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PubMed1995

Hemorrhagic principles in the venom of Bitis arietans, a viperous snake. I. Purification and characterization.

Two hemorrhagic principles (Bitis arietans hemorrhagin a and b: abbreviated as BHRa and BHRb) were purified from the venom of the viperous snake Bitis arietans (puff adder) by gel filtration, ion-exchange and absorption chromatography. A 10-fold purification was achieved for BHRa and 7-fold for BHRb with an overall yield of 6.4% of hemorrhagic activity. The hemorrhagins were homogeneous according to disc- and SDS-polyacrylamide gel electrophoresis and immunodiffusion. BHRa and BHRb consist of 623 and 685 amino-acid residues and their apparent molecular weights were 68,000 and 75,000, respectively. They were also immunologically distinct. The purified hemorrhagins express proteolytic activity with heat-denatured casein and hide powder azure. The proteolytic activity with heat-denatured casein was almost the same as that of the crude venom, but that with hide powder azure was less than one-tenth of that of the crude venom. The purified hemorrhagins were free of arginine esterase and phospholipase A2 activities and they are acid labile hemorrhagic toxins. Their hemorrhagic activity was inhibited by EDTA, cysteine and by polyvalent anti-snake serum, but not by phenylmethanesulfonyl fluoride or soybean trypsin inhibitor.

Amino Acids↗
PubMed1994

Plasminogen activator inhibitor type-1 interacts exclusively with the proteinase domain of tissue plasminogen activator.

Two different techniques have been used to study the complex formation of recombinant human plasminogen activator inhibitor type-1, PAI-1, with either recombinant human two-chain tissue plasminogen activator, tc tPA (EC 3.4.21.68), or the tPA deletion variants tc K2P, containing the kringle 2 domain and the proteinase domain, and P, containing only the proteinase domain. The same value for Kon, 2.10(7) M-1s-1 for binding of PAI-1 was found for the three tPA forms by direct detection of the complex formation in real time by surface plasmon resonance, BIAcore, or indirectly by monitoring the time course of the inhibition of tPA using the chromogenic substrate N-methylsulfonyl-D-Phe-Gly-Arg-4-pNA-acetate. Apparently, no conformational change is involved in the rate-limiting step, since the kon value was found to be independent of the temperature from 20 to 35 degrees C. By the BIAcore technique, it was found that the complex between PAI-1 and tPA covalently coupled to the surface, was stable at 25 degrees C, since no dissociation was seen in buffer. However, extended treatment with 1 M NH4OH destroyed the complex with t 1/2 = 5 h. The same kon values and complex composition were found by measuring either the binding of tPA to PAI-1 captured on the monoclonal antibody MAI-11 or the binding of PAI-1 to tPA captured on the monoclonal antibody 2:2 B10. Quantification of the complex composition between PAI-1 captured on the monoclonal antibody MAI-11 with either tPA, K2P or P gave a one-to-one ratio with the fraction of active PAI-1, consistent with the results from SDS-PAGE and the specific activity of PAI-1. The complexes of the three tPA forms with PAI-1 captured on a large surface of MAI-11 dissociated more rapidly from MAI-11, with the same apparent koff, kdis, = 2.10(-3) s-1, compared with 0.7-10(-3) s-1 for the dissociation of PAI-1 alone. In consistance, the Kd, calculated from the direct determination of the kon and koff for the association of different form of PAI-1 to a small surface of MAI-11, was found to be higher for PAI-1 in complex with tPA than for free active PAI-1. Apparently, upon complex formation, a change is induced in PAI-1 at the binding epitope for MAI-11.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Release of iron from diferric transferrin in the presence of rat liver plasma membranes: no evidence of a plasma membrane diferric transferrin reductase.

The transfer of iron from diferric transferrin to bathophenanthroline disulfonate was measured under varying conditions by spectrophotometry and EPR spectroscopy. Intact rat hepatocytes efficiently mediated the transfer of iron from human diferric transferrin to bathophenanthroline disulfonate. Isolated rat liver plasma membranes, in contrast, failed to facilitate the reaction at pH 7.4 in the presence of NADH, although the membranes were able to reduce ferricyanide and to oxidize NADH. Oxidation of NADH was stimulated by diferric transferrin. However, ferricyanide reductase and transferrin-stimulated NADH oxidase activities were apparently not linked to release of iron from transferrin. Our results, together with theoretical considerations, show that the ability (or inability) of intact cells or isolated plasma membranes to facilitate the transfer of iron from transferrin to strong diferric iron chelators does not allow interferences about the existence of an iron reduction step as part of the process of cellular uptake of iron from transferrin.

Apoproteins↗
PubMed1992

trans-5-prostaglandin E2 stimulates plasminogen activation by tissue-type plasminogen activator.

The effect of trans-5-prostaglandin E2 (trans-PGE2) on fibrinolysis was examined in vitro using synthetic chromogenic substrate S-2251. trans-PGE2 was found to enhance plasminogen (PLG) activation mediated by tissue-type plasminogen activator (tPA). The enhancing effect was dependent on the concentration of trans-PGE2. cis-PGE2 and the other PGs (PGE1 and PGI2) did not show such an effect as trans-PGE2, despite to the fact that their structures are similar to that of trans-PGE2. trans-Configuration around the double bond at the 5-position seems to be important in the enhancement of the fibrinolytic activity.

Amino Acid Sequence↗
PubMed1996

Fluorogenic and chromogenic enzyme substrates in culture media and identification tests.

Rapid detection and identification of microorganisms is extremely important in many fields of applied and research microbiology. In general, fluorogenic and chromogenic substrates have proved to be a powerful tool, utilizing specific enzymatic activities of certain microorganisms, either in parallel with or instead of traditional methods. By incorporation of synthetic fluorogenic or chromogenic substrates into primary selective media, enumeration and detection can be performed directly on the isolation plate. The introduction of many of these media and identification tests has led to improved accuracy and faster detection of target organisms, often reducing the need for isolation of pure cultures and confirmatory tests.

Chromogenic Compounds↗
PubMed1991

Plasma endotoxin concentrations in patients with alcoholic and non-alcoholic liver disease: reevaluation with an improved chromogenic assay.

Plasma endotoxin concentration was measured in 85 patients with alcoholic liver disease (alcoholic cirrhosis (n = 64), alcoholic hepatitis without cirrhosis (n = 11), fatty liver (n = 10), and in patients with non-alcoholic cirrhosis (n = 15]. Endotoxin concentration was determined with an improved chromogenic substrate assay, using individual standard curves for each plasma sample. In patients with alcoholic cirrhosis the mean endotoxin concentration was significantly higher than in patients with non-alcoholic cirrhosis (p less than 0.05). In addition, distinctly higher endotoxin concentrations (greater than 20 pg/ml) were more frequently observed in patients with alcoholic cirrhosis than in non-alcoholic cirrhosis (34.4 vs. 14.3%, p less than 0.05). Mean endotoxin concentration was not significantly higher in cirrhotics with ascites or esophageal varices as compared with the subgroup without ascites or esophageal varices. The endotoxin concentration did not correlate with serum bilirubin, prothrombin concentration or serum enzyme activities. In patients with alcoholic liver disease, however, endotoxin concentration revealed a negative correlation (p less than 0.05) with the concentration of high density lipoprotein cholesterol. On admission endotoxin concentrations in alcoholics with fatty liver were similarly elevated as observed in alcoholic cirrhosis. In six out of 12 patients with fatty liver or alcoholic hepatitis, in whom a second sample of plasma was investigated after 6 to 8 days, endotoxemia was no longer detectable; in the remaining patients, the endotoxin concentration decreased markedly. The results indicate that, irrespective of the stage of liver disease, alcohol abuse favours the development of endotoxemia. They support the hypothesis that gut-derived endotoxins might play a role in the initiation and aggravation of alcohol-induced liver disease.

Adult↗
PubMed1994

Ultrastructural evidence for synaptic inputs of enkephalinergic nerve terminals to target neurons in the rat arcuate nucleus.

The morphological support of interactions between enkephalins and three systems--beta-endorphin (beta-END), tyrosine hydroxylase (TH), or neuropeptide Y (NPY)--well represented in the arcuate nucleus, was examined by using an electron microscopic double immunostaining combining two sensitive chromogens, diaminobenzidine (DAB) and tetramethylbenzidine (TMB). The first step consisted of visualizing Metenkephalinergic terminals with DAB reaction product, and the second one involved detecting the antigens TH, beta-END, and NPY in their respective neurons with TMB reaction product. Ultrastructural analysis revealed enkephalinergic terminals presynaptic to TH-immunopositive cells and dendrites, principally in the dorsal portion of the arcuate nucleus. Enkephalinergic nerve terminals also contacted synaptically ventrolaterally located beta-END-immunoreactive cells. In the ventromedial arcuate nucleus, few synaptic contacts were observed between enkephalinergic boutons and NPY neurons, which were principally in close apposition with glial processes. Enkephalin-immunoreactive synapses were more frequently seen on TH-immunopositive neurons. This TH neuronal group is known to correspond to the dopaminergic tuberoinfundibular neurons implicated in the control of reproductive functions. The pattern of distribution of the different synapses within the arcuate nucleus (TH dorsal, beta-END ventrolaterally; NPY ventromedially) suggests that enkephalins may play a role in the neuroendocrine regulation of gonadotropin and prolactin secretion. The results provide evidence that enkephalins, in the arcuate nucleus, exert a postsynaptic action on the beta-END cells in addition to the presynaptic regulation previously demonstrated in the mediobasal hypothalamus, related to beta-END release. Moreover, the arcuate nucleus is a site of intercellular relationships between enkephalins and dopamine and between enkephalins and other peptides such as NPY.

3,3'-Diaminobenzidine↗
PubMed1993

Evidence of free radical damage in the central nervous system of guinea-pigs at the prolonged acute and early relapse stages of chronic relapsing experimental allergic encephalomyelitis.

Central nervous system tissue from guinea-pigs in various stages of chronic relapsing experimental allergic encephalomyelitis (CR-EAE) has been analysed for evidence of free radical damage using the thiobarbituric acid test as an indicator of oxidative changes. Levels of thiobarbituric acid-reactive materials in brain and spinal cord regions from prolonged acute and early relapse phases of CR-EAE were significantly higher than in central nervous system tissue from Freund's adjuvant control and normal uninoculated animals. In the late relapse phase of CR-EAE, levels of thiobarbituric acid-reactive materials in CNS samples were at control values. Fluorescence analysis of central nervous system tissue confirmed evidence of increased free radical damage in prolonged acute and early relapse stages of CR-EAE. The increased oxidative damage observed in the central nervous system in CR-EAE is discussed in relation to the role of macrophages and their oxidative burst.

Acute Disease↗
PubMed1991

Chromogenic peptide substrate assays and their clinical applications.

Chromogenic peptide substrates were first introduced into research laboratories in the early 1970s and were quickly utilised to develop assays for the determination of enzymes, proenzymes and inhibitors of the coagulation system. These assays were gradually introduced into coagulation and clinical chemistry laboratories as laboratory tools in the diagnosis and treatment of coagulation disorders. From the knowledge of the structures of the natural substrates attacked by enzymes other than those of the coagulation system or by synthesis and random screening, substrates for enzymes of the fibrinolytic, plasma and glandular kallikrein and complement systems were produced. These allowed various research groups to develop assays for components of these systems and subsequently led to the use of these assays in studies on various clinical conditions. Substrates for activated protein C ensured that assays for this enzyme and its inhibitors could be developed and introduced into the haematological routine. With the introduction of substrates for limulus lysate not only were assays for endotoxins in clinical samples produced but the control of all disposable products and injectables for endotoxin contamination can now be effected. Initially high costs and time-consuming manual assays were a hinderence to the general acceptance of the use of chromogenic peptide substrate assays and they were only used routinely in a few specialised laboratories. With the introduction of automated and microtitre plate methods however, these assays are are now available in most hospital laboratories. Since the first chromogenic peptide substrate was described thousands of articles have been published on the use of chromogenic substrate assays to measure proenzymes, enzyme activators, enzyme cofactors and inhibitors in blood and other body fluids in normal subjects and clinical material. We have endeavoured to cover as many of these as possible in this review.

Blood Chemical Analysis↗
PubMed1990

The linkage between adenosine nucleotide binding and amidase activity in human alpha-thrombin.

The amidase activity of human alpha-thrombin has been studied in the presence of the adenosine nucleotides AMP, ADP and ATP. At low concentrations, adenosine nucleotides increase thrombin activity up to 30%, while at high concentrations (greater than 5 mM) inhibition takes place up to 20%. Inhibition is progressively reduced by increasing substrate concentration. A simple, phenomenological description of the linkage between adenosine nucleotide binding and amidase activity of human alpha-thrombin is proposed and the free energy changes for the underlying reactions involved in the linkage scheme are resolved by global analysis of the experimental data. The linkage scheme assumes that thrombin activation is determined by a conformational transition due to binding of adenosine nucleotides to a regulatory site. Inhibition, on the other hand, would be a consequence of competitive binding to the catalytic site.

Adenosine Diphosphate↗
PubMed1992

Protease 'Clipsin' extract activity reflects demographic characteristics of human brain.

'Clipsin' and other peptide bond hydrolase activities that appear to be integral membrane proteins, including two implicated in the formation of the histological hallmarks of Alzheimer's disease, were assayed in the frontal and parietal cortex from a large (n = 45) series of postmortem human brains. Tissues were extracted sequentially with detergent-containing and low ionic strength buffers. The final extracts obtained with detergent-high ionic strength buffer were assayed for peptide-bond hydrolase activity using radiolabelled casein and four chromophore-linked peptide substrates. Hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and alpha-casein showed evidence of sensitivity to the presence of Alzheimer's disease (n = 22) and some of the seven other demographic features (postmortem delay; mode of death in the case of one substrate) of the brain samples considered. By contrast, activity towards carbobenzoxy(Z)-Leu-Leu-Glu-2-naphthylamide, Z-Val-Lys-Met-4-methyl-coumaryl-7-amide and Z-Val-Lys-Lys-Arg-4-methoxy-2-naphthylamide were independent of all factors. The results are discussed in terms of damage to a sub-population of protease-containing membranes.

Aged↗
PubMed1988

Interaction of bovine factor XIIa with an inhibitor from bovine plasma.

An inhibitor of factor XIIa has been purified from bovine plasma and characterized (Thornton, R.D. and Kirby, E.P. (1987) J. Biol. Chem. 262, 12714-12721). This inhibitor interacts with XIIa to form a very stable complex with a 1:1 stoichiometry. The active site of XIIa, located on the light chain, is directly involved in the interaction, and complex formation between factor XIIa inhibitor and XIIa can be blocked by diisopropyl fluorophosphate, corn trypsin inhibitor, or the chromogenic substrate S2302. Incubation of the complex with excess XIIa does not result in cleavage of the complex. The complex does not spontaneously dissociate and is stable to boiling, SDS, thiocyanate, acid, and hydroxylamine or Tris at pH 7-10. In addition to complex formation, a cleaved form of factor XIIa inhibitor can be observed. We suggest that the inhibitor is acting as a mechanism-based inactivator, using the criteria of time-dependent inactivation under pseudo-first-order conditions, 1:1 stoichiometry, active site involvement, kinetic protection by substrate or by an active site inhibitor, and partitioning between cleavage of factor XIIa inhibitor and inactivation by complex formation.

Animals↗
PubMed1994

Ineffectiveness of the inhibition of the main haemorrhagic metalloproteinase from Bothrops jararaca venom by its only plasma inhibitor, alpha 2-macroglobulin.

Observations that a haemorrhagic metalloproteinase (jararhagin) from Bothrops jararaca venom had less effect on platelets suspended in plasma than in washed platelet suspensions, suggested that plasma contains naturally occurring inhibitor(s) of this enzyme. By using radiolabelled jararhagin and crossed immunoelectrophoresis, we have demonstrated the binding of this enzyme to alpha 2-macroglobulin in plasma. SDS-PAGE analysis of this binding revealed the presence of radioactivity in four bands with relative molecular masses of 640, 570, 520 and 410 kDa; in addition a small amount of 47 kDa free enzyme was demonstrable. Reduced samples showed an additional non-complexed 90 kDa fragment of alpha 2-macroglobulin generated by jararhagin. These results are compatible with a model in which, upon multiple cleavages of alpha 2-macroglobulin, the enzyme becomes covalently bound to the inhibitor, and the two halves of the inhibitor become crosslinked. However, jararhagin activity was not completely inhibited even after long incubation (60 min) with a large (10-fold) molar excess of alpha 2-macroglobulin either in plasma or a purified alpha 2-macroglobulin preparation. Kinetic studies showed that inhibition was comparatively slow, although jararhagin readily cleaved alpha 2-macroglobulin in the bait region. Therefore, the ineffectiveness of the inhibition could have resulted from a low tendency of this proteinase to form covalent complexes with the inhibitor. We conclude that the pronounced haemorrhagic activity of jararhagin can be attributed to prolonged access of this enzyme to high molecular weight substrates, even in the presence of a large molar excess of alpha 2-macroglobulin.

Animals↗
PubMed1984

Application of the silver-gold intensified 3,3'-diaminobenzidine chromogen to the light and electron microscopic detection of the luteinizing hormone-releasing hormone system of the rat brain.

A highly sensitive, recently developed immunohistological method is introduced in the present study with special emphasis on the luteinizing hormone-releasing hormone system of the rat brain. The method utilizes the specific capability of the diaminobenzidine endproduct, a frequently used chromogen in immunocytochemistry, to produce and bind silver grains from a special physical developer, following suppression of the argyrophilia of the nervous tissue by thioglycolic acid. Metal deposition into the immunolabelled structures results in a real Golgi-like appearance of immunoreactive profiles. Specificity of this silver method was confirmed by ultrastructural analysis, which showed that unlabelled elements did not bind silver. Using this method, more immunoreactive neurons and fibres were visualized than compared with the results of the traditional peroxidase-antiperoxidase method. The luteinizing hormone-releasing hormone-synthesizing neurons proved to be fusiform, exhibiting either smooth or rough surfaced contour. Unlabelled terminals established axo-somatic synapses on labelled perikarya. The juxtaposition of immunoreactive luteinizing hormone-releasing hormone profiles suggest the possibility of self-regulation within the luteinizing hormone-releasing hormone system. The main advantages of the method are the increased sensitivity with preserved selectivity and wide applicability in different fields of neuroscience (peptide and transmitter immunocytochemistry, double labelling, horseradish peroxidase tract tracing, X-ray analysis).

3,3'-Diaminobenzidine↗
PubMed1986

A qualitative and quantitative analysis of the distributions of cells in the spinal cord and spinomedullary junction projecting to the thalamus of the rat.

Horseradish peroxidase retrograde transport has been used to locate and make a quantitative study of neurons of the rat spinal cord which project to the thalamus. Across the grey, labelled cells were found in the head and neck of the dorsal horn (in three locations, but not the substantia gelatinosa); in the region of the internal basilar nucleus of Ramón y Cajal; in the intermediate grey zone; in the lateral cervical nucleus and funiculus; and in the ventral horn. The latter contains the largest population of neurons projecting ipsilaterally. The most significant finding concerns the craniocaudal distribution of the cells. More than 50% of the thalamically projecting neurons are confined to the upper four cervical segments, where every population is represented. Some populations are continuous with thalamically projecting populations in the lower medulla. The cervical enlargement contains less than 5% of spinothalamic cells. The lumbar enlargement contains 33%, most of which are in the region of the internal basilar nucleus. This population is also marked in the upper cervical segments. Thus, in the rat, the origin of the spinothalamic tract is distributed along the cord in a very uneven manner. This may indicate that sensory information delivered to any one segment of the spinal cord by a primary afferent is not always relayed direct to the thalamus by a local second order neuron; that different parts of the body are not represented to the same extent in the spinothalamic system; or that these two factors combine to produce the observed distribution.

Afferent Pathways↗
PubMed1987

Brainstem projections to lumbar motoneurons in rat--II. An ultrastructural study by means of the anterograde transport of wheat germ agglutinin coupled to horseradish peroxidase and using the tetramethyl benzidine reaction.

The descending projections from the ventrolateral medial reticular formation to the lumbar motoneuronal cell groups were studied in rat at the ultrastructural level using the anterograde transport of wheat germ agglutinin coupled to horseradish peroxidase in combination with the chromogen tetramethyl benzidine. The tissue containing the horseradish peroxidase reaction products was processed for electron microscopy using the method of chemical dehydration. In the ultrathin sections of the lumbar motoneuronal cell groups reaction products were easily recognized by their electron-dense crystal-like structure. These crystals were mostly found in terminals (66%) and to a lesser extent in axons (10%) and dendrites (10%). In the lumbar motoneuronal cell groups six different types of terminals were distinguished. It was found that F-type terminal profiles most frequently contained crystals, whereas fewer S- and G-types did so. The various findings are basically the same as those of a similar ultrastructural autoradiographic study with [3H]leucine as a tracer, except for the labelling of dendrites, which is discussed. It is concluded that the horseradish peroxidase technique employed in the present study can be reliably used for anterograde tracing at the ultrastructural level. Its advantages and disadvantages in comparison with the electron microscopical autoradiographic technique are discussed.

Animals↗
PubMed1993

Selective labelling of primary sensory afferent terminals in lamina II of the dorsal horn by injection of Bandeiraea simplicifolia isolectin B4 into peripheral nerves.

The I-B4 isolectin from Bandeiraea simplicifolia exhibits specific binding to a subpopulation of rat dorsal root ganglion neurons of small diameter which terminate in the substantia gelatinosa of the dorsal horn. Recent double-labelling experiments in the rat have demonstrated that only primary afferents which innervate the skin are recognized by the I-B4 lectin [Plenderleith and Snow (1993) Neurosci. Lett. (in press)]. As the I-B4 lectin appears to bind selectively to a subset of small-diameter primary afferents with cutaneous peripheral projections, we sought to determine whether it could be used as a transganglionic tracer which selectively labels the spinal terminations of cutaneous afferents in superficial dorsal horn. We now report that the I-B4-horseradish peroxidase conjugate labels synaptic terminals in lamina II of the dorsal horn following the injection of the conjugate into the sciatic and saphenous nerves in the rat. Electron-microscopic examination of the dorsal horn revealed many examples of labelled synaptic terminals and unmyelinated axons, but in no cases was label observed in myelinated axons. No label was observed outside of the substantia gelatinosa; thus the I-B4 isolectin is unique among lectins used for transganglionic tracing in that it does not retrogradely label motoneurons. These results, together with previous studies of lectin binding properties of primary sensory afferents, suggest that injection of I-B4 conjugates into peripheral nerves enables the visualization of the central terminations of cutaneous C-fibres. Transganglionic labelling with the I-B4 isolectin from Bandeiraea simplicifolia should facilitate further examination of synaptic relationships of nociceptive cutaneous afferents in the superficial dorsal horn.

Afferent Pathways↗
PubMed1985

Stabilization of TMB reaction product for electron microscopic retrograde and anterograde fiber tracing.

Use of the highly sensitive tetramethylbenzidine (TMB) method of horseradish peroxidase histochemistry for electron microscopy has been limited by the solubility of the reaction product in aqueous and alcoholic solutions. We have found that following the TMB reaction with a diaminobenzidine-cobalt (DAB-Co) step causes the TMB crystals to become coated with DAB-Co. The resultant reaction complex is insoluble, and easily localized using electron microscopy. By systematically varying the pH at which the TMB reaction is run, the size and shape of the reaction complex can be controlled. The pH 4.0 reaction complex was the most suitable for electron microscopic identification of labeled structures less than 1.0 micron in diameter (e.g., axon terminals).

Animals↗