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31.
Maria Isabel Perez-Lopez Rudy Mendez-Reina Steve Trier Cornelia Herrfurth Ivo Feussner Adriana Bernal Manu Forero-Shelton Chad Leidy 《生物化学与生物物理学报:生物膜》2019,1861(5):978-987
Bacteria are often found in close association with surfaces, resulting in the formation of biofilms. In Staphylococcus aureus (S. aureus), biofilms are implicated in the resilience of chronic infections, presenting a serious clinical problem world-wide. Here, S. aureus biofilms are grown under flow within clinical catheters at 37 °C. The lipid composition and biophysical properties of lipid extracts from these biofilms are compared with those from exponential growth and stationary phase cells. Biofilms show a reduction in iso and anteiso branching compensated by an increase in saturated fatty acids compared to stationary phase. A drastic reduction in carotenoid levels is also observed during biofilm formation. Thermotropic measurements of Laurdan GP and DPH polarization, show a reduction of lipid packing at 37 °C for biofilms compared to stationary phase. We studied the effects of carotenoid content on DMPG and DPPG model membranes showing trends in thermotropic behavior consistent with those observed in bacterial isolates, indicating that carotenoids participate in modulating lipid packing. Additionally, bending elastic constant (kc) measurements using vesicle fluctuation analysis (VFA) show that the presence of carotenoids can increase membrane bending rigidity. The antimicrobial peptide Magainin H2 was less activity on liposomes composed of stationary phase compared to biofilms or exponential growth isolates. This study contributes to an understanding of how Staphylococcus aureus modulates the composition of its membrane lipids, and how those changes affect the biophysical properties of membranes, which in turn may play a role in its virulence and its resistance to different membrane-active antimicrobial agents. 相似文献
32.
Binding of bovine serum albumin to heparin determined by turbidimetric titration and frontal analysis continuous capillary electrophoresis 总被引:1,自引:0,他引:1
The association of proteins with glycosaminoglycans is a subject of growing interest, but few techniques exist for elucidating this interaction quantitatively. Here we demonstrate the application of capillary electrophoresis to the system of serum albumin (SA) and heparin (Hp). These two species form soluble complexes, the interaction increasing with reduction in pH and/or ionic strength (I). The acid-base property of Hp was characterized by potentiometric titration of ion-exchanged Hp. Conditions for complex formation with SA were qualitatively determined by turbidimetry, which revealed points of incipient binding (pH(c)) and phase separation (pH(phi)), both of which depend on I. At pH > pH(phi), i.e., prior to phase separation, frontal analysis continuous capillary electrophoresis was used to measure the concentration of free protein and to determine the protein-HP binding isotherm. The binding isotherms were well fit by the McGhee-von Hippel model to yield quantitative binding information in the form of intrinsic binding constants (K(obs)) and binding site size (n). The strong increase in K(obs) with decrease of pH or I could be explained on the basis of electrostatic interactions, considering the effects of protein charge heterogeneity. The value of n, independent of pH, was rationalized on the basis of size considerations. The implications of these findings for clinical applications of Hp and for its physiological behavior are discussed. 相似文献
33.
Dubin G Popowicz G Krajewski M Potempa J Dubin A Holak TA 《Journal of biomolecular NMR》2004,28(3):295-296
The increasing antibiotic resistance of an important human pathogen Staphylococcus aureus calls for the development of new therapeutic strategies. Staphylococcal cysteine proteases have been suggested as targets for such therapies. The recent discovery of staphostatins, specific protein inhibitors of these enzymes, gives prospects for the design and production of synthetic, low molecular weight analogs which might become drugs. We have decided to structurally characterize staphostatin A, a representative inhibitor of staphylococcal cysteine proteases, and to assess its binding mode to the target protease with the view of clarifying the specificity determinants. Here we report the (1)H, (15)N and (13)C NMR resonance assignments of staphostatin A. 相似文献
34.
Dubin G Stec-Niemczyk J Dylag T Silberring J Dubin A Potempa J 《Biological chemistry》2004,385(6):543-546
Staphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action and distinct protein fold has recently been discovered. In this report we describe the properties of Staphylococcus epidermidis staphostatin A (EcpB), a new member of the family. As for other staphostatins, the recombinant S. epidermidis staphostatin A exerted very narrow inhibitory specificity, limited to cysteine protease from the same species. The closely related proteases from S. aureus cleaved the inhibitor at the reactive site peptide bond and inactivated it. The EcpB homologue, S. aureus staphostatin A (ScpB), was also susceptible to proteolytic cleavage at the same site by non-target cysteine proteases. Conversely, S. aureus staphostatin B (SspC) was resistant to such proteolysis. The difference in the susceptibility of individual inhibitors to proteolytic cleavage at the reactive site suggests subtle variations in the mechanism of interaction with cysteine proteases. 相似文献
35.
Rzychon M Filipek R Sabat A Kosowska K Dubin A Potempa J Bochtler M 《Protein science : a publication of the Protein Society》2003,12(10):2252-2256
Staphostatins are the endogenous inhibitors of the major secreted cysteine proteases of Staphylococcus aureus, the staphopains. Here, we present the 1.4 A crystal structure of staphostatin B and show that the fold can be described as a fully closed, highly sheared eight-stranded beta-barrel. Thus, staphostatin B is related to beta-barrel domains that are involved in the inhibition or regulation of proteases of various catalytic types and to the superfamily of lipocalins/cytosolic fatty acid binding proteins. Unexpectedly for a cysteine protease inhibitor, staphostatin B is not significantly similar to cystatins. 相似文献
36.
Dubin G 《Acta biochimica Polonica》2003,50(3):715-724
Staphylococcus aureus is a human pathogen causing a wide range of diseases. Most staphylococcal infections, unlike those caused by other bacteria are not toxigenic and very little is known about their pathogenesis. It has been proposed that a core of secreted proteins common to many infectious strains is responsible for colonization and infection. Among those proteins several proteases are present and over the years many different functions in the infection process have been attributed to them. However, little direct, in vivo data has been presented. Two cysteine proteases, staphopain A (ScpA) and staphopain B (SspB) are important members of this group of enzymes. Recently, two cysteine protease inhibitors, staphostatin A and staphostatin B (ScpB and SspC, respectively) were described in S. aureus shedding new light on the complexity of the processes involving the two proteases. The scope of this review is to summarize current knowledge on the network of staphylococcal cysteine proteases and their inhibitors in view of their possible role as virulence factors. 相似文献
37.
Determination of molecular weight of heparin by size exclusion chromatography with universal calibration 总被引:3,自引:0,他引:3
The molecular weight (MW) of heparin can be accurately determined by size exclusion chromatography using "universal calibration." A universal calibration curve was constructed for Superose 12 with standard pullulan samples and verified using characterized ficoll fractions. This calibration yielded the correct MW of heparin as determined by light scattering, when the ionic strength of the mobile phase was maintained over 1.0M. Sodium poly(styrenesulfonate) samples were not suitable standards because of adsorption at high salt concentration and repulsion from the packing material at low ionic strength. The extraordinarily high charge density of heparin leads to the need for high salt concentration to screen such repulsions. 相似文献
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40.
Andrea Nühs Manu De Rycker Sujatha Manthri Eamon Comer Christina A. Scherer Stuart L. Schreiber Jean-Robert Ioset David W. Gray 《PLoS neglected tropical diseases》2015,9(9)
Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery. 相似文献