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1.
Zanen G Houben EN Meima R Tjalsma H Jongbloed JD Westers H Oudega B Luirink J van Dijl JM Quax WJ 《The FEBS journal》2005,272(18):4617-4630
Signal peptides that direct protein export in Bacillus subtilis are overall more hydrophobic than signal peptides in Escherichia coli. To study the importance of signal peptide hydrophobicity for protein export in both organisms, the alpha-amylase AmyQ was provided with leucine-rich (high hydrophobicity) or alanine-rich (low hydrophobicity) signal peptides. AmyQ export was most efficiently directed by the authentic signal peptide, both in E. coli and B. subtilis. The leucine-rich signal peptide directed AmyQ export less efficiently in both organisms, as judged from pulse-chase labelling experiments. Remarkably, the alanine-rich signal peptide was functional in protein translocation only in E. coli. Cross-linking of in vitro synthesized ribosome nascent chain complexes (RNCs) to cytoplasmic proteins showed that signal peptide hydrophobicity is a critical determinant for signal peptide binding to the Ffh component of the signal recognition particle (SRP) or to trigger factor, not only in E. coli, but also in B. subtilis. The results show that B. subtilis SRP can discriminate between signal peptides with relatively high hydrophobicities. Interestingly, the B. subtilis protein export machinery seems to be poorly adapted to handle alanine-rich signal peptides with a low hydrophobicity. Thus, signal peptide hydrophobicity appears to be more critical for the efficiency of early stages in protein export in B. subtilis than in E. coli. 相似文献
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Background
A preliminary survey showed half of the participating Royal Netherlands Air Force (RNLAF) F-16 fighter pilots to have nasal integument and osteocartilagenous disorders related to wearing in-flight oxygen masks.Aim
To make an inventory of these disorders and possible associated factors.Methods
All RNLAF F-16 pilots were requested to fill out a semi-structured questionnaire for a cross-sectional survey. Additionally, one squadron in The Netherlands and pilots in operational theater were asked to participate in a prospective study that required filling out a pain score after each flight. Pilot- and flight-related variables on all participants were collected from the RNLAF database. A linear mixed model was built to identify associated factors with the post-flight pain score.Results
The response rate to the survey was 83%. Ninety of the 108 participants (88%, 6 missing) reported tenderness, irritation, pain, erythema, skin lesions, callous skin, or swelling of nasal bridge integument or architecture. Seventy-two participants (71%, 6 missing) reported their symptoms to be troublesome after a mean of 6±3 out of 10 flights (0;10, 54 missing). Sixty-six pilots participated in scoring post-flight pain. Pain scores were significantly higher if a participant had ≥3 nasal disorders, after longer than average flights, after flying abroad, and after flying with night vision goggles (respectively +2.7 points, p = 0.003; +0.2 points, p = 0.027; +1.8 points, p = 0.001; +1.2 points p = 0.005). Longer than average NVG flights and more than average NVG hours per annum decreased painscores (respectively −0.8 points, p = 0.017; −0.04 points, p = 0.005).Conclusions
The majority of the RNLAF F-16 fighter pilot community has nasal disorders in the contact area of the oxygen mask, including pain. Six pilot- or flight-related characteristics influence the experienced level of pain. 相似文献3.
Houtenbos I Westers TM Stam AG de Gruijl TD Scheper RJ Ossenkoppele GJ van de Loosdrecht AA 《Cancer immunology, immunotherapy : CII》2003,52(7):455-462
PURPOSE: Immunotherapy holds promise as a new strategy for the eradication of residual cells in acute myeloid leukaemia (AML). Leukaemic antigen presenting cells (APCs) combining optimal antigen presentation and tumour antigenicity could be used as potent T cell activators. For clinical purposes it is desirable to culture APCs under serum-free conditions. Therefore, we compared morphological, immunophenotypical and functional outcome of the serum-free culture of AML-APCs to their serum-enriched culture. METHODS: AML blasts (n=19) were cultured in the presence of either a cytokine mix or calcium ionophore (CI) for 14 and 2 days, respectively, in FCS-containing medium (FCS), StemSpan serum-free medium (SP) and CellGro serum-free medium (CG). After culture relative yields were calculated and immunophenotypic analysis of APC markers was performed. The mixed leukocyte reaction (MLR) was used to determine T cell stimulating capacity. RESULTS: Serum-free culture of AML-APCs resulted in comparable morphology, relative yields and immunophenotype to serum-enriched culture. By comparing both serum-free media we observed a trend towards a more mature phenotype of CI-cultured AML-APCs in SP. MLR showed that serum-free cultured cells have equal T cell stimulatory capacity in comparison with serum-enriched culture. CONCLUSION: These data show that the serum-free culture of AML-APCs is feasible and that these APCs are comparable to serum-enriched cultured AML-APCs with regard to morphological, immunophenotypical and functional characteristics. These AML-APCs are suitable for the development of active specific immunisation protocols which meet the criteria for good clinical practise (GCP). 相似文献
4.
Fasehee H Westers H Bolhuis A Antelmann H Hecker M Quax WJ Mirlohi AF van Dijl JM Ahmadian G 《Proteomics》2011,11(19):3905-3913
Sortases of Gram-positive bacteria catalyze the covalent C-terminal anchoring of proteins to the cell wall. Bacillus subtilis, a well-known host organism for protein production, contains two putative sortases named YhcS and YwpE. The present studies were aimed at investigating the possible sortase function of these proteins in B. subtilis. Proteomics analyses revealed that sortase-mutant cells released elevated levels of the putative sortase substrate YfkN into the culture medium upon phosphate starvation. The results indicate that YfkN required sortase activity of YhcS for retention in the cell wall. To analyze sortase function in more detail, we focused attention on the potential sortase substrate YhcR, which is co-expressed with the sortase YhcS. Our results showed that the sortase recognition and cell-wall-anchoring motif of YhcR is functional when fused to the Bacillus pumilus chitinase ChiS, a readily detectable reporter protein that is normally secreted. The ChiS fusion protein is displayed at the cell wall surface when YhcS is co-expressed. In the absence of YhcS, or when no cell-wall-anchoring motif is fused to ChiS, the ChiS accumulates predominately in the culture medium. Taken together, these novel findings show that B. subtilis has a functional sortase for anchoring proteins to the cell wall. 相似文献
5.
Westers H Braun PG Westers L Antelmann H Hecker M Jongbloed JD Yoshikawa H Tanaka T van Dijl JM Quax WJ 《Applied and environmental microbiology》2005,71(4):1899-1908
Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the B. subtilis genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes, skfABCD, located in the prophage 1 region. Intact skfABCD genes are required not only for LipA production at wild-type levels by B. subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient B. subtilis strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor. 相似文献
6.
Margaret I Butler Jeremy Gray Timothy JD Goodwin Russell TM Poulter 《BMC evolutionary biology》2006,6(1):42-26
Background
We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal. 相似文献7.
Proteomics of Protein Secretion by Bacillus subtilis: Separating the “Secrets” of the Secretome
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Harold Tjalsma Haike Antelmann Jan D.H. Jongbloed Peter G. Braun Elise Darmon Ronald Dorenbos Jean-Yves F. Dubois Helga Westers Geeske Zanen Wim J. Quax Oscar P. Kuipers Sierd Bron Michael Hecker Jan Maarten van Dijl 《Microbiological reviews》2004,68(2):207-233
Secretory proteins perform a variety of important “remote-control” functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which ~90 extracellular proteins were identified. Analysis of these proteins disclosed various “secrets of the secretome,” such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only ~50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance. 相似文献
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J Daugherty TM Evans T Skillom LE Watson NP Money 《Fungal genetics and biology : FG & B》1998,24(3):354-363
Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press. 相似文献