Antisnake venom activity of ethanolic seed extract of Strychnos nux vomica Linn

The whole seed extract of S. nux vomica (in low doses) effectively neutralized Daboia russelii venom induced lethal, haemorrhage, defibrinogenating, PLA2 enzyme activity and Naja kaouthia venom induced lethal, cardiotoxic, neurotoxic, PLA2 enzyme activity. The seed extract potentiated polyvalent snake venom antiserum action in experimental animals. An active compound (SNVNF) was isolated and purified by thin layer chromatography and silica gel column chromatography, which effectively antagonised D. russelii venom induced lethal, haemorrhagic, defibrinogenating, oedema, PLA2 enzyme activity and N. kaouthia induced lethal, cardiotoxic, neurotoxic, PLA, enzyme activity. Polyvalent snake venom antiserum action was significantly potentiated by the active compound. Spectral studies revealed it to be a small, straight chain compound containing methyl and amide radicals. Detailed structure elucidation of the compound (SNVNF) is warranted before its clinical trials as a snake venom antagonist.     

ERK7 expression and kinase activity is regulated by the ubiquitin-proteosome pathway

ERK7 is a unique member of the extracellular signal-regulated kinase (ERK) subfamily of MAP kinases. Although ERK7 shares a TEY motif in the activation loop of the kinase, it displays constitutive activation, nuclear localization, and growth inhibitory properties that are regulated by its C-terminal domain. Because ERK7 is expressed at low levels compared with ERK2 and its activity is dependent upon its expression level, we investigated the mechanism by which ERK7 expression is regulated. We now show that ERK7 expression is regulated by ubiquitination and rapid proteosomal turnover. Furthermore, both the kinase domain and the C-terminal tail are independently degraded at a rate comparable with that of the intact protein. Analysis of a series of chimeras between ERK2 and ERK7 reveal that the N-terminal 20 amino acids of the kinase domain are a primary determinant of ERK7 degradation. Fusion of the N-terminal 20 amino acids is both necessary and sufficient to cause proteolytic degradation of both ERK2 and green fluorescent protein. Finally, ERK7 is stabilized by an N-terminal mutant of Cullin-1 suggesting that ERK7 is ubiquitinated by the Skip1-Cullin-F box complex. These results indicate that ERK7 is a highly regulated enzyme whose cellular expression and kinase activation level is tightly controlled by the ubiquitin-proteosome pathway.     

Hydration and packing effects on prion folding and beta-sheet conversion. High pressure spectroscopy and pressure perturbation calorimetry studies

The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we compare the stability against pressure and the thermomechanical properties of the alpha-helical and beta-sheet conformations of recombinant murine prion protein, designated as alpha-rPrP and beta-rPrP, respectively. High temperature induces aggregates and a large gain in intermolecular antiparallel beta-sheet (beta-rPrP), a conformation that shares structural similarity with PrP(Sc). alpha-rPrP is highly stable, and only pressures above 5 kilobars (1 kilobar = 100 MegaPascals) cause reversible denaturation, a process that leads to a random and turnrich conformation with concomitant loss of alpha-helix, as measured by Fourier transform infrared spectroscopy. In contrast, aggregates of beta-rPrP are very sensitive to pressure, undergoing transition into a dissociated species that differs from the denatured form derived from alpha-rPrP. The higher susceptibility to pressure of beta-rPrP can be explained by its less hydrated structure. Pressure perturbation calorimetry supports the view that the accessible surface area of alpha-rPrP is much higher than that of beta-rPrP, which explains the lower degree of hydration of beta-rPrP. Our findings shed new light on the mechanism of prion conversion and show how water plays a prominent role. Our results allow us to propose a volume and free energy diagram of the different species involved in the conversion and aggregation. The existence of different folded conformations as well as different denatured states of PrP may explain the elusive character of its conversion into a pathogenic form.     

Transgenic overexpression of BMP4 increases astroglial and decreases oligodendroglial lineage commitment

Bone morphogenetic proteins (BMPs) promote astrocytic differentiation of cultured subventricular zone stem cells. To determine whether BMPs regulate the astrocytic lineage in vivo, transgenic mice were constructed that overexpress BMP4 under control of the neuron-specific enolase (NSE) promoter. Overexpression of BMP4 was first detectable by Western analysis on embryonic day 16 and persisted into the adult. The overexpression of BMP4 resulted in a remarkable 40% increase in the density of astrocytes in multiple brain regions accompanied by a decrease in the density of oligodendrocytes ranging between 11 and 26%, depending on the brain region and the developmental stage. No changes in neuron numbers or the pattern of myelination were detected, and there were no gross structural abnormalities. Similar phenotypes were observed in three independently derived transgenic lines. Coculture of transgenic neurons with neural progenitor cells significantly enhanced astrocytic lineage commitment by the progenitors; this effect was blocked by the BMP inhibitor Noggin, indicating that the stimulation of astrogliogenesis was due to BMP4 release by the transgenic neurons. These observations suggest that BMP4 directs progenitor cells in vivo to commit to the astrocytic rather than the oligodendroglial lineage. Further, differentiation of radial glial cells into astrocytes was accelerated, suggesting that radial glia were a source of at least some of the supernumerary astrocytes. Therefore, BMPs are likely important mediators of astrocyte development in vivo.     

Effects of midazolam on the contraction and relaxation of segments of thoracic aorta stripped of endothelium and stimulated by adrenaline – experimental study in rabbits

To evaluate the effects of midazolam on the angiokinesis of segments of rabbits' thoracic aorta stripped of endothelium and stimulated by adrenaline.Two groups of aortic rings removed from albinic rabbits anesthetized with thiopental were used (Group I – 6 animals; Group II – 12 animals), stripped of endothelium, studied in an organ chamber, perfused by Krebs-Henseleit solution. The groups were stimulated by adrenaline, recording the maximum contraction and dT/dt at 12, 36, 60 and 120. When the plateau phase was reached, the vessel was washed with perfusion solution, recording relaxation at 2, 4 and 6. When the base values were reached, Group I underwent a new adrenergic stimulus; and Group II was stimulated with midazolam and then with adrenaline, and the same values were recorded. T test was applied as a statistical analysis when two variables were studied. When studying more than two variables the Anova test was used, supplemented by the Tuckey test.Group I did not show any significant difference between the two stimuli. Group II – the midazolam significantly reduced the maximum contraction induced by adrenaline (83.01 ± 4.11%) (p < 0.01). The dT/dt was reduced at 12 (57.06 ± 8.47%), and also at 36 (70.59 ± 5.26%). There was no significance at 60 and 120 (p < 0.01).The relaxation increased significantly at all measurements – at 2-adrenaline 39.31 ± 9.60%; adrenaline/midazolam: 44.06 ± 9.62% (p < 0.05). At 4-adrenaline: 53.08 ± 8.3%; adrenaline/midazolam: 61.68 ± 8.50% (p < 0.01). At 6-adrenaline: 76.26 ± 5.45%; adrenaline/midazolam: 84.20 ± 7.96% (p < 0.01).Midazolam significantly reduced the maximum contraction obtained by the adrenergic stimulus as well as the dT/dt in the initial phases of contraction. The relaxation speed also increased.     

Stroma-mediated granulocyte-macrophage colony-stimulating factor (GM-CSF) control of myelopoiesis: spatial organisation of intercellular interactions

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the major cytokines involved in control of haemopoiesis both in bone marrow and in extramedullar sites. Its biological activity depends upon the composition and physicochemical properties of the microenvironment provided by the supporting stroma. GM-CSF activity is modulated and controlled by the stromal heparan-sulphate proteoglycans, but their optimal interaction occurs only at low pH. We questioned whether the microenvironment organisation of the interface between stroma and haemopoietic cells provides such conditions. We studied myeloid progenitor proliferation in contact with bone marrow-derived and extramedullar stromas using electron microscopy and selective labelling of pericellular components. We present evidence that, upon interaction, the two cell types reorganise their interface both in shape and molecular composition. Haemopoietic cells extend projections that considerably increase the area of intercellular contact, and stromal cells form lamellipodia and carry out a redistribution of membrane-associated sialylated glycoconjugates and proteoglycans. Such rearrangements lead to extensive capping of negatively charged molecules at the interface between the supporting stroma and the haemopoietic cells, leading potentially to a local decrease in pH. Our results indicate that the distribution of negative charges at the cellular interface may be responsible for the selectivity of cell response to GM-CSF.Publication of the Millennium Institute for Tissue Bioengineering. The study was supported by PRONEX, CNPq and FINEP grants from the Brazilian Ministry of Science and Technology and a FAPERJ grant from the Rio de Janeiro State Government.     

Proline accumulation and glutamine synthetase activity are increased by salt-induced proteolysis in cashew leaves

In this study cashew (Anacardium occidentale) plants were exposed to a short- and long-term exposure to NaCl in order to establish the importance of the salt-induced proteolysis and the glutamine synthetase activity on the proline accumulation. The cashew leaf showed a prominent proline accumulation in response to salt stress. In contrast, the root tissue had no significant changes in proline content even after the drastic injury caused by salinity on the whole plant. The leaf proline accumulation was correlated to protease activity, accumulation of free amino acid and ammonia, and decrease of both total protein and chlorophyll contents. The leaf GS activity was increased by the salt stress whereas in the roots it was slightly lowered. Although the several amino acids in the soluble pool of leaf tissue have showed an intense increment in its concentrations in the salt-treated plants, proline was the unique to show a proportional increment from 50 to 100 mol m-3 NaCl exposure (16.37 to 34.35 mmol kg-1 DM, respectively). Although the leaf glutamate concentration increased in the leaves of the salt-stressed cashew plants, as compared to control, its relative contribution to the total amino acid decreased significantly in stressed leaves when compared to other amino acids. In addition, when the leaf discs were incubated with NaCl in the presence of exogenous precursors (Glu, Gln, Orn or Arg) involved in the proline synthesis pathways, the glutamate was unique in inducing a significant enhancement of the proline accumulation compared to those discs with precursor in the absence of NaCl. These results, together with the salt-induced increase in the GS activity, suggest an increase in the de novo synthesis of proline probably associated with the increase of the concentration of glutamate. Moreover, the prominent salt-induced proline accumulation in the leaves was associated with the higher salt-sensitivity in terms of proteolysis and salt-induced senescence as compared to the roots. In conclusion, the leaf-proline accumulation was due, at least in part, to the increase in the salt-induced proteolysis associated with the increments in the GS activity and hence the increase in the concentration of glutamate precursor in the soluble amino acid pool.     

Antibody isotype responses in Balb/c mice immunized with the cytoplasmic repetitive antigen and flagellar repetitive antigen of Trypanosoma cruzi

In the present report we analyzed the levels of IgG1, IgG2a, IgG2b and IgG3 isotypes from Balb/c mice immunized with cytoplasmic repetitive antigen (CRA), and flagellar repetitive antigen (FRA) of Trypanosoma cruzi. The immunization was done by subcutaneous route three times (20 days apart) and the analysis was performed 14 days after each treatment. CRA-immunized mice produced high levels of all IgG isotypes, mainly IgG3 and IgG1. FRA-immunization elicited only high levels of IgG1.     

Gamma-proteobacteria Aquicella lusitana gen. nov., sp. nov., and Aquicella siphonis sp. nov. infect protozoa and require activated charcoal for growth in laboratory media

Several isolates, belonging to two new species of the same novel genus of gamma-proteobacteria, were recovered from drilled well (borehole) and spa water at S?o Gemil in central Portugal. These organisms are phylogenetically most closely related to the strictly intracellular uncultured species of the genus Rickettsiella, which cause disease in arthropods, and to the facultatively intracellular species of the genus Legionella, some of which cause Legionnaires' disease and Pontiac fever. The S?o Gemil strains grew only on media containing charcoal, as is also true of the species of the genus LEGIONELLA: Unlike the vast majority of Legionella isolates, the new isolates did not require L-cysteine or ferric pyrophosphate for growth but like the legionellae had an absolute requirement for alpha-ketoglutarate. Strains SGT-39(T) and SGT-56 grew consistently between 30 and 43 degrees C, while strains SGT-108(T) and SGT-109 grew between 30 and 40 degrees C. The pH ranges for growth of these organisms were surprisingly narrow: strains SGT-39(T) and SGT-56 grew between pH 6.3 and 7.3, while strains SGT-108(T) and SGT-109 grew between pH 6.3 and 7.0. Both organisms proliferated in the amoeba Hartmannella vermiformis but did not grow in U937 human cells. Based on 16S rRNA gene sequence analysis and physiological, biochemical, and chemical analysis we describe two new species of one novel genus; one species is represented by strain SGT-39(T), for which we propose the name Aquicella lusitana, while strain SGT-108(T) represents a second species of the same genus, for which we propose the name Aquicella siphonis.     

Bradykinin B1 receptor stimulates the proximal tubule Na(+)-ATPase activity through protein kinase C pathway

Recently, our group described a B1-mediated stimulatory effect of des-Arg(9)-bradykinin (DABK) on the Na(+)-ATPase activity of proximal tubule basolateral membranes (BLM) [Biochim. Biophys. Acta 1431 (1999) 483.]. Data in the present report suggest the participation of a phosphatidylinositol-specific PLC (PI-PLC)/protein kinase C (PKC) pathway as the molecular mechanism of DABK-mediated stimulation of the Na(+)-ATPase activity since (i) 10(-8) M DABK activates PI-PLC activity; (ii) 10(-9) M U73122, a PI-PLC inhibitor, abolishes the effect of 10(-8) M DABK on the Na(+)-ATPase activity; (iii) 10(-8) M DABK increases phosphoprotein formation by 34%. This effect is completely reversed by 10(-7) M calphostin C, an inhibitor of PKC; (iv) 20 ng/ml TPA, an activator of PKC, and 10(-8) M DABK stimulate the Na(+)-ATPase activity in a similar and nonadditive manner. Furthermore, the effect of 10(-8) M DABK is completely reversed by calphostin C; (v) 10(-8) M DABK increases phosphoserine residue levels by 54%. This effect is completely reversed by 10(-7) M calphostin C.