Inhibition, by a protease inhibitor, of the solubilization of the F1-portion of the Mg2+-stimulated adenosine triphosphatase of Escherichia coli. (33/206)

The effects of two protease inhibitors on the solubilization of the membrane-bound Mg2+-adenosine triphosphatase (Mg-ATPase) of Escherichia coli were investigated. p-Aminobenzamidine prevented the solubilization of the Mg-ATPase during treatment of membranes with low-ionic-strength buffers containing ethylenediaminetetraacetic acid. p-Aminobenzamidine did not prevent subsequent solubilization of the Mg-ATPase by treatment of the membranes with chloroform. This method of solubilization yielded a preparation of similar apparent molecular weight but with a 10-fold-increased specific activity as compared with the Mg-ATPase solubilized by washing with low-ionic-strength buffer. However, in contrast to the latter preparation, the chloroform-solubilized Mg-ATPase did not reconstitute ATP-dependent energization of stripped membranes, which were prepared by low-ionic-strength washing in the absence of p-aminobenzamidine. Another protease inhibitor, epsilon-amino-n-caproic acid, did not effect the solubilization of the Mg-ATPase, but did inhibit the loss of activity occurring during concentration, by ultrafiltration, of the Mg-ATPase solublized by the low-ionic-strength treatment.  (+info)

Effects of epsilon-aminocaproiloaminoacids on the amidolytic activity of tissue plasminogen activator, urokinase and kallikrein. (34/206)

Effect of three epsilon-aminocaproylaminoacids with a significant antifibrinolytic activity on amidolytic activity of tissue plasminogen activator (t-PA), urokinase and kallikrein was examined. epsilon-Aminocaproyl-S-benzyl)-L-cysteine and epsilon-aminocaproyl-L-norleucine were weak inhibitors of kallikrein. Weak activation of t-PA activity was observed at high concentration of the tested compounds. Only one of the examined dipeptides was a weak inhibitor of amidolytic activity of urokinase.  (+info)

On the reduced intercellular adhesiveness of virally transformed BHK21 cells. (35/206)

Baby hamster kidney fibroblasts (BHK21 cells) transformed by polyoma or Rous sarcoma viruses aggregate less than the untransformed parental cells when incubated in growth medium in a gyratory shaker for 18-24 h. This difference can be measured by electronic particle counting, or by filtering aggregated suspensions of 32P-labelled cells through bolting fabric. The aggregation of transformed derivatives is not enhanced by the presence, during aggregation of epsilon-amino caproic acid, an inhibitor of plasmin activation. Some lines of transformed BHK21 cells do not appear less adhesive than untransformed cells in a short-term aggregation assay, and none adheres markedly less well when seeded onto homotypic cell sheets. The decreased aggregation of transformed cells is consistent with suggestions that LETS protein is involved in intercellular adhesion of fibroblasts as well as in attachment of cells to non-cellular substrates. If so, the short-term aggregation of freshly trypsinized cells may depend on secretion of LETS from an intracellular pool.  (+info)

Crystal structure of human arginase I at 1.29-A resolution and exploration of inhibition in the immune response. (36/206)

Human arginase I is a potential target for therapeutic intervention in diseases linked to compromised l-arginine homeostasis. Here, we report high-affinity binding of the reaction coordinate analogue inhibitors 2(S)-amino-6-boronohexanoic acid (ABH, Kd = 5 nM) and S-(2-boronoethyl)-l-cysteine (BEC, Kd = 270 nM) to human arginase I, and we report x-ray crystal structures of the respective enzyme-inhibitor complexes at 1.29- and 1.94-A resolution determined from crystals twinned by hemihedry. The ultrahigh-resolution structure of the human arginase I-ABH complex yields an unprecedented view of the binuclear manganese cluster and illuminates the structural basis for nanomolar affinity: bidentate inner-sphere boronate-manganese coordination interactions and fully saturated hydrogen bond networks with inhibitor alpha-amino and alpha-carboxylate groups. These interactions are therefore implicated in the stabilization of the transition state for l-arginine hydrolysis. Electron density maps also reveal that active-site residue H141 is protonated as the imidazolium cation. The location of H141 is such that it could function as a general acid to protonate the leaving amino group of l-ornithine during catalysis, and this is a revised mechanistic proposal for arginase. This work serves as a foundation for studying the structural and chemical biology of arginase I in the immune response, and we demonstrate the inhibition of arginase activity by ABH in human and murine myeloid cells.  (+info)

X-ray crystallographic analysis of 6-aminohexanoate-dimer hydrolase: molecular basis for the birth of a nylon oligomer-degrading enzyme. (37/206)

6-Aminohexanoate-dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII') that has only approximately 0.5% of the specific activity toward the 6-aminohexanoate-linear dimer, are encoded on plasmid pOAD2 of Arthrobacter sp. (formerly Flavobacterium sp.) KI72. Here, we report the three-dimensional structure of Hyb-24 (a hybrid between the EII and EII' proteins; EII'-level activity) by x-ray crystallography at 1.8 A resolution and refined to an R-factor and R-free of 18.5 and 20.3%, respectively. The fold adopted by the 392-amino acid polypeptide generated a two-domain structure that is similar to the folds of the penicillin-recognizing family of serine-reactive hydrolases, especially to those of d-alanyl-d-alanine-carboxypeptidase from Streptomyces and carboxylesterase from Burkholderia. Enzyme assay using purified enzymes revealed that EII and Hyb-24 possess hydrolytic activity for carboxyl esters with short acyl chains but no detectable activity for d-alanyl-d-alanine. In addition, on the basis of the spatial location and role of amino acid residues constituting the active sites of the nylon oligomer hydrolase, carboxylesterase, d-alanyl-d-alanine-peptidase, and beta-lactamases, we conclude that the nylon oligomer hydrolase utilizes nucleophilic Ser(112) as a common active site both for nylon oligomer-hydrolytic and esterolytic activities. However, it requires at least two additional amino acid residues (Asp(181) and Asn(266)) specific for nylon oligomer-hydrolytic activity. Here, we propose that amino acid replacements in the catalytic cleft of a preexisting esterase with the beta-lactamase fold resulted in the evolution of the nylon oligomer hydrolase.  (+info)

Isolation of a new antibiotic, alaremycin, structurally related to 5-aminolevulinic acid from Streptomyces sp. A012304. (38/206)

A new antibiotic, which is structurally related to 5-aminolevulinic acid, a precursor of heme biosynthesis, and named alaremycin, was isolated from the culture broth of an actinomycete strain through a random screening with the blue assay to detect the formation of anucleate cells in Escherichia coli. The producing strain was identified as Streptomyces sp. by morphological, physiological, chemical and genetic criteria. Alaremycin was purified from the culture supernatant by HP-20 hydrophobic-interaction chromatography, sequential solvent/water extraction in the acidic or alkaline pH range, and QMA cation-exchange chromatography. The chemical structure of alaremycin was determined as 5-acetamido-4-oxo-5-hexenoic acid by analyses of mass and NMR spectra. The antibacterial activity of alaremycin was enhanced in the presence of 5-aminolevulinic acid.  (+info)

The risk associated with aprotinin in cardiac surgery. (39/206)

BACKGROUND: The majority of patients undergoing surgical treatment for ST-elevation myocardial infarction receive antifibrinolytic therapy to limit blood loss. This approach appears counterintuitive to the accepted medical treatment of the same condition--namely, fibrinolysis to limit thrombosis. Despite this concern, no independent, large-scale safety assessment has been undertaken. METHODS: In this observational study involving 4374 patients undergoing revascularization, we prospectively assessed three agents (aprotinin [1295 patients], aminocaproic acid [883], and tranexamic acid [822]) as compared with no agent (1374 patients) with regard to serious outcomes by propensity and multivariable methods. (Although aprotinin is a serine protease inhibitor, here we use the term antifibrinolytic therapy to include all three agents.) RESULTS: In propensity-adjusted, multivariable logistic regression (C-index, 0.72), use of aprotinin was associated with a doubling in the risk of renal failure requiring dialysis among patients undergoing complex coronary-artery surgery (odds ratio, 2.59; 95 percent confidence interval, 1.36 to 4.95) or primary surgery (odds ratio, 2.34; 95 percent confidence interval, 1.27 to 4.31). Similarly, use of aprotinin in the latter group was associated with a 55 percent increase in the risk of myocardial infarction or heart failure (P<0.001) and a 181 percent increase in the risk of stroke or encephalopathy (P=0.001). Neither aminocaproic acid nor tranexamic acid was associated with an increased risk of renal, cardiac, or cerebral events. Adjustment according to propensity score for the use of any one of the three agents as compared with no agent yielded nearly identical findings. All the agents reduced blood loss. CONCLUSIONS: The association between aprotinin and serious end-organ damage indicates that continued use is not prudent. In contrast, the less expensive generic medications aminocaproic acid and tranexamic acid are safe alternatives.  (+info)

Cost-effectiveness of cell salvage and alternative methods of minimising perioperative allogeneic blood transfusion: a systematic review and economic model. (40/206)

OBJECTIVES: To compare patient outcomes, resource use and costs to the NHS and NHS Blood Transfusion Authority (BTA) associated with cell salvage and alternative methods of minimising perioperative allogeneic blood transfusion. DATA SOURCES: Electronic databases covering the period 1996-2004 for systematic reviews and 1994-2004 for economic evidence. REVIEW METHODS: Existing systematic reviews were updated with data from selected randomised controlled trials (RCTs) that involved adults scheduled for elective non-urgent surgery. Any resource use or cost data were extracted for potential use in populating an economic model. Relative risks or weighted mean difference of each outcome for each intervention were assessed, taking into account the number of RCTs included in each outcome and intervention and the presence of any heterogeneity. This allowed indirect comparison of the relative effectiveness of each intervention when the intervention is compared with allogeneic blood transfusion. A decision analytic model synthesised clinical and economic data from several sources, to estimate the relative cost-effectiveness of cell salvage for people undergoing elective surgery with moderate to major expected blood loss. The perspective of the NHS and patients and a time horizon of 1 month were used. The economic model was developed from reviews of effectiveness and cost-effectiveness and clinical experts. Secondary analysis explored the robustness of the results to changes in the timing and costs of cell salvage equipment, surgical procedure, use of transfusion protocols and time horizon of analysis. RESULTS: Overall, 668 studies were identified electronically for the update of the two systematic reviews. This included five RCTs, of which two were cell salvage and three preoperative autologous donation (PAD). Five published systematic reviews were identified for antifibrinolytics, fibrin sealants and restrictive transfusion triggers, PAD plus erythropoietin, erythropoietin alone and acute normovolaemic haemodilution (ANH). Twelve published studies reported full economic evaluations. All but two of the transfusion strategies significantly reduced exposure to allogeneic blood. The relative risk of exposure to allogeneic blood was 0.59 for the pooled trials of cell salvage (95% confidence interval: 0.48 to 0.73). This varied by the type and timing of cell salvage and type of surgical procedure. For cell salvage, the relative risk of allogeneic blood transfusion was higher in cardiac surgery than in orthopaedic surgery. Cell salvage had lower costs and slightly higher quality-adjusted life years compared with all of the alternative transfusion strategies except ANH. The likelihood that cell salvage is cost-effective compared with strategies other than ANH is over 50%. Most of the secondary analyses indicated similar results to the primary analysis. However, the primary and secondary analyses indicated that ANH may be more cost-effective than cell salvage. CONCLUSIONS: The available evidence indicates that cell salvage may be a cost-effective method to reduce exposure to allogeneic blood transfusion. However, ANH may be more cost-effective than cell salvage. The results of this analysis are subject to the low quality and reliability of the data used and the use of indirect comparisons. This may affect the reliability and robustness of the clinical and economic results. There is a need for further research that includes adequately powered high-quality RCTs to compare directly various blood transfusion strategies. These should include measures of health status, health-related quality of life and patient preferences for alternative transfusion strategies. Observational and tracking studies are needed to estimate reliably the incidence of adverse events and infections transmitted during blood transfusion and to identify the lifetime consequences of the serious hazards of transfusion on mortality, health status and health-related quality of life.  (+info)