Chromogenic Substrate (chromogenic + substrate)

Distribution by Scientific Domains


Selected Abstracts


Facile Synthesis of 2,4-Dinitrophenyl ,- D -Glycopyranosides as Chromogenic Substrates for ,-Glycosidases

CHEMBIOCHEM, Issue 7 2007
Hong-Ming Chen Dr.
Pure and simple. The efficient synthesis of chromogenic aryl glycoside substrates for N -acetyl-,-hexosaminidases, which were previously troublesome to prepare, is described (see scheme). This one-pot arylation/anomerisation sequence gives access to a range of aryl ,-glycosides, and minimizes decomposition of ,-linked intermediates in the case of the 2-acetamido sugars. [source]


B-domain deleted recombinant factor VIII preparations are bioequivalent to a monoclonal antibody purified plasma-derived factor VIII concentrate: a randomized, three-way crossover study

HAEMOPHILIA, Issue 2 2005
C. M. Kessler
Summary., Background:, Deletion of the B-domain of recombinant blood coagulation factor VIII (BDDrFVIII) increases the manufacturing yield of the product but does not impair in vitro or in vivo functionality. BDDrFVIII (ReFacto®) has been developed with the additional benefit of being formulated without human albumin. Objective:, The primary objective of this three-way crossover-design study was to compare the pharmacokinetic (PK) parameters of two BDDrFVIII formulations (one reconstituted with 5 mL of sterile water, the other reconstituted with 4 mL sodium chloride 0.9% USP) with those of a plasma-derived, full-length FVIII preparation (Hemofil® M) in patients with haemophilia A to determine bioequivalence. Methods:, A series of blood samples were collected over a period of 48 h after i.v. administration of each of the FVIII preparations. Plasma FVIII activity was determined using a validated chromogenic substrate assay. Plasma FVIII activity vs. time curves was characterized for a standard set of PK parameter estimates. Two parameter estimates, the maximum plasma concentration (Cmax) and the area under plasma concentration vs. time curves (AUCs), were used to evaluate bioequivalence. The two preparations were considered bioequivalent if the 90% confidence intervals for the ratio of geometric means for Cmax and AUCs fell within the bioequivalence window of 80% to 125%. Results/Conclusion:, Results show that each BDDrFVIII formulation is bioequivalent to Hemofil M and the two formulations of BDDrFVIII are bioequivalent to each other. [source]


Pharmacokinetics of factor VIII and factor IX

HAEMOPHILIA, Issue 2003
M. Morfini
Summary., A survey of principal pharmacokinetic (PK) studies on factor VIII (FVIII) and factor IX (FIX) plasma- and rDNA-derived concentrates, analysed by means of the PKRD program, has been performed. Notwithstanding the accurate definition of the study design, released in 1991 by the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (SSC-ISTH), a large variability of PK parameters has been pointed out. In the majority of the PK studies, the size of the population is small. In this situation, a careful individualization of haemophilia therapy is strongly recommended. The tailored prediction of loading and maintenance dosages and the need for strict control of trough FVIII/IX levels are mandatory not only to decrease the risk of bleeds but also to spare financial resources. Recently, the old problem of FVIII assay standardization has again become a concern among physicians, especially after the introduction of B-domain deleted rFVIII concentrate. The discrepancies between the widely used one-stage clotting assay and the chromogenic substrate assay seem to be solved by the introduction of a product-specific laboratory standard. [source]


Granulocyte elastase, matrix metalloproteinase-8 and prostaglandin E2 in gingival crevicular fluid in matched clinical sites in smokers and non-smokers with persistent periodontitis

JOURNAL OF CLINICAL PERIODONTOLOGY, Issue 5 2002
B. Söder
Abstract Background/aims: Smokers with persistent periodontitis may have granulocytes with impaired function. This study aimed to determine the levels of granulocyte elastase, matrix metalloproteinase-8 (MMP-8) and prostaglandin E2 (PGE2) in gingival crevicular fluid (GCF) in smokers and non-smokers with persistent periodontitis. Methods: We analyzed GCF from 70 matched sites in 29 periodontitis and 6 gingivitis sites in 34 subjects, 17 smokers, and 17 non-smokers. We also analyzed separately GCF from 28 of these subjects, 14 smokers and 14 non-smokers in 14 matched periodontitis sites. The following measurements were made: elastase complexed to ,1 -antitrypsin (EA-,1AT) and MMP-8 with ELISA, functional elastase with a chromogenic substrate, and PGE2 with radioimmunoassay (125I RIA). The significance of the findings was determined with Mann-Whitney test. Results: In the 29 matched periodontitis sites, smokers had significantly more functional elastase (p<0.005) and more EA-,1AT (p<0.05) than non-smokers. In the 14 matched periodontitis sites in 14 smokers and 14 non-smokers, the former had significantly more functional elastase than the latter (p<0.001). A significant correlation was found between EA-,1AT and MMP-8 in smokers (p<0.05) and non-smokers (p<0.001) and a positive correlation between levels of functional elastase and MMP-8 in non-smokers (r2=0.98; p<0.001). Conclusions: Granulocyte function seems to be impaired in smokers with persistent periodontitis. The cells react to the bacterial challenge by releasing serine proteases, which reflect the degradation of connective tissue. The risk of progression of the disease is therefore higher in smokers with persistent periodontitis than in non-smokers. Zusammenfassung Hintergrund, Ziele: Raucher mit bestehender Parodontitis haben möglicherweise Granulozyten mit beeinträchtigter Funktion. Diese Studie zielt auf die Bestimmung der Levels von Granulozytenelastase, Matrix-Metalloproteinase-8 (MMP-8) und Prostaglandin E2 (PGE2) in der krevikulären gingivalen Flüssigkeit (GCF) bei Rauchern und Nichtrauchern mit bestehender Parodontitis. Methoden: Wir analysierten GCF von 70 entsprechenden Flächen bei 29 Parodontitis und 6 Gingivitisflächen von 34 Personen, 17 Rauchern und 17 Nichtrauchern. Wir analysierten zusätzlich getrennt die GCF von 28 dieser Personen: 14 Raucher und 14 Nichtraucher von 14 entsprechenden parodontalen Flächen. Die folgenden Messungen wurden vorgenommen: Elastasekomplex zu ,1 -Antitrypsin (EA-,1AT) und MMP-8 mit ELISA, funktionelle Elastase mit chromogenem Substrat und PGE2 mit Radioimmunoassay (125I RIA). Die Signifikanz der Ergebnisse wurde mit dem Mann-Whitney Test bestimmt. Ergebnisse: In den 29 entsprechenden parodontalen Flächen hatten die Raucher signifikant mehr funktionelle Elastase (p<0.005) und mehr EA-,1At (p<0.05) als Nichtraucher. Bei den 14 entsprechenden parodontalen Flächen der 14 Raucher und 14 Nichtraucher hatten die ersten signifikant mehr funktionelle Elastase als die letzteren (p<0.001). Eine signifikante Korrelation wurde zwischen EA-,1AT und MMP-8 bei Rauchern (p<0.05) und Nichtrauchern (p<0.001) gefunden und eine positive Korrelation zwischen den Levels der funktionellen Elastase und MMP-8 bei Nichtrauchern (r2=0.98; p<0.001) festgestellt. Schlussfolgerungen: Die Granulozytenfunktion scheint bei Rauchern mit bestehender Parodontitis beeinträchtigt zu sein. Die Zellen reagieren auf die bakterielle Herausforderung durch Freisetzung von Serinproteasen, die die Degradation von Bindegewebe reflektiert. Das Risiko einer Progression dieser Erkrankung ist deshalb bei Rauchern mit bestehender Parodontitis höher als bei Nichtrauchern. Résumé Origine, but: Les fumeurs avec parodontite persistante pourraient avoir des granulocytes ayant des fonctions déréglées. Cette étude a eu pour but de déterminer les niveaux d'élastase granulocytaire, de la métallo-protéinase-8 de la matrice (MMP-8) et de la prostaglandine E2 (PGE2) dans le fluide créviculaire gingival (GCF) chez les fumeurs et les non-fumeurs avec parodontite persistante. Méthodes: Le GCF a été prélevé de 70 sites équivalents dans 29 parodontites et 6 sites avec gingivite chez 34 sujets, 17 fumeurs et 17 non-fumeurs. Le GCF de 28 de ces sujets a été analysé séparément, 14 fumeurs et 14 non-fumeurs dans 14 sites équivalents du point de vue parodontite. Les mesures suivantes ont été relevées: l'élastase avec ,1 -antitrypsine (EA-,1AT) et MMP-8 par ELISA, l'élastase fonctionnelle avec un substrat chromogénique, et PGE2 avec un essai radio-immunitaire (125I RIA). La signification de ces découvertes a été par l'utilisation du test de Mann-Whitney. Résultats: Dans les 29 sites équivalents, les fumeurs avaient significativement plus d'élastase functionnelle (p<0.005) et plus de EA-,1AT (p<0.05) que les non-fumeurs. Dans les 14 sites équivalents du point de vue parodontite, les 14 fumeurs avaient significativement plus d'élastase fonctionnelle que les 14 non-fumeurs (p<0.001). Une relation significative a étéétablie entre EA-,1AT et MMP-8 chez les fumeurs (p<0.05) et les non-fumeurs (p<0.001) et une relation positive entre les niveaux d'élastase fonctionnelle et de MMP-8 chez les non-fumeurs (r2=0.98; p<0.001). Conclusions: La fonction granulocytaire semble être altérée chez les fumeurs avec parodontite persistante. Les cellules réagissent à l'attaque bactérienne en relâchant des protéases sérine, ce qui démontre une dégradation du tissu conjonctif. Le risque de progression de la maladie est ainsi plus élevé chez les fumeurs avec parodontite persistante que chez les non-fumeurs. [source]


Application of an enhanced luminol chemiluminescence reaction using 4-[4,5-di(2-pyridyl)-1H -imidazol-2-yl]phenylboronic acid to photographic detection of horseradish peroxidase on a membrane

LUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 2 2001
Naotaka Kuroda
Abstract Photographic detection of horseradish peroxidase (HRP) on a membrane by the luminol,hydrogen peroxide,HRP chemiluminescence reaction using 4-[4,5-di(2-pyridyl)-1H -imidazol-2-yl]phenylboronic acid (DPPA) as an enhancer is described. The method is based on the long-lived chemiluminescence emission obtained by using DPPA. Under the optimum conditions, as little as 0.10,ng (ca. 2.3 fmol) and 0.20,ng (ca. 4.6 fmol) per spot of HRP on a membrane were detected as visible spots with exposure time of 60 and 10,min, respectively, by using an instant photographic film and a camera luminometer. The proposed method was highly sensitive and was successfully applied to the detection of HRP conjugates as an alternative to the colorimetric method using a chromogenic substrate in a commercially available assay kit of Western blotting. Copyright © 2001 John Wiley & Sons, Ltd. [source]


Studies on the aminopeptidase activities of Porphyromonas gingivalis

MOLECULAR ORAL MICROBIOLOGY, Issue 4 2001
D. Grenier
Porphyromonas gingivalis is an asaccharolytic bacterium that requires nitrogen substrates as carbon and energy sources. The aims of this study were to investigate the aminopeptidase activities of P. gingivalis and to evaluate the effect of aminopeptidase inhibitors on bacterial growth. Only arginine aminopeptidase and dipeptidyl aminopeptidase IV activities were detected. Experimental evidence was obtained suggesting that the Arg-gingipains of P. gingivalis can function as both an endopeptidase and an aminopeptidase. Firstly, the arginine aminopeptidase activity was found to be inhibited by leupeptin, a well-known inhibitor of Arg-gingipain activity. Secondly, a preparation of Arg-gingipain activity could hydrolyze the chromogenic substrate for arginine aminopeptidase. Lastly, a mutant of P. gingivalis constructed via gene disruption by use of suicide plasmids and deficient in both Arg-gingipain A and B was also devoid of arginine aminopeptidase activity. To investigate the key role of aminopeptidase activities in growth of P. gingivalis, aminopeptidase inhibitors were incorporated in the culture medium prior to inoculation. Bestatin and actinonin were the only ones to inhibit growth of P. gingivalis. Their mechanism of growth inhibition appears to be different but does not involve inhibition of the two major aminopeptidase activities (arginine aminopeptidase and dipeptidyl aminopeptidase IV). [source]


Identification of mutants in phosphorus metabolism

ANNALS OF APPLIED BIOLOGY, Issue 1 2001
JULIE C LLOYD
Summary Phosphorus availability is often limiting for plant growth. However, little is known of the pathways and mechanisms that regulate phosphorus (P) uptake and distribution in plants. We have developed a screen based on the induction of secreted root acid phosphatase activity by low-P stress to identify mutants of Arabidopsis thaliana with defects in P metabolism. Acid phosphatase activity was detected visually in the roots of A. thaliana seedlings grown in vitro on low-P medium, using the chromogenic substrate, 5-bromo-4-chloro-3-indolyl-phosphate (BCIP). In low-P stress conditions the roots of wild-type plants stained blue, as the induced root acid phosphatase cleaved BCIP to release the coloured product. Potential mutants were identified as having white, or pale blue, roots under these conditions. Out of approximately 79 000 T-DNA mutagenised seedlings screened, two mutants with reduced acid phosphatase staining were further characterised. Both exhibited reduced growth and differences in their P contents when compared to wild-type A. thaliana. The mutant with the most severe phenotype, pho3, accumulated high levels of anthocyanins and starch in a distinctive visual pattern within the leaves. The phenotypes of these mutants are distinct from two previously identified phosphorus mutants (phol and pho2) and from an acid phosphatase deficient mutant (pupl) of A. thaliana. This suggested that the screening method was robust and might lead to the identification of further mutants with the potential for increasing our understanding of P nutrition. [source]


Expression, purification, and characterization of pro-phenoloxidase-activating serine protease from Spodoptera litura

ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2009
Naresh Arora
Abstract One of the important trigger molecules for innate immunity is a serine protease that activates zymogen phenol oxidase (PPO). Central to wound healing response is the activation of phenol oxidase zymogen. Molecular characterization of phenol oxidase has been recently reported by us. Here, we report isolation, cloning, expression, and purification of prophenol oxidase activating enzyme 1 (slppae1) from polyphagous pest, Spodoptera litura. SLPPAE1 is induced within 6,h of physical injury. The structural features of the mature polypeptide are reminiscent of other lepidopteran PPAE in having a signal peptide, propeptide, and catalytically active polypeptide. The cDNA has been expressed in Sf21 cells using baculovirus expression vector. Fractionation of expressing Sf21 cells revealed its expression in the membranes. The recombinant protein was solubilized from membranes and purified by Ni-NTA affinity chromatography. The purified enzyme is catalytically active on chromogenic substrate, activates recombinantly expressed prophenol oxidase (PPO) of S. litura, and is sensitive to inhibition by aprotenin. N-terminal sequencing of processed phenol oxidase revealed 11,kDa propeptide instead of in-silico predicted 6,kDa polypeptide. © 2009 Wiley Periodicals, Inc. [source]


Secretion of proteases in serglycin transfected Madin,Darby canine kidney cells

FEBS JOURNAL, Issue 3 2006
Lillian Zernichow
Madin,Darby canine kidney (MDCK) cells, which do not normally express the proteoglycan (PG) serglycin, were stably transfected with cDNA for human serglycin fused to a polyhistidine tag (His-tag). Clones with different levels of serglycin mRNA expression were generated. One clone with lower and one with higher serglycin mRNA expression were selected for this study. 35S-labelled serglycin in cell fractions and conditioned media was isolated using HisTrap affinity chromatography. Serglycin could also be detected in conditioned media using western blotting. To investigate the possible importance of serglycin linked to protease secretion, enzyme activities using chromogenic substrates and zymography were measured in cell fractions and serum-free conditioned media of the different clones. Cells were cultured in both the absence and presence of phorbol 12-myristate 13-acetate (PMA). In general, enzyme secretion was strongly enhanced by treatment with PMA. Our analyses revealed that the clone with the highest serglycin mRNA expression, level of HisTrap isolated 35S-labelled serglycin, and amount of serglycin core protein as detected by western blotting, also showed the highest secretion of proteases. Transfection of serglycin into MDCK cells clearly leads to changes in secretion levels of secreted endogenous proteases, and could provide further insight into the biosynthesis and secretion of serglycin and potential partner molecules. [source]


The application of chromogenic media in clinical microbiology

JOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2007
J.D. Perry
Summary Since 1990, a wide range of chromogenic culture media has been made commercially available providing useful tools for diagnostic clinical microbiology. By the inclusion of chromogenic enzyme substrates targeting microbial enzymes, such media are able to target pathogens with high specificity. Examples of target pathogens include Staphylococcus aureus, Streptococcus agalactiae, Salmonella spp. and Candida spp. The inclusion of multiple chromogenic substrates into culture media facilitates the differentiation of polymicrobial cultures, thus allowing for the development of improved media for diagnosis of urinary tract infections and media for the enhanced discrimination of yeasts. The purpose of this review is to provide some insight into how such media work and appraise their utility in routine clinical diagnostics, in comparison with conventional media. [source]


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

LETTERS IN APPLIED MICROBIOLOGY, Issue 2 2005
L.N. Ten
Abstract 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. [source]


Increased activity of plasma and tissue kallikreins, plasma kininase II and salivary kallikrein in pemphigus foliaceus (fogo selvagem)

BRITISH JOURNAL OF DERMATOLOGY, Issue 4 2005
T.B. Rosatelli
Summary Background, Pemphigus foliaceus (PF) is an autoimmune blistering disease of unknown aetiology, which is endemic in Brazil. Although the pathogenesis of PF is still unknown, proteins of the contact system have been implicated. Objectives, As the components of the kinin system may interact with those of the contact system, in this study we evaluated the plasma levels of high-molecular-weight kininogen (HK) and low-molecular-weight kininogen (LK), and the activity of plasma kallikrein, tissue kallikrein and kininase II in plasma of patients with PF presenting with Nikolsky's sign. As kidneys and salivary glands are relevant sources of tissue kallikrein for plasma, we also evaluated urinary/salivary kallikrein and urinary kininase II activities. Methods, Fifteen patients and 15 age- and sex-matched controls were studied. Kininogen levels were determined by enzyme-linked immunosorbent assay, and the activities of kallikreins and kininase II were determined using selective chromogenic substrates. Results, Compared with controls, plasma HK levels were decreased (P = 0·031), whereas the activities of plasma kallikrein, tissue kallikrein and kininase II in plasma, and the activity of salivary kallikrein, were increased in patients (P < 0·001 for each comparison). Plasma levels of LK and the activities of urinary kallikrein and urinary kininase II were not significantly different from controls. Conclusions, Diminished levels of HK associated with increased activities of plasma kallikrein and kininase II indicate that the kinin system is activated at the systemic level in PF. As active plasma kallikreins may act on some proteins of the contact system, it is possible that the enzyme may contribute to blister formation. The further observation of an increased tissue kallikrein activity at the systemic and saliva levels may be interpreted as a systemic reflex of skin inflammation. Whether the activation of the kinin system is a cause or a consequence of blister formation needs further clarification. [source]