Improved DNA markers for efficient analysis of fragile X families

We report the characteristics of two new probes that detect BclI RFLPs useful for analysis of fragile X families. With these two probes and a single blot, 34% of women are heterozygous both for the proximal marker DXS105 (closer to the fragile X locus than the factor IX gene) and for the distal markers DXS52 or the factor VIII gene. Combined with the analysis of previously described polymorphic markers, it is possible to have a majority of families fully informative for flanking markers using a limited number of probes and restriction digests.     

Cyclooxygenase-2 is expressed frequently and early in Barrett's oesophagus and associated adenocarcinoma

AIMS: To establish the prevalence of cyclooxygenase-2 (COX-2) expression in a large series of resected Barrett's adenocarcinoma and associated preneoplastic lesions and to correlate this expression with clinicopathological data and prognosis. METHODS: COX-2 expression was assessed by immunohistochemistry in resected surgical specimens of 66 Barrett's adenocarcinomas and 32 cases of Barrett's mucosa (with dysplasia in 17 cases). RESULTS: Epithelial expression of COX-2 protein was increased in Barrett's mucosa compared with normal oesophagus. Epithelial expression of COX-2 was found in 91% of Barrett's specialized mucosa negative for dysplasia, 94% of Barrett's mucosa with dysplasia, and 97% of Barrett's adenocarcinoma. COX-2 expression was significantly higher in the well-differentiated adenocarcinomas when compared with the poorly differentiated tumours. There was no significant correlation between COX-2 expression and the other pathological features of the tumours. Survival analysis showed no significant prognostic value for COX-2. CONCLUSION: Our results confirm up-regulation of COX-2 in Barrett's oesophagus-metaplastic and dysplastic-and in Barrett's adenocarcinoma. Increased COX-2 expression did not differ during the progression from Barrett's oesophagus negative for dysplasia to Barrett's adenocarcinoma and is related to adenocarcinoma whose histology is well differentiated. This suggests that enhanced expression of COX-2 may occur early during Barrett's-associated neoplastic transformation.     

Inhibition of rat liver fibrogenesis through noradrenergic antagonism

The effect of adrenergic innervation and/or circulating catecholamines on the function of liver fibrogenic cells is poorly understood. Our aim was to investigate the effects of noradrenergic antagonism on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Two weeks of CCl4 induced an approximately 5-fold increase in the area of fibrosis as compared with controls. The addition of 6-hydroxydopamine (OHDA), a toxin that destroys noradrenergic fibers, decreased fibrosis by 60%. After 6 weeks of CCl4, the area of fibrosis increased about 30-fold in CCl4-treated animals and was decreased by 36% with OHDA. At 2 weeks, OHDA abrogated the CCl4-induced increase in mRNA level of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), an inhibitor of extracellular matrix degradation, and it greatly reduced it at 6 weeks. Finally, when rats treated with CCl4 for 2 weeks also received prazosin, an antagonist of alpha1-adrenergic receptors, fibrosis was decreased by 83%. In conclusion, destruction of noradrenergic fibers or antagonism of noradrenergic signaling through alpha1 receptors inhibited the development of liver fibrosis. Because adrenoreceptor antagonists have a very sound safety profile, they appear as attractive drugs to reduce liver fibrogenesis.     

Rapid molecular characterization of mutations leading to unstable hemoglobin β-chain variants

Summary Characterization of unstable hemoglobins by protein analysis is often difficult. However, it is facilitated by DNA analysis, especially in the case of hyperunstable -chain variants, which produce a -thalassemia phenotype. We have applied an efficient strategy to the detection of such variants at the DNA level, based on computer-designed denaturing gradient gel electrophoresis (DGGE) of amplified DNA fragments. This approach makes it possible to detect any anomaly in the -globin gene. We describe the use of the DGGE method for rapid characterization of -chain variants and report a new missense mutation in the -globin gene third exon, 127 CAG-CGG/Gln-Arg, which is responsible for the synthesis of a highly unstable hemoglobin.     

Effects and regulation of connective tissue growth factor on hepatic stellate cells

Connective tissue growth factor (CTGF) is a 38-kd protein involved in several human fibrotic disorders including atherosclerosis and skin and renal fibrosis. Although it has been shown that human and experimental liver fibrosis is associated with CTGF expression through up-regulation of CTGF mRNA by hepatic stellate cells (HSC), the role of CTGF in the liver has not yet been determined. The aim of the present study was to assess the effects of CTGF on rat primary HSC and its regulation in a well-established model of in vitro liver fibrogenesis. Incubation of primary HSC with recombinant CTGF induced a significant migratory (2.3-fold, 50 ng/ml CTGF) and proliferative effect (1.8-fold, 100 ng/ml CTGF). Type I collagen mRNA expression, as assessed by a real-time RT-PCR procedure, was also increased when cells were incubated in the presence of CTGF (2-fold, 50 ng/ml). Transforming growth factor-beta1 (TGF-beta1) strongly stimulated CTGF mRNA expression, a direct mechanism observed in the absence of any intermediate protein synthesis. Furthermore, spontaneous activation of HSC plated on plastic and stimulation by vascular endothelial growth factor, lipid peroxidation products (HNE, MDA), acetaldehyde, and platelet-derived growth factor (PDGF)-BB significantly up-regulated CTGF mRNA expression in HSC. PDGF-induced CTGF stimulation might be related in part to TGF-beta1 secretion because CTGF mRNA up-regulation observed after PDGF-BB stimulation was abrogated in the presence of neutralizing TGF-beta1 antibody. In conclusion, this study extends the role of CTGF in HSC activation and suggests that CTGF up-regulation might be a central pathway during HSC activation.