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31.
Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus
Type IV pili are expressed from a wide variety of Gram‐negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/GspF family members contain repeats of a conserved helix‐rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N‐terminal domain of Thermus thermophilus PilC. The N‐domain forms a dimer, adopting a six helix bundle structure with an up‐down‐up‐down‐up‐down topology. The monomers are related by a rotation of 170°, followed by a translation along the axis of the final α‐helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram‐negative bacteria. Site‐directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function. Proteins 2010. © 2010 Wiley‐Liss, Inc. 相似文献
32.
目的:建立肾衰康浓缩丸质量控制方法。方法:采用TLC鉴别肾衰康浓缩丸中大黄、丹参;采用HPLC-ELSD对制剂中黄芪甲苷进行含量测定,流动相为乙腈-水(30:70),流速:1 mL/min,柱温:28℃,漂移管温度:42℃,氮气:355kPa。结果:大黄、丹参的TLC鉴别斑点清晰、专属性强;黄芪甲苷在1.0μg-10.0μg之间呈良好的线性关系(r=0.9997,n=5),平均回收率为99.81%,RSD为1.73%(n=6)。结论:所建立的方法简便准确、专属性强、重现性好,可作为控制肾衰康浓缩丸质量的方法。 相似文献
33.
David Contreras Jaime Rodríguez Pablo Salgado Yuhui Qian 《Inorganica chimica acta》2011,374(1):643-646
A dihydroxybenzenes(DHB)-driven Fenton reaction was found to be more efficient than a simple Fenton reaction based on OH radical and activated species production. The reason for this enhanced reactivity by [Fe DHB] complexes is not well understood, but results suggest that it may be explained by the formation of oxidation species different from those formed during the classic Fenton reactions. In previous work, greater concentrations, and more sustained production of OH over time were observed in DHB driven Fenton reactions versus neat Fenton and Fenton-like reactions. In this work, chemiluminescence (CL) was monitored, and compared to OH production kinetics. The CL of the DHB-driven Fenton reaction was shorter than that for sustained production of OH. CL appears to have been caused by excited Fe(IV) species stabilized by the DHB ligands initially formed in the reaction. Formation of this species would have to have occurred by the reaction between OH and Fe(III) in a DHB complex. 相似文献
34.
Annikki Liakka Meeri Apaja-Sarkkinen Tuomo Karttunen Helena Autio-Harmainen 《Cell and tissue research》1991,263(2):245-252
Summary The immunohistochemical distribution of the basement membrane (BM) proteins, laminin and type IV collagen, and interstitial type III collagen was investigated in 12 fetal spleens at the 15th–38th gestational weeks (g.w.) and in spleens of 8 infants from term to 4 years. The results were compared with the distribution of the same proteins in adult human spleen. BM proteins were found to be abundantly present in the red pulp of all spleens during the whole of development. The content of type III collagen gradually decreased with advancing age and, in adult spleen, there were only occasional positively staining fibers in Billroth's cords. This finding indicates that the composition of reticular fibers in the red pulp of spleen is different from the reticular fibers elsewhere in lymphoreticular tissue. Early signs of ring fiber formation in the walls of venous sinuses were detectable at the 15th–19th g.w., although their more complete development occurred relatively late from the 36th g.w. onwards. Ring fibers contained both laminin and type IV collagen in all the investigated spleens. They never stained for type III collagen. The developing white pulp was positive for BM proteins, but showed no staining for type III collagen at the 15th g.w. At later ages, the white pulp stained similarly for both BM proteins and type III collagen. 相似文献
35.
Kathryn Patricia Haley Eric Joshua Blanz Jennifer Angeline Gaddy 《Journal of visualized experiments : JoVE》2014,(93)
Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the “pilus”5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses. 相似文献
36.
Qi Y Chuah ML Dong X Xie K Luo Z Tang K Liang ZX 《The Journal of biological chemistry》2011,286(4):2910-2917
FimX is a multidomain signaling protein required for type IV pilus biogenesis and twitching motility in the opportunistic pathogen Pseudomonas aeruginosa. FimX is localized to the single pole of the bacterial cell, and the unipolar localization is crucial for the correct assembly of type IV pili. FimX contains a non-catalytic EAL domain that lacks cyclic diguanylate (c-di-GMP) phosphodiesterase activity. It was shown that deletion of the EAL domain or mutation of the signature EVL motif affects the unipolar localization of FimX. However, it was not understood how the C-terminal EAL domain could influence protein localization considering that the localization sequence resides in the remote N-terminal region of the protein. Using hydrogen/deuterium exchange-coupled mass spectrometry, we found that the binding of c-di-GMP to the EAL domain triggers a long-range (∼ca. 70 Å) conformational change in the N-terminal REC domain and the adjacent linker. In conjunction with the observation that mutation of the EVL motif of the EAL domain abolishes the binding of c-di-GMP, the hydrogen/deuterium exchange results provide a molecular explanation for the mediation of protein localization and type IV pilus biogenesis by c-di-GMP through a remarkable allosteric regulation mechanism. 相似文献
37.
A. V. Vasil’ev V. I. Ivakhnenko S. A. Khotimchenko V. V. Korzh 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2008,2(3):306-310
Studies of Km for glutathione peroxidase (GPx) and activities of superoxide dismutase and GPx were carried out in liver and erythrocytes of rats kept on either the normal semisynthetic diet or a high-fat diet with increased content of Cu, Zn, Mn, and Se. The diet containing microelement additions caused the increase in TBH affinity of liver and erythrocyte GPx, as well as the decrease of liver SOD observed on the 14th day of the treatment of rats with the high-fat diet with additional increase of Cu, Zn, Mn, and Se. 相似文献
38.
Van Goethem S Van der Veken P Dubois V Soroka A Lambeir AM Chen X Haemers A Scharpé S De Meester I Augustyns K 《Bioorganic & medicinal chemistry letters》2008,18(14):4159-4162
To obtain selective and potent inhibitors of dipeptidyl peptidases 8 and 9, we synthesized a series of substituted isoindolines as modified analogs of allo-Ile-isoindoline, the reference DPP8/9 inhibitor. The influence of phenyl substituents and different P2 residues on the inhibitors’ affinity toward other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed. Within this series compound 8j was shown to be a potent and selective inhibitor of DPP8/9 with low activity toward DPP II. 相似文献
39.
Maria Zeydel Karen Puglia Mahboubeh Eghbali Jane Fant Sam Seifter Olga O. Blumenfeld 《Cell and tissue research》1991,265(2):353-359
Summary In the heart of the adult rat, fibroblasts are mainly responsible for the synthesis and deposition of the collagenous matrix. Because these cells in vitro may serve as an important model system for studies of collagen metabolism in heart tissue, we have cultured and characterized rat-heart fibroblasts from young adult and old animals. Conditions included use of media of different compositions with and without addition of ascorbate. Cell used were either cultured directly from fresh tissues or thawed previously frozen cells. Cultured cells were studied with respect to growth properties, morphology and ultrastructure and patterns of collagen. Heart fibroblasts generally resembled fibroblasts cultured from other tissues, but were more like skeletal muscle fibroblasts in that they deposited, in addition to type I collagen, type IV collagen and laminin. The fibroblasts showed a typical appearance in phase-contrast microscopy and electron microscopy. In the case of cells grown with added ascorbate, aligned collagen fibrils in the extracellular matrix showed a periodicity typical of type I collagen. The deposition of type I collagen occurred only in medium supplemented with ascorbate, and in that circumstance increased as a function of time past confluence; this was independent of the age of the animal from which the cells were obtained or of other changes of medium composition studied. Immunofluorescence studies with specific antibodies revealed that the cells deposited types I and IV collagens, laminin and fibronectin. In contrast to the case of type I collagen, the deposition of type IV collagen occurred in cells grown either with or without ascorbate. Direct observation of type IV collagen is consistent with the previous finding of type IV mRNA in cardiac fibroblasts in situ and in freshly isolated populations of these cells. 相似文献
40.
Christopher G. Ford Subramaniapillai Kolappan Hanh T. H. Phan Matthew K. Waldor Hanne C. Winther-Larsen Lisa Craig 《The Journal of biological chemistry》2012,287(43):36258-36272
Vibrio cholerae colonize the small intestine where they secrete cholera toxin, an ADP-ribosylating enzyme that is responsible for the voluminous diarrhea characteristic of cholera disease. The genes encoding cholera toxin are located on the genome of the filamentous bacteriophage, CTXφ, that integrates as a prophage into the V. cholerae chromosome. CTXφ infection of V. cholerae requires the toxin-coregulated pilus and the periplasmic protein TolA. This infection process parallels that of Escherichia coli infection by the Ff family of filamentous coliphage. Here we demonstrate a direct interaction between the N-terminal domain of the CTXφ minor coat protein pIII (pIII-N1) and the C-terminal domain of TolA (TolA-C) and present x-ray crystal structures of pIII-N1 alone and in complex with TolA-C. The structures of CTXφ pIII-N1 and V. cholerae TolA-C are similar to coliphage pIII-N1 and E. coli TolA-C, respectively, yet these proteins bind via a distinct interface that in E. coli TolA corresponds to a colicin binding site. Our data suggest that the TolA binding site on pIII-N1 of CTXφ is accessible in the native pIII protein. This contrasts with the Ff family phage, where the TolA binding site on pIII is blocked and requires a pilus-induced unfolding event to become exposed. We propose that CTXφ pIII accesses the periplasmic TolA through retraction of toxin-coregulated pilus, which brings the phage through the outer membrane pilus secretin channel. These data help to explain the process by which CTXφ converts a harmless marine microbe into a deadly human pathogen. 相似文献