Quantity - but not diversity - of secreted peptides and proteins increases with age in the tree frog Pithecopus nordestinus

Amphibians inhabit the terrestrial environment, a conquest achieved after several evolutionary steps, which were still insufficient to make them completely independent of the aquatic environment. These processes gave rise to many morphological and physiological changes, making their skin (and cutaneous secretion) rich in bioactive molecules. Among the tree frogs, the secretion is composed mainly of peptides; but alkaloids, proteins and steroids can also be found depending on the species. The most known class of biologically active molecules is the antimicrobial peptides (AMPs) that act against bacteria, fungi and protozoans. Although these molecules are well-studied among the hylids, AMPs ontogeny remains unknown. Therefore, we performed peptidomic and proteomic analyses of Pithecopus nordestinus (formerly Phyllomedusa nordestina) in order to evaluate the peptide content in post-metamorphosed juveniles and adult individuals. Methods: Cutaneous secretion of both life stages of individuals was obtained and analyzed by LC-MS/MS after reduction and alkylation of disulfide bonds or reduction, alkylation and hydrolysis by trypsin. Results: Differences in the TIC profile of juveniles and adults in both treatments were observed. Moreover, the proteomic data revealed known proteins and peptides, with slight differences in the composition, according to the life stage and the treatment. AMPs were identified, and bradykinin-potentiating peptides were observed in trypsin-treated samples, which suggests a protein source of such peptide (cryptide). Conclusion: In general, skin secretion contents were similar between juveniles and adults, varying in quantity, indicating that the different stages of life are reflected in the number of molecules and not on their diversity.(AU)

Quantity - but not diversity - of secreted peptides and proteins increases with age in the tree frog Pithecopus nordestinus

Amphibians inhabit the terrestrial environment, a conquest achieved after several evolutionary steps, which were still insufficient to make them completely independent of the aquatic environment. These processes gave rise to many morphological and physiological changes, making their skin (and cutaneous secretion) rich in bioactive molecules. Among the tree frogs, the secretion is composed mainly of peptides; but alkaloids, proteins and steroids can also be found depending on the species. The most known class of biologically active molecules is the antimicrobial peptides (AMPs) that act against bacteria, fungi and protozoans. Although these molecules are well-studied among the hylids, AMPs ontogeny remains unknown. Therefore, we performed peptidomic and proteomic analyses of Pithecopus nordestinus (formerly Phyllomedusa nordestina) in order to evaluate the peptide content in post-metamorphosed juveniles and adult individuals. Methods: Cutaneous secretion of both life stages of individuals was obtained and analyzed by LC-MS/MS after reduction and alkylation of disulfide bonds or reduction, alkylation and hydrolysis by trypsin. Results: Differences in the TIC profile of juveniles and adults in both treatments were observed. Moreover, the proteomic data revealed known proteins and peptides, with slight differences in the composition, according to the life stage and the treatment. AMPs were identified, and bradykinin-potentiating peptides were observed in trypsin-treated samples, which suggests a protein source of such peptide (cryptide). Conclusion: In general, skin secretion contents were similar between juveniles and adults, varying in quantity, indicating that the different stages of life are reflected in the number of molecules and not on their diversity.(AU)

Isolation and structural identification of a new T1-conotoxin with unique disulfide connectivities derived from Conus bandanus

Conopeptides are neuropharmacological peptides derived from the venomous salivary glands of cone snails. Among 29 superfamilies based on conserved signal sequences, T-superfamily conotoxins, which belong to the smallest group, include four different frameworks that contain four cysteines denominated I, V, X and XVI. In this work, the primary structure and the cysteine connectivity of novel conotoxin of Conus bandanus were determined by tandem mass spectrometry using collision-induced dissociation. Methods: The venom glands of C. bandanus snails were dissected, pooled, and extracted with 0.1% trifluoroacetic acid in three steps and lyophilized. The venom was fractionated and purified in an HPLC system with an analytical reversed-phase C18 column. The primary peptide structure was analyzed by MALDI TOF MS/MS using collision-induced dissociation and confirmed by Edman's degradation. The peptide's cysteine connectivity was determined by rapid partial reduction-alkylation technique. Results: The novel conotoxin, NGC1C2(I/L)VREC3C4, was firstly derived from de novo sequencing by MS/MS. The presence of isoleucine residues in this conotoxin was confirmed by the Edman degradation method. The conotoxin, denominated Bn5a, belongs to the T1-subfamily of conotoxins. However, the disulfide bonds (C1-C4/C2-C3) of Bn5a were not the same as found in other T1-subfamily conopeptides but shared common connectivities with T2-subfamily conotoxins. The T1-conotoxin of C. bandanus proved the complexity of the disulfide bond pattern of conopeptides. The homological analysis revealed that the novel conotoxin could serve as a valuable probe compound for the human-nervous-system norepinephrine transporter. Conclusion: We identified the first T1-conotoxin, denominated Bn5a, isolated from C. bandanus venom. However, Bn5a conotoxin exhibited unique C1-C4/C2-C3 disulfide connectivity, unlike other T1-conotoxins (C1-C3/C2-C4). The structural and homological analyses herein have evidenced novel conotoxin Bn5a that may require further investigation.(AU)

Isolation and structural identification of a new T1-conotoxin with unique disulfide connectivities derived from Conus bandanus

Background: Conopeptides are neuropharmacological peptides derived from the venomous salivary glands of cone snails. Among 29 superfamilies based on conserved signal sequences, T-superfamily conotoxins, which belong to the smallest group, include four different frameworks that contain four cysteines denominated I, V, X and XVI. In this work, the primary structure and the cysteine connectivity of novel conotoxin of Conus bandanus were determined by tandem mass spectrometry using collision-induced dissociation. Methods: The venom glands of C. bandanus snails were dissected, pooled, and extracted with 0.1% trifluoroacetic acid in three steps and lyophilized. The venom was fractionated and purified in an HPLC system with an analytical reversed-phase C18 column. The primary peptide structure was analyzed by MALDI TOF MS/MS using collision-induced dissociation and confirmed by Edman's degradation. The peptides cysteine connectivity was determined by rapid partial reduction-alkylation technique. Results: The novel conotoxin, NGC1C2(I/L)VREC3C4, was firstly derived from de novo sequencing by MS/MS. The presence of isoleucine residues in this conotoxin was confirmed by the Edman degradation method. The conotoxin, denominated Bn5a, belongs to the T1-subfamily of conotoxins. However, the disulfide bonds (C1-C4/C2-C3) of Bn5a were not the same as found in other T1-subfamily conopeptides but shared common connectivities with T2-subfamily conotoxins. The T1-conotoxin of C. bandanus proved the complexity of the disulfide bond pattern of conopeptides. The homological analysis revealed that the novel conotoxin could serve as a valuable probe compound for the human-nervous-system norepinephrine transporter. Conclusion: We identified the first T1-conotoxin, denominated Bn5a, isolated from C. bandanus venom. However, Bn5a...(AU)

Jingzhaotoxin-X, a gating modifier of Kv4.2 and Kv4. 3 potassium channels purified from the venom of the Chinese tarantula Chilobrachys jingzhao

The tarantula Chilobrachys jingzhao is one of the largest venomous spiders in China. In previous studies, we purified and characterized at least eight peptides from C. jingzhao venom. In this report, we describe the purification and characterization of Jingzhaotoxin-X (JZTX-X), which selectively blocks Kv4.2 and Kv4.3 potassium channels. Methods: JZTX-X was purified using a combination of cation-exchange HPLC and reverse-phase HPLC. The amino-acid sequence was determined by automated Edman degradation and confirmed by mass spectrometry (MS). Voltage-gated ion channel currents were recorded in HEK293t cells transiently transfected with a variety of ion channel constructs. In addition, the hyperalgesic activity of JZTX-X and the toxin´s effect on motor function were assessed in mice. Results: JZTX-X contained 31 amino acids, with six cysteine residues that formed three disulfide bonds within an inhibitory cysteine knot (ICK) topology. In whole-cell voltage-clamp experiments, JZTX-X inhibited Kv4.2 and Kv4.3 potassium channels in a concentration- and voltage-dependent manner, without affecting other ion channels (Kv1.1, 1.2, 1.3, 2.1, delayed rectifier potassium channels, high- and low-voltage-activated Ca2+ channels, and voltage-gated sodium channels Nav1.5 and 1.7). JZTX-X also shifted the voltage-dependent channel activation to more depolarized potentials, whereas extreme depolarization caused reversible toxin binding to Kv4.2 channels. JZTX-X shifted the Kv4.2 and Kv4.3 activities towards a resting state, since at the resting potential the toxin completely inhibited the channels, even in the absence of an applied physical stimulus. Intrathecal or intraplantar injection of JZTX-X caused a long-lasting decrease in the mechanical nociceptive threshold (hyperalgesia) but had no effect on motor function as assessed in the rotarod test. Conclusions: JZTX-X selectively suppresses Kv4.2 and Kv4.3 potassium channel activity in a concentration- and voltage-dependent manner and causes long-lasting mechanical hyperalgesia.(AU)

Jingzhaotoxin-X, a gating modifier of Kv4.2 and Kv4.3 potassium channels purified from the venom of the Chinese tarantula Chilobrachys jingzhao

Background:The tarantula Chilobrachys jingzhao is one of the largest venomous spiders in China. In previous studies, we purified and characterized at least eight peptides from C. jingzhao venom. In this report, we describe the purification and characterization of Jingzhaotoxin-X (JZTX-X), which selectively blocks Kv4.2 and Kv4.3 potassium channels.Methods:JZTX-X was purified using a combination of cation-exchange HPLC and reverse-phase HPLC. The amino-acid sequence was determined by automated Edman degradation and confirmed by mass spectrometry (MS). Voltage-gated ion channel currents were recorded in HEK293t cells transiently transfected with a variety of ion channel constructs. In addition, the hyperalgesic activity of JZTX-X and the toxin´s effect on motor function were assessed in mice.Results:JZTX-X contained 31 amino acids, with six cysteine residues that formed three disulfide bonds within an inhibitory cysteine knot (ICK) topology. In whole-cell voltage-clamp experiments, JZTX-X inhibited Kv4.2 and Kv4.3 potassium channels in a concentration- and voltage-dependent manner, without affecting other ion channels (Kv1.1, 1.2, 1.3, 2.1, delayed rectifier potassium channels, high- and low-voltage-activated Ca2+ channels, and voltage-gated sodium channels Nav1.5 and 1.7). JZTX-X also shifted the voltage-dependent channel activation to more depolarized potentials, whereas extreme depolarization caused reversible toxin binding to Kv4.2 channels. JZTX-X shifted the Kv4.2 and Kv4.3 activities towards a resting state, since at the resting potential the toxin...(AU)

Identification, expression and characterization of the recombinant Sol g 4. 1 protein from the venom of the tropical fire ant Solenopsis geminata

Fire ant venom is a complex mixture consisting of basic piperidine alkaloids, various biologically active peptides and protein components, including a variety of major allergenic proteins. Tropical fire ant Solenopsis geminata is an important stinging ant species that causes anaphylaxis and serious medical problems. Although the biological activities of allergenic venom proteins that are unique to ant venom, particularly Solenopsis 2 and 4, are still unknown, these proteins are believed to play important roles in mediating the effects of the piperidine derivatives in the venom. Methods: In the present study, the cDNA cloning, sequencing and three-dimensional structure of Sol g 4.1 venom protein are described. The recombinant Sol g 4.1 protein (rSol g 4.1) was produced in E. coli , and its possible function as a hydrophobic binding protein was characterized by paralyzing crickets using the 50% piperidine dose (PD50). Moreover, an antiserum was produced in mice to determine the allergenic properties of Sol g 4.1, and the antiserum was capable of binding to Sol g 4.1, as determined by Western blotting. Results: The molecular weight of Sol g 4.1 protein is 16 kDa, as determined by SDS-PAGE. The complete cDNA is 414 bp in length and contains a leader sequence of 19 amino acids. The protein consists of six cysteines that presumably form three disulfide bonds, based on a predicted three-dimensional model, creating the interior hydrophobic pocket and stabilizing the structure. The rSol g 4.1 protein was expressed in inclusion bodies, as determined by SDS-PAGE. Dialysis techniques were used to refold the recombinant protein into the native form. Its secondary structure, which primarily consists of α-helices, was confirmed by circular dichroism analysis, and the three-dimensional model was also verified. The results of allergenic analysis performed on mice showed that the obtained protein was predicted to be allergenically active. Moreover, we report on the possible role of the Sol g 4.1 venom protein, which significantly reduced the PD50 from 0.027 to 0.013% in paralyzed crickets via synergistic effects after interactions with piperidine alkaloids. Conclusions: The primary structure of Sol g 4.1 showed high similarity to that of venom proteins in the Solenopsis 2 and 4 family. Those proteins are life-threatening and produce IgE-mediated anaphylactic reactions in allergic individuals. The possible function of this protein is the binding of the interior hydrophobic pockets with piperidine alkaloids, as determined by the analysis of the structural model and PD50 test.(AU)

Identification, expression and characterization of the recombinant Sol g 4.1 protein from the venom of the tropical fire ant Solenopsis geminata

Background: Fire ant venom is a complex mixture consisting of basic piperidine alkaloids, various biologically active peptides and protein components, including a variety of major allergenic proteins. Tropical fire ant Solenopsis geminata is an important stinging ant species that causes anaphylaxis and serious medical problems. Although the biological activities of allergenic venom proteins that are unique to ant venom, particularly Solenopsis 2 and 4, are still unknown, these proteins are believed to play important roles in mediating the effects of the piperidine derivatives in the venom. Methods: In the present study, the cDNA cloning, sequencing and three-dimensional structure of Sol g 4.1 venom protein are described. The recombinant Sol g 4.1 protein (rSol g 4.1) was produced in E. coli , and its possible function as a hydrophobic binding protein was characterized by paralyzing crickets using the 50% piperidine dose (PD50). Moreover, an antiserum was produced in mice to determine the allergenic properties of Sol g 4.1, and the antiserum was capable of binding to Sol g 4.1, as determined by Western blotting. Results: The molecular weight of Sol g 4.1 protein is 16 kDa, as determined by SDS-PAGE. The complete cDNA is 414 bp in length and contains a leader sequence of 19 amino acids. The protein consists of six cysteines that presumably form three disulfide bonds, based on a predicted three-dimensional model, creating the interior hydrophobic pocket and stabilizing the structure. The rSol g 4.1 protein was expressed in inclusion bodies, as determined by SDS-PAGE. Dialysis techniques were used to refold the recombinant protein into the native form. Its secondary structure, which primarily consists of -helices, was confirmed by circular dichroism analysis, and the three-dimensional model was also verified. The results of allergenic analysis performed on mice showed that the...(AU)

Identification, expression and characterization of the recombinant Sol g 4.1 protein from the venom of the tropical fire ant Solenopsis geminata

Background: Fire ant venom is a complex mixture consisting of basic piperidine alkaloids, various biologically active peptides and protein components, including a variety of major allergenic proteins. Tropical fire ant Solenopsis geminata is an important stinging ant species that causes anaphylaxis and serious medical problems. Although the biological activities of allergenic venom proteins that are unique to ant venom, particularly Solenopsis 2 and 4, are still unknown, these proteins are believed to play important roles in mediating the effects of the piperidine derivatives in the venom. Methods: In the present study, the cDNA cloning, sequencing and three-dimensional structure of Sol g 4.1 venom protein are described. The recombinant Sol g 4.1 protein (rSol g 4.1) was produced in E. coli , and its possible function as a hydrophobic binding protein was characterized by paralyzing crickets using the 50% piperidine dose (PD50). Moreover, an antiserum was produced in mice to determine the allergenic properties of Sol g 4.1, and the antiserum was capable of binding to Sol g 4.1, as determined by Western blotting. Results: The molecular weight of Sol g 4.1 protein is 16 kDa, as determined by SDS-PAGE. The complete cDNA is 414 bp in length and contains a leader sequence of 19 amino acids. The protein consists of six cysteines that presumably form three disulfide bonds, based on a predicted three-dimensional model, creating the interior hydrophobic pocket and stabilizing the structure. The rSol g 4.1 protein was expressed in inclusion bodies, as determined by SDS-PAGE. Dialysis techniques were used to refold the recombinant protein into the native form. Its secondary structure, which primarily consists of -helices, was confirmed by circular dichroism analysis, and the three-dimensional model was also verified. The results of allergenic analysis performed on mice showed that the...

LECTINA RELACIONADA AO FIBRINOGÊNIO DO OURIÇO-DO-MAR Echinometra lucunter: ISOLAMENTO, SEQUÊNCIA DE AMINOÁCIDOS E ATIVIDADE ANTIMICROBIANA

Uma das primeiras linhas de defesa dos invertebrados está relacionada ao reconhecimento dos patógenos por receptores/proteínas de reconhecimento padrão (PRRs/PRPs) e moléculas de reconhecimento. Dentre elas, destacam-se as proteínas relacionadas ao fibrinogênio (FRePs) identificadas nos invertebrados e que, em sua maioria, atuam como PRPs desempenhando papéis importantes na aglutinação microbiana, lise e clearance bacteriano e na defesa antiparasitária. Uma lectina relacionada ao fibrinogênio chamada de ELL foi isolada do fluido celomático do ouriço-do-mar Echinometra lucunter por cromatografia de afinidade em goma de xantana. ELL é um dímero composto por subunidades de 25 kDa, unidas por uma ligação dissulfeto. A nova lectina foi inibida por glicoproteínas como mucinas (PSM II, PSM III e BSM) e tiroglobulina. A sequência de aminoácidos de ELL foi determinada por espectrometria de massas. O monômero de ELL consiste de 229 aminoácidos, incluindo sete cisteínas envolvidas em três ligações dissulfeto intracadeia conservadas e uma ligação dissulfeto intercadeia. A sequência completa de ELL possui motivos conservados e domínio de fibrinogênio na porção C-terminal típico de proteínas relacionadas ao fibrinogênio. Um modelo tridimensional de ELL foi previsto por homologia, revelando que a proteína possui dois domínios de reconhecimento a carboidratos, o DRC A está localizado na região N-terminal e parece interagir com N-acetil-manosamina e o DRC B encontra-se na porção C-terminal e pode ligar-se a N-acetil-galactosamina. Em ensaios de atividade biológica, ELL foi capaz de inibir o crescimento de bactérias Gram-positivas patogênicas, além de reduzir a biomassa e viabilidade celular do biofilme formado por bactérias Gram-positivas e Gram-negativas, sugerindo sua capacidade de reconhecer patógenos.

One of the first lines of defense for invertebrates is related to the recognition of pathogens by standard recognition receptors / proteins (PRRs / PRPs) and recognition molecules. Among them, fibrinogen-related proteins (FRePs) that are identified in invertebrates stand out, and that, in their majority, act as PRPs playing important roles in microbial agglutination, bacterial lysis and clearance and in antiparasitic defense. A fibrinogen-related lectin called ELL was isolated from the coelomic fluid of the sea urchin Echinometra lucunter by affinity chromatography on xanthan gum. ELL is a dimer composed of 25 kDa subunits, joined by a disulfide bond. The new lectin was inhibited by glycoproteins such as mucins (PSM II, PSM III and BSM) and thyroglobulin. The amino acid sequence of ELL was determined by mass spectrometry. The ELL monomer consists of 229 amino acids, including seven cysteines involved in three conserved intrachain disulfide bonds and an interchain disulfide bond. The complete ELL sequence has conserved motifs and fibrinogen domain in the typical C-terminal portion of fibrinogen-related proteins. A three-dimensional model of ELL was predicted by homology, revealing that the protein has two carbohydrate recognition domains, CRD A is located in the N-terminal region and appears to interact with N-acetyl-mannosamine and CRD B is found in the C-terminal and can bind to N-acetyl-galactosamine. In biological activity assays, ELL was able to inhibit the growth of pathogenic Gram-positive bacteria, in addition to reducing the biomass and cell viability of the biofilm formed by Gram-positive and Gram-negative bacteria, suggesting its ability to recognize pathogens.

Regulation of ovarian function: structural characterization and role of anti-müllerian hormone (AMH) / Regulação da função ovariana: caracterização estrutural e papel do hormônio anti-mülleriano (AMH)

Background: The anti-müllerian hormone (AMH) is a member of the transforming growth factor (TGF-) superfamily, which exerts important functions on local regulation of folliculogenesis. Although in vivo and in vitro studies suggest that AMH affects the primordial follicle assembly and activation, as well as the responsiveness of growing follicles to folliclestimulating hormone (FSH), the physiological mechanisms involved in these actions remain to be fully elucidated. Given the relevance of AMH in the folliculogenesis, this review aimed to describe the structural features, expression and the main biological effects of AMH on the follicular development. Review: Originally identified as a testicular product, AMH is responsible for regression of the Müllerian ducts during sexual differentiation of male embryos. In females, AMH is produced almost exclusively by granulosa cells of ovarian growing follicles, whose serum levels are positively related to the number of ovarian follicles, making AMH an excellent clinic marker of ovarian reserve. Along this work, it was shown aspects related to the structural characterization of AMH and its specific type II receptor (AMHRII). AMH is a glycoprotein dimer linked by disulfide bonds. The mature protein comprises two unequal domains: a long N-terminal domain (110-kDa) and a short C-terminal domain (25-kDa), responsible for the biological act

A foliculogênese é um evento complexo que envolve o recrutamento do pool de folículos primordiais, seguido por uma fase de crescimento e diferenciação. É sabido que para a sua regulação são requeridos vários fatores parácrinos e autócrinos, especialmente fatores de crescimento produzidos pelas células da granulosa. Dentre esses fatores, destaca-se o hormônio anti-mülleriano (AMH), uma glicoproteína membro da superfamília de fatores de crescimento transformantes (TGF-). Inicialmente o AMH foi estudado por seu papel regulatório no processo de diferenciação sexual masculina. [...]

Regulação da função ovariana: caracterização estrutural e papel do hormônio anti-mülleriano (AMH) / Regulation of Ovarian Function: Structural Characterization and Role of Anti-Müllerian Hormone (AMH)

Background: The anti-müllerian hormone (AMH) is a member of the transforming growth factor ß (TGF-ß) superfamily, which exerts important functions on local regulation of folliculogenesis. Although in vivo and in vitro studies suggest that AMH affects the primordial follicle assembly and activation, as well as the responsiveness of growing follicles to folliclestimulating hormone (FSH), the physiological mechanisms involved in these actions remain to be fully elucidated. Given the relevance of AMH in the folliculogenesis, this review aimed to describe the structural features, expression and the main biological effects of AMH on the follicular development. Review: Originally identified as a testicular product, AMH is responsible for regression of the Müllerian ducts during sexual differentiation of male embryos. In females, AMH is produced almost exclusively by granulosa cells of ovarian growing follicles, whose serum levels are positively related to the number of ovarian follicles, making AMH an excellent clinic marker of ovarian reserve. Along this work, it was shown aspects related to the structural characterization of AMH and its specific type II receptor (AMHRII). AMH is a glycoprotein dimer linked by disulfide bonds. The mature protein comprises two unequal domains: a long N-terminal domain (110-kDa) and a short C-terminal domain (25-kDa), responsible for the biological activity of the molecule. The AMHRII is an 82-kDa protein. Like others members of TGF-ß family, AMH signals through two types of serine/threonine kinase receptors called type I and type II, and two types of Smad proteins, receptor-regulated Smad (R-Smad) and common Smad, Smad4. However, the identity of the AMH type I receptor is not clear; three type I receptors of Bone Morphogenetic Proteins (BMPs), Alk2, Alk3 and Alk6 may transduce AMH signals. AMH expression was detected in granulosa cells of growing follicles and it decreases once FSH-dependent follicular growth has been initiated. Follicles showing signs of atresia also have decreased or no AMH expression, and expression is completely lost in corpora lutea. AMH is not found in primordial follicles, theca cells, oocytes or the interstitium. This specific expression pattern of AMH suggests a role in the two regulatory steps of folliculogenesis: the recruitment of primordial follicles and the sensitivity of large preantral and small antral follicles to FSH. Evidences obtained from studies with AMH-knockout mice suggest that AMH inhibits activation of primordial follicles into the growing pool, while at cyclic recruitment AMH lowers the FSH-sensitivity of follicles. In addition, AMH was recently implicated in the primordial follicle assembly. AMH was found to inhibit primordial follicle assembly and decrease the initial primordial follicle pool size in a rat ovarian organ culture. Conclusion: From this review, we can conclude that AMH plays a key role on the modulation of ovarian function in mammals. This substance is an important player in two checkpoints that regulate the efficiency of primordial follicle pool usage and the choice of the dominant follicle: activation and selection, respectively. However, it is necessary to perform additional studies that may provide a better understanding about the importance of AMH during folicullogenesis.

Efeitos da glutationa reduzida sobre os processos de capacitação e reação acrossômica in vitro em espermatozoides suínos refrigerados

Um estudo foi realizado objetivando-se avaliar os efeitos da glutationa reduzida (GSH) sobre a função espermática, durante a capacitação e posterior indução da reação acrossômica in vitro, em sêmen suíno refrigerado. Isso foi feito por meio da avaliação de vários marcadores de capacitação in vitro e outros parâmetros globais de qualidade de sêmen, tais como viabilidade, motilidade total, exocitose acrossomal induzida por progesterona, fragmentação de DNA, níveis de desordens lipídicas na membrana espermática, espécies reativas de oxigênio (ERO), resíduos de cisteínas livres em extratos de cabeça e cauda espermáticas e fosforilação prot eica de resíduos de tirosina. Um total de 62 ejaculados, provenientes de 35 reprodutores suínos saudáveis Pietrain, entre 2 e 3 anos de idade, foram usados nas diferentes análises deste trabalho. A adição de GSH, no meio de capacitação (CM), impediu a maioria das alterações concomitantes à capacitação. Além disso, o GSH provocou uma queda rápida e intensa da motilidade total (P<0,05), a qual foi mantida dur ante todo o período de incubação. Apesar destes resultados, o GSH não afetou o início da exocitose acrossomal in vitro induzida pela progesterona (IVAE) observada após 4h de incubação no CM. Os resultados mostraram que a capacitação in vitro de sêmen suíno está relacionada com um aumento significante tanto da quebra total das pontes dissulfeto quanto dos níveis de ERO intracellular. Esses fenômenos podem desempenhar um papel, na obtenção do estado capacitante da célula espermática, embora eles não sejam fundamentais na realização da posterior IVAE. Finalmente, a motilidade dos espermatozoides parece ser , parcialmente, controlada por mecanismos iônicos e de oxidação-redução, tal como indicou a incubação com GSH em condições separadas. Portanto es te estudo demonstra a existência de vias paralelas e separadas dentro dos eventos da capacitação espermática controladas pelo GSH e, assim, contribui para uma melhor compreensão desses fenômenos e traz um novo direcionamento para trabalhos futuros dentro dessa linha de pesquisa.

A study was conducted with the objective of evaluating the effects of reduced glutathione (GSH) over spermatic function during capacitation and posterior induction of acrosome reaction in vitro, in refrigerated pig semen. This was done by evaluating many in vitro capacitation markers and other global parameters of semen quality, such as feasibility, total motility, acrosome exocytosis induced by progesterone, DNA fragmentation, levels of lipid disorders in the spermatic membrane, reactive oxygen species (ROS), free cysteine residue in sperm head and tail extracts and protein phosphorylation of tyrosine residue. A total of 62 ejaculates derived from 35 healthy breeding Pietrain pigs, between 2 and 3 years of age, were used in the different analyses conducted in this work. The addition of GHS in the capacitation medium (CM) prevented most changes consequent to capacitation. In addition, the GSH caused quick fall and intense total motility (P < 0.05), which was maintained during the entire incubation period. Despite these results, the GH did not affect the beginning of in vitro acrosome exocytosis induced by progesterone (IVAE), observed after four hours of CM incubation. The results showed that in vitro pig semen capacitation is related to the significant increase of both total break of disulfide bonds and the levels of intracellular ROS. These phenomena can fulfill a role in obtaining the capacitating state of the spermatic cell, although they are not fundamental in performing the posterior IVAE. Finally, spermatozoid motility seems to be partially controlled by ionic and oxidation-reduction mechanisms, as was indicated by the GSH incubation in separate conditions. Therefore, this study demonstrates the existence of parallel and separated paths within the spermatic capacitation events controlled by the GSH, thus contributing for the better understanding of these phenomena, and brings a new direction for future works in this line of research.

Determination of disulfide bridges of two spider toxins: hainantoxin-III and hainantoxin-IV

Peptide toxins are usually highly bridged proteins with multipairs of intrachain disulfide bonds. Analysis of disulfide connectivity is an important facet of protein structure determination. In this paper, we successfully assigned the disulfide linkage of two novel peptide toxins, called HNTX-III and HNTX-IV, isolated from the venom of Ornithoctonus hainana spider. Both peptides are useful inhibitors of TTX-sensitive voltage-gated sodium channels and are composed of six cysteine residues that form three disulfide bonds, respectively. Firstly, the peptides were partially reduced by tris(2-carboxyethyl)-phosphine (TCEP) in 0.1 M citrate buffer containing 6 M guanidine-HCl at 40° C for ten minutes. Subsequently, the partially reduced intermediates containing free thiols were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and alkylated by rapid carboxamidomethylation. Then, the disulfide bonds of the intermediates were analyzed by Edman degradation. By using the strategy above, disulfide linkages of HNTX-III and HNTX-IV were determined as I-IV, II-V and III-VI pattern. In addition, this study also showed that this method may have a great potential for determining the disulfide bonds of spider peptide toxins.

Determination of disulfide bridges of two spider toxins: hainantoxin-III and hainantoxin-IV

Peptide toxins are usually highly bridged proteins with multipairs of intrachain disulfide bonds. Analysis of disulfide connectivity is an important facet of protein structure determination. In this paper, we successfully assigned the disulfide linkage of two novel peptide toxins, called HNTX-III and HNTX-IV, isolated from the venom of Ornithoctonus hainana spider. Both peptides are useful inhibitors of TTX-sensitive voltage-gated sodium channels and are composed of six cysteine residues that form three disulfide bonds, respectively. Firstly, the peptides were partially reduced by tris(2-carboxyethyl)-phosphine (TCEP) in 0.1 M citrate buffer containing 6 M guanidine-HCl at 40° C for ten minutes. Subsequently, the partially reduced intermediates containing free thiols were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and alkylated by rapid carboxamidomethylation. Then, the disulfide bonds of the intermediates were analyzed by Edman degradation. By using the strategy above, disulfide linkages of HNTX-III and HNTX-IV were determined as I-IV, II-V and III-VI pattern. In addition, this study also showed that this method may have a great potential for determining the disulfide bonds of spider peptide toxins.(AU)

Regulation of ovarian function: structural characterization and role of anti-müllerian hormone (AMH) / Regulação da função ovariana: caracterização estrutural e papel do hormônio anti-mülleriano (AMH)

Background: The anti-müllerian hormone (AMH) is a member of the transforming growth factor (TGF-) superfamily, which exerts important functions on local regulation of folliculogenesis. Although in vivo and in vitro studies suggest that AMH affects the primordial follicle assembly and activation, as well as the responsiveness of growing follicles to folliclestimulating hormone (FSH), the physiological mechanisms involved in these actions remain to be fully elucidated. Given the relevance of AMH in the folliculogenesis, this review aimed to describe the structural features, expression and the main biological effects of AMH on the follicular development. Review: Originally identified as a testicular product, AMH is responsible for regression of the Müllerian ducts during sexual differentiation of male embryos. In females, AMH is produced almost exclusively by granulosa cells of ovarian growing follicles, whose serum levels are positively related to the number of ovarian follicles, making AMH an excellent clinic marker of ovarian reserve. Along this work, it was shown aspects related to the structural characterization of AMH and its specific type II receptor (AMHRII). AMH is a glycoprotein dimer linked by disulfide bonds. The mature protein comprises two unequal domains: a long N-terminal domain (110-kDa) and a short C-terminal domain (25-kDa), responsible for the biological act

A foliculogênese é um evento complexo que envolve o recrutamento do pool de folículos primordiais, seguido por uma fase de crescimento e diferenciação. É sabido que para a sua regulação são requeridos vários fatores parácrinos e autócrinos, especialmente fatores de crescimento produzidos pelas células da granulosa. Dentre esses fatores, destaca-se o hormônio anti-mülleriano (AMH), uma glicoproteína membro da superfamília de fatores de crescimento transformantes (TGF-). Inicialmente o AMH foi estudado por seu papel regulatório no processo de diferenciação sexual masculina. [...]