Chromogenic microtitre tray assay for plasminogen.
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Chromogenic Compounds↗
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A modified chromogenic assay for the measurement of very low levels of factor VIII activity (FVIII:C).
A precise and sensitive chromogenic assay for the measurement of very low levels of factor VIII (FVIII) in plasma has been developed. The assay is based on modifications of a commercially available chromogenic assay. The modifications include reduction of sample final dilution factor and prolongation of the development period. The modified assay allows accurate and precise measurement of FVIII in the range of 0.001-0.02 IU mL(-1). The detection limit is 0.0005 IU mL(-1) and the quantitation limit is 0.0015 IU mL(-1). This assay can be used in research and to study the clinical efficacy of low circulating levels of FVIII in haemophilia A patients.
Calibration↗
Quantitative measurement of endotoxin in rainbow trout (Salmo gairdneri) serum by the chromogenic substrate method.
The amount of endotoxin in serum collected from normal rainbow trout (Salmo gairdneri) and trout inoculated with viable Vibrio anguillarum or lipopolysaccharide (LPS) extracted from bacteria was determined by the chromogenic substrate method. The mean values of endotoxin in four different groups of normal rainbow trout sera ranged from 31.9 to 65.3 pg/ml. When fish were inoculated with viable bacteria (1 x 10(8], they became septicaemic and a large amount of endotoxin ng/ml) was detected in the sera. In fish inoculated with a smaller number of bacteria the amount of endotoxin was several times higher than that of normal fish in spite of failure of bacterial isolation. Although the endotoxin level in serum increased rapidly (greater than 100 ng/ml) after intraperitoneal inoculation with purified V. anguillarum LPS (540 micrograms), no fish died during the experiment. The high level of endotoxin in normal rainbow trout and the resistance of trout to endotoxin are in striking contrast to those of mammalian and avian species.
Animals↗
Rapid methods for differentiating gram-positive from gram-negative aerobic and facultative anaerobic bacteria.
Different tests based on lysis by KOH and on reaction with fluorogenic and chromogenic substrates, L-alanine-4-nitroanilide (LANA); L-alanine-4-methoxy- beta-naphthylamide (MNA); 4-alanine-2-amidoacridone (AAA); L-alanine-7-amido- 4-methylcoumarin (AAMC); 8-anilino-1-naphthalene-sulphonic acid (ANS) were compared for their suitability to distinguish Gram-positive from Gram-negative bacteria. A concentration of 100 micrograms/ml was chosen for incorporating LANA, AAA, AAMC and ANS into the growth medium, based on sensitivity tests. MNA did not show any detectable reaction over a concentration range from 50 to 200 micrograms/ml, and led to inhibition of all bacteria at 200 micrograms/ml. In the examination of a total of 146 bacterial strains, including Yersinia enterocolitica, Bacillus cereus, and B. subtilis the KOH test was not comparable with the Gram staining. A good correlation with Gram staining was found between LANA, AAA and AAMC added to plate count agar on one hand, and LANA and AAMC impregnated paper strips on the other hand, thereby utilizing the aminopeptidase activity. Agar containing ANS showed detectable fluorescence with all Gram-negative strains, but with Staphylococcus aureus and Staph. epidermidis a weak reaction was also observed. AAMC was selected for a rapid paper strip test. With this substrate a pronounced blue fluorescence was obtained with Gram-negative colonies.
Animals↗
Evaluation of chromogenic media for the detection of Listeria species in food.
AIMS: The aim of this study was to evaluate the performance of chromogenic agars, Agar Listeria according to Ottaviani and Agosti (ALOA) and Rapid L. mono agar, compared with Oxford agar for the enumeration and detection of Listeria species in food. METHODS AND RESULTS: A total of 170 food samples were examined using the three plating media. Listeria species were isolated from 63 samples. In contrast to Oxford agar, detection of Listeria colonies on chromogenic media was as good after 24 h of incubation of plates as after 48 h. While there was no significant difference in recovery of Listeria monocytogenes on the three media, recovery of other Listeria species was significantly poorer on Rapid L. mono agar compared with Oxford and ALOA agars. Recovery of species other than L. monocytogenes was significantly improved by including a secondary enrichment stage in the detection method. CONCLUSIONS: Using chromogenic agars, presumptive identification of L. monocytogenes is possible after 24 h, compared with 3-4 days using Oxford agar. However, the poor detection of species other than L. monocytogenes on Rapid L. mono agar is a disadvantage of this medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information regarding the isolation of Listeria species other than L. monocytogenes from food using chromogenic plating media. This is important, as non-pathogenic Listeria species act as markers for the likelihood of presence of L. monocytogenes and allow preventive action to be taken to avoid its presence.
Agar↗
Evaluation of a new chromogenic agar for the detection of Listeria in food.
AIM: Rapid identification of Listeria in food is important in protecting consumers from infection. The development of chromogenic media such as agar Listeria according to Ottaviani and Agosti (ALOA) has allowed more rapid detection of Listeria monocytogenes, with presumptive identification of this pathogenic species after only 24 h of incubation. The aim of this study was to evaluate Oxoid chromogenic Listeria agar (OCLA) in comparison with ALOA and a traditional, nonchromogenic medium, Oxford agar. METHODS AND RESULTS: Media were compared using pure cultures, spiked food samples and naturally contaminated samples. Whilst development of typical colony morphology took 48 h on Oxford agar, Listeria spp. were frequently detected after 24 h of incubation on OCLA and ALOA. There was no significant difference in recovery between the two chromogenic media. CONCLUSIONS: Results indicate that OCLA gives equivalent recovery of Listeria spp. compared with ALOA. Whilst L. monocytogenes was frequently detected after 24 h of incubation, a 48-h incubation time was necessary to ensure detection of both L. monocytogenes and other Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that a commercially available chromogenic medium other than ALOA is appropriate for use in the international standard method. The commercial availability of more than one medium will facilitate the more widespread use of the method, thus increasing confidence in the ability to detect L. monocytogenes in food in the presence of other Listeria spp.
Agar↗
Core-shell CdS/Cd(OH)2 quantum dots: synthesis and bioconjugation to target red cells antigens.
We report a new and efficient methodology of labelling red blood cells, in order to investigate the expression of anti-A antigen, employing luminescent semiconductor nanocrystals. Highly luminescent and stable core-shell cadmium sulphide/cadmium hydroxide [CdS/CdS(OH)2] colloidal particles were obtained in the nanometre size range. The surface of these particles was characterized by using a monoclonal anti-A antibody via a one-step glutaraldehyde cross-linking procedure, followed by conjugation of the particles to red cells of blood groups A+, and O+. Laser scanning confocal microscopy images indicated that after conjugation for 30 min, A+ and erythrocytes presented different patterns of dual bright emission whereas the O+ group cells showed no emission. We suggest that this labelling procedure may be applied as a quantitative tool to investigate the distribution and expression of alloantigen in red blood cells.
Alloys↗
Amplification of the epidermal growth factor receptor in astrocytic tumours by chromogenic in situ hybridization: association with clinicopathological features and patient survival.
Chromogenic in situ hybridization (CISH) was used to detect amplification of the epidermal growth factor receptor (EGFR) gene in tissue microarrays of tumours derived from 287 patients with grade II-IV diffuse astrocytomas. Amplification was found in 32% of the tumours with a highly significant association with histological grade (4% in grade II, 21% in grade III and 39% in grade IV; P < 0.001). Amplification of the EGFR gene was more common in primary than in secondary glioblastomas (41%vs. 16%, P = 0.033). Overexpression of EGFR mRNA and protein (wild-type and vIII variant) was found to correlate with EGFR gene amplification (P = 0.028, P = 0.035 and P = 0.014 respectively), but wild-type EGFR protein was also frequently overexpressed in tumours without EGFR gene amplification. Patients with older age (P < 0.001) and tumours with lack of p53 overexpression (P = 0.03) and higher apoptosis rate (P < 0.001) had significantly more EGFR gene amplifications than their counterparts. No such correlation with apoptosis was found in glioblastomas. The survival of patients with EGFR gene-amplified grade III tumours was significantly shorter than in those with grade III non-amplified tumours (P = 0.03). No such difference was noted in glioblastomas (grade IV tumours). Our data verify the central role of EGFR in the pathobiology of astrocytic tumours, and highlight the advantages of CISH as a simple and practical assay to screen for EGFR gene amplification in astrocytic tumours.
Adolescent↗
Proteolytic activity during storage of platelets in plasma.
Proteolytic activity was studied in platelet concentrates (PC) stored in plasma at 22 degrees C. In experiment 1, two PC with a higher (A) and a lower (B) white cell concentration were prepared from each of nine donors by centrifugation. Aliquots of the cell-free plasma, PPP, were stored as a control. Samples for the assay of fibrinopeptide A (FPA), elastase, spontaneous proteolytic activity (SPA), kallikrein-inhibiting activity, thrombin-antithrombin complexes (TAT) and D-dimers were collected initially and on days 1, 3, 5 and 7 of storage. Consumption of glucose, pH and concentrations of lactate dehydrogenase (LDH) and ATP were determined to investigate the metabolic status of the PC. The decrease in pH correlated to the leucocyte count, r = -0.74, P < 0.001 and to the increase in LDH, r = -0.74, P < 0.01. The levels of elastase and the SPA were consistently low in the PPP bags. In the PC elastase had increased by day 5 and the SPA by day 3; the levels in PC A were significantly higher than in PC B, P < 0.01. The leucocyte count correlated with the elastase activity, r = 0.71, P < 0.01, and with the SPA, r = 0.65, P < 0.01. A minor increase in FPA was demonstrated while no TAT and D-dimers could be detected. The cause of the formation of FPA was studied in experiment 2; three bags of PC and four of PPP were prepared from each of 16 donors. To the PC and three of the PPP bags either hirudin, aprotinin or no enzyme inhibitor (control) was added.(ABSTRACT TRUNCATED AT 250 WORDS)
Antithrombin III↗
Synthesis of chromophoric dehydro analog of leucine enkephalin.
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Chromogenic Compounds↗
Solid-phase synthesis of peptide-4-nitroanilides.
A wide variety of Glu/Asp and Gln containing peptide-4-nitroanilides and other chromogenic peptidyl-arylamides could be quickly synthesized by a Fmoc-based solid-phase synthesis strategy employing the side-chain carboxyl groups for transient anchoring to the resin. Suitable synthons for this method, Fmoc-Glu-NH-Np and Fmoc-Asp-NH-Np, were prepared using a diphenylphosphinic chloride-mediated coupling reaction. Peptides of the common structure Suc-Ala-Phe-Pro-Xaa-NH-Np (Xaa = Glu/Asp, Gln) were synthesized and were shown to be substrates for the protease subtilisin Carlsberg (E.C.3.4.21.14a) and for peptidyl-prolyl cis/trans-isomerases (PPIases E.C. 5.2.1.8.). The method was extended to amino acids possessing a side chain missing an anchor for binding to the matrix. We synthesized Suc-Ala-Phe-Pro-Gln-Phe-NH-Np anchoring the dipeptide derivative Fmoc-Glu-Phe-NH-Np with the carboxyl group to Rink amide resin using standard SPPS procedures. Additionally this procedure allowed us the preparation of peptidyl-arylamides, utilizing the commercial available Fmoc-Glu-OAll as building block. A mixture of pentapeptide-4-nitroanilides with the general sequence Ala-Ala-Xaa-Pro-Gln-NH-Np was synthesized. Electrospray ionization mass spectrometry (ESI-MS) was used to evaluate the hydrolysis of the peptide mixture by the protease subtilisin Carlsberg. It could be shown that peptides with the hydrophobic amino acids Phe, Tyr, Leu and Val in the varied P3-position were most rapidly cleaved under the chosen conditions. Hydrolysis of the Gln-NH-Np bond in Ala-Ala-Pro-Pro-Gln-NH-Np has not been observed.
Anilides↗
Salivary collagenase, elastase- and trypsin-like proteases as biochemical markers of periodontal tissue destruction in adult and localized juvenile periodontitis.
The profile of salivary proteases and their cellular origin, with special reference to polymorphonuclear leukocytes and bacteria, was studied in localized juvenile periodontitis and compared with adult periodontitis and healthy controls. General proteolytic activity in saliva as well as collagenase, elastase-like and trypsin-like activity was measured. In addition, the sensitivity of salivary collagenase of patients with localized juvenile periodontitis to doxycycline inhibition was studied. The saliva of localized juvenile periodontitis patients contained low amounts of collagenase compared with adult periodontitis saliva, and almost all salivary collagenase was found to exist in endogenously active form, as was found to be the case also in adult periodontitis patients and healthy controls. The salivary collagenase of localized juvenile periodontitis patients was relatively insensitive to 100 mumol/l doxycycline but was completely inhibited by 600 mumol/l doxycycline, reflecting rather matrix metalloproteinase-1 (fibroblast-type) than matrix metalloproteinase-8 (polymorphonuclear leukocyte) enzyme. The saliva of localized juvenile periodontitis patients also contained low amounts of elastase-like activity compared with the saliva of untreated adult periodontitis patients. Scaling and root planing caused a significant decrease in elastase-like activity in the saliva of adult periodontitis patients. General proteolytic and trypsin-like activities were also low in the saliva of localized juvenile periodontitis patients. Furthermore, the reducing agent beta-mercaptoethanol did not activate or inhibit the salivary trypsin-like activity of localized juvenile periodontitis or adult periodontitis patients, although the reductant readily activated partially purified Porphyromonas gingivalis trypsin-like protease in a characteristic manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Aggressive Periodontitis↗
Fluorogenic substrates for bacterial aminopeptidase P and its analogs detected in human serum and calf lung.
A sensitive fluorimetric assay was developed for bacterial aminopeptidase P, based on intramolecularly quenched fluorogenic substrates. Two substrates were synthesized. Phe(NO2)-Pro-HN-CH2-CH2-NH-ABz (substrate I) and Phe(NO2)-Pro-Pro-HN-CH2-CH2-NH-ABz (substrate II), in which the Phe(NO2) group (rho-nitro-L-phenylalanyl) quenches the fluorescence of the ABz group (omicron-aminobenzoyl). Both substrates were readily cleaved by aminopeptidase P from Escherichia coli, releasing rho-nitro-L-phenylalanine and causing a proportional increase in fluorescence. Complete hydrolysis of the two substrates resulted in a 7.5-fold and 3.4-fold fluorescence increase, respectively. Applying this fluorogenic assay, we were able to detect and measure quantitatively amino-peptidase P-like activity in the human serum and calf-lung extracts. Substrate II was shown to be specifically cleaved by aminopeptidase P in these preparations, while substrate I was apparently cleaved by other enzymes as well. In both preparations, the enzyme activity was independent of Co2+ ions, and Pro-HN-CH2-CH2-NH-ABz (Cbz) was inhibitory. The kinetic constant Km was determined as 0.35 mM and 0.28 mM for the human serum and the calf-lung enzymes respectively. The enzyme activity was only slightly dependent on pH in the range 7.0-8.4.
Aminopeptidases↗
Actinomycin D, 1H NMR studies on intramolecular interactions and on the planarity of the chromophore.
The conformation of actinomycin D in acetone and chloroform solution at different temperatures has been studied by 1H NMR spectroscopy. At lower temperature the resonances due to the two chromophoric amino protons were observed. These signals exhibit very different resonance positions indicating a severely hindered rotation of the 2-amino group and the presence of a hydrogen-bond connecting the 2-amino and the 1-carbonyl groups. In 1H NMR spectra of partially 15N-enriched actinomycin D, the 1JN-H coupling constants at the 2-amino group were determined and a strong sp2 character for the 2-amino nitrogen was deduced. The strong amide character of the 2-amino group is caused by mesomerism involving the 1-carbonyl group. The amino proton signals are sensitive indicators for differences in the spatial relationship of the diverse parts of the actinomycin molecule. At lower temperatures a simultaneous and selective broadening of the alpha ring threonine and valine amide proton signals as well as of the 2-amino group resonance was observed, indicating the presence of one dynamic process in the molecule which slows down upon temperature reduction. A swinging motion of the N(10) nitrogen through the chromophore plane would explain this observation. The interpretation of these results requires the presence of a non-planar chromophoric system in the actinomycin molecule in acetone and chloroform solution. The possible implications of this non-planarity for the intercalation process and for the biological activity of the drug are discussed.
Amides↗
Intramolecular interactions, mesomerism and dynamics in actinomycin D studied by 15N NMR spectroscopy.
We present a detailed conformational study of 15N-labelled actinomycin D in different organic solvents using 1H, 15N and two-dimensional (2D) NMR techniques at 30.4 MHz and 50.6 MHz. The assignment of the threonine and valine 15N resonances to the individual residues on the alpha- or beta-lactone rings was achieved via heteronuclear shift-correlated 2D NMR experiments. The solvent perturbation studies allow an estimation of the solvent accessibility of the nitrogens and carbonyl groups. Evidence is presented that the pentapeptide rings of actinomycin D have different conformations in polar and in apolar solvents. The chromophoric N10 is efficiently solvent-protected, the solvent-dependence of its 15N resonance resulting from solvent interactions at other positions of the molecule and from solvent-dependent changes in the twisting of the chromophoric system. The chromophoric 2-amino nitrogen is shown to exhibit a strong sp2 character due to the formation of a conjugated system with the carbonyl group at C1. Such a conjugation requires a non-planar chromophoric ring system. Additionally, a hydrogen bond connecting the 2-amino and the 1-carbonyl group was detected. In some solvents, two resonances appear for the 2-amino nitrogen implying the presence of the 2-amino group in two different conformations. The possible implications of the non-planarity of the chromophore for the intercalation process and for the biological activity of the drug are discussed.
Chemical Phenomena↗
Studies on the effect of serine protease inhibitors on activated contact factors. Application in amidolytic assays for factor XIIa, plasma kallikrein and factor XIa.
Amidolytic assays have been developed to determine factor XIIa, factor XIa and plasma kallikrein in mixtures containing variable amounts of each enzyme. The commercially available chromogenic p-nitroanilide substrates Pro-Phe-Arg-NH-Np (S2302 or chromozym PK), Glp-Pro-Arg-NH-Np (S2366), Ile-Glu-(piperidyl)-Gly-Arg-NH-Np (S2337), and Ile-Glu-Gly-Arg-NH-Np (S2222) were tested for their suitability as substrates in these assays. The kinetic parameters for the conversion of S2302, S2222, S2337 and S2366 by beta factor XIIa, factor XIa and plasma kallikrein indicate that each active enzyme exhibits considerable activity towards a number of these substrates. This precludes direct quantification of the individual enzymes when large amounts of other activated contact factors are present. Several serine protease inhibitors have been tested for their ability to inhibit those contact factors selectively that may interfere with the factor tested for. Soybean trypsin inhibitor very efficiently inhibited kallikrein, inhibited factor XIa at moderate concentrations, but did not affect the amidolytic activity of factor XIIa. Therefore, this inhibitor can be used to abolish a kallikrein and factor XIa contribution in a factor XIIa assay. We also report the rate constants of inhibition of contact activation factors by three different chloromethyl ketones. D-Phe-Pro-Arg-CH2Cl was moderately active against contact factors (k = 2.2 X 10(3) M-1 s-1 at pH 8.3) but showed no differences in specifity. D-Phe-Phe-Arg-CH2Cl was a very efficient inhibitor of plasma kallikrein (k = 1.2 X 10(5) M-1 s-1 at pH 8.3) whereas it slowly inhibited factor XIIa (k = 1.4 X 10(3) M-1 s-1) and factor XIa (k = 0.11 X 10(3) M-1 s-1). Also Dns-Glu-Gly-Arg-CH2Cl was more reactive towards kallikrein (k = 1.6 X 10(4) M-1 s-1) than towards factor XIIa (k = 4.6 X 10(2) M-1 s-1) and factor XIa (k = 0.6 X 10(2) M-1 s-1). Since Phe-Phe-Arg-CH2Cl is highly specific for plasma kallikrein it can be used in a factor XIa assay selectively to inhibit kallikrein. Based on the catalytic efficiencies of chromogenic substrate conversion and the inhibition characteristics of serine protease inhibitors and chloromethyl ketones we were able to develop quantitative assays for factor XIIa, factor XIa and kallikrein in mixtures of contact activation factors.
Amino Acid Chloromethyl Ketones↗
Effects of protons on the thrombin-fibrinogen interaction.
Amidase activity of human alpha-thrombin toward the synthetic substrate Tosyl-Gly-Pro-Arg-NH-Ph and fibrinogen has been studied as a function of pH at t = 25 degrees C, under steady-state conditions. A viscosity-perturbation method allowed us to compute the equilibrium binding constant along with the rate constants for the acylation and deacylation reactions. The ionization constants for the groups affecting binding and hydrolysis of the synthetic substrate were measured by application of linkage thermodynamics principles. The binding of the synthetic substrate is controlled by two ionizable groups having pKa values of 7.5 and 8.7 in the free enzyme and 6.3 and 9.8 in the Michaelis adduct. These two groups were found to control the acylation process as well. Thrombin-fibrinogen interaction has been studied by measurements of steady-state hydrolysis of the synthetic substrate Phe-pipecolyl-Arg-NH-Ph in the presence of fibrinogen, used as a competitive inhibitor. This method allowed us to measure the Km of thrombin-fibrinogen interaction. The values of Km computed at different solution viscosities were used in order to calculate the equilibrium dissociation constant and both k2/k3 and k2/k-1 ratios. The same residues that were found to control binding of Tosyl-Gly-Pro-Arg-NH-Ph to alpha-thrombin, do modulate binding of fibrinogen as well. These residues shift their pKa values upon the formation of the Michaelis adduct from 7.5 to 5.7 and from 8.7 to 9.7, respectively. Furthermore the ratio kcat/Km as a function of pH has been obtained by HPLC measurements of fibrinopeptides release. The kcat/Km values along with the ratio k2/k-1, derived from viscometric experiments, allowed us to calculate the forward-rate constant, k+1, for the thrombin-fibrinogen interaction. The association process was found to depend on pH, namely in the alkaline region. The results for Tosyl-Gly-Pro-Arg-NH-Ph and fibrinogen are compared and discussed on the basis of the structural elements which differentiate the interactions of these substrates with human alpha-thrombin.
Amidohydrolases↗
Evidence for propeptide-assisted folding of the calcium-dependent protease of the cyanobacterium Anabaena.
The Ca(2+)-dependent protease of the cyanobacterium Anabaena variabilis is a cytoplasmic enzyme with a substrate specificity like trypsin. Its previously published DNA sequence [Maldener, I., Lockau, W., Cai, Y. & Wolk, C. P. (1991) Mol. Gen. Genet. 225, 113-120] contained a sequencing error. Here we report the corrected sequence which shows, that the Ca(2+)-protease belongs to the family of subtilases (subtilisin-like serine proteases). Consistent with its cytoplasmic localization, a pre-sequence is not found. The enzyme is produced as a precursor with a large amino-terminal propeptide. Expression of the pro-region and mature region (protease domain) in Escherichia coli cells in trans demonstrates that formation of the active enzyme requires the propeptide. The results demonstrate that propeptide-assisted protein folding also occurs with cytoplasmic enzymes, in support of the hypothesis that this mechanism is a widespread phenomenon.
Amino Acid Sequence↗