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911.
Cadherins are multidomain adhesion proteins whose interactions direct cell sorting during histogenesis. They determine cell adhesion specificity, but prior studies failed to identify physical differences that could underlie cell sorting. These single molecule studies identify kinetic and strength differences between different cadherins. They further demonstrate that the modular extracellular architecture of cleavage stage C-cadherin supports a multistate binding mechanism. These multiple bonds exhibit a kinetic hierarchy of strengths that map to the different cadherin domains. The outer two N-terminal domains of C-cadherin form two bound states with dissociation rates of 3.9 and 0.02 s(-1). The latter is 25-fold slower than between the corresponding epithelial cadherin segments. In addition to the two fast bonds, the five-domain fragment (CEC1-5) forms two additional stronger, longer-lived bonds with dissociation rates of 0.00039 and 0.00001 s(-1). We further quantified the lifetimes of bonds subject to a constant force, and thus identified multiple dissociation events with rates that agree quantitatively with the force spectroscopy data. The qualitative features are similar to those reported for epithelial cadherin. However, the significant differences in the dissociation rates of the outer domains, which include the specificity-determining region, suggest that kinetic differences may determine cadherin discrimination, rather than adhesion energies. 相似文献
912.
Little is known about how pH-buffering capacity affects phosphorus (P) solubilization by Penicillium bilaiae. This study compared solubilization of rock phosphate (RP) by P. bilaiae in nonbuffered (pH 5.0) and buffered (pH 7.0) media. Fungal growth reached the stationary phase around day 12 and was slightly enhanced in the buffered medium. The fungus reduced solution pH from 5.0 to 4.1 in the nonbuffered medium and from 7.0 to 4.9 in the buffered medium by day 12. Phosphorus concentrations increased after day 9 more in the buffered than in the nonbuffered media (53 and 5 mg P x L(-1), respectively, on day 12). On day 12, higher concentrations of citric and oxalic acids were detected in the buffered (2.0 and 1.2 g x L(-1), respectively) than nonbuffered media (0.5 and 0.04 g x L(-1), respectively). Solubilization of RP was simulated without P. bilaiae in solutions equivalent to the nonbuffered and buffered cultures of P. bilaiae grown with RP. After a 24 h incubation, the P concentrations were of similar magnitudes to those observed in the P. bilaiae culture (18 and 47 mg P x L(-1), respectively, in the nonbuffered and buffered media). Under increased pH-buffering conditions, the enhanced production of citric and oxalic acids led to significant RP solubilization. 相似文献
913.
Steussy CN Robison AD Tetrick AM Knight JT Rodwell VW Stauffacher CV Sutherlin AL 《Biochemistry》2006,45(48):14407-14414
Recent structural studies of the HMG-CoA synthase members of the thiolase superfamily have shown that the catalytic loop containing the nucleophilic cysteine follows the phi and psi angle pattern of a II' beta turn. However, the i + 1 residue is conserved as an alanine, which is quite unusual in this position as it must adopt a strained positive phi angle to accommodate the geometry of the turn. To assess the effect of the conserved strain in the catalytic loop, alanine 110 of Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was mutated to a glycine. Subsequent enzymatic studies showed that the overall reaction rate of the enzyme was increased 140-fold. An X-ray crystallographic study of the Ala110Gly mutant enzyme demonstrated unanticipated adjustments in the active site that resulted in additional stabilization of all three steps of the reaction pathway. The rates of acetylation and hydrolysis of the mutant enzyme increased because the amide nitrogen of Ser308 shifts 0.4 A toward the catalytic cysteine residue. This motion positions the nitrogen to better stabilize the intermediate negative charge that develops on the carbonyl oxygen of the acetyl group during both the formation of the acyl-enzyme intermediate and its hydrolysis. In addition, the hydroxyl of Ser308 rotates 120 degrees to a position where it is able to stabilize the carbanion intermediate formed by the methyl group of the acetyl-S-enzyme during its condensation with acetoacetyl-CoA. 相似文献
914.
Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. UvrABC endonuclease, encoded by the UvrA, UvrB, and UvrC genes can incise DNA containing bulky nucleotide adducts and intrastrand cross-links. UvrA, UvrB, and UvrC were cloned from Bacillus caldotenax (Bca)and UvrC from Thermatoga maritima (Tma), and recombinant proteins were overexpressed in and purified from Escherichia coli. Incision activities of UvrABC composed of all Bca-derived subunits (UvrABC(Bca)) and an interspecies combination UvrABC composed of Bca-derived UvrA and UvrB and Tma-derived UvrC (UvrABC(Tma)) were compared on benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide (BPDE)-adducted substrates. Both UvrABC(Bca) and UvrABC(Tma) specifically incised both BPDE-adducted plasmid DNAs and site-specifically modified 50-bp oligonucleotides containing a single (+)-trans- or (+)-cis-BPDE adduct. Incision activity was maximal at 55-60 degrees C. However, UvrABC(Tma) was more robust than UvrABC(Bca) with 4-fold greater incision activity on BPDE-adducted oligonucleotides and 1.5-fold greater on [(3)H]BPDE-adducted plasmid DNAs. Remarkably, UvrABC(Bca) incised only at the eighth phosphodiester bond 5' to the BPDE-modified guanosine. In contrast, UvrABC(Tma) performed dual incision, cutting at both the fifth phosphodiester bond 3' and eighth phosphodiester bond 5' from BPDE-modified guanosine. BPDE adduct stereochemistry influenced incision activity, and cis adducts on oligonucleotide substrates were incised more efficiently than trans adducts by both UvrABC(Bca) and UvrABC(Tma). UvrAB-DNA complex formation was similar with (+)-trans- and (+)-cis-BPDE-adducted substrates, suggesting that UvrAB binds both adducts equally and that adduct configuration modifies UvrC recognition of the UvrAB-DNA complex. The dual incision capabilities and higher incision activity of UvrABC(Tma) make it a robust tool for DNA adduct studies. 相似文献
915.
Methane- and Sulfur-Metabolizing Microbial Communities Dominate the Lost City Hydrothermal Field Ecosystem 总被引:2,自引:0,他引:2
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William J. Brazelton Matthew O. Schrenk Deborah S. Kelley John A. Baross 《Applied microbiology》2006,72(9):6257-6270
Hydrothermal venting and the formation of carbonate chimneys in the Lost City hydrothermal field (LCHF) are driven predominantly by serpentinization reactions and cooling of mantle rocks, resulting in a highly reducing, high-pH environment with abundant dissolved hydrogen and methane. Phylogenetic and terminal restriction fragment length polymorphism analyses of 16S rRNA genes in fluids and carbonate material from this site indicate the presence of organisms similar to sulfur-oxidizing, sulfate-reducing, and methane-oxidizing Bacteria as well as methanogenic and anaerobic methane-oxidizing Archaea. The presence of these metabolic groups indicates that microbial cycling of sulfur and methane may be the dominant biogeochemical processes active within this ultramafic rock-hosted environment. 16S rRNA gene sequences grouping within the Methylobacter and Thiomicrospira clades were recovered from a chemically diverse suite of carbonate chimney and fluid samples. In contrast, 16S rRNA genes corresponding to the Lost City Methanosarcinales phylotype were found exclusively in high-temperature chimneys, while a phylotype of anaerobic methanotrophic Archaea (ANME-1) was restricted to lower-temperature, less vigorously venting sites. A hyperthermophilic habitat beneath the LCHF may be reflected by 16S rRNA gene sequences belonging to Thermococcales and uncultured Crenarchaeota identified in vent fluids. The finding of a diverse microbial ecosystem supported by the interaction of high-temperature, high-pH fluids resulting from serpentinization reactions in the subsurface provides insight into the biogeochemistry of what may be a pervasive process in ultramafic subseafloor environments. 相似文献
916.
Rosenbluh J Kapelnikov A Shalev DE Rusnati M Bugatti A Loyter A 《Analytical biochemistry》2006,352(2):157-168
Solid phase assay systems such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and overlay gels are used to study processes of protein-protein interactions. The common principle of all these methods is that they monitor the binding between soluble and surface-immobilized molecules. Following the use of bovine serum albumin (BSA)-peptide conjugates or isolated synthetic peptides and the above-mentioned solid phase assay systems, the results of the current work demonstrate that positively charged peptides can interact with each other. Both the ELISA and SPR methods demonstrated that the binding process reached saturation with K(d) values ranging between 1 and 14 nM. No interaction was observed between BSA conjugates bearing positively charged peptides and conjugates bearing negatively charged peptides or with pure BSA molecules, strengthening the view that interaction occurs only between positively charged peptides. However, interactions between peptides in solution were not observed by nuclear magnetic resonance (NMR) or by native gel electrophoresis. It appears that for positively charged molecules to interact, one of the binding partners must be immobilized to a surface, a process that may lead to the exposure of otherwise masked groups or atoms. We discuss the relevance of our findings for the use of solid phase assay systems to study interactions between biomolecules. 相似文献
917.
Yeargin SW Casa DJ McClung JM Knight JC Healey JC Goss PJ Harvard WR Hipp GR 《Journal of strength and conditioning research / National Strength & Conditioning Association》2006,20(2):383-389
The purpose was to assess whether body cooling between 2 bouts of exercise in the heat enhances performance during the second exercise session. Using a random, crossover design, 15 subjects (3 women, 12 men; 28 +/- 2 years, 180 +/- 2 cm, 69 +/- 2.3 kg) participated in all 3 trials. Subjects ran 90 minutes on hilly trails in a hot environment (approximately 27 degrees C) before 12 minutes of either cold water immersion (CWI; 13.98 degrees C), ice water immersion (IWI; 5.23 degrees C), or a mock treatment (MT) of sitting in a tub with no water (29.50 degrees C). After immersion, subjects ran a 2-mile race. CWI had faster (p < 0.05) performance time (725 seconds) than MT (769 seconds). CWI and IWI had significantly (p < 0.05) lower rectal temperatures postimmersion than MT as well as postrace (p < 0.05). Heart rate also remained significantly lower (p < 0.05) during the CWI and IWI trials for the first half of the race. In conclusion, CWI enhances performance (6% improvement in race time) in the second bout of exercise, supporting its potential role as an ergogenic aid in athletic performance. 相似文献
918.
Rossi AG Sawatzky DA Walker A Ward C Sheldrake TA Riley NA Caldicott A Martinez-Losa M Walker TR Duffin R Gray M Crescenzi E Martin MC Brady HJ Savill JS Dransfield I Haslett C 《Nature medicine》2006,12(9):1056-1064
Apoptosis is essential for clearance of potentially injurious inflammatory cells and subsequent efficient resolution of inflammation. Here we report that human neutrophils contain functionally active cyclin-dependent kinases (CDKs), and that structurally diverse CDK inhibitors induce caspase-dependent apoptosis and override powerful anti-apoptosis signals from survival factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that the CDK inhibitor R-roscovitine (Seliciclib or CYC202) markedly enhances resolution of established neutrophil-dependent inflammation in carrageenan-elicited acute pleurisy, bleomycin-induced lung injury, and passively induced arthritis in mice. In the pleurisy model, the caspase inhibitor zVAD-fmk prevents R-roscovitine-enhanced resolution of inflammation, indicating that this CDK inhibitor augments inflammatory cell apoptosis. We also provide evidence that R-roscovitine promotes apoptosis by reducing concentrations of the anti-apoptotic protein Mcl-1. Thus, CDK inhibitors enhance the resolution of established inflammation by promoting apoptosis of inflammatory cells, thereby demonstrating a hitherto unrecognized potential for the treatment of inflammatory disorders. 相似文献
919.
Di A Brown ME Deriy LV Li C Szeto FL Chen Y Huang P Tong J Naren AP Bindokas V Palfrey HC Nelson DJ 《Nature cell biology》2006,8(9):933-944
Acidification of phagosomes has been proposed to have a key role in the microbicidal function of phagocytes. Here, we show that in alveolar macrophages the cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) participates in phagosomal pH control and has bacterial killing capacity. Alveolar macrophages from Cftr-/- mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria. Lysosomes from CFTR-null macrophages failed to acidify, although they retained normal fusogenic capacity with nascent phagosomes. We hypothesize that CFTR contributes to lysosomal acidification and that in its absence phagolysosomes acidify poorly, thus providing an environment conducive to bacterial replication. 相似文献
920.
Avent ND Madgett TE Lee ZE Head DJ Maddocks DG Skinner LH 《Expert reviews in molecular medicine》2006,8(13):1-20
The Rhesus (Rh) blood group system is expressed by a pair of 12-transmembrane-domain-containing proteins, the RhCcEe and RhD proteins. RhCcEe and RhD associate as a Rh core complex that comprises one RhD/CcEe protein and most likely two Rh-associated glycoproteins (RhAG) as a trimer. All these Rh proteins are homologous and share this homology with two human non-erythroid proteins, RhBG and RhCG. All Rh protein superfamily members share homology and function in a similar manner to the Mep/Amt ammonium transporters, which are highly conserved in bacteria, plants and invertebrates. Significant advances have been made in our understanding of the structure and function of Rh proteins, as well as in the clinical management of Rh haemolytic disease. This review summarises our current knowledge concerning the molecular biology of Rh proteins and their role in transfusion and pregnancy incompatibility. 相似文献