Amine coupling versus biotin capture for the assessment of sulfonamide as ligands of hCA isoforms.

This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% < 10%). The second corresponds to a capture of proteins on a streptavidin chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives.

Phenotype and HFE genotype in a population with abnormal iron markers recruited from an Endocrinology Department.

OBJECTIVE: The aim of this study was to describe HFE genotype in a population of patients with altered iron markers recruited in an Endocrinology Department and to define the possible phenotype-genotype relationships. METHODS: A total of 156 patients with high serum ferritin concentrations (>300 ng/ml) or transferrin saturation (>45%) (I group), and a control group of 106 healthy subjects (C group) underwent HFE genotyping (classical C282Y and H63D mutations). We also examined the main genetic features of subgroups in I according to the presence (D) or the absence (ND) of diabetes. RESULTS: (1) The genotypes were significantly different in the I and C groups (P<0.001), with an increased frequency of major 282Y allele in the I group (35% vs 7.5%), but not of minor 63D allele (17 vs 18.5%). (2) The genotype of D and ND groups also differed (P<0.0001), with a lower frequency of C282 heterozygosity (P<0.0001) in the D group, and a higher prevalence of H63D heterozygosity in the D vs ND groups (P<0.01). (3) The phenotypic comparison of D and ND groups also showed a higher mean body mass index, age, and serum ferritin concentration, as well as an increased proportion of males with increased liver enzymes in the D group. CONCLUSION: This population harboring abnormal iron markers had a different HFE genotype and a higher 282Y allele frequency than the control population. This suggests that blood iron markers could be checked in etiological investigations of metabolic disturbances to identify patients who should undergo genotyping, since approximately 20% were diagnosed with C282Y homozygosity.

Expression of progenitor cell markers during expansion of sorted human pancreatic beta cells.

Functional pancreatic beta cell mass is dynamic and although fully differentiated, beta cells are capable of reentering the cell cycle upon appropriate stimuli. Stimulating regeneration-competent cells in situ is clearly the most desirable way to restore damaged tissue. Regeneration by dedifferentiation and transdifferentiation is a potential source of cells exhibiting a more developmentally immature phenotype and a wide differentiation potential. In this context and to gain a better understanding of the transformation induced in human beta cells during forced in vitro expansion, we focused on identifying differences in gene expression along with phenotypical transformation between proliferating and quiescent human beta cells. FACS-purified beta cells from three different human pancreata were cultured during 3-4 months (8-10 subcultures) on HTB-9 cell matrix with hepatocyte growth factor. Gene expression profiling was performed on cells from each subculture on "in-house" pancreas-specific microarrays consisting of 218 genes and concomitant morphological transformations were studied by immunocytochemistry. Immunocytochemical studies indicated a shift from epithelial to neuroepithelial cell phenotype, including progenitor cell features such as protein gene product 9.5 (PGP 9.5), Reg, vimentin, and neurogenin 3 protein expression. The expression of 49 genes was downregulated, including several markers of endocrine differentiation while 76 were induced by cell expansion including several markers of progenitor cells. Their pattern also argues for the transdifferentiation of beta cells into progenitor cells, demonstrating neuroepithelial features and overexpressing both PBX1, a homeodomain protein that can bind as a heterodimer with PDX1 and could switch the nature of its transcriptional activity, and neurogenin 3, a key factor for the generation of endocrine islet cells. Our study of the machinery that regulates human beta cell expansion and dedifferentiation may help elucidate some of the critical genes that control the formation of adult pancreatic progenitor cells and hence design targets to modify their expression in view of the production of insulin-secreting cells.

Expression of apoptosis regulatory factors during myocardial dysfunction in endotoxemic rats.

OBJECTIVES: To document the time course of apoptosis pathway activation in sepsis and to determine whether Bcl-2 overexpression would improve endotoxin-induced myocardial dysfunction and mortality rate. DESIGN: Randomized, controlled trial. SETTING: Experimental laboratory. SUBJECTS: Male Sprague Dawley rats, wild-type C57BL/6 female mice, C57BL/6 female mice overexpressing Bcl-2. INTERVENTIONS: Hearts were isolated from rats treated with endotoxin (10 mg/kg, intravenously) to perform heart function, immunohistochemistry (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick 3'-end labeling, caspase 3), RNase protection assay, reverse transcriptase polymerase chain reaction, Western blotting (caspase 3), and radiolabeled annexin V studies. Twenty-four hours before endotoxin challenge (10 mg/kg, intravenously), rats were pretreated with saline or endotoxin (0.5 mg/kg, intraperitoneally), with or without parthenolide (1 mg/kg, intraperitoneally). Isolated hearts were used to test myocardial function. Mortality induced by endotoxin (10 mg/kg, intraperitoneally) was tested on wild-type or mice overexpressing Bcl-2. MEASUREMENTS AND MAIN RESULTS: Endotoxin-induced heart dysfunction was maximal at 4 and 8 hrs postinjection, started to improve, and was fully restored at 24 hrs after endotoxin treatment. Endotoxin also induced phosphatidylserine outer leaflet membrane exposure, caspase 3 activation, nuclear apoptosis, and changes in apoptosis gene expression. Bcl-2 overexpression induced by endotoxin pretreatment prevented endotoxin-induced myocardial dysfunction. Mice overexpressing Bcl-2 had dramatic improvement in survival rate compared with wild-type mice. CONCLUSIONS: These observations suggest that both death receptor and caspase-mediated apoptosis processes are activated in this sepsis model. Bcl-2 overexpression before endotoxin challenge prevents myocardial dysfunction in rats and improves survival rate in mice.

Selective alteration of gene expression in response to natural and synthetic retinoids.

BACKGROUND: Retinoids are very potent inducers of cellular differentiation and apoptosis, and are efficient anti-tumoral agents. Synthetic retinoids are designed to restrict their toxicity and side effects, mostly by increasing their selectivity toward each isotype of retinoic acids receptors (RARalpha,beta, gamma and RXRalpha, beta, gamma). We however previously showed that retinoids displayed very different abilities to activate retinoid-inducible reporter genes, and that these differential properties were correlated to the ability of a given ligand to promote SRC-1 recruitment by DNA-bound RXR:RAR heterodimers. This suggested that gene-selective modulation could be achieved by structurally distinct retinoids. RESULTS: Using the differential display mRNA technique, we identified several genes on the basis of their differential induction by natural or synthetic retinoids in human cervix adenocarcinoma cells. Furthermore, this differential ability to regulate promoter activities was also observed in murine P19 cells for the RARbeta2 and CRABPII gene, showing conclusively that retinoid structure has a dramatic impact on the regulation of endogenous genes. CONCLUSIONS: Our findings therefore show that some degree of selective induction or repression of gene expression may be achieved when using appropriately designed ligands for retinoic acid receptors, extending the concept of selective modulators from estrogen and peroxisome proliferator activated receptors to the class of retinoid receptors.