Nobuteru Usuda

Sept8 controls the binding of vesicle-associated membrane protein 2 to synaptophysin.

Septins, a conserved family of GTP/GDP‐binding proteins, are present in organisms as diverse as yeast and mammals. We analyzed the distribution of five septins, Sept6, Sept7, Sept8, Sept9 and Sept11, in various rat tissues by western blot analyses and found all septins to be expressed in brain. We also examined the developmental changes of expression of these septins in the rat brain and found that the level of Sept8 increased during post‐natal development. Morphological analyses revealed that Sept8 is enriched at pre‐synapses. Using yeast two‐hybrid screening, we identified vesicle‐associated membrane protein 2 (VAMP2), a soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE), as an interacting protein for Sept8. Synaptophysin is reported to associate with and recruit VAMP2 to synaptic vesicles and dissociate prior to forming the SNARE complex consisting of VAMP2, syntaxin and synaptosome‐associated protein of 25 kDa. We showed that Sept8 suppresses the interaction between VAMP2 and synaptophysin through binding to VAMP2. In addition, we found that Sept8 forms a complex with syntaxin1A, and the Sept8‐VAMP2 interaction is disrupted by synaptosome‐associated protein of 25 kDa. These results suggest that Sept8 may participate in the process of the SNARE complex formation and subsequent neurotransmitter release.

Characterization of a multidomain adaptor protein, p140Cap, as part of a pre-synaptic complex

p140Cap (Cas‐associated protein) is an adaptor protein considered to play pivotal roles in cell adhesion, growth and Src tyrosine kinase‐related signaling in non‐neuronal cells. It is also reported to interact with a pre‐synaptic membrane protein, synaptosome‐associated protein of 25 kDa, and may participate in neuronal secretion. However, properties and precise functions of p140Cap in neuronal cells are almost unknown. Here we show, using biochemical analyses, that p140Cap is expressed in rat brain in a developmental stage‐dependent manner, and is relatively abundant in the synaptic plasma membrane fraction in adults. Immunohistochemistry showed localization of p140Cap in the neuropil in rat brain and immunofluorescent analyses detected p140Cap at synapses of primary cultured rat hippocampal neurons. Electron microscopy further revealed localization at pre‐ and post‐synapses. Screening of p140Cap‐binding proteins identified a multidomain adaptor protein, vinexin, whose third Src‐homology 3 domain interacts with the C‐terminal Pro‐rich motif of p140Cap. Immunocomplexes between the two proteins were confirmed in COS7 and rat brain. We also clarified that a pre‐synaptic protein, synaptophysin, interacts with p140Cap. These results suggest that p140Cap is involved in neurotransmitter release, synapse formation/maintenance, and signaling.

Strain‐dependent viral dynamics and virus‐cell interactions in a novel in vitro system supporting the life cycle of blood‐borne hepatitis C virus

We developed an in vitro system that can be used for the study of the life cycle of a wide variety of blood‐borne hepatitis C viruses (HCV) from various patients using a three‐dimensional hollow fiber culture system and an immortalized primary human hepatocyte (HuS‐E/2) cell line. Unlike the conventional two‐dimensional culture, this system not only enhanced the infectivity of blood‐borne HCV but also supported its long‐term proliferation and the production of infectious virus particles. Both sucrose gradient fractionation and electron microscopy examination showed that the produced virus‐like particles are within a similar fraction and size range to those previously reported. Infection with different HCV strains showed strain‐dependent different patterns of HCV proliferation and particle production. Fluctuation of virus proliferation and particle production was found during prolonged culture and was found to be associated with change in the major replicating virus strain. Induction of cellular apoptosis was only found when strains of HCV‐2a genotype were used for infection. Interferon‐alpha stimulation also varied among different strains of HCV‐1b genotypes tested in this study. Conclusion: These results suggest that this in vitro infection system can reproduce strain‐dependent events reflecting viral dynamics and virus‐cell interactions at the early phase of blood‐borne HCV infection, and that this system can allow the development of new anti‐HCV strategies specific to various HCV strains. (HEPATOLOGY 2009.)

Phosphorylation of neuronal nitric oxide synthase at Ser847 in the nucleus intermediolateralis after spinal cord injury in mice

We previously demonstrated that Ca2+/calmodulin (CaM)-dependent protein kinase IIalpha (CaM-KIIalpha) can phosphorylate neuronal nitric oxide synthase (nNOS) at Ser847 and attenuate NOS activity in neuronal cells. In the present study we focused on chronological alteration in levels and cellular location of nNOS, phosphorylated (p)-Ser847-nNOS (NP847), CaM-KII and p-Thr286-CaM-KIIalpha following spinal cord injury (SCI) in mice. Western blot analysis showed nNOS to be significantly phosphorylated at Ser847 from 3 h after SCI, peaking at 24 h and gradually decreasing thereafter, and CaM-KII to be colocalized with nNOS after SCI. Immunohistochemical analysis revealed that SCI causes an increase in both NP847 and p-Thr286-CaM-KIIalpha in the nucleus intermediolateralis. These findings suggest that SCI induces p-Thr286-CaM-KIIalpha, which phosphorylates the nNOS at Ser847 in the nucleus intermediolateralis where NO is thought to play a role as a neurotransmitter in autonomic preganglionic neurons. Thus, the NP847 signaling pathway might be involved in the autonomic failure which occurs immediately after SCI.

Transcription coactivator PRIP, the Peroxisome Proliferator-Activated Receptor (PPAR)-interacting protein, is redundant for the function of nuclear receptors PPARα and CAR, the constitutive androstane receptor, in mouse liver

Disruption of the genes encoding for the transcription coactivators, peroxisome proliferator-activated receptor (PPAR)-interacting protein (PRIP/ASC-2/RAP250/TRBP/NRC) and PPAR-binding protein (PBP/TRAP220/DRIP205/MED1), results in embryonic lethality by affecting placental and multiorgan development. Targeted deletion of coactivator PBP gene in liver parenchymal cells (PBP(LIV-/-)) results in the near abrogation of the induction of PPARalpha and CAR (constitutive androstane receptor)-regulated genes in liver. Here, we show that targeted deletion of coactivator PRIP gene in liver (PRIP(LIV-/-)) does not affect the induction of PPARalpha-regulated pleiotropic responses, including hepatomegaly, hepatic peroxisome proliferation, and induction of mRNAs of genes involved in fatty acid oxidation system, indicating that PRIP is not essential for PPARalpha-mediated transcriptional activity. We also provide additional data to show that liver-specific deletion of PRIP gene does not interfere with the induction of genes regulated by nuclear receptor CAR. Furthermore, disruption of PRIP gene in liver did not alter zoxazolamine-induced paralysis, and acetaminophen-induced hepatotoxicity. Studies with adenovirally driven EGFP-CAR expression in liver demonstrated that, unlike PBP, the absence of PRIP does not prevent phenobarbital-mediated nuclear translocation/retention of the receptor CAR in liver in vivo and cultured hepatocytes in vitro. These results show that PRIP deficiency in liver does not interfere with the function of nuclear receptors PPARalpha and CAR. The dependence of PPARalpha- and CAR-regulated gene transcription on coactivator PBP but not on PRIP attests to the existence of coactivator selectivity in nuclear receptor function.

Immunohistochemical localization of mitochondrial fatty acid β-oxidation enzymes in Müller cells of the retina

The presence of a mitochondrial fatty acid β-oxidation system in the retina was shown by immunohistochemistry. Fatty acids are considered to serve as a major energy source metabolized by fatty acid β-oxidation together with glucose metabolized by glycolysis in the organs of the entire body, but almost nothing is known about this metabolic system in the retina. Adult rat retinae were subjected to immunofluorescence and immuno-electron microscopy for the localization of fatty acid β-oxidation enzymes, together with western blot analysis for quantitation of the amount of enzyme proteins and DNA microarray analysis for gene expression. All the enzymes examined were shown to be present in the retina, but in small amounts, with the amount of protein and gene expression in the retina being about 1/10 of those in the liver. Immunohistochemistry at light and electron microscopic levels revealed the enzymes to be more preferentially localized to the mitochondria of Müller cells than the retinal neurons. The Müller cells were isolated from the retina and confirmed for the presence of mitochondrial fatty acid β-oxidation enzymes. A mitochondrial fatty acid β-oxidation system was thus shown to be present in the retina heterogeneously.

Activation of endothelial nitric oxide synthase following spinal cord injury in mice

Endothelial nitric oxide synthase (eNOS) plays a neuroprotective role after cerebral ischemia through the production of NO, which enhances cerebral blood flow. However, precise details regarding activation of eNOS after spinal cord injury (SCI) largely remain to be elucidated. In the present study we investigated chronological alteration and cellular location of eNOS and phosphorylated (p)-eNOS at Ser(1177) following SCI in mice. Western blot analysis showed eNOS to be significantly phosphorylated at Ser(1177) from 1 to 2 days after mild SCI, with gradual decrease thereafter. Immunohistochemistry revealed the p-eNOS to be mainly expressed in the endothelial cells of microvessels within gray matter under these conditions. These findings suggest that mild SCI activates eNOS in the subacute stage, which increases spinal cord blood flow and may be involved in protective and repair responses.

Immunohistochemical Localization of Mitochondrial Fatty Acid β-Oxidation Enzymes in Rat Testis

The testis consists of two types of tissues, the interstitial tissue and the seminiferous tubule, which have different functions and are assumed to have different nutritional metabolism. The localization of enzymes of the mitochondrial fatty acid β-oxidation system in the testis was investigated to obtain a better understanding of nutrient metabolism in the testis. Adult rat testis tissues were subjected to immunoblot analysis for quantitation of the amounts of enzyme proteins, to DNA microarray analysis for gene expression, and to immunofluorescence and immunoelectron microscopy for localization. Quantitative analysis by immunoblot and DNA microarray revealed that enzymes occur abundantly in Leydig cells in the interstitial tissue but much less so in the seminiferous tubules. Immunohistochemistry revealed that Leydig cells in the interstitial tissue and Sertoli cells in the seminiferous tubules contain a full set of mitochondrial fatty acid β-oxidation enzymes in relatively plentiful amounts among the cells in the testis, but that this is not so in spermatogenic cells. This characteristic localization of the mitochondrial fatty acid β-oxidation system in the testis needs further elucidation in terms of a possible role for it in the nutritional metabolism of spermatogenesis.

High-contrast imaging of plastic-embedded tissues by phase contrast electron microscopy.

Phase contrast electron microscopy utilizing phase plates has been considered suitable for high-contrast observation of weak phase objects. This novel technique was newly applied to histochemically stained strong phase objects of osmificated biological specimens. Sections of various thicknesses, specifically stained for the Golgi apparatus by the ZIO technique using the heavy metals Zn and Os, were observed with a phase contrast electron microscope in Zernike and Hilbert imaging modes. Quantitative analysis of image contrast in real space and the power spectrum in Fourier space showed a high-contrast gain even for strong phase objects. This result clearly indicates that phase contrast electron microscopy can be effectively used not only for weak phase objects but also for strong phase objects in biology.

Activation of c-jun in the rat basilar artery after subarachnoid hemorrhage

Subarachnoid hemorrhage (SAH) initiates a series of cellular and molecular events, some of which involve a mitogen activated protein kinase, c-jun N-terminal kinase (JNK). However, precise details regarding activation of c-jun in the vessel wall after SAH largely remain to be elucidated. In this study, we therefore investigated the localization and time-dependent expression of c-jun in the rat basilar artery after SAH in a rat single-hemorrhage model featuring infusion of autologous arterial blood. Basilar arteries were obtained at 2, 6 and 12h and 1, 2, 4 and 7 days after SAH, as well as from controls. Western blot analysis with c-jun, phosphorylated c-jun at Ser(63), and actin antibodies revealed that c-jun was immediately phosphorylated at Ser(63) within 2h, thereafter gradually becoming dephosphorylated, while total c-jun and actin levels remained almost unchanged. Immunohistochemistry demonstrated phosphorylation of c-jun at Ser(63) to occur in smooth muscle cells of the basilar artery 2h after SAH. These results indicate that c-jun is activated in the basilar artery immediately after the onset of SAH, presumably resulting in transcription of immediate early genes and smooth muscle cell proliferation.