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PubMed1997

Kinetics of reciprocal pro-urokinase/plasminogen activation--stimulation by a template formed by the urokinase receptor bound to poly(D-lysine).

The two zymogens, plasminogen and pro-urokinase plasminogen activator (pro-uPA), constitute a system of reciprocal activation, since plasmin, generated by uPA-catalysed plasminogen activation, can activate pro-uPA to uPA. Two such zymogens, when mixed, will undergo autocatalytic, reciprocal activation resulting in generation of proteolytic activity. As an example of reciprocal zymogen activation, the plasminogen/pro-uPA system was analysed in terms of a kinetic model which describes the progression in activated enzymes. This model gave a detailed description of the progress curves in plasmin and uPA. It accounted for the effects of varying the concentration of the zymogens, and also for the effects of plasmin substrates and inhibitors in the reaction mixture. The model assumes non-significant zymogen activity. It did not, however, exclude that a very low initial proteolytic activity, accounting for maximally 0.01% of that obtained when pro-uPA is fully activated, could be attributed to a genuine pro-uPA activity. Binding of the uPA receptor (uPAR) to pro-uPA/uPA might affect separate steps of the reciprocal activation reaction, or it might induce a significant pro-uPA activity. To distinguish between these possibilities the effect of a recombinant soluble (residues 1-277) form of uPAR, uPAR-(1-277)-peptide, on reciprocal pro-uPA/plasminogen activation was studied. uPAR-(1-277)-peptide attenuated reciprocal zymogen activation, and the results suggested that this was due to a decreased accessibility of the pro-uPA/uPAR-(1-277)-peptide complex to activation by plasmin. The uPAR-(1-277)-peptide in the presence of poly(D-lysine) caused a 20-fold enhancement of reciprocal zymogen activation. Kinetic analysis of separate activation steps revealed that this was due to a threefold stimulation of plasminogen activation by uPA/uPAR-(1-277)-peptide combined with a sixfold stimulation of plasmin's activation of pro-uPA/uPAR-(1-277)-peptide. The results suggested that poly(D-lysine) provided a template for a catalytically favourable interaction between plasminogen/plasmin and the uPAR-(1-277)-peptide complex with pro-uPA/uPA. There was no indication of a significant uPAR-(1-277)-peptide-induced enhancement of pro-uPA activity.

Animals↗
PubMed2005

Comparison of standard phenotypic assays with a PCR method to discriminate Candida albicans and C. dubliniensis.

In 1995, Candida dubliniensis was described as a new species in the genus Candida. Its close relationship to C. albicans has proved problematic in the identification of C. dubliniensis in clinical specimens. The objective of this study was to determine if reproducible differentiation between both species can be obtained by phenotypic assays. Therefore, 100 strains from 86 patients with the ability to produce chlamydospores were examined with different methods including API ID 32 C, colour development on CHROMagar, chlamydospore formation on Staib agar, growth at different temperatures and germ tube formation at 39 degrees C. Additionally, polymerase chain reaction (PCR) was used as gold standard. Six of the investigated strains were C. dubliniensis. The results suggest that there is still no single phenotypic method satisfactory to distinguish between C. albicans and C. dubliniensis.

Agar↗
PubMed2006

Clinical and experimental evaluation of a new chromogenic medium (OCCA, Oxoid) for direct identification of Candida albicans, C. tropicalis and C. krusei.

Oxoid Chromogenic Candida Agar (OCCA) is a new commercial ready-to-use medium that contains chromogenic substrates for rapid detection and specific identification of Candida albicans, C. tropicalis and C. krusei. We evaluated the performance of this medium with 364 clinical specimens and 31 subcultures, and examined its ability to support the growth of small inocula of six reference strains. CHROMagar Candida was used as the reference medium. OCCA permitted the growth of most important yeasts, and readily discriminated among Candida spp. in clinical specimens, including mixed cultures.

Candida↗
PubMedDate not supplied

Endotoxaemia in patients with liver cirrhosis and upper gastrointestinal bleeding: detection by the chromogenic assay with plasma Tween 80 pretreatment.

A recently developed chromogenic endotoxin assay with plasma Tween 80 pretreatment was compared with the conventional dilution and heating method. Plasma endotoxin was measured in patients with liver cirrhosis and upper gastrointestinal (GI) bleeding by these methods. Plasma endotoxin concentration was calculated from an individual internal standard curve by adding three different standard endotoxin solutions to each sample. By the conventional heating method, added standard endotoxin gave different OD values in each sample and the slope of the standard curve showed interindividual variations. When sample plasma from chronic alcoholics was pretreated with 1% Tween 80 and ultrasonification after heating, the slope of standard curves was somewhat increased and interindividual variation was minimized. Significantly higher plasma endotoxin levels in cirrhotics with upper GI bleeding compared with those without upper GI bleeding was detected by this Tween 80 method. There was a strongly positive correlation between the endotoxin levels determined by this method and those determined by the perchloric acid method and endotoxin-specific substrate in patients with upper GI bleeding. Endotoxin levels, which were elevated 1-2 days after the bleeding, tended to decrease as patients recovered. In summary, the recovery of endogenous and exogenous endotoxin from plasma sample was increased by adding Tween 80 before the chromogenic substrate assay. Transient elevation of plasma endotoxin was demonstrated by this Tween 80 method in patients with liver cirrhosis and upper GI bleeding.

Adult↗
PubMedDate not supplied

Plasma endotoxin concentration and endotoxin binding capacity of plasma acute phase proteins in cirrhotics with variceal bleeding: an analysis by new methods.

Plasma endotoxin levels in 12 cirrhotics with bleeding from oesophageal varices and 50 cirrhotics without bleeding were measured by the chromogenic assay after the pretreatment of sample by perchloric acid (HClO4) and triethylamine. Endotoxin in cirrhotics with bleeding from varices was significantly higher than those without bleeding. In patients with bleeding, endotoxin increased for 3 days after the bleeding, first in the supernatant fraction and then in the precipitate fraction by HClO4 treatment. Peak plasma alpha 1-acid glycoprotein and haptoglobin were observed 3 days after the bleeding. Alpha 1-antitrypsin gradually increased for 14 days. Transferrin did not markedly change. The endotoxin-binding capacity of transferrin and alpha 1-acid glycoprotein increased immediately after bleeding and thereafter decreased, but that of alpha 1-antitrypsin tended to increase in the recovery period. In summary, the plasma endotoxin concentration and endotoxin-binding capacity of alpha 1-acid glycoprotein and transferrin were shown to have increased after bleeding from varices by this new method. There may be a close relationship between endotoxaemia and acute phase reaction in this situation.

Acute-Phase Proteins↗
PubMed1993

Glutamate-like immunoreactivity in retinal terminals of the mouse suprachiasmatic nucleus.

With a view to identifying the neurotransmitter content of retinal terminals within the mouse suprachiasmatic nucleus, a highly specific antiserum to glutaraldehyde-coupled glutamate was used in a postembedding immunogold procedure at the ultrastructural level. Retinal terminals were identified by cholera toxin--horseradish peroxidase transported anterogradely from the retina and reacted with tetramethyl benzidine/tungstate/H2O2, or by their characteristically pale and distended mitochondria with irregular cristae. Controls included model ultrathin sections containing high concentrations of various amino acids. Alternate serial sections were labelled with anti-glutamate and anti-gamma-aminobutyric acid (GABA). Data were analysed by computer-assisted image analysis. Density of glutamate labelling (gold particles per micron2) on whole retinal terminals was > 3 times higher than that on postsynaptic dendrites, and > 5 times higher than that on miscellaneous non-retinal non-glutamatergic terminals in the suprachiasmatic nucleus. The overall density of gold particles over retinal terminals was approximately 3 times higher than that over GABAergic terminals, in which glutamate-like immunoreactivity was mainly mitochondrial. Labelling of vesicles in retinal terminals was almost 5 times greater than the apparent labelling of vesicles in GABAergic terminals, underscoring the location of transmitter glutamate within synaptic vesicles in retinal terminals. In the retino-recipient region of the suprachiasmatic nucleus there was also a small population of non-retinal glutamatergic terminals. Their overall immunoreactivity was similar to or exceeded that of retinal terminals, but morphological features clearly distinguished between these two types of glutamate-containing terminals. The present results indicate that the vast majority of retinal terminals may use glutamate as a transmitter, in keeping with electrophysiological and neuropharmacological data from other sources. The possibility of cotransmitters within retinal terminals, suggested by the presence of dense-core vesicles among the glutamate-containing synaptic vesicles, has still to be addressed.

Animals↗
PubMed2006

Evaluation of three chromogenic media (MRSA-ID, MRSA-Select and CHROMagar MRSA) and ORSAB for surveillance cultures of methicillin-resistant Staphylococcus aureus.

Screening specimens were homogenised in saline 0.9% w/v before either direct inoculation or following enrichment in broth on three chromogenic media (MRSA-ID, CHROMagar MRSA and MRSA Select) and ORSAB medium for the detection of methicillin-resistant Staphylococcus aureus (MRSA). In total, 102 of 466 specimens yielded MRSA on at least one medium. After incubation for 16-18 h, the sensitivity was 51%, 59%, 47% and 65% on MRSA-ID, CHROMagar MRSA, ORSAB and MRSA Select, respectively, compared with 82%, 75%, 67% and 80%, respectively, after 42 h, and 93%, 95%, 79% and not tested, respectively, following broth enrichment. There were significantly more MRSA colonies on MRSA-Select after 16-18 h than on ORSAB or MRSA ID (p 0.001 and 0.0022, respectively), whereas there were more MRSA colonies after 42 h on MRSA-ID and MRSA-Select than on ORSAB (p 0.0004 and 0.012, respectively). The specificity of the media for identifying MRSA based on the colour of colonies after incubation for 16-18 h was 100%, 99%, 99% and 100%, respectively, compared with 98%, 97%, 98% and 98%, respectively, after 42 h, and 100%, 99%, 100% and not tested, respectively, following broth enrichment. The speed of detection (mean time to report a positive result) was 1.65, 1.72, 2.31 and 1.35 days, respectively. For each of the three media tested following enrichment, the use of an enrichment broth increased the detection rate of MRSA by 16-24%.

Chromogenic Compounds↗
PubMed1994

Rapid identification of pathogenic neisserias using the Identicult-Neisseria test.

A rapid enzymatic method using chromogenic substrates for the rapid identification of pathogenic neisseria (Identicult-Neisseria, Scott Laboratories Inc., CA, USA) was tested in parallel with the rapid carbohydrate utilization test (RCUT) and the Phadebact Monoclonal GC Test against 198 consecutive clinical isolates of oxidase-positive Gram-negative diplococci (118 Neisseria gonorrhoeae, 76 N. meningitidis and four N. lactamica). On initial testing the Identicult-Neisseria gave a 95% overall concordance (97.5% N. gonorrhoeae, 90.8% N. meningitidis) with the RCUT and Phadebact tests; the corresponding figures after repeat testing were 98% overall concordance (98.3% N. gonorrhoeae, 97.4% N. meningitidis). Two of the three strains of N. gonorrhoeae mis-identified as N. meningitidis on primary testing were also mis-identified on repeat testing. Seven strains of N. meningitidis were mis-identified on initial testing (six as Moraxella catarrhalis and one as N. lactamica) and two on repeat testing (both as Mor. catarrhalis). We conclude that the Identicult-Neisseria is not sufficiently reliable for the culture confirmation of gonococci and meningococci.

Agglutination Tests↗
PubMed1995

Evaluation of a new chromogenic agar medium for the identification of urinary tract pathogens.

This study evaluated the performance of CPS ID2 (bioMérieux) compared to that of conventionally used selective agar for the identification of bacteria responsible for urinary tract infections. This medium detects bacterial enzymes using chromogenic substrates. Two hundred and nineteen samples of urine were tested in order to evaluate new CPS ID2 in comparison to blood agar and MacConkey agar. According to our results, the CPS ID2 agar is an easy, rapid and sensitive method for the screening of colonies suspected of being Escherichia coli, reducing the number of biochemical tests needed. Also, enterococci and Proteae can be easily detected. Other micro-organisms require further identification. The greatest value of this medium is the accurate identification of polymicrobial cultures.

Agar↗
PubMed2004

Comparison of fluorogenic and chromogenic assay systems in the detection of Escherichia coli O157 by a novel polymyxin-based ELISA.

AIMS: Different indicator enzymes and fluorogenic or chromogenic substrates were compared as detector systems in a novel polymyxin-based enzyme-linked immunosorbent assay (ELISA) for Escherichia coli O157 lipopolysaccharide (LPS) antigens. METHODS AND RESULTS: An ELISA system was developed using polymyxin immobilized in the wells of a microtitre plate as a high-affinity adsorbent for E. coli O157 LPS antigens, which were immunoenzymatically detected using anti-E. coli O157 antibody-enzyme conjugates. With peroxidase as the indicator enzyme the fluorogenic substrates Amplex Red and QuantaBlu produced only slight improvement in the performance characteristics of the polymyxin-ELISA compared with the use of the chromogenic substrate tetramethylbenzidine (TMB). On the other hand, with alkaline phosphatase as the indicator enzyme a pronounced improvement in assay performance was noted using the fluorogenic substrate Attophos compared with the chromogenic substrate p-nitrophenylphosphate. CONCLUSIONS: The detection system exhibiting the best characteristics with respect to cost, ease of use and overall performance in the detection of E. coli O157 in enrichment cultures from a variety of solid foods was based on the use of peroxidase as the indicator enzyme with the chromogenic substrate TMB. SIGNIFICANCE AND IMPACT OF THE STUDY: The polymyxin-ELISA provides a rapid, simple and inexpensive assay system for the detection of E. coli O157 in foods.

Alkaline Phosphatase↗
PubMed2005

A plate assay for simultaneous screening of polysaccharide- and protein-degrading micro-organisms.

AIMS: To develop a plate assay for simultaneous screening of polysaccharide-degrading and protein-degrading micro-organisms. METHODS AND RESULTS: A plate assay, based on the visible solubilization of small substrate particles and the formation of haloes on Petri dishes, containing a mixture of diversely coloured insoluble polysaccharides and dye-labelled collagen as chromogenic substrates, was developed. This method was successfully applied for isolating the diverse polysaccharide- and/or protein-degrading bacteria from soil and sludge samples. Selected strains were identified using 16S rDNA partial sequencing; most of them belong to the genera Bacillus, Cellulomonas and Cellulosimicrobium. CONCLUSIONS: This novel approach provides unique and valuable information for direct primary screening when the target of selection is micro-organisms exhibiting protein-degrading activity, polysaccharide-degrading activity or a specific combination of them. SIGNIFICANCE AND IMPACT OF THE STUDY: This plate assay is convenient and easy to perform, rapid, and more adaptable for screening of a large number of samples, compared with other existing methods in the literature.

Actinomycetales↗
PubMed2005

Performance of a new chromogenic plating medium for the isolation of Listeria monocytogenes from marine environments.

AIMS: This study investigated the performance of a new chromogenic plating medium for the detection of Listeria monocytogenes from naturally contaminated samples obtained from marine environments in Morocco in comparison with the conventional plating media PALCAM and Oxford. METHODS: A total of 479 marine samples (sea water, sediment and mussels) were collected from 16 littoral sites in the region of Agadir (western centre of Morocco). They were examined for the presence of L. monocytogenes using a slight modification of the standardized French method (AFNOR V 08-055) for the detection of L. monocytogenes from food and three different isolation media: PALCAM, Oxford and a new chromogenic plating medium. RESULTS AND SIGNIFICANCE OF THE STUDY: The Oxford and the new chromogenic plating media were found relatively more efficient than the PALCAM medium for the isolation of L. monocytogenes (chi-square test, P < 0.05) from marine samples. However, the new chromogenic plating medium was significantly more selective for L. monocytogenes (P < 0.005) than the two other isolation media as 87.5% of the suspect colonies on this medium were indeed confirmed through identification of the isolates vs 12.7% for Oxford and only 3.8% for the PALCAM medium.

Agar↗
PubMed2006

Evaluation of a new chromogenic agar medium for isolation and identification of Group B streptococci.

AIMS: To evaluate a new chromogenic agar as a screening medium for the isolation of Group B streptococci from high vaginal swabs from pregnant women. METHODS AND RESULTS: The medium was evaluated with 195 high vaginal swabs referred for antenatal screening and compared with blood agar and Granada medium. The new chromogenic medium showed 100% sensitivity for the detection of Group B streptococci, and also showed a positive predictive value of 100%. Granada medium also showed excellent sensitivity and specificity and both media were superior to blood agar. CONCLUSIONS: The new chromogenic medium showed excellent performance for the detection of Group B streptococci from clinical samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first chromogenic medium described for the detection of Group B streptococci. The medium offers an effective and convenient alternative to conventional media, currently used in clinical laboratories.

Agar↗
PubMedDate not supplied

Targeting topoisomerase IIa in endometrial adenocarcinoma: a combined chromogenic in situ hybridization and immunohistochemistry study based on tissue microarrays.

Topoisomerase IIa is a nucleic enzyme that affects the topological structure of DNA and also is a target for chemotherapy (ie, anthracyclines). In this study, we coevaluated its protein expression with chromosome 17 and gene status. Using tissue microarrays, 40 cases of sporadic, primary endometrial adenocarcinomas, 5 cases of atypical hyperplasia, and 5 cases of benign hyperplasia were obtained and reembedded into two paraffin blocks with a core diameter of 1 mm. Immunohistochemistry combined with chromogenic in situ hybridization was performed in 2 and 5 microm sections, respectively. Finally using a semiautomated Image Analysis System, we evaluated the levels of Nuclear labeling index of topoisomerase IIa expression. Statistical analysis was performed by SPSS version 11.0 software. The results indicate that chromosome 17 instability (aneuploidy in 7/40 cases) and Topo IIa gene deregulation (amplification in 3/40 and deletion in 1/40 cases) are significant genetic events correlated with biologic behavior in endometrial adenocarcinoma. Because protein overexpression was observed in a significant proportion of the tumors (18/40), detection of the specific gene deregulation mechanism is a crucial process for application of targeted chemotherapies, which are characterized by different levels of cardiotoxicity and other serious effects.

Adenocarcinoma↗
PubMed2004

Collaborative field study on the utility of a BDD factor VIII concentrate standard in the estimation of BDDr Factor VIII:C activity in hemophilic plasma using one-stage clotting assays.

Advances in production technologies of factor (F)VIII concentrates during the last two decades has resulted in very pure and safe products. In assessment of recombinant FVIII:C, inconsistent assay values are found comparing one-stage assays with two-stage (e.g. amidolytic) methods. Such discrepancies have been quite prominent in the case of a B-domain deleted recombinant FVIII (BDDrFVIII, ReFacto). In order to alleviate this assay variance, a product-specific reference standard [the ReFacto Laboratory Standard (RLS)], was established for laboratory use with either one-stage clotting or chromogenic substrate assays for the measurement of FVIII:C in ReFacto-containing patient samples. The primary objective of the current study was to assess, under field laboratory conditions, the accuracy and precision of the one-stage clotting assay for the determination of FVIII:C in ReFacto-containing samples employing the new concentrate standard. A secondary goal was to assess whether use of the RLS would minimize the discrepancy between one-stage clotting and chromogenic substrate assays. Thirty-one clinical laboratories worldwide participated in the study of severe-hemophilic plasma (SHP) samples that had been spiked with ReFacto to target levels of 0.9, 0.6 and 0.2 IU mL(-1). FVIII:C levels were determined against both the RLS and the local in-house plasma standard (IHS). The results showed good agreement between laboratories in FVIII:C levels obtained by one-stage clotting assays utilizing the RLS, and a good degree of accuracy was found compared with the intended target values. Consistent with previously published data, a discrepancy of approximately 30% was observed between one-stage clotting and chromogenic potencies when the IHS was used as the calibrator. The discrepancy between one-stage and chromogenic assay methodologies was significantly reduced when the RLS was employed as calibrator in the one-stage assay. In conclusion, the study demonstrates that accurate and precise FVIII:C results can be obtained for ReFacto-containing SHP samples by clinical laboratories using a product-specific standard in one-stage clotting assays. In addition, the product-specific reference standard significantly reduced the discrepancy between the one-stage clotting and the chromogenic substrate assay for ReFacto.

Blood Coagulation↗
PubMed2005

Shiga toxin enhances functional tissue factor on human glomerular endothelial cells: implications for the pathophysiology of hemolytic uremic syndrome.

BACKGROUND: The pathogenesis of Shiga toxin (Stx)-mediated childhood hemolytic uremic syndrome (HUS) is not fully delineated, although current evidence implicates a prothrombotic state. We hypothesized that the tissue factor (TF) pathway plays a major role in the pathophysiology of HUS. MATERIALS AND METHODS: We measured cell surface TF activity in response to tumor necrosis factor-alpha (TNF-alpha) (20 ng mL(-1), 2-144 h), Stx-1 (10(-11) mol L(-1), 4-144 h), or their combination (TNF-alpha 22 h and Stx-1 for the last 0.5-4 h of TNF-alpha incubation) on human glomerular (microvascular) endothelial cells (HGECs) and human umbilical vein (macrovascular) endothelial cells (HUVECs). RESULTS AND CONCLUSIONS: We observed that while TNF-alpha caused an increase in cell surface TF activity on both cell types, the combination of TNF-alpha and Stx-1 differentially affected HGECs. On these cells, TF activity was increased further by 2.67 +/- 0.38-fold (n = 38, P < 0.001), consistent with our parallel observation that Stx-1 binds to HGECs but not to HUVECs. Anti-TF antibody abolished functional TF while anti-tissue factor pathway inhibitor antibody enhanced TF activity. Stx-1 alone did not induce TF activity on either cell type. Measurement of TF antigen levels and quantitative real-time polymerase chain reaction demonstrated that exposure to TNF-alpha markedly increased TF protein and TF mRNA for HGECs, but the exposure to the combination of TNF-alpha and Stx-1 did not increase further the amount of either TF protein or TF mRNA. We conclude that cytokine-activated HGECs, but not HUVECs, undergo a significant augmentation of cell surface TF activity following exposure to Stx, suggesting an important role for TF in the coagulopathy observed in HUS.

Cell Membrane↗
PubMed1997

Transmembrane potentials during high voltage shocks in ischemic cardiac tissue.

Transmembrane, voltage sensitive fluorescent dye (TMF) recording techniques have shown that high voltage shocks (HVS), typically used in defibrillation, produce either hyper- or depolarization of the transmembrane potential (TMP) when delivered in the refractory period of an action potential (AP) in normal cardiac tissue (NT). Further, HVS produce an extension of the AP, which has been hypothesized as a potential mechanism for electrical defibrillation. We examined whether HVS modify TMP of ischemic tissue (IT) in a similar manner. In seven Langendorff rabbit hearts, recordings of APs were obtained in both NT and IT with TMF using di-4-ANEPPS, and diacetylmonoxime (23 microM) to avoid motion artifacts. Local ischemia was produced by occlusion of the LAD, HVS of either biphasic (5 + 5 ms) or (3 + 2 ms) or monophasic shapes (5 ms) were delivered at varying times (20%-90%) of the paced AP. Intracardiac ECG and TMF recordings of the TMP were each amplified, recorded, and digitized at a frequency of 1 kHz. The paced AP in IT was triangular in shape with no obvious phase 3 plateau, typically seen in NT. There was normally a reduced AP amplitude (expressed as fractional fluorescence) in IT (2.6% +/- 1.79%) compared to 3.8% +/- 0.66% in NT, and shortened AP duration (137 +/- 42 vs 171 +/- 11 ms). One hundred-Volt HVS delivered during the refractory period of paced AP in IT in five rabbits, elicited a depolarization response of the TMP with an amplitude up to three times greater than the paced AP. This is in contrast to NT where the 100-V HVS produced hyperpolarization in four hearts, and only a slight depolarization response in one heart. These results suggest that HVS, typically delivered by a defibrillation shock, modify TMPs in a significantly different manner for ischemic cells, which may influence success in defibrillation.

Action Potentials↗