Pharmacological screening of plants recommended by folk medicine as anti-snake venom: I. Analgesic and anti-inflammatory activities

We have observed that several plants used popularly as anti-snake venom show anti-inflammatory activity. From the list prepared by Rizzini, Mors and Pereira some species have been selected and tested for analgesic activity (number of contortions) and anti-inflammatory activity (Evans blue dye diffusion - 1% solution) according to Whittle's technique (intraperitoneal administration of 0.1 N-acetic acid 0.1 ml/10 g) in mice. Previous oral administration of a 10% infusion (dry plant) or 20% (fresh plant) corresponding to 1 or 2 g/Kg of Apuleia leiocarpa, Casearia sylvestris, Brunfelsia uniflora, Chiococca brachiata, Cynara scolymus, Dorstenia brasiliensis, Elephantopus scaber, Marsypianthes chamaedrys, Mikania glomerata and Trianosperma tayuya demonstrated analgesic and/or anti-inflammatory activities of varied intensity

Protective role of peroxisome proliferator-activated receptor-β/δ in septic shock.

Rationale: Peroxisome proliferator activated receptor (PPAR)-beta/delta is a transcription factor that belongs to the PPAR nuclear hormone receptor family, but the role of PPAR-beta/delta in sepsis is unknown. Objectives: We investigated the role of PPAR-beta/delta in murine models of LPS-induced organ injury and dysfunction and cecal ligation and puncture (CLP)-induced polymicrobial sepsis. Methods: Wild-type (WT) and PPAR-beta/delta knockout (1(0) mice and C57BL/6 mice were subjected to LPS for 16 hours. C57BL/6 mice received the PPAR-beta/delta agonist GW0742 (0.03 mg/kg intravenously, 1 h after LPS) or GW0742 plus the PPAR-beta/delta antagonist GSK0660 (0.1 mg/kg intravenously, 30 min before LPS). CD-1 mice subjected to CLP received GW0742 or GW0742 plus GSK0660. Measurements and Main Results: In PPAR-beta/delta KO mice, endotoxemia exacerbated organ injury and dysfunction (cardiac, renal, and hepatic) and inflammation (lung) compared with WT mice. In C57BL/6 mice subjected to endotoxemia, GW0742 significantly (1) attenuated organ (cardiac and renal) dysfunction and inflammation (lung); (2) increased the phosphorylation of Akt and glycogen synthase kinase (GSK)-3 beta; (3) attenuated the increase in extracellular signal-regulated kinase (ERK)1/2 and signal transducer and activator of transcription (STAT)-3 phosphorylation; and (4) attenuated the activation of nuclear factor (NF)-kappa B and the expression of inducible nitric oxide synthase (iNOS). In CD-1 mice subjected to CLP, GW0742 improved 10-day survival. All the observed beneficial effects of GW0742 were attenuated by the PPAR-beta/delta antagonist GSK0660. Conclusions: PPAR-beta/delta protects against multiple organ injury and dysfunction, and inflammation caused by endotoxic shock and improves survival in polymicrobial sepsis by a mechanism that may involve activation of Akt and inhibition of GSK-3 beta and NF-kappa B.

The role of fixed delays in neuronal rate models

Fixed delays in neuronal interactions arise through synaptic and dendritic processing. Previous work has shown that such delays, which play an important role in shaping the dynamics of networks of large numbers of spiking neurons with continuous synaptic kinetics, can be taken into account with a rate model through the addition of an explicit, fixed delay. Here we extend this work to account for arbitrary symmetric patterns of synaptic connectivity and generic nonlinear transfer functions. Specifically, we conduct a weakly nonlinear analysis of the dynamical states arising via primary instabilities of the stationary uniform state. In this way we determine analytically how the nature and stability of these states depend on the choice of transfer function and connectivity. While this dependence is, in general, nontrivial, we make use of the smallness of the ratio in the delay in neuronal interactions to the effective time constant of integration to arrive at two general observations of physiological relevance. These are: 1 - fast oscillations are always supercritical for realistic transfer functions. 2 - Traveling waves are preferred over standing waves given plausible patterns of local connectivity.

Essential role of CD8 palmitoylation in CD8 coreceptor function.

To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.

The role of cytokines in Plasmodium vivax malaria

The cytokine tumor necrosis factor and other as yet unidentified factor(s) which together mediate the killing of intraerythrocytic malaria parasites are transiently elevated in sera during paroxysms in human Plasmodium vivax infections in non-immunes. These factors which included TNF and parasite killing factor(s) are associated with the clinical disease in malaria to the extent that their transient presence in infection sera coincided with paroxysms, the most pronounced clinical disturbances of P. vivax malaria and secondly because their levels were markedly lower in paroxysm sera of semi-immune patients who were resident of an endemic area. Further, a close parallel was obtained between serum TFN levels and changes in body temperature that occur during a P. vivax paroxysm in non-immune patients, suggesting a causative role for TNF in the fever in malaria. P. vivax rarely if ever cause complicated clinical syndromes. Nevertheles serum TFN levels reached in acutely ill P. vivax patients were as high as in patients suffering from cerebral complications of P. falciparum malaria as reported in studies from the Gambia. Cytokine profiles and other changes accompanying clinical disease in P. vivax and P. falciparum malaria are compared in this paper with a view to discussing the potential role of cytokines in the causation of disease in malaria.

Role of penicillin-binding proteins in Streptococcus gordonii

Résumé Streptococcus gordonii est une bactérie colonisatrice naturelle de la cavité buccale de l'homme. Bien que normalement commensale, elle peut causer des infections graves, telles que des bactériémies ou des endocardites infectieuses. La pénicilline étant un des traitements privilégiés dans de tels cas, l'augmentation rapide et globale des résistances à cet antibiotique devient inquiétante. L'étude de la physiologie et des bases génétiques de ces résistances chez S. gordonii s'avère donc importante. Les cibles moléculaires privilégiées de la pénicilline G et des β-lactames sont les penicilllin-binding proteins (PBPs). Ces enzymes associées à la membrane ont pour rôle de catalyser les réactions de transpeptidation et de transglycosylation, qui constituent les dernières étapes de la biosynthèse du peptidoglycan (PG). Elles sont définies comme classe A ou B selon leur capacité d'assurer soit les deux réactions, soit uniquement la transpeptidation. Les β-lactames inhibent le domaine transpeptidase de toutes les PBPs, entraînant l'inhibition de la synthèse du PG, l'inhibition de la croissance, et finalement la mort cellulaire. Chez les streptocoques, les PBPs sont aussi les premiers déterminants de la résistance à la pénicilline. De plus, elles sont impliquées dans la morphologie bactérienne, en raison de leur rôle crucial dans la formation du PG. Le but de ce travail était de caractériser les PBPs de S. gordonii et d'étudier leurs fonctions dans la vie végétative de la bactérie ainsi que durant le développement de la résistance à la pénicilline. Premièrement, des mutants auxquels il manque une ou deux PBP(s) ont été construits. Leur étude - au niveau physiologique, biochimique et morphologique - a montré le caractère essentiel ou dispensable de chaque protéine, ainsi que certaines de leurs fonctions potentielles. Deuxièmement, des mutants résistants à la pénicilline ont été générés. Leur caractérisation a montré l'importance des mutations dans les PBPs ainsi que dans d'autres gènes encore inconnus, de même que le rôle crucial des PBPs de classe A dans le développement de la résistance à la pénicilline. Des expériences supplémentaires sur des isolats résistants ont aussi prouvé que la résistance a un coût en terme de fitness, coût que S. gordonii parvient à compenser par des mécanismes d'adaptation. Finalement, les promoteurs des gènes des PBPs ont été déterminés et leur expression a été étudiée grâce au gène de luciférase. Il a ainsi été montré que la résistance à la pénicilline entraîne non seulement des altérations au niveau des protéines, mais aussi au niveau de la régulation des gènes. De plus, la pénicilline génère directement des modifications dans l'expression de PBPs spécifiques. Summary Streptococcus gordonii is a normal inhabitant of the human oral cavity and a pioneer colonizer of teeth. Although usually considered as a commensal, this organism can cause life-threatening infections such as bacteraemia or endocarditis. Since penicillin is one of the preferential treatments for such pathologies, the rapid and general increase of antibiotic resistance in the overall population becomes an issue. Thus, studying the physiologic and genetic bases of such a resistance in S. gordonii is of interest. The primary molecular targets of penicillin G and other β-lactams are the so called penicillin-binding proteins (PBPs). These are membrane-associated proteins that catalyze the last steps in peptidoglycan (PG) biosynthesis, namely transpeptidation and transglycosylation. Depending on their capacity to catalyze either reactions or only transpeptidation, they are considered as class A or class B PBPs, respectively. β-lactam antibiotics inhibit the transpeptidase domain of both of these classes of enzymes, resulting in inhibition of PG assembly, inhibition of bacterial growth, and ultimately leading to cell death. In streptococci, PBPs are also the primary determinants of penicillin-resistance. Moreover, because of their crucial role in PG formation, they are implicated in fundamental aspects of cell morphology. The goal of this work was thus to characterize S. gordonii PBPs and to explore their functions in terms of vegetative life and penicillin-resistance development. First, single and double PBP-inactivated mutants were generated and their effect on the bacterial physiology, cell wall biochemistry and ultrastructural morphology was assessed. This demonstrated the essentiality or dispensability of each protein for bacterial life. Second, penicillin-resistant mutants were generated by cyclic exposure to increasing concentrations of the drug. Characterization of these mutants pointed out the importance of both PBP and non-PBP mutations, as well as the crucial role of the class A PBPs in the development of penicillin-resistance. Further experiments on resistant isolates demonstrated the fitness cost of this resistance, but also the capacity of S. gordonii to adapt and regain the fitness of the wild-type. Finally, the promoters of PBP genes were determined and their expression was monitored using luciferase fusions. This showed that penicillin-resistance, in addition to modifications at the level of the protein, also triggered genetic alterations. Moreover, penicillin itself generated modifications in the expression of specific PBPs.

Mutagenesis and modelling of the alpha(1b)-adrenergic receptor highlight the role of the helix 3/helix 6 interface in receptor activation.

Computer simulations on a new model of the alpha1b-adrenergic receptor based on the crystal structure of rhodopsin have been combined with experimental mutagenesis to investigate the role of residues in the cytosolic half of helix 6 in receptor activation. Our results support the hypothesis that a salt bridge between the highly conserved arginine (R143(3.50)) of the E/DRY motif of helix 3 and a conserved glutamate (E289(6.30)) on helix 6 constrains the alpha1b-AR in the inactive state. In fact, mutations of E289(6.30) that weakened the R143(3.50)-E289(6.30) interaction constitutively activated the receptor. The functional effect of mutating other amino acids on helix 6 (F286(6.27), A292(6.33), L296(6.37), V299(6.40,) V300(6.41), and F303(6.44)) correlates with the extent of their interaction with helix 3 and in particular with R143(3.50) of the E/DRY sequence.

Nicotine-induced release of vasopressin in the conscious rat: role of opioid peptides and hemodynamic effects.

Nicotine has been shown to stimulate the release of vasopressin and to cause significant hemodynamic changes. The mechanisms leading to enhanced vasopressin secretion and the vascular consequences of the high plasma vasopressin levels during nicotine infusion have not yet been determined. Therefore, the purposes of the present study were 1) to examine in normal conscious rats the role of opioid peptides in the nicotine-induced increase in plasma vasopressin levels and 2) to assess the role of vasopressin in the hemodynamic effects of nicotine (20 micrograms/min for 15 min) using a specific V1 antagonist of the vascular actions of vasopressin. Plasma vasopressin levels were significantly increased in the nicotine-treated animals (39.5 +/- 10 vs. 3.7 +/- 0.6 pg/ml in the controls, P less than .01). Pretreatment with naloxone, an antagonist of opioids at their receptors, did not reduce the vasopressin levels (47.7 +/- 9 pg/ml). Nicotine also increased mean blood pressure (122.5 +/- 2.5 to 145.2 +/- 3.3 mm Hg, P less than .01) and decreased heart rate (461 +/- 6 to 386 +/- 14.5 beats/min, P less than .05). Administration of the vasopressin V1 antagonist before the nicotine infusion did not affect the systemic hemodynamics or the regional blood flow distribution, as assessed by radiolabeled microspheres. Thus, these results suggest that the nicotine-induced secretion of vasopressin is not mediated by opioid receptors and that the high plasma vasopressin levels do not exert any significant hemodynamic effect on cardiac output or blood flow distribution.

The role of egg antigens, cytokines in granuloma formation in murine schistosomiasis mansoni

The induction of granuloma formation by soluble egg antigens (SEA) of Schistosoma mansoni is accompanied by T cell-mediated lymphokine production that regulates the intensity of the response. In the present study we have examined the ability of SDS-PAGE fractioned SEA proteins to elicit granulomas and lymphokine production in infected and egg-immunized mice. At the acute stage of infection SEA fractions (<21, 25-30, 32-38, 60-66, 70-90, 93-125, and > 200 kD) that elicited pulmonary granulomas also elicited IL-2, IL-4 lymphokine production. At the chronic stage a diminished number of fractions (60-66, 70-90, 93-125, and > 200 kD) were able to elicit granulomas with an overall decrease in IL-2, IL-4 production. Granulomas were elicited by larval-egg crossreactive and egg-specific fractions at both the acute and chronic stage of the infection. Examination of lymphokine production from egg-immunized mice demonstrated that as early as 4 days IL-2 was produced by spleen cells stimulated with <21, 32-38, 40-46, 93-125, and >200 kD fractions. By 16 days, IL-2production was envoked by 8 of 9 fractions. IL-4 production at 4 days in response to all fractions was minimal while at 16 days IL-4 was elicited with the < 21, 25-30, 50-56, 93-125, and > 200 kD fractions. The present study reveals differences in the range of SEA fractions able to elicit granulomas and IL-2, IL-4 production between acute and chronic stages of infection. Additionally, this study demonstrates sequential (IL-2 followed by IL-4) lymphokine production during the primary egg antigen response.

The role of cytokines in the pathogenesis of hepatic granulomatous disease in Schistosoma mansoni infected mice

Cytokines are important in the cell-mediated response to Schistosoma mansoni eggs. We have found that Th2 cytokine responses (e.G. IL-4 and IL-5) are argumented after egg laying begins while the response (IL-2 and IFN-*) are down regulated in S. mansoni infected mice. Treatment of mice with anti-IL-5 monoclonal antibodies (Mab) suppressed the eosinophil response almost completley but did not affect granuloma size and slightly increased hepatic fibrosis. Anti-IL-4 treatment abolished IgE responses in infected mice and decreased hepatic fibrosis slightly. Anti-IFN-* treatment had no effect on hepatic pathology. Anti-IL-2 treatment decreased granuloma size significantly and decreased hepatic fibrosis markedly. Anti-IL-2 treatment dramatically decreased IL-5 secretion by splenic cells in vitro and decreased peripheral blood and tissue eosinophilia. In contrast IL-4 secretion was unaffected and serum IgE was normal or increased. IL-2 and IFN-* secretion by splenic cells of treated mice were slightly but not significantly increased suggesting that anti-IL-2 treatment affecting Th2 rather than Th1 responses.

Delinquency in male adolescents: the role of alexithymia and family structure

The purpose of this study was to examine the linkages between alexithymia and delinquency in male adolescents (age ranging from 14 to 18 years), and to investigate whether alexithymia was a good discriminatory factor for juvenile delinquency. Thirty-six offender adolescents and 46 non-offender control adolescents participated in the study and completed the 20-item Toronto Alexithymia Scale (TAS-20) (alexithymia), the Revised Children's Manifest Anxiety Scale (R-CMAS) (anxiety), the Liste d'Adjectifs Bipolaires et en Echelles de Likert (LABEL) (personality-Big Five) and demographic data. Findings revealed that the adolescents of the offender group scored high on alexithymia and that proportion of disrupted family structure in the offender group is higher than in the control group. Logistic regressions confirmed that alexithymia and family structure are the strongest discriminatory factors for juvenile delinquency. Limitations and clinical implications are discussed, and recommendations for future research are provided

The role of the Estrogen-Responsive B box protein in skin morphogenesis, wound repair and skin disease

SUMMARY: EBBP is a poorly characterized member of the RBCC/TRIM family (RING finger B-box coiled-coilltripartite motif). It is ubiquitously expressed, but particularly high levels are found in keratinocytes. There is evidence that EBBP is involved in inflammatory processes, since it can interact with pro-interleukin-1 ß (prolL-1 ß) in human macrophages and keratinocytes, and its downregulation results in reduced secretion of IL-1 ß. IL-1ß activation and secretion requires the proteolytic cleavage of prolL-1ß by caspase-1, which in turn is actìvated by a protein complex called the inflammasome. As it has been demonstrated that EBBP can bind two different proteins of the inflammasome (NALP-1 and caspase 1), we assumed that EBBP plays a role in the regulation of inflammation and that the inflammasome, which has as yet only been described in ínflammatory cells, may also exist in keratinocytes. Indeed, I could show in my thesis that the inflammasome components are expressed in human keratinócytes at the RNA and protein level and also in vivo in human epidermis. After irradiation with a physiological dose of UVB, keratinocytes activated prolL-1ß and secreted prolL-1 a, IL-1 ß, prolL-18 and inflammasome proteins, although all these proteins lack a classical signal peptide. The secretion was dependent on caspase-1 activity, but not on de novo protein synthesis. Knock-down of NALP1 and -3, caspase-1 and -5, EBBP and Asc strongly reduced the secretion of IL-1 ß, demonstrating that also in keratinocytes inflammasome proteins are directly involved in maturation of this cytokine. These results demonstrate for the first time the presence of an active inflammasome in non-professional immune cells. Moreover, they show that UV irradiation is a stimulus for inflammasome activation in keratinocytes. For the analysis of the ín vivo functions of EBBP, transgenic mice overexpressing EBBP in the epidermis were generated. To examine the influence of EBBP overexpression on inflammatory processes, we subjected the mice to different challenges, which induce inflammation. Wound-healing, UVB irradiation and delayed hypersensitivity were tested, but we did not observe any phenotype in the K14-EBBP mice. Besides, a conditional ebbp knockout mouse has been obtained, which will allow to determine the effects of EBBP gene deletion in different tissues and organs. RESUME: EBBP est un membre encore mal connu de la famille des RBCC/TRIM (RING finger B-box coiled-coil/tripartite motif). Il est exprimé de manière ubiquitaire, et en particulier dans les kératinocytes. EBBP étant capable d'interagir avec la prointerleukine-1 ß (prolL-1 ß) dans les macrophages et les kératinocytes humains et de réguler la sécrétion de l'IL-1 ß, il est très probable que cette protéine est impliquée dans l'inflammation. L'activation et la sécrétion de l'IL-1 ß requièrent le clivage protéolytique de son précurseur prolL-1ß par la caspase-1, qui est elle-même activée par un complexe protéique appelé l'inflammasome. Comme il a été démontré qu'EBBP peut lier deux protéines de l'inflammasome (NALP-1 et caspase-1), nous avons émis l'hypothèse qu'EBBP joue un rôle dans la régulation de l'inflammation et que l'inflammasome, jusqu'ici décrit exclusivement dans des cellules inflammatoires, existe dans les kératinocytes. En effet, j'ai pu montrer dans ma thèse que les composants de l'inflammasome sont exprimés dans les kératinocytes humains ainsi que in vivo dans l'épiderme humain. Après irradiation avec une dose, physiologique d'UVB, les kératinocytes activent la prolL-1 ß et sécrètent la prolL-1a, l'IL-1 ß, la prolL-18 et des protéines de l'inflammasome, bien que toutes ces protéines soient dépourvues de peptide signal. La sécrétion dépend de la caspase-1 mais pas de la synthèse protéique de novo. Le knock-down de NALP-1 et -3, des caspase-1 et -5, d'EBBP et d'Asc réduit de manière marquée la sécrétion d'IL-1 ß, démontrant que dans les kératinocytes également, les protéines de l'inflammasome sont impliquées directement dans la maturation de cette cytokine. Ces résultats démontrent pour la première fois la présence d'un inflammasome actif dans des cellules immunitaires non professionnelles. De plus, ils montrent que l'irradiation aux UV est un stimulus pour l'activation de l'inflammasome dans les kératinocytes. Pour l'analyse des fonctions d'EBBP in vivo, nous avons généré des souris transgéniques qui surexpriment EBBP dans l'épiderme. En vue d'examiner l'influence de la surexpression d'EBBP sur le processus inflammatoire, nous avons soumis ces souris à differents modèles d'inflammation. Nous avons testé cicatrisation, UVB et hypersensibilité retardée, mais n'avons pas observé de phénotype chez les souris transgéniques. En parallèle, nous avons également généré des souris knock-out pour ebbp qui devraient nous permettre de déterminer les effets de la suppression d'EBBP dans différents tissus et organes.

Role of the stress sigma factor RpoS in GacA/RsmA-controlled secondary metabolism and resistance to oxidative stress in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens biocontrol strain CHA0, the two-component system GacS/GacA positively controls the synthesis of extracellular products such as hydrogen cyanide, protease, and 2,4-diacetylphloroglucinol, by upregulating the transcription of small regulatory RNAs which relieve RsmA-mediated translational repression of target genes. The expression of the stress sigma factor sigmaS (RpoS) was controlled positively by GacA and negatively by RsmA. By comparison with the wild-type CHA0, both a gacS and an rpoS null mutant were more sensitive to H2O2 in stationary phase. Overexpression of rpoS or of rsmZ, encoding a small RNA antagonistic to RsmA, restored peroxide resistance to a gacS mutant. By contrast, the rpoS mutant showed a slight increase in the expression of the hcnA (HCN synthase subunit) gene and of the aprA (major exoprotease) gene, whereas overexpression of sigmaS strongly reduced the expression of these genes. These results suggest that in strain CHA0, regulation of exoproduct synthesis does not involve sigmaS as an intermediate in the Gac/Rsm signal transduction pathway whereas sigmaS participates in Gac/Rsm-mediated resistance to oxidative stress.

The role of multisensory memories in unisensory object discrimination.

Past multisensory experiences can influence current unisensory processing and memory performance. Repeated images are better discriminated if initially presented as auditory-visual pairs, rather than only visually. An experience's context thus plays a role in how well repetitions of certain aspects are later recognized. Here, we investigated factors during the initial multisensory experience that are essential for generating improved memory performance. Subjects discriminated repeated versus initial image presentations intermixed within a continuous recognition task. Half of initial presentations were multisensory, and all repetitions were only visual. Experiment 1 examined whether purely episodic multisensory information suffices for enhancing later discrimination performance by pairing visual objects with either tones or vibrations. We could therefore also assess whether effects can be elicited with different sensory pairings. Experiment 2 examined semantic context by manipulating the congruence between auditory and visual object stimuli within blocks of trials. Relative to images only encountered visually, accuracy in discriminating image repetitions was significantly impaired by auditory-visual, yet unaffected by somatosensory-visual multisensory memory traces. By contrast, this accuracy was selectively enhanced for visual stimuli with semantically congruent multisensory pasts and unchanged for those with semantically incongruent multisensory pasts. The collective results reveal opposing effects of purely episodic versus semantic information from auditory-visual multisensory events. Nonetheless, both types of multisensory memory traces are accessible for processing incoming stimuli and indeed result in distinct visual object processing, leading to either impaired or enhanced performance relative to unisensory memory traces. We discuss these results as supporting a model of object-based multisensory interactions.