We propose that, within the Igh C(H)-3'RR domain, Pax5 is responsible for both establishing and maintaining high rates of origin firing, mostly by controlling events downstream of the assembly of pre-replication complexes.     

Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells

Background

Fatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells.

Methods

We used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro.

Results

We demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus.

Conclusion

The H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease.