The norpurpureine alkaloid from Annona purpurea inhibits human platelet activation in vitro.

BACKGROUND: The leaves of Annona purpurea have yielded several alkaloids with anti-aggregation activities against rabbit platelets. This is promising in the search for agents that might act against platelets and reduce the incidence of cardiovascular diseases. Since significant differences in platelet function have been reported between human and animal platelets, a study focusing on the effect of A. purpurea extracts against human platelet activation is necessary. METHODS: The compounds in an A. purpurea ethanolic extract underwent bio-guided fractionation and were used for in vitro human platelet aggregation assays to isolate the compounds with anti-platelet activity. The bioactive compounds were identified by spectroscopic analysis. Additional platelet studies were performed to characterize their action as inhibitors of human platelet activation. RESULTS: The benzylisoquinoline alkaloid norpurpureine was identified as the major anti-platelet compound. The IC50 for norpurpureine was 80 µM against platelets when stimulated with adenosine 5'-diphosphate (ADP), collagen and thrombin. It was pharmacologically effective from 20 to 220 µM. Norpurpureine (220 µM) exhibited its in vitro effectiveness in samples from 30 healthy human donors who did not take any drugs during the 2 weeks prior to the collection. Norpurpureine also gradually inhibited granule secretion and adhesion of activated platelets to immobilized fibrinogen. At the intra-platelet level, norpurpureine prevented agonist-stimulated calcium mobilization and cAMP reduction. Structure-activity relationship analysis indicates that the lack of a methyl group at the nitrogen seems to be key in the ability of the compound to interact with its molecular target. CONCLUSION: Norpurpureine displays a promising in vitro pharmacological profile as an inhibitor of human platelet activation. Its molecular target could be a common effector between Ca2+ and cAMP signaling, such as the PLC-PKC-Ca2+ pathway and PDEs. This needs further evaluation at the protein isoform level.

Screening of Venezuelan medicinal plant extracts for cytostatic and cytotoxic activity against tumor cell lines.

There are estimated to be more than 20,000 species of plants in Venezuela, of which more than 1500 are used for medicinal purposes by indigenous and local communities. Only a relatively small proportion of these have been evaluated in terms of their potential as antitumor agents. In this study, we screened 308 extracts from 102 species for cytostatic and cytotoxic activity against a panel of six tumor cell lines using a 24-h sulphorhodamine B assay. Extracts from Clavija lancifolia, Hamelia patens, Piper san-vicentense, Physalis cordata, Jacaranda copaia, Heliotropium indicum, and Annona squamosa were the most cytotoxic, whereas other extracts from Calotropis gigantea, Hyptis dilatata, Chromolaena odorata, Siparuna guianensis, Jacaranda obtusifolia, Tapirira guianensis, Xylopia aromatica, Protium heptaphyllum, and Piper arboreum showed the greatest cytostatic activity. These results confirm previous reports on the cytotoxic activities of the above-mentioned plants as well as prompting further studies on others such as C. lancifolia and H. dilatata that have not been so extensively studied.

Silencing of rotavirus NSP4 or VP7 expression reduces alterations in Ca2+ homeostasis induced by infection of cultured cells.

Rotavirus infection of cells in culture induces major changes in Ca(2+) homeostasis. These changes include increases in plasma membrane Ca(2+) permeability, cytosolic Ca(2+) concentration, and total cell Ca(2+) content and a reduction in the amount of Ca(2+) released from intracellular pools sensitive to agonists. Various lines of evidence suggest that the nonstructural glycoprotein NSP4 and possibly the major outer capsid glycoprotein VP7 are responsible for these effects. In order to evaluate the functional roles of NSP4 and other rotavirus proteins in the changes in Ca(2+) homeostasis observed in infected cells, the expressions of NSP4, VP7, and VP4 were silenced using the short interfering RNA (siRNA) technique. The transfection of specific siRNAs resulted in a strong and specific reduction of the expression of NSP4, VP7, and VP4 and decreased the yield of new viral progeny by more than 90%. Using fura-2 loaded cells, we observed that knocking down the expression of NSP4 totally prevented the increase in Ca(2+) permeability of the plasma membrane and cytosolic Ca(2+) concentration measured in infected cells. A reduction in the levels of VP7 expression partially reduced the effect of infection on plasma membrane Ca(2+) permeability and Ca(2+) pools released by agonist (ATP). In addition, the increase of total Ca(2+) content (as measured by (45)Ca(2+) uptake) observed in infected cells was reduced to the levels in mock-infected cells when NSP4 and VP7 were silenced. Finally, when the expression of VP4 was silenced, none of the disturbances of Ca(2+) homeostasis caused by rotaviruses in infected cells were affected. These data altogether indicate that NSP4 is the main protein responsible for the changes in Ca(2+) homeostasis observed in rotavirus-infected cultured cells. Nevertheless, VP7 may contribute to these effects.

Expression of nonstructural rotavirus protein NSP4 mimics Ca2+ homeostasis changes induced by rotavirus infection in cultured cells.

Rotavirus infection modifies Ca(2+) homeostasis, provoking an increase in Ca(2+) permeation, the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyto)), and total Ca(2+) pools and a decrease in Ca(2+) response to agonists. A glycosylated viral protein(s), NSP4 and/or VP7, may be responsible for these effects. HT29 or Cos-7 cells were infected by the SA11 clone 28 strain, in which VP7 is not glycosylated, or transiently transfected with plasmids coding for NSP4-enhanced green fluorescent protein (EGFP) or NSP4. The permeability of the plasma membrane to Ca(2+) and the amount of Ca(2+) sequestered in the endoplasmic reticulum released by carbachol or ATP were measured in fura-2-loaded cells at the single-cell level under a fluorescence microscope or in cell suspensions in a fluorimeter. Total cell Ca(2+) pools were evaluated as (45)Ca(2+) uptake. Infection with SA11 clone 28 induced an increase in Ca(2+) permeability and (45)Ca(2+) uptake similar to that found with the normally glycosylated SA11 strain. These effects were inhibited by tunicamycin, indicating that inhibition of glycosylation of a viral protein other than VP7 affects the changes of Ca(2+) homeostasis induced by infection. Expression of NSP4-EGFP or NSP4 in transfected cells induced the same changes observed with rotavirus infection, whereas the expression of EGFP or EGFP-VP4 showed the behavior of uninfected and untransfected cells. Increased (45)Ca(2+) uptake was also observed in cells expressing NSP4-EGFP or NSP4, as evidenced in rotavirus infection. These results indicate that glycosylated NSP4 is primarily responsible for altering the Ca(2+) homeostasis of infected cells through an initial increase of cell membrane permeability to Ca(2+).

Molecular biology of rotavirus entry and replication.

Rotavirus is a nonenveloped, double-stranded, RNA virus belonging to the Reoviridae family and is the major etiological agent of viral gastroenteritis in young children and young animals. Remarkable progress in the understanding of the rotavirus cycle has been made in the last 10 years. The knowledge of viral replication thus far acquired is based on structural studies, the expression and coexpression of individual viral proteins, silencing of individual genes by siRNAs, and the effects that these manipulations have on the physiology of the infected cell. The functions of the individual rotavirus proteins have been largely dissected; however, the interactions between them and with cell proteins, and the molecular mechanisms of virus replication, are just beginning to be understood. These advancements represent the basis for the development of effective vaccination and rational therapeutic strategies to combat rotavirus infection and diarrhea syndromes. In this paper, we review and try to integrate the new knowledge about rotavirus entry, replication, and assembly, and pose some of the questions that remain to be solved.

Dengue virus induced changes in Ca2+ homeostasis in human hepatic cells that favor the viral replicative cycle.

The role of Ca2+ during dengue virus (DENV) replication is unknown; thus, changes in Ca2+ homeostasis in DENV infected human hepatic HepG2 and Huh-7 cells were analyzed. Infected HepG2 cells, but not Huh-7 cells, showed a significant increase in plasma membrane permeability to Ca2+, while both cell lines showed marked reduced levels of Ca2+ stored in the endoplasmic reticulum. While the expression levels of STIM1 and ORAI1 showed no changes, STIM1 and ORAI1 were shown to co-localized in infected cells, indicating activation of the store-operated Ca2+ entry (SOCE) pathway. Finally, manipulation in the infected cells of the intra and extracellular Ca2+ levels by chelators (BAPTA-AM and EGTA), SOC inhibitor (SKF96365), IP3 Receptor antagonist (2APB) or increase of extracellular [Ca2+], significantly reduced DENV yield, but not vesicular stomatitis virus yield, used as a control. These results show that DENV infection alters cell Ca2+ homeostasis and that such changes favor viral replication.

Intracellular disassembly of infectious rotavirus particles by depletion of Ca2+ sequestered in the endoplasmic reticulum at the end of virus cycle.

Rotavirus infection is characterized by a number of Ca(2+) dependent virus-cell interactions. The structure of rotavirus triple-layered particles (TLP) is dependent on Ca(2+) concentration. Acquisition of the capsid outer layer requires a high Ca(2+) concentration inside the ER. Infection modifies Ca(2+) homeostasis of the cell, increasing ER Ca(2+) content, which may be advantageous to virus replication. We studied the role of sequestered Ca(2+) on the stabilization of already mature viral particles within the ER. Thapsigargin (TG), a SERCA pump inhibitor, added for 30min at the end of infection depleted ER Ca(2+) and reduced the titer of already mature TLP accumulated in the cell. Another inhibitor, cyclopiazonic acid, and two Ca(2+) ionophores (A23187 and ionomycin) in the presence of EGTA had similar effects. TG eliminated the peak of radiolabeled TLP, increasing that of DLP in CsCl gradients. Electron microscopy revealed accumulation of clustered particles in the ER, which had lost their integrity. The [Ca(2+)] in the ER of infected cells is important for virus maturation and for maintaining the integrity of mature TLP. Viral particles in this compartment may be potentially infectious, already containing VP7 and VP4.

IFN-gamma alters the human sperm membrane permeability to Ca(2+).

Inflammation in the male genitourinary tract has been associated with the release of pro-inflammatory cytokines such as interferon-gamma (IFN-γ) and elevated reactive oxygen species, which affects spermatozoa capacitation, motility, and the acrosome reaction, along with functions regulated by the concentration of cytoplasmic Ca(2+) ([Ca(2+)]cyto). Though Ca(2+) signaling is of particular significance in sperm, the effect of IFN-γ intracellular calcium on these cells is still unknown. The present study evaluated the effect of IFN-γ on the [Ca(2+)]cyto and Ca(2+) permeability on human sperm. A cell suspension loaded with fura-2 was incubated with or without IFN-γ (from 0 to 2000 pg/ml) for 0, 30, 60, and 120 minutes, and the [Ca(2+)]cyto was measured. The permeability to Ca(2+) was evaluated by the change of the intracellular concentration following an extracellular Ca(2+) pulse. IFN-γ at low concentrations (≤ 500 pg/ml) did not affect the [Ca(2+)]cyto and Ca(2+) permeability of sperm. At a high concentration (2000 pg/ml), IFN-γ did not alter the [Ca(2+)](cyto), but significantly decreased the magnitude and velocity of Ca(2+) entry into the cell. This effect was dependent on incubation time and IFN-γ concentration. This alteration induced by IFN-γ was prevented by the simultaneous incubation of sperm with the antioxidant butylhydroxytoluene (BHT). In conclusion, in vitro, IFN-γ modifies Ca(2+) sperm membrane permeability, probably via lipid peroxidation. IFN-γ in high concentration, as observed in inflammation/infection, can affect [Ca(2+)](cyto) regulation and alter sperm fertilizing capacity.

Rotavirus infection of cells in culture induces activation of RhoA and changes in the actin and tubulin cytoskeleton.

Rotavirus infection induces an increase in [Ca(2+)](cyto), which in turn may affect the distribution of the cytoskeleton proteins in the infected cell. Changes in microfilaments, including the formation of stress fibers, were observed starting at 0.5 h.p.i. using fluorescent phalloidin. Western blot analysis indicated that RhoA is activated between 0.5 and 1 h.p.i. Neither the phosphorylation of RhoA nor the formation of stress fibers were observed in cells infected with virions pre-treated with an anti-VP5* non-neutralizing mAb, suggesting that RhoA activation is stimulated by the interaction of the virus with integrins forming the cell receptor complex. In addition, the structure of the tubulin cytoskeleton was also studied. Alterations of the microtubules were evident starting at 3 h.p.i. and by 7 h.p.i. when microtubules were markedly displaced toward the periphery of the cell cytoplasm. Loading of rotavirus-infected cells with either a Ca(2+) chelator (BAPTA) or transfection with siRNAs to silence NSP4, reversed the changes observed in both the microfilaments and microtubules distribution, but not the appearance of stress fibers. These results indicate that alterations in the distribution of actin microfilaments are initiated early during infection by the activation of RhoA, and that latter changes in the Ca(2+) homeostasis promoted by NSP4 during infection may be responsible for other alterations in the actin and tubulin cytoskeleton.

Dissecting the Ca²âº entry pathways induced by rotavirus infection and NSP4-EGFP expression in Cos-7 cells.

Rotavirus infection modifies Ca(2+) homeostasis provoking an increase in Ca(2+) permeation, cytoplasmic Ca(2+) concentration ([Ca(2+)](cyto)), total Ca(2+) pools and, a decrease of Ca(2+) response to agonists. These effects are mediated by NSP4. The mechanism by which NSP4 deranges Ca(2+) homeostasis is not yet known. It has been proposed that the increase in [Ca(2+)](cyto) is the result of Ca(2+) release from intracellular stores, thereby activating store-operated Ca(2+) entry (SOCE). We studied the mechanisms involved in the changes of Ca(2+) permeability of the plasma membrane elicited by rotavirus infection and NSP4 expression in Cos-7 cells loaded with fura-2 or fluo-4, using inhibitors and activators of different pathways. Total depletion of ER Ca(2+) stores induced by thapsigargin or ATP was not able to elicit Ca(2+) entry in mock-infected cells to the level attained with infection or NSP4-EGFP expression. The pathway induced by NSP4-EGFP expression or infection shows properties shared by SOCE: it can be inactivated by high [Ca(2+)](cyto), is permeable to Mn(2+) and inhibited by La(3+) and the SOC inhibitor 2-aminoethoxydiphenyl borate (2-APB). Contribution of the agonist-operated channels (AOCs) to Ca(2+) entry is small and not modified by infection. The plasma membrane permeability to Ca(2+) in rotavirus infected or NSP4-EGFP expressing cells is also blocked by KB-R7943, an inhibitor of the plasma membrane Na(+)/Ca(2+) exchanger (NCX), operating in its reverse mode. In conclusion, the expression of NSP4 in infected Cos-7 cells appears to activate the NCX in reverse mode and the SOCE pathway to induce increased Ca(2+) entry.

Heterogeneity of acid secretion induced by carbachol and histamine along the gastric gland axis and its relationship to [Ca2+]i.

The gastric glands of the mammalian fundic mucosa are constituted by different cell types. Gastric fluid is a mixture of acid, alkali, ions, enzymes, and mucins secreted by parietal, chief, and mucous cells. We studied activation of acid secretion using LysoSensor Yellow/Blue in conjunction with fluo 3 to measure changes in pH and Ca(2+) in isolated rabbit gastric glands. We evidenced a spatial heterogeneity in the amplitude of acid response along the gland axis under histamine and cholinergic stimulation. Carbachol induced a transitory pH increase before acidification. This relative alkalinization may be related to granule release from other cell types. Omeprazole inhibited the acid component but not the rise in pH. Histamine stimulated acid secretion without increase of lumen pH. We studied the relationship between Ca(2+) release and/or entry and H(+) secretion in glands stimulated by carbachol. Ca(2+) release was associated with a fast and transient components of H(+) secretion. We found a linear relationship between Ca(2+) release and H(+) secretion. Ca(2+) entry was associated with a second slow and larger component of acid secretion. The fast component may be the result of activation of Cl(-) and K(+) channels and hence H(+)/K(+) pumps already present in the membrane, whereas the slow component might be associated with translocation of H(+)/K(+) pumps to the canaliculi. In conclusion, with cholinergic stimulation, gastric glands secrete a mixture of acid and other product(s) with a pH above 4.2, both triggered by Ca(2+) release. Maintenance of acid secretion depends on Ca(2+) entry and perhaps membrane fusion.

Stomach lysozymes of the three-toed sloth (Bradypus variegatus), an arboreal folivore from the Neotropics.

Lysozymes are antimicrobial defences that act as digestive enzymes when expressed in the stomach of herbivores with pre-gastric fermentation. We studied this enzyme in the complex stomach of the three-toed sloth (Bradypus variegatus), a folivore with pre-gastric fermentation. Lysozymes were identified by SDS-PAGE and immunoblotting in all portions: diverticulum, pouch, glandular and muscular prepyloric area with 14.3 kDa of molecular mass. Purified lysozymes from all areas but the diverticulum were characterized by MALDI-TOF, optimal pH, optimal ionic strength, and specific activity. The differences observed suggested at least three isoforms. The optimal pHs were similar to the pH of the stomach portion where the enzymes were isolated. The lysozyme from the pouch (fermentation chamber) exhibited higher specific activity and concentration than the others. The specific activity of the enzyme from the acid muscular prepyloric portion was comparable to that reported in the cow abomasums; however, its concentration was lower than that observed in cow. This distinctive pattern of secretion/specific activity and overall low concentration suggests different roles for the lysozymes in this herbivore compared to Artiodactyla. We postulate that sloth stomach lysozymes may still be antimicrobial defences by protecting the microbial flora of the fermentation chamber against foreign bacteria.

Ca2+ permeability of the plasma membrane induced by rotavirus infection in cultured cells is inhibited by tunicamycin and brefeldin A.

Rotavirus infection of cultured cells induces a progressive increase in plasma membrane permeability to Ca2+. The viral product responsible for this effect is not known. We have used tunicamycin and brefeldin A to prevent glycosylation and membrane traffic and study the involvement of viral glycoproteins, NSP4 and/or VP7, in rotavirus-infected HT29 and MA104 cells. In infected cells, we observed an increase of plasma membrane Ca2+ permeability and a progressive depletion of agonist-releasable ER pools measured with fura 2 and an enhancement of total Ca2+ content measured as 45Ca2+ uptake. Tunicamycin inhibited the increase in membrane Ca2+ permeability, induced a depletion of agonist-releasable and 45Ca2+-sequestered pools. Brefeldin A inhibited the increase of Ca2+ permeability and the increase in 45Ca2+ uptake induced by infection. We propose that the glycosylated viral product NSP4 (and/or VP7) travels to the plasma membrane to form a Ca2+ channel and hence elevate Ca2+ permeability.

I, 2. Physiology and pathophysiology of the gut in relation to viral diarrhea.

Many advances have been made in the understanding of intestinal electrolyte transport from the molecular to the whole-tissue level. This chapter discusses the molecular mechanisms of intestinal epithelial ion transport processes, as well as the intra- and extracellular factors involved in their regulation, as a framework for the understanding of virus-induced gastroenteritis. Based on the present knowledge of the effects of rotavirus (RV) infection on the physiology of the intestine at different levels of organization, a working model for the pathogenesis of RV diarrhea is presented in the chapter. The understanding of the pathogenic processes of viral diarrheas may serve as the basis for a rational approach in the design of novel therapeutic strategies and the search for new antiviral drugs.

Requirement for vacuolar H+ -ATPase activity and Ca2+ gradient during entry of rotavirus into MA104 cells.

The mechanism by which rotavirus and other nonenveloped viruses enter the cell is still not clear. We have proposed an endocytosis model where the critical step for virus uncoating and membrane permeabilization is the decrease in Ca(2+) concentration in the endosome. In this paper, we monitored rotavirus entry by measuring alpha-sarcin-rotavirus coentry and infectivity in MA104 cells. The participation of endocytosis, acidification, and endosomal Ca(2+) concentration on virus entry was studied by inhibiting the endosomal H(+)-ATPase with bafilomycin A1 and/or increasing the extracellular calcium reservoir by addition of 10 mM CaEGTA. Rotavirus-alpha-sarcin coentry was inhibited by bafilomycin A1 and by addition of 10 mM CaEGTA. These effects were additive. These substances induced a significant inhibition of infectivity without affecting virus binding and postentry steps. These results are compatible with the interpretation that bafilomycin A1 and CaEGTA block rotavirus penetration from the endosome into the cytoplasm and support our hypothesis of a Ca(2+)-dependent endocytosis model.

Antibodies to rotavirus outer capsid glycoprotein VP7 neutralize infectivity by inhibiting virion decapsidation.

The rotavirus capsid is composed of three concentric protein layers. Proteins VP4 and VP7 comprise the outer layer. VP4 forms spikes, is the viral attachment protein, and is cleaved by trypsin into VP8* and VP5*. VP7 is a glycoprotein and the major constituent of the outer protein layer. Both VP4 and VP7 induce neutralizing and protective antibodies. To gain insight into the virus neutralization mechanisms, the effects of neutralizing monoclonal antibodies (MAbs) directed against VP8*, VP5*, and VP7 on the decapsidation process of purified OSU and RRV virions were studied. Changes in virion size were followed in real time by 90 degrees light scattering. The transition from triple-layered particles to double-layered particles induced by controlled low calcium concentrations was completely inhibited by anti-VP7 MAbs but not by anti-VP8* or anti-VP5* MAbs. The inhibitory effect of the MAb directed against VP7 was concentration dependent and was abolished by papain digestion of virus-bound antibody under conditions that generated Fab fragments but not under conditions that generated F(ab')(2) fragments. Electron microscopy showed that RRV virions reacted with an anti-VP7 MAb stayed as triple-layered particles in the presence of excess EDTA. Furthermore, the infectivity of rotavirus neutralized via VP8*, but not that of rotavirus neutralized via VP7, could be recovered by lipofection of neutralized particles into MA-104 cells. These data are consistent with the notion that antibodies directed at VP8* neutralize by inhibiting binding of virus to the cell. They also indicate that antibodies directed at VP7 neutralize by inhibiting virus decapsidation, in a manner that is dependent on the bivalent binding of the antibody.

Comparación del control de la secreción de ácido y de pepsinogeno por la célula oxintopéptica del anfibio / Compasison of the control of acid and pepsinogen secretion by oxyntopeptic all of amphibians

La secreción de pepsinógeno y de HCI en el estómago de anfibio tiene lugar en un solo tipo celular, la célula oxintopéptica. La distribución de pepsinógeno es heterógena en la mucosa gástrica de Bufo marinus y la mayor concentración se encuentra en el fundus. Las dos secreciones responden a los mismos secretagogos. La histamina produce la mayor respuesta en ambas secreciones, mientras que el carbacol, por sí solo, sin el componente de histamina endógena, tiene un efecto pequeño. La forskolina y el 8Br-cAMP tienen un efecto parecido al producido por la histamina. El ionóforo de Ca2+, A23187, y el análogo de diacilglicerol, el octanoilacilglicerol (OAG), tienen un efecto pequeño sobre ambas secreciones, como el carbacol. La adición conjunta de histamina y carbacol, o de forskolina y carbacol, o el OAG y 8Br-cAMP induce una respuesta potenciada en las dos secreciones. El análisis cuantitativo de las respuestas y el estudio de la relación entre ellas durante las estimulaciones con histamina y carbacol muestran un patrón de estimulación no paralela y desacoplada