The carbonylation and covalent dimerization of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity is inhibited by the radical scavenger tempol.

Tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) reduces tissue injury in animal models of various diseases via mechanisms that are not completely understood. Recently, we reported that high doses of tempol moderately increased survival in a rat model of ALS (amyotrophic lateral sclerosis) while decreasing the levels of oxidized hSOD1 (human Cu,Zn-superoxide dismutase) in spinal cord tissues. To better understand such a protective effect in vivo, we studied the effects of tempol on hSOD1 oxidation in vitro. The chosen oxidizing system was the bicarbonate-dependent peroxidase activity of hSOD1 that consumes H2O2 to produce carbonate radical, which oxidizes the enzyme. Most of the experiments were performed with 30 µM hSOD1, 25 mM bicarbonate, 1 mM H2O2, 0.1 mM DTPA (diethylenetriaminepenta-acetic acid) and 50 mM phosphate buffer at a final pH of 7.4. The results showed that tempol (5-75 µM) does not inhibit hSOD1 turnover, but decreases its resulting oxidation to carbonylated and covalently dimerized forms. Tempol acted by scavenging the carbonate radical produced and by recombining with hSOD1-derived radicals. As a result, tempol was consumed nearly stoichiometrically with hSOD1 monomers. MS analyses of turned-over hSOD1 and of a related peptide oxidized by the carbonate radical indicated the formation of a relatively unstable adduct between tempol and hSOD1-Trp32•. Tempol consumption by the bicarbonate-dependent peroxidase activity of hSOD1 may be one of the reasons why high doses of tempol were required to afford protection in an ALS rat model. Overall, the results of the present study confirm that tempol can protect against protein oxidation and the ensuing consequences.

The role of BKCa channels on hyperpolarization mediated by hyperosmolarity in human articular chondrocytes.

Chondrocytes, the only cell in cartilage, are subjected to hyperosmotic challenges continuously since extracellular osmolarity in articular cartilage increases in response to mechanical loads during joint movement. Hyperosmolarity can affect membrane transport, and it is possible that load modulates matrix synthesis through alterations in intracellular composition. In the present study, the effects of hyperosmotic challenges were evaluated using the whole-cell patch clamp technique, whole cell mode on freshly isolated human and bovine articular chondrocytes. In human chondrocytes, hypertonicity induced the activation of outward Ca(2+)-sensitive K(+) currents, which were inhibited by iberiotoxin and TEA-Cl. The current induced by hypertonic switching (osmolarity from 300 to 400 mOsm/l) caused cell hyperpolarization (from -39 mV to -70 mV) with a reversal potential of -96 ± 7 mV. These results suggest a role for Ca(2+)-activated K(+) channels in human articular chondrocytes, leading to hyperpolarization as a consequence of K(+) efflux through these channels. These channels could have a role in the articular chondrocyte's response to a hyperosmotic challenge and matrix metabolism regulation by load.

Aislamiento y caracterización de un péptido antibacteriano del veneno de Centruroides margaritatus / Isolation and characterization of antibacterial peptide from Centruroides margaritatus venom

En este trabajo se ha aislado y caracterizado parcialmente un péptido con actividad antibacteriana del veneno del escorpión Centruroides margaritatus (Gervais, 1841) (Scorpiones: Buthidae). Este péptido fue aislado a partirde 50 mg de veneno crudo, y la purificación fue inicialmente realizada por cromatografía de intercambio iónico en CM-Sephadex C-25, obteniéndose siete picos de proteína. El pico III de esta separación fue purificado porcromatografía de filtración en Sephadex G-75, obteniéndose dos picos de proteína, de los cuales el segundo mostró ser el péptido antibacteriano. Este péptido representa aproximadamente el 3% de la proteína total del veneno y por PAGE-SDS, se determinó que tiene un peso molecular de 7,3 kDa. El péptido antibacteriano inhibe el crecimiento de Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa y Serratia marcencens, en microplacas con medio mínimo de Davies, pero los ensayos en agar Müller-Hinton, no mostraron halos de inhibición significativos, concluyéndo se que el péptido tiene actividad bacteriostática pero no bactericida. Además, no mostró acción sobre Enterococcus faecalis, Escherichia coli, Salmonella choleraesuis y Klebsiella pneumoniae. El péptido antibacteriano también fue capaz de inhibir el crecimiento de Aspergillus níger y Candida albicans durante 48 horas, pero no tiene actividad hemolítica sobre eritrocitos humanos.

In this work, from the venom of Centruroides margaritatus (Gervais, 1841) (Scorpiones, Buthidae), one peptide with antibacterial activity was isolated and characterized. This peptide was isolated from 50 mg of wholevenom, the purification was initially performed by chromatography on CM-Sephadex C-25 obtaining protein peaks seven. The peak III of this separation was purified by gel filtration on Sephadex G-75, yielding protein peaks two, the second of which proved to be the antibacterial peptide. This peptide represents about 3 % of whole venom protein and by PAGE-SDS, was determinate it has 7,3 kDa of molecular weight. The antibacterial peptide inhibits the growth of Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa y Serratia marcencens, in microplates with minimal media Davies, but in assays in Muller-Hinton agar, showed no significant inhibition halos; concluding that peptide has bacteriostatic activity but not bactericidal activity. In addition has not activity on Enterococcus faecalis, Escherichia coli, Salmonella choleraesuis and Klebsiella pneumoniae. Antibacterial peptide also inhibited the growth of Aspergillus níger and Candida albicans during 48 hours, but has not hemolytic activity.

Pseudallescheria boydii releases metallopeptidases capable of cleaving several proteinaceous compounds.

Pseudallescheria boydii is an opportunistic filamentous fungus that causes serious infections in humans. Virulence attributes expressed by P. boydii are unknown. Conversely, peptidases are incriminated as virulence factors in several pathogenic fungi. Here we investigated the extracellular peptidase profile in P. boydii. After growth on Sabouraud for 7 days, mycelia of P. boydii were incubated for 20 h in PBS-glucose. The cell-free PBS-glucose supernatant was submitted to SDS-PAGE and 12 secretory polypeptides were observed. Two of these polypeptides (28 and 35 kD) presented proteolytic activity when BSA was used as a copolymerized substrate. The extracellular peptidases were most active in acidic pH (5.5) and fully inhibited by 1,10-phenanthroline, a zinc-metallopeptidase inhibitor. Other metallo-, cysteine, serine and aspartic proteolytic inhibitors did not significantly alter these activities. To confirm that these enzymes belong to the metallo-type peptidases, the apoenzymes were obtained by dialysis against chelating agents, and supplementation with different cations, especially Cu(2+) and Zn(2+), restored their activities. Except for gelatin, both metallopeptidases hydrolyzed various co-polymerized substrates, including human serum albumin, casein, hemoglobin and IgG. Additionally, the metallopeptidases were able to cleave different soluble proteinaceous substrates such as extracellular matrix components and sialylated proteins. All these hydrolyses were inhibited by 1,10-phenanthroline. Interestingly, Scedosporium apiospermum (the anamorph of P. boydii) produced a distinct extracellular peptidase profile. Collectively, our results demonstrated for the first time the expression of acidic extracellular metallopeptidases in P. boydii capable of degrading several proteinaceous compounds that could help the fungus to escape from natural human barriers and defenses.

A natural carrier effect and the generation of specific antibodies to biologically active peptides

Production of specific antibodies to haptens, especially antipeptides, without interference by carrier protein, is desirable. The bradykinin-potentiating peptides (BPPs) are a family of pyroglutamyl proline-rich oligopeptides with strong antihypertensive properties. In this work, the production of antibodies to BPPs by use of an efficient immunization protocol in mice genetically modified for the high antibody responsiveness (HIII line) is described. Although it was possible to induce antibody production by single-dose administration of free BPPs, higher antibody titers were obtained in mice preimmunized with carrier protein before administration of peptides conjugated to this carrier. Interestingly, both mouse groups had a higher titer of IgG1 than IgG2a isotypes, regardless of prior immunization with the carrier protein. However, a lower titer of IgG2a was observed in unprimed mice. A single band of about 27 kDa corresponding to the BPP precursor protein was recognized by these antibodies in the cytosol of the Bothrops jararaca venom gland. This work proposes an efficient immunization protocol based on classic studies described for the hapten-carrier effect for generating specific antibodies against biologically active peptides.

Thrombomodulin-independent activation of protein C and specificity of hemostatically active snake venom serine proteinases: crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator.

Protein C activation initiated by the thrombin-thrombomodulin complex forms the major physiological anticoagulant pathway. Agkistrodon contortrix contortrix protein C activator, a glycosylated single-chain serine proteinase, activates protein C without relying on thrombomodulin. The crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator determined at 1.65 and 1.54 A resolutions, respectively, indicate the pivotal roles played by the positively charged belt and the strategic positioning of the three carbohydrate moieties surrounding the catalytic site in protein C recognition, binding, and activation. Structural changes in the benzamidine-inhibited enzyme suggest a probable function in allosteric regulation for the anion-binding site located in the C-terminal extension, which is fully conserved in snake venom serine proteinases, that preferentially binds Cl(1-) instead of SO(4)(2-).

Involvement of a 50-kDa mRNP protein from Saccharomyces cerevisiae in mRNA binding to ribosomes.

A yeast 50-kDa mRNA-binding protein (50mRNP) is found selectively associated with the 48S and 80S initiation complexes. This protein is structurally related to the translational elongation factor EF-1alpha. The protein reacts with antibodies directed against EF-1alpha and, similarly, EF-1alpha recognizes antibodies against the 50mRNP protein. This is evidence that they share at least one epitope which allows a similar antigenic behavior. In addition, both proteins show similar cleavage patterns upon treatment with the endoproteinase Lys-C. A murine antibody raised against 50mRNP inhibits both 48S and 80S initiation complex formation. The inhibitory effect is relieved by preincubating anti-50mRNP with EF-1alpha. Antibody to EF-1alpha manifests a similar inhibitory pattern for the formation of 48S and 80S complexes. These data strongly suggest that 50mRNP is an EF-1alpha-like polypeptide essential for the formation of the above complexes.

Effect of gastrin-releasing peptide (GRP) and GRP antagonists on TSH secretion from rat isolated pituitaries.

It has previously been demonstrated that gastrin releasing peptide (GRP), a bombesin-like peptide, inhibited TSH release "in vivo". In this study, we have shown that GRP acts directly at the pituitary gland, inhibiting basal and TRH-stimulated TSH release from incubated rat anterior pituitary glands. This effect was observed at the highest GRP concentration (10(-5) M), but not at the lower concentrations (10(-7) and 10(-9) M). Incubation of the glands with two antagonists of GRP (d-Phe8-GRP and Gly6-GRP) induced an increase of basal TSH secretion. We suggest a physiological role of locally produced bombesin-like peptides in the control of TSH release. Another antagonist (Ala6-GRP) did not change TSH secretion. This result suggests the existence of different subtypes of GRP receptors in the anterior pituitary gland.