Polymorphisms of the Pfatpase 6 and Pfcrt gene and their relationship with the in vitro susceptibility to dihydroartemisinin and chloroquine of Plasmodium falciparum isolates from Abobo, Côte d'Ivoire.

As a result of widespread resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP), artemisinin-based combination therapy (ACT) has been recommended as a first-line anti-malarial regimen in Côte d'Ivoire since 2005. A thorough understanding of the molecular bases of P. falciparum resistance to existing drugs is therefore needed. The aims of this study were to analyze the in vitro sensitivity of P. falciparum field isolates from Abobo to CQ, pyronaridine (PYR) and dihydroartemisinine (DHA), and to investigate the polymorphisms associated with drug resistance. The standard in vitro drug sensitivity microtechnique recommended by the WHO was used to assess the sensitivity of Plasmodium falciparum isolates collected in December 2006. The Pfcrt haplotype 76 was analysed by PCR-RFLP while Pfatpase 6 amplification products were sequenced. Associations between drug sensitivity and parasite gene polymorphisms were evaluated with Cohen's kappa test. The correlation between the IC50 values for different drugs was assessed by the coefficient of determination (r²). Significance was assumed at p<0.05. Of 128 in vitro tests performed, 112 (87.5%) were successful. Of the isolates, 56.2% were resistant for CQ and 48% for PYR. One isolate (3.6%) demonstrated reduced DHA sensitivity (IC50 higher than 10 nM). The mutant K76T pfcrt codon, present in 90% of DNA fragments analyzed, was associated with CQ-R (ĸ=0.76). The N669Y (16.1%), D734Y (28.6%) and D734H (1.8%) isolates were found to have mutant Pfatpase6, however, these mutations were not associated with diminished DHA sensitivity (k=0.01). These high levels of antimalarial drug resistance in Abobo (Côte d'Ivoire) demand further studies of drug efficacy across the whole country.

Aging and obesity induce distinct gene expression adaptation in the liver of C57BL/6J mice.

BACKGROUND: Aging and obesity induce complex transcriptomic changes in the liver, promoting the development of insulin resistance and type 2 diabetes. In spite of an increasing amount of studies on the role of aging and nutrient excess in metabolic disorders, the specific molecular events leading to insulin resistance are still poorly understood. METHODS: This study presents a comparative analysis of hepatic gene expression profiles between young adult C57BL/6J mice fed with a low- or a high-fat diet for 1 and 12 months. We evaluated the expression of a defined set of genes implicated in glucose and lipid metabolism as well as key nuclear receptors and their target genes, IGF1 signaling and clock genes. RESULTS: Aging and short-term high-fat consumption induced insulin resistance, albeit through two distinct processes. Hepatic gene expression changes were more pronounced in the context of aging. We further analyzed expression profiles together with plasma parameters by principal component analysis with regard to diet condition. CONCLUSIONS: Our results suggest that in the liver of C57BL/6J mice, the molecular mechanisms underlying high-fat feeding or aging which mediated insulin resistance were not identical.

Mitral valve repair by Alfieri's technique does not limit exercise tolerance more than Carpentier's correction.

OBJECTIVE: The main goal of this study was to evaluate if the edge-to-edge mitral repair could be a limiting factor for exercise tolerance and to compare these results to those of classical techniques. METHODS: Between 2000 and 2002, 54 consecutive patients were operated on for mitral valve regurgitation (MR). Twenty-five patients were operated with Alfieri's technique (group A) and 29 patients with Carpentier's technique (group C). The mean age was 63.9 years in group A and 63.8 years in group C (p = 0.98). After a mean follow-up of 16.2+/-12 months, survivor patients were seen at the outpatient clinic, by the same physician for a clinical evaluation, an echocardiogram at rest and at peak exercise, and received a cardiorespiratory exercise testing with maximal oxygen uptake (VO2 max) recording. RESULTS: Clinical status improved with 0% of the patients in class NYHA III or IV in either group postoperatively versus 77% preoperatively. There was no significant MR in 80% of cases in group A versus 89.6% in group C (p = 0.54). The mean mitral valve area was 2.5 and 2.9 cm2 in groups A and C, respectively (p = 0.018). The mitral gradient at rest was 3.8 and 3.3 mmHg (p = 0.31) and the mitral gradient at peak exercise was 8.5 and 9.7 mmHg (p = 0.22) in groups A and C, respectively. Cardiorespiratory exercise testing showed a mean VO2 max of 73.7+/-15% of normal value in group A versus 79.6+/-13.1% in group C (p = 0.18). CONCLUSION: Alfieri's technique has the same efficiency on improvement of MR and clinical status than classical repair. Despite a higher restriction of mitral valve area at rest in group A, gradient and mean VO2 max at peak exercise were similar in both groups.

A cDNA macroarray resource for gene expression profiling in ruminant tissues involved in reproduction and production (milk and beef) traits.

cDNA arrays have proven to be useful tools to screen gene expression in many animal species including livestock species. A collaborative program was launched to construct a ruminant cDNA collection, representative of three tissues: Muscle, Embryo and Mammary gland, named MEM. This collection gathers clones mainly arising from 3 non-normalised cDNA libraries: a directed bovine muscle library, a 14-day-old bovine embryo library and a goat lactating mammary library. It is made up of 1896 clones (637 muscle, 882 embryo and 377 mammary cDNAs), selected after sequencing and bioinformatic analyses. Amplification products yielded from these clones as well as controls were printed onto Nylon membranes to generate macroarrays. Hybridisation with relevant cDNA targets allowed checking the location of about 50 cDNAs and the specificity of each sub-set of the repertoire. Macroarrays were hybridised with radiolabelled cDNA complex targets from five different tissues (muscle, embryo, mammary gland, adipose tissue and oocyte). Both somatic and germinal complex targets gave valid hybridisation signals with 45 to 80% of the printed probes. This specific cDNA collection now provides a powerful tool for transcriptomic studies with the ultimate objective to better understand physiological and metabolic functions in ruminants. It will be subsequently included into a forthcoming larger collection.

Effects of ceramide on aggrecanase activity in rabbit articular cartilage.

Ceramide participates in signal transduction of IL-1 and TNF, two cytokines likely involved in cartilage degradation in osteoarthritis. We previously showed that ceramide stimulates proteoglycan degradation, mRNA expression of matrix metalloproteinase (MMP)-1, -3, and -13, and pro-MMP-3 production in rabbit cartilage. Since aggrecan, the main cartilage proteoglycan, can be cleaved by metalloproteinases both of MMP and aggrecanase type, the aim of this study was to determine if ceramide stimulates aggrecanase action and, if that is the case, in which measure aggrecanase mediates the degradative effect of ceramide. To this end, antibodies were used against the C terminal aggrecan neoepitopes generated by aggrecanases (NITEGE(373)) and MMPs (DIPEN(341)). Ceramide C(2) at 10(-5) to 10(-4) M dose-dependently increased NITEGE signal, without changing that of DIPEN, in cultured explants of rabbit cartilage. The effects of 10(-4) M C(2) on NITEGE signal and proteoglycan degradation were similarly antagonized by the metalloproteinase inhibitor batimastat, with return to the basal level at 10(-6) M. These results show that, similarly to IL-1 and TNF, ceramide-induced aggrecan degradation is mainly due to aggrecanases. That no increase of MMP activity was detected, despite stimulation of MMP expression, was probably due to lack of proenzyme conversion to mature form, since addition of a MMP activator to C(2)-treated cartilage increased both DIPEN signal and proteoglycan degradation. These findings support the hypothesis that cytokine-induced ceramide could play a mediatory role in situations of increased degradation of cartilage matrix.

Effects of ceramide on apoptosis, proteoglycan degradation, and matrix metalloproteinase expression in rabbit articular cartilage.

Cartilage loss in osteoarthritis is characterized by matrix degradation and chondrocyte death. The lipid messenger ceramide is implicated in signal transduction of the catabolic cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1), as well as in apoptosis. The aim of this study was to examine the in vitro effects of ceramide on proteoglycan degradation, matrix-metalloproteinase (MMP) expression and activity, and chondrocyte apoptosis in rabbit articular cartilage. Cell-permeant ceramide C(2) stimulated proteoglycan degradation in cartilage explants starting from 3 x 10(-5) M, with 100% increase at the dose of 10(-4) M. This effect was probably due to MMPs since it was blocked by the MMP inhibitor batimastat. Furthermore, in isolated chondrocytes, C(2) stimulated the expression of MMP-1, 3, and 13 at the mRNA level, MMP activity, and MMP-3 production. Ceramide also caused chondrocyte apoptosis at doses ranging from 10(-5) to 10(-4) M. This study supports the hypothesis that ceramide might play a mediatory role in both matrix degradation and apoptosis in processes of cartilage loss such as those observed in osteoarthritis.

Ontogeny of extracellular matrix gene expression by rat lung cells at late fetal gestation.

Lung epithelial and mesenchymal cells are separated by a basement membrane. At late fetal gestation, this basement membrane in fenestrated, allowing epithelial cytoplasmic extensions to reach in close proximity of the interstitial fibroblast. The enzymes responsible for this focal basement membrane remodelling, and their cellular origin, remains to be defined. Basement membrane remodelling generally involves a special class of matrix-degrading enzymes, called metalloproteinases. Herein, we report that fetal lung cells originating from both tissue layers, mesoderm and endoderm, express the metalloproteinase genes, MMP-1 or interstitial collagenase, and MMP-3 or stromelysin. The inhibitor of metalloproteinases, TIMP-1, is mainly expressed in fetal lung fibroblasts. During late fetal development, MMP-1 mRNA expression in both cell types increases close to term (day 21, term = 22 days), while that of stromelysin and TIMP-1 remain constant. Both fibroblasts and epithelial cells express fibronectin (FN) mRNA. The expression of the FN gene in epithelial cells decreases slightly at the canalicular stage of lung development (days 19-20), whereas FN expression in fibroblasts is not changed with advancing gestation. Procollagen alpha 1 (I) mRNA is predominantly detected in fibroblasts whereas message for laminin B1 chain is primarily found in epithelial cells. Expression of procollagen alpha 1 (I) mRNA decreases in fibroblasts during the canalicular stage of fetal lung development compared to the pseudoglandular stage (day 18) but increases thereafter at the saccular stage (day 21) of development. Laminin B1 expression in epithelial cells declines with advancing gestation. These data are consistent with a process of basement membrane thinning during the canalicular stage, followed by metalloproteinase-mediated penetration. Further, a progressive reduction in laminin expression is consistent with progressive epithelial differentiation.

Inhibition of fibroblast growth by epithelial cells in fetal rat lung.

The canalicular and saccular stages of rat lung development are marked by thinning of mesenchymal tissue. Because cell-cell interactions are important for normal fetal lung development, we investigated whether this regression of mesenchymal tissue is controlled by fibroblast-epithelial cell interactions. Using flow cytometry, thymidine uptake into DNA, and cell doubling time, we observed an increase in the proportion of lung fibroblasts in the G0/G1 phase of the cell cycle with advancing gestation. Conditioned medium of epithelial cells from the canalicular stage of lung development, but not from the pseudoglandular and saccular stages, inhibited fetal lung fibroblast proliferation. Fetal lung epithelial cell growth was not affected by the epithelial cell-conditioned medium. The response of fibroblasts to this epithelial cell-derived growth-inhibitory activity was organ specific but not gestation dependent. The inhibitory effect of epithelial cell-conditioned medium on fibroblast proliferation was overcome by the addition of > 2% fetal bovine serum. The inhibition was not mediated by prostaglandins because 50 microM ibuprofen, a prostaglandin synthase inhibitor, did not block the elaboration of the inhibitory activity by fetal lung epithelial cells. Partial characterization of the fibroblast growth-inhibitory activity in epithelial cell-conditioned medium showed that it was trypsin labile, heat and acid insensitive, and lipid extractable. Its molecular weight appears to be > 3.5 and < 12.5 kD. Transforming growth factor-beta 1 and surfactant proteins B and C did not mimic the inhibitory effect of epithelial cell-conditioned medium. These data suggest that fetal lung epithelial cells elaborate a hydrophobic polypeptide that inhibits fetal lung fibroblast proliferation in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and characterization of type IV collagenases in rat lung cells during development.

Fetal type II epithelial cells rest on a basal lamina which separate them from the underlying connective tissue. At late fetal gestation, this basement membrane is occasionally disrupted, allowing epithelial cytoplasmic extensions to reach in close proximity of the interstitial fibroblast. The cellular source and enzymes responsible for the focal degradation of the basal membrane remains to be defined. In the present study, we examined the developmental expression of basement membrane associated type IV collagen and its degrading enzymes. Both fetal lung epithelial cells and fibroblasts expressed 72-kDa type IV collagenase and type IV (alpha 1) (alpha 2) collagen genes. Fibroblasts were enriched in the expression of 72-kDa type IV collagenase mRNA. Zymography showed that both cell types actively secrete 72- and 92-kDa type IV collagenases. Conditioned medium of fibroblasts contained more gelatin degrading activity than that of epithelial cells. At the time of appearance of basement membrane discontinuities (19-21 days, term = 22 days), 72-kDa type IV collagenase mRNA expression in fibroblasts increased while that of epithelial cells remained constant. Gelatinolytic activity of fibroblast conditioned medium also increased during this period. In contrast, type IV collagen gene expression decreased in both epithelial cells and fibroblasts. These data are compatible with a role for type IV collagenases in the genesis of basement membrane disruptions during late fetal lung development.

Culture of fetal alveolar epithelial type II cells in serum-free medium.

A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle's minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistence of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment.