Quantitative measurement of iNOS expression in melanoma, nasopharyngeal, colorectal, and breast tumors of Tunisian patients: comparative study and clinical significance.

Chronic inflammation increases the risk of development of human malignancies. iNOS is an enzyme dominantly expressed during inflammatory reactions and seems to play a critical role in tumorigenesis. Our aim was to assess the iNOS expression in four types of human tumors: breast, colorectal, nasopharyngeal, and melanoma, of Tunisian patients. The level of iNOS was measured by RT-QPCR in tumor specimens. We showed that the expression of iNOS was higher in breast compared to colorectal and nasopharyngeal tumors, whereas in melanoma, the level of iNOS expression was low. Significant associations were found when comparing the iNOS expression in cancers pairs such as melanoma versus colorectal (p < 0.0001), colorectal versus nasopharyngeal (p = 0.0072), and melanoma versus breast (p < 0.0001). Furthermore, iNOS expression correlated with the Breslow thickness, Clark level, and histological subtype in melanoma, while in nasopharyngeal carcinoma, significant association was seen with age at diagnosis, TNM, metastasis, response to treatment, and expression of COX-2. Furthermore, the expression of iNOS correlated with tumor size, TNM, tumor location, and histological type in colorectal cancer, and with tumor size, tumor stage, SBR grade, and triple negative cases in breast cancer. On the other hand, immunohistochemistry analysis shows that the expression of iNOS is observed in the stroma and tumor cells as well. Overall, our results highlight that iNOS is a reliable marker for advanced stage and aggressive behavior for the four types of cancer and might be a potential promising therapeutic target.

Characterization of C69R variant HBsAg: effect on binding to anti-HBs and the structure of virus-like particles.

Several variants of the major "a" determinant of the HBsAg, the main target of HBV neutralization by antibodies, have been described. However, mutations outside this region have not been as thoroughly investigated. During the genotyping of HBV from Tunisian patients with chronic hepatitis B, we identified a variant with a C69R substitution in the cytosolic loop of the S protein, resulting in a change in the hydrophobicity profile compared to the wild-type HBsAg. Wild-type and mutant HBsAgs were produced in Saccharomyces cerevisiae and recombinant proteins were tested for their ability to correctly self-assemble into virus-like particles (VLPs), and their ability to bind to HBs antibodies. The C69R substitution resulted in a decrease in binding to commercial anti-HBs antibodies, and although the variant appeared to assemble properly into VLPs, the average size of the particles was larger than that of the wild-type HBsAg. Prediction of the tertiary structure of the C69R mutant revealed a change in the first (aa 60-70) and the second loop (aa 110 to 120) compared to the wild-type protein. Furthermore, we showed by an isothermal titration calorimetry assay that the interaction between the wild-type HBsAg and the anti-HBs antibody was exothermic, whereas that with the mutant C69R was endothermic, indicating an effect on the binding affinity.

Negative control glucose dependent mediated by the PreS2 region on the translation efficiency of the reporter Sh-bleomycin gene in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is able to sense and respond to environmental changes such as the availability of carbon sources. In a previous work, we showed that the expression of the PreS2-S gene of HBV in yeast was negatively regulated at the translational level dependent of glucose. In this study, we show that the S mRNA is detected in the polysomes indicating its active translation, while the PreS2-S mRNA was mainly found in monosomes. Moreover, we used the gene reporter assay based on Zeocin resistance, to better characterize the PreS2 region responsible for this control. Two chimeric genes composed of the N- and C-terminal part of the PreS2 fused to the Sh-bleomycin gene conferring the resistance to Zeocin were expressed in yeast. We found that the strain expressing the N-terminal part of the PreS2 was sensitive to Zeocin on rich medium with 2% glucose. In contrast, the strain harbouring the C-terminal part of the PreS2 fused to the Sh-bleomycin grew on Zeocin, indicating that the Sh-bleomycin mRNA is efficiently translated, subsequently conferring resistance to Zeocin. Our data suggest the establishment of a translational control via the N-terminal part of the PreS2 mediated by the presence of 2% glucose in the media.

Extraction and purification of hepatitis B virus-like M particles from a recombinant Saccharomyces cerevisiae strain using alumina powder.

A recombinant hepatitis B surface antigen (HBsAg) has been produced in the yeast Saccharomyces cerevisiae and used as a vaccine against hepatitis B virus (HBV) infection. The present study aimed to optimize the extraction of recombinant virus-like particles (rVLPs) to develop a simple purification procedure based on gel filtration and high performance size-exclusion chromatography. The findings showed that disruption of yeast cells with alumina powder increased the yield of the total proteins (290mg/l) and rVLPs (1mg/l) compared to the values for glass beads (171mg/l and 0.5mg/l), as estimated by quantitative ELISA. The purification of rVLPs was performed by two consecutive gel filtration chromatographic steps, namely Sephacryl S-200 followed by SEC-250 HPLC. The purified M protein was analyzed by atomic force microscopy and shown to assemble in particles that were able to recognize HBV antibodies in the sera of patients with chronic hepatitis B, providing evidence for their immunoreactivity.

Yeasts as a tool for heterologous gene expression.

The yeasts Saccharomyces cerevisiae and Pichia pastoris are attractive hosts for production of human proteins. The main advantages offered by these systems are the well-developed and easily accessible genetic tools, rapid growth, the simple and inexpensive culture media, and many of the cellular and metabolic processes found in higher eukaryotes are conserved in both yeast species. In this chapter, we describe the production of two proteins of therapeutic interest: the human P53 tumor suppressor and the viral HBsAg in P. pastoris and S. cerevisiae using the strong and inducible promoters AOX1 and Gal10/Cyc1, respectively. Besides the production as a goal of both expressions, we also report on an unexpected result that has occurred in S. cerevisiae: The overexpression of human p53 induces yeast cell death with characteristic markers of apoptosis, such as the externalization of phosphatidylserines and DNA strand cleavage.

Expression of HBsAg and preS2-S protein in different yeast based system: a comparative analysis.

We have expressed both S and preS2-S genes coding for the hepatitis B small (S) and medium (M) proteins, respectively, in different yeast based expression systems and compared the production level of the recombinant proteins. In Saccharomyces cerevisiae, viral genes were expressed under the inducible Gal10/cyc1 and the constitutive PGK promoters using 2mu replicating vectors. We showed that the yield of S protein was higher than M protein under both inducible (14.27 vs 10.9 mg/l) and constitutive (9.18 vs 6.39 mg/l) conditions, respectively. In the methylotrophic yeast Pichia pastoris, the viral genes were expressed in GS115 (Mut(+): Methanol Utilizing) and KM71 (Mut(S): Methanol Utilizing Slow) under the control of the alcohol oxidase promoter (AOX1). In Mut(S) background, both S and preS2-S genes were expressed at higher levels than in Mut(+). In attempt to increase the yield of recombinant viral proteins in S. cerevisiae, we have co-expressed both inducible and constitutive vectors harboring the S or preS2-S genes leading to recombinant strains called UTS (containing pDP/S+pYePIT/S) and UTP (containing pDP/preS2-S+pYePIT/preS2-S). We showed that the recombinant S and preS2-S proteins were successfully detected and the production level reached 18.31 mg/l for the S and 13.22 mg/l for the M proteins. Our comparative study provides evidence that in small scale, S. cerevisiae is more suitable for HBsAg and preS2-S proteins production than P. pastoris under inducible rather than constitutive condition.