5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Skeletal Muscle Insulin Resistance and Absence of Inflammation Characterize Insulin-Resistant Grade I Obese Women

ContextObesity is associated with insulin-resistance (IR), the key feature of type 2 diabetes. Although chronic low-grade inflammation has been identified as a central effector of IR development, it has never been investigated simultaneously at systemic level and locally in skeletal muscle and adipose tissue in obese humans characterized for their insulin sensitivity.ObjectivesWe compared metabolic parameters and inflammation at systemic and tissue levels in normal-weight and obese subjects with different insulin sensitivity to better understand the mechanisms involved in IR development.Methods30 post-menopausal women were classified as normal-weight insulin-sensitive (controls, CT) and obese (grade I) insulin-sensitive (OIS) or insulin-resistant (OIR) according to their body mass index and homeostasis model assessment of IR index. They underwent a hyperinsulinemic-euglycemic clamp, blood sampling, skeletal muscle and subcutaneous adipose tissue biopsies, an activity questionnaire and a self-administrated dietary recall. We analyzed insulin sensitivity, inflammation and IR-related parameters at the systemic level. In tissues, insulin response was assessed by P-Akt/Akt expression and inflammation by macrophage infiltration as well as cytokines and IκBα expression.ResultsSystemic levels of lipids, adipokines, inflammatory cytokines, and lipopolysaccharides were equivalent between OIS and OIR subjects. In subcutaneous adipose tissue, the number of anti-inflammatory macrophages was higher in OIR than in CT and OIS and was associated with higher IL-6 level. Insulin induced Akt phosphorylation to the same extent in CT, OIS and OIR. In skeletal muscle, we could not detect any inflammation even though IκBα expression was lower in OIR compared to CT. However, while P-Akt/Akt level increased following insulin stimulation in CT and OIS, it remained unchanged in OIR.ConclusionOur results show that systemic IR occurs without any change in systemic and tissues inflammation. We identified a muscle defect in insulin response as an early mechanism of IR development in grade I obese post-menopausal women.     

LIMK1 regulates Golgi dynamics, traffic of Golgi-derived vesicles, and process extension in primary cultured neurons

In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.     

Diverse functions of RNase L and implications in pathology

The endoribonuclease L (RNase L) is the effector of the 2-5A system, a major enzymatic pathway involved in the molecular mechanism of interferons (IFNs). RNase L is a very unusual nuclease with a complex mechanism of regulation. It is a latent enzyme, expressed in nearly every mammalian cell type. Its activation requires its binding to a small oligonucleotide, 2-5A. 2-5A is a series of unique 5'-triphosphorylated oligoadenylates with 2'-5' phosphodiester bonds. By regulating viral and cellular RNA expression, RNase L plays an important role in the antiviral and antiproliferative activities of IFN and contributes to innate immunity and cell metabolism. The 2-5A/RNase L pathway is implicated in mediating apoptosis in response to viral infections and to several types of external stimuli. Several recent studies have suggested that RNase L could have a role in cancer biology and evidence of a tumor suppressor function of RNase L has emerged from studies on the genetics of hereditary prostate cancer.     

MAP6-F Is a Temperature Sensor That Directly Binds to and Protects Microtubules from Cold-induced Depolymerization

Microtubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease.     

Increased stability and antiviral activity of 2'-O-phosphoglyceryl derivatives of (2'-5')oligo(adenylate)

Metabolically stable analogues of (2'-5')oligo(adenylate), (2'-5')(A)n, might constitute a new class of antiviral agents as they mimic some of the effects of interferons. 2'-O-phosphoglyceryl derivatives of (2'-5')(A)n oligomers, (2'-5')(A)n-PGro have been synthesized by chemical modification of their terminal ribose residue. Such analogues are resistant to degradation by phosphodiesterases but remain sensitive to phosphatase activity, at least in cell-free extracts. In line with its increased stability, (2'-5')(A)n-PGro has a powerful antiviral activity against an RNA virus when microinjected with micropipettes into the cytoplasm of intact cells. This antiviral activity remains transient however, possibly as a consequence of degradation in intact cells. Since (2'-5')(A)n and its derivatives do not easily cross cell membranes, their possible use in antiviral chemotherapy is tightly linked with the development of vectors suitable for their administration in vivo.     

Regeneration of enzyme activity after western blot: activation of RNase L by 2-5A on filter--importance for its detection.

A rapid and convenient new procedure for detecting RNase L activity following Western blot by renaturation of the enzyme on the nitrocellulose sheets is described. This method allows the simultaneous analysis of enzymatic activity (e.g., cleavage of poly(uridylic acid)-3'-[32P]pCp) and RNase L binding to radioactivE probes (e.g., 2-5A-3'-[32P]pCp) in the same sample. Unlike previously published methods, this procedure eliminates interference from proteases or other RNases during the analysis of RNase L activity. The detection of RNase(s) L is also affected by the presence of endogenous 2-5A, 2-5A derivatives, or other possible "inhibitors" in cell extracts; this Western blot assay allows of RNase(s) L to be detected independently of intracellular 2-5A or analogs. Differences between the procedures used so far and this Western blot technique can indeed be demonstrated. It is shown with this Western blot assay that although RNase L has been described as a protein of 185-200 kDa under nondenaturating conditions, its 80-kDa (and 40-kDa) component is able to bind 2-5A and to cleave poly(uridylic acid) in a 2-5A-dependent way, independently of other subunit(s) or cofactor(s).