Substrate cleavage by HIV-1 proteinase.
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Amino Acid Sequence↗
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Results for “Chromogenic Compounds”
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A new perchloric acid treatment of human plasma for detection of endotoxin by an endotoxin-specific chromogenic test.
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Animals↗
Dynamics of kallikrein activity in different biological fluids of pregnant animals.
Kallikrein activity was measured in urine, saliva and blood plasma samples from pregnant cows by using the chromogenic substrates S-2266 and S-2302 (Kabi, Sweden). It was found that the kallikrein activity in these biological fluids showed a trend to an increase with a peak prior to parturition. At the same time the plasma prekallikrein levels decreased during pregnancy reaching the lowest values at the end of the gestation period.
Animals↗
Immunoassays for the determination of human tissue kallikrein (TK) in different body fluids based on monoclonal antibodies.
A monoclonal antibody produced against human tissue kallikrein was used to develop solid phase immunoassays for the determination of total immunoreactive tissue kallikrein, of the complex of tissue kallikrein with alpha 1-proteinase inhibitor, and of enzymatically active tissue kallikrein. The assays permit the specific determination of various forms of tissue kallikrein in body fluids and should be very useful in studies on the biological function of tissue kallikrein-kinin systems.
Amino Acid Sequence↗
Biological assay for tissue kallikrein: comparison with the synthetic substrate S2266.
A highly sensitive biological assay for tissue kallikrein is described, using human kininogen as substrate; and quantitation, by radioimmunoassay, of generated kinins. Using purified human urinary kallikrein as a reference standard we have correlated the kininogenase activity of kallikrein with amidase activity as measured by cleavage of the synthetic substrate S2266.
Animals↗
Standardization of reagents and methods used in cytological and histological practice with emphasis on dyes, stains and chromogenic reagents.
The need for the standardization of reagents and methods used in the histology laboratory is demonstrated. After definitions of dyes, stains, and chromogenic reagents, existing standards and standards organizations are discussed. This is followed by practical instructions on how to standardize dyes and stains through the preparation of reference materials and the development of chromatographic methods. An overview is presented of the problems concerned with standardization of the Romanowsky-Giemsa stain for cytological and histological application. Finally, the problem of how to convince routine dye and stain users of the need for standardization in their histology laboratories is discussed.
Animals↗
Cell-wall-associated proteinase of Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397: differential extraction, purification and properties of the enzyme.
Whole cells of Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 were able to hydrolyse alpha- and beta-caseins. Irrespective of the growth medium used, milk or De Man-Rogosa-Sharpe (MRS) broth, identical patterns of alpha- and beta-casein hydrolytic products, respectively, were visualized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A soluble proteinase present in cell-wall extracts was active on caseins and displayed the same hydrolytic patterns as whole cells. It was purified from cell-wall extract to homogeneity by ultrafiltration and ion exchange chromatography. The enzyme is a monomer with a molecular mass of 170 kDa, an optimum temperature of 42 degrees C and an optimum pH of 5.5. It was strongly activated by dithiothreitol and partially inhibited by E-64. These properties indicate that cysteine residues play an important role in the enzyme mechanism. The purified proteinase was not able to hydrolyse di- or tripeptides.
Amino Acid Sequence↗
Quantification of alachlor in water by a novel magnetic particle-based ELISA.
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Acetamides↗
Standardization of biological dyes and stains: pitfalls and possibilities.
The present paper gives a review of the actual state of standardization of biological dyes and stains. In a first part general information is given on practical problems encountered by the routine user of dyes with special emphasis on dye contamination. Some theoretical aspects of standardization are discussed. The second part of the paper gives more detailed information on commercial batches of hematoxylin-eosin-, Giemsa- and Papanicolaou-stains and on their standardization. Special problems arising with the application of image analysis techniques are briefly mentioned. User-oriented specifications for the standardization of dyes, stains and staining procedures are given. Fluorescent dyes and dyes used in chromogenic reagents such as the Feulgen-Schiff reaction are not included in this review.
Chromogenic Compounds↗
An ultrastructural double-labelling method: immunohistochemical localization of cell adhesion molecule L1 on HRP-labelled developing corticospinal tract axons in the rat.
A double electronmicroscopical (EM) staining was developed which enabled the ultrastructural localization of cell adhesion molecules on the outer axonal membrane of horseradish peroxidase (HRP)-labelled axons in the developing central nervous system (CNS). HRP was used to anterogradely trace outgrowing corticospinal tract (CST) axons in ten-day-old rats. After visualization of HRP using tetramethylbenzidine (TMB) as a chromogen and ammoniumheptamolybdate (AHM) as a stabilizer at pH 6.0 as described previously (Joosten et al. 1987, J Histochem Cytochem 35: 623-626) an additional diaminobenzindine (DAB)-Ni incubation was carried out for further stabilization. Subsequently a preembedding immunoperoxidase (DAB) staining was executed for detection of cell adhesion molecule L1. Using this procedure anterogradely HRP-labelled CST axons were recognizable by a granular black TMB-AHM-DABNi reaction product at the light microscopic (LM) level, which clearly contrasts to the relatively homogeneous brown L1-immunostaining. Electronmicroscopically HRP-labelled CST axons were characterized by the presence of an intracellular crystaloid TMB-AHM-DABNi reaction product which made identification of CST axons rather easy, whereas the L1-DAB precipitate could be noted on the outer axonal membrane of the HRP-labelled CST axons, marking the presence of the L1 cell adhesion molecule. In addition the procedure described in this report preserves ultrastructural details of developing neural tissue. In conclusion, the method presented can be employed in combined HRP-tracing and immunohistochemical electronmicroscopic studies.
Animals↗
Significance of urinary endotoxin concentration in patients with urinary tract infection.
Endotoxin is a component of the outer membrane of gram-negative rods (GNR). Since GNR are responsible for the majority of urinary tract infection (UTI), we measured the concentration of endotoxin in urine using chromogenic endotoxin-specific assay and examined its diagnostic utility in patients with suspected UTI. In all 18 urine samples with an endotoxin concentration exceeding 350 pg/ml and 2 samples with 10-350 pg/ml of endotoxin concentration, GNR were detected at a count of 10(4) cfu/ml. Negative for endotoxin were 3 samples of culture positive for gram-positive cocci (GPC), 2 samples containing various bacterial contaminants and all 37 samples with no growth on culture. Two urine samples collected 5 h after antibiotic dosage showed negative culture for GNR but a significant concentration of endotoxin. In an in vitro experiment, a residual concentration of antibiotic in urine inhibited bacterial growth, leading to a false-negative culture. These results suggest that chromogenic endotoxin assay is a reliable method for diagnosing UTI caused by GNR and detecting false-negative culture of GNR.
Adult↗
Observations on the early development of ascending spinal pathways. Studies using the North American opossum.
The development of ascending spinal pathways has been studied in the North American opossum using degeneration methods and the retrograde transport of horseradish peroxidase. Axons from caudal thoracic and/or lumbosacral levels of the spinal cord reach the lateral reticular nucleus, the inferior olivary complex, the reticular formation of the medulla and pons as well as the cerebellum very early in development. Innervation of the nucleus gracilis occurs somewhat later. Spinal axons grow into most of the caudal brain stem areas they occupy in the adult animal, including the nucleus gracilis, before there is convincing evidence that they reach the thalamus. Although spinal axons enter the cerebellum early in development their adult distribution with its characteristic discontinuities appears relatively late.
Afferent Pathways↗
A new family with classical factor X deficiency as demonstrated by electroimmunoassay.
A new family with classical factor X deficiency is described. The proposita is a 6 year old girl who presented with occasional epistaxis and a hematoma after an intramuscular injection. The main laboratory features consisted in a prolongation of partial thromboplastin, prothrombin and Stypven clotting times corrected by the addition of normal serum. Factor X activity varied between 3 and 6%. Factor X amydolytic activity was 15% of normal. Electroimmunoassay failed to show the presence of factor X antigen. No inhibitor was found in the proposita plasma. Parents and other family members showed intermediate levels of factor X activity and antigen and were considered to be heterozygotes. No consanguineity was found in the family.
Antigens↗
Chromogenic identification of promoters in Streptomyces lividans by using an ampC beta-lactamase promoter-probe vector.
A promoter-probe system, based on the ampC beta-lactamase gene of Escherichia coli, has been developed for the isolation and characterization of transcriptional signals in the gram-positive bacterium Streptomyces lividans. The promoter-probe vector, denoted pJAS14, has the SLP1.2 replicon with a copy number of four-five plasmids per cell. It contains a unique BamHI site just in front of the ampC ribosome-binding site, and upstream of this BamHI site a transcriptional terminator signal that prevents readthrough transcription from plasmid-borne promoters has been inserted. Using pJAS14, we have shot-gun cloned chromosomal DNA from S. lividans and S. lavendulae into the BamHI site, and isolated a number of promoter containing DNA fragments by the use of the chromogenic cephalosporin nitrocefin. On plates, we identified promoters of varying strengths and also with differences in nutritional and temporal expression. Using liquid cultures of S. lividans, it has been demonstrated that one promoter, denoted P1 (SEP8), as well as the ampC gene of E. coli, show activity corresponding to the vegetative growth of the cells. The P1 (SEP8) promoter was shown to be expressed also in E. coli, and it initiates RNA synthesis at exactly the same nucleotides in both S. lividans and E. coli. The promoter shows good homology to the E. coli promoter consensus sequence in both the -35 and -10 regions. Thus, this promoter is a representative of the SEP (Streptomyces E. coli-type promoter) class of promoters recently described (Jaurin and Cohen 1985). This indicates that an S. lividans RNA polymerase recognizes the same sequence determinants and chooses the point of initiation of RNA synthesis in the same way as the corresponding E. coli enzyme.
Base Sequence↗
Antithrombin III activity (residual thrombin activity) in plasma from non-medicated or heparinized horses.
Two synthetic substrate assays (fluorometric and chromogenic) were used to measure antithrombin-III (AT-III) activity (residual thrombin activity) in non-medicated and heparin (sodium) treated horses. In 18 non-medicated horses the fluorometric substrate assay (FSA) values were similar to previous reports but they reflected inconsistent trends and larger deviations in the heparin-treated groups (Group 2: 40 and 100 U/kg IV, n = 6; Group 3: 240 U/kg IV, n = 5; Group 4: 80 U/kg IV followed by 160 U/kg SC, n = 8) when compared to the chromogenic substrate assay (CSA) values. The CSA values for the 18 non-medicated horses indicated a higher AT-III activity (lower residual thrombin activity) than the FSA. AT-III activity was quantified in 18 non-medicated horses (29 mg/dl) and compared well with values for humans (30 mg/dl) and dogs (40 mg/dl). Plasma heparin concentrations, determined by the FSA, correlated well with the 'therapeutic range' (1.5 fold to 2.5 fold prolongation of the activated partial thromboplastin time (APTT) normal value) and values reported for humans. The effect of heparin therapy on AT-III activity in four treatment regimens was evaluated. AT-III activity was not significantly affected (with one exception) by a single dose of intravenous (IV) heparin (40 and 100 U/kg) nor by repeated subcutaneous (SC) injections of heparin (240 U/kg). A transient increase in residual thrombin activity was measured 12 h after an intravenous (80 U/kg) injection of heparin. Large doses of heparin (80 U/kg IV followed by 160 U/kg SC) given every 12 h produced a progressive prolongation of the APTT. In this group the APTT remained prolonged 48 h after the last treatment.
Animals↗
Investigation of proteases with chromogenic substrates after isoelectric focusing (IEF).
A technique is described for the detection of protease isoenzymes which is more sensitive than disc electrophoresis. Supernatants of crude rat and human organ homogenates are subjected to analytical isoelectric focusing (IEF) and the gel strips are finally incubated in histochemical media containing 4-methoxy-2-naphthylamine amino acids or peptides and diazonium salts for simultaneous or post-coupling. The incubation media are identical with those used for section histochemistry of proteases. This combination of IEF and proteases histochemistry yields excellent and reproducible data which cannot be obtained by protease histochemistry alone. Post-coupling delivers less and more diffuse bands than simultaneous coupling. For simultaneous coupling, Fast Blue B and Fast Black K are the most suitable diazonium salts. More bands are found in agarose gels compared with polyacrylamide. Sex-differences exist for endopeptidases in the submandibular gland, but are absent in other rat organs. Despite their uniform membrane localization in tissue sections, aminopeptidase (AP) A and M and dipeptidylpeptidase (DPP) IV and gamma-glutamyltranspeptidase (GGT) show striking heterogeneous band patterns depending on the investigated organ. The similar band patterns of APA and APM can be specified by the use of activators or inhibitors. In rat kidney, up to 26 bands are obtained with DPP II and IV substrates, 3 for APA and APM and up to 12 for GGT. DPP IV of human liver is different from that in rat liver.
Animals↗
Isoelectric focusing (IEF) and band detection with fluorogenic protease substrates.
In a previous paper, combined dye histochemistry and analytical isoelectric focusing (IEF) of supernatants from organ homogenates have been shown to yield good results for the detection of protease isoenzymes. Difficulties arise when the protease to be studied is partially or completely inhibited by diazonium salts and when synthetic peptide substrates different from 4-methoxy-2-naphthylamine (MNA) amino acids and peptides are to be used. In this paper a technique is described in which cellulose acetate foils impregnated with MNA, 7-amino-4-methyl-coumarin (AMC) and 7-amino-4-trifluoromethylcoumarin (AFC) substrates are overlaid on electrophoresis strips after IEF. After incubation, the foils are viewed with an UV-lamp and photographed. The MNA, AMC and AFC peptides are equally suitable for fluorescence band detection. Using this technique, occasionally protease isoenzymes are found which are more sensitive towards diazonium salts, e.g. aminopeptidase A and M. Sometimes it is also possible to detect thiolproteases which is not the case when employing dye histochemistry.
Aminopeptidases↗
Prekallikrein activation in human, bovine, and rabbit plasmas: presence of an inhibitor in bovine plasma.
Contact of human plasma with a negatively charged surface such as dextran sulfate activates prekallikrein to kallikrein, which releases the vasoactive peptide bradykinin from high-molecular-weight kininogen. The dextran sulfate-induced activation of prekallikrein at 0 degree C (assayed by its amidolytic activity on the chromogenic substate S-2302) could not be observed in either bovine or rabbit plasmas when compared to human plasma. Neither bovine nor rabbit plasma inhibited the amidolytic activity of contact-activated human plasma at 0 degrees C. The activation of prekallikrein in human plasma was significantly inhibited by the addition of bovine plasma but not by rabbit plasma. Bovine plasma (0.025 units, 1 unit = 1 ml of plasma) caused 68.8% inhibition of prekallikrein activation. Eighty percent of the inhibitory property of bovine plasma was present in the greater than 30,000-molecular-weight fraction. These results indicate the presence of an inhibitor(s) of prekallikrein activation in bovine plasma.
Animals↗