RESUMO
The study designed to evaluated the activity of pancreatic exocrine enzymes in diabetic male rats induced by alloxan. The hyperglycaemia was induced in forty-five rats after fasting of the animals for 24 hours by single intraperitoneal (i.p) injection of Alloxan100mg/kg B.W., three days after injection fasting blood glucose was measured when the concentration higher than 150mg/dL, were considered as hyperglycemic/ diabetes. A total of sixty adult male rats (45 diabetes and 15 non- diabetes) divided in to two groups as follows. The first group serves as control groups (15 animals) will be single i.p injection with distilled water. the second group diabetic groups (45 animals from first experiment) were subdivided into three subgroups as following (15 for each). Group (G1), Group (G2) and Group (G3) serves as 20, 40- and 60-days diabetic animals respectively. The blood samples collection were take through cardiac puncture technique from each rat for each period days for measurement the following parameters: (Serum glucose, total protein, insulin, cholesterol, albumin, triglyceride, LDL-C, HDL-C and VLDL-C) concentration, the rats pancreatic tissue were be taken for measured tissue pancreatic lipase, amylase, and trypsin concentration. The results demonstrate a significant increase in serum glucose concentration and a decrease in serum insulin and total protein in the diabetic group as compared with the control rats' group in all experimental days. The results showed a significant rise in serum total cholesterol concentration within the diabetic group when compared with the control group at day 20 and 60. Meanwhile, a significant increase in serum triglyceride and LDL concentration and a significant decrease in serum HDL concentration within the diabetic group when compared with the control rats group at day 20, 40 and 60. But the serum VLDL concentration depicted a significant increase in the group of diabetic when compared with the control rats group at day 40 and 60. The value of pancreatic tissue protease activity clarified there was a significant decrease of protease action in the group of diabetic rats when compared with the control rats group on both day 20 and day 60. And a significant decrease in amylase activity in the diabetic groups when compared with the group of control rats in both day 20, 40 and day 60. While the results of pancreatic tissue lipase show there were non-significant changes within the diabetics group when compared with the control group. In conclusion, the exocrine pancreatic function is very frequently and severely altered in diabetes mellitus male rats and the metabolic disorder effect of diabetes mellitus was manifested by hyperlipidemic and hypoprotenimic .
Assuntos
Ratos , Ratos , Coleta de Amostras Sanguíneas/veterinária , Diabetes Mellitus Experimental/complicações , Glândulas Exócrinas/enzimologiaRESUMO
Snake venom metalloproteinases (SVMPs) are zinc-dependent enzymes responsible for most symptoms of human envenoming. Like matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAM) proteins, SVMPs are synthesized as zymogens, and enzyme activation is regulated by hydrolysis of their prodomain, but the processing of SVMPs is still unclear. In this study, we attempted to identify the presence of prodomain in different compartments of snake venom glands as zymogens or in the free form to elucidate some mechanism involved in SVMP activation. Using antibodies obtained by immunization with a recombinant prodomain, bands of zymogen molecular mass and prodomain peptides were detected mostly in gland extracts all along the venom production cycle and in the venom collected from the lumen at the peak of venom production. Prodomain was detected in secretory cells mostly in the secretory vesicles near the Golgi. We hypothesize that the processing of SVMPs starts within secretory vesicles and continues in the lumen of the venom gland just after enzyme secretion and involves different steps compared to ADAMs and MMPs but can be used as a model for studying the relevance of peptides resulting from prodomain processing and degradation for controlling the activity of metalloproteinases.
Assuntos
Venenos de Crotalídeos/enzimologia , Metaloproteases/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Répteis/metabolismo , Sequência de Aminoácidos , Animais , Bothrops/anatomia & histologia , Bothrops/metabolismo , Ativação Enzimática , Glândulas Exócrinas/citologia , Glândulas Exócrinas/enzimologia , Feminino , Metaloproteases/química , Dados de Sequência Molecular , Precursores de Proteínas/química , Transporte Proteico , Proteínas de Répteis/química , Homologia de Sequência de AminoácidosRESUMO
In Latin America, Lutzomyia longipalpis is the main vector of the protozoan parasite Leishmania infantum, which is the causal agent of American Visceral Leishmaniasis. This insect uses male-produced pheromones for mate recognition. Elucidation of pheromone biogenesis or its regulation may enable molecular strategies for mating disruption and, consequently, the vector's population management. Motivated by our recent results of the transcriptomic characterization of the L. longipalpis pheromone gland, we performed a proteomic analysis of this tissue combining SDS-PAGE, and mass spectrometry followed by an integrative data analysis. Considering that annotated genome sequences of this sand fly are not available, we designed an alternative workflow searching MS/MS data against two customized databases using three search engines: Mascot, OMSSA and ProLuCID. A total of 542 proteins were confidently characterized, 445 of them using a Uniref100-insect protein database, and 97 using a transcript translated database. In addition, use of PEAKS for de novo peptide sequencing of MS/MS data confirmed ~90% identifications made with the combination of the three search engines. Our results include the identification of six of the seven enzymes of the mevalonate-pathway, plus the enzymes involved in sesquiterpenoid biosynthesis, all of which are proposed to be involved in pheromone production in L. longipalpis. BIOLOGICAL SIGNIFICANCE: L. longipalpis is the main vector of the protozoan parasite L. infantum, which is the causal agent of American Visceral Leishmaniasis. One of the control measures of such disease is focused on vector population control. As this insect uses male-produced pheromones for mate recognition, the elucidation of pheromone biogenesis or its regulating process may enable molecular strategies for mating disruption and, consequently, this vector's population management. On this regard, in this manuscript we report expression evidence, at the protein level, of several molecules potentially involved in the pheromone production of L. longipalpis. Our results include the identification of the mevalonate-pathway enzymes, plus the enzymes involved in sesquiterpenoid biosynthesis, all of which are proposed to be involved in pheromone production in L. longipalpis. In addition, considering that the annotated genome sequences of this sand fly are not yet available, we designed an alternative workflow searching MS/MS data against proteomic and transcript translated customized databases, using three search engines: Mascot, OMSSA, and ProLuCID. In addition, a de novo peptide sequencing software (PEAKS) was used to further analyze the MS/MS data. This approach made it possible to identify and annotate 542 proteins for the pheromone gland of L. longipalpis. Importantly, all annotated protein sequences and raw data are available for the research community in protein repositories that provide free access to the data.
Assuntos
Dípteros/enzimologia , Glândulas Exócrinas/enzimologia , Proteínas de Insetos/biossíntese , Insetos Vetores/enzimologia , Ácido Mevalônico/metabolismo , Atrativos Sexuais/metabolismo , Animais , Dípteros/genética , Dípteros/parasitologia , Feminino , Regulação Enzimológica da Expressão Gênica , Proteínas de Insetos/genética , Insetos Vetores/genética , Insetos Vetores/parasitologia , Leishmania infantum , Leishmaniose Visceral , Masculino , Espectrometria de Massas , Atrativos Sexuais/genéticaRESUMO
The paraphyletic family Colubridae comprises several species of rear-fanged snakes with toxin-secreting Duvernoy's gland, some of them able to cause human envenomation with systemic and/or local damage. In this work we have explored some aspects of biochemical composition and activity of the venoms of five species from Colubridae family from Brazil. Taken together our results suggest distinct features in colubrid venoms, which could be related to the presence of still unknown toxins.
Assuntos
Colubridae/fisiologia , Venenos de Serpentes/química , Animais , Brasil , Carboidratos/química , Colorimetria , Dieta , Eletroforese em Gel de Poliacrilamida , Glândulas Exócrinas/química , Glândulas Exócrinas/enzimologia , Fibrinogênio/química , Fibrinogênio/metabolismo , Hemorragia/induzido quimicamente , Peptídeo Hidrolases/análise , Fosfolipases A2/análise , Comportamento Predatório , Proteínas/química , Venenos de Serpentes/enzimologia , Especificidade da Espécie , Especificidade por SubstratoRESUMO
Biochemical studies revealed that the activity of some hydrolytic enzymes from the venom glands of honey bee Apis mellifera was higher in workers of 14 days of age than in those of 40 days. Among these enzymes, the highest activity was recorded for acid phosphatase, which was cytochemically detected throughout the length of the secretory filament and surrounding the canaliculi of the distal region of the reservoir. The acid phosphatase was considered to be a typical secretion product, since it was present in the cytoplasm as well as in the canaliculi of the secretory cells.
Assuntos
Venenos de Abelha/enzimologia , Abelhas/enzimologia , Abelhas/crescimento & desenvolvimento , Glândulas Exócrinas/enzimologia , Glândulas Exócrinas/crescimento & desenvolvimento , AnimaisRESUMO
Gyroxin is one of main serine proteases of Crotalus durissus terrificus venom, representing about 2% of the protein content in the crude venom. It is a 33 kDa glycoprotein with 3.8% by weight of sugar moiety. This toxin induces hemotoxicity in mice and a neurological condition called barrel rotation syndrome. In the present work, we report the molecular cloning of five new nucleotide sequences from a cDNA library of the venom glands of a single specimen of C. d. terrificus. These sequences have been analyzed in silico with respect to their cDNA organization and similarity with other snake venom serine proteases (SVSPs). We also describe a rapid and efficient method for screening vectors for mammalian cell expression, based on the fact that SVSPs are difficult-to-express toxins due to the presence of several disulfide bonds and glycosylation in their structures. Thus, one of the Gyroxin cDNAs was subcloned into pSectag2 HygroA and pED vectors and used to transfect COS-7 cells. Expression of the functional recombinant Gyroxin isoform was achieved with this cell line with esterase activity in the conditioned culture medium, as revealed by immunoblot of secreted protein and standard anti-crotalic serum from Butantan Institute.
Assuntos
Venenos de Crotalídeos/biossíntese , DNA Complementar/biossíntese , Glândulas Exócrinas/química , Serina Endopeptidases/biossíntese , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Chlorocebus aethiops , Clonagem Molecular , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/genética , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Esterases/química , Esterases/metabolismo , Glândulas Exócrinas/enzimologia , Biblioteca Gênica , Vetores Genéticos , Camundongos , Peso Molecular , Plasmídeos/genética , Proteínas Recombinantes/genética , Serina Endopeptidases/genéticaRESUMO
Sphingomyelinase D (SMase D) present in the venoms of Loxosceles spiders is the principal component responsible for local and systemic effects observed in the loxoscelism. By using "expressed sequencing tag", it was possible to identify, in a L. laeta venom gland library, clones containing inserts coding for proteins with similarity to SMase D. One of these clones was expressed and the recombinant protein compared with the previously characterized SMase I from L. laeta, in terms of their biological, biochemical and structural properties. The new recombinant protein, SMase II, possesses all the biological properties ascribed to the whole venom and SMase I. SMase II shares 40% and 77% sequence similarity with SMase I and Lb3, respectively; the latter, a SMase D isoform from L. boneti, catalytically inactive. Molecular modeling and molecular dynamics simulations were employed to understand the structural basis, especially the presence of an additional disulfide bridge, in an attempt to account for the observed differences in SMases D activity.
Assuntos
Glândulas Exócrinas/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Venenos de Aranha/enzimologia , Sequência de Aminoácidos , Western Blotting , Soluções Tampão , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Eritrócitos/efeitos dos fármacos , Citometria de Fluxo , Hemólise/efeitos dos fármacos , Humanos , Indicadores e Reagentes , Modelos Moleculares , Dados de Sequência Molecular , Necrose/induzido quimicamente , Necrose/patologia , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Proteínas Recombinantes/química , Pele/patologiaRESUMO
This study documents effects of the toxic dinoflagellate Gymnodinium catenatum, a producer of paralytic shellfish poison, on juvenile farmed (5.9+/-0.39 cm) giant lions-paw scallop Nodipecten subnodosus. Scallops were fed bloom concentrations of toxic dinoflagellate G. catenatum for 7 h. The effect of the toxic dinoflagellate in different tissues was determined by analysis of antioxidant enzymes (catalase, superoxide dismutase, gluthathione peroxidase), thiobarbituric acid reactive substances (lipid peroxidation), and hydrolytic enzymes (proteases, glycosidases, phosphatases, lipases, and esterases). Histopathological photos record the effects of the toxic dinoflagellate in various tissues. The results show that juvenile lions-paw scallops produce pseudo-feces, partially close their shell, increase melanization, and aggregate hemocytes. Several enzymes were affected and could serve as biological markers. In general, the adductor muscle was not affected. In the digestive gland, some enzymes could be the result of defensive and digestive processes. Gills and mantle tissue were markedly affected because these sites respond first to toxic dinoflagellates, leading to the idea that proteolytic cascades could be involved.
Assuntos
Dinoflagellida/metabolismo , Hidrolases/metabolismo , Oxirredutases/metabolismo , Pectinidae , Venenos/toxicidade , Saxitoxina/toxicidade , Animais , Biomarcadores/metabolismo , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/enzimologia , Sistema Digestório/patologia , Dinoflagellida/crescimento & desenvolvimento , Glândulas Exócrinas/efeitos dos fármacos , Glândulas Exócrinas/enzimologia , Glândulas Exócrinas/patologia , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Brânquias/patologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Melaninas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Pectinidae/efeitos dos fármacos , Pectinidae/enzimologia , Pectinidae/crescimento & desenvolvimento , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Testes de Toxicidade AgudaRESUMO
Hydrolytic enzymes from hypopharyngeal gland extracts of newly emerged, nurse and foraging workers of two eusocial bees, Scaptotrigona postica, a native Brazilian stingless bee, and the Africanized honey bee (Apis mellifera) in Brazil, were compared. The hypopharyngeal gland is rich in enzymes in both species. Fifteen different enzymes were found in the extracts, with only a few quantitative differences between the species. Some of the enzymes present in the extracts may have intracellular functions, while others seem to be digestive enzymes. Scaptotrigona postica, had lower beta-glucosidase and higher lipase esterase activities than A. mellifera. The differences may be due to different feeding habits and behavioral peculiarities of the two species.
Assuntos
Abelhas/enzimologia , Glândulas Exócrinas/enzimologia , Hidrolases/isolamento & purificação , Hipofaringe/enzimologia , Animais , Feminino , Hidrolases/classificaçãoRESUMO
Hydrolytic enzymes from hypopharyngeal gland extracts of newly emerged, nurse and foraging workers of two eusocial bees, Scaptotrigona postica, a native Brazilian stingless bee, and the Africanized honey bee (Apis mellifera) in Brazil, were compared. The hypopharyngeal gland is rich in enzymes in both species. Fifteen different enzymes were found in the extracts, with only a few quantitative differences between the species. Some of the enzymes present in the extracts may have intracellular functions, while others seem to be digestive enzymes. Scaptotrigona postica, had lower beta-glucosidase and higher lipase esterase activities than A. mellifera. The differences may be due to different feeding habits and behavioral peculiarities of the two species.
Assuntos
Animais , Feminino , Abelhas/enzimologia , Glândulas Exócrinas/enzimologia , Hidrolases/isolamento & purificação , Hipofaringe/enzimologia , Hidrolases/classificaçãoRESUMO
Three isotrypsins from digestive gland of Penaeus vannamei were purified and characterized by molecular, biochemical and kinetic parameters. Purified isotrypsins A, B, and C are glycoproteins with molecular masses between 30.2 and 32.9 kDa, and, therefore similar to other trypsins. The isoelectric points are anionic and different among the three isotrypsins: pH 3.5 for isotrypsin A, pH 3.0 for isotrypsin B, and pH 4.5 for isotrypsin C. Differences in the NH(2)-terminal amino acid sequences allowed us to define three different protein entities that match isotrypsins previously deduced by cDNA. Isoform C has higher physiological efficiency and specific activity, lower K(m), and requires higher concentrations of Ca(+2) to reach the same activity as the other two isotrypsins.
Assuntos
Penaeidae/enzimologia , Tripsina/isolamento & purificação , Tripsina/metabolismo , Animais , Estabilidade Enzimática , Glândulas Exócrinas/enzimologia , Glicosilação , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Isoenzimas , Cinética , Temperatura , Tripsina/químicaRESUMO
Using immunoelectronmicroscopy we analyzed qualitative and quantitatively the intracellular distribution of bothropasin, hemorrhagic factor 2 (HF2) and hemorrhagic factor 3 (HF3) in the venom secretory cells from adult snakes in the active (7 days after venom extraction) and in the resting (without venom extraction for 40 days) stages of protein synthesis. Glands from the newborn Bothrops jararaca were also studied. The results lead to the conclusion that all the secretory cells and the secretory pathway in the cells are qualitatively alike in regard to their content of the three metalloproteases. Secretory cells from the resting glands, unlike the active ones and the newborn glands, did not present immunolabeling in the narrow intracisternal spaces of the rough endoplasmic reticulum (RER). The label intensity for bothropasin was greater than that for the other proteins in the adults. HF3 and HF2 labeling densities in the newborn were higher than in the adults and HF3 labeling was not different from that of bothropasin. Co-localization of the three metalloproteases was detected in the RER cisternae of the active gland secretory cells, implying that mixing of the proteases before co-packaging into secretory vesicles occurs at the beginning of protein synthesis in the RER cisternae.
Assuntos
Bothrops , Glândulas Exócrinas/enzimologia , Metaloendopeptidases/análise , Peçonhas/enzimologia , Fatores Etários , Animais , Animais Recém-Nascidos , Venenos de Crotalídeos/análise , Glândulas Exócrinas/ultraestrutura , Microscopia Imunoeletrônica , Especificidade por SubstratoRESUMO
In the present investigation, in order to dispute the rational criticism against the presence of proteolytic enzymes in the electrostimulated venom obtained from spiders of the genus Loxosceles, as a consequence of contamination with abdominal secretions, venoms of L. intermedia and L. laeta were directly collected from venom glands by microdissection and gentle homogenization. Gel electrophoresis stained by silver method carried out to compare L. intermedia electrostimulated venom and venom gland extract demonstrated no significant differences in protein profile. Zymogram analysis of L. intermedia venom gland extract detected a gelatinolytic activity in the 32-35 kDa region. The inhibitory effect of 1,10-phenanthroline on this proteolytic activity further supported its metalloprotease nature. In proteolytic digestion experiments L. intermedia venom gland extract was also able to cleave purified fibronectin and fibrinogen. The inhibitory effect of 1,10-phenanthroline on these degrading activities confirmed the presence of metalloproteases in the venom. In addition, when purified fibrinogen was incubated with L. intermedia abdominal extract, the fibrinogenolysis was completely different, generating low mass fragments that ran away from the gel, a proteolytic event not blocked by 1,10-phenanthroline. Zymogram experiments using L. laeta venom gland extracts further detected a gelatinolytic band at 32-35 kDa, also inhibited by 1,10-phenanthroline, confirming the presence of metalloproteases in both species.
Assuntos
Estimulação Elétrica/métodos , Glândulas Exócrinas/enzimologia , Metaloendopeptidases/metabolismo , Venenos de Aranha/enzimologia , Aranhas , Animais , Dissecação , Eletroforese em Gel de Poliacrilamida , Glândulas Exócrinas/metabolismo , Glândulas Exócrinas/ultraestrutura , Fibrinólise/efeitos dos fármacos , Humanos , Metaloendopeptidases/antagonistas & inibidores , Microscopia Eletrônica de Varredura , Fenantrolinas/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Venenos de Aranha/metabolismoRESUMO
The present study reports the localization of acid phosphatase in the hypopharyngeal gland cells from workers (newly-emerged, nurse and forager), queens (newly-emerged and laying) and males (newly-emerged and mature for mating) of the Brazilian stingless bee, Scaptotrigona postica. The phosphatase activity varied in intensity and localization depending on the individual class, physiological age and the substrate used. In newly-emerged workers, the phosphatase-positive sites suggest the involvement of the enzyme with cellular differentiation that occurs in the presecretory phase, in nurse workers with protein synthesis and in forager workers with changes in cellular activity or glandular regression. In males mature for mating and laying queens, the positive sites are related to secretory activity, showing that the gland maintains some activity in spite of the regressive aspect. Of the substrates used, beta-glycerophosphate gave the least specific localization.