RESUMO
The aim of this study was to evaluate the effect of the non-selective phosphodiesterase inhibitor 3- isobutyl-1-methyl-xanthine (IBMX) given concomitantly with eCG on reproductive performance of ewes. In trial 1, eighty ewes were allocated into 8 groups (AC, A1, A2, A3 and DC, D1, D2, D3). Oestruses were synchronized by progestagen sponges. At sponges removal all ewes of A groups received 400 IU of eCG. Concomitantly with eCG, ewes of groups A1, A2 or A3 received IMBX at the dose of 2.5, 5.0 or 25.0 mg, respectively. Ewes of D1, D2 or D3 groups received only IBMX at the same doses, respectively; in each case, ewes of AC or DC groups were controls (C). In trial 2, one hundred eighteen ewes were allocated into 4 groups (BC1, B1 and BC2, B2). Oestruses were synchronized by progestagen sponges. At sponges removal ewes of BC1 or B1 received 200 IU of eCG, while ewes of BC2 or B2 groups received 100 IU. At the same time only ewes of B1 or B2 groups received 2.5 mg IBMX. In both trials, 48 hours after hormonal treatment, ewes were mated. In trial 1, pregnancy or lambing rate did not differ among A or D groups (P > 0.05). Total lambs per ewe were significantly higher in A1 compared to AC, A2, A3 or D1 groups (P < 0.05), but did not differ among D groups (P > 0.05). In trial 2, pregnancy or lambing rate did not differ among B groups (P > 0.05). Total lambs per ewe were significantly higher (P < 0.05) in B1 and B2 compared to BC1 and BC2 groups, respectively. These results indicate that IBMX combined with eCG at the end of an oestrus synchronization treatment improves litter size probably by increasing ovulation rate in ewes. This latter effect seems to be mainly dependent on the amount of gonadotrophins used.(AU)
Assuntos
Animais , Eletrocardiografia , Diester Fosfórico Hidrolases/análise , Diester Fosfórico Hidrolases/isolamento & purificação , Ovinos/embriologia , Comportamento Reprodutivo , PrenhezRESUMO
The aim of this study was to evaluate the effect of the non-selective phosphodiesterase inhibitor 3- isobutyl-1-methyl-xanthine (IBMX) given concomitantly with eCG on reproductive performance of ewes. In trial 1, eighty ewes were allocated into 8 groups (AC, A1, A2, A3 and DC, D1, D2, D3). Oestruses were synchronized by progestagen sponges. At sponges removal all ewes of A groups received 400 IU of eCG. Concomitantly with eCG, ewes of groups A1, A2 or A3 received IMBX at the dose of 2.5, 5.0 or 25.0 mg, respectively. Ewes of D1, D2 or D3 groups received only IBMX at the same doses, respectively; in each case, ewes of AC or DC groups were controls (C). In trial 2, one hundred eighteen ewes were allocated into 4 groups (BC1, B1 and BC2, B2). Oestruses were synchronized by progestagen sponges. At sponges removal ewes of BC1 or B1 received 200 IU of eCG, while ewes of BC2 or B2 groups received 100 IU. At the same time only ewes of B1 or B2 groups received 2.5 mg IBMX. In both trials, 48 hours after hormonal treatment, ewes were mated. In trial 1, pregnancy or lambing rate did not differ among A or D groups (P > 0.05). Total lambs per ewe were significantly higher in A1 compared to AC, A2, A3 or D1 groups (P 0.05). In trial 2, pregnancy or lambing rate did not differ among B groups (P > 0.05). Total lambs per ewe were significantly higher (P < 0.05) in B1 and B2 compared to BC1 and BC2 groups, respectively. These results indicate that IBMX combined with eCG at the end of an oestrus synchronization treatment improves litter size probably by increasing ovulation rate in ewes. This latter effect seems to be mainly dependent on the amount of gonadotrophins used.
Assuntos
Animais , Diester Fosfórico Hidrolases/análise , Diester Fosfórico Hidrolases/isolamento & purificação , Eletrocardiografia , Ovinos/embriologia , Comportamento Reprodutivo , PrenhezRESUMO
Bites by spiders from Loxosceles genus often lead to a wide variance in envenomation profile of patients and diagnosis is difficult due to the number of diseases that mimic loxoscelism. In such a context, it is of interest to consider the design of standardized recombinant colorimetric antibodies for diagnosis and specific detection of individual circulating toxins in biological fluids of envenomed patients. We have previously prepared a monoclonal murine IgG (LiMab7) that reacts with Loxosceles intermedia venom components of 32-35kDa and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a colorimetric bifunctional protein consisting in the corresponding single-chain antibody fragment (scFv) fused to alkaline phosphatase (AP) of Escherichia coli. The immune tracer was tested in two different types of immunoassays and it proved to be efficient in both. Thus, this recombinant fusion protein (scFv-LiMab7/AP) can be used for rapid and specific immunotitration of L. intermedia venom with a linear range of 39-20000ng/mL and a detection limit of 39ng/mL without any cross-reaction.
Assuntos
Aranha Marrom Reclusa/fisiologia , Imunoensaio/métodos , Neurotoxinas/análise , Diester Fosfórico Hidrolases/análise , Pele/metabolismo , Picada de Aranha/diagnóstico , Venenos de Aranha/análise , Fosfatase Alcalina/genética , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Diagnóstico Diferencial , Humanos , Camundongos , Neurotoxinas/imunologia , Diester Fosfórico Hidrolases/imunologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Padrões de Referência , Sensibilidade e Especificidade , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Pele/patologia , Picada de Aranha/imunologia , Venenos de Aranha/imunologiaRESUMO
In this work, an anti-loxoscelic serum was produced by immunizing horses with a recombinant dermonecrotic protein from Loxosceles intermedia (rLiD1). Anti-rLiD1 antibodies were able to recognize different species of Loxosceles venoms by Western Blot and ELISA. The efficacy of anti-rLiD1 serum against the toxic effects of Loxosceles laeta (Peru) venom was tested, showing that anti-rLiD1 serum can neutralize those effects. This study confirms that recombinant proteins can be good candidates to replace crude venoms for antivenom production.
Assuntos
Antivenenos/imunologia , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Proteínas Recombinantes/metabolismo , Venenos de Aranha/química , Animais , Antivenenos/farmacologia , Western Blotting , Brasil , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Cavalos , Testes de Neutralização , Peru , Diester Fosfórico Hidrolases/análise , Especificidade da Espécie , Venenos de Aranha/enzimologiaRESUMO
Bioactive lipid molecules as lysophosphatidic acid (LPA), prostaglandins (PG) and endocannabinoids are important mediators of embryo implantation. Based on previous published data we became interested in studying the interaction between these three groups of lipid derivatives in the rat uterus during the window of implantation. Thus, we adopted a pharmacological approach in vitro using LPA, DGPP (a selective antagonist of LPA3, an LPA receptor), endocannabinoids' receptor selective antagonists (AM251 and AM630) and non selective (indomethacin) and selective (NS-398) inhibitors of cyclooxygenase-1 and 2 enzymes. Cyclooxygenase isoforms participate in prostaglandins' synthesis. The incubation of the uterus from rats pregnant on day 5 of gestation (implantation window) with LPA augmented the activity and the expression of fatty acid amide hydrolase, the main enzyme involved in the degradation of endocannabinoids in the rodent uteri, suggesting that LPA decreased endocannabinoids' levels during embryo implantation. It has been reported that high endocannabinoids are deleterious for implantation. Also, LPA increased PGE2 production and cyclooxygenase-2 expression. The incubation of LPA with indomethacin or NS-398 reversed the increment in PGE2 production, suggesting that cyclooxygenase-2 was the isoform involved in LPA effect. PGs are important mediators of decidualization and vascularization at the implantation sites. All these effects were mediated by LPA3, as the incubation with DGPP completely reversed LPA stimulatory actions. Besides, we also observed that endocannabinoids mediated the stimulatory effect of LPA on cyclooxygenase-2 derived PGE2 production, as the incubation of LPA with AM251 or AM630 completely reversed LPA effect. Also, LPA augmented via LPA3 decidualization and vascularization markers. Overall, the results presented here demonstrate the participation of LPA3 in the process of implantation through the interaction with other groups of lipid molecules, prostaglandins and endocannabinoids, which prepare the uterine milieu for embryo invasion during the window of implantation.
Assuntos
Implantação do Embrião , Endocanabinoides/metabolismo , Lisofosfolipídeos/metabolismo , Prostaglandinas/metabolismo , Amidoidrolases/metabolismo , Animais , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Feminino , Diester Fosfórico Hidrolases/análise , Diester Fosfórico Hidrolases/metabolismo , Gravidez , Ratos , Ratos Wistar , Receptores de Ácidos Lisofosfatídicos/análise , Receptores de Ácidos Lisofosfatídicos/metabolismo , Útero/irrigação sanguínea , Útero/metabolismoRESUMO
Phosphorus (P)-limited plants produce higher amounts of root phosphatases, but research has mostly focused on phosphomonoesterases (PMEs). Because phosphate diesters can form a significant proportion of organic P in wetlands, we aimed to determine whether wetland plants produce both root PMEs and root phosphodiesterases (PDEs), and, if so, what factors influence activities of these enzymes. We measured the activities of root PMEs and PDEs colorimetrically in a wide range of macrophytes from natural and P-enriched wetlands. Hydrolyzable P in sediments was analyzed using commercially available PMEs and PDEs. In all species, both root PMEs and PDEs were always present, and their activities were closely correlated. Sedges and broadleaved emergents had the highest activity of both enzymes, while those of floating-leaved plants were the lowest. Redundancy analysis revealed close association between root enzymes and the proportion of monoesterase- and diesterase-hydrolyzable dissolved unreactive P. Both enzymes were positively correlated with root tissue N : P ratio. Both plant and sediment traits were important when explaining differences in enzyme activities. Although the activities are related to ambient P regime, the relationship was not close enough to use root enzymes as reliable predictors of dissolved unreactive P that is hydrolyzed by sediment phosphomono- and diesterases.
Assuntos
Diester Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fósforo/metabolismo , Raízes de Plantas/enzimologia , Brotos de Planta/enzimologia , Belize , Nitrogênio/análise , Nitrogênio/metabolismo , Diester Fosfórico Hidrolases/análise , Monoéster Fosfórico Hidrolases/análise , Fósforo/análise , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Plantas/enzimologia , Plantas/metabolismo , Solo/química , Microbiologia do Solo , Áreas AlagadasRESUMO
The venom of Loxosceles spiders produces severe dermonecrotic damage, intravascular hemolysis, systemic alterations and risk of death. Clostridium perfringens is present in the microbial flora of the fangs and venom glands of Loxosceles intermedia. Its inoculation with the venom may infect the wound site and exacerbate the dermonecrotic damage. This anaerobic bacterium is widely distributed in nature and capable of damage with similar characteristics and severity to the spider venom. In this study we isolated and characterized species of Clostridium from the fangs and venom glands of Loxosceles laeta, including C. perfringens. The sensitivity patterns of different isolates of C. perfringens were evaluated by minimum inhibitory concentration against penicillin, ampicillin, erythromycin, gentamicin, chloramphenicol, clindamycin and tetracycline, under anaerobic conditions, using the method of microdilution in broth. Strain C. perfringens H28 showed resistance to penicillin, ampicillin, tetracycline and chloramphenicol. Resistance to penicillin and ampicillin was mediated by beta-lactamase. In vivo evaluation of dermonecrosis in rabbits using L. laeta venom co-inoculated with isolate C. perfringens H28 produced an increase in the area of dermonecrotic lesions in the presence of penicillin and tetracycline, but not with gentamicin. Antibiotic therapy Loxosceles poisoning should be re-evaluated, considering the existence of multi-resistant strains of C. perfringens.
Assuntos
Antibacterianos/farmacologia , Clostridium perfringens/isolamento & purificação , Glândulas Exócrinas/microbiologia , Diester Fosfórico Hidrolases/efeitos adversos , Picada de Aranha/microbiologia , Venenos de Aranha/efeitos adversos , Aranhas/microbiologia , Dente/microbiologia , Animais , Antivenenos/administração & dosagem , Clostridium perfringens/efeitos dos fármacos , Clostridium perfringens/patogenicidade , Expressão Gênica , Injeções Intradérmicas , Masculino , Necrose/induzido quimicamente , Resistência às Penicilinas/efeitos dos fármacos , Resistência às Penicilinas/genética , Penicilinas/farmacologia , Diester Fosfórico Hidrolases/administração & dosagem , Diester Fosfórico Hidrolases/análise , Coelhos , Pele/efeitos dos fármacos , Picada de Aranha/tratamento farmacológico , Venenos de Aranha/administração & dosagem , Venenos de Aranha/análise , Tetraciclina/farmacologia , Resistência a Tetraciclina/efeitos dos fármacos , Resistência a Tetraciclina/genética , beta-Lactamases/metabolismoRESUMO
Loxosceles intermedia spider venom was subjected to proteomic analysis through a MudPIT shot-gun approach to identify the protein composition. Were identified 39 proteins which seem to responsible by the lesion of different types of tissues, to some physiopathological actions and by the prevention of structural damage to the toxin structures.
Assuntos
Diester Fosfórico Hidrolases/análise , Proteínas/análise , Venenos de Aranha/análise , Aranhas , Animais , Humanos , Espectrometria de Massas , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/toxicidade , Proteínas/metabolismo , Proteínas/toxicidade , Proteoma/análise , Mordeduras de Serpentes/fisiopatologia , Venenos de Aranha/metabolismo , Venenos de Aranha/toxicidadeRESUMO
Xerostomia is commonly caused by antidepressant drugs and ATP can influence the saliva production. Adenosine is the product of extracellular hydrolysis of adenine nucleotides in submandibular gland cells, which occurs by the action of ectonucleotidases. In this study, we have evaluated the effect of three different antidepressants in ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP1-3) activities in cultured cells from salivary glands. Rats received imipramine (10mg/ml), fluoxetine (20mg/ml) or moclobemide (30mg/ml) by oral gavage. The drugs were administered once a day for 14 days. Our results have shown that the hydrolysis of p-nitrophenyl-5'-thymidine monophosphate increased in all treatments. These effects were not consequence of transcriptional control of E-NPP1-3 genes. The results reported here can highlight the importance of ectonucleotidases in the most common side effect caused by antidepressant therapy.
Assuntos
Antidepressivos/farmacologia , Diester Fosfórico Hidrolases/efeitos dos fármacos , Pirofosfatases/efeitos dos fármacos , Glândula Submandibular/enzimologia , Animais , Antidepressivos de Segunda Geração/farmacologia , Antidepressivos Tricíclicos/farmacologia , Células Cultivadas , Fluoxetina/farmacologia , Hidrólise , Imipramina/farmacologia , Masculino , Moclobemida/farmacologia , Diester Fosfórico Hidrolases/análise , Fosforilação , Pirofosfatases/análise , Ratos , Ratos Wistar , Proteínas e Peptídeos Salivares/análise , Proteínas e Peptídeos Salivares/efeitos dos fármacos , Glândula Submandibular/citologia , Glândula Submandibular/efeitos dos fármacos , Timidina Monofosfato/análogos & derivados , Timidina Monofosfato/análise , Fatores de TempoRESUMO
In the present study the recombinant form (recLiD1) of a dermonecrotic protein present in the Brazilian brown spider Loxosceles intermedia venom was expressed in Escherichia coli cells and purified by reversed-phase HPLC using a C8 Vydac column. About 25.8mg of purified recLiD1 was produced from a litre of bacterial culture. SDS/PAGE and immunoblot analysis of the recombinant protein revealed an apparent molecular weight of 32-35kDa. The later result was confirmed by mass spectrometry (32,758Da). recLiD1 displayed dermonecrotic and platelet aggregation activities which were qualitatively similar to that displayed by the crude L. intermedia venom. However, very low sphingomyelinase D enzymatic activity and complement-dependent haemolytic activities were observed. recLiD1 immunized BALB/c mice developed an antibody response. Anti-recLiD1 antibodies recognized L. intermedia venom in an indirect enzyme-linked immunosorbent assay (ELISA) and cross-reacted with crude venoms from L. intermedia, L. gaucho and L. laeta. An in vivo protection assay carried out 5 weeks after the end of the immunization protocol showed that 75% of the vaccinated mice could resist the challenge by 2.5LD(50) of L. intermedia venom. To characterize epitopes associated with protective antibodies, we prepare sets of immobilized synthetic 15 mer overlapping peptides covering the complete amino acid sequences of the recLiD1. Antibodies revealed one antigenic region in the N-terminal part of the toxin. The amino acid sequence of this epitope was found in several dermonecrotic proteins and some of its residues have been implicated with the active site of the toxin.
Assuntos
Diester Fosfórico Hidrolases/toxicidade , Proteínas Recombinantes/toxicidade , Serina Endopeptidases/toxicidade , Pele/efeitos dos fármacos , Venenos de Aranha/toxicidade , Animais , Epitopos/química , Hemólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Espectrometria de Massas , Camundongos , Peso Molecular , Necrose/induzido quimicamente , Necrose/patologia , Diester Fosfórico Hidrolases/análise , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/imunologia , Agregação Plaquetária/efeitos dos fármacos , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Serina Endopeptidases/química , Serina Endopeptidases/imunologia , Pele/patologia , Venenos de Aranha/química , Venenos de Aranha/imunologiaRESUMO
By studying Loxosceles intermedia (Brown spider) venom we were able to detect a proteolytic action on fibronectin and fibrinogen but an inability to degrade full length laminin, type I and type IV collagens. By studying enzyme inhibitors we observed that divalent metal chelators as EDTA and 1,10-phenanthroline completely blocked this cleaving action whereas serine-protease inhibitors, thiol-protease inhibitor and acid-protease inhibitor showed little or no effect on the proteolytic activity of the venom indicating involvement of a metalloproteinase. Zymogram analysis of venom detected a 35 kDa molecule with gelatinolytic activity. The metalloproteinase nature was further supported by its sensitivity to 4-aminophenyl mercuric acetate (APMA) treatment which decreased its molecular weight to 32 kDa, inhibition of its gelatinolytic effect by 1,10-phenanthroline and its elution from gelatin-sepharose affinity beads. In addition, zymogram experiments using fibronectin and fibrinogen as substrates detected a fibronectinolytic and fibrinogenolytic band at 28 kDa which changed its electrophoretic mobility to 20 kDa band after organomercurial treatment. The inhibitory effect of 1,10 phenanthroline and APMA sensitivity on this proteolytic effect confirmed the presence of a second metalloproteinase in the venom. The data presented herein describe two invertebrate metalloproteinases in L. intermedia venom with different specificities one gelatinolytic and another, fibronectinolytic and fibrinogenolytic, probably involved in the harmful effects of the venom.
Assuntos
Metaloendopeptidases/isolamento & purificação , Venenos de Aranha/análise , Animais , Fibrinogênio/metabolismo , Fibronectinas/metabolismo , Gelatina/metabolismo , Metaloendopeptidases/metabolismo , Diester Fosfórico Hidrolases/análise , Venenos de Aranha/enzimologiaRESUMO
1. "In vitro" incubation of red blood cells with phosmetoxon induced crenated and invaginated forms. 2. [32P] phosphate incorporation was greater in membranes from erythrocytes exposed to 300 nM phosmetoxon for 10 min than in control cells. 3. The highest incorporation was for phosphatidylinositol (PI), followed by phosphatidylinositol phosphate (PIP) and phosphatidylinositolbiphosphate (PIP2). 4. An activation of phosphatidylinositol (PI) kinase was detected with 150 and 300 nM of the pesticide, while there was no change in poliphosphoinositides (PPI) phosphodiesterase activity. 5. Results suggest an association between changes in PI kinase activity, the phosphorylation cycle of phosphatidylinositols and alterations in erythrocyte morphology induced by phosmetoxon.
Assuntos
Eritrócitos/efeitos dos fármacos , Fosmet/farmacologia , Fosfatidilinositóis/metabolismo , 1-Fosfatidilinositol 4-Quinase , Tamanho Celular/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/metabolismo , Humanos , Técnicas In Vitro , Lipídeos/análise , Microscopia Eletrônica de Varredura , Fosmet/metabolismo , Fosfoinositídeo Fosfolipase C , Diester Fosfórico Hidrolases/análise , Fosforilação/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/análiseRESUMO
Seven species of rattlesnakes (genus Crotalus) from Baja, Mexico, were investigated for venom yield, protein content, lethal toxicity, 'Mojave toxin' content and hemorrhagic, esterase (BAEE), phosphodiesterase and protease activities. Venom yield was lowest in C. catalinensis and C. enyo enyo and highest in C. ruber ruber. All venoms exhibited esterase and phosphodiesterase activities, except that three (of 14) specimens of C. e. enyo lacked esterase activity. Protease activity was extremely low or absent in adult C. e. enyo, adult C. mitchellii mitchellii, adult C. viridis caliginis and juvenile C. r. ruber venoms. The venom of C. m. mitchellii was the only venom lacking hemorrhagic activity. This venom also had the highest lethal toxicity (i.p. LD50 0.13 - 0.24 mg/kg) in mice, and was the only venom that exhibited a toxin antigenically related to 'Mojave toxin'.