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At least 955 records · Page 53Linked to original sources
PubMed2007

Evaluation of mannitol salt agar, CHROMagar Staph aureus and CHROMagar MRSA for detection of meticillin-resistant Staphylococcus aureus from nasal swab specimens.

Mannitol salt agar (MSA), CHROMagar Staph aureus (CSA) and CHROMagar MRSA (CSA-MRSA) were evaluated with nasal surveillance specimens for their ability to detect Staphylococcus aureus and meticillin-resistant S. aureus (MRSA). CSA was found to be more sensitive than MSA in detecting S. aureus (98 versus 84.3 %; P=0.03). CSA and CSA-MRSA were equivalent in the ability to detect MRSA at 24 h (89.7 versus 87.2 %) and at 48 h (94.9 versus 94.9 %). When combined with Staphaurex slide confirmation testing, both CSA and CSA-MRSA were highly specific (100 %) media for detecting MRSA from nasal swab specimens.

Agar↗
PubMed1998

Substrate specificity of barley cysteine endoproteases EP-A and EP-B.

The cysteine endoproteases (EP)-A and EP-B were purified from green barley (Hordeum vulgare L.) malt, and their identity was confirmed by N-terminal amino acid sequencing. EP-B cleavage sites in recombinant type-C hordein were determined by N-terminal amino acid sequencing of the cleavage products, and were used to design internally quenched, fluorogenic peptide substrates. Tetrapeptide substrates of the general formula 2-aminobenzoyl-P2-P1-P1'-P2'-tyrosine(NO2)-aspartic acid, in which cleavage occurs between P1 and P1', showed that the cysteine EPs preferred phenylalanine, leucine, or valine at P2. Arginine was preferred to glutamine at P1, whereas proline at P2, P1, or P1' greatly reduced substrate kinetic specificity. Enzyme cleavage of C hordein was mainly determined by the primary sequence at the cleavage site, because elongation of substrates, based on the C hordein sequence, did not make them more suitable substrates. Site-directed mutagenesis of C hordein, in which serine or proline replaced leucine, destroyed primary cleavage sites. EP-A and EP-B were both more active than papain, mostly because of their much lower Km values.

Amino Acid Sequence↗
PubMed1997

Regulation of pigmentation in cultured skin substitutes by cytometric sorting of melanocytes and keratinocytes.

Unpredictable pigmentation in cultured skin substitutes (CSS) is an anatomic deficiency after wound treatment and can require years to normalize. Variable numbers of human melanocytes (HM) survive in cultures of human keratinocytes (HK) as demonstrated by focal areas of pigmentation in CSS after healing. The purposes of this study were to deplete HM from HK cultures and to regulate the numbers of HM contained in CSS. A highly pigmented HM cell strain was chosen for these studies to emphasize the differences in light scattering between HK and HM by flow cytometry. Cytometric gates were set with selective cultures of HM and HK and were used to sort a mixed population of HK + 4% HM. After sorting, CSS were prepared from human fibroblasts attached to collagen-glycosaminoglycan sponges combined with cells from the HK + 4% HM (pre-treatment control), the sorted HK (experimental), or sorted HK + 3% HM (post-treatment positive control) subpopulations and grafted to athymic mice. Grafted wounds were assessed for 6 wk by planimetry for area of pigment and by a Minolta Chromameter for color density and hue in situ. Histology and staining of HLA-ABC were performed at 6 wk. Data from percent pigmented area and chromameter measurements identified quantitative and statistically significant decreases in color of healed skin after flow cytometric separation of HK and HM. Therefore, a purified HK subpopulation depleted of HM was isolated by flow cytometry that generated healed skin with reduced pigmentation. These results suggest that HM can be selectively depleted from HK cultures and then added to cultured skin substitutes at specific densities to generate predictable pigmentation for improved function and cosmesis in healed wounds.

Animals↗
PubMed1995

Purification and characterization of a fibrinogen-degrading protease in Bacteroides fragilis strain YCH46.

A novel fibrinogenolytic protease was purified from Bacteroides fragilis strain YCH46. The protease was extracted from cells by ultrasonic treatment and was purified 425-fold with a recovery of 2.1% by sequential procedures using azocasein as a substrate. The purified protease showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an estimated molecular weight of 100 kDa, which was consistent with the value obtained by gel filtration, indicating a monomeric native structure. Its optimal pH, Km, and Vmax for azocasein were 7.5, 0.2%, and 286 U/min/mg, respectively. The protease activity was completely inhibited by addition of 1 mM Hg2+, Cu2+, Zn2+, diisopropyl fluorophosphate, N-ethylmaleimide or p-chloromercuribenzoate but not by the inhibitors of metalloprotease or aspartic protease, suggesting that the enzyme is a serine-thiol-like protease. The protease hydrolyzed azocasein, casein, fibrinogen, gelatin, and azocoll, but not bovine serum albumin, ovalbumin, fibrin, fibronectin, immunoglobulins, transferrin, hemoglobin or types I, III, and IV collagen. The enzyme also hydrolyzed the chromogenic substrates alanyl-alanine p-nitroanilide, L-valyl-alanine p-nitroanilide, alanyl-alanyl-valyl-alanine p-nitroanilide, and glycyl-proline p-nitroanilide, but was inert toward L-alanine p-nitroanilide, alanyl-alanyl-alanine p-nitroanilide, and N-alpha-benzoyl-DL-arginine p-nitroanilide. The protease completely hydrolyzed the alpha-chain of fibrinogen at 37 C within 10 hr and at the same time the time required for clotting of protease-treated fibrinogen by thrombin was prolonged. The fibrinogenolytic activity of a crude extract of B. fragilis was stronger than that of other species of the Bacteroides fragilis group tested: B. ovatus, B. distasonis, B. eggerthii, B. uniformis, and B. thetaiotaomicron. These results suggest that the fibrinogenolytic protease is an important biological factor in Bacteroides infection.

Amino Acid Sequence↗
PubMed1998

Studies of Escherichia coli cultured on Rainbow Agar O157 with particular reference to enterohaemorrhagic Escherichia coli (EHEC).

Rainbow Agar O157 is designed for the rapid isolation and identification of enterohaemorrhagic Escherichia coli (EHEC), particularly O157, characterised by black colonies. Five-hundred-eighty-five E. coli strains, including O157, O111 and O113 serogroups from many sources were examined on Rainbow Agar O157. EHEC O157 could readily be isolated and recognized uniquely by typical black colonies. Some other EHEC also stand out as blue-black, whereas O113 and some other EHEC strains were mauve, red or pink and indistinguishable from SLT-negative strains of E. coli.

Agar↗
PubMed2004

Quantum dots targeted to the assigned organelle in living cells.

Fluorescent nanocrystal quantum dots (QDs) have the potential to be applied to bioimaging since QDs emit higher and far longer fluorescence than conventional organic probes. Here we show that QDs conjugated with signal peptide obey the order to transport the assigned organelle in living cells. We designed the supermolecule of luminescent QDs conjugated with nuclear- and mitochondria-targeting ligands. When QDs with nuclear-localizing signal peptides were added to the culture media, we can visualize the movements of the QDs being delivered into the nuclear compartment of the cells with 15 min incubation. In addition, mitochondrial signal peptide can also transport QDs to the mitochondria in living cells. In conclusion, these techniques have the possibility that QDs can reveal the transduction of proteins and peptides into specific subcellular compartments as a powerful tool for studying intracellular analysis in vitro and even in vivo.

Amino Acid Sequence↗
PubMed1980

Immunochemical analysis of active and inactive antithrombin III.

Antithrombin III (AT) levels from normal and AT deficiency persons were measured by electroimmunoassay (EIA) and the results compared with a chromogenic assay (S2238). Discrepant results were obtained when plasma and serum were compared using one antiserum, and therefore did not always relate to functional activity. Another antiserum, however, when used was capable of differentiating active AT from inactive AT complexed with its proteases and demonstrated close correlation with all samples tested (r=0.97). The specificity of the antisera and consequent anomalous results were elucidated when purified human thrombin was added to plasma samples and subsequently reanalysed. Quantitative differences observed when serum samples were compared by single radial immunodiffusion and electroimmunoassay with one antiserum, illustrates the fundamental principle differences between the two methods. These results give some insight as to why previous anomalies with AT immunoassays have occurred. They also indicate that plasma and not serum should be used as clinical test material. AT antisera should be capable of recognizing and distinguishing free AT from AT/protease complexes if the results obtained by electroimmunoassay are to correlate with functional activity.

Antithrombin III↗
PubMed1981

Comparative studies on human activators of plasminogen.

Agents activating human plasminogen have been prepared from a variety of human sources, namely plasma, blood vessel endothelium and tissue. Their physical and functional properties have been found to differ from that of the urinary activator, urokinase. Antiserum to urokinase, while it quenched the activity of urokinase itself, had no effect on the activity of the plasma, endothelial or tissue activators.

Antifibrinolytic Agents↗
PubMed1981

An evaluation of chromogenic substrates in the control of oral anticoagulant therapy.

A comparison has been made between the prothrombin time test using British Comparative Thromboplastin (BCT) and four modified amidolytic assays in the assessment of laboratory control of oral anticoagulant administration in short-term and long-term patients. The results were also correlated with the APTT and specific assays for factors VII and X based on established coagulation techniques. The chromogenic substrate assays did not give as close agreement with the BCT as the clotting assays for factors VII and X. A combination of the amidolytic factor II and X results did not improve the correlation. Agreement with the BCT was better in long-term patients with the substrates but there was no obvious advantage between the factor II and X amidolytic methods in this group. The development of a specific amidolytic assay for factor VII should be considered based on the evidence of this study although its reliability for oral anticoagulant control would need to be validated by clinical trial. In the interim, it may be anticipated that thromboplastins showing less sensitivity than BCT to factor VII would give a better correlation with the amidolytic assays.

Acenocoumarol↗
PubMed1990

Revised recommendations for the measurements of the serum iron in human blood. Iron Panel of the International Committee for Standardization in Haematology.

Certain modifications to the previously published ICSH recommendations (1978) have been approved by the Iron Panel of the International Committee for Standardization in Haematology (ICSH). They include substitution of the chromogen bathophenanthroline sulphonate with ferrozine or ferene, which are more sensitive and cheaper, and a reduction in the volume of the test sample from 2 ml to 0.5 ml.

Blood Chemical Analysis↗
PubMed1993

The behaviour of different factor VIII concentrates in a chromogenic factor X-activating system.

A chromogenic factor Xa generation method has been developed for comparing the co-factor activity of factor VIII concentrates at physiological factor VIII concentrations (1 iu/ml). In the presence of thrombin all concentrates gave similar rapid rates of factor Xa generation, but in the absence of thrombin there were major differences between the rates of Xa generation between different products. High purity products, particularly those prepared by monoclonal antibody purification from plasma and recombinant sources, gave more rapid Xa generation than most intermediate-purity products. There was a very strong correlation between the rate of Xa generation and the difference in factor VIII potency by one-stage and two-stage assays. These results suggest the possible presence of small amounts of activated factor VIII in some concentrates, but differences in von Willebrand factor content could also contribute towards the different rates of factor Xa generation observed.

Blood Chemical Analysis↗
PubMed1995

A chromogenic assay for activated protein C resistance.

Resistance to activated protein C (APC) diagnosed on the basis of prolongation of clotting time in an activated partial thromboplastin time (aPTT) assay is now considered a major cause of inherited thrombophilia. The majority of patients with APC resistance carry a factor V molecule with a point mutation at one APC cleavage site (Arg506Gln) which prevents the optimal inactivation of activated factor V by APC. To overcome the limitations of aPTT-based assays in the diagnosis of APC resistance, we have developed a chromogenic assay which is based on the capacity of APC to limit the generation of factor Xa by inactivating factor VIIIa in plasma. The ratio of the factor Xa amidolytic activity in a sample without APC to its factor Xa activity with the addition of APC reflects the response of the plasma coagulation system to APC. The normal range in 44 healthy individuals was 1.62-2.06. APC response ratios as measured by the chromogenic assay correlated with ratios measured by the aPTT assay and were below the normal range in 23/24 individuals with Arg506Gln mutant factor V from three different families with familial thrombosis and from 11 unrelated asymptomatic individuals. In reconstitution experiments, purified factor V corrected the decreased APC response in plasma samples from patients with the Arg506Gln mutation as well as with factor V deficiency, and increased the APC response in normal plasma, whereas the addition of activated factor V had no enhancing effect.

Anticoagulants↗
PubMed2007

In vitro kinetics of factor VIII activity in patients with mild haemophilia A and a discrepancy between one-stage and two-stage factor VIII assay results.

In some mild haemophilia A patients (discrepant haemophilia), factor VIII coagulant activity (FVIII:C) levels, by one-stage assay are more than double than those by two-stage assay. This may be due to the longer incubation times (10-12 min) in the two-stage assay. This study aimed to determine the time course of the activation phase of the two-stage assay, using both classical coagulation and chromogenic detection methods. In both systems, for equivalent patients (equivalent FVIII:C levels by one-stage and two-stage assays, n = 6, all different mutations), similar FVIII:C results were obtained with short- or long-incubation times. In contrast, plasma from discrepant patients (n = 8, five different mutations) showed higher FVIII:C at shorter incubation times than after longer incubation times. In the chromogenic assay, FVIII:C levels were higher after incubation for 2 min (23-56%, mean 41%) than after 10 min (19-41%, mean 29%). In the classical coagulation assay, FVIII:C levels were higher at shorter incubation times (21-64%, mean 37%) than with the longer incubation times usually used (13-29%, mean 23%). These time-course experiments have verified that the longer incubation time used in the two-stage assay is at least partly responsible for the lower FVIII:C measured by that assay in discrepant haemophilia.

Blood Coagulation Tests↗
PubMed1982

Oral anticoagulant control with a chromogenic substrate: calibration, precision and cost.

Prothrombin time ratios, and bioassays and chromogenic (S2222) assays for factors VII and X, were obtained on 23 newly anticoagulated patients and 23 patients on long term oral anticoagulants. Factor levels were plotted against prothrombin time ratios. For factor VII, a different relationship was apparent for newly anticoagulated and for stabilized patients by both assay methods, but for factor X the same relationship was found. Chromogenic factor-X levels were calibrated against prothrombin time, and a prescribing exercise showed reasonable agreement between doses based on prothrombin time ratios and on assays for both clinical groups; factor-VII levels could not be used because of the different relationship between newly anti-coagulated and long-term patients. Despite the high precision of reading the chromogenic end point, chromogenic assays were not more precise than bioassays. With S2222, the cost of automated anticoagulant control would be more expensive than control by manual prothrombin times.

Administration, Oral↗
PubMed1983

Monitoring heparin therapy--a role for the chromogenic assay.

Heparin therapy was evaluated by simultaneous determinations of the activated partial thromboplastin time (APTT), and plasma heparin levels by chromogenic assay. These tests showed good correlation (r = 0.73) in patients on heparin alone. However, in the presence of concurrent warfarin administration, r fell to 0.37, reflecting an additional warfarin effect upon the APTT. Furthermore, the studies confirmed the increased heparin requirement in patients with pulmonary embolization.

Biological Assay↗
PubMed1983

A comparison of antithrombin III procedures.

Antithrombin III (AT III) is the most potent physiologic inactivator of thrombin and other serine proteases in the blood clotting mechanism. Hereditary deficiency of this protein is associated with recurrent deep-vein thrombosis that begins in late adolescence. Untreated, this disease may lead to early death from recurrent and massive pulmonary emboli. Attempts to identify groups of patients who are the most likely to develop thromboembolic disease because of an acquired deficiency of AT III have been frustrated by the lack of standardization of the assays and the inability to compare results of the different AT III assays. The functional assays and immunoelectrophoretic determinations do not measure the same component. In order to compare the ability of current AT III procedures to determine levels of AT III in various disease states, we used immunoelectrophoretic, chromogenic, and clottable assays to measure the AT III of patients with congenital AT III deficiency and of patients with possible acquired AT III deficiency.

Antithrombin III↗