Subunit-specific Regulation of N-Methyl-d-aspartate (NMDA) Receptor Trafficking by SAP102 Protein Splice Variants

Synapse-associated protein 102 (SAP102) is a scaffolding protein abundantly expressed early in development that mediates glutamate receptor trafficking during synaptogenesis. Mutations in human SAP102 have been reported to cause intellectual disability, which is consistent with its important role during early postnatal development. SAP102 contains PDZ, SH3, and guanylate kinase (GK)-like domains, which mediate specific protein-protein interactions. SAP102 binds directly to N-methyl-d-aspartate receptors (NMDARs), anchors receptors at synapses, and facilitates transduction of NMDAR signals. Proper localization of SAP102 at the postsynaptic density is essential to these functions. However, how SAP102 is targeted to synapses is unclear. In the current study we find that synaptic localization of SAP102 is regulated by alternative splicing. The SAP102 splice variant that possesses a C-terminal insert (I2) between the SH3 and GK domains is highly enriched at dendritic spines. We also show that there is an intramolecular interaction between the SH3 and GK domains in SAP102 but that the I2 splicing does not influence SH3-GK interaction. Previously, we have shown that SAP102 expression promotes spine lengthening. We now find that the spine lengthening effect is independent of the C-terminal alternative splicing of SAP102. In addition, expression of I2-containing SAP102 isoforms is regulated developmentally. Knockdown of endogenous I2-containing SAP102 isoforms differentially affect NMDAR surface expression in a subunit-specific manner. These data shed new light on the role of SAP102 in the regulation of NMDAR trafficking.     

GnRH-like proteins in cows: concentrations during corpora lutea development and selective localization in granulosal cells

Gonadotropin-releasing hormone (GnRH)-like proteins with anti-gonadotropic properties were recently discovered in the ovaries of several species, including humans. Since neither GnRH receptors nor GnRH are in bovine ovarian tissue, we examined, in the present studies, whether concentrations of GnRH-like proteins varied during development of the corpus luteum (CL) and whether GnRH-like proteins were selectively localized in ovarian cells of cows. For these studies, GnRH-like proteins were extracted from various ovarian and nonovarian tissues and fluids and fractionated for hydrophobic interaction chromatography. A highly specific and sensitive radioreceptor assay (RRA) was used to quantify concentrations of GnRH-like proteins. The major findings of these studies demonstrated that 1) the amount of GnRH-like proteins in the corpus luteum (CL) was proportional to the weight of the CL; 2) the concentration of GnRH-like proteins in luteal tissue decreased during development of the CL; 3) GnRH-like proteins were in ovarian and numerous nonovarian tissues, but were not in the heart, plasma, or follicular fluid; 4) the retention time for GnRH-like proteins following high-pressure liquid chromatography (HPLC) varied with the tissue source; and 5) compared with all other tissues, the greatest concentration of GnRH-like proteins was in granulosal cells. We concluded that the concentration of GnRH-like proteins in luteal cells decreased during development of the CL, and that a specific GnRH-like protein was selectively localized in bovine granulosal cells.     

Effects of prostaglandin E1 or E2 (PGE1; PGE2) on luteal function and binding of luteinizing hormone in nonpregnant ewes

Two studies were conducted to determine the effects of PGE1 or PGE2 on luteal function and binding of luteinizing hormone (LH) to luteal cell membranes in nonpregnant ewes. In Study I, ewes (n=5 per group) received an injection of vehicle (VEH) or 333 micrograms of PGE1 or PGE2 into the tissue surrounding the ovarian vascular pedicle (intrapedicle) on day 7 postestrus. Systemic progesterone concentrations of PGE1-treated ewes were greater (P less than 0.01) than those of VEH-treated ewes at 24 and 48 hr after injection. For PGE2-treated ewes, progesterone concentrations were greater (P less than 0.01) than for VEH-treated ewes only at 24 hr. Neither PGE1 nor PGE2 affected luteal weights or LH binding capacity at 48 hr. Treatment with PGE1, however, increased (P less than 0.10) endogenously bound LH at this time. In Study II, ewes (n=5 per group) received an intrapedicle injection of VEH, or 10 mg of PGE1 or PGE2 on day 8 postestrus. Systemic progesterone concentrations in PGE1-treated ewes were less (P less than 0.01) than for VEH-treated ewes at 24 hr, but by 72 hr were not different from those of VEH-treated ewes. For PGE2-treated ewes, systemic progesterone declined steadily to reach low values by 72 hr. Prostaglandin E2 had no effect on luteal binding of LH at 72 hr, whereas PGE1 increased (P less than 0.05) LH binding capacity and endogenously bound LH. Although PGE2 had no apparent affect on luteal binding of LH in these studies, PGE1 may enhance the function of ovine corpora lutea by stimulating an increase in their binding of LH and capacity to bind LH when the CL receives a luteolytic signal.     

Mechanisms for the antigonadotropic action of the ovarian gonadotropin-releasing hormone-binding inhibitor protein/histone H2A on ovarian cells.

A GnRH-binding inhibitor (GnRH-BI) was recently purified from bovine ovaries. On the basis of amino acid composition and partial sequence analysis this antigonadotropic GnRH-BI was identified as histone H2A. In the present study the mechanism for the antigonadotropic action of histone H2A was examined and compared to that of GnRH and poly-L-lysine. The potential sites examined were the receptor-coupled pathway of second message synthesis including receptor binding of hormone, G protein activation, and adenylyl cyclase activation. Histone H2A inhibited (ID50 = 2 microM) the binding of hCG by membrane receptors from luteinized rat ovaries in a noncompetitive and dose-dependent manner. The binding of FSH by membrane receptors from immature rat ovaries was not inhibited by histone H2A. Binding of GnRH by pituitary membrane receptors was inhibited by histone H2A, and the ID50 of 8 microM was similar to that previously observed for GnRH binding sites in rat ovarian membranes. No high-affinity binding of histone H2A by rat ovarian membranes was detected. Near-maximal doses of histone H2A (7 microM), poly-L-lysine (10 microM), and GnRH (1 microM) inhibited LH-stimulated cAMP production in isolated rat luteal cells. Inhibition by H2A and poly-L-lysine was larger than by GnRH. Furthermore, histone H2A and poly-L-lysine inhibited cholera toxin (CT)-stimulated cAMP production, but GnRH did not. Like GnRH, neither histone H2A nor poly-L-lysine inhibited forskolin (FK)-stimulated cAMP production. In isolated rat granulosa cells, histone H2A and poly-L-lysine inhibited FSH-, CT-, and FK-stimulated cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Comparison of the Use of In Vitro-Transcribed and Native rRNA for the Quantification of Microorganisms in the Environment

Abstract Nearly full-length, small subunit (SSU) rRNA was transcribed in vitro from clones of SSU rDNA genes. Comparing the use of in vitro-transcribed and native rRNA indicated that, when in vitro-transcribed rRNA was used as a standard for quantitative hybridizations with oligonucleotide probes, the population was consistently underestimated. The population abundance was expressed as a percentage of specific target SSU rRNA (determined with a specific oligonucleotide probe), relative to the total SSU rRNA (measured with a universal probe). Differences in hybridization signals could be related to specific probe target locations and rRNA denaturation conditions, suggesting that higher order structure is important in quantitative membrane hybridizations. Therefore, in vitro-transcribed rRNA cannot always be used for the absolute quantification of microbial populations, but can be employed as a standard to quantify shifts in population abundance over time, and to compare community structure in various environments.     

The guanosine 5'-diphosphate D-mannose: guanosine 5'-diphosphate L-galactose epimerase of Chlorella pyrenoidosa. Chemical synthesis of guanosine 5'-diphosphate L-galactose and further studies of the enzyme and the reaction it catalyzes.

An enzyme from extracts of the green alga Chlorella pyrenoidosa that catalyzes the reversible epimerization of guanosine 5′-diphosphate d-mannose to guanosine 5′-diphosphate l-galactose was further purified. The substrate guanosine 5′-diphosphate l-galactose was made chemically by the morpholidate procedure. An improved method was developed for the synthesis of an intermediate in that process, β-l-galactopyranosyl phosphate, via an orthoester of l-galactose. Various characteristics of the enzyme and the reaction it catalyzes were studied. A new method using gas-liquid chromatography was introduced for following the course of the reaction with unlabeled substrates.     

Control of ascorbic acid efflux in rat luteal cells: role of intracellular calcium and oxygen radicals

In luteal cells, prostaglandin (PG)F_2a mobilizes intracellular calcium concentration ([Ca]_i), generates reactive oxygen species (ROS), depletes ascorbic acid (AA) levels, inhibits steroidogenesis, and ultimately induces cell death. We investigated the hypothesis that [Ca]_i mobilization stimulates ROS, which results in depletion of cellular AA in rat luteal cells. We used a self-referencing AA-selective electrode that noninvasively measures AA flux at the extended boundary layer of single cells and fluorescence microscopy with fura 2 and dichlorofluorescein diacetate (DCF-DA) to measure [Ca]_i and ROS, respectively. Menadione, a generator of intracellular superoxide radical (), PGF_2a, and calcium ionophore were shown to increase [Ca]_i and stimulate intracellular ROS. With calcium ionophore and PGF_2a, but not menadione, the generation of ROS was dependent on extracellular calcium influx. In unstimulated cells there was a net efflux of AA of 121.5 ± 20.3 fmol · cm^–1 · s^–1 (mean ± SE, n = 8), but in the absence of extracellular calcium the efflux was significantly reduced (10.3 ± 4.9 fmol · cm^–1 · s^–1; n = 5, P < 0.05). PGF_2a and menadione stimulated AA efflux, but calcium ionophore had no significant effect. These data suggest two AA regulatory mechanisms: Under basal conditions, AA efflux is calcium dependent and may represent recycling and maintenance of an antioxidant AA gradient at the plasma membrane. Under luteolytic hormone and/or oxidative stress, AA efflux is stimulated that is independent of extracellular calcium influx or generation of ROS. Although site-specific mobilization of calcium pools and ROS cannot be ruled out, the release of AA by PGF_2a-stimulated luteal cells may occur through other signaling pathways. luteolysis; apoptosis; self-referencing microelectrode     

Eicosatetraynoic and eicosatriynoic acids, lipoxygenase inhibitors, block meiosis via antioxidant action

We previously showed thatnordihydroguaiaretic acid (NDGA) and other antioxidants inhibit theresumption of meiosis in oocyte-cumulus complexes (OCC) and denudedoocytes (DO). Because NDGA is well known to be an inhibitor oflipoxygenases (LOX), we assessed whether other LOX inhibitors influencespontaneous germinal vesicle breakdown (GVBD) in OCC and DO.Spontaneous GVBD in rat OCC obtained from preovulatory follicles wassignificantly and reversibly inhibited by the minimum effective dosesof 80 and 100 µM 5,8,11,14-eicosatetraynoic acid (ETYA) and5,8,11-eicosatriynoic acid (ETI), respectively. In DO, GVBD wassignificantly inhibited by 100 µM ETYA or ETI. The minimum effectiveconcentrations of ETYA and ETI for inhibition of GVBD in either OCC orDO are ~30- to 50-fold higher than the concentrations necessary toinhibit LOX activity by 50% in intact cells. Because we previouslyshowed that NDGA and other antioxidants inhibit the spontaneousresumption of meiosis, we assessed whether ETYA and ETI may actsimilarly as scavengers of reactive oxygen species (ROS).Luminol-amplified chemiluminescence showed that 50 µM of either ETYAor ETI markedly and significantly reduced ROS generated with 10 mM2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH). Moreover,incubation of DO with 30 mM AAPH reversed the inhibition of GVBDproduced by 100 µM ETYA or ETI. These findings support the conclusionthat ETYA and ETI inhibit oocyte maturation by acting as antioxidantsrather than by inhibiting LOX.

     

Perennial Biomass Grasses and the Mason–Dixon Line: Comparative Productivity across Latitudes in the Southern Great Plains

Understanding latitudinal adaptation of switchgrass (Panicum virgatum L.) and Miscanthus (Miscanthus?×?giganteus J. M. Greef & Deuter ex Hodk. & Renvoize) to the southern Great Plains is key to maximizing productivity by matching each grass variety to its optimal production environment. The objectives of this study were: (1) to quantify latitudinal variation in production of representative upland switchgrass ecotypes (Blackwell, Cave-in-Rock, and Shawnee), lowland switchgrass ecotypes (Alamo, Kanlow), and Miscanthus in the southern half of the US Great Plains and (2) to investigate the environmental factors affecting yield variation. Leaf area and yield were measured on plots at 10 locations in Missouri, Arkansas, Oklahoma, and Texas. More cold winter days led to decreased subsequent Alamo switchgrass yields and increased subsequent upland switchgrass yields. More hot-growing season days led to decreased Kanlow and Miscanthus yields. Increased drought intensity also contributed to decreased Miscanthus yields. Alamo switchgrass had the greatest radiation use efficiency (RUE) with a mean of 4.3 g per megajoule intercepted PAR and water use efficiency (WUE) with a mean of 4.5 mg of dry weight per gram of water transpired. The representative RUE values for other varieties ranged from 67 to 80 % of Alamo’s RUE value and 67 to 87 % of Alamo’s WUE. These results will provide valuable inputs to process-based models to realistically simulate these important perennial grasses in this region and to assess the environmental impacts of production on water use and nutrient demands. In addition, it will also be useful for landowners and companies choosing the most productive perennial grasses for biofuel production.