Dopamine receptor repertoire of human granulosa cells

Background  

High levels of dopamine (DA) were described in human ovary and recently evidence for DA receptors in granulosa and luteal cells has been provided, as well. However, neither the full repertoire of ovarian receptors for DA, nor their specific role, is established. Human granulosa cells (GCs) derived from women undergoing in vitro fertilization (IVF) are an adequate model for endocrine cells of the follicle and the corpus luteum and were therefore employed in an attempt to decipher their DA receptor repertoire and functionality.     

A structural insight into the inhibition of human and Leishmania donovani ornithine decarboxylases by 1-amino-oxy-3-aminopropane

The critical role of polyamines in key processes such as cell growth, differentiation and macromolecular synthesis makes the enzymes involved in their synthesis potential targets in the treatment of certain types of cancer and parasitic diseases. Here we present a study on the inhibition of human and Leishmania donovani ODC (ornithine decarboxylase), the first committed enzyme in the polyamine biosynthesis pathway, by APA (1-amino-oxy-3-aminopropane). The present study shows APA to be a potent inhibitor of both human and L. donovani ODC with a K(i) value of around 1.0 nM. We also show that L. donovani ODC binds the substrate, the co-enzyme pyridoxal 5'-phosphate and the irreversible inhibitor alpha-difluoromethylornithine (a curative agent of West African sleeping sickness) with less affinity than human ODC. We have also determined the three-dimensional structure of human ODC in complex with APA, which revealed the mode of the inhibitor binding to the enzyme. In contrast with earlier reports, the structure showed no indication of oxime formation between APA and PLP (pyridoxal 5'-phosphate). Homology modelling suggests a similar mode of binding of APA to L. donovani ODC. A comparison of the ODC-APA-PLP structure with earlier ODC structures also shows that the protease-sensitive loop (residues 158-168) undergoes a large conformational change and covers the active site of the protein. The understanding of the structural mode of APA binding may constitute the basis for the development of more specific inhibitors of L. donovani ODC.     

The role of phosphoinositide 3-kinase C2alpha in insulin signaling

The members of the class II phosphoinositide 3-kinase (PI3K) family can be activated by several stimuli, indicating that these enzymes can regulate many intracellular processes. Nevertheless, to date, there has been no definitive identification of their in vivo product, their mechanism(s) of activation, or their precise intracellular roles. By metabolic labeling, we here identify phosphatidylinositol 3-phosphate as the sole in vivo product of the insulin-dependent activation of PI3K-C2alpha, confirming the emerging role of such a phosphoinositide in signaling. We demonstrate that activation of PI3K-C2alpha involves its recruitment to the plasma membrane and that activation is mediated by the GTPase TC10. This is the first report showing a membrane targeting-mediated mechanism of activation for PI3K-C2alpha and that a small GTP-binding protein can activate a class II PI3K isoform. We also demonstrate that PI3K-C2alpha contributes to maximal insulin-induced translocation of the glucose transporter GLUT4 to the plasma membrane and subsequent glucose uptake, definitely assessing the role of this enzyme in insulin signaling.     

Nitric oxide acts as an antioxidant and delays programmed cell death in barley aleurone layers

Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of alpha-amylase. alpha-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective antioxidant in aleurone cells. The amounts of CAT and SOD, two enzymes that metabolize ROS, are greatly reduced in aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and SOD. We speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.     

TBC1D24, an ARF6-Interacting Protein, Is Mutated in Familial Infantile Myoclonic Epilepsy

Idiopathic epilepsies (IEs) are a group of disorders characterized by recurrent seizures in the absence of detectable brain lesions or metabolic abnormalities. IEs include common disorders with a complex mode of inheritance and rare Mendelian traits suggesting the occurrence of several alleles with variable penetrance. We previously described a large family with a recessive form of idiopathic epilepsy, named familial infantile myoclonic epilepsy (FIME), and mapped the disease locus on chromosome 16p13.3 by linkage analysis. In the present study, we found that two compound heterozygous missense mutations (D147H and A509V) in TBC1D24, a gene of unknown function, are responsible for FIME. In situ hybridization analysis revealed that Tbc1d24 is mainly expressed at the level of the cerebral cortex and the hippocampus. By coimmunoprecipitation assay we found that TBC1D24 binds ARF6, a Ras-related family of small GTPases regulating exo-endocytosis dynamics. The main recognized function of ARF6 in the nervous system is the regulation of dendritic branching, spine formation, and axonal extension. TBC1D24 overexpression resulted in a significant increase in neurite length and arborization and the FIME mutations significantly reverted this phenotype. In this study we identified a gene mutation involved in autosomal-recessive idiopathic epilepsy, unveiled the involvement of ARF6-dependent molecular pathway in brain hyperexcitability and seizures, and confirmed the emerging role of subtle cytoarchitectural alterations in the etiology of this group of common epileptic disorders.     

In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery

We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5) was constructed to express green fluorescent protein (GFP) and a small interfering RNA (siRNA) targeting mammalian target of rapamycin (mTOR). The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4-L6) of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200) and small-diameter neurons (nociceptors). The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.     

The F-box protein Dia2 regulates DNA replication

Ubiquitin-mediated proteolysis plays a key role in many pathways inside the cell and is particularly important in regulating cell cycle transitions. SCF (Skp1/Cul1/F-box protein) complexes are modular ubiquitin ligases whose specificity is determined by a substrate-binding F-box protein. Dia2 is a Saccharomyces cerevisiae F-box protein previously described to play a role in invasive growth and pheromone response pathways. We find that deletion of DIA2 renders cells cold-sensitive and subject to defects in cell cycle progression, including premature S-phase entry. Consistent with a role in regulating DNA replication, the Dia2 protein binds replication origins. Furthermore, the dia2 mutant accumulates DNA damage in both S and G2/M phases of the cell cycle. These defects are likely a result of the absence of SCF(Dia2) activity, as a Dia2 DeltaF-box mutant shows similar phenotypes. Interestingly, prolonging G1-phase in dia2 cells prevents the accumulation of DNA damage in S-phase. We propose that Dia2 is an origin-binding protein that plays a role in regulating DNA replication.     

TRAIL/DR5 pathway promotes AKT phosphorylation,skeletal muscle differentiation,and glucose uptake

TNF-related apoptosis-inducing ligand (TRAIL) is a protein that induces apoptosis in cancer cells but not in normal ones, where its effects remain to be fully understood. Previous studies have shown that in high-fat diet (HFD)-fed mice, TRAIL treatment reduced body weight gain, insulin resistance, and inflammation. TRAIL was also able to increase skeletal muscle free fatty acid oxidation. The aim of the present work was to evaluate TRAIL actions on skeletal muscle. Our in vitro data on C2C12 cells showed that TRAIL treatment significantly increased myogenin and MyHC and other hallmarks of myogenic differentiation, which were reduced by Dr5 (TRAIL receptor) silencing. In addition, TRAIL treatment significantly increased AKT phosphorylation, which was reduced by Dr5 silencing, as well as glucose uptake (alone and in combination with insulin). Our in vivo data showed that TRAIL increased myofiber size in HFD-fed mice as well as in db/db mice. This was associated with increased myogenin and PCG1α expression. In conclusion, TRAIL/DR5 pathway promotes AKT phosphorylation, skeletal muscle differentiation, and glucose uptake. These data shed light onto a pathway that might hold therapeutic potential not only for the metabolic disturbances but also for the muscle mass loss that are associated with diabetes.Subject terms: Insulin signalling, Mechanisms of disease