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51.
Eva Garcia-Gonzalez Lena Poppinga Anne Fünfhaus Gillian Hertlein Kati Hedtke Agata Jakubowska Elke Genersch 《PLoS pathogens》2014,10(7)
Paenibacillus larvae, the etiological agent of the globally occurring epizootic American Foulbrood (AFB) of honey bees, causes intestinal infections in honey bee larvae which develop into systemic infections inevitably leading to larval death. Massive brood mortality might eventually lead to collapse of the entire colony. Molecular mechanisms of host-microbe interactions in this system and of differences in virulence between P. larvae genotypes are poorly understood. Recently, it was demonstrated that the degradation of the peritrophic matrix lining the midgut epithelium is a key step in pathogenesis of P. larvae infections. Here, we present the isolation and identification of PlCBP49, a modular, chitin-degrading protein of P. larvae and demonstrate that this enzyme is crucial for the degradation of the larval peritrophic matrix during infection. PlCBP49 contains a module belonging to the auxiliary activity 10 (AA10, formerly CBM33) family of lytic polysaccharide monooxygenases (LPMOs) which are able to degrade recalcitrant polysaccharides. Using chitin-affinity purified PlCBP49, we provide evidence that PlCBP49 degrades chitin via a metal ion-dependent, oxidative mechanism, as already described for members of the AA10 family. Using P. larvae mutants lacking PlCBP49 expression, we analyzed in vivo biological functions of PlCBP49. In the absence of PlCBP49 expression, peritrophic matrix degradation was markedly reduced and P. larvae virulence was nearly abolished. This indicated that PlCBP49 is a key virulence factor for the species P. larvae. The identification of the functional role of PlCBP49 in AFB pathogenesis broadens our understanding of this important family of chitin-binding and -degrading proteins, especially in those bacteria that can also act as entomopathogens. 相似文献
52.
2-Deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose have been prepared by tritiation of the corresponding unlabeled 2-fluoro sugars. The tritiated 2-fluoro sugars are phosphorylated and activated by UTP and by GTP to yield UDP-2-deoxy-2-fluoro-D-[3H]glucose, UDP-2-deoxy-2-fluoro-D-[3H]mannose, GDP-2-deoxy-2-fluoro-D-[3H]glucose and GDP-2-deoxy-2-fluoro-D-[3H]mannose in both cell types. The nucleotide derivatives could also be labeled in the nucleotide moiety by feeding the cells with [14C]uridine or [14C]guanosine in the presence of unlabeled 2-fluoro sugar. No evidence was obtained for metabolic steps in which the six-carbon chain of 2-fluoro sugars was not preserved. No epimerisation of the label to 2-deoxy-2-fluoro-D-[3H]galactose could be observed by radioactive gas-liquid chromatography of the enzymatic cleavage products of the different 2-fluoro sugar metabolites isolated from either cell type. Yeast and chick embryo cells both incorporate 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose specifically into glycoproteins, although this incorporation is very low when compared to the incorporation of 2-deoxy-D-[3H]glucose. 相似文献
53.
The metabolism of the nonsteroidal antiinflammatory drug flobufen, 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, was studied in primary cultures of human hepatocytes prepared by two-step collagenase perfusion of livers from four donors. Racemic flobufen or its individual enantiomers, R-(+)- and S-(-)-flobufen were used as substrates. Aliquots of culture medium were collected during 24-h incubation. The time-dependent disappearance of flobufen enantiomers and the formation of metabolites (stereoisomers of dihydroflobufen (DHF)) in hepatocytes were measured by chiral HPLC. The reduction of flobufen in human hepatocytes was stereoselective ((+)-R-flobufen was preferentially metabolized) and stereospecific ((2R;4S)-DHF and (2S;4S)-DHF stereoisomers were mostly formed). Although the structure of flobufen is different from the profens (2-arylpropionates), flobufen undergoes chiral inversion in human hepatocytes. The inversion of R-(+)-flobufen to S-(-)-flobufen predominates. The individual DHF stereoisomers were incubated in hepatocyte cultures and their biotransformation studied. The unidirectional chiral inversion of (2S;4S)-DHF to (2R;4S)-DHF and (2R;4R)-DHF to (2S;4R)-DHF was observed. Stereoselective oxidation of the DHFs to flobufen was also detected. Thus, flobufen metabolism in primary cultures of human hepatocytes is much more complicated (via chiral inversion and DHF re-oxidation) than was presumed from a preliminary achiral point of view. 相似文献
54.
Deacylated pulmonary surfactant protein SP-C transforms from alpha-helical to amyloid fibril structure via a pH-dependent mechanism: an infrared structural investigation 下载免费PDF全文
Bovine pulmonary surfactant protein C (SP-C) is a hydrophobic, alpha-helical membrane-associated lipoprotein in which cysteines C4 and C5 are acylated with palmitoyl chains. Recently, it has been found that the alpha-helix form of SP-C is metastable, and under certain circumstances may transform from an alpha-helix to a beta-strand conformation that resembles amyloid fibrils. This transformation is accelerated when the protein is in its deacylated form (dSP-C). We have used infrared spectroscopy to study the structure of dSP-C in solution and at membrane interfaces. Our results show that dSP-C transforms from an alpha-helical to a beta-type amyloid fibril structure via a pH-dependent mechanism. In solution at low pH, dSP-C is alpha-helical in nature, but converts to an amyloid fibril structure composed of short beta-strands or beta-hairpins at neutral pH. The alpha-helix structure of dSP-C is fully recoverable from the amyloid beta-structure when the pH is once again lowered. Attenuated total reflectance infrared spectroscopy of lipid-protein monomolecular films showed that the fibril beta-form of dSP-C is not surface-associated at the air-water interface. In addition, the lipid-associated alpha-helix form of dSP-C is only retained at the surface at low surface pressures and dissociates from the membrane at higher surface pressures. In situ polarization modulation infrared spectroscopy of protein and lipid-protein monolayers at the air-water interface confirmed that the residual dSP-C helix conformation observed in the attenuated total reflectance infrared spectra of transferred films is randomly or isotropically oriented before exclusion from the membrane interface. This work identifies pH as one of the mechanistic causes of amyloid fibril formation for dSP-C, and a possible contributor to the pathogenesis of pulmonary alveolar proteinosis. 相似文献
55.
Albrecht E Kolisek M Viergutz T Zitnan R Schweigel M 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2008,178(3):285-295
In this study, we have studied the expression, localization, and functionality of vacuolar-type H+-ATPase (vH+-ATPase) and Na+/K+-ATPase in the bovine rumen epithelium. Compared with the intracellular pH (pHi) of control rumen epithelial cells (REC; 7.06 ± 0.07), application of inhibitors selective for vH+-ATPase (foliomycin) and Na+/K+-ATPase (ouabain) reduced pHi by 0.10 ± 0.03 and 0.18 ± 0.03 pH-units, respectively, thereby verifying the existence of both functional proteins. Results
from qRT-PCR and immunoblotting clearly confirm the expression of vH+-ATPase B subunit in REC. However, the amount of Na+/K+-ATPase mRNA and protein is tenfold and 11-fold of those of vH+-ATPase subunit B, respectively, reflecting a lower overall abundance of the latter in REC. Na+/K+-ATPase immunostaining has revealed the protein in the plasma membrane of all REC from the stratum basale to stratum granulosum,
with the highest abundance in basal cells. In contrast, the vH+-ATPase B subunit has been detected in groups of cells only, mainly localized in the stratum spinosum and stratum granulosum
of the epithelium. Furthermore, vH+-ATPase has been detected in the cell membrane and in intracellular pools. Thus, functional vacuolar-type H+ pumps are expressed in REC and probably play a role in the adaptation of epithelial transport processes. 相似文献
56.
S. R. Schmolke K. Broeg S. Zander V. Bissinger P. D. Hansen N. Kress B. Herut E. Jantzen G. Krüner A. Sturm W. Körting H. von Westernhagen 《Helgoland Marine Research》1999,53(3-4):257-266
A comprehensive database, containing biological and chemical information, collected in the framework of the bilateral interdisciplinary
MARS project (”biological indicators of natural and man-made changes in marine and coastal waters”) during the years 1995–1997
in the coastal environment of the North Sea, was subjected to a multivariate statistical evaluation. The MARS project was
designated to combine a variety of approaches and to develop a set of methods for the employment of biological indicators
in pollution monitoring and environmental quality assessment. In total, nine ship cruises to four coastal sampling sites were
conducted; 765 fish and 384 mussel samples were analysed for biological and chemical parameters. Additional information on
the chemical background at the sampling sites was derived from sediment samples, collected at each of the four sampling sites.
Based on the available chemical data in sediments and black mussel (Mytilus edulis) a pollution gradient between the selected sites, was established. The chemical body burden of flounder (Platichthys flesus) from these sites, though, did not reflect this gradient equally clear. In contrast, the biological information derived from
measurements in fish samples displayed significant a regional as well as a temporal pattern. A multivariate bioindicator data
matrix was evaluated employing a factor analysis model to identify relations between selected biological indicators, and to
improve the understanding of a regional and temporal component in the parameter response. In a second approach, applying the
k-means algorithm on the data matrix, two significantly different clusters of samples, characterised by the current health
status of the fish, were extracted. Using this classification a temporal, and in the second order, a less pronounced spatial
effect was evident. In particular, during July 1996, a clear sign of deteriorating environmental conditions was extracted
from the biological data matrix.
Received: 20 June 1999 / Received in revised form: 8 November 1999 / Accepted: 8 November 1999 相似文献
57.
58.
Huber M Bahr I Krätzschmar JR Becker A Müller EC Donner P Pohlenz HD Schneider MR Sommer A 《Molecular & cellular proteomics : MCP》2004,3(1):43-55
In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex trade mark, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state. 相似文献
59.
Photosynthesis inhibition in algae (Chlorella) and plant (spinach) chloroplasts by quaternary ammonium salts of heptacaine {N-[2-(2-heptyloxyphenylcarbamoyloxy)-ethyl]-N-alkylpiperidinium
bromides} depended on the alkyl chain length of the alkyl substituent and showed good correlations with theoretical hydrophobic
fragment constants as well as with experimentally determined physico-chemical parameters, namely extraction constants and
surface activities.
Communicated by. Z. ŠESTáK 相似文献
60.
Pauline Scherdel Jean-Fran?ois Salaün Marie-No?lle Robberecht-Riquet Laura Reali Gabriella Páll Elke J?ger-Roman Manuel Praena Crespo Marilena Moretto Margareta Seher-Zupan?i? Sigurlaug Agustsson the European Confederation of Primary Care Paediatricians research group Martin Chalumeau 《PloS one》2013,8(8)