Antioxidant activity and protective effect of Clitoria ternatea flower extract on testicular damage induced by ketoconazole in rats.

BACKGROUND: Ketoconazole (KET), an antifungal drug, has adverse effects on the male reproductive system. Pre-treatments with antioxidant plant against testicular damage induced by KET are required. The flowers of Clitoria ternatea (CT) are proven to have hepatoprotective potential. However, the protective effect on KET-induced testicular damage has not been reported. OBJECTIVE: To investigate the protective effect of CT flower extracts with antioxidant activity on male reproductive parameters including sperm concentration, serum testosterone level, histopathology of the testis, and testicular tyrosine phosphorylation levels in rats induced with KET. METHODS: The antioxidant activity of CT flower extracts was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. Male rats were treated with CT flower extracts (10, 50, or 100 mg/kg BW) or distilled water via a gastric tube for 28 d (preventive period: Days 1-21) and induced by KET (100 mg/kg BW) via intraperitoneal injection for 7 d (induction period: Days 22-28). After the experiment, all animals were examined for the weights of the testis, epididymis plus vas deferens and seminal vesicle, serum testosterone levels, sperm concentration, histological structures and diameter of testis, and testicular tyrosine phosphorylation levels by immunoblotting. RESULTS: The CT flower extracts had capabilities for DPPH scavenging and high reducing power. At 100 mg/kg BW, the extract had no toxic effects on the male reproductive system. Significantly, in CT+KET groups, CT flower extracts (50 and 100 mg/kg BW) alleviated the reduction of reproductive organ weight parameters, testosterone levels, and sperm concentration. In addition, CT flower extracts gave protection from testicular damage in KET-induced rats. Moreover, in the CT100+KET group, CT flower extracts significantly enhanced the expression of a testicular 50-kDa tyrosine phosphorylated protein compared with that of other groups. CONCLUSIONS: C. ternatea flower extracts possessing antioxidant activity are not harmful to the male reproductive system and can protect against testicular damage in KET-induced rats.

FABP7 expression in normal and stab-injured brain cortex and its role in astrocyte proliferation.

Reactive gliosis, in which astrocytes as well as other types of glial cells undergo massive proliferation, is a common hallmark of all brain pathologies. Brain-type fatty acid-binding protein (FABP7) is abundantly expressed in neural stem cells and astrocytes of developing brain, suggesting its role in differentiation and/or proliferation of glial cells through regulation of lipid metabolism and/or signaling. However, the role of FABP7 in proliferation of glial cells during reactive gliosis is unknown. In this study, we examined the expression of FABP7 in mouse cortical stab injury model and also the phenotype of FABP7-KO mice in glial cell proliferation. Western blotting showed that FABP7 expression was increased significantly in the injured cortex compared with the contralateral side. By immunohistochemistry, FABP7 was localized to GFAP(+) astrocytes (21% of FABP7(+) cells) and NG2(+) oligodendrocyte progenitor cells (62%) in the normal cortex. In the injured cortex there was no change in the population of FABP7(+)/NG2(+) cells, while there was a significant increase in FABP7(+)/GFAP(+) cells. In the stab-injured cortex of FABP7-KO mice there was decrease in the total number of reactive astrocytes and in the number of BrdU(+) astrocytes compared with wild-type mice. Primary cultured astrocytes from FABP7-KO mice also showed a significant decrease in proliferation and omega-3 fatty acid incorporation compared with wild-type astrocytes. Overall, these data suggest that FABP7 is involved in the proliferation of astrocytes by controlling cellular fatty acid homeostasis.

Diacylglycerol kinase beta accumulates on the perisynaptic site of medium spiny neurons in the striatum.

Following activation of Gq protein-coupled receptors, phospholipase C yields a pair of second messengers, i.e. diacylglycerol (DAG) and inositol 1,4,5-trisphosphate. The former activates protein kinase C and the latter mobilizes Ca(2+) from intracellular store. DAG kinase (DGK) then phosphorylates DAG to produce another second messenger (phosphatidic acid). Of 10 mammalian DGK isozymes, DGKbeta is expressed in dopaminergic projection fields with the highest level in the striatum and its particular splice variant is differentially expressed in patients with bipolar disorder. To gain molecular anatomical evidence for its signaling role, we investigated the cellular expression and subcellular localization of DGKbeta in the striatum of rat brain. DGKbeta was expressed in medium spiny neurons constituting the striatonigral and striatopallidal pathways, whereas striatal interneurons were below the detection threshold. DGKbeta was distributed in somatodendritic elements of medium spiny neurons and localized in association with the smooth endoplasmic reticulum and plasma membrane or in the narrow cytoplasmic space between them. In particular, DGKbeta exhibited dense accumulation at perisynaptic sites on dendritic spines forming asymmetrical synapses. The characteristic anatomical localization was consistent with exclusive enrichment of DGKbeta in the microsomal and postsynaptic density fractions. Intriguingly, DGKbeta was very similar in immunohistochemical and immunochemical distribution to Gq-coupled receptors, such as metabotropic glutamate receptors 1 and 5, and also to other downstream molecules involving DAG metabolism, such as phospholipase C beta and DAG lipase. These findings suggest that abundant DGKbeta is provided to perisynaptic sites of medium spiny neurons so that it can effectively produce phosphatidic acid upon activation of Gq-coupled receptors and modulate the cellular state of striatal output neurons.

Prominent expression and activity-dependent nuclear translocation of Ca2+/calmodulin-dependent protein kinase Idelta in hippocampal neurons.

Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) including CaMKI, II and IV, are thought to regulate a variety of neuronal functions. Unlike CaMKII, which is regulated by autophosphorylation, CaMKI as well as CaMKIV are activated by CaMKK. In this study, we examined the cellular and subcellular localization of CaMKIdelta, a recently identified fourth isoform of CaMKI, in the mature brain. In situ hybridization analysis demonstrated wide expression of CaMKIdelta mRNA in the adult mouse brain with prominent expression in the hippocampal pyramidal cells. FLAG-tagged CaMKIdelta was localized at the cytoplasm and neurites without nuclear immunoreactivity in approximately 80% of the transfected primary hippocampal neurons. The stimulation with either KCl depolarization or glutamate triggered the nuclear localization of FLAG-tagged CaMKIdelta by two-fold with a peak at 1 min. In contrast, the catalytically inactive mutants of CaMKIdelta remained cytoplasmic without nuclear translocation during KCl depolarization, indicating the requirement of its activation for the nuclear translocation. Furthermore, we showed that immunoprecipitated CaMKIdelta could phosphorylate cAMP response element binding protein (CREB)alphain vitro and that the over-expression of CaMKIdelta enhanced GAL4-CREB-luciferase activity in PC12 cells stimulated by KCl depolarization. Our present study provides the first evidence for the possible involvement of CaMKIdelta in nuclear functions through its nuclear translocation in response to stimuli that trigger intracellular Ca2+ influx.

p55 protein is a member of PSD scaffold proteins in the rat brain and interacts with various PSD proteins.

p55 is a membrane-associated guanylate kinase (MAGuK) family member that consists of a single PDZ followed by SH3, HOOK and guanylate kinase (GuK or GK) domains. We investigated rat p55 (r-p55) in the brain. r-p55 mRNA was expressed widely in various tissues and in various regions of the brain. r-p55 protein was also expressed widely in various rat tissues, including brain and erythrocytes. The protein was enriched in the synaptic plasma membrane and postsynaptic density (PSD) fractions of the forebrain. An immunocytochemical study using cultured cortical neurons suggested postsynaptic localization of r-p55 protein. Pull-down assay showed that r-p55 protein interacted with r-p55 itself and various PSD proteins, such as PSD-95, SAP97, GKAP, CASK, GRIP, neuroligin, cadherin, tubulin, actin, alpha-internexin, neurofilament-L and Ca(2+)/calmodulin-dependent protein kinase II, through its PDZ, SH3, HOOK or GK domains. The interaction with PSD-95 was found to occur between the PDZ domains of PSD-95 and the HOOK and GK domains of r-p55 protein. These findings, together with the presence of r-p55 puncta in a period of early synaptogenesis, suggest that r-p55 protein functions as one of postsynaptic scaffold component in an early stage of synaptogenesis in the brain. r-p55 protein may form a basic structure, which interlinks diverse functional molecules of the PSD necessary for postsynaptic signaling and synaptic adhesion.

Cloning and characterization of a novel variant (mM-rdgBbeta1) of mouse M-rdgBs, mammalian homologs of Drosophila retinal degeneration B gene proteins, and its mRNA localization in mouse brain in comparison with other M-rdgBs.

We report the cloning, characterization and localization in the brain of a novel isoform termed mM-rdgBbeta1 (mouse type of mammalian retinal degeneration Bbeta1 protein) in comparison with the localization of three known mammalian homologs (M-rdgBbeta, M-rdgB1, M-rdgB2). mM-rdgBbeta1 cDNA contains a sequence of 119 bp as a form of insertion in the open reading frame of the known mM-rdgBbeta, and encodes a protein of 269 amino acids with a calculated molecular mass of 31.7 kDa, different from the molecular mass of 38.3 kDa of mM-rdgBbeta. It also contains a phosphatidylinositol transfer protein (PITP)-like domain similar to the known three homologs, as well as D-rdgB. The recombinant mM-rdgBbeta1 protein shows the specific binding activity to phosphatidylinositol but not to other phospholipids. This novel molecule is localized not only in the cytoplasm but also in the nucleus, different from the cytoplasmic localization of mM-rdgBbeta. In in situ hybridization analysis, the gene expression for mM-rdgBbeta1 in the brain, though weak, is rather confined to the embryonic stage, different from wider expression of mM-rdgBbeta in the gray matters of pre- and post-natal brains. Taken together, mM-rdgBbeta1 is suggested to play a role in the phosphoinositide-mediated signaling in the neural development.