Catherine Bisbal

A 37 kDa 2-5A binding protein as a potential biochemical marker for chronic fatigue syndrome.

PURPOSE Recent studies have revealed abnormalities in the ribonuclease L pathway in peripheral blood mononuclear cells of patients with the chronic fatigue syndrome. We conducted a blinded study to detect possible differences in the distribution of 2-5A binding proteins in the cells of patients with chronic fatigue syndrome and controls. PATIENTS AND METHODS We studied 57 patients with chronic fatigue syndrome and 53 control subjects (28 healthy subjects and 25 patients with depression or fibromyalgia). A radioactive probe was used to label 2-5A binding proteins in unfractionated peripheral blood mononuclear cell extracts and to compare their distribution in the three groups. RESULTS A 37 kDa 2-5A binding polypeptide was found in 50 (88%) of the 57 patients with chronic fatigue syndrome compared with 15 (28%) of the 53 controls (P < 0.01). When present, the amount of 37 kDa protein was very low in the control groups. When expressed as the ratio of the 37 kDa protein to the 80 kDa protein, 41 (72%) of the 57 patients with chronic fatigue syndrome had a ratio > 0.05, compared with 3 (11%) of the 28 healthy subjects and none of the patients with fibromyalgia or depression. CONCLUSION The presence of a 37 kDa 2-5A binding protein in extracts of peripheral blood mononuclear cells may distinguish patients with chronic fatigue syndrome from healthy subjects and those suffering from other diseases.

The 2′-5′ Oligoadenylate/RNase L/RNase L Inhibitor Pathway Regulates Both MyoD mRNA Stability and Muscle Cell Differentiation

ABSTRACT The 2′-5′ oligoadenylate (2-5A)/RNase L pathway is one of the enzymatic pathways induced by interferon. RNase L is a latent endoribonuclease which is activated by 2-5A and inhibited by a specific protein known as RLI (RNase L inhibitor). This system has an important role in regulating viral infection. Additionally, variations in RNase L activity have been observed during cell growth and differentiation but the significance of the 2-5A/RNase L/RLI pathway in these latter processes is not known. To determine the roles of RNase L and RLI in muscle differentiation, C2 mouse myoblasts were transfected with sense and antisense RLI cDNA constructs. Importantly, the overexpression of RLI in C2 cells was associated with diminished RNase L activity, an increased level of MyoD mRNA, and accelerated kinetics of muscle differentiation. Inversely, transfection of the RLI antisense construct was associated with increased RNase L activity, a diminished level of MyoD mRNA, and delayed differentiation. In agreement with these data, MyoD mRNA levels were also decreased in C2 cells transfected with an inducible RNase L construct. The effect of RNase L activity on MyoD mRNA levels was relatively specific because expression of several other mRNAs was not altered in C2 transfectants. Therefore, RNase L is directly involved in myoblast differentiation, probably through its role in regulating MyoD stability. This is the first identification of a potential mRNA target for RNase L.

The 2–5A/RNase L/RNase L Inhibitor (RNI) Pathway Regulates Mitochondrial mRNAs Stability in Interferon α-treated H9 Cells

Interferon α (IFNα) belongs to a cytokine family that exhibits antiviral properties, immuno-modulating effects, and antiproliferative activity on normal and neoplasic cells in vitro and in vivo. IFNα exerts antitumor action by inducing direct cytotoxicity against tumor cells. This toxicity is at least partly due to induction of apoptosis. Although the molecular basis of the inhibition of cell growth by IFNα is only partially understood, there is a direct correlation between the sensitivity of cells to the antiproliferative action of IFNα and the down-regulation of their mitochondrial mRNAs. Here, we studied the role of the 2–5A/RNase L system and its inhibitor RLI in this regulation of the mitochondrial mRNAs by IFNα. We found that a fraction of cellular RNase L and RLI is localized in the mitochondria. Thus, we down-regulated RNase L activity in human H9 cells by stably transfecting (i) RNase L antisense cDNA or (ii) RLI sense cDNA constructions. In contrast to control cells, no post-transcriptional down-regulation of mitochondrial mRNAs and no cell growth inhibition were observed after IFNα treatment in these transfectants. These results demonstrate that IFNα exerts its antiproliferative effect on H9 cells at least in part via the degradation of mitochondrial mRNAs by RNase L.

Grape Polyphenols Prevent Fructose- Induced Oxidative Stress and Insulin Resistance in First-Degree Relatives of Type 2 Diabetic Patients

OBJECTIVE To assess the clinical efficacy of nutritional amounts of grape polyphenols (PPs) in counteracting the metabolic alterations of high-fructose diet, including oxidative stress and insulin resistance (IR), in healthy volunteers with high metabolic risk. RESEARCH DESIGN AND METHODS Thirty-eight healthy overweight/obese first-degree relatives of type 2 diabetic patients (18 men and 20 women) were randomized in a double-blind controlled trial between a grape PP (2 g/day) and a placebo (PCB) group. Subjects were investigated at baseline and after 8 and 9 weeks of supplementation, the last 6 days of which they all received 3 g/kg fat-free mass/day of fructose. The primary end point was the protective effect of grape PPs on fructose-induced IR. RESULTS In the PCB group, fructose induced 1) a 20% decrease in hepatic insulin sensitivity index (P < 0.05) and an 11% decrease in glucose infusion rate (P < 0.05) as evaluated during a two-step hyperinsulinemic-euglycemic clamp, 2) an increase in systemic (urinary F2-isoprostanes) and muscle (thiobarbituric acid–reactive substances and protein carbonylation) oxidative stress (P < 0.05), and 3) a downregulation of mitochondrial genes and decreased mitochondrial respiration (P < 0.05). All the deleterious effects of fructose were fully blunted by grape PP supplementation. Antioxidative defenses, inflammatory markers, and main adipokines were affected neither by fructose nor by grape PPs. CONCLUSIONS A natural mixture of grape PPs at nutritional doses efficiently prevents fructose-induced oxidative stress and IR. The current interest in grape PP ingredients and products by the global food and nutrition industries could well make them a stepping-stone of preventive nutrition.

A newly discovered function for RNase L in regulating translation termination.

The antiviral and antiproliferative effects of interferons are mediated in part by the 2′-5′ oligoadenylate–RNase L RNA decay pathway. RNase L is an endoribonuclease that requires 2′-5′ oligoadenylates to cleave single-stranded RNA. In this report we present evidence demonstrating a role for RNase L in translation. We identify and characterize the human translation termination factor eRF3/GSPT1 as an interacting partner of RNase L. We show that interaction of eRF3 with RNase L leads to both increased translation readthrough efficiency at premature termination codons and increased +1 frameshift efficiency at the antizyme +1 frameshift site. On the basis of our results, we present a model describing how RNase L is involved in regulating gene expression by modulating the translation termination process.

Antioxidants and glucose metabolism disorders.

Purpose of reviewRecent evidence suggests that oxidative stress is a cornerstone of the metabolic mechanisms by which overfeeding leads to insulin resistance. This review is an update of the most recent arguments in favor of this theory and of the possible role of antioxidants. Recent findingsReactive oxidative species (ROS) are produced by multiple pathways within the cell and are essential for many cellular functions. ROS production is balanced by enzymatic and nonenzymatic antioxidant systems. The perturbation of the pro-oxidant/antioxidant balance can lead to increased oxidative damage of macromolecules, a phenomenon known as ‘oxidative stress’. ROS are involved both in insulin signal transduction and in insulin resistance when produced in excess. Overfeeding, saturated fatty acids, and obesity play a key role in the excessive production of ROS. However, a diet rich in fruits and vegetables, and therefore antioxidants, has demonstrated beneficial effects against oxidative damages and insulin resistance. SummaryExperimental data are in favor of a beneficial role of antioxidants in glucose metabolism, but clinical data in humans are more controversial. Even if a diet rich in fruits and vegetables could provide an optimal mix of antioxidants, it remains unclear whether supplementation with antioxidants alone can reproduce the same effect.

Dietary antioxidants: Do they have a role to play in the ongoing fight against abnormal glucose metabolism?

Overfeeding, an increased intake of saturated fatty acids, and sugary foods are key dietary changes that have occurred in recent decades in addition to the emergence of the obesity epidemic. In addition to an increase in energy storage as fat, these dietary changes are accompanied by an increase in mitochondrial macronutrient oxidation, leading to an excessive free radical production and, hence, oxidative stress. The latter has long been considered a central mechanism linking nutrient overload, insulin resistance, the metabolic syndrome, and diabetes. However, food, through fruit and vegetable consumption, also can be a great source of antioxidants that protect the body against oxidative damage and insulin resistance and thus help cope with the metabolic backlash of the energy-dense Westernized diet. Experimental data are in favor of the beneficial role conveyed by antioxidants in glucose metabolism, but clinical data in humans remain controversial. This review therefore aimed to sort out any underlying discrepancies and provide an overall clear view of the role of antioxidants in the ongoing fight against abnormal glucose metabolism.

Regulation of mitochondrial mRNA stability by RNase L is translation-dependent and controls IFN α -induced apoptosis

Interferons (IFNs) inhibit the growth of many different cell types by altering the expression of specific genes. IFNs activities are partly mediated by the 2′-5′ oligoadenylates-RNase L RNA decay pathway. RNase L is an endoribonuclease requiring activation by 2′-5′ oligoadenylates to cleave single-stranded RNA. Here, we present evidence that degradation of mitochondrial mRNA by RNase L leads to cytochrome c release and caspase 3 activation during IFNα-induced apoptosis. We identify and characterize the mitochondrial translation initiation factor (IF2mt) as a new partner of RNase L. Moreover, we show that specific inhibition of mitochondrial translation with chloramphenicol inhibits the IFNα-induced degradation of mitochondrial mRNA by RNase L. Finally, we demonstrate that overexpression of IF2mt in human H9 cells stabilizes mitochondrial mRNA, inhibits apoptosis induced by IFNα and partially reverses IFNα-cell growth inhibition. On the basis of our results, we propose a model describing how RNase L regulates mitochondrial mRNA stability through its interaction with IF2mt.

Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

Skeletal muscle maintenance and repair involve several finely coordinated steps in which pluripotent stem cells are activated, proliferate, exit the cell cycle and differentiate. This process is accompanied by activation of hundreds of muscle-specific genes and repression of genes associated with cell proliferation or pluripotency. Mechanisms controlling myogenesis are precisely coordinated and regulated in time to allow the sequence of activation/inactivation of genes expression. Muscular differentiation is the result of the interplay between several processes such as transcriptional induction, transcriptional repression and mRNA stability. mRNA stability is now recognized as an essential mechanism of control of gene expression. For instance, we previously showed that the endoribonuclease L (RNase L) and its inhibitor (RLI) regulates MyoD mRNA stability and consequently muscle differentiation. We now performed global gene expression analysis by SAGE to identify genes that were down-regulated upon activation of RNase L in C2C12 myogenic cells, a model of satellite cells. We found that RNase L regulates mRNA stability of factors implicated in the control of pluripotency and cell differentiation. Moreover, inappropriate RNase L expression in C2C12 cells led to inhibition of myogenesis and differentiation into adipocytes even when cells were grown in conditions permissive for muscle differentiation. Conversely, over-expression of RLI allowed muscle differentiation of myogenic C2C12 cells even in non permissive conditions. These findings reveal the central role of RNase L and RLI in controlling gene expression and cell fate during myogenesis. Our data should provide valuable insights into the mechanisms that control muscle stem cell differentiation and into the mechanism of metaplasia observed in aging or muscular dystrophy where adipose infiltration of muscle occurs.

Biological Activities of Oligonucleotides Linked to Poly(L-Lysine)

Abstract We have developped a method to couple oligonucleotides to poly(L-lysine). This tool has been tested with anti m-RNA synthetic oligodeoxyribonucleotides and 2′-5′(A)n and allows oligonucleotides to enter intact cells.