Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K-i = 3.29 nM) and human cathepsin L (K-i= 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan. (C) 2011 Elsevier Inc. All rights reserved

Identification of a selenium-dependent glutathione peroxidase in the blood-sucking insect Rhodnius prolixus

The selenium-dependent glutathione peroxidase (SeGPx) is a well-studied enzyme that detoxifies organic and hydrogen peroxides and provides cells or extracellular fluids with a key antioxidant function. The presence of a SeGPx has not been unequivocally demonstrated in insects. In the present work, we identified the gene and studied the function of a Rhodnius prolixus SeGPx (RpSeGPx). The RpSeGPx mRNA presents the UGA codon that encodes the active site selenocysteine (Sec) and a corresponding Sec insertion sequence (SECIS) in the 3' UTR region. The encoded protein includes a signal peptide, which is consistent with the high levels of GPx enzymatic activity in the insect's hemolymph, and clusters phylogenetically with the extracellular mammalian GPx03. This result contrasts with all other known insect GPxs, which use a cysteine residue instead of Sec and cluster with the mammalian phospholipid hydroperoxide GPx04. RpSeGPx is widely expressed in insect organs, with higher expression levels in the fat body. RNA interference (RNAi) was used to reduce RpSeGPx gene expression and GPx activity in the hemolymph. Adult females were apparently unaffected by RpSeGPx RNAi, whereas first instar nymphs showed a three-day delay in ecdysis. Silencing of RpSeGPx did not alter the gene expression of the antioxidant enzymes catalase, xanthine dehydrognase and a cysteine-GPx, but it reduced the levels of the dual oxidase and NADPH oxidase 5 transcripts that encode for enzymes releasing extracellular hydrogen peroxide/superoxide. Collectively, our data suggest that RpSeGPx functions in the regulation of extracellular (hemolymph) redox homeostasis of R. prolixus. (C) 2015 Elsevier Ltd. All rights reserved

FcgammaRIIB inhibits the development of atherosclerosis in low-density lipoprotein receptor-deficient mice

The immune processes associated with atherogenesis have received considerable attention during recent years. IgG FcRs (FcgammaR) are involved in activating the immune system and in maintaining peripheral tolerance. However, the role of the inhibitory IgG receptor FcgammaRIIB in atherosclerosis has not been defined. Bone marrow cells from FcgammaRIIB-deficient mice and C57BL/6 control mice were transplanted to low-density lipoprotein receptor-deficient mice. Atherosclerosis was induced by feeding the recipient mice a high-fat diet for 8 wk and evaluated using Oil Red O staining of the descending aorta at sacrifice. The molecular mechanisms triggering atherosclerosis was studied by examining splenic B and T cells, as well as Th1 and Th2 immune responses using flow cytometry and ELISA. The atherosclerotic lesion area in the descending aorta was ~5-fold larger in mice lacking FcgammaRIIB than in control mice (2.75 +/- 2.57 versus 0.44 +/- 0.42%; p < 0.01). Moreover, the FcgammaRIIB deficiency resulted in an amplified splenocyte proliferative response to Con A stimulation (proliferation index 30.26 +/- 8.81 versus 2.96 +/- 0.81%, p < 0.0001) and an enhanced expression of MHC class II on the B cells (6.65 +/- 0.64 versus 2.33 +/- 0.25%; p < 0.001). In accordance, an enlarged amount of CD25-positive CD4 T cells was found in the spleen (42.74 +/- 4.05 versus 2.45 +/- 0.31%; p < 0.0001). The plasma Ab and cytokine pattern suggested increased Th1 and Th2 immune responses, respectively. These results show that FcgammaRIIB inhibits the development of atherosclerosis in mice. In addition, they indicate that absence of the inhibiting IgG receptor cause disease, depending on an imbalance of activating and inhibiting immune cells

Identification and Structural-Functional Analysis of Cyclin-Dependent Kinases of the Cattle Tick <i>Rhipicephalus (Boophilus) microplus</i>

<div><p>Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases essential for cell cycle progression. Herein, we describe the participation of CDKs in the physiology of <i>Rhipicephalus microplus</i>, the southern cattle tick and an important disease vector. Firstly, amino acid sequences homologous with CDKs of other organisms were identified from a <i>R. microplus</i> transcriptome database <i>in silico</i>. The analysis of the deduced amino acid sequences of CDK1 and CDK10 from <i>R. microplus</i> showed that both have caspase-3/7 cleavage motifs despite their differences in motif position and length of encoded proteins. CDK1 has two motifs (DKRGD and SAKDA) located opposite to the ATP binding site while CDK10 has only one motif (SLLDN) for caspase 3–7 near the ATP binding site. Roscovitine (Rosco), a purine derivative that inhibits CDK/cyclin complexes by binding to the catalytic domain of the CDK molecule at the ATP binding site, which prevents the transfer of ATP's γphosphoryl group to the substrate. To determine the effect of Rosco on tick CDKs, BME26 cells derived from <i>R. microplus</i> embryo cells were utilized <i>in vitro</i> inhibition assays. Cell viability decreased in the Rosco-treated groups after 24 hours of incubation in a concentration-dependent manner and this was observed up to 48 hours following incubation. To our knowledge, this is the first report on characterization of a cell cycle protein in arachnids, and the sensitivity of BME26 tick cell line to Rosco treatment suggests that CDKs are potential targets for novel drug design to control tick infestation.</p></div

CDK-gene read counts on RNA-Seq of <i>R. microplus</i> tissues.

<p>CDK-gene read counts on RNA-Seq of <i>R. microplus</i> tissues.</p

Hematoxylin-eosin staining of the BME26 cell line upon exposure to roscovitine BME26 cells after 24 h of incubation with different concentrations of roscovitine were stained with Hematoxylin and Eosin.

<p>A) Control with addition of 0.1% DMSO; B) rosco 150 µM; C) rosco 200 µM. The cells were observed under a light microscope. Magnification: 10X. The scale corresponds to 100 µm. Pictures are representative of 3 independent experiments in triplicates. D) Graphic represents the number of cells in each treatment quantified by image J in three different fields. On all tested concentrations, values of roscovitine groups were significantly different (One-way analysis of variance—ANOVA, <i>p</i><0.05) as compared to the control groups.</p

Identification of Rm-CDKs in BME26 cells.

<p>RT-PCR of BME26 cells showing the transcription of Rm-CDK1, 2, 5, 7, 8, 10, 11 and 14 in BME26 cells. ELF1A was used as a positive control. WM – weight marker; ELF – constitutive gene (108 bp); 1 – CDK1 (148 bp); 10 – CDK10 (155 bp); 2 – CDK2 (149 bp); 5 – CDK5 (151 bp); 7 – CDK7 (147 bp); 8 – CDK8 (152 bp); 9 – CDK9 (155 bp); 11 – CDK11 (150 bp); 14 – CDK14 (149 bp).</p

A, B: Top pose obtained by docking of roscovitine with Rm-CDK1 comparative model (yellow carbon atoms).

<p>Hydrogen atoms have been omitted for a better view. Hydrogen bonds are depicted in blue dashed lines.</p

MTT viability assay of BME26 cells.

<p>BME26 cells were incubated with different concentrations of roscovitine (a CDK inhibitor) or DMSO 0.1% (control) for 24 h (gray bar) and 48 h (grid bar) and then cell viability was determined by the MTT assay. After incubation the medium was removed and added MTT for 2 h. Reaction was read in spectrophotometer at 570 nm. Graph represents three independent experiments in triplicate. On all tested concentrations, with the exception of roscovitine 75 µM for 24 h, values of roscovitine groups were significantly different (One-way analysis of variance—ANOVA, <i>p</i><0.05) as compared to the control groups.</p