Modulation of Serine Proteinases and Metalloproteinases During Morphogenic Glial-Endothelial Interactions

Abstract: The regulation of microvessel formation and the expression of CNS-specific endothelial properties are attributed to perivascular astroglia. Specific proteolytic pathways mediate processes such as tissue remodeling, differentiation, invasion, and metastasis. We used a coculture system in which C6 glial cells induce CNS microvascular endothelial cells to form capillary-like structures to examine the role of plasminogen activators and collagenases in CNS microvessel morphogenesis. Fibrin zymography revealed the presence of high-molecular weight urokinase-type plasminogen activator (uPA), low-molecular weight uPA, and uPA/inhibitor complexes within endothelial cultures and cocultures. Gelatin zymography revealed the presence of 92-, 72-, and 62-kDa type IV collagenases within endothelial cultures and cocultures. uPA activity was confirmed by incubating the extracts with amiloride, an inhibitor of uPA. Collagenase activity was confirmed by incubating the gels with EDTA, an inhibitor of metalloproteinases. Quantitative densitometry showed a six- to eightfold decrease in coculture uPA during capillary-like structure formation. Substantially less change in type IV 72-kDa procollagenase activity was seen in cocultures during capillary-like structure formation, but active type IV 62-kDa collagenase activity was significantly increased during capillary-like structure formation. These findings establish that uPA and activated type IV collagenase activity specifically regulates morphogenic endothelial responses to glial interactions and suggest mechanisms by which microvessels respond within the CNS.     

An early cretaceous section in the Kircaova area (Berdiga Limestone,NE-Turkey) and its correlation with platform carbonates in W-Slovenia

Summary In the Kale (Gümüshane) area in the North Eastern Turkey, platform carbonates of the Berdiga Limestone were deposited during Late Jurassic-Early Cretaceous time in environments varying from intertidal to fore reef. The sequence shows extensive lateral and vertical alterations and interfingering of different facies types. In the upper part of the Berdiga Limestone in the Kircaova area a bituminous thin-bedded to platy limestone and shale 5 to 6 m thick occurs at the Early/Late Aptian boundary. It is underlain by limestones rich in silica nodules of up to 10 cm size. A facies analysis of a section about 70 m thick including the bituminous interval was carried out in 1994/95 at the SW border of the Kircaova area close to the road from L?rikas to Kale. The limestones consist mainly of packstones and grainstones locally rich in calcareaous algae and forminifera. Fragments of molluscs and echinoids as well as some ostracods and calcispheres occur. Some sponges, corals, and beds rich in molluscs occur in minor amounts in the middle part of the section which is characterized by intertidal to shallow subtidal facies. Algae and foraminifera indicate a Barremian-Early Aptian age of the lower part and Late Aptian age of the upper part of the section (e.g.Salpingoporellamuehlbergii, Salpingoporella aff.melitae, Clypeina solkani, Novalesia producta), divided by the bituminous limestones. In West Slovenia (close to the Italian border) a complete Cretaceous section occurs at Sabotin mountain containing Aptian beds with comparable faunal composition. In contrast to the Berdiga Limestone, in Slovenia at the rim of the dinaric platform a patch reef about 50 m in thickness is developed which is also covered by a bituminous limestones (black shale) marking the Early/Late Aptian boundary. Faunal elements in Slovenia arePalorbitolina lenticularis, Cuneolina laurentii, Orbitolina (Mesorbitolina) texana andSalpingopoprella dinarica. The bituminous limestone appears to be a marker horizon. At both locations it is locally rich in characeans probably indicating a regressive maximum before another transgression began in the Late Aptian/Albian as world-wide drowning event. Possibly the occurrence of the bituminous limestone (black shale) is associated with volcanic activity during the Aptian. If so it could be used as a chronostratigraphic marker horizon in both areas analyzed.     

The Novel Extracellular Cyclophilin A (CyPA) - Inhibitor MM284 Reduces Myocardial Inflammation and Remodeling in a Mouse Model of Troponin I -Induced Myocarditis

Cyclophilins are a group of highly conserved cytosolic enzymes that have a peptidylprolyl cis/trans isomerase activity. Cyclophilin A (CyPA) can be secreted in the extracellular space by inflammatory cells and upon cell death. The presence of CyPA in patients with non-ischemic cardiomyopathy is associated with poor clinical prognosis. Here, we investigated the inhibition of extracellular CyPA in a mouse model of troponin I-induced autoimmune myocarditis using the strictly extracellular CyPA-inhibitor MM284. Since A/J mice develop severe inflammation and fibrosis after immunization with murine cardiac troponin I (mcTn I), we used this model to analyze the effects of an extracellular CyPA inhibition. As extracellular CyPA-inhibitor we used the recently described CsA-derivate MM284. In vitro studies confirmed that MM284 inhibits CyPA-induced monocytic migration and adhesion. A/J mice immunized with mcTnI were treated with MM284 or vehicle every second day. After 28 days, we found a considerable reduction of myocardial injury and fibrosis. Further analysis revealed a reduced myocardial presence of T-cells and macrophages compared to control treated animals. Whereas MMP-9 expression was reduced significantly by MM284, we observed no significant reduction of inflammatory cytokines such as IL-6 or TNFα. Extracellular CyPA plays an important role in autoimmune myocarditis for myocardial damage and fibrosis. Our data suggest a new pharmacological approach for the treatment of myocardial inflammation and reduction of cardiac fibrosis by inhibition of extracellular CyPA.     

The Influence of Mark-Recapture Sampling Effort on Estimates of Rock Lobster Survival

Five annual capture-mark-recapture surveys on Jasus edwardsii were used to evaluate the effect of sample size and fishing effort on the precision of estimated survival probability. Datasets of different numbers of individual lobsters (ranging from 200 to 1,000 lobsters) were created by random subsampling from each annual survey. This process of random subsampling was also used to create 12 datasets of different levels of effort based on three levels of the number of traps (15, 30 and 50 traps per day) and four levels of the number of sampling-days (2, 4, 6 and 7 days). The most parsimonious Cormack-Jolly-Seber (CJS) model for estimating survival probability shifted from a constant model towards sex-dependent models with increasing sample size and effort. A sample of 500 lobsters or 50 traps used on four consecutive sampling-days was required for obtaining precise survival estimations for males and females, separately. Reduced sampling effort of 30 traps over four sampling days was sufficient if a survival estimate for both sexes combined was sufficient for management of the fishery.     

Effects of intraperitoneally injected selenium and vitamin E in rats anesthetized with halothane.

Halothane, commonly used for anesthetizing humans and animals, is one of the most important volatile anesthetics and may cause the formation of free radicals during its biotransformation. Free radicals may lead to degeneration of liver cells. Vitamin E and glutathione peroxidase (GSH-Px) containing selenium are two natural antioxidants, and these may protect the cellular lipid and lipoproteins against oxidative damage caused by free radicals. Therefore, the purposes of the present study were to investigate the probable protective effects of intraperitoneally administered Se and vitamin E on liver enzymes and to determine some other hematological parameters in the halothane anesthesia of rats. All rats were randomly divided into five groups. The first group was used as a control, and physiological saline (0.9%) was intraperitoneally injected into these animals as a placebo. The second group was used as an anesthesia control group and was only anesthetized with halothane for two hours. The third group received intraperitoneally administered Se (Na2SeO3, 0.3 mg/200 g body weight), the fourth group vitamin E (dl-alpha-tocopheryl acetate, 100 mg/kg body weight), and the fifth group a Se plus vitamin E combination (Na2SeO3, 0.3 mg/200 g body weight + dl-alpha-tocopheryl acetate, 100 mg/kg body weight). The activities of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, triglycerides, erythrocyte counts, the packet-cell volume, hemoglobin concentrations and neutrophyle rates significantly increased (p < 0.05 to p < 0.01) after halothane anesthesia and returned to near control levels after Se, vitamin E and Se plus vitamin E injections. The values of cholesterol, total protein, white blood cell counts and lymphocyte rates significantly decreased (p < 0.05 to p < 0.01) in the anesthesia control group. However, the levels of albumin, total bilirubin, creatinine, the mean corpuscular volume, the mean corpuscular hemoglobin, and the mean corpuscular hemoglobin concentration were not statistically influenced. In conclusion, we have determined that halothane anesthesia affected some liver enzymes and some other biochemical and hematological parameters. Se, vitamin E and their combination may prevent the increase of liver enzymes after halothane anesthesia. Based upon these results, Se and vitamin E may play an important role in the indication of hepatic cellular injury produced by halothane.     

Sex differences in modulating blood brain barrier permeability by NO in pentylenetetrazol-induced epileptic seizures

Susceptibility to epilepsy as well as BBB dysfunction in some pathological conditions varies depending on sex difference. It has recently been shown that systemically given NO donor and antagonists modify the nature of seizures induced by PTZ (pentylenetetrazol) differently in male and female rats. This study investigates the role of NO on BBB permeability in PTZ seizures with sex differences using NO donor, sodium nitroprusside (SNP), and NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). Nitrite+nitrate levels as indices of NO generation in the brain were also assessed. L-NAME prolonged seizure latency in male rats, seizure intensity and seizure duration were lessened. L-NAME depicted opposite effects in seizure nature in female rats. SNP prolonged seizure latency, while seizure intensity and duration were lessened only in female rats. L-NAME in male rats increased L-NAME use in female rats (not in male rats) which resulted in a more leaky BBB especially in midbrain, thalamus, hippocampus, corpus striatum and cerebellum whereas SNP use in male rats and not in female rats resulted in pronounced BBB opening in all brain regions studied than PTZ per se. L-NAME while decreasing nitrite+nitrate levels in male rat brains, acted in an opposite fashion in females. SNP use depicted an inverse picture with respect to L-NAME, with an opposite action in different sexes. This study reveals that NO effect on BBB in PTZ-induced seizures depends unequivocally on sex difference. The sex-dependent action of NO in seizures and in CNS pathologies warrants further investigation.     

Purification,Characterization, and Substrate Specificity of a Novel Highly Glucose-Tolerant β-Glucosidase from Aspergillus oryzae

Aspergillus oryzae was found to secrete two distinct β-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3′,4′,5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total β-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant β-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-β-d-glucoside as the substrate, we found that the enzyme was optimally active at 50°C and pH 5.0 and had a specific activity of 1,066 μmol min⁻¹ mg of protein⁻¹ and a K_m of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (K_i, 1.36 M) or glucono-δ-lactone (K_i, 12.5 mM), another powerful β-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal β-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1→3)- and (1→6)-β-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-β-d-glucosides in a grape must (pH 2.9, 90 g of glucose liter⁻¹). Other flavor precursors (benzyl- and 2-phenylethyl-β-d-glucosides) and prunin (4′,5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel β-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.β-Glucoside glucohydrolases, commonly called β-glucosidases, catalyze the hydrolysis of alkyl- and aryl-β-glucosides, as well as diglucosides and oligosaccharides. These enzymes are widely used in various biotechnological processes, including the production of fuel ethanol from cellulosic agricultural residues (4, 27, 48) and the synthesis of useful β-glucosides (21, 38). In the flavor industry, β-glucosidases are also key enzymes in the enzymatic release of aromatic compounds from glucosidic precursors present in fruits and fermentating products (13, 39). Indeed, many natural flavor compounds, such as monoterpenols, C-13 norisoprenoids, and shikimate-derived compounds, accumulate in fruits as flavorless precursors linked to mono- or diglycosides and require enzymatic or acidic hydrolysis for the liberation of their fragrances (41, 45). Finally, β-glucosidases can also improve the organoleptic properties of citrus fruit juices, in which the bitterness is in part due to a glucosidic compound, naringin (4′,5,7-trihydroxyflavanone-7-rhamnoglucoside), whose hydrolysis requires, in succession, an α-rhamnosidase and a β-glucosidase (33).It is now well-established that certain monoterpenols of grapes (e.g., linalol, geraniol, nerol, citronelol, α-terpineol, and linalol oxide), which are linked to diglycosides, such as 6-O-α-l-rhamnopyranosyl-, 6-O-α-l-arabinofuranosyl-, and 6-O-β-d-apiofuranosyl-β-d-glucosides, contribute significantly to the flavor of wine (15, 44). The enzymatic hydrolysis of these compounds requires a sequential reaction; first, an α-l-rhamnosidase, an α-l-arabinofuranosidase, or a β-d-apiofuranosidase cleaves the (1→6) osidic linkage, and then, the flavor compounds are liberated from the monoglucosides by the action of a β-glucosidase (18, 19). Unlike acidic hydrolysis, enzymatic hydrolysis is highly efficient and does not result in modifications of the aromatic character (16). However, grape and yeast glucosidases exhibit limited activity on monoterpenyl-glucosides during winemaking, and a large fraction of the aromatic precursors remains unprocessed (9, 16, 35). The addition of exogenous β-glucosidase during or following fermentation has been found to be the most effective way to improve the hydrolysis of the glycoconjugated aroma compounds in order to enhance wine flavor (2, 14, 39, 40). The ideal β-glucosidase should function and be stable at a low pH value (pH 2.5 to 3.8) and should be active at a high concentration of glucose (10 to 20%) and in the presence of 10 to 15% ethanol. However, most microbial β-glucosidases are very sensitive to glucose inhibition (4, 12, 47), as well as to inhibition by glucono-δ-lactone, another powerful β-glucosidase inhibitor produced by grape-attacking fungi which can be found in wine must at concentrations up to 2 g/liter (10).The need for more suitable enzymes has led us and other workers to search for novel β-glucosidases with the desired properties. Recently, we showed that an extracellular glucose-tolerant and pH-stable β-glucosidase can be produced by Aspergillus strains (17). However, the enzyme of interest represented only a minor fraction of total β-glucosidase activity, and the major form was highly sensitive to glucose inhibition. Aspergillus oryzae appeared to be the best producer of the minor form when it was grown on quercetin (3,3′,4′,5,7-pentahydroxyflavone), a phenolic flavonoid found in plant cell walls. This paper presents further data on the production and characterization of this novel highly glucose-tolerant β-glucosidase (designated HGT-BG) purified from the extracellular culture filtrate of A. oryzae grown on quercetin.     

Combined Mass Mapping and Biochemical Characterization of Grape β-Glycosidase-enriched Extract

A beta-glucosidase enzyme activity was enriched from skins of ripe grape berry by cell wall fractionation, hydrophobic interaction and cation-exchange chromatographies. This enriched enzyme extract contained several beta-glycosidase activities hydrolyzing a wide range of synthetic and natural monoglycosides and diglycosides, as well as a beta-fructosidase activity. The enzyme extract was further characterized by two-dimensional gel electrophoresis coupled to peptide mass fingerprinting of eight spots using MALDI-TOF mass spectrometry. No beta-glucosidase but a beta-fructosidase associated to the relevant spot at 66 kDa/pI 5.1 was identified. Taken together all results issued from the biochemical characterization, the substrate specificity and the mass spectrometry-based identification of this enriched enzyme extract, we propose that this protein could be a specific beta-fructosidase isoform associated with a broad spectrum of beta-glycosidase activities in grape berry skin and involved in cell wall modifications which occur during the ripening-induced thickness of the grape.