Abstract Fifteen polar extracts from leaf, seed, pod, stem, flower and root of Crotalaria spectabilis were prepared using aqueous systems, based on the principles of green chemistry, and showed different protease inhibitor (PI) activities on trypsin, papain, pepsin and the extracellular L. amazonensis serine protease (LSPIII). The most pronounced inhibitory effect on LSPIII was observed in leaf (CS-P), root, stem, flower (CS-FPVPP) and pod (CS-VA) extracts. Crotalaria extracts exhibited low cytotoxicity on macrophages; however, they decreased the viability of L. amazonensis promastigotes and amastigotes, as observed in leaf (CS-AE, CS-P, CS-T and CS-PVPP), seed (CS-ST), flower and root (CS-RA) extracts. CS-P was chosen to study PI and secondary metabolites and a 10-12 kDa protein, analyzed by mass spectrometry, was identified as a serine PI homologous with papaya latex serine PI. Glycosylated flavonoids, such as quercetins, vitexin and tricin were the major secondary metabolites of CS-P. The presence of PIs in C. spectabilis is a new finding, especially in other organs than seeds since PIs have been reported only in seed legumes. Besides, this is the first report of antileishmanial activity of C. spectabilis extracts and the identification of serine polypeptide PI and glycosylated flavonoids from leaf.

Resumo Ornithodoros mimon é um carrapato argasídeo parasita de morcegos, aves e marsupiais, além de ser bastante agressivo aos humanos. O conhecimento dos transcritos presentes no intestino dos carrapatos auxilia no entendimento do papel de moléculas vitais no processo de digestão e na relação parasito-hospedeiro, além de fornecer também informações sobre a evolução dos artrópodes hematófagos. Desta maneira, o presente estudo teve como objetivo conhecer e identificar as principais moléculas expressas no intestino de uma espécie de carrapato argasídeo após o repasto sanguíneo, através de uma análise transcritômica descritiva do intestino de fêmeas ingurgitadas de O. mimon, utilizando um sequenciamento de RNA de nova geração da plataforma 454. Além de inferir a relação filogenética de carrapatos através de um conjunto de dados transcritômicos. O transcriptoma do intestino revelou diversos transcritos associados com a digestão da hemoglobina, como proteinases das classes serino, cisteína, aspártica e metalo. Registramos a presença de um transcrito de uma cisteína peptidase do tipo catepsina O em carrapatos. A inferência filogenética baseada em conjunto de dados transcritos homólogos tem uma resolução topológica similar a de outros conjuntos de dados moleculares. Foram depositados no banco de dados gênico público 2213 transcritos de O. mimon. Os achados obtidos no presente estudo podem contribuir para compreensão dos importantes processos, como digestão, nutrição e imunidade dos carrapatos da família Argasidae, além de fornecer informações sobre a filogenia da ordem Ixodida.

Abstract Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also harmful to humans. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to know and ascertain the main molecules expressed in the gut of argasid after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon using 454-based RNA sequencing. The gut transcriptome analysis reveals several transcripts associated with hemoglobin digestion, such as serine, cysteine, aspartic proteases and metalloenzymes. The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. We recorded the presence a cathepsin O peptidase-coding transcript in ticks. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on mitochondrial genome and nuclear rRNA sequences. We deposited 2,213 sequence of O. mimon to the public databases. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity.

BACKGROUND Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells. OBJECTIVES To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters. METHODS Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed. FINDINGS In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1β, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters. MAIN CONCLUSIONS L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function.

In patients affected by differentiated thyroid cancer, the whole-body scan (WBS) with 131-radioiodine, especially when performed after a therapeutic activity of131I, represents a sensitive procedure for detecting thyroid remnant and/or metastatic disease. Nevertheless, a wide spectrum of potentially pitfalls has been reported. Herein we describe a 63-year-old woman affected by follicular thyroid cancer, who was accidentally found to have an abdominal mass at post-dose WBS (pWBS). pWBS showed abnormal radioiodine uptake in the upper mediastinum, consistent with lymph-node metastases, and a slight radioiodine uptake in an abdominal focal area. Computed tomography revealed an inhomogeneous mass in the pelvis, previously unrecognized. The lesion, surgically removed, was found to be a typical dermoid cyst of the ovary, without any evidence of thyroid tissue. By immunohistochemistry, a moderate expression of the sodium-iodine symporter (NIS) was demonstrated in the epithelial cells, suggesting a NIS-dependent uptake of radioiodine by the cyst.

Gastrointestinal anisakiasis is a parasitic infection occurring in people that consume raw or inadequately cooked fish or squid. It is frequently characterized by severe epigastric pain, nausea and vomiting caused by the penetration of the larvae into the gastric wall. Acute gastric anisakiasis with severe chest discomfort is rarely reported in Italy. On the other hand, gastro-allergic anisakiasis with rash, urticaria and isolated angioedema or anaphylaxis is a clinical entity that has been described only recently. Also, if patients usually develop symptoms within 12 hours after raw seafood ingestion, not always endoscopic exploration can promptly identify the Anisakis larvae. Moreover, some authors consider the prevailing allergic reaction as a natural and effective defense against the parasitic attack. We report two cases of peculiar manifestations of anisakiasis in both acute and chronic forms (severe chest discomfort and anaphylactoid reaction).

Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania) amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME) as substrate, phenylmethylsulphone fluoride (PMSF) and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK) as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate), but also in a crude plasma membrane fraction (2.0-fold). Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP), with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

Isolou-se uma lectina a partir do extrato salino da fava dos frutos de Caesalpinia tinctoria mediante dialofiltração e cromatografia de afinidade em coluna de quitina. A lectina purificada foi caracterizada parcialmente com relação às suas propriedades bioquímicas e estruturais, e classificada como um glicopeptídio contendo 8,3 % de carboidratos, aglutinando hemácias do grupo ABO. Sua composição em aminoácidos foi caracterizada por grande número de resíduos ácidos e hidrofóbicos, e sua massa molecular, estimada em 12,5 kDa. A análise do grupo N-terminal mostrou a homogeneidade da lectina, cuja seqüência dos 21 resíduos iniciais é a seguinte: D¹-V-P-A-Y-V-Y-V-H-F10-G-F-G-E-E-H-R-D-V-F 20-D. O espectro de dicroísmo circular da lectina na região do UV distante indicou a presença de 10 % de a-hélice; 38 % de folhas b; 19 % de voltas b; 28 % de estruturas desordenadas e 6 % da conformação P II (hélice poli-L-prolina II).

A lectin was isolated from the pod saline extract of Caesalpinia tinctoria by dialoconcentration on Centripep-10 and affinity chromatography on chitin column. The purified lectin was partially characterized with respect to its biochemical and structural properties. It contains 8.3 % of carbohydrate and exhibited an agglutinating activity against human erythrocytes (ABO groups). Its amino acid composition was characterized by a great number of acidic and hydrophobic residues and the estimated molecular mass was 12.5 kDa. The presence of only one N-terminal amino acid sequence (D¹-V-P-A-Y-V-Y-V-H-F10-G-F-G-E-E-H-R -D-V-F20-D), showed the homogeneity of the purified lectin. The far-ultraviolet circular dichroism (CD) spectrum of lectin indicated that it contains 10 % a-helix, 38 % b-sheet, 28 % unordered form and 6 % of P II (poly-L-proline II helix conformation).

The four dominant outer membrane proteins (46, 38, 33 and 28 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a semi-purified preparation of vesicle membranes of a Neisseria meningitidis (N44/89, B:4:P1.15:P5.5,7) strain isolated in Brazil. The N-terminal amino acid sequence for the 46 kDa and 28 kDa proteins matched that reported by others for class 1 and 5 proteins respectively, whereas the sequence (25 amino acids) for the 38 kDa (class 3) protein was similar to class 1 meningococcal proteins. The sequence for the 33 kDa (class 4) was unique and not homologous to any known protein.

A distribuição das proteínas superficiais de Toxoplasma gondii radiodinadas, foram estudadas usando a técnica de separação de fases e a capacidade de ligação em coluna de fenil-Sepharose. Oito polipeptídeos com peso molecular entre 22 e 180.000 distribuíram-se exclusivamente na fase rica em detergente enquanto seis polipeptídeos com PM entre 15.00 e 76.000 distribuíram-se exclusivamente na fase pobre em detergente. Dois polipeptídeos com 15.00 e 70.000 distribuem-se em ambas as fases. Todos os polipeptídeos presentes na fase rica em detergente foram retidos por coluna de fenil-Sepharose e isolados em dois picos de acordo com sua hidrofobicidade relativa. Dois polipeptídeos hidrofóbicos com PM 60.000 e 66.000 reconhecidos por soro humano, foram isolados pela associação das duas técnicas. Os nossos resultados mostram que as proteínas de superfície do T. gondii possuem diferentes graus de hidrofobicidade e que o emprego da cromatografia de interação hidrofóbica após extração com Triton X-114, pode ser um importante método de isolamento de proteínas de membrana.

The distribution of the surface proteins of toxoplasma gondii radiodinated were studied using the phase separation technique and ability of binding in the phenyl-Sepharose column. Eight polypeptides with Mr 22 to 180 distributed exclusively in the detergent rich-phase, while six polypeptides with mol. wt. 15,000 to 76,000 distributed exclusively in the detergent poor-phase. Twopolypeptides with 15,000 and 70,000 distributed on both phase. All the polypeptides present in the detergent rich-phase binding in the phenyl-Sepharose column, and can be isolated in two peak according with their relative hydrophobicities.two polypeptides hydrophobic with Mr 60 and 66 recognized by human serum were isolated by the association of the two technique. Our result showed that the surface proteins of t. gondii present different degrees of hydrophobicity and that the use of hydrophobic interaction chromatography after Triton X-114 extraction may be an important isolation method of membrane proteins.