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Results for “Chromogenic Compounds”

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PubMed1988

A new chromogenic assay for direct detection of staphylocoagulase.

Tube test for detection of staphylococcal coagulase, despite of many disadvantages, is commonly used in clinical microbiology for identification of Staphylococcus aureus. In this paper a new chromogenic method for detection of the coagulase directly in staphylococcal cultures is described and evaluated on the basis of a comparison with the standard tube assay. The chromogenic assay appeared to be as sensitive as the tube test but results of the former one can be read in a few hours without any apparatus.

Chromogenic Compounds↗

PubMed2002

[Use of MPO chromogenic culture medium for routine processing of urine cultures].

BACKGROUND: The chromogenic culture medium, MPO, was compared to culture on CLED (cystein, lactose, electrolyte-deficient) agar for the detection, enumeration and identification of urinary tract pathogens. METHODS: A total of 1,080 clinical urine specimens were assessed. All samples were inoculated in MPO and CLED using the calibrated loop method. RESULTS: Among 145 positive urine samples, 171 strains of bacteria were isolated (111 Escherichia coli, 26 Enterococcus spp., 12 Proteus spp., 10 Enterobacteriaceae from the Klebsiella-Enterobacter-Serratia group, 5 Pseudomonas aeruginosa, 4 Streptococcus agalactiae, 3 Staphylococcus spp. and 4 Candida albicans. For all samples, enumeration of microorganisms was comparable with the two media studied. Identification was also similar, except for 6 cases in which Enterococcus spp. were only detected with the chromogenic medium. CONCLUSIONS: Overall urine culture results with MPO chromogenic medium were similar to those obtained with CLED, making it a feasible alternative to the standard medium. Moreover, use of a chromogenic technique implies a significant reduction in workload, since additional tests to identify the microorganisms isolated are not needed in most cases.

Bacteria↗

PubMed2004

[Reliability of MPO chromogenic culture medium for presumptive identification of uropathogenic microorganisms].

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Bacteria↗

PubMedDate not supplied

[Evaluation of xylose galactosidase (XG) culture medium for the isolation of enteropathogens].

INTRODUCTION: The chromogenic medium, XG, was evaluated and compared to conventional media for the isolation of Salmonella spp., Shigella spp., Yersinia enterocolitica and Aeromonas spp. METHODS: A total of 1226 human stool samples were inoculated on XG, MacConkey agar, Salmonella-Shigella agar (SS), selenite broth, blood-ampicillin agar and cefsulodin-Irgasan-novobiocin agar (CIN). RESULTS: The 235 positive cultures included the following: 229 Salmonella spp., 3 Shigella spp., 2 Yersinia enterocolitica and one Aeromonas spp. Among the 229 containing Salmonella spp., 100 were detected on both XG and conventional media and the 129 remaining were detected only on conventional media; recovery of Salmonella spp. on conventional media was significantly higher with respect to XG medium (p < 0.005). The 3 isolates of Shigella spp. were obtained on XG, the 2 isolates of Yersinia enterocolitica were recovered on CIN agar and the single isolate of Aeromonas spp. was obtained both on XG and blood-ampicillin agar. Colonies suspected to be some of the enteropathogens investigated were present in 791 of the negative stool samples. Among these false-positives 441 (35.9%) were obtained from XG, 142 (11.6%) after selenite enrichment, 132 (10.8%) from MacConkey agar and 76 (6.2%) from SS agar. Most of the false-positive isolates obtained on XG medium were consistent with Salmonella spp. (n = 408). CONCLUSIONS: XG chromogenic medium showed low sensitivity (64%) and specificity (69%) for the detection of Salmonella spp. Recovery of Shigella spp. on XG medium in three samples may have been due to the immediate processing of the samples. We conclude that XG chromogenic medium can not be recommended as an alternative to currently used conventional media.

Aeromonas↗

PubMed1997

Presence of denatured hemoglobin deposits in diseased temporomandibular joints.

PURPOSE: The purpose of this study was to test the hypotheses that hemoglobin recovered by arthrocentesis of the superior joint space of symptomatic human temporomandibular joints (TMJs) is "old" hemoglobin that was not iatrogenically introduced by the arthrocentesis procedure and that it exists primarily in a non-native or denatured conformation state that may be sufficient to catalyze a reaction leading to the formation of damaging free radicals. PATIENTS AND METHODS: Twelve patients diagnosed with a unilateral articular disk displacement with TMJ arthralgia were included in this study. A superior joint space arthrocentesis was performed in the affected TMJ, and outflow lavage volumes were collected in serial 2-mL fractions. alpha-Hemoglobin/albumin ratios were determined for each collected fraction by densitometric analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, 3,3',5,5'-tetramethylbenzidine (TMB) assays were used to determine the conformation state of the recovered hemoglobin. RESULTS: High alpha-hemoglobin/albumin ratios relative to that of serum (at least 10 times greater) were observed in several collected fractions of TMJ lavage fluid in all subjects studied. Because the tissue half-life of hemoglobin is significantly longer than that of albumin, these findings indicate that much of the hemoglobin recovered by arthrocentesis of symptomatic TMJs represents "old" hemoglobin that was present in the joint before the procedure. Furthermore, based on reactivity in the TMB assay, we estimate that up to 89% of the alpha-hemoglobin present in TMJ lavage fluid samples exists in a denatured state. CONCLUSIONS: These results indicate that a significant amount of hemoglobin recovered by arthrocentesis of symptomatic TMJs exists in a denatured state and was present in the joint before arthrocentesis. Recent studies suggest that denatured hemoglobin may contribute redox active iron that can catalyze a reaction, leading to the formation of damaging free radicals. Such a process may represent one of the earliest molecular events involved in the pathogenesis of degenerative TMJ disease.

Albumins↗

PubMed1998

Evidence for multiple enzyme site involvement in the modulation of thrombin activity by products of prothrombin proteolysis.

Kinetic evidence is presented for the interaction of prothrombin with several distinctive topological regions of the thrombin molecule. Modulations of thrombin catalytic activity on the protein substrates prothrombin and prethrombin 1 are demonstrated that involve the fragment 1 and fragment 2 portions. The inhibitory effects are demonstrably non-competitive. In addition to exhibiting non-competitive inhibition, fragment 2 is capable of enhancing proteolysis by thrombin; and therefore to react with a second region of the enzyme. On the basis of the crystallographic studies of the complex between fragment 2 and thrombin (Arni et al., Biochemistry 32 (1992) 4727), this activating site is proposed to be associated with exosite II. The allosteric switch between procoagulant and anticoagulant activities identified from studies by Di Cera (Dang et al., Proc. Natl. Acad. Sci USA 92 (1995) 5977) could be 'thrown' by a macromolecular effector that is generated during thrombin formation--a plausible mechanism for switching that deserves further investigation.

Allosteric Site↗

PubMed2003

A subset of nociceptin/orphanin FQ receptor-expressing neurons in the anterior hypothalamic area, as revealed in mice with lacZ reporter gene.

Nociceptin/orphanin FQ (N/OFQ) is an endogenous peptide agonist for the opioid receptor homolog, N/OFQ receptor, and serves for the central control of autonomic functions. Morphological details including the cell types that may account for such N/OFQ functions, however, remain unclear. By using X-gal histochemistry for the detection of receptor-expressing cells at both light and electron microscopic levels, we examined the hypothalamus from the receptor-deficient mice bearing a lacZ insertional mutation in the N/OFQ receptor gene. The N/OFQ receptor reflected by lacZ expression was seen at high levels in the anterior hypothalamic area. With electron microscopy, lacZ expression was observed in a subset of neurons showing large cell size and indented nucleus.

Animals↗

PubMed1998

Effect of antibodies against beta-N-acetylhexosaminidase on reproductive efficiency of the bovine tick Boophilus microplus.

A polyclonal antibody (anti-HEX) was developed against a soluble N-acetylhexosaminidase (HEX) isolated from larval extracts of Boophilus microplus. Purified hexosaminidase was strongly inhibited by the IgG fraction of this antibody. The antibody inhibited the hexosaminidase activity of other sources, such as haemolymph and larval membranes. The antibody reacted with different antigens in the tick haemolymph, but did not recognize any antigen in saliva, as seen by immunoblot analysis. The anti-HEX was inoculated into fully engorged B. microplus females, resulting in a decrease in oviposition of approximately 26%, relative to the effect of pre-immune IgG. These data show the potential of the use of this tick enzyme as an antigen in vaccine development.

Animals↗

PubMed2003

Spectroscopic studies of the interaction of bichromophoric cyanine dyes with DNA. Effect of ionic strength.

Spectroscopic characteristics of a cyanine dye with two chromophores (biscyanine dye, BCD) in aqueous solutions and effects of NaCl and DNA upon these characteristics have been studied by optical absorption, circular dichroism (CD) and fluorescence spectroscopies. In homogeneous solutions, BCD is characterized by intense optical absorption (epsilon =1.33 x 10(5) M(-1) x cm(-1)) and weak fluorescence (phi(fl)=0.018) in the wavelength region greater than 600 nm. The dye forms H-aggregates at low concentrations (10(-6) M). NaCl stimulates the formation of both H- and J-aggregates of the dye at much lower dye concentrations, while DNA in low concentrations (<10(-6) M) stimulated the formation of just J-aggregates on the surface of the DNA molecule. Higher DNA concentrations induce the dye to disaggregate, and there exists an equilibrium between three dye forms: free monomers, J-aggregates and bound monomers, the maximum content of J-aggregates was observed at [DNA]/[BCD]=0.6+/-0.2 and total disaggregation at [DNA]/[BCD]=190+/-20. J-aggregates are characterized by phi(fl)=0.05 and bound monomers by phi(fl)=0.44. In the presence of NaCl, total disaggregation was observed at [DNA]/[BCD]=570+/-10 due to competition between Na(+) and the dye molecules for DNA electronegative binding sites.

Carbocyanines↗

PubMed1999

Thalamic input to parvalbumin-immunoreactive GABAergic interneurons: organization in normal striatum and effect of neonatal decortication.

The neocortex and thalamus send dense glutaminergic projections to the neostriatum. The neocortex makes synaptic contact with spines of striatal projection neurons, and also targets a distinct class of GABAergic interneurons immunoreactive for the calcium-binding protein parvalbumin. We determined whether the parafascicular thalamic nucleus also targets striatal parvalbumin-immunoreactive interneurons. The anterograde tracer biotinylated dextranamine was injected into the parafascicular nucleus of adult rats. Double-labeled histochemistry/immunohistochemistry revealed overlapping thalamic fibers and parvalbumin-immunoreactive neurons in the neostriatum. Areas of overlap within the sensorimotor striatum were analysed by electron microscopy. Of 311 synaptic boutons originating from the parafascicular nucleus, 75.9% synapsed with unlabeled dendrites, 22.5% with unlabeled spines, and 1.3% had parvalbumin-immunoreactive dendrites as a postsynaptic target. Only 4% of all asymmetric synapses on parvalbumin-immunoreactive dendrites were derived from the parafascicular nucleus. A separate group of animals underwent bilateral neocortical deafferentation on the third postnatal day, prior to injection of anterograde tracer into the parafascicular nucleus of adult animals. These experiments were performed with the dual purpose of (i) reducing the possibility that thalamic inputs to parvalbumin-immunoreactive neurons are the result of transsynaptic uptake of tracer by a thalamo-cortico-striatal route, and (ii) determining whether competitive interactions between developing corticostriatal and thalamostriatal fibers may account for the relatively sparse thalamic input onto parvalbumin-immunoreactive interneurons. In decorticates, 219 striatal synaptic contacts derived from the parafascicular nucleus, out of which 77.2% were on unlabeled dendrites, 20.9% were upon unlabeled spines, and 0.9% targeted parvalbumin-immunoreactive dendrites. We conclude that the thalamic parafascicular nucleus indeed sends synaptic input to parvalbumin-immunoreactive striatal neurons. Parafascicular nucleus inputs to striatal parvalbumin-immunoreactive interneurons are sparse in comparison to other asymmetric inputs, most of which are likely to be of cortical origin. The synaptic profile of thalamostriatal inputs to parvalbumin-immunoreactive neurons and unlabeled elements is unchanged following neonatal decortication. This suggests that competitive interaction between developing thalamostriatal and corticostriatal projections is not a major mechanism determining synaptic input to striatal subpopulations.

Animals↗

PubMed1985

[Limulus test using a chromogenic method: application to the control of pyrogens in blood derivatives].

We report here the application of the LAL Test to a chromogenic substrate to detect endotoxins in Human Blood Products. In order to reduce the cost, we used a microplate procedure with the Multiskan Reader. Quantitative results in the range of 0,01 to 0,1 ng/ml allowed for a good correlation with the Rabbit Pyrogen test. For 20% albumins and 4% albumins, the mean endotoxins levels of non pyrogenic lots were 0,38 +/- 0,18 and 0,09 +/- 0,03 ng/ml. All the lots which passed the Rabbit Pyrogen test had endotoxins levels lower than 1 ng/ml and 0,2 ng/ml, respectively. We can use this test for other plasma derivatives; Gammaglobulines and PPSB are easily tested. Dried Concentrated Antihemophilic Factor and Dried plasma contain citrate which inhibits the reaction. Dilution and Addition of Calcium Chloride overcome this inhibition. For dried plasma, we should destroy plasma inhibitors by heating at 75 degrees C. This sensitive and reproductible in vitro assay improves the control of pyrogenecity in Human Blood Products.

Blood Transfusion↗

PubMed2003

Identification and characterization of four proteases produced by Streptococcus suis.

Streptococcus suis is an important worldwide swine pathogen. In this study, we investigated the production of proteases by S. suis serotype 2. Proteases were identified and characterized using chromogenic and fluorogenic assays and zymography. An Arg-aminopeptidase with a molecular mass of 55 kDa was found to be both cell-associated and extracellular. Cell-associated chymotrypsin-like and caseinase activities, belonging to the serine- and metalloprotease classes respectively, were also detected. Lastly, a dipeptidyl peptidase IV (DPP IV) with a molecular mass of 70 kDa was detected in both whole cells and culture supernatants of S. suis serotype 2. Arg-aminopeptidase, caseinase and DPP IV activities were detected in all strains of S. suis serotype 2 tested whereas the chymotrypsin-like activity was only detected in European virulent strains of serotype 2. The optimum pH for all four proteases was between 6 and 8, and the optimum temperature ranged from 25 to 42 degrees C. This is the first report on the production of proteases by S. suis. Further investigations will determine the possible contribution of these proteases in the pathogenicity of S. suis serotype 2.

Aminopeptidases↗

PubMed1998

Accurate insertional inactivation of lacZalpha: construction of pTrueBlue and M13TrueBlue cloning vectors.

Color selection plasmid and phage vectors based on insertional inactivation of the lacZalpha gene fragment have been in wide use for nearly two decades. The originals (the pUC and M13mp series) and all subsequent derivatives of these vectors contain the same mechanism for insertional inactivation of lacZalpha placing the sites for gene interruption between the initiator ATG codon and codon 7, a region that encodes a functionally nonessential part of the lacZalpha-complementation peptide. Numerous observations found in the literature and made in our laboratory suggest that the color selection process in these vectors does not function reliably and that erroneous conclusions about the success of cloning experiments are frequently drawn. We have studied the efficiency of lacZalpha inactivation by performing insertions at different intervals along the entire length of the proximal 60-amino-acid (aa)-coding region of the lacZ gene (lacZalpha). Our results show that accurate and reliable inactivation of lacZalpha occurs only when insertions are made within the DNA region that encodes aa 11-36. Based on these results, novel color selection plasmid and phage vectors, pTrueBlue and M13TrueBlue, have been constructed. Application of these vectors for gap-free shotgun sequencing of genomic DNA is discussed.

Amino Acid Sequence↗

PubMed2003

Detection of pathogenic Listeria spp. in zoo animal faeces: use of immunomagnetic separation and a chromogenic isolation medium.

Faecal samples, collected from 200 healthy animals in Antwerp Zoo, were examined for the presence of pathogenic Listeria spp. A two-stage standard isolation (ISO) method was combined with immunomagnetic separation (IMS). ALOA agar, a chromogenic isolation medium, differentiating Listeria spp. on the basis of beta-glucosidase and phosphatidylinositol-specific phospholipase C (PIPLC) activity, was compared with PALCAM agar. Confirmation of the isolates was based on conventional biochemical tests and a disc test, which detects a specific aminopeptidase produced by all Listeria spp. except Listeria monocytogenes. Listeria spp. were isolated from 42 (21.0%), L. monocytogenes from 14 (7.0%), and Listeria ivanovii from two (1.0%) faecal samples. The application of IMS after primary enrichment detected pathogenic Listeria spp. in 12 (6.0%) samples. The ISO method, combining primary and secondary enrichment, detected pathogenic Listeria spp. in 15 (7.5%) samples. The sensitivity of IMS compared to the ISO method was 73.3% and the specificity was 99.5%. ALOA agar was superior to PALCAM agar for isolation of Listeria spp. The disc test identified all L. monocytogenes isolates. IMS after primary enrichment was a suitable screening method, but secondary enrichment increased the number of positive samples.

Animals↗

PubMed1996

Changes in sperm-bound amidase activity suggest subtle damage to ram sperm acrosomes by freezing/thawing, not detected by light microscopy.

We have measured sperm-bound amidase activity in fresh, cooled and frozen/thawed ram spermatozoa, in order to study if freezing and thawing led to some degree of acrosome damage of motile/viable spermatozoa not detected by optical methods. This assay was based on the fact that membrane damage would result in an increased access of the enzyme substrate to the sperm acrosome. Semen was collected from adult Australian Merino rams, and spermatozoa were washed by centrifugation through a Ficoll solution. Sperm-bound amidase activity was measured in whole spermatozoa using the protease substrate benzoyl-arginyl-p-nitroanilide (BAPNA). Acrosomal status was also assessed by light microscopy after Giemsa staining. Most amidase activity was shown to be sperm-bound, as only a minor fraction of the enzyme activity was release into the medium after induced damage. Simultaneous assessment of sperm-bound amidase activity and the percentage of spermatozoa with microscopically evident acrosomal damage, after mild sonication for different times, showed a high correlation between both parameters (r = 0.97, p < 0.001). In separate experiments, fresh, cooled and frozen/thawed semen samples were filtered through Sephadex G-10 to obtain a subpopulation of motile, mostly acrosome-intact spermatozoa. As controls, spermatozoa from the same samples to which extensive acrosome damage was induced were evaluated. Slow cooling to 4 degrees C had no effect on amidase activity or percent acrosomal damage with respect to fresh samples. Freezing and thawing resulted in a sperm population that, after filtration through Sephadex, had a low percentage of acrosome damage (9.4%, vs. 2.1% for fresh filtered controls), which was 11% of that obtained after extensive acrosome damage (83%). However, amidase activity in these samples was markedly increased, showing values of activity that were 56% of those obtained in extensively damaged spermatozoa. This effect was not due to an alteration in the enzyme kinetics. We conclude that sperm-bound amidase activity is useful to detect subtle changes, provoked by a standard freezing/thawing procedure, in the permeability of acrosomes from ram spermatozoa which are not detected by direct observation of the acrosomes after Giemsa staining.

Acrosome↗

PubMed2000

Antithrombin activity of medicinal plants of the Azores.

A chromogenic bioassay was utilized to determine the antithrombin activity of methylene chloride and methanol extracts prepared from 50 plants of Azores. Extracts of the six plants Hedychium gardneranum, Tropaeolum majus, Gunnera tinctoria, Hedera helix, Festuca jubata and Laurus azorica demonstrated activity of 78% or higher in this bioassay system.

Azores↗

PubMed1985

Quantitative determination of bacterial endotoxin in middle ear effusions by chromogenic substrate method.

It is known that bacterial endotoxin on the outer surface of most gram-negative bacteria (GNB) is not only biologically active material, but also the modulator of the immune response. The earlier experiment documented that the endotoxin of killed Haemophilus influenzae was responsible for induction of chronic otitis media with effusion (OME). Recent study by DeMaria et al. determined the endotoxin of human middle ear effusion (MEE) by means of limulus amebocyte lysate assay. It was concluded the endotoxin was present in a high percentage of the effusion in human tympanic cavities. The present study was undertaken to quantitatively determine the endotoxin in human MEE by use of chromogenic substrate technique which is more sensitive and accurate than limulus assay. Sixty samples of mucoid and serous effusion were subjected to chromogenic substrate method. It was revealed that the mucoid effusion showed a significantly high level of endotoxin at 282 pg/ml in average, though the serous effusion contained only 35.9 pg/ml. It is assumed that the bacterial endotoxin may greatly contribute to the pathogenesis of otitis media with mucoid effusion.

Adolescent↗

PubMed1997

Quantitation of taurolidine decomposition in polymer solutions by chromotropic acid formaldehyde assay method.

A sensitive, selective, and specific assay was needed to study the degradation kinetics of taurolidine and its stabilization by polyvinylpyrrolidone (PVP). The purpose of the present study was to evaluate the usefulness of the chromotropic acid method and other formaldehyde or amine derivatization methods. The methods evaluated included formaldehyde derivatization with chromotropic acid, acetylacetone, 4-amino-5-hydrazino-3-mercapto-1,2,4-triazole, semicarbazide hydrochloride, or 2,4-dinitrophenylhydrazine and taurolidine decomposition product derivatization with dansylchloride or 7-chloro-4-nitrobenz-2-oxa-1,3-diazole chloride. Results indicated that the chromotropic acid method provided sufficient selectivity, reproducibility and sensitivity. It was able to quench taurultam decomposition and avoided PVP interference. The method was optimized by performance based selection of reagent lots, appropriate reagent storage and preparation, and controlled derivatization conditions. In conclusion, the optimized chromotropic method was the most appropriate method for quantitating taurolidine decomposition.

Anti-Infective Agents↗