Biochemical characterization of the cAMP-dependent protein kinase regulatory subunit-like protein from Trypanosoma equiperdum, detection of its inhibitory activity, and identification of potential interacting proteins.

An enriched fraction of an inhibitor of both the catalytic subunit of the cAMP-dependent protein kinase (PKA) from pig heart and a Trypanosoma equiperdum PKA catalytic subunit-like protein (TeqC-like) was obtained from the soluble fraction of T. equiperdum parasites after three consecutive purification steps: sedimentation through a linear 5-20% sucrose gradient, diethylaminoethyl-Sepharose anion-exchange chromatography, and Bio-Sil Sec-400-S size-exclusion high-performance liquid chromatography. The inhibitor was identified as the T. equiperdum PKA regulatory subunit-like protein (TeqR-like) on the basis of Western blot and mass spectrometry analyses, and behaved as an uncompetitive or anti-competitive inhibitor of the parasite TeqC-like protein, with respect to a fluorescently labeled substrate (kemptide, sequence: LRRASLG), showing a Ki of 1.17 µM. The isolated protein possesses a molecular mass of 57.54 kDa, a Stokes radius of 3.64 nm, and a slightly asymmetric shape with a frictional ratio f/fo = 1.43. As revealed during the purification steps and by immunoprecipitation experiments, the TeqR-like and TeqC-like proteins were not associated forming a heterooligomeric complex, differing from traditional PKA subunits. Co-immunoprecipitation results followed by mass spectrometry sequencing identified two isoforms of the parasite heat-shock protein 70, α-tubulin, and ß-tubulin as candidates that interact with the TeqR-like protein in T. equiperdum.

Browplasminin, a condensed tannin with anti-plasmin activity isolated from an aqueous extract of Brownea grandiceps Jacq. flowers.

ETHNOPHARMACOLOGICAL RELEVANCE: Following Venezuelan traditional medicine, females with heavy menstrual blood loss (menorrhagia) drink Brownea grandiceps Jacq. flowers (BG) decoctions to reduce the bleeding. In a previous study, we demonstrated that BG aqueous extract (E) possesses a potent anti-fibrinolytic activity capable of inhibiting plasmin, the main serine-protease that degrades fibrin. It is widely known that plasmin inhibitors are often used as anti-fibrinolytics to reduce bleeding during surgeries with high risk of blood loss such as cardiac, liver, vascular, tooth extraction and large orthopedic procedures, as well as for menorrhagia treatments. The aim of this work was to isolate and characterize from BGE the compound responsible for the reported anti-fibrinolytic activity. MATERIALS AND METHODS: A decoction of BG was prepared; then it was homogenized, centrifuged and lyophilized to obtain BGE. Subsequently the extract was fractionated by gel filtration and reverse phase using HPLC and the active compound was characterized by MALDI-ToF MS. The kinetic parameters of anti-plasmin activity were evaluated by an amidolytic assay using a chromogenic substrate; also the anti-plasmin activity was estimated by fibrin plate method. Data were analyzed by nonparametric statistics. RESULTS: The active compound was a condensed tannin denominated Browplasminin, which is capable of inhibiting the plasmin activity in a dose-dependent manner when measured in fibrin plates or by the amidolytic activity method; it also has a minor effect on the FXa activity. However, it does not affect the activity of other serine-proteases such as trypsin, t-PA or u-PA. Browplasminin consists predominately of heteroflavan-3-ols of catechin with B-type linkages, and extents up to heptadecamers (~ 5000Da), with hexose residues attached to the polymer that presents a high degree of galloylation. Its IC50 for plasmin was 47.80µg/mL and for FXa was 237.08µg/mL, while the Ki were 0.76 and 61.61µg/mL for plasmin and FXa, respectively. CONCLUSIONS: The overall outcome of this study suggests that Browplasminin could be responsible for reducing heavy menstrual bleeding in women because its kinetic parameters showed that is a good plasmin inhibitor.

Protein content of the Hylesia metabus egg nest setae (Cramer [1775]) (Lepidoptera: Saturniidae) and its association with the parental investment for the reproductive success and lepidopterism.

Hylesia metabus is a neotropical moth possessing toxic setae, which once in contact with the skin cause a severe dermatitis to humans known as lepidopterism. The only known function of the setae in the life cycle is to provide protection during the mating and egg-hatching stages. Approximately 65% of the protein content of the setae is a cluster of five proteases (28-45kDa) showing sequence homology to other S1A serine proteases. The N-glycans of a 40kDa protease are a mixture of neutral and sulfated G0F structures. The sulfated N-glycans have an important role in triggering the inflammatory response typical of lepidopterism while the proteolytic activity may promote the erosion of blood vessels and tissues causing focal hemorrhages. The presence of Chitinase and a 30kDa lipoprotein is probably related to the antifungal defense. In addition, chitin digestion of the setae may potentiate the inflammatory reaction caused by the toxins due to the formation of chitin adjuvants fragments. The combined effect of proteases and a chitinase may dissuade predating arthropods, by damaging their exoskeletons. Vitellogenin, a bacteriostatic protein, is able to recognize pathogen-associated patterns, which suggests its possible role in protecting the embryonated eggs from pathogenic microorganisms. SIGNIFICANCE: The present study is the first report describing the different protein species present in the urticating egg nest setae of the neotropical moth Hylesia metabus - the most harmful of the Hylesia moths - causing a severe urticating dermatitis in humans known as lepidopterism. A distinctive feature of the venom is the presence of five different S1A serine proteases probably used to guarantee a more efficient degradation of a wider number of protein substrates. This work confirms that the presence of sulfated N-glycans is not an isolated finding since its presence has been demonstrated in two different proteases affirming that this PTM is of importance for the activation of the inflammatory response typical of lepidopterism. Additionally, this study gives useful information on the defense mechanisms used for protection of its progeny vs. vertebrate predators, fungus, bacteria or other arthropods such as ants. The proteins detected in the egg nest should be seen as an extended parental effort made by the females in order to achieve an optimal reproductive success, thus compensating for the considerable loss of progeny during the larval stages that seriously limits the number of sexually mature adults reaching the reproductive phase.

Structural characterization and biological implications of sulfated N-glycans in a serine protease from the neotropical moth Hylesia metabus (Cramer [1775]) (Lepidoptera: Saturniidae).

Contact with the urticating setae from the abdomen of adult females of the neo-tropical moth Hylesia metabus gives rise to an urticating dermatitis, characterized by intense pruritus, generalized malaise and occasionally ocular lesions (lepidopterism). The setae contain a pro-inflammatory glycosylated protease homologous to other S1A serine proteases of insects. Deglycosylation with PNGase F in the presence of a buffer prepared with 40% H2 (18)O allowed the assignment of an N-glycosylation site. Five main paucimannosidic N-glycans were identified, three of which were exclusively α(1-6)-fucosylated at the proximal GlcNAc. A considerable portion of these N-glycans are anionic species sulfated on either the 4- or the 6-position of the α(1-6)-mannose residue of the core. The application of chemically and enzymatically modified variants of the toxin in an animal model in guinea pigs showed that the pro-inflammatory and immunological reactions, e.g. disseminated fibrin deposition and activation of neutrophils, are due to the presence of sulfate-linked groups and not on disulfide bonds, as demonstrated by the reduction and S-alkylation of the toxin. On the other hand, the hemorrhagic vascular lesions observed are attributed to the proteolytic activity of the toxin. Thus, N-glycan sulfation may constitute a defense mechanism against predators.

Comparative proteomic analysis of growth hormone secretagogue A233 treatment of murine macrophage cells J774A.2 indicates it has a role in antiviral innate response.

BACKGROUND: Growth hormone secretagogues (GHS), among other factors, regulate the release of GH. The biological activity of the secretagogue peptide A233 as a promoter of growth and innate immunity in teleost fish has previously been demonstrated, but its role in the immune system of mammals is not well understood. METHODS: The effect of the peptide was investigated in J774A.2 macrophage cells using a comparative proteomics approach after 6 and 12 h of peptide stimulation. RESULTS: The functional analysis of differentially modulated proteins showed that A233 peptide treatment appears to promote activation and ROS-dependent cytotoxic functions in macrophages and enhanced expression of antiviral protein complexes such as MAVS. In accordance with this hypothesis, we found that A233 treatment enhanced superoxide anion production and the IFN-γ level in J774A.2 cells and mouse splenocytes, respectively, and reduced viral load in a dengue virus mouse model of infection. CONCLUSIONS: The growth hormone secretagogue A233 peptide promotes activation of ROS-dependent cytotoxic functions and exerts immunomodulatory effects that enable an antiviral state in a dengue virus mouse model. GENERAL SIGNIFICANCE: The increase of IFN-γ level and the differential modulation of antiviral proteins by the A233 peptide suggest that the molecule could activate an innate immune response with a possible further impact in the treatment of acute and chronic diseases.

Desmielinización in vitro inducida por inyección endoneural de suero y líquido céfalo-raquídeo de pacientes con síndrome de Guillain Barré, evidenciada por difracción de rayos X / Demyelination in vitro induced by serum and cerebrospinal fluid in patients with an acute stage of Guillain-Barre was demostrated by X rays diffraction

Estudiamos la desmielinización inducida por suero y líquido céfalo-raquídeo de 12 pacientes con síndrome de Guillain-Barre en fase aguda, inyectados en el nervio ciático derecho de ratas Lewis. En siete casos el material se había almacenado congelado a -70ºC hasta su uso, en los otros cinco, el material se utilizó inmediatamente después de su obtención; como control se usó el nervio ciático contra-lateral. Además, se inyectó un grupo de ratas con solución salina y otro con suero de personas sanas para comprobar que la inyección "per se" y la inoculación de suero humano normal no produce daño de la mielina. Para demostrar la desmielinización se usó la técnica de difracción de rayos X que mide la distancia de repetición D entre dos lamelas contiguas de mielina y el porcentaje de mielina total. Los nervios inyectados con solución salina y suero de personas sanas no mostraron variaciones en relación a los controles, como tampoco los nervios inyectados con suero y líquido céfalo-raquídeo de pacientes con síndrome de Guillain-Barré congelado previamente. En los nervios inyectados con suero en fresco, el valor de D fue de 173,3 A en comparación con 171,3 A de los controles; el porcentaje de mielina total fue de 0,51 por ciento en relación a los controles (0,62 por ciento). La D de los nervios inyectados con líquido céfalo-raquídeo en fresco en promedio fue mayor (174,3 A) y el porcentaje de mielina total fue menor (0,54 por ciento) que el valor de los controles (171,3 A y 0,62 por ciento respectivamente)