Cloning and characterization of the

Analysis and Evaluation of Conceptual Models of Nursing

Two neuronal G proteins are involved in chemosensation of the Caenorhabditis elegans Dauer-inducing pheromone.

Mammalian cells utilize multiple signaling mechanisms to protect against the osmotic stress that accompanies plasma membrane ion transport, solute uptake, and turnover of protein and carbohydrates (Schliess, F., and Haussinger, D. (2002) Biol. Chem. 383, 577-583). Recently, osmotic stress was found to increase synthesis of bisdiphosphoinositol tetrakisphosphate ((PP)2-InsP4), a high energy inositol pyrophosphate (Pesesse, X., Choi, K., Zhang, T., and Shears, S. B. (2004) J. Biol. Chem. 279, 43378-43381). Here, we describe the purification from rat brain of a diphosphoinositol pentakisphosphate kinase (PPIP5K) that synthesizes (PP)2-InsP4. Partial amino acid sequence, obtained by mass spectrometry, matched the sequence of a 160-kDa rat protein containing a putative ATP-grasp kinase domain. BLAST searches uncovered two human isoforms (PPIP5K1 (160 kDa) and PPIP5K2 (138 kDa)). Recombinant human PPIP5K1, expressed in Escherichia coli, was found to phosphorylate diphosphoinositol pentakisphosphate (PP-InsP5) to (PP)2-InsP4 (Vmax = 8.3 nmol/mg of protein/min; Km = 0.34 microM). Overexpression in human embryonic kidney cells of either PPIP5K1 or PPIP5K2 substantially increased levels of (PP)2-InsP4, whereas overexpression of a catalytically dead PPIP5K1(D332A) mutant had no effect. PPIP5K1 and PPIP5K2 were more active against PP-InsP5 than InsP6, both in vitro and in vivo. Analysis by confocal immunofluorescence showed PPIP5K1 to be distributed throughout the cytoplasm but excluded from the nucleus. Immunopurification of overexpressed PPIP5K1 from osmotically stressed HEK cells (0.2 M sorbitol; 30 min) revealed a persistent, 3.9 +/- 0.4-fold activation when compared with control cells. PPIP5Ks are likely to be important signaling enzymes.

Purification, sequencing, and molecular identification of a mammalian PP-InsP5 kinase that is activated when cells are exposed to hyperosmotic stress

The Dopamine D2 Receptor Gene

Ethanol is a widely used drug, excessive consumption of which could lead to medical conditions with diverse symptoms Ethanol abuse causes dysfunction of memory, attention, speech and locomotion across species Dopamine signaling plays an essential role in ethanol dependent behaviors in animals ranging from C elegans to humans We devised an ethanol dependent assay in which mutants in the dopamine autoreceptor, dop-2, displayed a unique sedative locomotory behavior causing the animals to move in circles while dragging the posterior half of their body Here, we identify the posterior dopaminergic sensory neuron as being essential to modulate this behavior We further demonstrate that in dop-2 mutants, ethanol exposure increases dopamine secretion and functions in a DVA interneuron dependent manner DVA releases the neuropeptide NLP-12 that is known to function through cholinergic motor neurons and affect movement Thus, DOP-2 modulates dopamine levels at the synapse and regulates alcohol induced movement through NLP-12

/pdf/increased-dopaminergic-neurotransmission-results-in-ethanol-524lw3aep4.pdf

Increased dopaminergic neurotransmission results in ethanol dependent sedative behaviors in Caenorhabditis elegans.

Perturbations in synaptic function could affect the normal behavior of an animal, making it important to understand the regulatory mechanisms of synaptic signaling. Previous work has shown that in Caenorhabditis elegans an immunoglobulin superfamily protein, RIG-3, functions in presynaptic neurons to maintain normal acetylcholine receptor levels at the neuromuscular junction (NMJ). In this study, we elucidate the molecular and functional mechanism of RIG-3. We demonstrate by genetic and BiFC (Bi-molecular Fluorescence Complementation) assays that presynaptic RIG-3 functions by directly interacting with the immunoglobulin domain of the nonconventional Wnt receptor, ROR receptor tyrosine kinase (RTK), CAM-1, which functions in postsynaptic body-wall muscles. This interaction in turn inhibits Wnt/LIN-44 signaling through the ROR/CAM-1 receptor, and allows for maintenance of normal acetylcholine receptor, AChR/ACR-16, levels at the neuromuscular synapse. Further, this work reveals that RIG-3 and ROR/CAM-1 function through the β-catenin/HMP-2 at the NMJ. Taken together, our results demonstrate that RIG-3 functions as an inhibitory molecule of the Wnt/LIN-44 signaling pathway through the RTK, CAM-1.

https://www.genetics.org/content/genetics/206/3/1521.full.pdf

Regulation of WNT Signaling at the Neuromuscular Junction by the Immunoglobulin Superfamily Protein RIG-3 in Caenorhabditis elegans.

Cell surface immunoglobulin superfamily (IgSF) proteins play important roles in the development and function of the nervous system . Here we define the role of a Caenorhabditis elegans IgSF protein, RIG-3, in the function of the AVA command interneuron. This study reveals that RIG-3 regulates the abundance of the glutamate receptor subunit, GLR-1, in the AVA command interneuron and also regulates reversal behavior in C. elegans. The mutant strain lacking rig-3 (rig-3 (ok2156)) shows increased reversal frequency during local search behaviors. Genetic and behavioral experiments suggest that RIG-3 functions through GLR-1 to regulate reversal behavior. We also show that the increased reversal frequency seen in rig-3 mutants is dependent on the increase in GLR-1 abundance at synaptic inputs to AVA, suggesting that RIG-3 alters the synaptic strength of incoming synapses through GLR-1. Consistent with the imaging experiments, altered synaptic strength was also reflected in increased calcium transients in rig-3 mutants when compared to wild-type control animals. Our results further suggest that animals lacking rig-3 show increased AVA activity, allowing the release of FLP-18 neuropeptide from AVA, which is an activity-dependent signaling molecule. Finally, we show that FLP-18 functions through the neuropeptide receptor, NPR-5, to modulate reversal behavior in C. elegans.

https://www.genetics.org/content/genetics/214/1/135.full.pdf

Control of Locomotory Behavior of Caenorhabditis elegans by the Immunoglobulin Superfamily Protein RIG-3

The perception of our surrounding environment is an amalgamation of stimuli detected by sensory neurons. In Caenorhabditis elegans, olfaction is an essential behavior that determines various behavioral functions such as locomotion, feeding and development. Sensory olfactory cues also initiate downstream neuroendocrine signaling that controls aging, learning, development and reproduction. Innate sensory preferences toward odors (food, pathogens) and reproductive pheromones are modulated by 11 pairs of amphid chemosensory neurons in the head region of C. elegans Amongst these sensory neurons, the ASI neuron has neuroendocrine functions and secretes neuropeptides, insulin-like peptide (DAF-28) and the TGF-β protein, DAF-7. Its expression levels are modulated by the presence of food (increased levels) and population density (decreased levels). A recent study has shown that EXP-1, an excitatory GABA receptor regulates DAF-7/TGF-β levels and participates in DAF-7/TGF-β-mediated behaviors such as aggregation and bordering. Here, we show that exp-1 mutants show defective responses toward AWC-sensed attractive odors in a non-autonomous manner through ASI neurons. Our dauer experiments reveal that in daf-7 mutants, ASI expressed EXP-1 and STR-2 (a G-protein-coupled receptor; GPCR) that partially maintained reproductive growth of animals. Further, studies suggest that neuronal connections between ASI and AWC neurons are allowed at least partially through ASI secreted DAF-7 or through alternate TGF- β pathway/s regulated by EXP-1 and STR-2. Together, our behavioral, genetic and imaging experiments propose that EXP-1 and STR-2 integrate food cues and allow the animals to display DAF-7/TGF-β neuroendocrine dependent or independent behavioral responses contributing to chemosensensory and developmental plasticity.

/pdf/dauer-formation-in-c-elegans-is-modulated-through-awc-and-3wqoe3jpd1.pdf

Dauer Formation in C. elegans Is Modulated through AWC and ASI-Dependent Chemosensation.

Inositol pyrophosphates (PPx{square}InsPs) are of key interest, since they are known to participate in multiple physiological processes from lower eukaryotes to humans. However, limited knowledge is available for their role in plants and especially in crops. In this study, two diphosphoinositol pentakisphosphate kinase PPIP5K wheat homologs, TaVIH1 and TaVIH2 were identified and characterized for their spatio-temporal expression along with their physiological functions. The biochemical assay demonstrated the presence of active VIH-kinase domains as evident from the InsP6 phosphorylation activity. The yeast complementation assays showed differential function, where only TaVIH2-3B was capable of rescuing the growth defects of yeast vip1{Delta} genotype. Reporter assays with TaVIH2-promoter in Arabidopsis displayed strong GUS expression in response to dehydration stress and Pi-starvation with similar observations noted at the transcriptional level. In an attempt to identify VIH2 function, a yeast two hybrid screen of wheat library resulted in the identification of multiple interacting proteins primarily associated with cell-wall. One such interactor of wheat VIH2-3B was identified to be a Fasciclin-like arabinogalactan protein (FLA6) that was confirmed by pull-down assay. Systematic analysis of transgenic Arabidopsis overexpressing TaVIH2-3B protein showed robust growth and enhanced relative water content when compared to controls. Biochemical analysis of their cell-wall components in the shoots resulted in increased accumulation of polysaccharides such as cellulose, arabinogalactan and arabinoxylan, whereas Atvih2-3 mutant showed decrease in some of these components. Overall, our results provide novel insight into the functional role of inositol pyrophosphate kinases that modulate cell-wall components so as to provide tolerance towards the dehydration stress.

/pdf/wheat-inositol-pyrophosphate-kinase-tavih2-3b-interacts-with-40z31utdu0.pdf

Wheat inositol pyrophosphate kinase (TaVIH2-3B) interacts with Fasciclin-like arabinogalactan (FLA6) protein and alters the plant cell-wall composition

Pratima Pandey

Papers

Increased dopaminergic neurotransmission results in ethanol dependent sedative behaviors in Caenorhabditis elegans.

Regulation of WNT Signaling at the Neuromuscular Junction by the Immunoglobulin Superfamily Protein RIG-3 in Caenorhabditis elegans.

Control of Locomotory Behavior of Caenorhabditis elegans by the Immunoglobulin Superfamily Protein RIG-3

Dauer Formation in C. elegans Is Modulated through AWC and ASI-Dependent Chemosensation.

Wheat inositol pyrophosphate kinase (TaVIH2-3B) interacts with Fasciclin-like arabinogalactan (FLA6) protein and alters the plant cell-wall composition