Immunohistochemical Distribution of RGS7 Protein and Cellular Selectivity in Colocalizing with Gαq Proteins in the Adult Rat Brain

Abstract : Regulators of G protein signaling (RGS) proteins serve as potent GTPase-activating proteins for the heterotrimeric G proteins αi/o and αq/11. This study describes the immunohistochemical distribution of RGS7 throughout the adult rat brain and its cellular colocalization with Gαq/11, an important G protein-coupled receptor signal transducer for phospholipase Cβ-mediated activity. In general, both RGS7 and Gαq/11 displayed a heterogeneous and overlapping regional distribution. RGS7 immunoreactivity was observed in cortical layers I-VI, being most intense in the neuropil of layer I. In the hippocampal formation, RGS7 immunoreactivity was concentrated in the strata oriens, strata radiatum, mossy fibers, and polymorphic cells, with faint to nondetectable immunolabeling within the dentate gyrus granule cells and CA1-CA3 subfield pyramidal cells. Numerous diencephalic and brainstem nuclei also displayed dense RGS7 immunostaining. Dual immunofluorescence labeling studies with the two protein-specific antibodies indicated a cellular selectivity in the colocalization between RGS7 and Gαq/11 within many discrete brain regions, such as the superficial cortical layer I, hilus area of the hippocampal formation, and cerebellar Golgi cells. To assess the ability of Gαq/11-mediated signaling pathways to modulate dynamically RGS expression, primary cortical neuronal cultures were incubated with phorbol 12,13-dibutyrate, a selective protein kinase C activator. A time-dependent increase in levels of mRNA for RGS7, but not RGS4, was observed. Our results provide novel information on the region- and cell-specific pattern of distribution of RGS7 with the transmembrane signal transducer, Gαq/11. We also describe a possible RGS7-selective neuronal feedback adaptation on Gαq/11-mediated pathway function, which may play an important role in signaling specificity in the brain.     

2-Aminoethyl diphenylborinate (2-APB) analogues: Regulation of Ca signaling

In order to obtain compounds with modified 2-APB activities, we synthesized number of 2-APB analogues and analyzed their inhibitory activities for SOCE. The IC₅₀ of 2-APB for SOCE inhibition is 3 μM while IC₅₀ of some of our 2-APB analogues range 0.1–10 μM. The adducts of amino acids with diphenyl borinic acid have strong inhibitory activities. By using these compounds, we will be able to regulate intracellular Ca²⁺ concentration and consequent cellular processes more efficiently than with 2-APB.     

Regulator of complement activation (RCA) gene cluster in <Emphasis Type="Italic">Xenopus tropicalis</Emphasis>

Genome and expressed sequence tag information of Xenopus tropicalis suggested that short-consensus repeat (SCR)-containing proteins are encoded by three genes that are mapped within a 300-kb downstream of PFKFB2, which is a marker gene for the regulator of complement activation (RCA) loci in human and chicken. Based on this observation, we cloned the three cDNAs of these proteins using 3′- or 5′-RACE technique. Since their primary structures and locations of the proximity to the PFKFB2 locus, we named them amphibian RCA protein (ARC) 1, 2, and 3. Expression in human HEK293 or CHO cells suggested that ARC1 is a soluble protein of Mr ∼67 kDa, ARC2 is a membrane protein with Mr 44 kDa, and ARC3 a secretary protein with a putative transmembrane region. They were N-glycosylated during maturation. In human and chicken RCA clusters, the order in which genes for soluble, GPI-anchored, and membrane forms of SCR proteins are arranged is from the distant to proximity to the PFKFB2 gene. However, the amphibian ARC1, 2, and 3 resembled one another and did not reflect the same order found in human and chicken RCA genes. This may be due to self-duplication of ARCs to form a family, and it evolved after the amphibia separated from the ancestor of the amniotes, which possessed soluble, GPI-anchored, and membrane forms of SCR protein members. Taken together, frog possesses a RCA locus, but the constitution of the ARC proteins differs from that of the amniotes with a unique self-resemblance.     

Modulation of subfamily B/R4 RGS protein function by 14-3-3 proteins

Regulator of G protein signalling (RGS) proteins are primarily known for their ability to act as GTPase activating proteins (GAPs) and thus attenuate G protein function within G protein-coupled receptor (GPCR) signalling pathways. However, RGS proteins have been found to interact with additional binding partners, and this has introduced more complexity to our understanding of their potential role in vivo. Here, we identify a novel interaction between RGS proteins (RGS4, RGS5, RGS16) and the multifunctional protein 14-3-3. Two isoforms, 14-3-3β and 14-3-3ε, directly interact with all three purified RGS proteins and data from in vitro steady state GTP hydrolysis assays show that 14-3-3 inhibits the GTPase activity of RGS4 and RGS16, but has limited effects on RGS5 under comparable conditions. Moreover in a competitive pull-down experiment, 14-3-3ε competes with Go for RGS4, but not for RGS5. This mechanism is further reinforced in living cells, where 14-3-3ε sequesters RGS4 in the cytoplasm and impedes its recruitment to the plasma membrane by G protein. Thus, 14-3-3 might act as a molecular chelator, preventing RGS proteins from interacting with G, and ultimately prolonging the signal transduction pathway. In conclusion, our findings suggest that 14-3-3 proteins may indirectly promote GPCR signalling via their inhibitory effects on RGS GAP function.     

Starch biosynthesis,its regulation and biotechnological approaches to improve crop yields

Structurally composed of the glucose homopolymers amylose and amylopectin, starch is the main storage carbohydrate in vascular plants, and is synthesized in the plastids of both photosynthetic and non-photosynthetic cells. Its abundance as a naturally occurring organic compound is surpassed only by cellulose, and represents both a cornerstone for human and animal nutrition and a feedstock for many non-food industrial applications including production of adhesives, biodegradable materials, and first-generation bioethanol. This review provides an update on the different proposed pathways of starch biosynthesis occurring in both autotrophic and heterotrophic organs, and provides emerging information about the networks regulating them and their interactions with the environment. Special emphasis is given to recent findings showing that volatile compounds emitted by microorganisms promote both growth and the accumulation of exceptionally high levels of starch in mono- and dicotyledonous plants. We also review how plant biotechnologists have attempted to use basic knowledge on starch metabolism for the rational design of genetic engineering traits aimed at increasing starch in annual crop species. Finally we present some potential biotechnological strategies for enhancing starch content.