Signals regulating muscle formation in the limb during embryonic development

Classically, somites have been the preparation of choice for the study of muscle development, while the limb bud is the preferred model of axis formation. Nevertheless, the limb bud offers some experimental advantages for muscle studies. This review describes the successive events involved in limb muscle formation during embryonic development, the properties of the key marker molecules and resumes our current knowledge of the signalling pathways involved.     

Homozygosity mapping of a Weill-Marchesani syndrome locus to chromosome 19p13.3-p13.2

Weill-Marchesani syndrome (WMS) is a rare disease characterized by short stature, brachydactyly, joint stiffness, and characteristic eye abnormalities, including microspherophakia, ectopia lentis, and glaucoma. Both autosomal recessive and autosomal dominant modes of inheritance have been described in association with WMS. We have performed a genome-wide search in two large consanguineous families of Lebanese and Saudian origin consistent with an autosomal recessive mode of inheritance. Here, we report the linkage of the disease gene to chromosome 19p13.3-p13.2 (Zmax=5.99 at theta=0 at locus D19S906). A recombination event between loci D19S905 and D19S901 defines the distal boundary, and a second recombination event between loci D19S221 and D19S840 defines the proximal boundary of the genetic interval encompassing the WMS gene (12.4 cM). We hope that our ongoing studies will lead to the identification of the disease-causing gene.     

Identification of two novel ACTH-responsive genes encoding manganese-dependent superoxide dismutase (SOD2) and the zinc finger protein TIS11b [tetradecanoyl phorbol acetate (TPA)-inducible sequence 11b

ACTH is the major trophic factor regulating and maintaining adrenocortical function, affecting such diverse processes as steroidogenesis, cell proliferation, cell migration, and cell survival. We used differential display RT-PCR to identify genes that are rapidly induced by ACTH in the bovine adrenal cortex. Of 42 PCR products differentially amplified from primary cultures of bovine adrenocortical cells treated with 10 nM ACTH, six identified mRNAs that were confirmed by Northern blot analysis to be induced by ACTH. Four of these amplicons encoded noninformative repetitive sequences. Of the other two sequenced amplicons, one encoded a partial sequence for mitochondrial manganese-dependent superoxide dismutase (SOD2), an enzyme that is likely to protect adrenocortical cells from the cytotoxic effects of radical oxygen species generated during steroid biosynthesis. The second was identified as TIS11b (phorbol-12-myristate-13-acetate-inducible sequence 11b)/ERF-1/cMG, a member of the CCCH double-zinc finger protein family. SOD2 induction by ACTH was independent of extracellular steroid concentration or oxidative stress. SOD2 and TIS11b mRNA expressions were rapidly induced by ACTH, reaching a maximal level after 8 h and 3 h of treatment, respectively. These ACTH effects were mimicked by forskolin but appeared independent of cortisol secretion. Upon ACTH treatment, induction of TIS11b expression closely followed the previously characterized peak of vascular endothelial growth factor (VEGF) expression. Transfection of a TIS11b expression plasmid into 3T3 fibroblasts induced a decrease in the expression of a reporter gene placed upstream of the VEGF 3'-untranslated region, indicating that TIS11b may be an important regulator of VEGF expression through interaction with its 3'-untranslated region.     

Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3)

Secretion of cytolytic granules content at the immunological synapse is a highly regulated process essential for lymphocyte cytotoxicity. This process requires the rapid transfer of perforin containing lytic granules to the target cell interface, followed by their docking and fusion with the plasma membrane. Defective cytotoxicity characterizes a genetically heterogeneous condition named familial hemophagocytic lymphohistiocytosis (FHL), which can be associated with perforin deficiency. The locus of a perforin (+) FHL subtype (FHL3), observed in 10 patients, was mapped to 17q25. This region contains hMunc13-4, a member of the Munc13 family of proteins involved in vesicle priming function. HMunc13-4 mutations were shown to cause FHL3. HMunc13-4 deficiency results in defective cytolytic granule exocytosis, despite polarization of the secretory granules and docking with the plasma membrane. Expressed tagged hMunc13-4 localizes with cytotoxic granules at the immunological synapse. HMunc13-4 is therefore essential for the priming step of cytolytic granules secretion preceding vesicle membrane fusion.     

Aluminum speciation studies in biological fluids. Part 9. A quantitative investigation of aluminum(III)-glutamate complex equilibria and their potential implications for aluminum metabolism and toxicity

As a nonessential element, aluminum is likely to be toxic both at low usual dietary levels in the long run (chronic toxicity) and at high therapeutic levels in shorter periods of time (acute toxicity). In both situations, aluminum toxicity is a direct function of aluminum bioavailability, which is itself dependent on Al(3+) solubility and charge neutralization. Dietary acids, by their intrinsic acidity and coordinating capacity, can extend the pH range, thus the section of the gastrointestinal tract, within which the Al(3+) ion remains soluble, and also help Al(3+) diffusion across the intestinal epithelium through the formation of neutral complex species. The present work examines the impact of glutamic acid, an essential amino acid also widely used in industrial food and drinks, on aluminum speciation in the gastrointestinal tract and blood plasma. Complex formation between the Al(3+) ion and glutamate has first been investigated through potentiometric titrations, complex stoichiometries being then checked by ESI mass spectrometry and NMR measurements. A series of mono- and polynuclear species has been characterized, whose influence on aluminum distribution in vivo has been assessed by computer simulation. The capacity of glutamate to maintain Al(3+) ions in solution under normal dietary conditions is predicted to be intermediate between glycine-like amino acids and succinate on the one hand, and tartrate and malate on the other hand, its Al(3+) neutralization effect being similar to that of succinate, tartrate and malate. These results, which point to a potential aggravating role of glutamate on aluminum gastrointestinal absorption, substantiate recent observations made on rats. In spite of the moderate effect expected from glutamate on aluminum bioavailability under most aluminum-based therapies investigated, attention is therefore called to the risk of glutamic acid ingestion simultaneously to any aluminum therapeutic form. Incidentally, the former implication of 'the' aluminum glutamate complex in the transfer of aluminum through the blood-brain barrier of aluminum loaded rats may effectively be attributed to one of the species characterized here, but is of no significance at all to aluminum contamination in humans, even at most extreme levels.     

Real-time PCR for chloroquine sensitivity assay and for pfmdr1-pfcrt single nucleotide polymorphisms in Plasmodium falciparum

Plasmodium falciparum drug resistance is a major problem in malaria endemic areas. Molecular markers and in vitro tests have been developed to study and monitor drug resistance. However, none, used alone, can provide sufficient data concerning the level of drug resistance and to issue precise guidelines for drug use policies in endemic areas. We propose real-time PCR for the simultaneous detection of pfcrt and pfmdr1 genes mutations and to determine the half-maximal inhibitory response (IC(50)) of antimalarial drug. Using hybridization probes and SybrGreen technology on LightCycler instrument, point mutations of pfcrt and pfmdr1 genes have been successfully detected in 161 human blood samples and determination of IC values was applied to chloroquine-sensitive and chloroquine-resistant strains. Moreover, mixed infections caused by P. falciparum clones with wild-type or mutant alleles could be efficiency separated. The aim of this study was not to provide definitive data concerning the rate of mutations in an endemic area, but to describe a powerful method allowing the quantification of DNA for IC(50) determination and the detection of major pfmdr1 and pfcrt mutations.     

The Fanconi anemia pathway and the DNA interstrand cross-links repair

Fanconi anemia (FA) is a genetic cancer-predisposition syndrome characterized by bone marrow failure and cellular and chromosomal hypersensitivity to DNA cross-linking agents. Seven FA genes have been isolated and their products associate to form a pathway that interacts functionally or physically with several DNA-damage response proteins involved in cell cycle checkpoints and/or DNA repair. These proteins include BLM, ATM, BRCA1, XPF and the MRE11/RAD50/NBS1 complex. In spite of several recent striking progresses in the biochemistry and the molecular biology of the disorder, the precise function(s) of the FA proteins remain(s) poorly determined. However, several recent data indicate that the FA pathway could be involved in the coordination of both cell cycle checkpoints and DNA repair.     

Born small for gestational age: increased risk of type 2 diabetes,hypertension and hyperlipidaemia in adulthood

The metabolic and cardiovascular complications associated with reduced fetal growth have been identified in the past 10 years. These include cardiovascular disease and the insulin resistance syndrome, comprising dyslipidaemia and impaired glucose tolerance or type 2 diabetes, and they appear to result from the initial development of insulin resistance. Although the mechanism underlying the development of insulin resistance associated with reduced fetal growth remains unclear, there is some evidence that adipose tissue plays a key role. Over the past decade, several hypotheses have been proposed to explain this unexpected association. Each points to either a detrimental fetal environment, genetic susceptibility or an interaction between the two. Although yet to be confirmed, the hypothesis suggesting that the association could be the consequence of genetic-environmental interactions is at present the most attractive.     

Low birth weight: effect on insulin sensitivity and lipid metabolism

The metabolic and cardiovascular complications associated with reduced fetal growth have been identified during the past 10 years. These complications that encompass cardiovascular diseases and insulin resistance syndrome consist of dyslipidemia, impaired glucose tolerance or type 2 diabetes and appear to result from the initial development of insulin resistance. The association of reduced fetal growth with the other parameters of the syndrome X appear less constant than with insulin resistance and the expression and/or the age of onset seem to depend on the degree of genetic predisposition of the population. Although the mechanisms underlying the development of the insulin resistance associated with reduced fetal growth remain unclear, some evidence argues in favor of a key role of the adipose tissue. Several hypotheses have been proposed over the past 10 years to understand this unexpected association. Each of them points to either a detrimental fetal environment or genetic susceptibilities or interactions between these two components as playing a critical role in this context. Although not confirmed, the hypothesis suggesting that this association could be the consequence of genetic/environmental interactions remains at the moment the most attractive.