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PubMed1998

Spectrophotometric determination of some catecholamine drugs using sodium bismuthate.

A novel spectrophotometric method is described for the determination of epinephrine (EP) and norepinephrine (NE). The method is based on the development of a red colour with sodium bismuthate, as a sensitive chromogenic reagent, in aqueous medium at pH 3. Oxidation of these catecholamines produces aminochrome derivatives which can be measured spectrophotometrically at 486.0 nm. Calibration graphs are linear in the range 4.8-800 (micromol l(-1)) for epinephrine bitartarate and 4.8-600 (micromol l(-1)) for norepinephrine bitartarate with detection limits of 0.26 (micromol l(-1)) and 2.46 (micromol l(-1)) for epinephrine and norepinephrine bitartarate salts, respectively. The method has successfully been applied to determination of these catecholamines in pharmaceutical preparations.

Antioxidants↗

PubMed2000

"Room temperature" use of CHROMagar Candida.

The color of colonies of 9 Candida species was examined on the chromogenic medium CHROMagar Candida incubated for 24-72 h at 25 degrees C, 30 degrees C or 37 degrees C. Colors and colony forms characteristic of C. albicans, C. dubliniensis, C. krusei and C. tropicalis were formed most rapidly and with the deepest hues at 37 degrees C. After 48 h incubation at 25 degrees C, 9 of 48 C. albicans isolates gave pink colonies instead of the green colonies characteristic for the species, and the blue-purple colony color characteristic of C. tropicalis isolates was not formed until 48 h at 25 degrees C. Incubation of the chromogenic medium at temperatures below 30 degrees C cannot be recommended for reliable presumptive identification of Candida spp., and pink colonies of C. glabrata would not be reliably distinguished from pink colonies formed by other species under any of the incubation conditions used.

Candida↗

PubMed1998

Evaluation of CHROMagar Candida for presumptive identification of clinically important Candida species.

CHROMagar Candida is a recently described and differential medium for the isolation and the presumptive identification of clinically important yeasts. We evaluated it with 262 yeast strains from clinical specimens, including 173 Candida albicans, 21 Candida tropicalis, 8 Candida krusei, 49 Candida glabrata, and 12 strains of other yeast species. Strains were presumptively identified on the basis of colony color and texture. These observations were compared with conventional identification results. Candida albicans was identified correctly in 170 (98%) of the 173 strains. A total of 46 of the 205 specimens that were plated on CHROMagar contained mixed cultures of yeast. Thirty-seven (80%) of these mixed cultures were not detected in the original specimens. CHROMagar Candida was useful for the rapid presumptive identification of Candida albicans and facilitated the recognition of mixed cultures. For other yeast species, it may provide additional information to laboratories that do not regularly perform identifications beyond the germ tube test.

Candida albicans↗

PubMedDate not supplied

Detection of aminopeptidases in Enterobacteriaceae by three simple chromogenic tests.

The action of 907 strains belonging to the various genera and species of the family Enterobacteriaceae were assayed on three chromogenic substrates used for the detection of aminopeptidases. The observed patterns of reactions may be useful for the biochemical identification of some serovars and biovars, particularly in the genus Shigella.

Aminopeptidases↗

PubMedDate not supplied

[Quantitative determination of bacterial endotoxins by the chromogenic limulus method: critical analysis and study of interactions between 3 divalent cations].

The reproducibility of the limulus amoebocyte lysate (LAL) test with the use of a chromogenic substrate was evaluated in the presence of 5 bacterial endotoxins (2 from Escherichia coli, 1 from Salmonella, 1 from Serratia marcescens and 1 from Vibrio cholerae) and the FDA reference endotoxin EC5. Endotoxins from E. coli 055B5, S. marcescens and V. cholerae were significantly more active than the reference endotoxin (p less than 0.01). Addition of Mg2+ activated the LAL chromogenic reaction (optimum = 160 mmol/l added), whereas Ca2+ in concentrations above 5 mmol/l inhibited the reaction. As little as 0.3 mmol/l Zn2+ strongly inhibited the reaction. Inhibition by Zn2+ was partly suppressed by addition of 160 mmol/l Mg2+. These divalent cations modified the LAL chromogenic reaction when added in the first step of the reaction (incubation with endotoxin and LAL reagent). The LAL chromogenic reaction was not modified by divalent cations when these were added in the second step of the reaction (with the chromogenic substrate).

Animals↗

PubMed2000

Hydrogen peroxide release from human eosinophils on fibronectin: scopoletin enhances eosinophil activation.

We have examined the release of H(2)O(2) from PAF or TNFalpha-stimulated human eosinophils on fibronectin (FN)-coated polystyrene plates. H(2)O(2) release was measured by the standard scopoletin-horseradish peroxidase (SCOP-HRP) method and compared with that measured by a new microplate fluorescent assay for H(2)O(2) using a novel HRP substrate A6550. We observed that the SCOP-HRP method gave a 25-fold higher estimate of H(2)O(2) release from eosinophils than did the A6550-HRP method. Microscopic examination of PAF or TNFalpha-stimulated eosinophils in buffer alone or A6550-HRP reaction mixture showed that the cells remained generally round, while eosinophils in SCOP-HRP reaction mixture were spread on the fibronectin-coated surface. Measurement of the cellular ATP content after PAF-stimulation showed that only eosinophils activated in SCOP-HRP had a 50% fall in ATP content. This supported our conclusion that measurement of H(2)O(2) release from eosinophils in SCOP-HRP reaction mixture is problematic since the SCOP-HRP system activates eosinophils. However, we also found that A6550-HRP, when present throughout the incubation, resulted in a lower estimate of H(2)O(2) release than expected. The method used to detect eosinophil H(2)O(2) release greatly influences the absolute amount of H(2)O(2) detected.

Adenosine Triphosphate↗

PubMed2003

Listeria: growth, phenotypic differentiation and molecular microbiology.

The identification of Listeria species is based on a limited number of biochemical markers, among which absence or presence of hemolysis and arylamidase are used to differentiate between L. monocytogenes and L. innocua. The CAMP (Christie, Atkins, Munch-Petersen) test must be interpreted with caution. Chromogenic media are based on both the specific chromogenic detection of phosphatidylinositol phospholipase C and the xylose fermentation and give specific and direct identification of L. monocytogenes and L. ivanovii. Isolates of L. monocytogenes with atypical properties require tools of molecular biology for final identification. Serotyping, although not allowing speciation, serves a useful purpose for confirming the genus diagnosis Listeria. Polymerase chain reaction is particularly useful when prior administration of antimicrobial agents compromises culture. For clinical specimens the importance of trying to isolate the pathogen as a prerequisite for an epidemiological work-up and finally for prevention of further cases cannot be overstressed.

Animals↗

PubMed1989

Few-minutes tests for the identification of group A streptococci and enterococci with chromogenic substrates.

The usefulness of paper strip tests for rapid identification of Streptococcus pyogenes and enterococci cultured on blood agar plates was investigated. The paper strips used contain dried chromogenic substrates for pyrrolidonyl peptidase (PYRase) and beta-glucosidase (beta-Gluc). Material from only a few colonies needed to be applied to the test strips. The reactions could be read after two min (PYRase) and 5-7 min (beta-Gluc), respectively. Results from testing 503 streptococcal strains were evaluated. The reactions proved very useful for rapid differentiation of S. pyogenes and enterococci from human sources. Nearly all strains of these streptococcal species showed positive reactions in the PYRase test whereas only the enterococci (E. faecalis and E. faecium) were positive for both enzymes.

Chromogenic Compounds↗

PubMed1987

Monitoring heparin and haemostasis during reconstruction of the abdominal aorta.

In spite of its unpredictable kinetics, heparin is still not generally monitored during peripheral vascular surgery. To evaluate heparin levels and neutralisation, plasma heparin concentrations were measured using a chromogenic substate method during 20 consecutive operations on the Abdominal Aorta. This was combined with measuring activated partial thromboplastin time (APTT), thrombin time (ThT), prothrombin time (PT), antithrombin-III (AT-III) and fibrinogen concentration. Heparin concentration 5 min after administration and the elimination rate showed a wide variation. Using a standard dosage for all patients resulted in plasma heparin levels that are potentially too low in some patients. The APTT and ThT were found to be unsuitable for an exact calculation of heparin levels. Protamine administration based on the surgeon's judgement of haemostasis was inadequate. Furthermore an intraoperative decrease of AT-III and fibrinogen was seen in eight patients. It is advisable and possible to have direct monitoring of heparin concentration during peripheral vascular surgery.

Aorta, Abdominal↗

PubMed2003

Molecular and functional maturation of the murine cardiac conduction system.

The rhythmic heartbeat reflects a highly coordinated electrical wave, initiated and propagated throughout the myocardium by the specialized cardiac conduction system (CCS). Conduction system abnormalities affect millions of individuals, with symptoms from merely bothersome palpitations to fainting or even sudden death. Despite years of investigation, knowledge of the genes and molecules that direct CCS development and function are incomplete. In 1977, Viragh and Challice published the first in a series of articles describing the morphologic development of the murine CCS. The framework established by those reports now can be revisited in light of recent data. Imaging the developing CCS-using genetically engineered mice combined with functional assays, including optical mapping of cardiac electrical excitation-reveal some of the earliest events in CCS formation and function. These findings build on the experiments of Viragh and Challice, leading to a more integrated picture of conduction system development in the mammalian embryo.

Animals↗

PubMed2003

Clinical evaluation of resilient denture liners. Part 2: Candida count and speciation.

PURPOSE: The purpose of this study was to count and to speciate Candida isolated from 2 resilient denture liners, Molloplast-B and MPDS-SL. MATERIALS AND METHODS: A group of 20 patients each had 1 maxillary denture and 2 mandibular dentures fabricated. One mandibular denture was lined with Molloplast-B, and 1 was lined with MPDS-SL. Each denture was used for 3 months. At the end of the 3-month period, the mandibular denture was surrendered, and a 5 x 5-mm circular resilient liner sample was obtained from the tissue surface of the lingual flange. Samples were processed, and Candida was isolated and counted. Speciation of Candida was performed using CHROMagar Candida and API 20C AUX strips. RESULTS: Molloplast-B had, on average, 5 times as many CFU/sample as MPDSL-SL, but this difference was not significant (p = 0.26). A sign test gave a similar nonsignificant trend (p = 0.057). CHROMagar identified several Candida species, and confirmation was made using API 20C AUX strips. One patient was lost to follow-up. Of 19 Molloplast-B samples, 7 had no growth, 4 grew C. albicans, 3 grew C. parapsilosis, 2 grew C. glabrata, 1 grew C. tropicalis, 2 grew a Trichosporon spp., and 2 grew a nonidentifiable colony. The analogous counts for 19 MPDS-SL samples were 10, 4, 1, 3, 0, 1, and 1 (p = 0.45 for culture positively, exact McNemar test). CONCLUSIONS: Candida growth on Molloplast-B was not significantly different from growth on MPDS-SL. Several yeast species were cultured from each material. The rates of culture-positive testing did not differ between the 2 resilient denture liners.

Adult↗

PubMed1997

Expression of smooth-muscle actin in cultured cells from human plasmacytoid myoepithelioma.

OBJECTIVE: The definition of plasmacytoid myoepithelioma, a neoplasm exhibiting myoepithelial differentiation, has been recently questioned. To better understand the histogenesis of this neoplasm, we searched for myoepithelial markers in histologic sections of plasmacytoid myoepithelioma and in a cell line (M1) derived from this same neoplasm. STUDY DESIGN: Expression of vimentin, pan-keratin (AE-3) and smooth-muscle actin was studied by immunohistochemistry in paraffin-embedded tissue and by immunofluorescence in M1 cells. RESULTS: Plasmacytoid myoepithelioma tumor sections showed vimentin and AE-3 reactivity, but evidence of smooth-muscle actin was not seen. The cell line derived from this tumor also produced vimentin and cytokeratin. In addition, all cultured cells expressed smooth-muscle actin. CONCLUSION: We demonstrated that cells derived from a case of plasmacytoid myoepithelioma appear to show full myoepithelial differentiation in vitro. Thus, they are myoepithelial-like cells in nature. The lack of myogenous differentiation in vivo could be due to an inhibitory process mediated by the extracellular matrix.

3,3'-Diaminobenzidine↗

PubMed2002

Actions of two serine proteases from Trimeresurus jerdonii venom on chromogenic substrates and fibrinogen.

Jerdonobin and jerdofibrase are two serine proteases purified from the venom of Trimeresurus jerdonii. The Michaelis constant K(m) and the catalytic rate constant K(cat) of jerdonobin or jerdofibrase on three chromogenic substrates, H-D-Pro-Phe-Arg-pNA (S2302), H-D-Phe-pipecolyl-Arg-pNA (S2238), and H-D-Val-Leu-Lys-pNA (S2251) were obtained from lineweaver-Burk plots. Jerdofibrase could hydrolyze all three substrates, but jerdonobin had no detectable activity on S2251, suggesting a relatively broader substrate specificity for jerdofibrase than jerdonobin. By SDS-PAGE, jerdofibrase preferentially degraded Bbeta-chain of fibrinogen. It also degraded Aalpha-chain of fibrinogen with relatively slow activity, but did not act on the gamma-chain. In contrast, jerdonobin did not degrade fibrinogen within 12 h. Fibrinopeptides liberation test, identified by HPLC, showed jerdonobin released fibrinopeptide A and a small amount of fibrinopeptide B. Unlike jerdonobin, jerdofibrase mainly released fibrinopeptide B. These results indicate that the two enzymes differ in their ability to hydrolyze chromogenic substrates and in their actions on fibrinogen.

Animals↗

PubMed2006

New and simple plate test for screening relative transfructosylation activity of fungi.

Several microorganisms are reported to have transfructosylation activity due to fructosyltransferase and/or fructofuranosidase activities. However, the search for other fungi with higher transfructosylation activity remains a challenge. So, a presumptive and indirect colorimetric plate assay for the evaluation of transfructosylation activity in fungi was developed which involved the simultaneous determination in the same plate of glucose and fructose released from sucrose. The method entailed the (a) glucose oxidase-peroxidase coupled reaction using phenol and 4-aminoantipyrine for determination of glucose; and (b) fructose dehydrogenase oxidation in the presence of a tetrazolium salt for determination of fructose. The presence of enzymes with transfructosylation activity was identified by the formation of pink (presence of glucose) and blue (presence of fructose) halos around the fungal colony. In conclusion, the results showed that the method is suitable for screening a large number of fungi due to its simplicity, reproducibility and rapidity and also gives a relative quantitative idea of the transfructosylation activity of different fungi species.

Ampyrone↗

PubMed2001

Novel methods for biocatalyst screening.

There have been a number of recent advances in catalysis assays applicable for screening biocatalyst libraries in high-throughput format. These include instrumental assays such as high-performance liquid chromatography, mass spectrometry, capillary electrophoresis and IR-thermography, reagent-based assays producing spectroscopic signals (UV/VIS or fluorescence) in response to reaction progress, and assays based on fluorogenic or chromogenic substrates. These fluorogenic substrates enable the assaying of a variety of enzymes in enantioselective and stereoselective manner, including alcohol dehydrogenases, aldolases, lipases, amidases, epoxide hydrolases and phosphatases.

Carrier Proteins↗

PubMed1997

Tract-tracing in the nervous system of vertebrates using horseradish peroxidase and its conjugates: tracers, chromogens and stabilization for light and electron microscopy.

Surprisingly the first generation of tract-tracing techniques based on intra-axonal transport, that is the methods utilizing the uptake and transport of horseradish peroxidase (HRP), still rank among the most widely used neuroanatomical tracing techniques. The success of these methods can be ascribed to several characteristics. They are fast and easy to implement. Complicated injection apparatus is unnecessary. The reaction products are visualized through simple histochemical reactions, and they are permanent or can be stabilized into permanency. Most usefully, the reaction products are visible in the light and electron microscopes. HRP (mol. wt. 44 kDa) is extracted from the roots of the horseradish plant (Cochlearia armoracia L.). Uptake of HRP into cells occurs via a passive process of endocytosis. Since lectins like wheat germ agglutinin (WGA) and bacterial toxin fragments (subunit B of cholera toxin (CTB)) strongly induce active, receptor-mediated uptake mechanisms, conjugates of these substances with HRP have been successfully applied in sensitive tract-tracing HRP and its conjugates are transported both in anterograde and retrograde direction. Retrograde transport occurs in small vesicles that are incorporated in lysosome-like vacuoles and in the Golgi apparatus. These vesicles differ in membrane properties from the anterogradely transported HRP vesicles. The retrogradely transported vesicles tend to fuse and thus accumulate HRP at high densities, facilitating the visibility of the final reaction product. The anterogradely transported HRP does not accumulate directly in lysosome-like bodies and is distributed diffusely and therefore often requires specific visualization methods. HRP and WGA-HRP may therefore be used in anterograde and retrograde transneuronal (multineuron) transport studies. Even in fixed material, labeling through diffusion of HRP can provide details on neural connections. Visualization of transported HRP is achieved by means of using the oxidative enzymatic activity of HRP to precipitate a chromogen according to the following reaction: [symbol: see text] The final reaction product may be soluble in buffer or ethanol and may require stabilization to prevent fading. In this protocol we discuss the widely used chromogens 3,3'-diaminobenzidine tetrahydrochloride (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), benzidine dihydrochloride (BDHC) and p-phenylenediamine dihydrochloride with pyrocatechol (PPD-PC). Other possible chromogens, not discussed here, are 4-chloro-1-naphthol (4C1N), 3-amino-9-ethylcarbazole (AEC) and o-phenylenediamine (OPD). The visualization of the reaction product can be further improved by intensification with metal salts. At the light microscopic level (LM) this intensification enables color differentiation between distinct markers. In the present protocol we provide an up-to-date guideline for the application of HRP and its conjugates in tracing with special emphasis on electron microscopic (EM) visualization. Some modifications for stabilization and of metal intensification to enhance visibility are incorporated in conjunction with specific methods for multiple labeling in combined tract-tracing experiments.

Animals↗

PubMed2002

Spectrophotometric studies on the simultaneous determination of cadmium and mercury with 4-(2-pyridylazo)-resorcinol.

A new method for direct spectrophotometric determination of cadmium with 4-(2-pyridylazo)-resorcinol is reported. Absorption maximum, molar absorptivity and Sandell's sensitivity of the 1:1 (M:L) complex are 510 nm, 2.5 x 10(5) l mol(-1) cm(-1) and 3.55 ng cm(-2), respectively. A linear calibration graph is obtained up to 4.49 microg ml(-1). The zero-crossing measurement technique is found suitable for the direct measurement of the first-derivative value at the specified wavelengths. Cadmium(II) (0.42-9.2 microg ml(-1)) and mercury(II) (0.35-7.4 microg ml(-1)) in different ratios have been determined simultaneously. A critical evaluation of the proposed method is performed by statistical analysis of the experimental data. The developed method was applied to the simultaneous spectrophotometric determination of Cd and Hg in some synthetic mixtures and was found to give satisfactory results.

Biophysical Phenomena↗

PubMed2002

Facile colorimetric methods for the quantitative determination of tetramisole hydrochloride.

A facile, rapid and sensitive methods for the determination of tetramisole hydrochloride in pure and in dosage forms are described. The procedures are based on the formation of coloured products with the chromogenic reagents alizarin blue BB (I), alizarin red S (II), alizarin violet 3R (III) and alizarin yellow G (IV). The coloured products showed absorption maxima at 605, 468, 631 and 388 nm for I-IV, respectively. The colours obtained were stable for 24 h. The colour system obeyed Beer's law in the concentration range 1.0-36, 0.8-32, 1.2-42 and 0.8-30 microg ml(-1) respectively. The results obtained showed good recoveries with relative standard deviations of 1.27, 0.96, 1.13 and 1.35%, respectively. The detection and determination limits were found to be 1.0 and 3.8, 1.2 and 4.2, 1.0 and 3.9 and finally 1.4 and 4.8 ng ml(-1) for I-IV complexes, respectively. Applications of the method to representative pharmaceutical formulations are represented and the validity assessed by applying the standard addition technique, which is comparable with that obtained using the official method.

Anthraquinones↗