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Biomedical subjects

K Ohno

Publications and source records attributed to K Ohno.

At least 73 records · Page 4Linked to original sources
PubMed2001

Developmental changes in gephyrin and collybistin mRNA expressions in the rat olfactory bulb.

GABA and glycine are inhibitory neurotransmitters in the olfactory bulb. Recently, gephyrin, a receptor-associated peripheral membrane protein, and collybistin, a gephyrin-binding protein have been shown to be pivotal for the formation of postsynaptic glycine and GABA(A) receptor clusters. In this study, we, thus, examined expressions of gephyrin and collybistin mRNAs in the developing olfactory bulb using in situ hybridization. Although collybistin has two splice variants, we found only collybistin 1 isoform mRNA in the developing olfactory bulb. On the first postnatal day (P1), mitral cells abundantly expressed gephyrin and collybistin 1 mRNAs. However, their expressions were weak or not detected in the other regions. On P3, we first detected both transcripts in granule cells. It was on P7 when periglomerular cells first showed hybridization signals for gephyrin and collybistin mRNAs. The sequence of their expressions was well correlated with that of the maturation of individual neurons, suggesting that gephyrin and collybistin 1 mRNA expressions are induced when neurons arrive at their final destinations and ready for synaptogenesis, and that they are indispensable to make efficient inhibitory synapses in the developing rat olfactory bulb.

Animals↗
PubMed2001

Accumulation of cholera toxin and GM1 ganglioside in the early endosome of Niemann-Pick C1-deficient cells.

We investigated intracellular trafficking of GM1 ganglioside in Niemann-Pick C1 (NPC1)-deficient Chinese hamster ovary cells [NPC1(-) cells] by using cholera toxin (CT) as a probe. Both the holotoxin and the B subunit (CTB) accumulated in GM1-enriched intracellular vesicles of NPC1(-) cells. CTB-labeled vesicles contained the early endosome marker Rab5 but not lysosome-associated membrane protein 2 and were not labeled with either Texas red-transferrin or Lysotracker, indicating that they represent early endosomes. Similarly, CT accumulated in intracellular vesicles of human NPC fibroblasts that contained both Rab5 and early endosomal antigen 1. CTB accumulation in NPC1(-) cells was abolished by expression of wild-type NPC1 but not by mutant proteins with a mutation either in the NPC domain or the sterol-sensing domain. A part of these mutant NPC1 proteins expressed in NPC1(-) cells was localized on CTB-labeled vesicles. U18666A treatment of "knock in" cells [NPC1(-) cells that stably expressed wild-type NPC1] caused CTB accumulation similar to that in NPC1(-) cells, and a part of wild-type NPC1was localized on CTB-labeled vesicles in drug-treated cells. Finally, CT tracer experiments in NPC1(-) cells revealed retarded excretion of internalized toxin into the culture medium and an increase in the intracellular release of A subunits. In accordance with the latter result, CT was more effective in stimulating cAMP formation in NPC1(-) than in wild-type cells. These results suggest that transport of CT/GM1 complexes from the early endosome to the plasma membrane depends on the function of NPC1, whereas transport to the Golgi apparatus/endoplasmic reticulum does not.

Animals↗
PubMed2001

TNF-alpha-induced cell death in feline immunodeficiency virus-infected cells is mediated by the caspase cascade.

TNF-alpha induced apoptosis in a feline fibroblastic cell line (CRFK) infected with FIV but not in its uninfected control. In this study, to understand the molecular basis of the different susceptibilities to TNF-alpha between FIV-infected and uninfected cells, we examined the expression of TNF receptors and the activation of the caspase and NF-kappaB pathways. Expression levels of TNFR I and TNFR II mRNAs were similar between uninfected and FIV-infected CRFK cells. To understand the role of caspases in TNF-alpha-induced apoptosis, we examined the effect of three different classes of caspase inhibitors, Z-VAD-FMK, Ac-YVAD-CMK, and Z-DEVD-FMK, on the TNF-alpha-induced apoptosis in FIV-infected cells. Pretreatment with each of these caspase inhibitors protected FIV-infected CRFK cells from TNF-alpha-induced cell death. Moreover, one of the caspase substrates, poly(ADP-ribose) polymerase, was shown to be cleaved after TNF-alpha treatment in FIV-infected CRFK cells but not in uninfected CRFK cells. Electrophoretic mobility shift assay using an NF-kappaB motif oligonucleotide and promoter assay using an NF-kappaB luciferase reporter construct indicated that TNF-alpha treatment had induced activation of NF-kappaB in both FIV-infected and uninfected CRFK cells. The present study indicates that TNF-alpha-induced apoptosis in FIV-infected CRFK cells is mediated by the activation of the caspase cascade, but not by either upregulation of TNF receptor or inhibition of NF-kappaB.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Differential expression patterns of three glutamate transporters (GLAST, GLT1 and EAAC1) in the rat main olfactory bulb.

Glutamate is the main neurotransmitter in the olfactory bulb. Therefore, glutamate transporters, which regulate the concentration of extracellular glutamate, might play pivotal roles in odor processing. In this study, we examined expressions of three glutamate transporters (GLAST, GLT1 and EAAC1) in the olfactory bulb using in situ hybridization and immunohistochemistry. EAAC1 mRNA was expressed in neurons, such as periglomerular cells, tufted cells, mitral cells and granule cells as shown before in other brain areas. In contrast, GLAST and GLT1 were found in glial cells throughout the olfactory bulb, with intenser expressions in the glomerular layer, external plexiform layer and internal plexiform layer where glutamatergic synapses are concentrated. In addition, using double staining immunohistochemistry we clearly showed that GLAST and GLT1 were expressed in astrocytes. Furthermore, we found that GLAST was also intensely expressed in the subependymal layer where precursor cells exist. These results suggest each glutamate transporter plays its unique role not only in glutamatergic neurotransmission but also in cell differentiation and migration in the olfactory bulb.

Amino Acid Transport System X-AG↗
PubMed2001

Molecular cloning of feline interferon-gamma-inducing factor (interleukin-18) and its expression in various tissues.

Interleukin-18 (IL-18) is a cytokine with potent interferon-gamma-inducing activity, and plays an important biologic role in the enhancement of the activity of natural killer cells and cytotoxic T-lymphocytes. In this study, feline IL-18 cDNA was cloned and characterized to establish a basis for the prospective cytokine therapy in small animal practice. The nucleotide sequence of feline IL-18 cDNA obtained in this study was 712bp long and contained its entire open reading frame encoding 192 amino acid residues. The predicted amino acid sequence of feline IL-18 cDNA showed 77.2, 84.8, 60.2 and 62.6% similarity with those of human, dog, rat and mouse counterparts, respectively. The feline IL-18 cDNA included a putative cleavage site of IL-1beta-converting enzyme (ICE) and IL-1 signature-like sequences identified in human and mouse IL-18 cDNAs. Expression of IL-18 mRNA was detected in various tissues including spleen, liver and cerebrum in the cat.

Amino Acid Sequence↗
PubMed2001

Induction of intestinal cytochrome P450 (CYP3A) by rifampicin in beagle dogs.

Both male and female beagle dogs (four dogs/sex) were orally treated with rifampicin (Rif) at the dose of 10 mg/kg/day for 7 days and an additional eight dogs (four dogs/sex) were used as a control. The inducible effect of Rif on intestinal cytochrome P450, especially CYP3A enzyme, was investigated by measuring microsomal testosterone 6beta-hydroxylation (6beta-OHT) activity, immunoblot and ELISA analysis. In male dogs, microsomal 6beta-OHT activity in the duodenum, upper, middle and lower part of the jejunum and the ileum of the control was 229, 204, 194, 129 and 57 pmol/min/mg protein, while the activity of the Rif-treated dogs significantly increased to 456, 486, 430, 192 and 138 pmol/min/mg protein, respectively. The activity of intestinal 6beta-OHT in the control and Rif-treated female dogs showed almost similar levels to those observed in the corresponding male dogs. The activity of intestinal 6beta-OHT in both control and Rif-treated dogs was specifically inhibited by anti-CYP3A12 antiserum. The apparent K(m) value for 6beta-OHT activity in all sections of the small intestine was comparable with that in the liver, and no significant changes were observed in between control and Rif-treated dogs. In both control and Rif-treated dogs, immunoblotting of intestinal microsomes with anti-CYP3A12 antiserum produced a band indistinguishable from that of purified CYP3A12 or of immunoreactive CYP3A12 in liver microsomes. A significant increase in intestinal CYP3A content by Rif treatment was quantitatively verified by the ELISA analysis and the magnitude of its increase correlated well with that of 6beta-OHT activity elevation. Furthermore, the results of immunohistochemistry using the anti-CYP3A12 antiserum indicated that CYP3A protein was specifically distributed in epithelial cells throughout the small intestine and appeared to be predominant at the apical side of villus cells. These results demonstrate that Rif induces not only hepatic CYP3A12 but also intestinal CYP3A in dogs.

Administration, Oral↗
PubMed2001

Niemann-Pick C1 disease: correlations between NPC1 mutations, levels of NPC1 protein, and phenotypes emphasize the functional significance of the putative sterol-sensing domain and of the cysteine-rich luminal loop.

To obtain more information of the functional domains of the NPC1 protein, the mutational spectrum and the level of immunoreactive protein were investigated in skin fibroblasts from 30 unrelated patients with Niemann-Pick C1 disease. Nine of them were characterized by mild alterations of cellular cholesterol transport (the "variant" biochemical phenotype). The mutations showed a wide distribution to nearly all NPC1 domains, with a cluster (11/32) in a conserved NPC1 cysteine-rich luminal loop. Homozygous mutations in 14 patients and a phenotypically defined allele, combined with a new mutation, in a further 10 patients allowed genotype/phenotype correlations. Premature-termination-codon mutations, the three missense mutations in the sterol-sensing domain (SSD), and A1054T in the cysteine-rich luminal loop all occurred in patients with infantile neurological onset and "classic" (severe) cholesterol-trafficking alterations. By western blot, NPC1 protein was undetectable in the SSD missense mutations studied (L724P and Q775P) and essentially was absent in the A1054T missense allele. Our results thus enhance the functional significance of the SSD and demonstrate a correlation between the absence of NPC1 protein and the most severe neurological form. In the remaining missense mutations studied, corresponding to other disease presentations (including two adults with nonneurological disease), NPC1 protein was present in significant amounts of normal size, without clear-cut correlation with either the clinical phenotype or the "classic"/"variant" biochemical phenotype. Missense mutations in the cysteine-rich luminal loop resulted in a wide array of clinical and biochemical phenotypes. Remarkably, all five mutant alleles (I943M, V950M, G986S, G992R, and the recurrent P1007A) definitively correlated with the "variant" phenotype clustered within this loop, providing new insight on the functional complexity of the latter domain.

Adolescent↗
PubMed2001

E2F-dependent transcription of the raf proto-oncogene during Drosophila development.

D-raf, a Drosophila homolog of the raf proto-oncogene, has diverse functions throughout development and is transcribed in a wide range of tissues, with high levels of expression in the ovary and in association with rapid proliferation. The expression pattern resembles those of S phase genes, which are regulated by E2F transcription factors. In the 5'-flanking region of D-raf, four sequences (E2F sites 1-4) similar to the E2F recognition sequence were found, one of them (E2F site 3) being recognized efficiently by Drosophila E2F (dE2F) in vitro. Transient luciferase expression assays confirmed activation of the D-raf gene promoter by dE2F/dDP. Expression of Draf-lacZ was greatly reduced in embryos homozygous for the dE2F mutation. These results suggest that dE2F is likely to be an important regulator of D-raf transcription.

Animals↗
PubMed2001

Developmental changes in GABA transporter (GAT1 and GAT3) mRNA expressions in the rat olfactory bulb.

Developmental expressions of GABA transporters 1 and 3 (GAT1 and GAT3) were investigated in the rat olfactory bulb by using in situ hybridization histochemistry. We found that the expression of GAT1 and GAT3 mRNAs was dramatically changed in the granule cell layer (GCL), external plexiform layer (EPL) and glomerular layer (GL) during postnatal development. Among bulbar neurons, granule cells and periglomerular cells are GABAergic and they are localized in the GCL and GL, respectively. In the EPL, granule cells make GABAergic synapses with mitral cell dendrites. Thus, the changes seen in the GCL, EPL and GL seemed related to the development of the GABAergic system in the olfactory bulb. On the whole, our results demonstrated that expression patterns of GAT1 and GAT3 mRNAs have become similar to adult patterns on postnatal day 14 (P14), suggesting that GABA transporters may play a pivotal role in GABAergic neurotransmission after P14. However, expression patterns of GAT1 and GAT3 mRNAs in early postnatal days were quite distinct from those in adulthood. For example, in the GCL, immature granule cells already exhibited strong levels of GAT1 mRNA on P1 and the expression level was higher than that of granule cells in adulthood. GAT3 mRNA was strongly expressed in presumable radial glial cells surrounding the subependymal layer. In the EPL, few signals for the two transcripts were detected on P1 but they were markedly increased by P14. Our results indicate that GAT1 and GAT3 may play important roles in the development of the GABAergic system in the olfactory bulb.

Animals↗
PubMed2001

Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans.

Choline acetyltransferase (ChAT; EC ) catalyzes the reversible synthesis of acetylcholine (ACh) from acetyl CoA and choline at cholinergic synapses. Mutations in genes encoding ChAT affecting motility exist in Caenorhabditis elegans and Drosophila, but no CHAT mutations have been observed in humans to date. Here we report that mutations in CHAT cause a congenital myasthenic syndrome associated with frequently fatal episodes of apnea (CMS-EA). Studies of the neuromuscular junction in this disease show a stimulation-dependent decrease of the amplitude of the miniature endplate potential and no deficiency of the ACh receptor. These findings point to a defect in ACh resynthesis or vesicular filling and to CHAT as one of the candidate genes. Direct sequencing of CHAT reveals 10 recessive mutations in five patients with CMS-EA. One mutation (523insCC) is a frameshifting null mutation. Three mutations (I305T, R420C, and E441K) markedly reduce ChAT expression in COS cells. Kinetic studies of nine bacterially expressed ChAT mutants demonstrate that one mutant (E441K) lacks catalytic activity, and eight mutants (L210P, P211A, I305T, R420C, R482G, S498L, V506L, and R560H) have significantly impaired catalytic efficiencies.

Adult↗
PubMed2001

Intracluster anionic oligomerization of acrylic ester molecules initiated by electron transfer from an alkali metal atom.

Stabilities and intracluster reactions have been investigated by photoionization mass spectrometry for clusters composed of an alkali metal atom (M; Na and K) and acrylic ester molecules, CH(2)=CHCO(2)R, such as methyl acrylate (MA; R = CH(3)) and ethyl acrylate (EA; R = C(2)H(5)). The following two features are commonly observed in the photoionization mass spectra of M(CH(2)=CHCO(2)R)(n): (1) The ion with n = 3 is clearly observed as a magic number. (2) Fragmented cluster ions with the loss of ROH, [M(CH(2)=CHCO(2)R)(n) - ROH] are detected only for n = 3. These features are both explained by an intracluster oligomerization reaction initiated by electron transfer from the metal atoms. The magic number trimer is concluded to have the stable structure of cyclohexane derivatives as a result of oligomerization. The fragmentation reaction is explained by Dieckmann cyclization after anionic oligomerization to produce another isomer of the trimer. The intracluster electron transfer is also supported by theoretical calculation for Na(MA) based on density functional theory.

Journal Article↗
PubMed2001

Site-specific phosphorylation of tau accompanied by activation of mitogen-activated protein kinase (MAPK) in brains of Niemann-Pick type C mice.

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol in most tissues and progressive neurodegeneration with the formation of neurofibrillary tangles. Neurofibrillary tangles are composed of paired helical filaments (PHF), a major component of which is the hyperphosphorylated tau. In this study we used NPC heterozygous and NPC homozygous mouse brains to investigate the molecular mechanism responsible for tauopathy in NPC. Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1, AT-180, and AT-100) revealed site-specific phosphorylation of tau at Ser-396 and Ser-404 in the brains of NPC homozygous mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases such as glycogen synthase kinase-3beta and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of neurons, the perikarya of which strongly immunostained with PHF-1, exhibited polymorphorous cytoplasmic inclusion bodies and multi-concentric lamellar-like bodies. Importantly, the accumulation of intracellular cholesterol in NPC mouse brains was determined to be a function of age. From these results we conclude that abnormal cholesterol metabolism due to the genetic mutation in NPC1 may be responsible for activation of the mitogen-activated protein kinase-signaling pathway and site-specific phosphorylation of tau in vivo, leading to tauopathy in NPC.

Age Factors↗
PubMed2001

SSCP analysis by RT-PCR for the prenatal diagnosis of Niemann-Pick disease type C.

The molecular prenatal diagnosis of Niemann-Pick disease type C (NPC) is presented. The proband with a late infantile type of NPC was a compound heterozygote of a paternal missense mutation, T529G, and a maternal 2 bp deletion at nt 350 of the NPC1 gene. These mutations were detected by single-strand conformation polymorphism (SSCP) analysis of RT-PCR products. When the proband was aged 4 years 3 months, prenatal diagnosis for the second child was performed using both biochemical and molecular methods. SSCP analysis for the parental mutations using cDNA from cultured amniotic fluid cells revealed the absence of both mutations and the fetus was diagnosed as being unaffected. This diagnosis was supported by a normal level of cholesterol esterification using cultured amniotic fluid cells. After the child's birth, when he was 21 months old, the diagnosis was confirmed by SSCP analysis of genomic DNAs of his family. This analysis also revealed a unique variation of intron 13, IVS13+753-758 del TTTTTT, that was shared only by the proband and the father, and was suspected as being linked to the T529G missense mutation. A combination of both biochemical and molecular analyses is very useful and reliable for prenatal diagnosis of Niemann-Pick disease type C.

Adult↗
PubMed2001

Simple and low-cost tele-nuclear medicine conference system with the e-mail protocol.

PURPOSE: Because of the recent innovative growth in computer technology, digital imaging, and the Internet, we can take advantage of these facilities for education and clinical work in nuclear medicine. We developed a tele-nuclear medicine conference system with electronic mail (e-mail) on the Internet. METHODS: Twenty-one physicians (20 radiologists, 1 neurologist), 6 technologists and 2 medical students in six university hospitals (Japan 5, Canada 1), 5 local hospitals in Japan participated in this project. We used digital still cameras (330 k pixels) equipped with a floppy disk drive and 10 x optical zoom to digitize images with JPEG compression (640 x 480 matrix). The images were attached to e-mail messages (containing a brief description of each case). The mail was sent simultaneously to all members on the mailing list. Scintigram and SPECT images as well as other radiological images were sent by e-mail. Reply mails about each case were sent to all members via the mailing list. RESULTS: During a period of 6 months, 18 cases (tumor/infection: 7, bone: 6, cardiovascular: 1, neurology; 3, endocrine: 1) with 144 e-mails (average 5.6/case) were submitted to the conference. The average period of discussion was 15.6 days. The number of attached images was 1 to 9 (average, 4.2/e-mails). JPEG compression rate was 1/10 to 1/20. The quality of the images was good enough for discussion. Some cases required additional images for further discussion. CONCLUSION: Our tele-nuclear medicine conference with an electronic mailing list and digital camera was simple and low-cost. The conference system was useful for education and clinical work.

Canada↗
PubMed2001

Tuberous sclerosis: epileptogenicity and multimodal presurgical evaluations.

We report on a patient with tuberous sclerosis-related epilepsy who benefited from surgical treatment. Various presurgical evaluations, including positron emission tomography (PET), made it possible for us to localize the epileptic focus accurately. In this paper, we stress the importance of performing multimodal evaluations to determine which tubers really possess epileptogenicity. In addition, the implications of PET in tuberous sclerosis-related epilepsy are described.

Cerebral Cortex↗
PubMed2001

Indications for surgical treatment of funnel chest by chest radiograph.

Forty-seven children with funnel chest (FC) who underwent sternal elevation and 210 normal children were examined to determine the indications for surgical treatment using the vertebral index (VI) and frontosagittal index (FSI). In normal children VI gradually increased and FSI gradually decreased with age. Both indices changed significantly at 3 years of age. Although the VI of FC patients decreased significantly from 33.8 +/- 7.6 (n=40) to 24.4 +/- 3.9 (n=38) postoperatively (P < 0.0001), it was significantly larger than that of normal children over 3 years of age (20.2 +/- 2.2, n=150) (P < 0.0001), and although the FSI of FC patients increased significantly from 22.0 +/- 7.0 (n=40) to 34.5 +/- 6.5 (n=38) postoperatively (P < 0.0001), it was significantly smaller than that of normal children over 3 years of age (41.1 +/- 4.0, n=150) (P < 0.0001). Since many patients had a thin and flat chest despite excellent correction, their postoperative indices were not normal. There was a correlation between VI and FSI in normal children and a high degree of correlation between VI and FSI both before and after operation in FC patients. We conclude that a VI of more than 27 and/or a FSI of less than 29 are indications for surgical treatment based on the mean VI + 3SD and FSI - 3SD of normal children over 3 years of age. These values are almost equal to the mean VI - SD and FSI + SD of patients with physical, cosmetic, and/or psychological disturbances. However, it is not necessary to measure both indices simultaneously. Postoperative VI and FSI did not always reflect the degree of chest-wall depression in FC patients because of their flat chests.

Adolescent↗
PubMed2001

Delineation of the critical interval for the familial exudative vitreoretinopathy gene by linkage and haplotype analysis.

Familial exudative vitreoretinopathy (FEVR) is an ocular disorder characterized by deficient vascularization of the peripheral retina and causes visual loss attributable to various types of retinal detachment. The locus of the gene responsible for the autosomal dominant form of FEVR (EVR1) has been assigned to 11q13-23. However, a detailed evaluation of the critical region has not been made. We present the results of linkage analysis of the EVR1 locus on 11q13-23 in 43 individuals belonging to seven unrelated families of Japanese origin. Multipoint analysis has shown that six families out of the seven are linked with 11q13-23 markers. Haplotype analysis reveals that the putative region is probably flanked by polymorphic markers D11S1362 and CHLC.GATA30G01, which are approximately 200 kb apart, although the recombination events in small families such as presented in this study should be interpreted cautiously.

Chromosomes, Human, Pair 11↗