Alternative splicing is a process by which a single stretch of genomic DNA yields several mRNAs encoding different proteins. Once believed to be a marginal phenomenon, alternative splicing now appears to be widespread among higher organisms and to be behind a large repertoire of human diseases. It involves a flexible mechanism for selecting splice sites, based on regulatory sequences recognized by cognate trans-acting protein factors (stimulatory SR proteins, or their antagonists). This RNA-protein interaction provides two types of targets for therapeutic manipulation. Masking regulatory RNA sequences with an antisense strategy is the most obvious, and encouraging results are beginning to accrue. Our lab is currently developing an entirely new approach in which activating proteins are targeted by small chemical molecules. A large screening program has been conducted with the chemical library from the Curie Institute. Several molecules (all indole derivatives) were found to counter the stimulatory effects of individual activating proteins, and have been selected for further development.

Since last years, the proteasome has emerged as a real and exciting target for anticancer therapy. Velcade (bortezomib, formerly known as PS341) remains the first selective proteasome inhibitor that has demonstrated significant preclinical activity in several tumor models and a significant efficacy in patients with refractory or relapsed multiple myeloma, resulting in an accelerated approval in US and Europe in such a setting. The major biological effect of bortezomib is the inhibition of the nuclear transcription factor NFkappaB, with subsequent inhibition of the growth tumor cells, induction of apoptosis, inhibition of angiogenesis and of cellular adhesion. The better understanding of the role of proteasome in the regulation of tumor cell growth has led to the development of new therapeutic approaches, notably in patients with multiple myeloma but also seems to hold interesting promises in other hematologic malignancies and solid tumors. This review provides a summary of the rationale for using proteasome inhibitors and an update on available and ongoing clinical studies involving human malignancies.

The neural crest-derived mesectoderm gives rise to physiologic apoptosis areas in early vertebrate embryos. Certain teratologic agents increase this phenomenon. The purpose of this work was to detect caspase 3 (which is associated with the apoptosis cascade) and p53 in cell death areas, both during physiological apoptosis and during apoptosis induced by three agents (retinoic acid, methyl-triazene, irradiation). Antibody revelation was performed using the aBC peroxidase kit. Quantifications were also performed on histological sections. We observed caspase 3 uptake on some apoptotic and preapoptotic cells in control embryos, and in the embryos exposed to the three teratogens. Immunoreactivity generally preceded the development of cytological features of apoptosis. However, p53 was expressed only in the embryos exposed to ionizing radiation and methyl-triazene (an alkylating agent), but not significantly in embryos exposed to retinoic acid. The present results throw some light on apoptosis mechanisms in several teratologic conditions.

Ras proteins belong to the monomeric GTPases familly. They control cell growth, differentiation, proliferation, and survival. Ras mutations are frequently found in human cancers and play a fundamental role in tumorigenesis. Ras requires localization to the plasma membrane to exert its oncogenic effects. This subcelllular localization is dependent of protein farnesylation which is a post translational modification catalysed by the farnesyl transferase enzyme. Farnesyl transferase Inhibitors (FTI) were then designed ten to twelve years ago to inhibit ras processing and consequently the growth of ras mutated tumor. Preclinical data show that FTIs inhibit cell proliferation and survival in vitro and in vivo of a wide range of cancer cell lines, many of which contain wild type ras suggesting that mutated Ras is not the only target of the FTIs effects. Four FTIs went then through clinical trials and three of then are still developed in the clinic. Phase I et II clinical trials confirmed a relevant antitumor activity and a low toxicity. Phase III clinical trials are currently undergoing for both solid and hematologic tumors. The expected results should allow to define the position of FTIs as anticancer drugs, particularly in combination with conventional chemotherapy, hormone therapy, radiotherapy or any other new targeted compound.

Glycemia is a physiological parameter tightly regulated for an optimal energetic supply to the organism, in spite of variable tissular glucose needs. Physiopathological alteration of glycemic regulation leads to dysfunctions of many cell types. For example, diabetes considerably increases morbidity and mortality linked to cardiovascular pathologies and constitute nowadays a serious public health problem. Many in vivo and in vitro studies have investigated the impact of extracellular glucose concentration on smooth muscle and endothelial cells. Glycemia regulates expression and activity of proteins implicated in various processes, such as vasodilation (eNOS), cellular adherence (ICAM-1, VCAM-1), glucose transport (GLUT-1) or free radical generation. Nuclear receptors of the PPAR (peroxisome proliferator-activated receptors) family which are implicated in glucose and lipid metabolism control, seem to have direct vascular actions, in the regulation of cellular functions by extracellular glucose, reinforcing their status of pharmacological targets for preservation and improvement of vascular function. More general processes, such as cellular proliferation and cell death, are also influenced by glucose concentration. Concerning the contractile function, hypoglycemia and hyperglycemia modulate vascular reactivity while acting on the vasoactive substances level and the cellular response to these molecules. In particular they act on variation of ionic channels (K+, Ca2+) activity or by interfering with some signaling pathways (NO). For example, the age-dependant vasodilation and endothelial calcium influx induced by elastin peptide are modulated by extracellular glucose levels. In conclusion, abnormal chronic variations of circulating glucose levels seem to be directly responsible for endothelial and smooth muscle cell dysfunction in the pathogenesis of cardiovascular abnormalities of patients presenting glycemia dysregulations.

GnRH is the native decapeptide which initiates the reproductive cascade. It is synthesized in a loose network of hypothalamic neurons and released into the hypothalamo-pituitary portal blood system in a pulsatile manner. The main physiologic actions of GnRH include the synthesis and release of LH and FSH. Analogs are synthetic versions of GnRH with various amino acid substitutions. These substitutions serve to increase their half-life and to increase their affinity for the GnRH receptor. There are two types of analog: GnRH agonists and GnRH antagonists. GnRH agonists behave like GnRH and are initially stimulatory ("flare up"). GnRH antagonists block the effects of GnRH and are inhibitory. When GnRH antagonists bind to the GnRH receptor they do not initiate the normal cascade of intracellular events, they prevent GnRH from gaining access to the receptor and prevent the above cascade from occuring. Consequently there is no "Flare Effect" and levels of LH and FSH begin immediately to fall. GnRH antagonists do not cause GnRH receptor downregulation: the pituitary remains responsive to GnRH or GnRH agonist administration. The degree of suppression of circulating LH and FSH is dependent on circulating levels of the GnRH antagonist. Administration of GnRH antagonist produces suppression of endogenous LH and FSH at all phases of the cycle. The degree of suppression is dependent on the amount of GnRH antagonist administered. The suppression of endogenous LH and FSH produced by GnRH antagonist can be overridden by GnRH or GnRH agonist.

Bacterial translocation leading to sepsis is increased by obstructive jaundice(OJ). Antithrombin III (ATIII) mediates the promotion of prostaglandin release, an inhibitor of leucocyte activation and downregulator of many proinflammatory cytokines. We investigated the effect of ATIII on histopatology and villus morphology of small intestine.

Because the GnRH receptor plays a paramount role within the reproductive axis, the understanding of the molecular apparatus that governs the tissue-specific expression and regulation of this gene must lead to a better knowledge of the physiology and the physiopathology of the gonadotrope function. To elucidate these mechanisms, we have used two complementary in vivo and in vitro approaches. Firstly, we have isolated the pituitary promoter of the rat GnRH receptor gene and investigated its activity using transient transfection into two gonadotrope-derived cell lines, the alphaT3-1 and the LbetaT2 cell lines. We have thus defined a primary set of transcription factors involved in the tissue-specific expression of the GnRH receptor gene. These include the steroidogenic factor-1 (SF-1) which plays a decisive role while functionally interacting with proteins related to the GATA and LIM homeodomain families of transcription factors. In addition, we highlighted the critical implication of SF-1 and its functional interaction with a CREB-related factor in the stimulatory action of PACAP (Pituitary Adenylate Cyclase Activating Polypeptide) on promoter activity. These results have led us to analyze the activity of this promoter by transgenesis in the mouse using human placental alkaline phosphatase as a reporter gene. In agreement with the in vitro data, the pituitary promoter was found to confer gonadotrope-specific activity in the pituitary. It was also able to direct transgene expression in several areas of the central nervous system known to express the endogenous GnRH receptor, in particular in the hippocampo-septal complex. Some of these tissue do not express SF-1, suggesting that, in vivo, its role would not be as decisive as suggested by the in vitro experiments. Surprisingly, during pituitary ontogenesis, the transgene is expressed as early as E 13.5 whereas SF-1 is not yet present in the pituitary. Thus, in vivo, SF-1 would not be necessary for the activation of the GnRH receptor gene during the early developmental stages in the pituitary. These results are consistent with data obtained following general or pituitary-specific knockout of the gene encoding SF-1, suggesting that the GnRH receptor is expressed despite the absence of this factor. Identifying the factors responsible for the activation of the GnRH receptor gene at these early developmental stages should make it possible to refine the role of SF-1, not only in gene regulation but more generally, in the physiology and the physiopathology of the gonadotrope function.

To study the functional contributions of the 5-HT4 receptor subtype of serotonin (5-HT), we have generated knockout mice lacking the 5-HT4 receptor gene. The male mutant mice exhibit a hyposensitivity to anorexic stress. Our recent data indicate that the pharmacological inactivation, using a systemic injection of the 5-HT4 receptor antagonist RS39604 (0.5 mg/kg), suppressed restraint stress-induced anorexia in wild-type female mice. In parallel, the same treatment reduced the 3,4-N-methylenedioxymethamphetamine (" ecstasy", 10 mg/kg)-induced anorexia in male wild-type mice. Our neurochemical analyses suggest that the mechanisms underlying feeding disorders in 5-HT4 receptor knockout mice are related to a lesser efficacy of 5-HT (hypothalamus, nucleus accumbens), leptin and the cocaine-amphetamine related transcript to reduce food intake following stress.

Duchenne muscle dystrophy results from the absence of dystrophin, a cytoskeletal protein of the muscle fibre. Dystrophin plays an essential role in the integrity of the membrane-associated protein complexes connected to the extracellular matrix. On chromosome 6 is located the gene of a protein presenting 80 % homology with dystrophin : utrophin, which is expressed at the neuromuscular junction. The review examines if utrophin can replace dystrophin and correct the structural and functional characteristics of the myopathy, and how the improvements can be quantitatively expressed. In transgenic mice, deficient in dystrophin, but overexpressing large quantities of utrophin, the latter is found on structures where dystrophin is normally located, histological signs of necrosis disappear and the recovery of functional disorders, specially affecting the mechanical properties of the muscle fibres, can be complete. The review examines also several ways of obtaining overexpression of utrophin in adult mdx mice, such as conditioned expression of the utrophin transgene (using a tetracycline-sensitive transactivator), transfection with viral vectors containing the utrophin cDNA (complete or truncated), actions on factor(s) controlling utrophin expression at the neuromuscular junction (heregulin, 4 N-acetylgalactosamine), and pharmacological ways of inducing expression (NO, arginine). Though partial improvements of the myopathy status have been obtained by these various approaches, they remain limited by their localized action and/or by the moderate level of utrophin expression obtained. Further researchs to overcome these limitations are urgently needed in order to transform the very promising effect of utrophin overexpression into a real treatment of Duchenne myopathy.

We describe an abnormal chromatin clumping phenomenon (ACC) with nuclear loss of segmentation on circulating neutrophils in three patients receiving mycophenolate mofetil (Cellcept). It is worth being aware of this rare adverse event because of its unknown origin and of its possible clinical consequences.