Comparison of the subcellular distribution of G-proteins in hepatocytes in situ and in primary cultures

The subcellular localization of the heterotrimeric G-proteins in hepatocytes in situ was compared to that in hepatocytes in primary culture. The ability of various ligands to activate adenylyl cyclase (AC) in membrane preparations was also investigated. In hepatocytes in situ the G proteins were mainly localized at the plasma membrane while in hepatocytes in culture they were predominantly cytoplasmic. The localization of the G-proteins in hepatocytes in situ correlates with their role in signal transduction. In homogenates prepared from the cultured cells, ligands which stimulate AC via G_sα were without effect, which was consistent with the localization of G_sα in the cytoplasmic and nuclear compartments. The “relocalization” of the G proteins to the cytoplasm when cells are cultured suggests that transmembrane signalling may be regulated by cell differentiation and cell-cell and cell-extracellular matrix interactions. © 1996 Wiley-Liss, Inc.     

B cell receptor-stimulated mitochondrial phospholipase A2 activation and resultant disruption of mitochondrial membrane potential correlate with the induction of apoptosis in WEHI-231 B cells

Cross-linking of the Ag receptors on the immature B cell lymphoma, WEHI-231, leads to growth arrest and apoptosis. We now show that although commitment to such B cell receptor (BCR)-mediated apoptosis correlates with mitochondrial phospholipase A(2) activation, disruption of mitochondrial function, and ATP depletion, it is executed independently of caspase activation. First, we demonstrate a pivotal role for mitochondrial function in determining B cell fate by showing up-regulation of cytosolic phospholipase A(2) expression, induction of mitochondrial phospholipase A(2) activity, arachidonic acid-mediated collapse of mitochondrial transmembrane inner potential (Delta psi(m)), and depletion of cellular ATP under conditions of apoptotic, but not proliferative, signaling via the BCR. Importantly, disruption of Delta psi(m), ATP depletion, and apoptosis can be prevented by rescue signals via CD40 or by Delta psi(m) stabilizers such as antimycin or oligomycin. Second, we show that commitment and postmitochondrial execution of BCR-mediated apoptosis are not dependent on caspase activation by demonstrating that such apoptotic signaling does not induce release of cytochrome c from the mitochondria or activation of effector caspases, as evidenced by poly(ADP-ribose) polymerase or Bcl-x(L) cleavage. Indeed, apoptotic signaling via the BCR in WEHI-231 B cells does not stimulate the activation of caspase-3 and, consistent with this, BCR-mediated disruption of Delta psi(m) and commitment to apoptosis take place in the presence of caspase inhibitors. In contrast, BCR signaling induces the postmitochondrial activation of cathepsin B, and resultant apoptosis is blocked by the cathepsin B inhibitor, (23,35)trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester (EST) suggesting a key role for this executioner protease in Ag receptor-driven apoptosis of WEHI-231 immature B cells.     

Biochemical, genetic and physiological characterization of venom components from two species of scorpions: Centruroides exilicauda Wood and Centruroides sculpturatus Ewing

Current literature concerning the taxonomic names of two possibly distinct species of scorpions from the genus Centruroides (sculpturatus and/or exilicauda) is controversial. This communication reports the results of biochemical, genetic and electrophysiological experiments conducted with C. exilicauda Wood of Baja California (Mexico) and C. sculpturatus Ewing of Arizona (USA). The chromatographic profile fractionation of the soluble venom from both species of scorpions is different. The N-terminal amino acid sequence for nine toxins of C. exilicauda was determined and compared with those from C. sculpturatus. Lethality tests conducted in mice support the idea that C. exilicauda venom should be expected to be medically less important than C. sculpturatus. Thirteen genes from the venomous glands of the scorpion C. exilicauda were obtained and compared with previously published sequences from genes of the species C. sculpturatus. Genes coding for cytochrome oxidase I and II of both species were also sequenced. A phylogenetic tree was generated with this information showing important differences between them. Additionally, the results of electrophysiological assays conducted with the venom from both species on the Ca(2+)-dependent K(+)-channels, showed significant differences. These results strongly support the conclusion that C. exilicauda and C. sculpturatus are in fact two distinct species of scorpions.     

FcgammaRIIb-mediated negative regulation of BCR signalling is associated with the recruitment of the MAPkinase-phosphatase, Pac-1, and the 3'-inositol phosphatase, PTEN

The low-affinity receptor for IgG, FcgammaRIIb, negatively regulates B cell antigen receptor (BCR)-mediated proliferative signalling. FcgammaRIIb has been reported to mediate this inhibition by uncoupling the BCR from the RasMAPkinase pathway. We now show that FcgammaRIIb-mediated negative feedback inhibition also correlates with induction of an Erk-associated phosphatase activity that reflects the rapid association of Erk and the MAPkinase phosphatase, Pac-1, and dephosphorylation and inactivation of ErkMAPkinase. This mechanism of abrogating ongoing ErkMAPkinase signalling therefore provides a rationale for rapid immune-complex-mediated feedback inhibition of active antigen-driven B cell responses. In addition, FcgammaRIIb signalling also induces the recruitment and activation of the 3'-inositol phosphatase, PTEN, which by antagonising PI 3kinase activity and inhibiting BCR-coupling to the anti-apoptotic kinase, Akt, provides an additional mechanism for FcgammaRIIb-mediated negative regulation of BCR-coupling to ErkMAPkinase, cell survival and proliferation.     

The Purruja mangrove, Golfito, Costa Rica: a management model

The Purruja mangrove (Golfito, Costa Rica) has an estimated area of 70 ha. A socio-biological research was the ground to set initial goals to manage the resources and to identify the mangrove biological condition. Community participation and the local organization were key factors in developing an integrated model for the management of natural resources. Constant monitoring and institutional networks were the other two factors to manage the mangrove. The constant profesional support was a tool to facilitate the acomplishment of goals and to establish an institucional network to promote local group iniciatives for collaborative management of the Purruja mangrove.     

Synaptotagmin VII restricts fusion pore expansion during lysosomal exocytosis

Synaptotagmin is considered a calcium-dependent trigger for regulated exocytosis. We examined the role of synaptotagmin VII (Syt VII) in the calcium-dependent exocytosis of individual lysosomes in wild-type (WT) and Syt VII knockout (KO) mouse embryonic fibroblasts (MEFs) using total internal reflection fluorescence microscopy. In WT MEFs, most lysosomes only partially released their contents, their membrane proteins did not diffuse into the plasma membrane, and inner diameters of their fusion pores were smaller than 30 nm. In Syt VII KO MEFs, not only was lysosomal exocytosis triggered by calcium, but all of these restrictions on fusion were also removed. These observations indicate that Syt VII does not function as the calcium-dependent trigger for lysosomal exocytosis. Instead, it restricts the kinetics and extent of calcium-dependent lysosomal fusion.     

Faecal pellets of Reeves’ muntjac,<Emphasis Type="Italic"> Muntiacus reevesi</Emphasis>: defecation rate,decomposition period,size and weight

Captive muntjac fed on vegetation had a daily defecation rate of 7 to 8.2.pellet groups. Even in the cold month of February 97.6% of pellets decayed within 80 days but for 8 other months the range was 4 to 23 days. Emphasis is given to the need for site-specific information when decay rate is required to assess population size from dung counts. The morphology, dimensions, weight and group size of pellets are described as an aid to distinguish them from the faeces of other cervids.     

Stability of enzymes and proteins in dried glassy systems: effect of simulated sunlight conditions

The purpose of the present work was to study the effects of simulated sunlight conditions on enzyme inactivation and structural damage in dehydrated glassy systems. Freeze-dried samples containing different enzymes (lactase, invertase, lysozyme and amyloglucosidase) were exposed to light using a medium-pressure metal halide HPA 400 W lamp. After 1 h of light exposure, the samples showed a significant reduction (more than 50%) in the denaturation peak area as analyzed by DSC, and this could be attributed to protein denaturation. For most of the pure enzymes, the loss of enzymic activity after 1 h of light exposure was around 50%. In the case of enzymes included in anhydrous model systems (trehalose, raffinose, maltodextrin, and dextran), the remaining activity also decreased dramatically during the light treatment. We showed that the light exposure in dehydrated systems generated both the loss of enzymic activity and structural changes such as denaturation (observed by DSC) and protein fragmentation and aggregation (observed by electrophoresis). Overall, we can conclude that a short exposure to the light produces dramatic changes in the enzymic activity in dehydrated systems with or without protective matrices.     

S-allylmercaptocysteine scavenges hydroxyl radical and singlet oxygen <Emphasis Type="Italic">in vitro</Emphasis> and attenuates gentamicin-induced oxidative and nitrosative stress and renal damage <Emphasis Type="Italic">in vivo</Emphasis>

Background  

Oxidative and nitrosative stress have been involved in gentamicin-induced nephrotoxicity. The purpose of this work was to study the effect of S-allylmercaptocysteine, a garlic derived compound, on gentamicin-induced oxidative and nitrosative stress and nephrotoxicity. In addition, the in vitro reactive oxygen species scavenging properties of S-allylmercaptocysteine were studied.