Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic signaling

Sonic hedgehog (Shh) is a crucial regulator of organ development during embryogenesis. We investigated whether intramyocardial gene transfer of naked DNA encoding human Shh (phShh) could promote a favorable effect on recovery from acute and chronic myocardial ischemia in adult animals, not only by promoting neovascularization, but by broader effects, consistent with the role of this morphogen in embryogenesis. After Shh gene transfer, the hedgehog pathway was upregulated in mammalian fibroblasts and cardiomyocytes. This resulted in preservation of left ventricular function in both acute and chronic myocardial ischemia by enhanced neovascularization, and reduced fibrosis and cardiac apoptosis. Shh gene transfer also enhanced the contribution of bone marrow-derived endothelial progenitor cells to myocardial neovascularization. These data suggest that Shh gene therapy may have considerable therapeutic potential in individuals with acute and chronic myocardial ischemia by triggering expression of multiple trophic factors and engendering tissue repair in the adult heart.     

Neuroglycan C, a novel member of the neuregulin family

Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan expressed predominantly in the brain that possesses an EGF-like extracellular domain. The goal of the present study was to determine whether NGC may activate ErbB tyrosine kinases. A recombinant human NGC extracellular domain induced tyrosine phosphorylation of ErbB2 and ErbB3 as well as cell growth of the human breast tumor cell lines, T47D and MDA-MB-453. In vitro pull-down assay revealed that NGC could directly bind to a recombinant ErbB3-immunoglobulin Fc fusion protein (ErbB3-Fc) but not to ErbB1-Fc, ErbB2-Fc or ErbB4-Fc. A newly established anti-ErbB3 neutralizing monoclonal antibody (#5C3) almost completely blocked NGC-induced ErbB activation in MDA-MB-453 cells. Taken together, these data indicate that NGC is an active growth factor and a direct ligand for ErbB3 and that NGC transactivates ErbB2. Thus, NGC should be classified as the sixth member (neuregulin-6) of the neuregulin family.     

Repression of tax expression is associated both with resistance of human T-cell leukemia virus type 1-infected T cells to killing by tax-specific cytotoxic T lymphocytes and with impaired tumorigenicity in a rat model

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Although the viral transactivation factor, Tax, has been known to have apparent transforming ability, the exact function of Tax in ATL development is still not clear. To understand the role of Tax in ATL development, we introduced short-interfering RNAs (siRNAs) against Tax in a rat HTLV-1-infected T-cell line. Our results demonstrated that expression of siRNA targeting Tax successfully downregulated Tax expression. Repression of Tax expression was associated with resistance of the HTLV-1-infected T cells to Tax-specific cytotoxic-T-lymphocyte killing. This may be due to the direct effect of decreased Tax expression, because the Tax siRNA did not alter the expression of MHC-I, CD80, or CD86. Furthermore, T cells with Tax downregulation appeared to lose the ability to develop tumors in T-cell-deficient nude rats, in which the parental HTLV-1-infected cells induce ATL-like lymphoproliferative disease. These results indicated the importance of Tax both for activating host immune response against the virus and for maintaining the growth ability of infected cells in vivo. Our results provide insights into the mechanisms how the host immune system can survey and inhibit the growth of HTLV-1-infected cells during the long latent period before the onset of ATL.     

Neuroglycan C,A Brain-Specific Chondroitin Sulfate Proteoglycan,Interacts with Pleiotrophin,A Heparin-Binding Growth Factor

Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that promotes neurite outgrowth. To identify the ligand of NGC, we applied a detergent-solubilized membrane fraction of fetal rat brains to an NGC-immobilized affinity column. Several proteins were eluted from the column including an 18 kDa-band protein recognized by an anti-pleiotrophin antibody. The binding of pleiotrophin (PTN) to NGC was confirmed by a quartz crystal microbalance method and had a Kd of 8.7 nM. PTN bound to the acidic amino acid cluster of the NGC extracellular domain. In addition, PTN bound to both chondroitin sulfate-bearing NGC and chondroitinase-treated NGC prepared from the neonatal rat brain. These results suggest that NGC interacts with PTN.     

Suppressive effect of exposure to Chenopodium album var. centrorubrum on the feeding response of the tortoise beetle, Cassida nebulosa, to spinach

Depending upon preceding dietary exposure, the tortoise beetle, Cassida nebulosa L. (Coleoptera: Chrysomelidae), exhibits various feeding responses to spinach, Spinacia oleracea L. (Chenopodiaceae). Adult tortoise beetles reared as adults on the main host Chenopodium album var. centrorubrum Makino (CAC) (Chenopodiaceae) eat little spinach, whereas those reared on spinach accept this plant as food. In this study, we investigated the suppressive effect of exposure to CAC on the feeding response of tortoise beetles to spinach. To investigate the effect of chemical factors in CAC, we provided beetles with Gomphrena globosa L. (Amaranthaceae) as the base food and exposed them to a CAC extract, because bioassays have indicated that exposure to G. globosa did not affect the feeding response of beetles to spinach. Adults reared on G. globosa treated with CAC extract consumed significantly less than adults reared on untreated G. globosa . Thus, we determined that exposure to CAC components is responsible for the suppressive effect on the feeding response to spinach observed in the beetles. In a choice feeding test with membrane filter discs, we found that adults reared on G. globosa treated with CAC extract exhibited a greater feeding deterrent response to the spinach deterrent than adults reared on untreated G. globosa leaves. Therefore, the suppression of spinach consumption due to exposure to CAC is probably caused by the development of a response to the feeding deterrents present in spinach.     

Discriminating formation of HNO from other reactive nitrogen oxide species

Nitroxyl (HNO) exhibits unique pharmacological properties that often oppose those of nitric oxide (NO), in part due to differences in reactivity toward thiols. Prior investigations suggested that the end products arising from the association of HNO with thiols were condition-dependent, but were inconclusive as to product identity. We therefore used HPLC techniques to examine the chemistry of HNO with glutathione (GSH) in detail. Under biological conditions, exposure to HNO donors converted GSH to both the sulfinamide [GSONH2] and the oxidized thiol (GSSG). Higher thiol concentrations generally favored a higher GSSG ratio, suggesting that the products resulted from competitive consumption of a single intermediate (GSNHOH). Formation of GSONH2 was not observed with other nitrogen oxides (NO, N2O3, NO2, or ONOO(-)),indicating that it is a unique product of the reaction of HNO with thiols. The HPLC assay was able to detect submicromolar concentrations of GSONH2. Detection of GSONH2 was then used as a marker for HNO production from several proposed biological pathways, including thiol-mediated decomposition of S-nitrosothiols and peroxidase-driven oxidation of hydroxylamine (an end product of the reaction between GSH and HNO) and NG-hydroxy-l-arginine (an NO synthase intermediate). These data indicate that free HNO can be biosynthesized and thus may function as an endogenous signaling agent that is regulated by GSH content.     

Phosphorylation and activation of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) by Ca2+/calmodulin-dependent protein kinase I (CaMKI)

Nuclear Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) is an enzyme that dephosphorylates and downregulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs) as well as AMP-dependent protein kinase. In our previous study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing and translocated to cytosol in a proteasome inhibitor-sensitive manner. In the present study, we found that zCaMKP-N is regulated by phosphorylation at Ser-480. When zCaMKP-N was incubated with the activated CaMKI, time-dependent phosphorylation of the enzyme was observed. This phosphorylation was significantly reduced when Ser-480 was replaced by Ala, suggesting that CaMKI phosphorylates Ser-480 of zCaMKP-N. Phosphorylation-mimic mutants, S480D and S480E, showed higher phosphatase activities than those of wild type and S480A mutant in solution-based phosphatase assay using various substrates. Furthermore, autophosphorylation of CaMKII after ionomycin treatment was more severely attenuated in Neuro2a cells when CaMKII was cotransfected with the phosphorylation-mimic mutant of zCaMKP-N than with the wild-type or non-phosphorylatable zCaMKP-N. These results strongly suggest that phosphorylation of zCaMKP-N at Ser-480 by CaMKI activates CaMKP-N catalytic activity and thereby downregulates multifunctional CaMKs in the cytosol.     

Synaptic vesicles are capable of synthesizing the VGLUT substrate glutamate from α-ketoglutarate for vesicular loading

Synaptic vesicle loading of glutamate is a pivotal step in glutamate synaptic transmission. The molecular machinery responsible for this step is comprised of v-type proton-pump ATPase and a vesicular glutamate transporter. Recent evidence indicates that synaptic vesicles are endowed with glycolytic ATP-synthesizing enzymes, providing energy for immediate use by vesicle-bound proton-pump ATPase. In this study, we provide evidence that synaptic vesicles are also capable of synthesizing the vesicular glutamate transporter substrate glutamate, from α-ketoglutarate and l-aspartate (as the amino group donor); glutamate thus produced is taken up into vesicles. We also report a finding that α-ketoglutarate-derived glutamate uptake into synaptic vesicles and aspartate aminotransferase are inhibited by 2,3-pyrazinedicarboxylate. Evidence is given that this is a selective inhibitor for aspartate aminotransferase. These observations provide insight into understanding the nerve endings' mechanism for high efficiency in glutamate transmission. Finding this inhibitor may have implications for further experimentation on the role of α-ketoglutarate-derived glutamate in glutamate transmission.     

Tributyltin induces oxidative stress and neuronal injury by inhibiting glutathione S-transferase in rat organotypic hippocampal slice cultures

Tributyltin (TBT) has been used as a heat stabilizer, agricultural pesticide and antifouling agents on ships, boats and fish-farming nets; however, the neurotoxicity of TBT has recently become a concern. TBT is suggested to stimulate the generation of reactive oxygen species (ROS) inside cells. The aim of this study was to determine the mechanism of neuronal oxidative injury induced by TBT using rat organotypic hippocampal slice cultures. The treatment of rat hippocampal slices with TBT induced ROS production, lipid peroxidation and cell death. Pretreatment with antioxidants such as superoxide dismutase, catalase or trolox, suppressed the above phenomena induced by TBT, indicating that TBT elicits oxidative stress in hippocampal slices, which causes neuronal cell death. TBT dose-dependently inhibited glutathione S-transferase (GST), but not glutathione peroxidase or glutathione reductase in the cytosol of rat hippocampus. The treatment of hippocampal slices with TBT decreased the GST activity. Pretreatment with reduced glutathione attenuated the reduction of GST activity and cell death induced by TBT, indicating that the decrease in GST activity by TBT is involved in hippocampal cell death. When hippocampal slices were treated with sulforaphane, the expression and activity of GST were increased. Notably, TBT-induced oxidative stress and cell death were significantly suppressed by pretreatment with sulforaphane. These results indicate that GST inhibition could contribute, at least in part, to the neuronal cell death induced by TBT in hippocampal slices. This study is the first report to show the link between neuronal oxidative injury and the GST inhibition elicited by TBT.     

Complement C1q activates canonical Wnt signaling and promotes aging-related phenotypes

Wnt signaling plays critical roles in development of various organs and pathogenesis of many diseases, and augmented Wnt signaling has recently been implicated in mammalian aging and aging-related phenotypes. We here report that complement C1q activates canonical Wnt signaling and promotes aging-associated decline in tissue regeneration. Serum C1q concentration is increased with aging, and Wnt signaling activity is augmented during aging in the serum and in multiple tissues of wild-type mice, but not in those of C1qa-deficient mice. C1q activates canonical Wnt signaling by binding to Frizzled receptors and subsequently inducing C1s-dependent cleavage of the ectodomain of Wnt coreceptor low-density lipoprotein receptor-related protein 6. Skeletal muscle regeneration in young mice is inhibited by exogenous C1q treatment, whereas aging-associated impairment of muscle regeneration is restored by C1s inhibition or C1qa gene disruption. Our findings therefore suggest the unexpected role of complement C1q in Wnt signal transduction and modulation of mammalian aging.