[Developmental regulation of sensory and motor neurons by LIM/homeodomain-transcription factors].
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Animals↗
Biomedical subjects
H Okamoto
Publications and source records attributed to H Okamoto.
[Study of mechanisms for the axonal pathfinding of the trigeminal motor neurons by using transgenic zebrafish].
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Animals↗
[Molecular virology of TT virus (TTV)].
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Animals↗
FGF signaling and the anterior neural induction in Xenopus.
We previously showed that FGF was capable of inducing Xenopus gastrula ectoderm cells in culture to express position-specific neural markers along the anteroposterior axis in a dose-dependent manner. However, conflicting results have been obtained concerning involvement of FGF signaling in the anterior neural induction in vivo using the same dominant-negative construct of Xenopus FGF receptor type-1 (delta XFGFR-1 or XFD). We explored this issue by employing a similar construct of receptor type-4a (XFGFR-4a) in addition, since expression of XFGFR-4a was seen to peak between gastrula and neurula stages, when the neural induction and patterning take place, whereas expression of XFGFR-1 had not a distinct peak during that period. Further, these two FGFRs are most distantly related in amino acid sequence in the Xenopus FGFR family. When we injected mRNA of a dominant-negative version of XFGFR-4a (delta XFGFR-4a) into eight animal pole blastomeres at 32-cell stage, anterior defects including loss of normal structure in telencephalon and eye regions became prominent as examined morphologically or by in situ hybridization. Overexpression of delta XFGFR-1 appeared far less effective than that of delta XFGFR-4a. Requirement of FGF signaling in ectoderm for anterior neural development was further confirmed in culture: when ectoderm cells that were overexpressing delta XFGFR-4a were cocultured with intact organizer cells from either early or late gastrula embryos, expression of anterior and posterior neural markers was inhibited, respectively. We also showed that autonomous neuralization of the anterior-type observed in ectoderm cells that were subjected to prolonged dissociation was strongly suppressed by delta XFGFR-4a, but not as much by delta XFGFR-1. It is thus indicated that FGF signaling in ectoderm, mainly through XFGFR-4, is required for the anterior neural induction by organizer. We may reconcile our data to the current "neural default model," which features the central roles of BMP4 signaling in ectoderm and BMP4 antagonists from organizer, simply postulating that the neural default pathway in ectoderm includes constitutive FGF signaling step.
Animals↗
Expression of kininogen genes by rat cardiomyocytes.
To determine the existence of the kallikrein-kinin system in the heart, we have studied in vitro and in vivo whether rat heart expresses kininogens (KGNs). The reverse transcription-polymerase chain reaction (RT-PCR) for KGN mRNAs demonstrated that the cardiac tissue of adult male rats expresses T-KGN mRNA but not high-molecular-weight (H-) KGN mRNA. An intravenous injection of lipopolysaccharide (LPS) resulted in a significant increase in T-KGN mRNA levels of rat heart within 12 h. Polyacrylamide gel electrophoresis of cDNA products generated by RT-PCR from heart mRNA using primers specific for either T- or low-molecular-weigh (L-) KGN revealed that rat heart expressed not only T-KGN gene but also L-KGN gene, and that LPS injection exclusively stimulated the expression of T-KGN but not of L-KGN gene. T-KGN mRNA was also detected in cultured myocytes derived from fetal rat heart, and the expression was markedly enhanced by an addition of LPS to cultures. These results demonstrated that rat cardiomyocytes are the source of T- and L-KGNs but not of H-KGN, and that their expression of T-KGN mRNA is stimulated by LPS, probably via LPS-receptor CD14.
Animals↗
The RING finger motif of photomorphogenic repressor COP1 specifically interacts with the RING-H2 motif of a novel Arabidopsis protein.
The constitutive photomorphogenic 1 (COP1) protein of Arabidopsis functions as a molecular switch for the seedling developmental fates: photomorphogenesis under light conditions and skotomorphogenesis in darkness. The COP1 protein contains a cysteine-rich zinc-binding RING finger motif found in diverse groups of regulatory proteins. To understand the role of the COP1 RING finger in mediating protein-protein interaction, we have performed a yeast two-hybrid screen and isolated a novel protein with a RING-H2 motif, a variant type of the RING finger. This protein, designated COP1 Interacting Protein 8 (CIP8), is encoded by a single copy gene and localized to cytosol in a transient assay. In addition to the RING-H2 motif, the predicted protein has a C4 zinc finger, an acidic region, a glycine-rich cluster, and a serine-rich cluster. The COP1 RING finger and the CIP8 RING-H2 domains are sufficient for their interaction with each other both in vitro and in yeast, whereas neither motif displayed significant self-association. Moreover, site-directed mutagenesis studies demonstrated that the expected zinc-binding ligands of the RING finger and RING-H2 fingers are essential for their interaction. Our findings indicate that the RING finger motif, in this case, serves as autonomous protein-protein interaction domain. The allele specific effect of cop1 mutations on the CIP8 protein accumulation in seedlings indicates that its stability in vivo is dependent on the COP1 protein.
Amino Acid Sequence↗
The entire nucleotide sequence of a TT virus isolate from the United States (TUS01): comparison with reported isolates and phylogenetic analysis.
A nonenveloped and single-stranded DNA virus designated TT virus (TTV) has been reported from Japan in association with hepatitis of unknown etiology. Very recently, the prototype TTV isolate (TA278) of genotype 1 is proven to have a circular genome with 3852 nucleotides. A TTV isolate (TUS01) was recovered from a blood donor in the United States, and its entire circular nucleotide sequence of 3818 nucleotides was determined. It possessed two open reading frames coding for 761 and 156 amino acids, respectively. TUS01 shared 60.5% of the nucleotide sequence with the TA278 isolate from Japan that was longer by 35 nt. The sequence of the noncoding region of 1203 nt was conserved with a similarity of 83.4%. Sequence preservation was much lower for the two open reading frames; nucleotide and amino acid sequences were 54.8 and 37.0% similar, respectively, for one and 55.5 and 38.8% similar for the other. By comparison of a partial sequence of 222 nucleotides among 239 TTV isolates available from various countries, at least 11 genotypes with sequence divergence of >30% were recognized. TUS01 was deduced to be of genotype 11, which has not been reported before. Conserved sequences in the noncoding region could be used as primers for sensitively detecting TTV DNA by polymerase chain reaction. Divergent sequences in coding regions would be useful as primers for distinguishing various TTV genotypes.
Amino Acid Sequence↗
Marked genomic heterogeneity and frequent mixed infection of TT virus demonstrated by PCR with primers from coding and noncoding regions.
A nonenveloped, single-stranded, and circular DNA virus designated TT virus (TTV) has been reported in association with hepatitis of unknown etiology. TTV has a wide sequence divergence (approximately 52%), by which it is classified into at least 16 genotypes separated by an evolutionary distance of >0.30. Therefore, the detection of TTV DNA by polymerase chain reaction would be influenced by primers deduced from conserved or divergent regions of the genome. Of the 30 sera from healthy individuals, up to 17% tested positive with primers deduced from coding region, much less frequently than up to 93% testing positive with primers from noncoding region. These differences were not attributable to the sensitivity of detection, because a cloned TTV DNA of genotype 1a was detected sensitively (up to 1 copy per test) with primers deduced from either the coding or the noncoding region of the same genotype. Sera testing positive only with noncoding region primers, or those showing higher titers with noncoding than coding region primers, contained TTV DNA strains with sequence divergence of 47-53% from the TA278 isolate of genotype 1a within the N22 region spanning 222-231 nucleotides. Some of the sera contained two or three TTV DNA strains of distinct genotypes. These results indicate TTV strains with extremely high sequence divergence prevailing in healthy individuals and frequent mixed infection with TTV strains of distinct genotypes.
Amino Acid Sequence↗
EDG3 is a functional receptor specific for sphingosine 1-phosphate and sphingosylphosphorylcholine with signaling characteristics distinct from EDG1 and AGR16.
AGR16/H218/EDG5 and EDG1 are functional receptors for lysosphingolipids, whereas EDG2 and EGD4 are receptors for lysophosphatidic acid (LPA). The present study demonstrates that EDG3, the yet poorly defined member of the EDG family G protein-coupled receptors, shows identical agonist specificity, but distinct signaling characteristics, compared to AGR16 and EDG1. Overexpression of EDG3 conferred a specific [32P]S1P binding, which was displaced by S1P and sphingosylphosphorylcholine (SPC), but not by LPA or other related lipids. In cells overexpressing EDG3, S1P induced inositol phosphate production and [Ca2+]i increase in a manner only partially sensitive to pertussis toxin (PTX), which was similar to the case of AGR16, but quite different from the case of EDG1, in which the S1P-induced responses were totally abolished by PTX. EDG3 also mediated activation of mitogen-activated protein kinase (MAPK) in PTX-sensitive and Ras-dependent manners, as in the cases of EDG1 and AGR16, although EDG3 and EDG1 were more effectively coupled to activation of MAPK, compared to AGR16. Additionally, EDG3 mediated a decrease in cellular cyclic AMP content, like EDG1, but contrasting with AGR16 which mediated an increase in cyclic AMP. These and previous results establish that EDG1, AGR16 and EDG3 comprise the lysosphingolipid receptor subfamily, each showing distinct signaling characteristics.
Animals↗
Study of the association between the serotonin transporter gene regulatory region polymorphism and personality traits in a Japanese population.
Recently a relationship between serotonin transporter transcriptional control region (5-HTTLPR) polymorphism and anxiety related personality traits in Caucasians was reported. We performed PCR of DNAs from the blood for determining the 5-HTTLPR genotypes of 191 Japanese subjects, which were medical staff and students, and obtained Revised NEO Personality Inventory (NEO-PI-R) and the Temperament and Character Inventory (TCI) in 144 subjects. The association was analyzed by one-way ANOVA. The present study demonstrated that allelic frequency of 5-HTTLPR (s allele frequency was 0.785) in our subjects was considerably different from that in Caucasians. No significant differences were found in the anxiety-related personality traits among genotypes, while cooperativeness in TCI showed a significant difference among genotypes. The property of 5-HTTLPR may not be reflected directory on the personality inventories.
Adult↗
Correlation of antiangiogenic and antitumor efficacy of N-biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA), an orally-active, selective matrix metalloproteinase inhibitor.
The antiangiogenic activity and antitumor efficacy of a newly developed matrix metalloproteinase (MMP) inhibitor were examined. N-biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA) potently inhibits MMP-2, -9, and -14, but not MMP-1, -3, or -7. In contrast, (-)BPHA, an enantiomer of BPHA, was inactive against all MMPs tested. Daily oral administration of 200 mg/kg BPHA, but not (-)BPHA in mice resulted in potent inhibition of tumor-induced angiogenesis, primary tumor growth, and liver metastasis. The growth inhibition activity of BPHA was 48% and 45% in a B16-BL6 melanoma and F2 hemangio-endothelioma model, respectively. BPHA also showed 42% inhibition of the liver metastasis of C-1H human colon carcinoma cells. These results indicate that selective MMP inhibition is correlated with antiangiogenic and antitumor efficacy and that the selective MMP inhibitor BPHA has therapeutic potential.
Animals↗
CD38 disruption impairs glucose-induced increases in cyclic ADP-ribose, [Ca2+]i, and insulin secretion.
Increases in [Ca2+]i in pancreatic beta cells, resulting from Ca2+ mobilization from intracellular stores as well as Ca2+ influx from extracellular sources, are important in insulin secretion by glucose. Cyclic ADP-ribose (cADPR), accumulated in beta cells by glucose stimulation, has been postulated to serve as a second messenger for intracellular Ca2+ mobilization for insulin secretion, and CD38 is thought to be involved in the cADPR accumulation (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). Here we created "knockout" (CD38(-/-)) mice by homologous recombination. CD38(-/-) mice developed normally but showed no increase in their glucose-induced production of cADPR in pancreatic islets. The glucose-induced [Ca2+]i rise and insulin secretion were both severely impaired in CD38(-/-) islets, whereas CD38(-/-) islets responded normally to the extracellular Ca2+ influx stimulants tolbutamide and KCl. CD38(-/-) mice showed impaired glucose tolerance, and the serum insulin level was lower than control, and these impaired phenotypes were rescued by beta cell-specific expression of CD38 cDNA. These results indicate that CD38 plays an essential role in intracellular Ca2+ mobilization by cADPR for insulin secretion.
ADP-ribosyl Cyclase↗
The novel sphingosine 1-phosphate receptor AGR16 is coupled via pertussis toxin-sensitive and -insensitive G-proteins to multiple signalling pathways.
In the present study, we determined the agonist specificity and the signalling mechanisms of a putative sphingosine 1-phosphate (S1P) receptor, AGR16. In CHO cells transiently transfected with an AGR16 expression vector, but not in cells transfected with an empty vector, the addition of a low concentration of S1P (1 nM) caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i) by mobilization of Ca2+ from both intra- and extra-cellular pools. To determine the spectrum of agonists for AGR16, we employed K562 cells, which in the naive state do not respond at all to either S1P or structurally related lipids with an increase in [Ca2+]i. In K562 cells stably expressing AGR16, S1P and sphingosylphosphorylcholine (SPC) dose-dependently increased [Ca2+]i with half-maximal values of 3 nM and 100 nM respectively. In CHO cells stably expressing AGR16 (CHO-AGR16), but not in parental CHO cells, we observed specific binding of [32P]S1P, which was displaced by unlabelled S1P and SPC. In CHO-AGR16 cells, but not in parental CHO cells, S1P stimulated the production of inositol phosphates and Ca2+ mobilization which was only 30% inhibited by pertussis toxin (PTX), different from the case of the recently identified S1P receptor EDG1. Also in CHO-AGR16 cells, but not in CHO cells, S1P at higher concentrations activated mitogen-activated protein kinase (MAPK) in a PTX-sensitive and Ras-dependent manner. S1P also induced the activation of two stress-activated MAPKs, c-Jun N-terminal kinase and p38, in a manner that was totally insensitive to PTX. In CHO-AGR16 cells, S1P induced stress-fibre formation, with an increase in myosin light chain phosphorylation, in a PTX-insensitive and Rho-dependent manner. S1P also induced an increase in the cellular cAMP content in CHO-AGR16 cells, which contrasts sharply with the case of EDG1. These results establish that the S1P receptor AGR16 is coupled via both PTX-sensitive and -insensitive G-proteins to multiple effector pathways.
Animals↗
Distinct genotypes of a nonenveloped DNA virus associated with posttransfusion non-A to G hepatitis (TT virus) in plasma and peripheral blood mononuclear cells.
TT virus (TTV) is a nonenveloped, single-stranded DNA virus with little sequence homology to known viruses, and associated with elevated transaminase levels in the patients with posttransfusion hepatitis of unknown etiology. The DNA of TTV was detected, by semi-nested polymerase chain reaction, in peripheral blood mononuclear cells (PBMC) from the 30 healthy individuals with circulating virus in plasma. A sequence of 222 bases was determined on 6-10 TTV DNA clones each from plasma and 6 clones -each from PBMC from eight individuals selected at random from this group. TTV can be classified into genotypes separated by an evolutionary distance > 0.30, which can be divided further into subtypes separated by that of 0.15. Three individuals possessed two different TTV variants of distinct genotypes, with predominant genotypes different between plasma and PBMC. Another possessed TTV of the same genotype in both the plasma and PBMC, but clones with a subtype not seen in plasma were observed in PBMC. A third individual had TTV variants with or without a deletion mutation, and those with the deletion mutation abounded only in PBMC. The remaining three individuals were infected with TTV with the same sequence both in plasma and PBMC. These results indicate that TTV variants with phylogenetic differences could infect the same individual, and that some variants would have a predilection for PBMC. It remains to be seen, however, if TTV replicates in PBMC or whether it has been sequestered before its evolution in the host.
Base Sequence↗
Effect of interferon on a nonenveloped DNA virus (TT virus) associated with acute and chronic hepatitis of unknown etiology.
An unenveloped DNA virus named TT virus (TTV) has been reported in association with acute and chronic hepatitis of unknown etiology. The effect of interferon on TTV was evaluated in the patients with chronic hepatitis C who were coinfected with TTV. TTV DNA was determined by a polymerase chain reaction with heminested primers in the 96 patients with chronic hepatitis C who received interferon-alpha (516 million units in 26 weeks) and followed for 24 months thereafter. TTV DNA was detected in 31 (32%) patients before therapy. TTV DNA became undetectable during interferon therapy and remained absent in 14 (45% of the 31 patients) through 24 months thereafter. The four patients with pretreatment TTV DNA titer > or =10(3)/ml did not respond. These results indicate that TTV is sensitive to interferon, and the response would be inversely correlated with pretreatment viral titers.
Acute Disease↗
Production of interleukin-7 and interleukin-15 by fibroblast-like synoviocytes from patients with rheumatoid arthritis.
OBJECTIVE: To examine the ability of fibroblast-like synoviocytes in rheumatoid arthritis (RA) to produce interleukin-7 (IL-7) and IL-15, and the ability of these cytokines to induce the proliferation of synovium-infiltrating T cells. METHODS: Messenger RNA (mRNA) and protein levels of IL-7 and IL-15 in synovial tissue cells and fibroblast cell lines were determined by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. T cell-enriched populations from RA synovial tissues were isolated by deleting adherent cells after a 14-hour incubation in plastic dishes or by expanding T cells during a 14-day incubation of tissue cells with IL-2 alone, and their proliferative responses to IL-7, IL-15, and IL-2 were measured by 3H-thymidine incorporation. RESULTS: Freshly isolated cells from RA synovial tissues more strongly expressed mRNA for both IL-7 and IL-15 compared with the cells from osteoarthritis tissues, and could spontaneously release greater amounts of these cytokine proteins in culture. Fibroblast cell lines prepared from RA patients were able to produce large amounts of IL-15 and small amounts of IL-7 at both the transcriptional and protein levels, and their cytokine production was significantly elevated when stimulated with IL-1 and tumor necrosis factor alpha. Purified synovial tissue macrophages spontaneously released IL-15 but not IL-7, and synovial T cells did not produce either cytokine. IL-7 and IL-15, similar to IL-2, stimulated the proliferation of synovial tissue T cells from RA patients; IL-7 was less potent than IL-15 or IL-2. CONCLUSION: These results indicated that fibroblast-like synoviocytes are an important source of the cytokines with IL-2-like activity, IL-15 and IL-7, in RA joints, and that IL-15 may be mainly responsible for local T cell activation and expansion in the presence of deficient IL-2 production by T cells.
Arthritis, Rheumatoid↗
Detection of retroperitoneal hemorrhage by transesophageal echocardiography during cardiac surgery.
PURPOSE: To present a case of massive retroperitoneal hemorrhage during cardiopulmonary bypass (CPB) which was detected using transesophageal echocardiography (TEE). CLINICAL FEATURE: A 50-yr-old man suffering from severe mitral regurgitation (MR) was admitted for mitral valvuloplasty. After the beginning of CPB, the volume in the reservoir was noticed to be gradually decreasing. Although venous cannulation had been properly performed, TEE showed an echo free space around the liver, the spleen and in front of the abdominal aorta showed intraabdominal hemorrhage. After cardiac surgery, emergency laparotomy revealed about 5,000 ml of blood in the retroperitoneal space probably as a result of femoral artery cannulation prior to CPB. Hemostasis was achieved, and the patient made complete cardiac and neurological recovery. Retrospective review of the TEE imaging revealed that the kidneys were surrounded by blood bilaterally confirming the diagnosis of retroperitoneal hemorrhage. CONCLUSION: Retroperitoneal hemorrhage during CPB is rare, but may be lethal. Transesophageal echocardiography is a useful monitor not only to evaluate cardiac performance, but also to detect unexpected intraabdominal bleeding during cardiac surgery.
Abdomen↗
Short inverted-repeat transposable elements in teleost fish and implications for a mechanism of their amplification.
Angel is the first miniature inverted-repeat transposable element (MITE) isolated from fish. Angel elements are imperfect palindromes with the potential to form stem-loop structures in vitro. Despite sequence divergence of elements of up to 55% within and between species, their inverted repeat structures have been maintained, implying functional importance. We estimate that there are about 10(3)-10(4) Angels scattered throughout the zebrafish genome, evidence that this family of transposable elements has been significantly amplified over the course of evolution. Angel elements and Xenopus MITEs carry common sequence motifs at their termini, indicating common origin and/or related mechanisms of transposition. We present a model in which MITEs take advantage of the basic cellular mechanism of DNA replication for their amplification, which is dependent on the characteristic inverted repeat structures of these elements. We propose that MITEs are genomic parasites that transpose via a DNA intermediate, which forms by a folding-back of a single strand of DNA, that borrow all of the necessary factors for their amplification from products encoded in the genomes in which they reside. DNA polymorphisms in different lines of zebrafish were detected by PCR using Angel-specific primers, indicating that such elements, combined with other transposons in vertebrate genomes, will be useful molecular tools for genome mapping and genetic analyses of mutations.
Animals↗