A Cytotoxicity Assay as an Alternative to the Murine Model for the Potency Testing of Bothrops jararaca Venom and Antivenom: An Intralaboratory Pre-validation Study.

Antivenom therapy is the only specific treatment for snakebite envenomation, and antivenom potency determination is key in the efficacy assurance quality control process. Nowadays, this process relies on the in vivo murine model - thus, the development of alternative in vitro methods is imperative. In the current study, the principle of the proposed method is the ability of Bothrops venom to induce cytotoxic effects in Vero cells, and the capacity to evaluate the inhibition of this cytotoxicity by the respective antivenom. After exposure to the venom/antivenom, the relative proportions of adherent (viable) cells were evaluated by direct staining with Coomassie Blue. The optical density (OD) of the lysed cell eluate was directly proportional to the number of adherent cells. This cytotoxicity-based alternative method could represent a potential candidate for validation as a replacement for the current in vivo test. The in vitro-determined cytotoxicity of the Brazilian Bothrops reference venom (expressed as the 50% effective concentration; EC50) was 3.61 µg/ml; the in vitro-determined 50% inhibitory concentration (IC50) of the Brazilian Bothrops reference antivenom was 0.133 µl/ml. From these two values, it was possible to calculate the potency of the reference antivenom. The results from the assays exhibited a good linear response, indicating that the method could be a potential candidate replacement method for use in antivenom quality control prior to lot release, subject to further validation.

Aphaereta pallipes (Hymenoptera: Braconidae) and Dirhinus anthracia (Hymenoptera: Chalcididae) associated with Peckia (Euboettcheria) collusor (Diptera: Sarcophagidae) in Brazil.

Flies of the Sarcophagidae family are widely distributed in the world, occupying different habitats. Some species have a high degree of synanthropy and are therefore often found in households in the urban environment. In Brazil, there is still little information related to the natural enemies of these insects in the urban environment, where population control is strictly chemical. Therefore, larvae and pupae of Peckia (Euboettcheria) collusor (Curran and Walley) (Diptera: Sarcophagidae) species were exposed in an urbanized location, and the presence and prevalence of parasitoids in the natural control of these immature stages was evaluated. We report for the first time the species Aphaereta pallipes (Say) (Hymenoptera: Braconidae) and Dirhinus anthracia Walker (Hymenoptera: Chalcididae), associated with P. (E.) collusor, highlighting the importance of these parasitoids in natural control in the urban environment, in addition to expanding the list of hosts for both parasitoid species and the distribution of this parasitoid-host interaction for Brazil and the Neotropical region.

First record of Brachymeria amenocles (Hymenoptera: Chalcididae) associated with larvae of Peckia (Peckia) chrysostoma (Diptera: Sarcophagidae) in Brazil.

In this work, we report for the first time the occurrence of the parasitoid wasp Brachymeria amenocles (Walker, 1846) (Hymenoptera: Chalcididae) associated with the larvae of the flesh fly, Peckia (Peckia) chrysostoma (Wiedemann, 1830) (Diptera: Sarcophagidae), in Brazil. This parasitoid species was collected together with the species Brachymeria podagrica (Fabricius, 1787), in the municipality of Rio de Janeiro, geographically located in the Southeast region of Brazil, in larvae of the same host species reared in the same attraction substrate, behaving as solitary parasitoids. A trap containing putrefying sardine was exposed for 48 h, in the upper part of a house, located in Vila Isabel, an urban area. The larvae collected were reared in the laboratory without control of environmental conditions. A total of three adult parasitoid wasps of the species B. podagrica, and nine adult parasitoid wasps of the species B. amenocles emerged from 12 host pupae, resulting in a prevalence of parasitoidism of 4.8% and 14.5%, respectively. Developing parasitoids at the pupal phase were observed in another 34 host pupae dissected.

Ecdysone modulates both ultrastructural arrangement of hindgut and attachment of Trypanosoma cruzi DM 28c to the rectum cuticle of Rhodnius prolixus fifth-instar nymph.

Studies on the effects of azadirachtin treatment, ecdysone supplementation and ecdysone therapy on both the ultrastructural organization of the rectum in 5th-instar nymph of Rhodnius prolixus and the ex vivo attachment behavior of Trypanosoma cruzi under these experimental conditions were carried out. Control insects had a typical and significant organization of the rectum cuticle consisted of four main layers (procuticle, inner epicuticle, outer epicuticle, and wax layer) during the entire period of the experiment. Both azadirachtin treatment and ecdysone supplementation avoid the development of both outer epicuticle and wax layer. Oral therapy with ecdysone partially reversed the altered organization and induce the development of the four main rectal cuticle layers. In the same way, the ex vivo attachment of T. cruzi to rectal cuticle was blocked by azadirachtin treatment but ecdysone therapy also partially recovered the parasite adhesion rates to almost those detected in control insects. These results point out that ecdysone may be a factor responsible - directly or indirectly - by the modulation of rectum ultrastructural arrangement providing a superficial wax layer to the attachment followed by metacyclogenesis of T. cruzi in the rectum of its invertebrate hosts.

New Law of Brazilian Biodiversity: Legal Aspects and Impact in the Field of Biotechnology.

Access to genetic resources (GR) and/or traditional knowledge associated with genetic resources (ATK) has been regulated in Brazil since 2001. The law 13,123 / 2015 determined a significant change in the theme, mainly on the rules of distribution of benefits obtained for conservation and sustainable use of biodiversity, the access to technology and technology transfer, the exploitation of products or reproductive material from the GR or ATK and consignment to the outside of part or all the living or dead organism shipped for GR. The implementation of international treaties on GR and ATK for research, biotechnological development and bioprospecting have been causing difficulties for Brazilian researchers, mainly due to the lack of information and dissemination available for compliance with the legislation. In this work, the members of the Committee for Access to Genetic Resources and Associated Traditional Knowledge of the Federal Fluminense University (UFFGEN) - Brazil, and collaborators performed a critical reflection on the new law, helping Brazilian researchers with information necessary to understand the changes made by the new legislation, especially in the field of Biotechnology associated with Brazilian Biodiversity.

The activity of IKCa and BKCa channels contributes to insulin-mediated NO synthesis and vascular tone regulation in human umbilical vein.

Insulin regulates the l-arginine/nitric oxide (NO) pathway in human umbilical vein endothelial cells (HUVECs), increasing the plasma membrane expression of the l-arginine transporter hCAT-1 and inducing vasodilation in umbilical and placental veins. Placental vascular relaxation induced by insulin is dependent of large conductance calcium-activated potassium channels (BKCa), but the role of KCa channels on l-arginine transport and NO synthesis is still unknown. The aim of this study was to determine the contribution of KCa channels in both insulin-induced l-arginine transport and NO synthesis, and its relationship with placental vascular relaxation. HUVECs, human placental vein endothelial cells (HPVECs) and placental veins were freshly isolated from umbilical cords and placenta from normal pregnancies. Cells or tissue were incubated in absence or presence of insulin and/or tetraethylammonium, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole, iberiotoxin or NG-nitro-l-arginine methyl ester. l-Arginine uptake, plasma membrane polarity, NO levels, hCAT-1 expression and placenta vascular reactivity were analyzed. The inhibition of intermediate-conductance KCa (IKCa) and BKCa increases l-arginine uptake, which was related with protein abundance of hCAT-1 in HUVECs. IKCa and BKCa activities contribute to NO-synthesis induced by insulin but are not directly involved in insulin-stimulated l-arginine uptake. Long term incubation (8 h) with insulin increases the plasma membrane hyperpolarization and hCAT-1 expression in HUVECs and HPVECs. Insulin-induced relaxation in placental vasculature was reversed by KCa inhibition. The results show that the activity of IKCa and BKCa channels are relevant for both physiological regulations of NO synthesis and vascular tone regulation in the human placenta, acting as a part of negative feedback mechanism for autoregulation of l-arginine transport in HUVECs.

Deletion of the adenosine A2A receptor increases the survival rate in a mice model of polymicrobial sepsis.

We aim to investigate the role of A2A receptor in peritonitis-related sepsis by injection of a fecal solution (FS) as a model of polymicrobial infection. C57/black J6 wild-type (WT) and A2A-deficient mice (A2AKO) were exposed to sepsis induced by intraperitoneal injection of a FS (FS-induced peritonitis) or instead was injected with saline buffer (Sham). Survival rate and sepsis score were measured up to 48 h. The presence of bacteria in tissue homogenates was analyzed. Telemetry and speckle laser Doppler were used for systemic blood pressure and peripheral blood perfusion analysis, respectively. Histological analysis and identification of active caspase 3 were performed in selected organs, including the liver. The survival rate of A2AKO mice exposed to FS-induced peritonitis was significantly higher, and the sepsis score was lower than their respective WT counterpart. Injection of FS increases (50 to 150 folds) the number of colonies forming units in the liver, kidney, blood, and lung in WT mice, while these effects were significantly attenuated in A2AKO mice exposed to FS-induced peritonitis. A significant reduction in both systolic and diastolic blood pressure, as well as in the peripheral perfusion was observed in WT and A2AKO mice exposed to FS-induced peritonitis. Although, these last effects were significantly attenuated in A2AKO mice. Histological analysis showed a large perivascular infiltration of polymorphonuclear in the liver of WT and A2AKO mice exposed to FS-induced peritonitis, but again, this effect was attenuated in A2AKO mice. Finally, high expression of active caspase 3 was found only in the liver of WT mice exposed to FS-induced peritonitis. The absence of the A2A receptor increases the survival rate in mice exposed to polymicrobial sepsis. This outcome was associated with both hemodynamic compensation and enhanced anti-bacterial response.

Improvement of endothelial function by Gunnera tinctoria extract with antioxidant properties.

BACKGROUND: Gunnera tinctoria has been collected by Mapuche-Pewenche people for food and medicinal purposes. The high polyphenol content of methanolic extract from G. tinctoria leaves with chemical constituents such as ellagic acid and quercetin derivatives suggests its application to prevent endothelial dysfunction and oxidative stress. The aim of this study was to provide evidence of the protective effect of this extract on endothelial function by reducing oxidative stress induced by high D-glucose and H2O2, as well as by stimulating nitric oxide (NO) levels in human umbilical vein endothelial cells (HUVECs). RESULTS: A methanolic extract with a high content of polyphenols (520 ± 30 mg gallic acid equivalents/g dry extract) was obtained from G. tinctoria leaves. Its main constituent was ellagic acid. The results of Ferric reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays of the extract confirmed its antioxidant activity by inhibition pathway of radical species. The incubation of HUVECs with the extract decreased the apoptosis and reactive oxygen species (ROS) synthesis induced by high extracellular concentration of D-glucose or hydrogen peroxide. The extract increased endothelial NO levels and reduced vasoconstriction in human placental vessels. CONCLUSIONS: This study provides evidence about the antioxidant and endothelial protective properties of methanolic G. tinctoria leaf extract. The extract improves the availability of NO in HUVECs, inhibiting the production of ROS and vasoconstriction.

Exploring the Insecticide and Acaricide Potential of Development Regulators obtained from Restinga vegetation from Brazil.

As a part of our continuing search for insect and arthropod development regulators from Brazilian restinga vegetation of the Rio de Janeiro State, crude extracts, purified fractions and essential oils were submitted to screening tests seeking for biological activities on the development of the insects Rhodnius prolixus, Dysdercus peruvianus, Oncopeltus fasciatus and Rhipicephalus (Boophilus) microplus. Up to now, 102 secondary metabolites have been detected in the fractions, among them monoterpenes, sesquiterpenes and two triterpenes which were obtained from the species, Eugenia sulcata, Pilocarpus spicatus, Manilkara subsericea, Myrciaria floribunda and Zanthoxylum caribaeum. These secondary plant metabolites are considered of interest for the use of studies related to arthropod endocrinology, vector-parasite interaction system, and population control of vector insect and agricultural pest. The observed biological activities were surprisingly high, involving increased mortality, molting and metamorphosis inhibition, paralysis, corporeal deformities, apparition of permanent nymphs, of adultoids and juvenoids, partial or total inhibition of oviposition and egg hatching. These compounds are now being studied further to determine if they may or may not be useful in controlling insect populations and/or interfere with the life cycle and vector transmission of parasites to animal and human populations.

Lipoproteins from vertebrate host blood plasma are involved in Trypanosoma cruzi epimastigote agglutination and participate in interaction with the vector insect, Rhodnius prolixus.

Chagas disease, infecting ca. 8 million people in Central and South America, is mediated by the protozoan parasite, Trypanosoma cruzi. The parasite is transmitted by the bite of blood sucking triatomine insects, such as Rhodnius prolixus, that had previously fed on parasite-infected vertebrate blood and voided their contaminated feces and urine into the wound. The stages of the parasite life cycle in both the insect vector and human host are well-known, but determinants of infection in the insect gut are complex and enigmatic. This paper examines the possible role of the R. prolixus gut agglutinins in the parasite life cycle. The results, derived from gut extracts made from R. prolixus fed on various diets with different vertebrate blood components, and cross adsorption experiments, showed for the first time that R. prolixus has two distinct gut agglutinins originating from their vertebrate blood meal, one for T. cruzi (the parasite agglutinin, PA) and the other for the erythrocytes (the hemagglutinin, HA). Again, uniquely, the results also demonstrate that these two agglutinins are derived, respectively, from the plasma and erythrocyte components of the vertebrate blood. Subsequent experiments, examining in more detail the nature of the plasma components forming the T. cruzi PA, used fractionated extracts of the vertebrate plasma (high density lipoprotein, HDL; low density lipoprotein, LDL, and delipidated plasma) in agglutination assays. The results confirmed the identity of the PA as a high density lipoprotein (HDL) in the plasma of the vertebrate blood meal which agglutinates parasites in the R. prolixus gut. In addition, the use of single or double labeled HDL in fluorescence and confocal microscopy showed the interaction of the labeled HDL with the parasite surface and its internalization at later times. Finally, results of T. cruzi parasitization of R. prolixus, incorporating various vertebrate blood components, resulted in highly significant increases in infectivity in the presence of HDL from the 2nd day of infection, thus confirming the important role of this molecule in T. cruzi infection of R. prolixus.

A novel PDE1A coupled to M2AChR at plasma membranes from bovine tracheal smooth muscle.

Muscarinic antagonists, via muscarinic receptors increase the cAMP/cGMP levels at bovine tracheal smooth muscle (BTSM) through the inhibition of phosphodiesterases (PDEs), displaying a similar behavior of vinpocetine (a specific-PDE1 inhibitor). The presence of PDE1 hydrolyzing both cyclic nucleotides in BTSM strips was revealed. Moreover, a vinpocetine and muscarinic antagonists inhibited PDE1 located at plasma membranes (PM) fractions from BTSM showing such inhibition, an M(2)AChR pharmacological profile. Therefore, a novel Ca(2+)/CaM dependent and vinpocetine inhibited PDE1 was purified and characterized at PM fractions from BTSM. This PDE1 activity was removed from PM fractions using a hypotonic buffer and purified some 38 fold using two columns (Q-Sepharose and CaM-agarose). This PDE1 was stimulated by CaM and inhibited by vinpocetine showing two bands in PAGE-SDS (56, 58 kDa) being the 58 kDa identified as PDE1A by Western blotts. This PDE1A activity was assayed with [(3)H]cGMP and [(3)H]cAMP exhibiting a higher affinity as Km (µM) for cGMP than cAMP but being close values with V(max) cAMP/cGMP ratio of 1.5. The co-factor Mg(2+) showed similar K(A) (mM) for both cyclic nucleotides. Vinpocetine showed similar inhibition concentration 50% (IC(50) of 4.9 and 4.6 µM) for cAMP and cGMP, respectively. CaM stimulated the cyclic nucleotides hydrolysis by PDE1A exhibiting similar activation constant as K(CaM), in nM range. The original finding was the identification and purification of a vinpocetine and muscarinic antagonist-inhibited and CaM-activated PM-bound PDE1A, linked to M(2)AChR. A model of this novel signal transducing cascade for the regulation of cyclic nucleotides levels at BTSM is proposed.

Muscarinic drugs regulate the PKG-II-dependent phosphorylation of M3 muscarinic acetylcholine receptors at plasma membranes from airway smooth muscle.

Muscarinic agonists induce the activation of the airway smooth muscle (ASM) leading to smooth muscle contraction, important in asthma. This activation is mediated through M2/M3 muscarinic acetylcholine receptors (mAChRs). Muscarinic receptor activity, expressed as [(3)H]QNB binding at plasma membranes from bovine tracheal smooth muscle (BTSM), increased with cGMP and was augmented significantly cGMP plus ATP but diminished with the PKG-II inhibitor, Sp-8-pCPT-cGMPS. The [(3)H]-QNB binding was accelerated by okadaic acid, (OKA), a protein phosphatase (PPase) inhibitor. These two results indicated the involvement of a membrane-bound PPase. Moreover, a cGMP-dependent-[(32)P]γATP phosphorylation of plasma membranes from BTSM was stimulated at low concentrations of muscarinic agonist carbamylcholine (CC). However, higher amounts of CC produced a significant decrement of [(32)P]-labeling. A selective M3mAChR antagonist, 4-DAMP produced a dramatic inhibition of the basal and CC-dependent [(32)P]-labeling. The [(32)P] labeled membrane sediments were detergent solubilized and immunoprecipitated with specific M2/M3mAChR antibodies. The M3mAChR immuno-precipitates exhibited the highest cGMP-dependent [(32)P]-labeling, indicating it is a PKG-II substrate. Experiments using synthetic peptides from the C-terminal of the third intracellular loop (i3) of both M2mAChR (356-369) and M3mAChR (480-493) as external PKG-II substrates resulted in the i3M3-peptide being heavily phosphorylated. These results indicated that PKG-II phosphorylated the M3mAChR at the i3M3 domain ((480)MSLIKEKK(485)), suggesting that Ser(481) may be the target. Finally, this phosphorylation site seems to be regulated by a membrane-bound PPase linked to muscarinic receptor. These findings are important to understand the role of M3mAChR in the patho-physiology of ASM involved in asthma and COPD.

Protective practices against zoonotic infections among rural and slum communities from South Central Chile.

BACKGROUND: Despite well-recognized recommendations to reduce human exposure to zoonotic pathogens, the use of personal and herd-level protective practices is inconsistent in communities where human interactions with animals are common. This study assessed household-level participation in rodent- (extermination, proper food storage, trash disposal), occupational- (preventive veterinary care, boot-wearing, glove-wearing), and garden-associated (restricting animal access, boot-wearing, glove-wearing) protective practices in farms, villages, and slums in the Los Rios region, Chile, where zoonotic pathogens are endemic. METHODS: Questionnaires administered at 422 households across 12 communities recorded household-level socio-demographic characteristics and participation in nine protective practices. Household inclusion in the analysis of occupational practices required having livestock and a household member with occupational exposure to livestock (n = 127), and inclusion in analysis of garden practices required having a garden and at least one animal (n = 233). The proportion of households participating in each protective practice was compared across community types through chi-square analyses. Mixed effects logistic regression assessed household-level associations between socio-demographic characteristics and participation in each protective practice. RESULTS: Most households (95.3 %) reported participation in rodent control, and a positive association between the number of rodent signs in a household and rodent extermination was observed (OR: 1.75, 95 % CI: 1.41, 2.16). Occupational protective practices were reported in 61.8 % of eligible households; household size (OR: 1.63, 95 % CI: 1.17, 5.84) and having children (OR: 0.22, 95 % CI: 0.06, 0.78) were associated with preventive veterinary care. Among eligible households, 73.8 % engaged in protective practices when gardening, and species diversity was positively associated with wearing boots (OR: 1.27, 95 % CI: 1.03, 1.56). Household-level participation in all three protective practices within any exposure category was limited (<10.4 %) and participation in any individual protective practice varied considerably within and across community types. CONCLUSIONS: The levels of participation in protective practices reported in this study are consistent with descriptions in the literature of imperfect use of methods that reduce human exposure to zoonotic pathogens. The wide differences across communities in the proportion of households participating in protective practices against human exposure to zoonotic pathogens, suggests that future research should identify community-level characteristics that influence household participation in such practices.

Biocontrol evaluation of extracts and a major component, clusianone, from Clusia fluminensis Planch. & Triana against Aedes aegypti.

Studies evaluated the effects of hexanic extracts from the fruits and flowers of Clusia fluminensis and the main component of the flower extract, a purified benzophenone (clusianone), against Aedes aegypti. The treatment of larvae with the crude fruit or flower extracts from C. fluminensis did not affect the survival ofAe. aegypti (50 mg/L), however, the flower extracts significantly delayed development of Ae. aegypti. In contrast, the clusianone (50 mg/L) isolate from the flower extract, representing 54.85% of this sample composition, showed a highly significant inhibition of survival, killing 93.3% of the larvae and completely blocking development of Ae. aegypti. The results showed, for the first time, high activity of clusianone against Ae. aegypti that both killed and inhibited mosquito development. Therefore, clusianone has potential for development as a biopesticide for controlling insect vectors of tropical diseases. Future work will elucidate the mode of action of clusianone isolated from C. fluminensis.

Effects of azadirachtin on the biology of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) adult female, the main vector of American visceral leishmaniasis.

The effects of azadirachtin A added to the sucrose diet of the adult females on the mortality, oviposition, and hatching of the sand fly vector of American visceral leishmaniasis Lutzomyia longipalpis (Lutz & Neiva, 1912) were investigated. Concentrations of 0.1, 1.0, and 10.0 microg/mg of azadirachtin significantly increased insect mortality in comparison with control insects. The same dose also significantly reduced oviposition but not hatching. After a long development period, significantly fewer adult insects were obtained from eggs hatching by azadirachtin-treated females in a dose-response manner. These results indicate that azadirachtin is a potent sterilizer that could be used against the development of Lu. longipalpis populations and as a tool for studying physiological and biochemical processes in phlebotomine species.

Development of an insecticidal nanoemulsion with Manilkara subsericea (Sapotaceae) extract.

BACKGROUND: Plants have been recognized as a good source of insecticidal agents, since they are able to produce their own defensives to insect attack. Moreover, there is a growing concern worldwide to develop pesticides with low impact to environment and non-target organisms. Hexane-soluble fraction from ethanolic crude extract from fruits of Manilkara subsericea and its triterpenes were considered active against a cotton pest (Dysdercus peruvianus). Several natural products with insecticidal activity have poor water solubility, including triterpenes, and nanotechnology has emerged as a good alternative to solve this main problem. On this context, the aim of the present study was to develop an insecticidal nanoemulsion containing apolar fraction from fruits of Manilkara subsericea. RESULTS: It was obtained a formulation constituted by 5% of oil (octyldodecyl myristate), 5% of surfactants (sorbitan monooleate/polysorbate 80), 5% of apolar fraction from M. subsericea and 85% of water. Analysis of mean droplet diameter (155.2 ± 3.8 nm) confirmed this formulation as a nanoemulsion. It was able to induce mortality in D. peruvianus. It was observed no effect against acetylcholinesterase or mortality in mice induced by the formulation, suggesting the safety of this nanoemulsion for non-target organisms. CONCLUSIONS: The present study suggests that the obtained O/A nanoemulsion may be useful to enhance water solubility of poor water soluble natural products with insecticidal activity, including the hexane-soluble fraction from ethanolic crude extract from fruits of Manilkara subsericea.

Cyclic GMP regulates M3AChR activity at plasma membranes from airway smooth muscle.

Muscarinic acetylcholine receptors MAChRs from Bovine Tracheal Smooth Muscle (BTSM) plasma membranes are responsible for the cGMP rise and signal-amplitude peaks associated with smooth muscle contraction present in bronchial asthma. These MAChRs bind [(3)H]QNB and exhibit the classic G Protein Coupled-Receptor (GPCR) behavior towards muscarinic agonist and antagonists that is sensitive to sensitive to GTP analogs. Interestingly, the [(3)H]QNB binding activity was stimulated by cGMP and ATP, and was enhanced by IBMX and Zaprinast, inhibitors of cGMP-PDE. Cyclic GMP plus ATP affected the agonist-antagonist muscarinic binding activities. Thus, the high affinity agonist (Carbamylcholine) binding sites disappeared, whereas, 4-DAMP, a M3 selective antagonist displayed an additional high affinity-binding site. In contrast, non-selective (atropine) and M2-selective (methoctramine and gallamine) antagonists revealed one low binding site. Moreover, the 4-DAMP-mustard alkylation of the MAChRs blocked the cGMP effect indicating that the M3AChR is the main receptor target of cGMP. Interestingly, these cGMP effects were potentiated by an activator (Sp-8-pCPT-cGMPS), and diminished by an inhibitor (Rp-8-pCPT-CGMPS), of cGMP-dependent protein kinase (PKG-II), which was detected by Western blotting using specific PKG II antibodies. Finally, plasma membrane M3AChRs were phosphorylated in a cGMP-dependent manner and this novel post-translational reversible modification at M3AChRs may act as a feedback mechanism to terminate the cGMP dependent muscarinic signal transduction cascades at the sarcolema of BTSM.

Characterisation of a cryptic plasmid from an Antarctic bacterium Pedobacter cryoconitis strain BG5.

A cryptic plasmid, pMWHK1 recovered from an Antarctic bacterium Pedobacter cryoconitis BG5 was sequenced and characterised. The plasmid is a circular 6206bp molecule with eight putative open reading frames designated as orf1, orf2, orf3, orf4, orf5, orf6, orf7 and orf8. All the putative open reading frames of pMWHK1 are found to be actively transcribed. Proteins encoded by orf2 and orf4 are predicted to be responsible for the mobilization and replication of the plasmid respectively. orf4 shares 55% and 61% identities with the theta-type Rep proteins from two strains of Riemerella anatipestifer. This suggests that pMWHK1 could be a member of the theta-type replicating plasmid. The origin of replication is located within the AT-rich region upstream of orf4. orf5 and orf6 encode bacterial toxin-antitoxin proteins predicted to maintain plasmid stability. orf3 encodes an entry exclusion protein that is hypothetically involved in reducing the frequency of DNA transfer through conjugation. orf1, orf7 and orf8 encode proteins with unknown functions. Plasmid, pMWHK1 is stably maintained in P. cryoconitis BG5 at 20°C.

Impaired A2A adenosine receptor/nitric oxide/VEGF signaling pathway in fetal endothelium during late- and early-onset preeclampsia.

To investigate whether fetal endothelial cell proliferation and migration are modulated by the A2A adenosine receptor (A2AAR), nitric oxide (NO) and the vascular endothelial growth factor (VEGF) signaling pathway, we isolated human umbilical vein endothelial cells from normal pregnancy (n = 23), preterm delivery (n = 4), and late-onset (LOPE, n = 10) and early-onset preeclampsia (EOPE, n = 8). We used the non-selective adenosine receptor agonist (NECA) and the selective agonist (CGS-21680) and/or selective antagonist (ZM-241385) for A2AAR. Also, the nitric oxide synthase (NOS) inhibitor, L-NAME, was used in co-incubation with CGS-21680. Compared to normal pregnancy, EOPE exhibited low cell proliferation and migration associated with reduced expressions of A2AAR and VEGF and NO synthesis (i.e., total and phosphorylated serine(1177) endothelial NOS and nitrite formation). In contrast, LOPE exhibited the opposite behavior in all these markers compared to normal pregnancy or EOPE. Cell proliferation and migration were increased by CGS-21680 (or NECA) in all analyzed groups (EOPE>LOPE>normal pregnancy) compared to their respective basal conditions, an effect that was associated with high NO and VEGF synthesis and blocked by ZM-241385 with significantly different IC50 for each group (EOPE>LOPE>normal pregnancy). The differences seem independent of gestational age. L-NAME blocked the CGS-21680-mediated cell proliferation and migration in normal pregnancy and LOPE (IC50 = 36.2 ± 2.5 and 8.6 ± 2.2 nM, respectively) as well as the VEGF expression in normal pregnancy. Therefore, the A2AAR/NO/VEGF signaling pathway exhibits a pro-angiogenic effect in normal pregnancies and LOPE, whereas impairment in this pathway seems related to the reduced angiogenic capacity of the fetal endothelium in EOPE.