Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque

BACKGROUND: The CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis), a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38. RESULTS: A cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i) surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii) detection of ~42 and 84 kDa proteins by Western blot and (iii) the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop. CONCLUSION: This multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme

Identification and Characterization of Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma Cell Lines

Background/Aims: Head and neck squamous cell carcinoma (HNSCC) ranks sixth worldwide for tumor-related mortality. A subpopulation of tumor cells, termed cancer stem cells (CSCs), has the ability to support cancer growth. Therefore, profiling CSC-enriched populations could be a reliable tool to study cancer biology. Methods: We performed phenotypic characterization of 7 HNSCC cell lines and evaluated the presence of CSCs. CSCs from Hep-2 cell line and HNSCC primary cultures were enriched through sphere formation and sphere-forming cells have been characterized both in vitro and in vivo. In addition, we investigated the expression levels of Nicotinamide N-methyltransferase (NNMT), an enzyme overexpressed in several malignancies. Results: CSC markers were markedly expressed in Hep-2 cell line, which was found to be highly tumorigenic. CSC-enriched populations displayed increased expression of CSC markers and a strong capability to form tumors in vivo. We also found an overexpression of CSC markers in tumor formed by CSC-enriched populations. Interestingly, NNMT levels were significantly higher in CSC-enriched populations compared with parental cells. Conclusion: Our study provides an useful procedure for CSC identification and enrichment in HNSCC. Moreover, results obtained seem to suggest that CSCs may represent a promising target for an anticancer therapy

[310-POS]: Potential role of Klotho protein in the pathophysiology of preeclampsia

OBJECTIVES: An aging-suppressor gene, klotho, is a candidate factor for vascular disease because its deficiency leads to impaired endothelium-dependent vasodilation and impaired angiogenesis. The aim was to verify a possible relation among the expression of the klotho gene, single nucleotide polymorphisms (SNP) in the promoter region, and placenta aging. METHODS: Placentas were collected from normal pregnancies (n=34) and pregnancies complicated by Preeclampsia (n=34), matched for gestational age. Klotho mRNA and protein were determined using Real-Time PCR and Western blot, respectively. SNPs (i.e.: -744delA, and -395A/G) were investigated using allele-specific PCR. Expression of pluripotency markers (i.e.: Nanog, and Oct-4) and telomere length measurement were assessed using Real-Time PCR. RESULTS: Real-Time PCR analyses demonstrated a significant down-regulation of Klotho ( 83%; p=0.005) in patients with Preeclampsia versus Controls. Results of Western Blot agreed with Real-Time PCR ones. Polymorphism analysis results suggest that -744delA allele is associated with 3-fold increased risk for preeclampsia. Real-Time PCR investigation revealed a significant down-regulation of pluripotency markers in pathological group. CONCLUSIONS: Klotho expression is decreased in preeclamptic pregnancies. Further data are required to confirm the role of this protein in pathophysiology of preeclampsia and the possible link to long term outcomes

In vitro structural aspects of the human trophoblastic cell

Maternal- fetal exchanges are mainly regulated by trophoblast, which displays an active role during embryo growth. Trophoblast organization into a syncytial layer involves structural and functional steps that may be monitored and better elucidated by "in vitro" studies. In light of this, we have carried out morphological and biochemical analyses in order to evaluate 1) the syncytiotrophoblast formation in culture (48 h, 5-30 days) the Na+/K+ATPase activity and 3) the plasmalemmal microviscosity changes occurring during "in vitro" trophoblast production. Morphological and biochemical modulations have been pointed out