Snake venom hyaluronidase: An evidence for isoforms and extracellular matrix degradation

The present study attempts to establish the isoforms of hyaluronidase enzyme and their possible role in the spreading of toxins during envenomation. Screening of venoms of 15 snakes belonging to three different families revealed varied hyaluronidase activity in ELISA-like assay, but with relatively similar pH and temperature optima. The zymograms of individual venoms showed varied activity banding patterns and indicated the presence of at least two molecular forms of the enzyme. During envenomation, activity of hyaluronidase is considered crucial for the spreading of toxins and is presumed to distort the integrity of extracellular matrix through the degradation of hyaluronic acid in it. This property has been addressed through localization of hyaluronic acid in human skin and muscle tissue sections using the probe, biotinylated hyaluronic acid binding protein. Faint and discontinuous staining pattern of hyaluronidase treated tissue sections over intense staining of untreated tissue sections confirm the selective degradation of hyaluronic acid in extracellular matrix and thus provide an evidence for the spreading property of the enzyme.     

Cytokine polymorphisms in silicosis and other pneumoconioses

Silicosis and coal workers' pneumoconiosis are complex multifactorial lung diseases whose etiopathogenesis are not well defined. It is generally accepted that fibrotic lung disorders are mediated by macrophage-derived cytokines and growth factors. There is evidence showing a crucial role for tumor necrosis factor- (TNF-) and interleukin-1 (IL-1) in inflammation caused by silica dust and in the transition from simple to progressive massive fibrosis. In this review we discuss genetic polymorphisms responsible for regulating the production of these proinflammatory cytokines and their role in modifying silicosis severity.     

Comparison of the antisecretory and antiulcer activity of epidermal growth factor,urogastrone and transforming growth factor alpha and its derivative in rodents in vivo

This study investigates the effects of epidermal growth factor (EGF), urogastrone (UG) and transforming growth factor-alpha (TGF) and its derivative on dimaprit- and pentagastrin-induced gastric acid secretion and on acidified ethanol (AE)-evoked ulcer formation in anaesthetized rats. EGF, TGF and UG administered subcutaneously (s.c.) 30 min before dimaprit inhibited gastric acid secretion. Against pentagastrin-stimulated secretion, TGF inhibited, while EGF and UG potentiated, acid secretion dose-dependently. Intraduodenal (i.d.) administration of TGF and UG had no effect, while EGF potentiated, both secretagogue-induced acid secretion in the same dosage schedule. Administration of either EGF, UG or TGF i.v. bolus, in response to continuous infusion of dimaprit resulted in a significant (p < 0.05–p < 0.001) inhibition of acid secretion which was transient and returned to normal within 30–45 min for UG while it slowly returned to normal for EGF and TGF. The truncated form of TGF (amino acids 34–43) did not show any antisecretory effect when administered parenterally. Acidified ethanol produced gastric haemorrhagic lesions in the rat 1 h after oral administration. The gastric mucosal protective effects of TGF, EGF and UG administered either orally or s.c. 30 min before the administration of AE were dose-dependent against this model of ulcer induction. Indomethacin (Indo), administered 15 min before AE to inhibit prostanoids biosynthesis, significantly (p < 0.001) reduced the cytoprotective effects of TGF, EGF and UG and aggravated the ulcer index when administered s.c. The results show that PGs may be involved in mediating the protective effects of the three growth factors. Administration of NG-nitro-L argininemethylester (L-NAME) 15 min prior to TGF, EGF and UG s.c. or orally, significantly (p < 0.001) decreased the degree of ulcer indices and was able to reduce the protective effects of TGF, EGF and UG, thus including the role of NO in mediating the protective effects of these growth factors. In conclusion, these results have demonstrated that EGF, UG and TGF have a short and reversible inhibitory effect on dimaprit-stimulated gastric acid secretion and each is effective parenterally but not orally. UG and EGF potentiated, while, TGF inhibited pentagastrin-stimulated acid secretion. In addition, TGF seems to lose its activity when it is truncated from the C terminus. The present study also suggests that EGF, UG and TGF are equally effective against AE-induced gastric ulcer and bring about their cytoprotective action through their reduction of acid secretion and through PG and NO pathways.     

Nodule ultrastructure and initial growth of Anadenanthera peregrina (L.) Speg. var. falcata (Benth.) altschul plants infected with rhizobia

The anatomy and ultrastructure of root nodules of Anadenanthera peregrina var. falcata (Leguminosae-Mimosoideae) were analysed, as was plant growth. To ensure that nodules developed, seedlings were inoculated with a mixture of six strains of rhizobia. Nodules were produced that differed in appearance-and probably also effectiveness-but their structure was similar and they showed characteristics typical of indeterminate nodules, such as persistent meristematic tissue and a gradient of cells at different stages of development. Many starch grains were present in inner cortex cells and interstitial cells of infected tissue. Infected cells were densely packed with bacteroids, which contained many poly-beta-hydroxybutyrate granules. The high incidence of these granules, together with high levels of starch accumulation in interstitial cells, suggested low N2-fixation efficiency of the rhizobia isolates used for inoculation. In the symbiosomes of early-senescent infected cells, reticulum-like structures, small vesicles and a fibrillar material were observed; these may be related to bacteroid degradation. In the cytoplasm of late-senescent infected cells, many vesicles and membrane-like structures were observed, probably associated with membrane degradation of bacteroids and peribacteroids. The total biomass of plants inoculated with rhizobia was low and their xylopodia and shoots had low levels of N compared with noninoculated plants fertilized with ammonium nitrate. However, inoculated plants did not show N-deficiency symptoms and grew better than non-inoculated plants without N fertilization. These growth results, together with ultrastructural observations of nodules, suggest that nitrogen fixation of rhizobia isolates associated with Anadenanthera peregrina var. falcata roots is poor.     

L-Lysine alpha-oxidase: physicochemical and biological properties

This review summarizes data on the properties of L-lysine -oxidase, an enzyme that belongs to the group of oxidases of L-amino acids. This enzyme acts virtually only on L-lysine with a rather low K _m yielding -keto--aminocaproic acid. The decrease in the level of the essential amino acid L-lysine and the formation of hydrogen peroxide during the reaction possibly provide the basis for the unique properties of L-lysine -oxidase: cytotoxic, antitumor, antimetastatic, antiinvasive, antibacterial, and antiviral activities, as well as an immunomodulating effect. Native L-lysine -oxidase and its immobilized forms are promising tools for determination of concentration of L-lysine in various biological materials.     

On the mechanism of interaction of organic solvents with the active site of alpha-chymotrypsin

Kinetic behavior of -chymotrypsin in the reaction of hydrolysis of the N-acetyl-L-tyrosine derivatives was investigated in non-denaturing water–dimethylsulfoxide and water–ethanol mixtures. Similar specific interactions between the two solvents and the active site of -chymotrypsin were shown to result in similar kinetic effects. It is proposed that the changes in the active site structure of the enzyme caused by the interaction with the organic solvents (conformational isomer formation) resulted in two parallel processes—acceleration of the acyl-enzyme formation step and a decrease in the deacylation rate. The possible molecular mechanism of this phenomenon and an adequate kinetic model describing the data are discussed.     

Specific binding of integrin alphaIIbbeta3 to RGD peptide immobilized on a nitrilotriacetic acid chip: a surface plasmon resonance study

Nitrilotriacetic acid has been routinely used in protein purification for its high affinity for His-tagged protein in the presence of Ni²⁺. Here we reported a type of nitrilotriacetic acid chip (NTA-chip) prepared by transferring NTA-DOGS containing a lipid monolayer to a 50 nm thick gold layer deposited on a glass slide. The surface binding ability of His-tagged protein and regeneration of NTA chip were characterized using a synthetic polypeptide P1 (His-His-His-His-His-His--aminohexanoic-Gly-Gly-Arg-Gly-Asp-Ser). The effect of divalent cations on integrin binding affinity for RGD ligand was investigated after P1 had been immobilized onto the sensor chip. The results show that the NTA-chip is a useful tool to immobilize His-tagged protein on the chip surface, and can provide a functional orientation for further investigation. The results also show that removing of Ca²⁺ bound on low affinity sites or adding of Mn²⁺ can increase the binding ability of integrin.     

Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.

This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO(*)) detection system we observed NO(*) production in H9c2 cells. As revealed by confocal microscopy, NO(*) fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C(green) value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO(*) in mitochondria. Conversely, the presence of NO(*) in mitochondria decreased following cells pretreatment with l-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C(green) value to 0.02. This work confirms that the presence of NO(*) in mitochondria can be modulated in response to different fluxes of NO(*).