Characterization of a novel cystatin type 2 from Rhipicephalus microplus midgut.

The Rhipicephalus (Boophilus) microplus is an exclusive bovine ectoparasite responsible for the transmission of pathogens that decrease meat, leather and milk productions. Cattle vaccination is an alternative to control tick infestations, but the discovery of potential antigens is still a challenge for researchers. Recently, our group performed a midgut transcriptome of engorged R. microplus tick, and out of 800 ESTs sequences one cystatin-coding sequence was identified and named Rmcystatin-4. In order to understand the physiological role of Rmcystatin-4, the aim of this work was the expression, purification and functional characterization of a novel type 2 cystatin from the tick R. microplus. Rmcystatin-4 gene expression was identified mostly in tick midgut suggesting its possible role in blood digestion control. Our data showed that rRmcystatin-4 was successfully expressed in active form using Pichia pastoris system and the purified inhibitor presented high selectivity to BmCl-1 (Ki = 0.046 nM). Moreover, rRmcystatin-4 was able to impaired BmCl-1 activity towards bovine hemoglobin.

Identification of cystatin SN as a novel biomarker for pancreatic cancer.

Cystatin SN (cystatin 1, CST1) is a member of the cystatin superfamily that inhibits the proteolytic activity of cysteine proteases. CST1 is a tumor biomarker that provides useful information for the diagnosis of esophageal, gastric, and colorectal carcinomas. However, the significance of CST1 in pancreatic cancer is unknown. The aim of this study was to assess whether CST1 is a potential biomarker for early diagnosis of malignant pancreatic neoplasms. Microarray analysis of mRNA extracted from pancreatic cancer and pancreatic normal tissues was performed. Bioinformatics revealed that CST1 was one of the highest expressed genes on the array in pancreatic cancer, compared with normal tissue. In addition, the upregulation of CST1 in pancreatic cancer and several pancreatic cancer cell lines was confirmed using real-time PCR (RT-PCR), immunohistochemistry, and Western blotting. Next, CST1 knockdown using siRNA reduced the expression of the proliferation-related proteins p-AKT and PCNA significantly, as well as colony formation and xenograft development in vitro. Consistent with this, CST1 mRNA overexpression was correlated closely with malignancy-associated proteins such as PCNA, cyclin D1, cyclin A2, and cyclin E in pancreatic cancer cell lines. In conclusion, our data suggest that CST1 might contribute to the proliferation of pancreatic cancer cells and could be a potential biomarker for the early detection of pancreatic cancer.

Caffeine increases the expression of cystatin SN in human submandibular acinar-like HSG cells.

OBJECTIVE: The study aimed at evaluating in vitro the effect of caffeine on expression of cystatin SN, a potential marker of sensitivity to bitterness in humans. METHODS: Differentiation of human submandibular gland (HSG) cells was induced by culturing cells on Matrigel. Caffeine cytotoxicity was assessed over 3 days by the Resazurin test. Finally, effects of 5, 50 and 100µM caffeine exposure on cystatin SN expression were explored over 3 days by ELISA. RESULTS: At concentrations relevant to human adult plasma levels (5, 50 and 100µM), caffeine did not affect cell viability whether cells were differentiated or not. Cystatin SN levels were overall higher in differentiated cells and increased with time in both conditions. There was a significant (p<0.001) effect of caffeine on cystatin SN expression specifically in differentiated cells. CONCLUSIONS: The HSG cell line proved to be a relevant tool to study in vitro the effect of caffeine at concentrations consistent with dietary intake in human subjects. The results suggest that salivary cystatin SN abundance may depend on caffeine intake, with possible consequences on taste sensitivity.

Analysis of the lung transcriptome in Mycobacterium tuberculosis-infected mice reveals major differences in immune response pathways between TB-susceptible and resistant hosts.

Using whole genome microarrays, we compared changes in gene expression patterns in the lungs of TB-resistant A/Sn and TB-susceptible I/St mice at day 14 following infection with Mycobacterium tuberculosis H37Rv. Analyses of differentially expressed genes for representation of gene ontology terms and activation of regulatory pathways revealed interstrain differences in antigen presentation, NK, T and B cell activation pathways. In general, resistant A/Sn mice exhibited a more complex pattern and stronger activation of host defense pathways compared to the TB-susceptible I/St mouse strain. In addition, in I/St mice elevated activation of genes involved in neutrophil response was observed and confirmed by quantitative RT-PCR and histopathology. Furthermore, a specific post infection upregulation of cysteine protease inhibitors was found in susceptible I/St mice.

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.

Novel expression of CST1 as candidate senescence marker.

Senescent cells exhibit altered expression of numerous genes. Identifying the significance of the changes in gene expression may help advance our understanding of the senescence biology. Here, we report on the consistent and strong upregulation of CST1 expression during cellular senescence, independent of the initial trigger. CST1 expression at both the messenger RNA and protein levels was barely detected in control cells, which included early passage proliferating, quiescent, or immortal human fibroblasts and various human tumor cell lines. Immunoblotting and immunofluorescence cytochemical studies further suggest that CST1 accumulates intracellularly, within vesicular structures. We discuss these results in light of the known function of CST1 as a potent inhibitor of lysosomal cysteine proteases.