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At least 613 records · Page 34Linked to original sources
PubMed1999

A chromogenic substrate assay kit for factor XII: evaluation and use for the measurement of factor XII levels in cardiopulmonary bypass patients.

Factor XII levels were determined in plasma samples from 75 patients before undergoing aortocoronary bypass grafting and from 40 healthy age-matched donors by using a microtitre plate adaptation of a new chromogenic peptide substrate assay kit for factor XII. The chromogenic peptide substrate assay values for factor XII correlated well with those obtained in clotting (r=0.90; y= 15.811+0.8236x) and immunochemical (r=0.88; y=17.90+0.817x) assays in the normal donor samples. Factor XII levels in the patients were significantly lower than those in the normal donors (83.3+/-23.2% versus 103.4+/-23.1: p=0.004), and nine patients (12%) had factor XII values below 50% compared with only one of the normal donors (2.5%). Factor XII levels and kallikrein-like activities (a measure of contact system activation) were followed before, during, and one day after cardiopulmonary bypass in 20 patients. Factor XII levels were significantly reduced, and kallikrein-like activities significantly elevated after 5 and 30 minutes cardiopulmonary bypass. One day after cardiopulmonary bypass both factor XII levels and kallikrein-like activities were significantly lower than preoperation values.

Adult↗
PubMed1999

Evaluation of the automated coagulation analyzer SYSMEX CA 6000.

In the present study the coagulation analyzer SYSMEX CA 6000 (TOA Medical Electronics Co., Kobe, Japan), an analyzer equipped with a photooptical clot detection unit and a cap-piercing system, was evaluated with respect to its technical characteristics in the determination of standard coagulation tests (prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen, and antithrombin) and in the determination of coagulation single factor activities. In the normal and in the pathological range the intraassay coefficients of variation (CV) and interassay CV for most parameters were below 5% (exceptions: intraassay CV 5.4% for prolonged thrombin time; intraassay CV 9.26% and interassay CV 10.7% for decreased antithrombin; interassay CV 5.62% for fibrinogen in the normal range, intraassay CV 10.1% for fibrinogen greater than 7.0 g/L; intraassay CV 6.36% and interassay CV 11.7% for decreased fibrinogen; interassay CV 11.6% for prolonged activated partial thromboplastin time; interassay CV 6.12% for decreased factor VII). Interference studies with lipemic, icteric, and hemolytic samples showed just minor influences of these abnormal sample characteristics on prothrombin time, activated partial thromboplastin time, fibrinogen, and antithrombin measurements when compared to the results obtained by using mechanical clot detection (STA, Stago Diagnostica, Asnieres-Sur-Seine, France). No carryover was detected in alternating measurements of heparinized (3 U/mL unfractionated heparin) and normal plasma samples. Measurement of the activities of clotting factors V, VII, VIII, and IX showed a good correlation (r=0.993 to r=0.977) between SYSMEX CA 6000 and STA. Our results demonstrate that using SYSMEX CA 6000 analyzer basal routine coagulation testing as well as specialized tests for single factor activities can be performed with satisfactory precision; in particular, the cap-piercing system has no negative effect on the performance of the analyzer.

Adult↗
PubMed2000

A new sensitive chromogenic substrate assay of tissue factor pathway inhibitor type 1.

The present assay is a modification of our previously published two-stage chromogenic substrate assay of tissue factor pathway inhibitor type-1 (TFPI) activity [1]. In the first stage, the reaction mixture was made with factor VIIa (FVIIa) molecules in excess of tissue factor (TF) binding sites and contained diluted plasma, recombinant FVIIa (10 nM), recombinant TF (1/400 vol/vol), bovine factor Xa (1,1 nM), I-2882(R) (100 microg/ml), and CaCl(2) (10 mM). The fibrin polymerisation inhibitor I-2882(R) was added to the reaction mixture to prevent formation of cross-linked fibrin. In the second stage, residual TF/FVIIa catalytic activity was measured by the addition of a substrate mixture that contained bovine factor X and a chromogenic substrate (S-2222(R)). Standard curves were constructed using serial dilutions (0-1%) of pooled normal plasma. The dose-response relationship for serial dilutions of plasma was linear. The intra-assay coefficient of variations (CVs) for pre- and postheparin plasma samples (i.e., normal and high TFPI levels) were 1.7% and 9.9%, respectively; the inter-assay CVs were 10.0% and 19. 7%, respectively. The effect of variation in antithrombin activity on the assay was approximately 5%. The present assay correlated fairly well with our previously published assay (r=0.82, n=100) and with a commercial TFPI activity assay (Actichrome(R) TFPI Activity Assay, American Diagnostica, Greenwich, CT, USA; r=0.90, n=100), as well as with an antigen assay for TFPI total antigen (Imubind(R), American Diagnostica; r=0,96, n=100). Altman and Bland plots revealed that our previous assay underestimated TFPI activity at high TFPI levels (i.e., postheparin TFPI samples) compared with the other methods.

Amides↗
PubMed1997

Use of peroxidase substrate Vector VIP for multiple staining in light microscopy.

The study of the distribution of a fiber input to a particular brain area and the visualization of the anatomical relationships of that input with both projection- and interneurons, requires a triple-staining that allows the unequivocal distinction of each of the three components in one and the same histological section. In this regard, we investigated the properties of a recently introduced peroxidase chromogen, VIP (V-VIP; Vector Labs) in combination with two traditional substrates, standard diaminobenzidine (DAB, brown precipitate) and nickel-enhanced DAB (DAB-Ni, black). In rats, the anterograde tracer biotinylated dextran amine (BDA) and the retrograde tracer fluorogold (FG) were injected in the perirhinal cortex and hippocampus, respectively. Transported BDA was detected with an avidin-biotin-peroxidase complex, whereas the transported FG was detected via a PAP method. Tracing with BDA and FG was combined with parvalbumin- or calbindin-immunocytochemistry. We compared various combinations and staining sequences. The best results were obtained with a staining sequence comprising first the BDA stain with DAB-Ni as chromogen, second the FG protocol with the chromogen DAB and finally, parvalbumin- or calbinding-immunocytochemistry using the chromogen V-VIP. The order with which the chromogens were applied appeared to be critical. Partial or even total loss of V-VIP reaction product has been observed after standard dehydration in ethanol. As an alternative, a quick dehydration procedure in toluene yields much better staining. Colour separation is excellent and the sensitivity is high. This procedure may also be used for detection of any other combination of three different labels, taking the usual care to avoid cross-reactivity between antibodies.

3,3'-Diaminobenzidine↗
PubMed1996

A semi-automated, microplate version of the SOS Chromotest for the analysis of complex environmental extracts.

Environmental monitoring for genotoxicity requires that a large number of measurements be made across space and time. This requirement demands a rapid and efficient bioassay system. The SOS Chromotest is a rapid, efficient bacterial system for the detection of DNA damaging agents. Over 100 publications have described its use on a variety of samples. Relatively few studies have used the test to examine complex mixtures. Effective testing of complex samples poses a variety of problems. Although solutions have been proposed, few have validated the resulting protocol. In this work we present a semi-automated microplate version of the SOS Chromotest for the examination of complex mixtures. Experiments were conducted to determine the optimal cell concentration, exposure time, substrate conversion time and S9 enzyme concentration. The performance of the method was evaluated using 6 reference genotoxins and 3 complex mixtures. The complex mixtures examined are extracts of diesel particulate matter, urban dust and coal tar. The results obtained indicate that optimal responses often require fewer cells (approximately equal to 5-10 x 10(6) CFU/ml) and a longer exposure (3 h) than that recommended in the original protocol. Interfering effects of colored and turbid samples are removed using centrifugation and initial optical density readings taken 60 min after cell resuspension and lysis. The performance of the established protocol was evaluated using mitomycin C and benzo[a]pyrene results for 207 microplates and solvent control results for 293 microplates. The results indicate that the established method is accurate, sensitive and precise. Coefficient of variation on mean SOSIP values for mitomycin C and benzo[a]pyrene are < 5%. Solvent control data indicate that the standard threshold for determination of a positive response (induction factor > 1.5) is excessively conservative. All liquid transfers were automated using the Biomek automated laboratory workstation. Automation permits a throughput of up to 72 samples per day and maintains excellent precision and accuracy.

Automation↗
PubMed1997

Dopamine increases in cat carotid body during excitation by carbon monoxide: implications for a chromophore theory of chemoreception.

Studies of dopamine (DA) release were conducted with 10 perfused/superfused cat carotid bodies using shallow recessed Nafion polymer-coated microsensors (tips approximately 5 microns). Simultaneous measurements of tissue DA and neuronal discharge (ND) from the sinus nerve were made after switching from normoxic, normocapnic control perfusate (20% O2, 5% CO2, balance N2) to a normoxic, normocapnic perfusate equilibrated with a high tension (> 550 Torr) of carbon monoxide (CO). When high PCO perfusate was delivered in the dark, ND increased from a baseline of 89 +/- 24 (SE) impulses/s, to a peak excitation of 374 +/- 44 impulses/s within 15-30 s. Excitation then diminished to a plateau of 281 +/- 36 impulses/s within 1-2 min. Both peak and plateau ND were significantly above baseline (P < 0.05). Average tissue DA values increased above basal levels by +7.2 +/- 1.0 and +5.6 +/- 0.6 microns, respectively, during the peak and plateau ND phases (P < 0.05). Bright light restored the chemosensory activity to baseline, but had no effect on DA. Both chemosensory excitation and tissue DA responses to high CO in the dark were diminished in 3 carotid bodies perfused with Ca(2+)-free solutions. Responses were reduced even further with Ca2+ chelator (EGTA) in the perfusate. The results suggest that the effect of high PCO on DA release and chemosensory excitation are dependent on Ca2+ in the media, but the two events are not coupled.

Action Potentials↗
PubMed1998

Agrobacterium tumefaciens beta-glucosidase is also an effective beta-xylosidase, and has a high transglycosylation activity in the presence of alcohols.

Agrobacterium tumefaciens beta-glucosidase, Cbg1 was extensively characterised and found to be a retaining aryl-glucosidase and an aryl-xylosidase. Cbg1s specificity for p-nitrophenyl beta-d-xylopyranoside was 73% that for p-nitrophenyl beta-d-glucopyranoside when measured by the ratio kcat/Km. The enzyme also hydrolysed p-nitrophenyl beta-d-fucopyranoside, and p-nitrophenyl beta-d-galactopyranoside with moderate efficiency. The enzyme released only terminal glucose from p-nitrophenyl beta-cellobioside and had a 20 000-fold preference for its natural substrate coniferin over cellobiose as indicated by the ratio kcat/Km. The enzyme was activated in the presence of 20 mM 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, and 1-octanol. In the case of butanol this activation was investigated and shown to be due to transglycosylation activity with over 80% of p-nitrophenyl beta-d-glucopyranoside being converted to 1-butyl beta-d-glucopyranoside in the presence of Cbg1 and 100 mM 1-butanol.

Agrobacterium tumefaciens↗
PubMed1999

Thrombin mutants with altered enzymatic activity have an impaired mitogenic effect on mouse fibroblasts and are inefficient modulators of stellation of rat cortical astrocytes.

We produced recombinant human thrombin mutants to investigate the correlation between the thrombin enzyme and mitogenic activity. Single amino acid substitutions were introduced in the catalytic triad (H43N, D99N, S205A, S205T), in the oxy-anion binding site (G203A) and in the anion binding exosite-1 region (R73E). Proteins were produced as prethrombin-2 mutants secreted in the culture medium of DXB11-derived cell lines. All mutants were activated by ecarin to the corresponding thrombin mutants; the enzymatic activity was assayed on a chromogenic substrate and on the procoagulant substrate fibrinogen. Mutations S205A and G203A completely abolished the enzyme activity. Mutations H43N, D99N and S205T dramatically impaired the enzyme activity toward both substrates. The R73E mutation dissociated the amidolytic activity and the clotting activity of the protein. The ability of thrombin mutants to induce proliferation was investigated in NIH3T3 mouse fibroblasts and rat cortical astrocytes. The ability of the thrombin mutants to revert astrocyte stellation was also studied. The mitogenic activity and the effect on the astrocyte stellation of the thrombin mutants correlated with their enzymatic activity. Furthermore the receptor occupancy by the inactive S205A mutant prevented the thrombin effects providing strong evidence that a proteolytically activated receptor is involved in cellular responses to thrombin.

3T3 Cells↗
PubMed2000

Application of a chromogenic medium and the PCR method for the rapid confirmation of L. monocytogenes in foodstuffs.

Detection of Listeria monocytogenes in foodstuffs by conventional cultivation methods carried out according to EN ISO guidelines is rather time-consuming. Therefore, two alternative methods were applied for rapid confirmation of L. monocytogenes in foodstuffs. Inoculum from liquid selective broth was plated on PALCAM and OXFORD agar and on chromogenic agar medium RAPID L. mono. Suspect colonies from PALCAM were confirmed according to EN ISO standards and by the multiplex PCR method. In total, 990 samples of foodstuffs were investigated and 63 strains of L. monocytogenes were isolated. The chromogenic medium RAPID L. mono provided results comparable to PCR, it is easier to handle and provides considerable financial savings.

Chromogenic Compounds↗
PubMed2003

Evaluation of Chromocult coliform agar for the detection and enumeration of Enterobacteriaceae from faecal samples from healthy subjects.

The purpose of this study was to examine the use of Chromocult agar medium for isolation and enumeration of Enterobacteriaceae from human faecal samples, to compare it to MacConkey agar and to evaluate its usefulness as a possible alternative selective medium in human faecal studies. The medium was shown to be effective in identifying Escherichia coli and coliforms in faeces without the need for extensive accompanying biochemical tests for confirmation of identity. A positive correlation (r=0.86) was found between the recovery of Enterobacteriaceae on the two media, and no significant difference (P>0.05) between overall mean bacterial counts for the whole study group or at different intervals of faecal collection were observed. Chromocult agar is an effective replacement for MacConkey agar in human faecal studies and has the advantage of differentiating E. coli from other coliforms.

Agar↗
PubMed2000

New developments in chromogenic and fluorogenic culture media.

This review describes some recent developments in chromogenic and fluorogenic culture media in microbiological diagnostic. The detection of beta-D-glucuronidase (GUD) activity for enumeration of Escherichia coli is well known. E. coli O157:H7 strains are usually GUD-negative and do not ferment sorbitol. These characteristics are used in selective media for these organisms and new chromogenic media are available. Some of the new chromogenic media make the Salmonella diagnostic easier and faster. The use of chromogenic and fluorogenic substrates for detection of beta-D-glucosidase (beta-GLU) activity to differentiate enterococci has received considerable attention and new media are described. Rapid detection of Clostridium perfringens, Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus are other application of enzyme detection methods in food and water microbiology.

Chromogenic Compounds↗
PubMed1998

Effect of water activity on hydrolytic enzyme production by Fusarium moniliforme and Fusarium proliferatum during colonisation of maize.

The effect of different water availabilities (water activity, aw; 0.98-0.93) and time (up to 15 days) on the production of seven hydrolytic enzymes by strains of F. moniliforme and F. proliferatum during early colonisation of gamma-irradiated living maize grain were examined in this study. Both the total activity (micromol 4-nitrophenol min(-1) g(-1) maize) and specific activity (nmol 4-nitrophenol min(-1) microg(-1) protein) were quantified using chromogenic p-nitrophenyl substrates. The dominant three enzymes produced by the fungi on whole colonised maize kernels were alpha-D-galactosidase, beta-D-glucosidase, and N-acetyl-beta-D-glucosaminidase. The other four enzymes were all produced in much lower total amounts and in terms of specific activity (beta-D-fucosidase, alpha-D-mannosidase, beta-D-xylosidase and N-acetyl-alpha-D-glucosaminidase), similar to that in uncolonised control maize grain. There were significant increases in the total production of the three predominant enzymes between 3-15 days colonisation, and between 3-6 days in terms of specific activity when compared to untreated controls. The total and specific activity of the alpha-D-galactosidase, beta-D-glucosidase and N-acetyl-beta-D-glucosaminidase, were maximum at 0.98 aw with significantly less being produced at 0.95 and 0.93 aw, with the exception of the total activity of alpha-D-galactosidase which was similar at both 0.95 and 0.93 aw. Single factors (time, aw, and inoculation treatment), two- and three- way interactions were all statistically significant for the three dominant enzymes produced except for specific activity of beta-D-glucosidase (two and three-way interactions) and for total activity of alpha-D-galactosidase in the time x aw treatment. This study suggests that these hydrolytic enzymes may play an important role in enabling these important fumonisin-producing Fusarium spp. to rapidly infect living maize grain over a wide aw range.

Acetylglucosaminidase↗
PubMed1988

Growth dependent enzymatic profiles of some gram-negative nonfermentative bacteria of clinical significance.

A total of 734 strains of gram-negative nonfermentative bacteria (46 species and biochemically defined groups) of the genera Pseudomonas, Alcaligenes, Bordetella, Moraxella, Acinetobacter, Agrobacterium, and Flavobacterium were investigated for their ability to hydrolyze 25 different chromogenic substrates. All tests were carried out in growth-stimulating media. Results were read photometrically and evaluated automatically following a 24-h incubation. Many of the 46 different species and biochemical groups exhibited uniform patterns of enzyme production. Some of the enzyme tests may serve as additional valuable tools for differential diagnosis of the organisms investigated. In combination with other biochemical tests, qualitative enzyme demonstration tests can facilitate the identification of gram-negative nonfermentative bacteria.

Acinetobacter↗