Expression and activation of matrix metalloproteinase-2 and -9 in rat brain after transient focal cerebral ischemia.

Matrix metalloproteinases (MMPs) degrade the extracellular matrix and carry out key functions during development and after injury. By means of zymography, Western blot and immunohistochemistry, we studied MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in rat brain after focal cerebral ischemia. The control rat brain showed constitutive MMP-2 and, to a lesser extent, MMP-9, which were mainly present as prozymogens. MMP-2 protein was located in the cell body of neurons, glia, and endothelium, whereas MMP-9 was associated to neurons and myelinated fibre tracts. Ischemia greatly increased MMP activation in two temporal waves, in the first one, MMP-9 protein was induced from 4 h to 4 days, and also a small and short-lasting increase in MMP-2 was detected at 4 h. The second wave showed a massive increase in MMP-2 protein expression and activation by day 4, which was compatible with abundant MMP-2 in reactive microglia/macrophages. Our results are compatible with progressive induction of MMP-9 proform, likely in neurons, shortly after ischemia. For MMP-2, the results suggest a discrete production immediately after reperfusion, while a very enhanced expression and activation of MMP-2 attributable to microglia/macrophages occurs on day 4, and it might contribute to the phagocytic action of these reactive cells.

Environmental toxicity monitoring using electrochemical biosensing systems.

Environmental monitoring faces the challenge of measuring an increasing number of analytes at ever decreasing concentrations. Since not all species of a given analyte have the same detrimental impact on the environment, new analytical devices and techniques are required to distinguish between the different species of a pollutant or different groups of pollutants. This paper describes analytical techniques based on biomaterials that are toxically sensitive to pollutants. This approach permits the biomonitoring of certain compounds by looking at their toxic properties. Although these techniques are based on a sound analytical strategy, their applications are limited because most of the interactions between the biological material and the analyte are irreversible. Additionally, the immobilised biological material has a limited stability. Several biomonitoring strategies based on electrochemical biosensing are discussed here and how to recover the bioactivity of biosensing system, both in discrete and automated procedures, is also reviewed.

(Amino)(aryl)carbenes: stable singlet carbenes featuring a spectator substituent.

Several (amino)(aryl)carbenes have been shown to be stable at room temperature in solution and in the solid state. Electroneutrality of the carbene center is ensured by the amino group, which has both pi-donor and final sigma-acceptor electronic character. The aryl group remains a spectator substituent, as shown by single-crystal x-ray analysis and by its chemical behavior. Because only one electron-active substituent is needed, numerous stable carbenes will become accessible, which will open the way for new synthetic developments and applications in various fields.

Estimation of gelatinase content in rat brain: effect of focal ischemia.

Matrix metalloproteinases degrade the extracellular matrix and are involved in a variety of diseases, including inflammatory diseases of the central nervous system. Here we estimated the content of gelatinase in rat brain under control conditions and 4 h after transient focal ischemia using gelatinolytic extraction and zymographic analysis. We also examined the expression of the MMP-9 and MMP-2 proteins by Western blot. Using the zymographic apparent gelatinase activity we estimated that brain gelatinase content was 0.44 ng/mg of protein. Ischemia induced a 1.7-fold increase at 4 h, thus showing an early MMP response to the ischemic injury. The main increase was seen for the MMP-9 proform, which was accompanied by enhanced MMP-9 protein expression. We suggest that basal cerebral MMP-9 and MMP-2 activities are involved in the maintenance of the extracellular matrix and prevent substrate accumulation, while enhanced postischemic MMP activity before cell death may contribute to edema formation and blood-brain barrier breakdown.

Pancreatitis induces HSP72 in the lung: role of neutrophils and xanthine oxidase.

The aim of this work was to evaluate the systemic Hsp72 expression in rat lung and liver in vivo in a model of acute pancreatitis and investigate the possible involvement of xanthine oxidase and neutrophils in this process. Pancreatitis was induced by intraductal administration of 5% sodium taurocholate and samples of lung and liver were obtained 1 and 3 h later. In some groups of rats circulating xanthine oxidase was inhibited with oxypurinol, and neutrophil recruitment was blocked with a monoclonal antibody against P-selectin. Hsp72 expression was assessed by means of Western blot and immunohistochemistry. Results showed Hsp72 induction in lung, but not in liver, shortly after pancreatitis. Hsp72-induced expression was located in bronchial epithelium, alveolar macrophages, infiltrating neutrophils, and blood vessels. Oxypurinol and the antibody against P-selectin prevented pancreatitis-induced lung Hsp72 overexpression suggesting that Hsp72 induction is mediated by neutrophil infiltration into the lungs.

Flow injection immunoanalysis based on a magnetoimmunosensor system.

A new immunosensor integrated to a flow system has been developed. It is based on magnetic immunoparticles immobilized on a solid-state transducer using a magnetic field. The described technique renews the immunoparticles reproducibly for each analysis allowing a good measurement precision. The developed experimental approach permits the implementation of an automated immunoassay that is quick (analytical cycle < 30 min) and sensitive in the micromolar concentration range. The system was applied to the determination of rabbit immunoglobulin G as an analyte model.

Development of electrochemical immunosensing systems with renewable surfaces.

The repeated use of immunochemically modified solid phases in electrochemical immunosensor analysis is the driving interest of this work. Two new strategies have been developed. One of these strategies is aimed at the development of a manual methodology. It comprises the construction of amperometric immunosensors based on rigid biocomposites. These biocomposites are formed by a conducting polymer composite matrix that acts as a reservoir of an immobilized immunologic material. The surface of the biocomposite can be renewed by a simple polishing procedure. The second strategy involves the design of an automatic methodology. It features an immunochemical analytical system using flow injection techniques. The potentiometric detection uses a solid phase formed by immunologic reagents immobilized in magnetic particles. These particles are fixed to the sensor with the use of a magnetic field. The renewal of the reactive surface is achieved by the release and activation of the restraining magnetic field and the manipulation of the flow. The analytical properties of these immunosensors were evaluated measuring RIgG using a competitive technique and measuring GaRIgG with a sandwich methodology. The labelling enzymes of the immunoconjugates were peroxidase in amperometric measurements and urease in potentiometric measurements.

Stimulation of aryl metabolite production in the basidiomycete Bjerkandera sp. strain BOS55 with biosynthetic precursors and lignin degradation products.

Aryl metabolites are known to have an important role in the ligninolytic system of white rot fungi. The addition of known precursors and aromatic acids representing lignin degradation products stimulated the production of aryl metabolites (veratryl alcohol, veratraldehyde, p-anisaldehyde, and 3-chloro-p-anisaldehyde) in the white rot fungus Bjerkandera sp. strain BOS55. The presence of manganese (Mn) is known to inhibit the biosynthesis of veratryl alcohol (T. Mester, E. de Jong, and J.A. Field, Appl. Environ. Microbiol. 61:1881-1887, 1995). A new finding of this study was that the production of the other aryl metabolites, p-anisaldehyde and 3-chloro-p-anisaldehyde, was also inhibited by Mn. We attempted to bypass the Mn-inhibited step in the biosynthesis of aryl metabolites by the addition of known and suspected precursors. Most of these compounds were not able to bypass the inhibiting effect of Mn. Only the fully methylated precursors (veratrate, p-anisate, and 3-chloro-p-anisate) provided similar concentrations of aryl metabolites in the presence and absence of Mn, indicating that Mn does not influence the reduction of the benzylic acid group. The addition of deuterated benzoate and 4-hydroxybenzoate resulted in the formation of deuterated aryl metabolites, indicating that these aromatic acids entered into the biosynthetic pathway and were common intermediates to all aryl metabolites. Only deuterated chlorinated anisyl metabolites were produced when the cultures were supplemented with deuterated 3-chloro-4-hydroxybenzoate. This observation combined with the fact that 3-chloro-4-hydroxybenzoate is a natural product of Bjerkandera spp. (H. J. Swarts, F. J. M. Verhagen, J. A. Field, and J. B. P. A. Wijnberg, Phytochemistry 42:1699-1701, 1996) suggest that it is a possible intermediate in chlorinated anisyl metabolite biosynthesis.

Validation of an automatic urea analyser used in the continuous monitoring of hemodialysis parameters.

The validation of an automatic urea analyser used in the monitoring of hemodialysis processes is reported. The analyser can indirectly determine dialysis parameters as dialysis delivery (KT/V) and protein catabolism (PCRn). These parameters are useful for the prescription and optimization of hemodialysis. The analyser, based on a previously-reported flow-injection analytical biosystem, was connected on-line to the effluent of a dialysis machine during several hemodialysis sessions. The urea concentration data were continuously processed and dialysis parameters were obtained in quasi real time by means of the integration of an adjusted time-dependent exponential function. These values were compared with those obtained by applying the methods traditionally employed in hospital laboratories. The evaluation comprised 24 data sets from several patients of different gender and age. No significant differences were found between the KT/V and PCRn results obtained with the usual method and those results produced by the analyser proposed here.

Bronchial perforation and bronchoesophageal fistulas: tuberculous origin in children.

A case of bronchial perforation and two cases of bronchoesophageal fistula of tuberculous oritin are presented. The lesions were radiologically identified by bronchography in one patient and esophagography in the other two. One patient with bronchoesophageal fistula died. His lesion had been erroneously considered to be congenital. At surgery, widespread pulmonary tuberculosis was found. The other two patients responded to antituberculous chemotherapy. In one of them, a follow-up barium esophagogram showed a large esophageal diverticulum located where a previous fistulous opening had closed.