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PubMed1986

Factors affecting the ultrastructural pattern of anterograde labeling in axon terminals with HRP.

Comparisons of anterograde labeling of axon terminals originating from short and long projection neurons were made in the hypoglossal nucleus. Injections of dilute and concentrated horseradish peroxidase (HRP) or wheat germ-agglutinin-horseradish peroxidase (WGA-HRP) were made via a glass micropipette into the nucleus reticularis parvocellularis (RPc = short projection neurons) and the Spinal V trigeminal complex (Sp. V = long projection neurons). Axon terminals in the hypoglossal nucleus, a common projection site of the two efferent systems, were evaluated ultrastructurally using diaminobenzidine (DAB) as the chromogen for the cobalt-glucose oxidase (CO-GOD) method of HRP labeling. Labeled axon terminals from these two sources demonstrated different distribution patterns of the reaction product. For the short pathway, high concentrations of the tracers resulted in diffuse, agranular labeling in the majority of axon terminals. Dilute concentrations of the tracers were associated with membrane-bound, granular type of labeling. All anterograde labeling of terminals of long projection neurons (Sp. V) was membrane-bound and granular irrespective of the tracer concentration. The length of the pathway and the concentration of the enzyme tracers are factors that affect the pattern of anterograde label in axon terminals of hypoglossal afferents.

3,3'-Diaminobenzidine↗
PubMed1990

A new chromogen for use in HRP-tract tracing and double-label immunocytochemistry.

A new chromogen, Indophane Blue (IB) was tested for use in HRP-tract tracing and double-label immunocytochemistry. Although TMB was slightly more sensitive and delineated individual labeled fibers better than IB in tract-tracing experiments, there were two advantages to using IB over TMB. First, the IB reaction could be performed at a pH of 6.0, rather than the pH of 3.3 required for TMB, resulting in much better preservation of tissue. Second, there was no crystalline artifact as has been observed with TMB. In the double-label immunocytochemistry experiments, the combination of diaminobenzidine for the first chromogen and IB as the second, provided excellent contrast.

Animals↗
PubMed1992

Evidence for dorsal root projection to somatostatin-immunoreactive structures in laminae I-II of the spinal dorsal horn.

In order to determine if somatostatin (SOM)-immunoreactive (I) cell bodies and processes in lamina (L) II of the rat spinal cord receive dorsal root input, the latter were anterogradely labeled by wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). SOM-I structures were demonstrated by immunohistochemistry. Cell bodies labeled transscellularly or transsynaptically by WGA-HRP and immunohistochemically for SOM were present in L II. In addition, a L I cell was double labeled. These results suggest that some dorsal root axons innervate SOM-I neurons in L I-II of the spinal cord. In addition to confirming immunohistochemical observations in published reports, we have revealed SOM-I central terminals in the type II glomerulus. Further, a SOM-I CI-terminal, presumed to be of primary afferent origin, contacted a SOM-I dendrite in L II. Since SOM has been implicated in nociceptive function in the dorsal horn, it is possible that some of the SOM-I structures identified are involved in nociceptive processing.

Animals↗
PubMed1991

Location and identification of substituents with free amino group in lipopolysaccharides of gram-negative bacteria with the use of chromophore labelling.

Interaction of ten different lipopolysaccharides (LPS) with 2,4-dinitrofluorobenzene yielded quantitatively yellow dinitrophenyl derivatives (DNP-LPS) to show the presence of substituents with free amino group. The DNP-LPS samples were degraded with 1% acetic acid, and after removal of lipid A precipitates the supernatants were separated on a Sephadex G-25 column to give coloured polysaccharide, oligosaccharide and monomeric fractions monitored at lambda DNP = 365 nm. The coloured materials, including DNP-derivative of lipid A, were dephosphorylated with hydrofluoric acid followed by identification of the released DNP-amines by thin layer chromatography (TLC) on silica gel. Subsequently, the dephosphorylated materials were hydrolysed with hydrochloric acid followed by TLC analysis. The approach allowed to detect, locate and identify the substituents with free amino group within the LPS molecules. Moreover, two types of core structures within LPS preparation from one strain were discovered for five microorganisms.

Amino Acids↗
PubMed1991

Improved methods for the detection of beta-galactosidase activity in colonies of Escherichia coli using a new chromogenic substrate: VBzTM-gal (2-(2-(4-(beta-D-galactopyranosyloxy)-3-methoxyphenyl)-vinyl)-3- methylbenzothiazolium toluene-4-sulphonate).

The use of a new substrate 2-(2-(4-(beta-D-galactopyranosyloxy)-3- methoxyphenyl)-vinyl)-3-methylbenzothiazolium toluene-4-sulphonate (VB-zTM-gal) is described for the detection of beta-galactosidase activity in colonies of wild type and mutant strains of Escherichia coli. On enzymic hydrolysis this substrate, which is soluble in water, released a chromophore which is red at pH 7 and bound to cellulose and nitrocellulose. The best procedure for the detection of activity was to grow colonies on standard nitrocellulose membranes (pore size 0.45 microns) laid onto an agar plate and to float the membranes over a solution of the substrate. Coloured colonies developed within 3 min, which were stable at 4 degrees C for several days, and this identified the expression of beta-galactosidase activity. This was found to be more specific than methods using triphenyltetrazolium or Eosin Methylene Blue media, and more economical than methods using X-gal (5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside). VBzTM-gal should have applications in gene cloning technology and in the detection of coliform organisms in polluted water.

Chromogenic Compounds↗
PubMed1992

The detection of lipase activity in bacteria using novel chromogenic substrates.

The propionate (Pro), decanoate (Dec) and laurate (Lau) esters of 5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline++ +-3-acetic acid were assessed as substrates for lipase and esterase. On hydrolysis these substrates yield an intensely red coloured phenol which could be assayed at 505 nm. The Pro ester was an effective substrate for porcine esterase and was hydrolysed at a rate 20 times greater than the Lau and Dec esters. Conversely, Pseudomonas lipase had a high activity towards the Lau and Dec esters, especially in the presence of bovine serum albumin, but little activity towards the Pro ester. The Dec and Lau were used to detect lipolytic activity in Pseudomonas strains associated with milk spoilage. For this purpose, the substrates were absorbed onto filter paper disks, which were placed over bacterial colonies growing on agar plates; activity was indicated by bright red colouration of discs within 2 h. Escherichia coli colonies hydrolysed the Pro but not the Lau or Dec esters.

Benzoates↗
PubMed1984

Perchloric acid treatment and use of chromogenic substrate in the Limulus test: application to veterinary diagnosis.

The Limulus test was carried out for endotoxin determination using a synthetic chromogenic substrate after perchloric acid(PCA) treatment to remove inhibiting substances in the blood. PCA treatment completely removed non-specific amidolytic activity and other factors which interfered with the Limulus test. The recovery of added endotoxin was about 100% irrespective of animal species. Endotoxin levels in PCA-treated blood plasma of healthy domestic animals were invariably below 1 pg ml-1. Many of the cows with gangrenous mastitis, from which Gram-negative rods were isolated, showed high endotoxin levels ranging from 22.8 to 138.0 pg ml-1. Endotoxin was also detected in the blood of cows affected with salmonellosis, colibacillosis, Haemophilus somnus infection and urethritis associated with Proteus.

Animals↗
PubMed1983

A comparison of four methods for detecting beta-lactamase in anaerobic bacteria.

Four methods for detecting beta-lactamase activity in anaerobic bacteria were evaluated and compared to a nitrocefin saturated filter paper test. The methods studied were Cefinase, PADAC, Beta Lactam, and the slide iodometric technique. Fifty anaerobes were tested including species of Bacteroides, Fusobacterium, and Clostridium. The percentages of agreement with the nitrocefin test are: Cefinase 100%, PADAC 96%, slide iodometric 78%, and Beta Lactam 72%. Positive nitrocefin tests have been shown to correlate with a penicillin MIC greater than 0.78 micrograms/ml.

Bacteria, Anaerobic↗
PubMed1995

Development, characterization, and initial evaluations of S1. A new chromogenic cephalosporin for beta-lactamase detection.

A novel, chromogenic cephalosporin reagent (S1) for beta-lactamase testing was produced that shares physicochemical characteristics with nitrocefin (formerly 87/312). S1 and nitrocefin in a disk-testing format for beta-lactamase performed at 100% agreement for detecting enzyme-producing isolates of Bacteroides fragilis group, Haemophilus influenzae, Moraxella catarrhalis, Neisseria gonorrhoeae, Staphylococcus aureus, and selected Enterobacteriaceae. The time required to achieve an initial color change or a strong positive reaction was comparable for both chromogenic reagents for all organisms except the Gram-positive species. S1 reaction times were approximately 50% faster than nitrocefin for beta-lactamase-positive enterococci and S. aureus. These results from the developmental studies and a commercially prepared disk lot indicate that S1 is a promising beta-lactamase disk test reagent with the ability to detect all significant enzyme-producing species strains, some significantly earlier than the nitrocefin disk method.

Bacteria↗
PubMed1990

A clinical comparison of the performance of four blood glucose reagent strips.

The widespread use of visually read blood glucose reagent strips for initiation of emergent treatment of hypoglycemia and hyperglycemia has produced concern over the accuracy of this method. This study evaluated the accuracy of Chemstrip bG (Bio-Dynamics, Boehringer Mannheim, Indianapolis, IN), Dextrostix (Ames, Miles Laboratories, Elkhart, IN), Glucostix (Ames), and Visidex II (Ames) as compared with hospital laboratory values in an emergency department (ED) setting. Blood samples from 96 ED patients were tested for glucose concentration by each of the four strips and by the hospital laboratory. Each strip was evaluated for sensitivity, specificity, correlation coefficient (r), 95% confidence intervals, and kappa statistic (kappa, a measure of agreement between nonparametric data) using laboratory values as reference. In addition, six observers scored each strip for ease of interpretation using an ordinal scale of 1 (poor) to 4 (excellent). From the samples, no patients were hypoglycemic (less than or equal to 60 mg/dL), 83 were euglycemic (greater than 60 and less than 160 mg/dL), and 13 were hyperglycemic (greater than or equal to 160 mg/dL). Results suggest that over the range of glucose concentration sampled, there is good to excellent correlation with laboratory values for all strips except Dextrostix. The lower r value for Dextrostix is in part artifact due to limitation of its range of measurement to less than or equal to 250 mg/dL. Decreased accuracy for all strips in the hyperglycemic range may have been attributable to small sample size. Chemstrip bG and Visidex II were found to be subjectively easier to interpret.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Glucose↗
PubMed1986

The reaction of 2-thiobarbituric acid with biologically active alpha,beta-unsaturated aldehydes.

The standard assay for lipid peroxidation is the measurement of the pink, 532 nm absorbing chromogen which is formed upon reaction of 2-thiobarbituric acid (TBA) with the lipid peroxidation product malonaldehyde (MDA). The present studies indicate that the toxic lipid peroxidation product trans-4-hydroxynonenal and its dehydration product trans,trans-nonadienal react with TBA to form chromogens which absorb maximally at 530 and 532 nm, respectively. Other biologically active alpha,beta-unsaturated aldehydes, such as acrolein and crotonaldehyde, short-chain homologs of alkenals formed during lipid peroxidation, and trans,trans-muconaldehyde, a novel diene dialdehyde, react with TBA to form products which absorb maximally at 495 nm. The molar extinction coefficients of the aldehyde:TBA chromogens formed were found to vary widely, suggesting that only small contributions to the 532 nm absorption by TBA adducts of reactive aldehydes other than MDA may be encountered during the use of the TBA assay.

Aldehydes↗
PubMed1994

Assessment of myeloperoxidase activity in renal tissue after ischemia/reperfusion.

We have shown that a photometric assay of myeloperoxidase derived from rat blood polymorphonucleocytes employing 3,3',5,5'-tetramethylbenzidine as substrate is more sensitive than an established assay employing o-dianisidine. We went on to demonstrate that rat renal tissue is capable of inhibiting peroxidase activity. This activity approached 100% when the rat renal supernate was incubated at 60 degree C for 2 h and the assay was conducted in the presence of a 10-fold higher concentration of hydrogen peroxide (H2O2). Rat kidneys undergoing 45 min ischaemia and 1,3 and 6 h reperfusion in vivo, exhibited significant increases in myeloperoxidase activity, indicating tissue polymorphonucleocyte accumulation. Monoclonal antibodies against rat intercellular adhesion molecule 1 (ICAM-1) and CD18 of beta 2-integrins administered both 5 min before a period of 45 min renal ischaemia (20 micrograms/kg i.v.) and at the commencement of 1 h reperfusion (20 micrograms/kg i.v.) reduced renal tissue polymorphonucleocyte accumulation. However, similar treatment with the parent murine antibody immunoglobulin G1 (IgG1) and an unrelated murine antibody, IgG2a, also significantly reduced renal tissue polymorphonucleocyte accumulation. In conclusion, we demonstrate that the rat renal suppression of peroxidase activity can be overcome by a combination of heat inactivation and the provision of excess assay H2O2. In addition, the available evidence suggests that murine monoclonal antibodies against rat adhesion molecules may exert non-specific actions in our model of renal ischaemia/reperfusion in vivo.

Animals↗
PubMed1993

Methods for measurement of antibody/antigen affinity based on ELISA and RIA.

Various enzyme-linked immunosorbent assay or radioimmunoassay methods are currently used to quantify the antibody-antigen interaction. Only those based on indirect competition--enzyme-linked immunosorbent assay or radioimmunoassay--can provide the real thermodynamic affinity of the antibody for its antigen. They can be applied to a variety of experimental situations, some of which are reviewed here.

Antibodies, Monoclonal↗
PubMed1992

lacZ transduced human breast cancer xenografts as an in vivo model for the study of invasion and metastasis.

A number of human cancer cell lines have been described as being invasive and metastatic in immune incompetent animals. However, it is difficult to assess metastatic spread of a subcutaneously injected or inoculated cell line, since an exact detection of all microfoci of human tumour cells in the animals by usual histological procedures would require extensive sectioning of the whole animal. To overcome this problem, we transduced human breast cancer cells with a replication-defective Moloney murine leukaemia retroviral vector (M-MuLV) containing both neoR (neomycin resistance) and lacZ genes. The resulting cell lines were selected for antibiotic (G418) resistance, and cell-sorted for lacZ expression. lacZ continued to be expressed in cultured cells for at least 20 passages without further G418 selection. The lacZ gene codes for beta-D-galactosidase, and cells expressing this gene stain blue with the chromogenic substrate X-gal. The lacZ-expressing cells retained the pre-transduction ability to traverse Matrigel in vitro, to form subcutaneous tumours in nude mice, and to grow invasively with the formation of metastases. X-gal staining showed high specificity, staining the tumour cells but not the surrounding mouse tissue on either whole tissue blocks or histological sections. The staining procedure was highly sensitive, allowing detection of microfoci of human cancer cells, and quantitative estimation of the metastatic capacity of the cells. These results indicate that lacZ transduction of human tumour cells is a powerful means of studying human cancer cell invasion and metastases in vivo.

Animals↗
PubMed1995

On the conformation of Phe78 of a chromoprotein antibiotic, neocarzinostatin.

A structure of neocarzinostatin, an antitumor chromoprotein antibiotic, has been built using X-ray crystallographic data and NMR data, particularly NOE data observed between the apoprotein and the chromophore. Chemical shift changes of protons of the chromophore upon binding to the apoprotein indicated that the aromatic plane of Phe52 has the conformation almost perpendicular to the C-2-C-3 triple bond of the core of the chromophore while Phe78 takes multiple conformations in solution although one of the stable conformations has been assigned for Phe78 in a crystal structure.

Antibiotics, Antineoplastic↗
PubMed1990

The potency of tissue-type plasminogen activator (TPA) determined with chromogen and clot-lysis assays.

An assessment was made of two methods for determining the potency of tissue-type plasminogen activator (TPA). A chromogenic microtitre plate assay was established which contained TPA, plasminogen, a synthetic plasmin substrate (H-D-valyl-L-leucyl-L-lysyl-p-nitroaniline dihydrochloride, S2251) and any one of the following stimulators: native fibrinogen, enzymatic and chemical digests of fibrinogen, poly-D-lysine (PDL) and chemical derivatives of the latter. The chromogen assay was compared with an automated clot-lysis (turbidimetric) assay for sensitivity, reproducibility and validity for potency determination. Reference preparations of TPA were titrated in both assays: in the chromogen assay the dose-response curves were non-parallel, whereas parallelism was observed in the clot-lysis assay. Thus, the chromogen assay was restricted in its applicability and disqualified from any routine regulatory use. The potency of individual lots of recombinant (r)TPA could only be estimated in International Units (IU) of TPA activity with the automated clot-lysis assay and the potency values obtained (IU/vial) were in remarkably close agreement with the manufacturers' values.

Chromogenic Compounds↗
PubMed1990

Evaluation of the use of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA).

Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain maximum sensitivity for both systems. Similar sensitivities were found using fluorogenic and chromogenic substrates. Tetramethyl benzidine substrate for horse-radish peroxidase enzyme conjugates was found to attain the highest sensitivity levels for chromogenic assays (0.12 ng IgG/ml and 1.0 ng/ml FMDV respectively), after 10 min incubation. Of the two fluorogenic enzyme/substrates studied, B-galactosidase was the most sensitive but required extended incubation times (2-3 h) as compared with chromogenic systems. Special microplates for fluoro-immunoassay (FIA) were compared with conventional microplates and no advantage was found to justify their use. An alkaline phosphatase anti-guinea-pig conjugate was used to confirm the equivalence of fluorogenic and chromogenic substrates in terms of sensitivity. A comparison of the amount of signal generated using various concentrations of enzyme in the absence of antigen was made for two different alkaline phosphatase conjugates to obtain theoretical sensitivity limits. One possible advantage of fluorogenic substrates is that high binding ratio can improve the confidence in discrimination of positive results.

Animals↗