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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. (J Histochem Cytochem 58:195–206, 2010)

Severe subarachnoid hemorrhage (SAH) induces dysfunction of endothelial nitric oxide synthase (eNOS), resulting in severe vasospasm. Clinically, however, some portions of cerebral arteries may show only mild vasospasm. Although severe vasospastic arteries after SAH have been intensively studied, activity of eNOS associated with the mild form of the disease has received less attention. The purpose of the present study was to clarify molecular mechanisms underlying the regulation of eNOS activity in mild vasospastic arteries after SAH. In a rat single-hemorrhage model, basilar arteries were obtained up to 7 days after SAH. Western blot analysis was used to study the temporal profiles of eNOS, phosphorylated (p)-eNOS at Ser(1177) or Thr(495), inducible NOS (iNOS), AMP-activated protein kinase alpha (AMPK alpha, p-AMPK alpha at Thr(172)Akt, p-Akt at Ser(473), cyclic AMP-dependent protein kinase (PKA), and p-PKA at Thr(197) in basilar arteries. Immunohistochemical studies were performed to examine the spatial expression patterns of p-eNOS at Ser(1177) and p-AMPK alpha at Thr(172). Western blot analysis showed eNOS to be significantly phosphorylated at Ser(1177) from 1 to 2 days after SAH, accompanied by upregulation of iNOS and AMPK, while activation states of Akt and PKA did not show significant change. Immunohistochemistry revealed phosphorylation of eNOS and AMPK alpha in endothelial cells of the basilar artery. SAH might thus induce temporary activation of AMPK alpha, which phosphorylates eNOS at Ser(1177) in endothelial cells of mild vasospastic basilar arteries. This signal transduction may play an important role in controlling cerebral blood flow after SAH.

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.)

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.

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 Ser1177 following SCI in mice. Western blot analysis showed eNOS to be significantly phosphorylated at Ser1177 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.

The lipid droplet (LD) is an organelle that is used for the storage of neutral lipids. It dynamically moves through the cytoplasm, interacting with other organelles, including the endoplasmic reticulum (ER). These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The hepatitis C virus (HCV) is a causative agent of chronic liver diseases. HCV capsid protein (Core) associates with the LD, envelope proteins E1 and E2 reside in the ER lumen, and the viral replicase is assumed to localize on ER-derived membranes. How and where HCV particles are assembled, however, is poorly understood. Here, we show that the LD is involved in the production of infectious virus particles. We demonstrate that Core recruits nonstructural (NS) proteins and replication complexes to LD-associated membranes, and that this recruitment is critical for producing infectious viruses. Furthermore, virus particles were observed in close proximity to LDs, indicating that some steps of virus assembly take place around LDs. This study reveals a novel function of LDs in the assembly of infectious HCV and provides a new perspective on how viruses usurp cellular functions.

Vinexin is an adaptor protein that is supposed to play pivotal roles in cell adhesion, cytoskeletal organization and signaling. At least three splice variants, vinexinalpha, beta and gamma, have so far been reported. In spite of the possible importance of vinexin, the properties and functions of vinexin in neuronal cells are almost unknown. Here we show that vinexin isoforms are expressed in rat brain in a developmental stage-dependent manner, and that vinexinalpha is relatively abundant in the telencephalon regions of the adult rat brain. An immunohistochemical study showed the localization of vinexinalpha in neurons and glia in the rat brain. In primary cultured rat hippocampal neurons, vinexin was found to be present at synapses and filopodia in growth cones by immunofluorescent analyses. Biochemical fractionation revealed the distribution of vinexin in synaptosomes. Nerve terminal localization of vinexin was confirmed by electron microscopy. Vinexinbeta is reported to be phosphorylated by extracellular signal-regulated kinase (ERK) at Ser189, which is equivalent to Ser593 of vinexinalpha. We thus constructed a site- and phosphorylation state-specific antibody to monitor the ERK-mediated phosphorylation of vinexin. In immunofluorescent analyses, the phosphorylation was observed at synapses formed among cultured rat hippocampal neurons and it was reduced by treatment of the cells with PD98059. In an immunoelectron microscopic examination, the phosphorylation signal was mainly detected on the postsynaptic side of synapses in the rat hippocampal neurons. As active ERK was co-localized with vinexin in synapses, the ERK signal is likely to be involved in the regulation of vinexin-dependent cellular processes in synapses. On the other hand, the phosphorylation was hardly detected in neurons cultured for 3 days, suggesting the presence of a yet unidentified regulatory mechanism of vinexin at the growth cone.

We applied the Hilbert differential contrast phase electron microscopy technique for the first time to mammalian cells, Ptk2 cells. Intracellular architectures such as the cytoskeletal network, membranous organelles, and mitochondria were observed without prior removal of cell membranes or extraction of soluble proteins. The attachment of mitochondria and membrane organelles with microtubules were observed. Microtubules were depolymerized by nocodazole treatment as expected. Thus, Hilbert differential contrast phase electron microscopy of vitrified cells is a nano-scale molecular imaging technique that opens up new vistas for exploring the supramolecular organization of the mammalian cell.

Transforming growth factor-β (TGF-β) is an anti-inflammatory cytokine and is expressed in the injured spinal cord. TGF-β signals through receptors to activate Smad proteins, which translocate into the nucleus. In the present study, we investigated the chronological alterations and cellular locations of the TGF-β/Smad signaling pathway following spinal cord injury (SCI) in mice. ELISA analysis showed that the concentration of interleukin-6 (IL-6) in injured spinal cords significantly increases immediately after SCI, while the concentration of TGF-β gradually increased after SCI, peaked at 2 days, and then gradually decreased. Immunohistochemical studies revealed that Smad3 was mainly expressed in neurons of the spinal cord. Phosphorylated Smad3 at the C-terminus (p-Smad3C) was stained within the motor neurons in the anterior horn, while phosphorylated Smad3 at the linker regions (p-Smad3L) was expressed in astrocytes within gray matter. These findings suggest that SCI induces gradual increases in TGF-β and induces different activation of p-Smad3C and p-Smad3L. Phosphorylated Smad3C might be involved in neuronal degeneration after SCI, and p-Smad3L may play a role in glial scar formation by astrocytes.

We previously demonstrated that calmodulin-dependent protein kinase IIα (CaM-KIIα) phosphorylates nNOS at Ser 847 in the hippocampus after forebrain ischemia; this phosphorylation attenuates NOS activity and might contribute to resistance to post-ischemic damage. We also revealed that cyclic AMP-dependent protein kinase (PKA) could phosphorylate nNOS at Ser 1412 in vitro . In this study, we focused on chronological and topographical changes in the phosphorylation of nNOS at Ser 1412 after rat forebrain ischemia. The hippocampus and adjacent cortex were collected at different times, up to 24 h, after 15 min of forebrain ischemia. NOS was partially purified from crude samples using ADP agarose gel. Neuronal NOS, phosphorylated ( p )-nNOS at Ser 1412 , PKA, and p -PKA at Thr 197 were studied in the rat hippocampus and cortex using Western blot analysis and immunohistochemistry. Western blot analysis revealed that p -nNOS at Ser 1412 significantly increased between 1 and 6 h after reperfusion in the hippocampus, but not in the cortex. PKA was cosedimented with nNOS by ADP agarose gel. Immunohistochemistry revealed that phosphorylation of nNOS at Ser 1412 and PKA at Thr 197 occurred in the subgranular layer of the dentate gyrus. Forebrain ischemia might thereby induce temporary activation of PKA at Thr 197 , which then phosphorylates nNOS at Ser 1412 in the subgranular layer of the dentate gyrus.

Schnurri-2 (Shn-2), an nuclear factor-κB site-binding protein, tightly binds to the enhancers of major histocompatibility complex class I genes and inflammatory cytokines, which have been shown to harbor common variant single-nucleotide polymorphisms associated with schizophrenia. Although genes related to immunity are implicated in schizophrenia, there has been no study showing that their mutation or knockout (KO) results in schizophrenia. Here, we show that Shn-2 KO mice have behavioral abnormalities that resemble those of schizophrenics. The mutant brain demonstrated multiple schizophrenia-related phenotypes, including transcriptome/proteome changes similar to those of postmortem schizophrenia patients, decreased parvalbumin and GAD67 levels, increased theta power on electroencephalograms, and a thinner cortex. Dentate gyrus granule cells failed to mature in mutants, a previously proposed endophenotype of schizophrenia. Shn-2 KO mice also exhibited mild chronic inflammation of the brain, as evidenced by increased inflammation markers (including GFAP and NADH/NADPH oxidase p22 phox), and genome-wide gene expression patterns similar to various inflammatory conditions. Chronic administration of anti-inflammatory drugs reduced hippocampal GFAP expression, and reversed deficits in working memory and nest-building behaviors in Shn-2 KO mice. These results suggest that genetically induced changes in immune system can be a predisposing factor in schizophrenia.

The signal transducer and activator of transcription 1 (STAT1) has been reported to be associated with neuronal cell death after cerebral ischemia. On the contrary, STAT3 has been revealed to regulate cell survival. We examined the chronological alteration and cellular localization of phosphorylated (p)-JAK1, p-STAT1 and p-STAT3 following mild spinal cord injury (SCI) in mice. Western blot analysis indicated that JAK1 is significantly phosphorylated, accompanied by the phosphorylation of STAT1 at Tyr(701) within a similar timeframe. Immunofluorescence staining indicated that signal transduction of STAT3 is introduced into the nucleus of the neurons within the anterior horns; however, in mirror sections, that of STAT1 is limited to the cytoplasm. These findings suggest that STAT3 signal is predominantly transduced into the nucleus and plays a stronger role in neuronal survival than STAT1. Modulation of the functional balance between STAT1 and STAT3 might determine the survival or death of neurons after SCI.

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 Muller cells than the retinal neurons. The Muller 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.

The signal transducer and activator of transcription 1 (STAT1) is one of the most important signaling molecule transducing signals from the cell surface in response to cytokines or growth factors. Subarachnoid hemorrhage (SAH) results in production of cytokines and growth factors in the CSF. We here investigated whether this signaling molecule is activated in the rat basilar artery after SAH. In a rat single-hemorrhage model of SAH, basilar arteries were obtained at various times until 7 days after SAH. Western blot analysis with phosphorylated ( p )-STAT1 at Tyr 701 , p -STAT1 at Ser 727 , STAT1, and actin antibodies was performed. The expression of STAT1 and p -STAT1 at Tyr 701 in basilar arteries was also examined by immunohistochemistry. Intracisternal injection of interleukin-6 (IL-6), hydrogen peroxide, or hydroxyl radical scavenger was conducted to examine for phosphorylation of STAT1. Western blot analysis showed STAT1 to be significantly phosphorylated at Tyr 701 and Ser 727 within 2 h of SAH and to gradually decrease thereafter. Immunohistochemistry revealed this phosphorylation of STAT1 to occur in the outer membranes of the basilar artery. Intracisternal injection of hydrogen peroxide, but not IL-6, also significantly increased phosphorylation of STAT1 at Tyr 701 . Hydroxyl radical scavenger significantly reduced phosphorylation of STAT1. These results indicate that reactive oxygen species, produced in the CSF after SAH, activates STAT1 molecule in the outer membranes of basilar arteries. This STAT1 signaling might contribute to morphological arterial wall changes in cerebral vasospasm.

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.

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.

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.