Spatial Distribution and Biochemical Characterization of Serine Peptidase Inhibitors in the Venom of the Brazilian Sea Anemone Anthopleura cascaia Using Mass Spectrometry Imaging.

Sea anemones are known to produce a diverse array of toxins with different cysteine-rich peptide scaffolds in their venoms. The serine peptidase inhibitors, specifically Kunitz inhibitors, are an important toxin family that is believed to function as defensive peptides, as well as prevent proteolysis of other secreted anemone toxins. In this study, we isolated three serine peptidase inhibitors named Anthopleura cascaia peptide inhibitors I, II, and III (ACPI-I, ACPI-II, and ACPI-III) from the venom of the endemic Brazilian sea anemone A. cascaia. The venom was fractionated using RP-HPLC, and the inhibitory activity of these fractions against trypsin was determined and found to range from 59% to 93%. The spatial distribution of the anemone peptides throughout A. cascaia was observed using mass spectrometry imaging. The inhibitory peptides were found to be present in the tentacles, pedal disc, and mesenterial filaments. We suggest that the three inhibitors observed during this study belong to the venom Kunitz toxin family on the basis of their similarity to PI-actitoxin-aeq3a-like and the identification of amino acid residues that correspond to a serine peptidase binding site. Our findings expand our understanding of the diversity of toxins present in sea anemone venom and shed light on their potential role in protecting other venom components from proteolysis.

Digestive enzymes and sphingomyelinase D in spiders without venom (Uloboridae).

Spiders have distinct predatory behaviours selected along Araneae's evolutionary history but are mainly based on the use of venom for prey paralysis. Uloboridae spiders have lost their venom glands secondarily during evolution. Because of this, they immobilise their prey by extensively wrapping, and digestion starts with the addition of digestive fluid. During the extra-oral digestion, the digestive fluid liquefies both the prey and the AcSp2 spidroins from the web fibres. Despite the efficiency of this process, the cocktail of enzymes involved in digestion in Uloboridae spiders remains unknown. In this study, the protein content in the midgut of Uloborus sp. was evaluated through enzymatic, proteomic, and phylogenetic analysis. Hydrolases such as peptidases (endo and exopeptidases: cysteine, serine, and metallopeptidases), carbohydrases (alpha-amylase, chitinase, and alpha-mannosidase), and lipases were biochemically assayed, and 50 proteins (annotated as enzymes, structural proteins, and toxins) were identified, evidencing the identity between the digestive enzymes present in venomous and non-venomous spiders. Even enzymes thought to be unique to venom, including enzymes such as sphingomyelinase D, were found in the digestive system of non-venomous spiders, suggesting a common origin between digestive enzymes and enzymes present in venoms. This is the first characterization of the molecules involved in the digestive process and the midgut protein content of a non-venomous spider.

Spiders' digestive system as a source of trypsin inhibitors: functional activity of a member of atracotoxin structural family.

Spiders are important predators of insects and their venoms play an essential role in prey capture. Spider venoms have several potential applications as pharmaceutical compounds and insecticides. However, transcriptomic and proteomic analyses of the digestive system (DS) of spiders show that DS is also a rich source of new peptidase inhibitor molecules. Biochemical, transcriptomic and proteomic data of crude DS extracts show the presence of molecules with peptidase inhibitor potential in the spider Nephilingis cruentata. Therefore, the aims of this work were to isolate and characterize molecules with trypsin inhibitory activity. The DS of fasting adult females was homogenized under acidic conditions and subjected to heat treatment. After that, samples were submitted to ion exchange batch and high-performance reverse-phase chromatography. The fractions with trypsin inhibitory activity were confirmed by mass spectrometry, identifying six molecules with inhibitory potential. The inhibitor NcTI (Nephilingis cruentata trypsin inhibitor) was kinetically characterized, showing a KD value of 30.25 nM ± 8.13. Analysis of the tertiary structure by molecular modeling using Alpha-Fold2 indicates that the inhibitor NcTI structurally belongs to the MIT1-like atracotoxin family. This is the first time that a serine peptidase inhibitory function is attributed to this structural family and the inhibitor reactive site residue is identified. Sequence analysis indicates that these molecules may be present in the DS of other spiders and could be associated to the inactivation of prey trypsin (serine peptidase) ingested by the spiders.

Spatial distribution and biochemical characterization of serine peptidase inhibitors in the venom of the brazilian sea anemone anthopleura cascaia using mass spectrometry imaging

Sea anemones are known to produce a diverse array of toxins with different cysteine-rich peptide scaffolds in their venoms. The serine peptidase inhibitors, specifically Kunitz inhibitors, are an important toxin family that is believed to function as defensive peptides, as well as prevent proteolysis of other secreted anemone toxins. In this study, we isolated three serine peptidase inhibitors named Anthopleura cascaia peptide inhibitors I, II, and III (ACPI-I, ACPI-II, and ACPI-III) from the venom of the endemic Brazilian sea anemone A. cascaia. The venom was fractionated using RP-HPLC, and the inhibitory activity of these fractions against trypsin was determined and found to range from 59% to 93%. The spatial distribution of the anemone peptides throughout A. cascaia was observed using mass spectrometry imaging. The inhibitory peptides were found to be present in the tentacles, pedal disc, and mesenterial filaments. We suggest that the three inhibitors observed during this study belong to the venom Kunitz toxin family on the basis of their similarity to PI-actitoxin-aeq3a-like and the identification of amino acid residues that correspond to a serine peptidase binding site. Our findings expand our understanding of the diversity of toxins present in sea anemone venom and shed light on their potential role in protecting other venom components from proteolysis.

Spiders digestive system as a source of trypsin inhibitors: functional activity of a member of atracotoxin structural family

Spiders are important predators of insects and their venoms play an essential role in prey capture. Spider venoms have several potential applications as pharmaceutical compounds and insecticides. However, transcriptomic and proteomic analyses of the digestive system (DS) of spiders show that DS is also a rich source of new peptidase inhibitor molecules. Biochemical, transcriptomic and proteomic data of crude DS extracts show the presence of molecules with peptidase inhibitor potential in the spider Nephilingis cruentata. Therefore, the aims of this work were to isolate and characterize molecules with trypsin inhibitory activity. The DS of fasting adult females was homogenized under acidic conditions and subjected to heat treatment. After that, samples were submitted to ion exchange batch and high-performance reverse-phase chromatography. The fractions with trypsin inhibitory activity were confirmed by mass spectrometry, identifying six molecules with inhibitory potential. The inhibitor NcTI (Nephilingis cruentata trypsin inhibitor) was kinetically characterized, showing a KD value of 30.25 nM ± 8.13. Analysis of the tertiary structure by molecular modeling using Alpha-Fold2 indicates that the inhibitor NcTI structurally belongs to the MIT1-like atracotoxin family. This is the first time that a serine peptidase inhibitory function is attributed to this structural family and the inhibitor reactive site residue is identified. Sequence analysis indicates that these molecules may be present in the DS of other spiders and could be associated to the inactivation of prey trypsin (serine peptidase) ingested by the spiders.

Digestive enzymes and sphingomyelinase D in spiders without venom (Uloboridae)

Spiders have distinct predatory behaviours selected along Araneae’s evolutionary history but are mainly based on the use of venom for prey paralysis. Uloboridae spiders have lost their venom glands secondarily during evolution. Because of this, they immobilise their prey by extensively wrapping, and digestion starts with the addition of digestive fluid. During the extra-oral digestion, the digestive fluid liquefies both the prey and the AcSp2 spidroins from the web fibres. Despite the efficiency of this process, the cocktail of enzymes involved in digestion in Uloboridae spiders remains unknown. In this study, the protein content in the midgut of Uloborus sp. was evaluated through enzymatic, proteomic, and phylogenetic analysis. Hydrolases such as peptidases (endo and exopeptidases: cysteine, serine, and metallopeptidases), carbohydrases (alpha-amylase, chitinase, and alpha-mannosidase), and lipases were biochemically assayed, and 50 proteins (annotated as enzymes, structural proteins, and toxins) were identified, evidencing the identity between the digestive enzymes present in venomous and non-venomous spiders. Even enzymes thought to be unique to venom, including enzymes such as sphingomyelinase D, were found in the digestive system of non-venomous spiders, suggesting a common origin between digestive enzymes and enzymes present in venoms. This is the first characterization of the molecules involved in the digestive process and the midgut protein content of a non-venomous spider.

Bioinformatic analyses to uncover genes involved in trehalose metabolism in the polyploid sugarcane.

Trehalose-6-phosphate (T6P) is an intermediate of trehalose biosynthesis that plays an essential role in plant metabolism and development. Here, we comprehensively analyzed sequences from enzymes of trehalose metabolism in sugarcane, one of the main crops used for bioenergy production. We identified protein domains, phylogeny, and in silico expression levels for all classes of enzymes. However, post-translational modifications and residues involved in catalysis and substrate binding were analyzed only in trehalose-6-phosphate synthase (TPS) sequences. We retrieved 71 putative full-length TPS, 93 trehalose-6-phosphate phosphatase (TPP), and 3 trehalase (TRE) of sugarcane, showing all their conserved domains, respectively. Putative TPS (Classes I and II) and TPP sugarcane sequences were categorized into well-known groups reported in the literature. We measured the expression levels of the sequences from one sugarcane leaf transcriptomic dataset. Furthermore, TPS Class I has specific N-glycosylation sites inserted in conserved motifs and carries catalytic and binding residues in its TPS domain. Some of these residues are mutated in TPS Class II members, which implies loss of enzyme activity. Our approach retrieved many homo(eo)logous sequences for genes involved in trehalose metabolism, paving the way to discover the role of T6P signaling in sugarcane.

Structural characterization of L-Galactose Dehydrogenase: an essential enzyme for vitamin C biosynthesis

In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learnt concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase (SoGDH from spinach), from the D-mannose/L-galactose (Smirnoff Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homologue from camu-camu, a super-accumulator of vitamin C found in the Peruvian amazon. Both enzymes are monomers in solution, have a pH optimum of 7 and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (β/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favours the oxidation reaction which ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes towards a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.

Bioinformatic analyses to uncover genes involved in trehalose metabolism in the polyploid sugarcane

Trehalose-6-phosphate (T6P) is an intermediate of trehalose biosynthesis that plays an essential role in plant metabolism and development. Here, we comprehensively analyzed sequences from enzymes of trehalose metabolism in sugarcane, one of the main crops used for bioenergy production. We identified protein domains, phylogeny, and in silico expression levels for all classes of enzymes. However, post-translational modifications and residues involved in catalysis and substrate binding were analyzed only in trehalose-6-phosphate synthase (TPS) sequences. We retrieved 71 putative full-length TPS, 93 trehalose-6-phosphate phosphatase (TPP), and 3 trehalase (TRE) of sugarcane, showing all their conserved domains, respectively. Putative TPS (Classes I and II) and TPP sugarcane sequences were categorized into well-known groups reported in the literature. We measured the expression levels of the sequences from one sugarcane leaf transcriptomic dataset. Furthermore, TPS Class I has specific N-glycosylation sites inserted in conserved motifs and carries catalytic and binding residues in its TPS domain. Some of these residues are mutated in TPS Class II members, which implies loss of enzyme activity. Our approach retrieved many homo(eo)logous sequences for genes involved in trehalose metabolism, paving the way to discover the role of T6P signaling in sugarcane.

Structural and functional characterization of the glutathione peroxidase-like thioredoxin peroxidase from the fungus Trichoderma reesei.

Glutathione peroxidases (GPx) are a family of enzymes with the ability to reduce organic and inorganic hydroperoxides to the corresponding alcohols using glutathione or thioredoxin as an electron donor. Here, we report the functional and structural characterization of a GPx identified in Trichoderma reesei (TrGPx). TrGPx was recombinantly expressed in a bacterial host and purified using affinity. Using a thioredoxin coupled assay, TrGPx exhibited activity of 28 U and 12.5 U in the presence of the substrates H2O2 and t-BOOH, respectively, and no activity was observed when glutathione was used. These results indicated that TrGPx is a thioredoxin peroxidase and hydrolyses H2O2 better than t-BOOH. TrGPx kinetic parameters using a pyrogallol assay resulted at Kmapp = 11.7 mM, Vmaxapp = 10.9 IU/µg TrGPx, kcat = 19 s-1 and a catalytic efficiency of 1.6 mM-1 s-1 to H2O2 as substrate. Besides that, TrGPx demonstrated an optimum pH ranging from 9.0-12.0 and a half-life of 36 min at 80 °C. TrGPx 3D-structure was obtained in a reduced state and non-catalytic conformation. The overall fold is similar to the other phospholipid-hydroperoxide glutathione peroxidases. These data contribute to understand the antioxidant mechanism in fungi and provide information for using antioxidant enzymes in biotechnological applications.

Structural and functional characterization of the glutathione peroxidase-like thioredoxin peroxidase from the fungus Trichoderma reesei

Glutathione peroxidases (GPx) are a family of enzymes with the ability to reduce organic and inorganic hydroperoxides to the corresponding alcohols using glutathione or thioredoxin as an electron donor. Here, we report the functional and structural characterization of a GPx identified in Trichoderma reesei (TrGPx). TrGPx was recombinantly expressed in a bacterial host and purified using affinity. Using a thioredoxin coupled assay, TrGPx exhibited activity of 28 U and 12.5 U in the presence of the substrates H2O2 and t-BOOH, respectively, and no activity was observed when glutathione was used. These results indicated that TrGPx is a thioredoxin peroxidase and hydrolyses H2O2 better than t-BOOH. TrGPx kinetic parameters using a pyrogallol assay resulted at Kmapp = 11.7 mM, Vmaxapp = 10.9 IU/μg TrGPx, kcat = 19 s−1 and a catalytic efficiency of 1.6 mM−1 s−1 to H2O2 as substrate. Besides that, TrGPx demonstrated an optimum pH ranging from 9.0–12.0 and a half-life of 36 min at 80 °C. TrGPx 3D-structure was obtained in a reduced state and non-catalytic conformation. The overall fold is similar to the other phospholipid-hydroperoxide glutathione peroxidases. These data contribute to understand the antioxidant mechanism in fungi and provide information for using antioxidant enzymes in biotechnological applications.

Identity and role of the non-conserved acid/base catalytic residue in the GH29 fucosidase from the spider Nephilingis cruentata

a-L-Fucosidases are widely occurring enzymes that remove fucose residues from N- and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all a-L-fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda a-L-fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active a-L-fucosidase from the spider Nephilingis cruentata (NcFuc) with a Km value for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2'fucosyllactose and also lacto-N-difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower kcat of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower kcat value of E59A. This was supported by the 57-fold increase in the kcat of E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new fucosidases.

First characterization of fucosidases in spiders

l-fucose is a constituent of glycoconjugates in different organisms. Fucosidases catalyze the removal of fucose residues, and have been correlated to different physiological and pathological processes, such as fertilization, cancer, fucosidosis, and digestion in molluscs and ticks. An alpha-L-fucosidase sequence was identified from the transcriptome and proteome from the midgut diverticula of the synanthropic spider Nephilingis cruentata. In this article, we describe the isolation of this alpha-L-fucosidase and the characterization of its activity using substrates and inhibitors demonstrating different specificities among fucosidases. The enzyme had a K-m of 32 and 400 mu M for 4-methylumbelliferyl alpha-L-fucopyranoside and 4-nitrophenyl alpha-L-fucopyranoside, respectively; and was unable to hydrolyze fucoidan. Nephilingis cruentata alpha-L-fucosidase was inhibited competitively by fucose and fuconojyrimycin. The fucosidase had two distinct pH optima even in the isolated form, due to oligomerization dependent on pH, as previously described to other fucosidases. Alignment and molecular homology modeling of the protein sequence with other fucosidases indicated that the active sites and catalytic residues were different, including residues involved in acid/base catalysis. Phylogenetic analysis showed, for the first time, gene-duplication events for fucosidases in Arachnida species. All these data reveal that studies on fucosidases in organisms distinct from bacteria, fungi, and humans are important.

Identity and role of the non-conserved acid/base catalytic residue in the GH29 fucosidase from the spider Nephilingis cruentata

a-L-Fucosidases are widely occurring enzymes that remove fucose residues from N- and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all a-L-fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda a-L-fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active a-L-fucosidase from the spider Nephilingis cruentata (NcFuc) with a Km value for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2'fucosyllactose and also lacto-N-difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower kcat of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower kcat value of E59A. This was supported by the 57-fold increase in the kcat of E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new fucosidases.

First characterization of fucosidases in spiders

l-fucose is a constituent of glycoconjugates in different organisms. Fucosidases catalyze the removal of fucose residues, and have been correlated to different physiological and pathological processes, such as fertilization, cancer, fucosidosis, and digestion in molluscs and ticks. An alpha-L-fucosidase sequence was identified from the transcriptome and proteome from the midgut diverticula of the synanthropic spider Nephilingis cruentata. In this article, we describe the isolation of this alpha-L-fucosidase and the characterization of its activity using substrates and inhibitors demonstrating different specificities among fucosidases. The enzyme had a K-m of 32 and 400 mu M for 4-methylumbelliferyl alpha-L-fucopyranoside and 4-nitrophenyl alpha-L-fucopyranoside, respectively; and was unable to hydrolyze fucoidan. Nephilingis cruentata alpha-L-fucosidase was inhibited competitively by fucose and fuconojyrimycin. The fucosidase had two distinct pH optima even in the isolated form, due to oligomerization dependent on pH, as previously described to other fucosidases. Alignment and molecular homology modeling of the protein sequence with other fucosidases indicated that the active sites and catalytic residues were different, including residues involved in acid/base catalysis. Phylogenetic analysis showed, for the first time, gene-duplication events for fucosidases in Arachnida species. All these data reveal that studies on fucosidases in organisms distinct from bacteria, fungi, and humans are important.

Data set of optimal parameters for colorimetric red assay of epoxide hydrolase activity.

The data presented in this article are related to the research article entitled "Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization" [1]. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. This article describes the optimal parameters for the colorimetric red assay to determine the enzymatic activity, with an emphasis on the characterization of the kinetic parameters, pH optimum and thermal stability of this enzyme. The effects of reagents that are not resistant to oxidation by sodium periodate on the reactions can generate false positives and interfere with the final results of the red assay.

Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization.

Epoxide hydrolases (EHs) are enzymes that are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EHs have biotechnological potential in chiral chemistry. We report the cloning, purification, enzymatic activity, and conformational analysis of the TrEH gene from Trichoderma reesei strain QM9414 using circular dichroism spectroscopy. The EH gene has an open reading frame encoding a protein of 343 amino acid residues, resulting in a molecular mass of 38.2kDa. The enzyme presents an optimum pH of 7.2, and it is highly active at temperatures ranging from 23 to 50°C and thermally inactivated at 70°C (t1/2=7.4min). The Michaelis constants (Km) were 4.6mM for racemic substrate, 21.7mM for (R)-(+)-styrene oxide and 3.0mM for (S)-(-)-styrene oxide. The kcat/Km analysis indicated that TrEH is enantioselective and preferentially hydrolyzes (S)-(-)-styrene oxide. The conformational stability studies suggested that, despite the extreme conditions (high temperatures and extremely acid and basic pHs), TrEH is able to maintain a considerable part of its regular structures, including the preservation of the native cores in some cases. The recombinant protein showed enantioselectivity that was distinct from other fungus EHs, making this protein a potential biotechnological tool.

Characterization of alpha-L-fucosidase and other digestive hydrolases from Biomphalaria glabrata

Schistosoma mansoni is one of the major agents of the disease Schistosomiasis, which is one of the major global public health concerns. Biomphalaria glabrata is an obligate intermediate mollusc host of S. mansoni. Although the development of S. mansoni occurs in the snail hepatopancreas, studies that focus on this organ remain limited. In this study, we biochemically identified five distinct carbohydrases (amylase, maltase, alpha-glucosidase, trehalase, and alpha-L-fucosidase), lipases, and peptidases in the B. glabrata hepatopancreas and focused on the isolation and characterization of the activity of alpha-L-fucosidase. The isolated alpha-L-fucosidase has a molecular mass of 141 kDa, an optimum pH of 5.8, and is inhibited by Tris, fucose, and 1-deoxyfuconojirimycin. B. glabrata alpha-L-fucosidase is an exoglycosidase that can hydrolyze the natural substrate fucoidan to fucose residues. It presented K-m values of 48.4 mu M to 4-Methylumbelliferyl alpha-L-fucopyranoside and 0.55 mM to p-nitrophenyl-alpha-L-fucopyranoside. Thus, alpha-L-fucosidase has a high activity in the hepatopancreas of B. glabrata, and the differential expression of this enzyme between susceptible and resistant strains indicates that besides its digestive role, alpha-L-fucosidase may also be important in host/parasite interactions.

Functional characterisation of vizottin, the first factor Xa inhibitor purified from the leech Haementeria vizottoi.

The strategic position of factor Xa (FXa) in blood coagulation makes it a compelling target for the development of new anticoagulants. Blood-sucking animals have in their salivary glands mixtures of anticoagulants, which could be used for designing novel antithrombotic compounds. Herein, we describe Vizottin, the first FXa inhibitor from the salivary complex of the leech Haementeria vizottoi . Vizottin was purified by gel filtration and reverse-phase chromatography, and shown to have anticoagulant effects in human plasma, prolonging the recalcification time in a dose-dependent manner (IC50 40 nM). Vizottin induced blood incoagulability in FX-deficient plasma, whereas in normal and reconstituted plasma, Vizottin doubled the prothrombin time at 160 nM. This peptide competitively inhibited human FXa (K(i) 2 nM) like FXa inhibitors from other leeches, albeit via a distinct mechanism of action. At high concentrations, vizottin inhibited the amidolytic activity of factor VIIa/tissue factor (IC50 96.4 nM). Vizottin inhibited FXa in the prothrombinase complex and Gla-domainless FXa. Moreover, vizottin did not interfere with FX activation induced by RVV-X, a known enzyme that requires the Gla-domain of FX for activation. Competition experiments in the presence of FXa and GGACK-FXa (active site blocked) demonstrated that the inhibition of FXa by vizottin is through binding to the active site rather than an exosite. This novel inhibitor appears to exert its inhibitory effects through direct binding to the active site of FXa in a time-dependent manner, but not involving a tight-binding model. In this context, vizottin is a promising model for designing novel anticoagulants for the treatment of thrombotic diseases.

Dimorfismo sexual alar em Aedes scapularis (Diptera: Culicidae) / Wing sexual dimorphism in Aedes scapularis (Diptera: Culicidae)

A detecção do sexo de mosquitos da família Culicidae é importante em estudos faunísticos e epidemiológicos, pois somente as fêmeas possuem competência vetora para patógenos. O dimorfismo sexual de genitália e de apêndices cefálicos é, em geral, facilmente visível em culicídeos. As asas também podem ser dimórficas e assim poderiam complementar o procedimento de sexagem. No entanto, tal distinção não é facilmente notável à observação direta. Visando descrever formalmente o dimorfismo sexual alar em Aedes scapularis, um culicídeo vetorialmente competente para arbovírus e filárias, asas de machos e fêmeas foram comparadas usando-se métodos de morfometria geométrica e análise estatística multivariada. Nestas análises, populações dos municípios São Paulo e Pariquera-Açu (Estado de São Paulo) foram amostradas. A forma das asas mostrou evidente dimorfismo sexual, o que permitiu um índice de acurácia de 100 por cento em testes-cegos de reclassificação, independentemente da origem geográfica. Já o tamanho alar foi sexualmente dimórfico apenas na população de São Paulo. Aparentemente, a forma alar é evolutivamente mais estável que o tamanho, interpretação que está de acordo com a teoria de Dujardin (2008b), de que a forma alar de insetos seria composta por caracteres genéticos quantitativos e pouco influenciada por fatores não-genéticos, enquanto que o tamanho alar seria predominantemente determinado por plasticidade decorrente de influências ambientais.

The sex in mosquitoes (Culicidae) is relevant for faunistic and epidemiological surveys because only females are competent to transmit pathogens. Sexual dimorphism comprising genitalia and cephalic appendages is ordinarily visible in culicids. Wings may be also dimorphic and thus be complementary to traditional sexing methods. Nevertheless, such dimorphism is not easily noticeable to direct observation. Aiming to formally describe the sexual dimorphism in Aedes scapularis, a mosquito species competent for virus and filariae, wings of males and females were compared using geometric morphometrics and multivariate statistics. We sampled populations from São Paulo and Pariquera-Açu municipalities (State of São Paulo). Concerning wing shape, the sexual dimorphism was evident from blind reclassification tests which were 100 percent accurate, independently of the geographical origin of samples. On the other hand, wing size was sexually dimorphic only in the population from Sao Paulo. Presumably, wing shape is evolutionarily more stable than its size, an interpretation which is in accordance with the theory posed by Dujardin (2008b). That theory indicates wing shape would be composed of genetically-determined quantitative characters, and marginally influenced by non-genetic factors, whereas wing size would be more labile and predominantly influenced by the environment.