PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Chromogenic Compounds”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 739 records · Page 41Linked to original sources
PubMed2003

New chromogenic dipeptide substrate for continuous assay of the D-alanyl-D-alanine dipeptidase VanX required for high-level vancomycin resistance.

A direct continuous UV-Vis spectrophotometric assay has been developed for VanX, a D-alanyl-D-alanine aminodipeptidase necessary for vancomycin resistance. This method is based on the hydrolysis of the alternative substrate D-alanyl-alpha-(R)-phenylthio-glycine D-Ala-D-Gly(S-Ph)-OH (H-DAla-DPsg-OH, 5a). Spontaneous decomposition of the released phenylthioglycine generates thiophenol, which is quantified using Ellman's reagent. The dipeptide behaved as an excellent substrate of VanX, exhibiting Michaelis-Menten kinetics with a kcat of 76 +/- 5/s and a KM of 0.83 +/- 0.08 mm (kcat = 46 +/- 3/s and KM = 0.11 +/- 0.01 mm for D-Ala-D-Ala). Determination of the kinetic parameters of the previously reported mechanism-based inhibitor D-Ala-D-Gly(SPhip-CHF2)-OH (H-D-Ala-DPfg-OH, 5c) [Araoz, R., Anhalt, E., René, L., Badet-Denisot, M.-A., Courvalin, P. & Badet, B. (2000) Biochemistry 39, 15971-15979] using the substrate reported in the present study yielded values of Kirr of 22 +/- 1 microM and kinact of 9.3 +/- 0.4/min in good agreement with values previously obtained in our laboratory (Kirr = 30 +/- 1 mm; kinact = 7.3 +/- 0.3/min). In addition, inhibition by the competing substrate D-Ala-D-Ala resulted in determination of a Ki = 70 +/- 6 microM close to the previously reported KM value. These results demonstrate that the present assay is a convenient, rapid and sensitive tool in the search for VanX inhibitors.

Bacterial Proteins↗
PubMed2001

Studies on the aminopeptidase activities of Porphyromonas gingivalis.

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).

Adhesins, Bacterial↗
PubMed2001

The purification and characterization of an 88-kDa Porphyromonas endodontalis 35406 protease.

A Porphyromonas endodontalis ATCC 35406 protease was purified from Triton X-114 cell extracts by preparative SDS-PAGE followed by electroelution. The purified enzyme exhibits a molecular size of 88 kDa and was dissociated into two polypeptides of 43 and 41 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The protease (pH optimum 7.5-8.0) degraded the extracellular matrix proteins fibrinogen and fibronectin. Collagen IV was also degraded at 37 degrees C but not at 28 degrees C. The protease also cleaved the bioactive peptide angiotensin at amino acid residue phenylalanine-8 and tyrosine-4 but failed to hydrolyze bradykinin, vasopressin and synthetic chromogenic substrates with phenylalanine or tyrosine at the P1 position. In addition, two peptidases were detected in P. endodontalis cells: a proline aminopeptidase that remained associated with the cell pellet after detergent extraction and peptidase/s that partitioned into the Triton X-114 phase after phase separation and degraded the bioactive peptides bradykinin and vasopressin. These P. endodontalis peptidases and proteases may play an important role in both the nutrition and pathogenicity of these assacharolytic microorganisms. The inactivation of bioactive peptides and degradation of extracellular matrix proteins by bacterial enzymes may contribute to the damage of host tissues accompanied with endodontic infections.

Aminopeptidases↗
PubMed2001

Prevalence of yeast among children in Nigeria and the United States.

Fungal infections have gained considerable importance over the last decade as a result of significant increase in the incidence of opportunistic and systemic candidosis. Although Candida albicans is the predominant causative agent of candidosis, particularly oral disease, recently an epidemiological trend has been observed where other less pathogenic species of Candida, including the newly characterized species Candida dubliniensis, are emerging as significant opportunistic pathogens. The present study aimed to screen for the presence of C. dubliniensis and to compare the recovery of yeast species from 30 seemingly healthy and 30 HIV-positive children in the United States, as well as from 64 malnourished Nigerian children. Oral samples were cultured for fungal growth, and all germ tube and chlamydospore positive isolates were tested for ability to grow at 45 degrees C to differentiate between C. albicans and C. dubliniensis. All isolates were speciated based on colony color production on CHROMagar medium and sugar assimilation profiles. Among the 30 HIV-positive children, 15 (50%) were positive for fungus; 12 were positive for C. albicans, with one of the latter also positive for Candida glabrata, and three were found to harbor C. dubliniensis. Among the 30 non-HIV-positive children, five C. albicans and four C. dubliniensis isolates were recovered. No C. dubliniensis isolates were recovered from the Nigerian group. However, eight other different yeast species were recovered from 31 (48.4%) of the 64 Nigerian children sampled, with six of them growing a combination of species. In comparing the data from the Nigerian and United States children, the frequency of yeasts in the malnourished Nigerian group was considerably higher. The most striking difference between the two groups was in the variety of the usually less encountered and less pathogenic yeast species recovered from the Nigerian population. The findings support previously reported observations that there may be intrinsic differences between different populations sampled and that malnutrition might favor the presence of yeast species other than C. albicans.

AIDS-Related Opportunistic Infections↗
PubMed2002

Interleukin-1beta, tumor necrosis factor-alpha levels and neutrophil elastase activity in peri-implant crevicular fluid.

The aim of this study was to determine interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) levels and neutrophil elastase (NE) activity in peri-implant crevicular fluid (PICF) of smoker and nonsmoker patients, and to investigate their relationships with clinical parameters. A total of 42 endosseous root-form dental implants of 14 patients were clinically examined by modified Plaque index (PI), modified Gingival index (GI) and probing depth (PD). Smoking habits of the patients were recorded. PICF of implants were collected by Periopaper strips and IL-1beta, TNF-alpha levels were determined by enzyme-linked immunosorbent assay (ELISA). NE was analyzed with a neutrophil specific chromogenic substrate, N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide. The cytokine and enzyme levels in PICF were expressed as total amount/activity and as concentrations. NE activity in PICF significantly correlated with GI and PD, and IL-1beta levels with GI and PICF volume (P < 0.05). The correlations were stronger when the PICF levels were expressed as total IL-1beta amount and as total NE activity. The implants with inflamed gingiva (GI > 1) had higher levels of IL-1beta and NE activity than implants with noninflamed or slightly inflamed gingiva (GI 3 mm) was greater than the implants with shallow pockets (PD <or= 3 mm) (P < 0.05). The implants of smoker patients had significantly lower PICF NE activity and IL-1beta levels, and significantly higher TNF-alpha levels than the implants of nonsmokers (P < 0.05). The findings of the present study indicate that NE activity and IL-1beta levels in PICF may be used to measure implant health status of patients who do not smoke.

Adult↗
PubMed1999

Tobacco smoking and gingival neutrophil activity in young adults.

The influence of smoking on the activity of the gingival neutrophils in young periodontally healthy adults was studied. The neutrophil activity was measured in terms of the gingival crevicular fluid (GCF) levels of elastase, lactoferrin (LF), alpha-1-antitrypsin (alpha-1-AT), alpha-2-macroglobulin (alpha-2 MG) and protein. 30 healthy dental students with no clinical signs of periodontitis, 15 smokers (8 women and 7 men) aged 20-32 years and 15 non-smokers (7 women and 8 men) aged 22-31 years, volunteered to take part in the investigation. The gingival inflammation was registered at 6 sites and the GCF volume was collected from the same sites. The GCF volume was measured with a Periotron 6000. The elastase activity was measured with a chromogenic low molecular substrate and the LF alpha-1-AT, alpha-2-MG levels were determined with ELISA. The protein concentration was measured by the Bradford method. The results showed a statistically significantly lower GCF volume among smokers as compared to non-smokers. No significant difference was found in the elastase activity/microl of the GCF supernatant between smokers and non-smokers but there was a large inter-individual variation. Nor did the concentrations of LF, alpha-1-AT, alpha-2-MG and protein per microl GCF differ significantly between the 2 groups. The results suggest that the influence of smoking on the examined factors associated with neutrophil activity is limited under healthy or slightly inflamed gingival conditions giving only small amounts of GCF.

Adult↗
PubMed1999

Increased release of elastase from in vitro activated peripheral neutrophils in patients with adult periodontitis.

The main object of this study was to determine if there was a difference between patients with adult periodontitis and healthy controls in the release of elastase. We also wanted to test the release of alpha-1-antitrypsin and lactoferrin from in vitro-activated peripheral neutrophils. A leukocyte-rich preparation from venous blood was made by lysing the red blood cells. The leukocytes were stimulated for 1 h at 37 degrees C with opsonized Staphylococcus aureus and the released elastase was measured with a chromogenic substrate. The release of elastase after stimulation with bacteria was significantly higher in patients than in controls. The amounts of elastase from unstimulated cells, i.e., both released extracellularly and extracted from the pellet, were similar in the 2 groups. However, after stimulation, the amount of elastase in the patient group, but not in the control group, was significantly increased. Similar releases of alpha-1-antitrypsin (AIAT) and lactoferrin were found in both groups of subjects. In conclusion, this study shows that peripheral neutrophils from patients with adult periodontitis release more active elastase after in vitro activation compared to healthy controls. The release of A1AT and lactoferrin showed no differences, indicating that the increased elastase activity was not due to a impaired inhibition by A1AT and that the differences in degranulation were limited to the primary granula.

Adult↗
PubMed2002

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

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 alpha1-antitrypsin (EA-alpha1AT) 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-alpha1AT (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-alpha1AT 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.

Case-Control Studies↗
PubMed2001

Dipeptidyl-peptidase II and cathepsin B activities in amelogenesis of the rat incisor.

A body of published evidence suggests that a significant portion of enamel matrix protein synthesized by ameloblasts localises in the lysosomal-endosomal organelles of these enamel organ cells. Little is known regarding the lysosomal proteolytic activities during amelogenesis. The aims of this study were to detect and measure the activities of lysosomal peptidases cathepsin B (E.C. 3.4.22.1) and dipeptidyl-peptidase II (E.C. 3.4.14.2) in the enamel organ of the rat incisor and to ascertain whether rat enamel matrix proteins are degraded by these peptidases in vitro. Whole enamel organs were dissected from rat mandibular incisors. Enamel protein was also collected from the rat teeth. Analysis indicated that the rat incisor enamel organs contained specific activities of both dipeptidyl-peptidase II and cathepsin B at levels comparable with those of kidney which is rich in both these lysosomal peptidases. Gel electrophoresis and immunoblotting demonstrated that both cathepsin B and dipeptidyl-peptidase II were able to substantially degrade the rat enamel proteins in vitro. Based on these observations, we propose that lysosomal proteases have roles in amelogenesis in the intracellular degradation of amelogenins.

Amelogenesis↗
PubMed2002

Gene transfer mediated by different viral vectors following direct cannulation of mouse submandibular salivary glands.

The salivary gland has been suggested as an accessible organ for gene transfer to express recombinant proteins locally in the saliva, as well as for secretion to the blood circulation. The aim of this study was to evaluate the efficiency of gene transfer to salivary glands using different viral vectors: adenovirus, vaccinia, herpes simplex type 1 (HSV), and two retroviral vectors (murine leukemia virus (MuLV) and lentivirus). We show, by in situ staining and beta-galactosidase reporter activity assay, that the adenoviral and vaccinia vectors were able to deliver the reporter gene efficiently to acinar and duct cells. The HSV vector was less efficient and infected only the acinar cells. The lentiviral vector infected acinar and duct cells, but at a relatively low efficiency. The MuLV vector did not infect the salivary gland unless cell proliferation was induced. Host immune responses to viral infection, inflammation, apoptosis and lymphocyte infiltration, in the transduced glands, were assessed. The DNA viral vectors induced local lymphocyte infiltration and apoptosis. In contrast, the retroviral vectors did not induce an immune response. Our results describe the outcome of salivary gland infection with each of the five different viral vectors and indicate their advantages and limitations for transferring genes to the salivary glands.

Adenoviridae↗
PubMed2001

Crevicular fluid elastase levels in relation to periodontitis and metabolic control of diabetes.

The purpose of this study was to investigate the associations between gingival crevicular fluid (GCF) elastase levels, clinical measures of periodontal status, and metabolic control of diabetes in insulin dependent (type 1) diabetes (IDDM) and non-insulin dependent (type 2) diabetes (NIDDM) patients. Sixty patients were recruited from the Diabetes Center at the University of California in San Francisco. Thirty subjects were type 1 diabetics and 30 subjects were type 2 diabetics. Metabolic control was evaluated by glycosylatted hemoglobin (HbA1c) levels. Demographic information was obtained using a structured interview with the subjects. Clinical measurements and GCF samples were taken from the mesio-buccal surfaces of 2 premolars and 2 molars from the most diseased sextant. GCF elastase was determined by measurement of p-Nitroanalide resulting from hydrolysis of elastase specific peptide. Crevicular fluid elastase levels were significantly correlated with gingival index, bleeding index, probing depth and attachment level in both type 1 and type 2 diabetes groups (0.01 <p < 0.001). HbA1c levels were not correlated with clinical measurements and GCF elastase. The results suggest that GCF elastase. age and smoking are risk indicators for periodontitis in patients with diabetes mellitus, and periodontal status is not associated with the duration and metabolic control of diabetes.

Adult↗
PubMed2003

Root surface debridement and endotoxin removal.

OBJECTIVE: This study assessed associations between the number of standardized scaling strokes and the reduction of endotoxin on the root surface. BACKGROUND: Therapy of periodontally involved teeth attempts removal of accretions by scaling and root planing. The amount of mechanical therapy required to free the root surface from etiologic factors remains unknown. METHODS: Twenty-four extracted human caries-free single rooted teeth with at least 5 mm attachment loss were used. A region of interest (ROI) which contained subgingival calculus was defined on the root surface of each tooth. Standardized force instrumentation was applied using a force-measuring curet. Fifty working strokes were applied to every ROI. Forces applied were recorded. The force recordings were converted from Millivolts into Newtons (N). After every unit of 5 strokes, presence of calculus was evaluated and scaling debris was collected. Endotoxin concentration was determined in the debris samples. RESULTS: The endotoxin values for strokes 1-5 were statistically significantly greater than the values from all other stroke intervals. Complete calculus removal occurred after a mean of 9.3 strokes. The endotoxin concentration reached a minimal level with concentrations of 0.03-0.306 EU/ml after calculus removal was complete. CONCLUSION: These findings suggest that completion of calculus removal coincides with endotoxin levels associated with clinically healthy teeth.

Chromogenic Compounds↗
PubMed1980

Plasminogen activator in cultured Lewis lung carcinoma cells measured by chromogenic substrate assay.

A chromogenic substrate assay for the plasminogen activator (PA) activity of Lewis lung carcinoma cells has been developed. The cells were incubated with plasminogen, the activation of which to plasmin was measured by the amidolysis of the chromogenic substrate S-2251. This was routinely performed as a 4h serum-free assay, but a variation lasting 24 h, in medium supplemented with plasminogen-free inhibitor-reduced serum, produced similar results. The assay also detected PA released into the medium. PA activity was proportional to cell density, and the assay was non-toxic to the cells. Assays were performed on cultures derived from primary and metastatic tumours. Host cells were effectively eliminated from such cultures but, because of an initial phase of tumour-cell death, PA assays were not carried out until cultures became established. No consistent difference was detected between PA levels in primary and metastatic cultures. However, these cultures were shown to be atypical of the parent tumour; they grew slowly when reinjected at the primary site, and their metastatic potential was impaired.

Animals↗
PubMed1987

Pharmacokinetics of streptokinase in patients based on amidolytic activator complex activity.

The purpose of this study was to determine the pharmacokinetics of intravenous streptokinase in humans. Five patients with myocardial infarction, six patients with venous thromboembolism, and two normal volunteers were studied. The patients with myocardial infarction received 500,000 U over 30 minutes, the patients with venous thromboembolism received 250,000 U over 30 minutes followed by 100,000 U/hr over 16 to 78 hours, and the normal volunteers received 100,000 U over 15 minutes. Plasma streptokinase levels were measured based on the amidolytic activity of the streptokinase-plasminogen complex on the chromogenic substrate S-2251. Pharmacokinetic parameters were: biologic half-life 82 +/- 25 minutes, total clearance 10.8 +/- 8.8 ml/min, and apparent volume of distribution 1.10 +/- 0.71 l. Streptokinase levels declined progressively during the continuous, prolonged infusion in the patients with venous thromboembolism over 60 hours of treatment. We conclude that there are distinct time-dependent changes in the pharmacokinetics of streptokinase during continuous intravenous infusion and that this phenomenon is likely to be associated with progressively decreasing thrombolytic efficacy.

Adult↗
PubMed2006

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization.

Chromogenic (CISH) and fluorescent (FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes (BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12;15)(p12;q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.

Adenoma, Pleomorphic↗