Biomedical subjects

Kazuyuki Nakajima

Publications and source records attributed to Kazuyuki Nakajima.

17 recordsLinked to original sources

PubMed2006

Expression of the mRNA and protein of serine racemase in primary cultures of rat neurons.

Real-time quantitative PCR, Western blot and in situ hybridization techniques were employed to clarify the presence of serine racemase in the primary cultures of rat neurons. We have detected both serine racemase mRNA and protein in the cultured neurons. Both the mRNA and the protein levels in the neurons are higher than those in the astrocytes. Sequential detection of serine racemase mRNA and MAP2 immunoreactivity also revealed that serine racemase and MAP2 are co-localized in the cultured neurons. These data are the first to demonstrate that a substantial amount of serine racemase exists in the cultured neurons.

Animals↗

PubMed2006

In vitro proliferation of axotomized rat facial nucleus-derived activated microglia in an autocrine fashion.

Transection of rat adult facial nerve leads to an increase in the number of activated microglia in the facial nucleus (FN), with a peak in proliferation 3 days after transection. To investigate the characteristics of these activated microglia, we isolated the cells with high purity from axotomized FN (axFN) 3 days after transection according to the previously reported procedure for explant culture. The isolated microglia exhibited immunocytochemical properties similar to those in vivo, and their numbers increased approximately five- to sevenfold over a period of 10 days without the addition of any mitogens, suggesting that self-reproduction was occurring. Actually, the microglia actively incorporated bromodeoxyuridine (BrdU) and strongly expressed an S-phase-specific protein marker, proliferating cell nuclear antigen (PCNA). To examine the mechanism underlying this proliferation, the expression of the mitogens and specific receptors of the microglia were analyzed in conditioned medium (CM) and cells. Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-CSF (GM-CSF) were detected in the CM as well as in the cells. Their specific receptor proteins, c-Fms and GMCSFRalpha, were also detected in the cell homogenate. These proliferating microglia were not found to produce deleterious factors for neurons. In summary, the microglia isolated from the axFN were found to be proliferative in an autocrine fashion and to have some cellular properties in common with those observed in vivo.

Animals↗

PubMed2006

Differential suppression of endotoxin-inducible inflammatory cytokines by nuclear factor kappa B (NFkappaB) inhibitor in rat microglia.

The molecular mechanism by which the deleterious cytokines interleukin 1 beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) are induced in endotoxin-stimulated microglia was investigated from the viewpoint of signal transduction. Neither cytokine is produced in nonstimulated rat microglia, but both are remarkably induced by stimulation with endotoxin lipopolysaccharide (LPS). LPS-inducible IL-1beta was significantly suppressed by pretreatment with the nuclear factor kappa B (NFkappaB) inhibitor ammonium pyrrolidine dithiocarbamate (APDC), but TNFalpha was not. APDC was actually confirmed to suppress the degradation of IkappaBalpha and IkappaBbeta in microglia, indicating a role for the inhibitor of NFkappaB activation. Taken together, these results suggest that the induction of IL-1beta and TNFalpha in endotoxin-stimulated microglia is differentially regulated at the level of NFkappaB activation.

Animals↗

PubMed2006

Nonparticipation of nuclear factor kappa B (NFkappaB) in the signaling cascade of c-Jun N-terminal kinase (JNK)- and p38 mitogen-activated protein kinase (p38MAPK)-dependent tumor necrosis factor alpha (TNFalpha) induction in lipopolysaccharide (LPS)-stimulated microglia.

The molecular mechanism of cytotoxic cytokine tumor necrosis factor alpha (TNFalpha) induction in microglia remains to be clarified. We have previously reported that p38 mitogen-activated protein kinase (p38MAPK) is an important signaling molecule for the induction of TNFalpha in lipopolysaccharide (LPS)-stimulated microglia. Recently, we have shown that c-Jun N-terminal kinase (JNK) is associated with the induction of TNFalpha. Furthermore, using an NFkappaB inhibitor (SN50), we discovered that activation of nuclear factor kappaB (NFkappaB) may also be linked to TNFalpha induction. We therefore examined the relationship between NFkappaB and the two MAPKs (p38MAPK and JNK) in the signaling cascade of TNFalpha induction in LPS-stimulated microglia. NFkappaB inhibitor SN50 decreased the induction of TNFalpha under the suppressed NFkappaB activation. However, SN50 was found to prevent the activation of MKK3/6-p38MAPK and MKK4-JNK pathways. On the other hand, the other NFkappaB inhibitor ammonium pyrrolidine dithiocarbamate (APDC) neither prevented the activation of p38MAPK and JNK nor inhibited TNFalpha induction in LPS-stimulated microglia, although it was confirmed to serve as an NFkappaB inhibitor. These results suggest that both MKK3/6-p38MAPK and MKK4-JNK pathways are important signaling cascades leading to the induction of TNFalpha in LPS-stimulated microglia, but that NFkappaB itself is not required for this induction.

Animals↗

PubMed2005

Axotomy-dependent urokinase induction in the rat facial nucleus: possible stimulation of microglia by neurons.

The phenomenon in which urokinase-type plasminogen activator (uPA) is induced in the axotomized facial nucleus suggests an interaction between injured motoneurons and microglia. We examined the relation of neurons and microglia to the induction of uPA in vitro. The amount of uPA released from a co-culture of neurons and microglia was much greater than the addition of that from each alone, suggesting the occurrence of an interaction between the two. The analysis of conditioned neuronal medium (CNM)-effects on microglia and conditioned microglial medium (CMM)-effects on neurons revealed that microglia enhance uPA release in response to CNM, rather than vice versa. Characterization of the CNM-effect on microglia demonstrated that CNM enhances not only uPA release but also the specific activity of acid phosphatase and 5'-nucleotidase in microglia. The profile of microglial activation caused by CNM was quite different from that caused by lipopolysaccharide (LPS)-activation. These results suggest that a specific soluble constituent(s) derived from neurons activates microglia by a mechanism different from LPS. As a candidate molecule for the microglial activation, brain-derived neurotrophic factor was detected in the CNM. Thus, uPA induction in the axotomized facial nucleus may be explained by a neuronal stimulus leading to uPA induction in microglia.

Acrylic Resins↗

PubMed2005

Halolactibacillus halophilus gen. nov., sp. nov. and Halolactibacillus miurensis sp. nov., halophilic and alkaliphilic marine lactic acid bacteria constituting a phylogenetic lineage in Bacillus rRNA group 1.

Eleven novel strains of marine-inhabiting lactic acid bacteria that were isolated from living and decaying marine organisms collected from a temperate area of Japan are described. The isolates were motile with peritrichous flagella and non-sporulating. They lacked catalase, quinones and cytochromes. Fermentation products from glucose were lactate, formate, acetate and ethanol. Lactate yield as percentage conversion from glucose was affected by the pH of the fermentation medium: approximately 55 % at the optimal growth pH of 8.0, greater than approximately 70 % at pH 7.0 and less than approximately 30 % at pH 9.0. The molar ratio of the other three products was the same at each cultivation pH, approximately 2 : 1 : 1. Carbohydrates and related compounds were aerobically metabolized to acetate and pyruvate as well as lactate. The isolates were slightly halophilic, highly halotolerant and alkaliphilic. The optimum NaCl concentration for growth was 2.0-3.0 % (w/v), with a range of 0-25.5 %. The optimum pH for growth was 8.0-9.5, with a range of 6.0-10.0. The G+C content of the DNA was 38.5-40.7 mol%. The isolates constituted two genomic species (DNA-DNA relatedness of less than 41 %) each characterized by sugar fermentation profiles. The cell-wall peptidoglycan of both phenotypes contained meso-diaminopimelic acid. The major cellular fatty acids were C(16 : 0) and a-C(13 : 0). Comparative sequence analysis of the 16S rRNA genes revealed that these isolates represent novel species constituting a phylogenetic unit outside the radiation of typical lactic acid bacteria and an independent line of descent within the group composed of the halophilic/halotolerant/alkaliphilic and/or alkalitolerant species in Bacillus rRNA group 1, with 94.8-95.1 % similarity to the genus Paraliobacillus, 93.7-94.1 % to the genus Gracilibacillus and 93.8-94.2 % to Virgibacillus marismortui. On the basis of possession of physiological and biochemical characteristics common to typical lactic acid bacteria within Bacillus rRNA group 1, chemotaxonomic characteristics and phylogenetic independence, a new genus and two species, Halolactibacillus halophilus gen. nov., sp. nov. and Halolatibacillus miurensis sp. nov., are proposed. The type strains are Halolactibacillus halophilus M2-2T (=DSM 17073T=IAM 15242T=NBRC 100868T=NRIC 0628T) (G+C content 40.2 mol%) and Halolactibacillus miurensis M23-1T (=DSM 17074T=IAM 15247T=NBRC 100873T=NRIC 0633T) (G+C content 38.5 mol%).

Animals↗

PubMed2004

Enhancement of urokinase-type plasminogen activator (uPA) secretion, but not that of substrate plasminogen (PGn), by rat microglia stimulated with neuronal conditioned medium.

Assuming the presence of intercellular interactions between injured motoneurons and microglia in the axotomized facial nucleus, we investigated the effects of neuronal conditioned medium (NCM) on the release of urokinase-type plasminogen activator (uPA) from microglia. Zymography revealed that NCM markedly enhanced the release of uPA from microglia, although NCM itself did not contain a significant amount of uPA. In contrast, the secretion of plasminogen (PGn), a substrate of uPA, was not promoted by the NCM treatment. The specific effect of NCM was found to be quite distinct from that of microglial activators, interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS), which reduce uPA release from microglia. In summary, Neuron-derived soluble molecules appear to stimulate microglia to enhance the production/release of uPA, but not that of PGn, by a mechanism independent of the activation reaction by IFN-gamma or LPS.

Animals↗

PubMed2004

Protein kinase C alpha requirement in the activation of p38 mitogen-activated protein kinase, which is linked to the induction of tumor necrosis factor alpha in lipopolysaccharide-stimulated microglia.

Activated microglia have been suggested to produce a cytotoxic cytokine, tumor necrosis factor alpha (TNF alpha), in many pathological brains. Thus, determining the molecular mechanism of this induction and suppression has been the focus of a great deal of research. Using lipopolysaccharide (LPS) as an experimental inducer of TNF alpha, we investigated the regulatory mechanism by which TNFalpha is induced or suppressed in microglia. We found that LPS-induced TNF alpha is suppressed by pretreatment with the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580. Similar suppression was achieved by pretreatment with specific protein kinase C (PKC) inhibitors, Gö6976, myristoylated pseudosubstrate (20-28), and bisindolylmaleimide. These results suggest that PKC alpha activity as well as p38MAPK activity is associated with TNF alpha induction in LPS-stimulated microglia. The requirement of PKC alpha in LPS-dependent TNFalpha induction was verified in PKC alpha-downregulated microglia which could be induced by phorbol-12-myristate-13-acetate pretreatment. Simultaneously, PKC alpha was found to be requisite for the activation of p38MAPK in LPS-stimulated microglia. In addition, the PKC alpha levels in the LPS-stimulated microglia were observed to decrease in response to the p38MAPK inhibitor, indicating that the PKC alpha levels are regulated by the p38MAPK activity. We therefore concluded that PKC alpha and p38MAPK are interactively linked to the signaling cascade inducing TNFalpha in LPS-stimulated microglia, and that in this cascade, PKC alpha is requisite for the activation of p38MAPK, leading to the induction of TNF alpha.

Animals↗

PubMed2004

Microglia: neuroprotective and neurotrophic cells in the central nervous system.

Microglia are currently accepted as sensor cells in the central nervous system that respond to injury and brain disease. The main function of microglia is believed to be brain defense, as they are known to scavenge invading microorganisms and dead cells, and also to act as immune or immunoeffector cells. However, microglia are also thought to contribute to the onset of or to exacerbate neuronal degeneration and/or inflammation in many brain diseases by producing deleterious factors including superoxide anions, nitric oxide and inflammatory cytokines. Nonetheless, microglia have also been shown to act neuroprotectively by eliminating excess excitotoxins in the extracellular space. Moreover, there is accumulating evidence that microglia produce neurotrophic and/or neuroprotective molecules; in particular, it has been suggested that they promote neuronal survival in cases of brain injury. In general, the question of whether microglia act as neurotoxic cells or as neuroprotective cells in vivo has gained much recent attention. In this paper, we provide a review of findings indicating that the microglia are basically neurotrophic/neuroprotective cells in the nervous system. In addition, the mechanism by which neurotrophic microglia become oriented to a neurotoxic state is discussed.

Animals↗

PubMed2003

Suppression of lipopolysaccharide-dependent tumor necrosis factor alpha induction in rat microglia, in which protein kinase C alpha is down-regulated.

Microglia are thought to produce a cytotoxic cytokine, tumor necrosis factor alpha (TNF alpha), in the pathological brain. Thus, the mechanism that suppresses the induction of TNF alpha in microglia is of interest. We found that lipopolysaccharide (LPS)-induced TNF alpha is strongly suppressed by pretreatment with specific protein kinase C (PKC) inhibitors, Gö6976 and bisindolylmaleimide, suggesting that PKC alpha plays a role in the signaling cascade of TNF alpha induction. Therefore, the association of PKC alpha with TNF alpha induction was investigated in PKC alpha-deficient microglia prepared by pretreatment with phorbol-12-myristate-13-acetate (PMA). PMA-pretreated microglia strongly decreased the TNF alpha induction in the absence of PKC alpha when activated with LPS. The lack of PKC alpha in the PMA-treated microglia was confirmed by determining conventional PKC activity. Taken together, the present study demonstrated that PKC alpha is a requisite PKC isoform in the signaling cascade of TNF alpha induction in LPS-stimulated microglia.

Animals↗

PubMed2003

Alpha1,3-fucosyltransferase IX (Fut9) determines Lewis X expression in brain.

The expression of the Lewis X (Lex) carbohydrate structure in brain is developmentally regulated and is thought to play a role in cell-cell interaction during neuronal development. Mice possess three functional alpha1,3-fucosyltransferase genes: Fut4, Fut7, and Fut9. Fut7 is known to have no activity to synthesize Lex. In the present study, the relative activities of Fut4 and Fut9 for Lex synthesis were determined using recombinant enzymes. Fut9 exhibited very strong activity for oligosaccharide acceptors and glycolipid acceptors, that is, more than 10- and 100-fold, respectively, than that of Fut4. Furthermore, both cerebrum and cerebellum at various stages of development (E17, P0, P7, P30, P100) expressed 15-100 times more Fut9 transcript than Fut4 transcript. Neurons and astrocytes in primary culture also expressed 10-15 times more Fut9 than Fut4 transcript. Moreover, alpha1,3-Fut activity toward a polylactosamine chain in homogenates of brain tissues and primary cultured cells showed a pattern typical of Fut9, not Fut4. The developmental profile of activity for the synthesis of Lex was well correlated with that of Fut9 transcript. Immunohistochemistry with anti-Fut9 monoclonal antibody revealed the distribution of the Lex structure. These results showed that Fut9 is the most responsible enzyme for the synthesis of Lex in brain.

Aging↗

PubMed2003

Activation of microglia with lipopolysaccharide leads to the prolonged decrease of conventional protein kinase C activity.

The activation of microglia by lipopolysaccharide (LPS) results in the induction of harmful factors including nitric oxide and tumor necrosis factor alpha (TNFalpha). Such microglial activation was suggested to be mediated by PKC activity based on the results of an inhibitor experiment. To clarify the relationship between microglial activation and PKC activity, conventional PKC (cPKC) activity was measured by enzyme-linked immunosorbent assay (ELISA) in LPS-activated microglia. LPS stimulation caused a time- and dose-dependent decrease (70%) of specific activity of cPKC, ascribed to the decreasing amounts of PKCalpha. However, the remaining PKC activity (30%) was sustained despite longer incubation or higher LPS concentration. Therefore, it is suggested that LPS-stimulated microglia require priming by PKC activation for the induction of harmful factors, while only a part (30%) of original PKC activity is sufficient for durable microglial activation.

Animals↗

PubMed2003

Marinilactibacillus psychrotolerans gen. nov., sp. nov., a halophilic and alkaliphilic marine lactic acid bacterium isolated from marine organisms in temperate and subtropical areas of Japan.

A novel marine lactic acid rod bacterium has been described for eight strains isolated from living and decomposing marine organisms collected from temperate and subtropical areas of Japan. The isolates were Gram-positive, catalase-negative, non-sporulating and motile with peritrichous flagella. They were slightly halophilic, highly halotolerant and alkaliphilic; the optimum NaCl concentration for growth was 2.0-3.75% (w/v) with a range from 0 to 17.0-20.5% (depending on the strain); the optimum pH was between 8.0 and 9.5 with a range from 6.0 to 10.0. They were psychrotolerant, growing well at -1.8 degrees C with a maximum at 40-45 degrees C and the optimum at 37-40 degrees C. Lactate yields were 87-100% per consumed glucose; the residual products were formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1. The product composition was markedly affected by the pH of fermentation medium; at higher pH, the yield of lactate decreased (60-65% at pH 9.0) and that of other products increased conversely. The cell-wall peptidoglycan type was type A4beta, Orn-D-Glu, whereas that of the genus Alkalibacterium, the phylogenetically closest lactic acid bacterium, was type A4beta, Orn-D-Asp. The major cellular fatty acids were C16 : 0, C16 : 1delta9, C18 : 0 and C18 : 1delta9 (oleic acid). The G + C content of the DNA was 34.6-36.2 mol%. The eight isolates were phenotypically homogeneous and formed a single genomic species. The 16S rRNA gene sequence analysis indicated that the isolates constituted an independent phylogenetic lineage within the radiation of lactic acid bacteria with 96.2% similarity to the genus Alkalibacterium. The secondary structure and the nucleotide sequence of the V6 region of the 16S rRNA were characteristic of the organism among other related lactic acid genera. On the bases of phenotypic and phylogenetic distinctness, the organism was proposed to belong to a new genus and species, Marinilactibacillus psychrotolerans gen. nov., sp. nov. The type strain, M13-2(T) (G + C = 36.2 mol%), has been deposited in the IAM, NBRC, NCIMB and NRIC culture collections as IAM 14980(T), NBRC 100002(T), NCIMB 13873(T) and NRIC 0510(T), respectively.

Animals↗

PubMed2002

Spatial and temporal aspects of Ca2+ signaling mediated by P2Y receptors in cultured rat hippocampal astrocytes.

ATP produces a variety of Ca2+ responses in astrocytes. To address the complex spatio-temporal Ca2+ signals, we analyzed the ATP-evoked increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured rat hippocampal astrocytes using fura-2 or fluo-3 based Ca2+ imaging techniques. ATP at less than 10 nM produced elementary Ca2+ release event "puffs" in a manner independent of extracellular Ca2+. Stimulation with higher ATP concentrations (3 or 10 micro M) resulted in global Ca2+ responses such as intercellular Ca2+ wave. These Ca2+ responses were mainly mediated by metabotropic P2Y receptors. ATP acting on both P2Y1 and P2Y2 receptors produced a transient Ca2+ release by inositol 1,4,5-trisphosphate (InsP3). When cells were stimulated with ATP much longer, the transient [Ca2+]i elevation was followed by sustained Ca2+ entry from the extracellular space. This sustained rise in [Ca2+]i was inhibited by Zn2+ (<10 micro M), an inhibitor of capacitative Ca2+ entry (CCE). CCE induced by cyclopiazonic acid or thapsigargin and Ca2+ entry evoked by ATP share the same pharmacological profile in astrocytes. Taken together, the hierarchical Ca2+ responses to ATP were observed in hippocampal astrocytes, i.e., puffs, global Ca2+ release by InsP3, and CCE in response to depletion of InsP3-sensitive Ca2+ stores. It should be noted that these Ca2+ signals and their modulation by Zn2+ could occur in the hippocampus in situ since both ATP and Zn2+ are rich in the hippocampus and could be released by excitatory stimulation.

Adenosine Triphosphate↗

PubMed2002

Cloning and characterization of a new human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase, designated pp-GalNAc-T13, that is specifically expressed in neurons and synthesizes GalNAc alpha-serine/threonine antigen.

To date, 10 members of the UDP-N-acetyl-alpha-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T) family have been cloned and analyzed in human. In this study, we cloned and analyzed a novel human pp-GalNAc-T from an NT2 cell cDNA library, and we named it pp-GalNAc-T13. In amino acid sequences, pp-GalNAc-T13 was highly homologous, showing 84.3% identity, to pp-GalNAc-T1. Real time PCR analysis revealed pp-GalNAc-T13 to be highly and restrictively expressed in the brain and present at very low or undetectable levels in other tissues, in contrast to the ubiquitous expression of pp-GalNAc-T1. pp-GalNAc-T13 was abundantly expressed in all neuroblastoma cells examined and primary cultured neurons but not in glioblastoma cells and primary cultured astrocytes. pp-GalNAc-T13 exhibited much stronger activity to transfer GalNAc to mucin peptides, such as Muc5Ac and MUC7, than did pp-GalNAc-T1. In addition, pp-GalNAc-T13 differed in substrate specificity to pp-GalNAc-T1. pp-GalNAc-T13 was able to form a triplet Tn epitope, three consecutive GalNAc-Ser/Thr structures, on peptides encoded in syndecan-3, a proteoglycan expressed in neurons. pp-GalNAc-T13-deficient mice have been established in a previous work. Immunohistochemical study showed a remarkable decrease in Tn antigen expression in the cerebellum of the pp-GalNAc-T13 knockout mouse. pp-GalNAc-T13 would be a major enzyme responsible for the synthesis of O-glycan and specifically the Tn antigen epitope in neurons.

Amino Acid Sequence↗

PubMed2002

Ceramide activates microglia to enhance the production/secretion of brain-derived neurotrophic factor (BDNF) without induction of deleterious factors in vitro.

In analyzing the regulation of neurotrophin production/secretion from microglia, C8-ceramide (D-erythro-sphingosine, N-octanoyl-) was found to induce secretion of brain-derived neurotrophic factor (BDNF) from microglia in vitro. In the present study, the action of C8-ceramide in secreting neurotrophic and harmful factors was investigated and compared with the effects of lipopolysaccharide (LPS). C8-ceramide as well as LPS enhanced the production/secretion of BDNF but, different from LPS, did not induce tumor necrosis factor alpha, interleukin-1beta, or nitric oxide. The C8-ceramide-induced BDNF release was significantly suppressed by protein kinase C (PKC) inhibitor, bisindolylmaleimide, which targets PKC isoforms, alpha, beta, gamma, delta and epsilon. However, it was not suppressed by a specific inhibitor of PKCalpha. Furthermore, PKCbeta and gamma were undetected in the microglia. Therefore, PKCdelta and/or epsilon appear to be functioning PKC isoforms. In contrast, none of the mitogen-activated protein kinases (MAPKs) and none of the transcription factors, including the cAMP response element-binding transcription factor (CREB) and nuclear factor kappaB (NFkappaB) were activated in the microglia in response to C8-ceramide. These results indicate that ceramide-induced BDNF release in microglia is mediated by a signaling pathway associated with PKCdelta and/or epsilon, but not with activation of MAPKs, CREB and NFkappaB.

Animals↗

PubMed2002

Cell transplantation to the brain with microglia labeled by neuropathogenic retroviral vector system.

A8 virus (A8-V) is a molecular clone of the neuropathogenic FrC6 virus derived from the Friend murine leukemia virus (F-MuLV). The A8-V infects endothelia and microglia in the brain. We constructed a gene transfer system with the A8-V gene. Pseudotyped virus carrying the surface protein of A8-V (A8-SU) transduced the beta-glactosidase gene incorporated in the retroviral vector efficiently to cultured microglial cells derived from newborn rats. Ex vivo gene transferred microglial cells were then injected into the right hemisphere of 3-day-old and 3-week-old rat brains. All of the rats examined at 4 weeks after the injection contained the labeled microglial cells in the brain (7/7 and 5/5 of the rats injected at 3 days and 3 weeks, respectively). None of the rats showed pathological changes in the whole body investigated, including the central nervous system, 4 weeks after transplantation of the labeled microglial cells.

Animals↗