Vancomycin-induced deletion of the methicillin resistance gene mecA in Staphylococcus aureus.
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OBJECTIVE: To elucidate factors that contribute to the development of vancomycin resistance in methicillin-resistant Staphylococcus aureus (MRSA). METHODS: Forty-nine MRSA isolates were subjected to passage selection with vancomycin to isolate mutants with reduced susceptibility to vancomycin. One mutant was chosen for detailed molecular and biochemical characterization. RESULTS: Five vancomycin-resistant mutants (vancomycin MICs, 6-12 mg/L) were obtained in vitro from five MRSA parent isolates. Upon acquisition of vancomycin resistance, all mutants showed a concomitant decrease in oxacillin resistance. In one particular MRSA strain, selection for vancomycin resistance repeatedly produced deletions and rearrangements, including loss of the mecA gene. Pleiotropic phenotypical changes, such as yellow pigment formation, loss of haemolysis, thickened cell wall, increased resistance to lysostaphin and reduced cell wall turnover were observed in this mutant. CONCLUSION: Acquisition of vancomycin resistance in one MRSA strain triggered mecA deletion suggesting that this deletion, coupled to other rearrangements and/or mutations, may be responsible for the increased vancomycin resistance phenotype. (+info)
Effect of phosphatidylinositol and inside-out erythrocyte vesicles on autolysis of mu- and m-calpain from bovine skeletal muscle.
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The finding that phospholipid micelles lowered the Ca2+ concentration required for autolysis of the calpains led to a hypothesis suggesting that the calpains are translocated to the plasma membrane where they interact with phospholipids to initiate their autolysis. However, the effect of plasma membranes themselves on the Ca2+ concentration required for calpain autolysis has never been reported. Also, if interaction with a membrane lowers the Ca2+ required for autolysis, the membrane-bound-calpain must autolyze itself, because it would be the only calpain having the reduced Ca2+ requirement. This implies that the autolysis is an intramolecular process, although several studies have shown that autolysis of the calpains in an in vitro assay and in the absence of phospholipid is an intermolecular process. Inside-out vesicles prepared from erythrocytes had no effect on the Ca2+ concentration required for autolysis of either mu- or m-calpain, although phosphatidylinositol (PI) decreased the Ca2+ concentration required for autolysis of the same calpains. The presence of a substrate for the calpains, beta-casein, reduced the rate of autolysis of both mu- and m-calpain both in the presence and in the absence of PI, suggesting that mu- and m-calpain autolysis is an intermolecular process in the presence of PI just as it is in its absence. Because IOV have no effect on the Ca2+ concentration required for calpain autolysis, association with the plasma membrane, at least with erythrocyte plasma membranes, does not initiate calpain autolysis by reducing the Ca2+ concentration required for autolysis as suggested by the membrane-activation hypothesis. Interaction with a membrane may serve to bind calpains to their substrates rather than promoting autolysis. (+info)
Revising the role of the pneumococcal vex-vncRS locus in vancomycin tolerance.
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Vancomycin is used increasingly to treat invasive infections caused by multidrug-resistant Streptococcus pneumoniae. Although no vancomycin-resistant strains have been isolated to date, tolerant strains that fail to die rapidly and that cause relapsing disease have been described. The vex123-pep27-vncRS locus, consisting of an ABC transporter, a presumed signaling peptide, and a two-component system, respectively, has been implicated in vancomycin tolerance. Recent findings, however, challenged this model. The data presented here indicate that erythromycin in the growth medium induces a vancomycin-tolerant phenotype and that loss of function of Pep27 or VncRS does not alter autolysis. However, a role for the ABC transporter encoded by the vex123 genes in tolerance was confirmed. A vex3 mutant was considerably more tolerant to vancomycin treatment than the wild-type strain T4, and the strength of the phenotype depended on the orientation of the resistance cassette used to construct the mutant. Microarray results suggested a number of genes that might be involved in tolerance in the vex3 mutant. Although the exact function and regulation of the vex123-pep27-vncRS locus remains to be determined, several factors influence the autolysis behavior of S. pneumoniae, including the bacterial capsule, erythromycin, and the lytA and vex3 gene products. (+info)
A comparison of rapid bovine spongiform encephalopathy testing methods on autolyzed bovine brain tissue.
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In 1999, the European Union (EU) approved 3 rapid methods for the testing of bovine brain samples for the presence of bovine spongiform encephalopathy (BSE). The evaluation that led to the approval did not include an analysis of autolyzed material. Member states of the EU have active surveillance programs for BSE, which target fallen stock as well as other categories of cattle. Autolysis is a common feature of fallen stock samples because there can be a considerable delay between death and collection of samples. Therefore, it is important to know whether these tests perform optimally on autolyzed samples. The Veterinary Laboratories Agency (VLA) selected 250 positive fallen stock samples. These had been detected during routine testing using the Prionics-Check Western blot and confirmed as BSE cases by immunohistochemistry or electron microscopy. Samples were graded according to the degree of autolysis and then tested by the 3 methods: Prionics-Check Western blot, Platelia test, and Enfer test. All 3 methods correctly classified the samples as positive BSE cases, therefore alleviating doubt about their ability to do so. Subsequent EU validation exercises, such as those conducted in 2002--2003, have included the testing of autolyzed material. It is important that all new methods be evaluated on autolyzed tissue before approval for official use. (+info)
mu-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans.
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mu-Calpain and calpain-3 are Ca2+-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that mu-calpain becomes proteolytically active in the presence of 2-200 microM Ca2+. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca2+ concentration levels ([Ca2+]i; approximately 1 microM) than the levels at which mu-calpain activation occurs. We have demonstrated the Ca2+-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca2+] levels (0, 2.5, 10, and 25 microM). Autolysis of calpain-3 was found to occur across a [Ca2+] range similar to that for mu-calpain, and both calpains displayed a seemingly higher Ca2+ sensitivity in human than in rat muscle homogenates, with approximately 15% autolysis observed after 1-min exposure to 2.5 microM Ca2+ in human muscle and almost none after 1- to 2-min exposure to the same [Ca2+]i level in rat muscle. During muscle activity, [Ca2+]i may transiently peak in the range found to autolyze mu-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s "all-out" cycling, n = 8; and 70% VO2 peak until fatigue, n = 3) on the amount of autolyzed mu-calpain or calpain-3 in human muscle. No significant autolysis of mu-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca2+]i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo. (+info)
The functional significance of the autolysis loop in protein C and activated protein C.
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The autolysis loop of activated protein C (APC) is five residues longer than the autolysis loop of other vitamin K-dependent coagulation proteases. To investigate the role of this loop in the zymogenic and anticoagulant properties of the molecule, a protein C mutant was constructed in which the autolysis loop of the protein was replaced with the corresponding loop of factor X. The protein C mutant was activated by thrombin with approximately 5-fold higher rate in the presence of Ca2+. Both kinetics and direct binding studies revealed that the Ca2+ affinity of the mutant has been impaired approximately 3-fold. The result of a factor Va degradation assay revealed that the anticoagulant function of the mutant has been improved 4-5-fold in the absence but not in the presence of protein S. The improvement was due to a better recognition of both the P1-Arg506 and P1-Arg306 cleavage sites by the mutant protease. However, the plasma half-life of the mutant was markedly shortened due to faster inactivation by plasma serpins. These results suggest that the autolysis loop of protein C is critical for the Ca(2+)-dependence of activation by thrombin. Moreover, a longer autolysis loop in APC is not optimal for interaction with factor Va in the absence of protein S, but it contributes to the lack of serpin reactivity and longer half-life of the protease in plasma. (+info)
Analysis of rugae in burn victims and cadavers to simulate rugae identification in cases of incineration and decomposition.
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The most challenging situations in Forensic Odonto-Stomatology are mass disasters, where the forensic dentist is usually confronted with charred human remains or heavily decomposed or fragmented bodies. This article determines the extent of preservation of palatal rugae for use as an alternative identification tool in such situations, using a study group comprising burn victims and cadavers simulating forensic cases of incineration and decomposition. The thermal effects and the decomposition changes on the palatal rugae of burn victims with panfacial third degree burns and human cadavers in storage were respectively assessed and graded on a new scale. Ninety three percent of burn victims and 77% of human cadavers had Grade 0 changes (normal). When changes were noted, they were less pronounced than the generalized body involvement of burns in burn victims and the generalized body decomposition of human cadavers. (+info)
Functional analysis of the YvrGHb two-component system of Bacillus subtilis: identification of the regulated genes by DNA microarray and northern blot analyses.
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YvrGHb is a novel two-component system in Bacillus subtilis. The yvrG and yvrHb genes are considered to encode sensor kinase and response regulator respectively. We found that the YvrGHb system positively regulates the 7 transcriptional units (wprA, wapA-yxxG, dltABCDE, sunA, sunT-bdbA-yolJ-bdbB, yvrI-yvrHa, and sigX-rsiX), and negatively regulates the lytABC operon. wprA, wapA, lytB, and lytC encode the main cell surface proteins of B. subtilis. Furthermore, SigX [extracytoplasmic function sigma factor (ECF sigma factor)] relating to the cell surface homeostatic functions, was regulated by this system. The yvrGHb null mutant showed the unusual autolysis and higher susceptibility to the four kinds of antibiotics (aztreonam, cefepime, bacitracin, and fosfomycin). These results indicate that the YvrGHb system is important to maintain the overall state of the cell surface through transcriptional regulation. (+info)