Biomedical subjects
K Inoue
Publications and source records attributed to K Inoue.
[Radiation induced cardiomyopathy].
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[Primary tumors of the cardiac septum].
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[Psychiatric problems of patients rescued during Hanshin earthquake--with special reference to those cared at ICU].
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[ATP receptor and intracellular signaling].
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Cloning and expression of a cDNA encoding the beta-subunit (30-kDa subunit) of bovine brain platelet-activating factor acetylhydrolase.
Bovine brain platelet-activating factor (PAF) acetylhydrolase isoform Ib is a heterotrimeric enzyme. Its gamma-subunit (which, formerly, we called the 29-kDa subunit) acts as a catalytic subunit, whereas the alpha-subunit (45 kDa) is the bovine homolog of the product of human LIS-1, the causative gene of Miller-Dieker lissencephaly, indicating that this intracellular PAF acetylhydrolase plays a key role in brain development. In the current study, we cloned the cDNA for the beta-subunit (30 kDa) of bovine brain PAF acetylhydrolase Ib. The predicted 229-amino acid sequence was homologous (63.2% identity) to that of the gamma-subunit, especially (86% identity) in the catalytic and PAF receptor homologous domains. The recombinant beta-protein produced in Escherichia coli showed significant PAF acetylhydrolase activity. A mutant protein, in which Ser48, which corresponds to the active serine residue of the gamma-subunit, was replaced with cysteine showed no enzymatic activity, suggesting Ser48 is the active serine residue. Although the beta- and gamma-subunits form a heterocomplex in the native enzyme, both recombinant beta- and gamma-proteins exist as a homodimer. The purified recombinant beta-protein was labeled readily with [1,3-H]diisopropyl fluorophosphate, whereas the beta-subunit in the native complex was only labeled with higher concentrations of [1,3-3H]diisopropyl fluorophosphate to a lesser extent than the gamma-subunit. Combined with our previous data, the present study demonstrated that bovine brain PAF acetylhydrolase Ib is a unique enzyme possessing two catalytic subunits and another, possibly regulatory, subunit.
Molecular mechanisms of Spemann's organizer formation: conserved growth factor synergy between Xenopus and mouse.
Mesoderm induction assays in Xenopus have implicated growth factors such as activin, Vg1, Xwnt-8, and noggin as important in directing the formation of dorsal mesoderm (Spemann's organizer). Because these growth factors are structurally very different, they presumably act through distinct cell surface receptors that initiate different intracellular signaling cascades. A consequence of all of these signaling pathways, however, seems to be the induction of goosecoid (gsc) gene expression. To understand how integration of these different signaling pathways results in formation of Spemann's organizer, we sought to identify growth factor-responsive elements within the gsc promoter. Through microinjection of reporter genes we have identified two cis-acting elements, a distal element (DE) and a proximal element (PE), that are required for activin/BVg1 and Wnt induction, respectively. We have shown that the DE mediates activin induction in the absence of protein synthesis and therefore constitutes the first activin response element identified to interpret transforming growth factor-beta (TGF-beta) superfamily member signaling directly. Using a reporter gene construct containing a multimerized DE, we find that an activin/BVg1-type signaling cascade is active throughout the vegetal hemisphere and marginal zone but not in the animal hemisphere. We demonstrate further that both the distal and proximal elements are essential for high-level transcription of the gsc gene, specifically in dorsal mesoderm, strongly suggesting that establishment of Spemann's organizer requires synergistic input from activin/BVg1-like and Wnt signaling pathways. Finally, mechanisms of establishing the organizer are likely to be conserved throughout vertebrate evolution.
Far-infrared spectroscopy study of an uncoupled mode in a two-dimensional photonic lattice.
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Age-associated changes of mitochondrial translation and respiratory function in mouse brain.
We examined age-associated changes of respiratory enzyme activities and protein synthesis in mitochondria isolated from mouse brain with high oxidative activities. Cytochrome c oxidase activity increased unexpectedly with aging, while the mitochondrial translational activities showed two phases of alterations: they increased progressively up to 21 weeks after birth followed by a gradual decrease with aging. Results showed that these changes were not due to the change in mitochondrial DNA (mtDNA) copy number or the accumulation of deletion mutations in mtDNA. These observations suggest that the common feature of age-associated changes in both human and mouse mitochondrial functions is limited to the decrease in mitochondrial translational activity. Therefore, mouse brain can be used as a model to understand the relationships between aging and mitochondrial function by examining the cause of decrease in mitochondrial translation activity.
[Microwave coagulation therapy of hepatocellular carcinoma].
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A novel phosphatidylserine-binding peptide motif defined by an anti-idiotypic monoclonal antibody. Localization of phosphatidylserine-specific binding sites on protein kinase C and phosphatidylserine decarboxylase.
A monoclonal anti-idiotypic antibody, Id8F7, previously shown to bind to a phosphatidylserine (PS)-specific binding site on protein kinase C (PKC) has been used to identify a 12-amino acid consensus sequence shared by PKC and phosphatidylserine decarboxylase (PSD). The 14-amino acid synthetic peptide derived from the corresponding region of PSD (amino acids 351-364 of the enzyme from Chinese hamster ovary cells) bound effectively and specifically to PS, and that derived from rat PKC gamma (amino acids 227-240) bound weakly but specifically to PS. Analysis of binding of Id8F7 to various synthetic peptides revealed that the consensus sequence motif, FXFXLKXXXKXR, is responsible for the interaction with both Id8F7 and PS. The results suggest that the conserved amino acid residues represent a basic structural motif for the specific interaction with PS, and the corresponding regions of PKC and PSD form the PS-specific binding sites of these enzymes.
Molecular characterization of two forms of nontoxic-nonhemagglutinin components of Clostridium botulinum type A progenitor toxins.
The entire sequences of the type A nontoxic-nonhemagglutinin gene and an adjacent open reading frame designated as orf 22-a, which are located between the neurotoxin and the HA-35 genes were determined. SDS-PAGE and N-terminal amino acid sequence analyses of the purified type A progenitor toxins (12S, 16S and 19S) indicate that the nontoxic-nonhemagglutinins of 16S and 19S are single peptides of approximately 120k, but that of 12S has a cleavage at the site between Pro-144 and Phe-145 of this protein.
Adult-onset spinocerebellar dysfunction caused by a mutation in the gene for the alpha-tocopherol-transfer protein.
BACKGROUND: Patients with isolated vitamin E deficiency have an impaired ability to incorporate alpha-tocopherol into lipoproteins in the liver and usually have symptoms and signs of spinocerebellar dysfunction before adolescence. Accumulated evidence suggests that the alpha-tocopherol-transfer protein, which is presumed to function in the intracellular transport of alpha-tocopherol, is abnormal in these patients. METHODS: We studied a patient from an isolated Japanese island who began to have ataxia, dysarthria, and sensory disturbances in the sixth decade of life. His serum vitamin E concentration was low (1.2 micrograms per milliliter [2.8 mumol per liter]). Exons of his gene for the alpha-tocopherol-transfer protein were analyzed by DNA sequencing. We also screened an additional 801 inhabitants of the island for the mutation. Both the normal and mutant alpha-to-copherol-transfer proteins were expressed in COS-7 cells and studied by immunoblot analysis and assay for alpha-tocopherol-transfer activity. RESULTS: The patient was homozygous for a point mutation that replaces histidine (CAT) with glutamine (CAG) at position 101 of the gene for the alpha-tocopherol-transfer protein. When expressed in COS-7 cells, the missense mutation produced a functionally defective alpha-tocopherol-transfer protein with approximately 11 percent of the transfer activity of the wild-type protein. Of the 801 island inhabitants examined, 21 were heterozygous for the His101Gln mutation. In all affected subjects, including the patient, this mutation cosegregated with an intron-sequence polymorphism. The heterozygotes were phenotypically normal and had serum vitamin E concentrations that were on average 25 percent lower than those of normal subjects (mean [+/- SD], 7.5 +/- 2.2 vs. 10.1 +/- 2.8 micrograms per milliliter [17.4 +/- 5.1 vs. 23.4 +/- 6.5 mumol per liter]; P = 0.002). CONCLUSIONS: alpha-Tocopherol-transfer protein is a determinant of serum vitamin E concentrations. An abnormality in this protein is a cause of spinocerebellar dysfunction.
Intramedullary spinal cord germinoma.
STUDY DESIGN: A very rare case of intramedullary spinal cord germinoma is described. OBJECTIVE: To increase knowledge about the pathogenesis and treatment of intramedullary spinal cord germinoma. SUMMARY OF BACKGROUND DATA: Primary intramedullary spinal cord germinoma is very rare and only two cases have been previously reported. METHODS: A case of intramedullary spinal cord germinoma with complete paraplegia is presented. The diagnosis was determined by biopsy. RESULTS: After local irradiation of 46 Gy, the tumor disappeared completely. CONCLUSION: Irradiation therapy effectively treated this tumor, as shown radiologically, although the patient remained paraplegic.
Novel nonsense proteolipid protein gene mutation as a cause of X-linked spastic paraplegia in twin males.
We report a third mutation of the proteolipid protein gene in male Japanese patients with X-linked spastic paraplegia. Although the proteolipid protein gene encodes two myelin proteins, proteolipid protein and DM 20, our W144X mutation resides in the latter part of exon 3 (exon 3B), which is spliced out in DM 20. This mutation may reserve the function of DM 20. Findings in our patients support that this form of spastic paraplesia is allelic to Pelizaeus-Merzbacher disease and that the mild clinical phenotype of this disorder may be related to a mutation within exon 3B of the PLP gene.
Transcription factor repression and activation of the human acetylcholinesterase gene.
Acetylcholinesterase in man is encoded by a single gene, ACHE, located on chromosome 7q22. In this study, the transcription start sites and major DNA promoter elements controlling the expression of this gene have been characterized by structural and functional studies. Immediately upstream of the first untranslated exon of the gene are GC-rich sequences containing consensus binding sites for several transcription factors, including Sp1, EGR-1 and AP2. In vitro transcription studies and RNase protection analyses of mRNA isolated from human NT2/D1 teratocarcinoma cells reveal that two closely spaced transcription cap sites are located at a consensus initiator (Inr) element similar to that found in the terminal transferase gene. Transient transfection of mutant genes shows that removal of three bases of this initiator sequence reduces promoter activity by 98% in NT2/D1 cells. In vitro transcription studies and transient transfection of a series of 5' deletion mutants of the ACHE promoter linked to a luciferase reporter show an Sp1 site at -71 to be essential for promoter activity. Purified Sp1 protein protects this site from DNase cleavage during in vitro footprinting experiments. A conserved AP2 consensus binding site, located between the GC box elements and the Inr, is protected by recombinant AP2 protein in DNase footprinting experiments, induces a mobility shift with AP2 protein and AP2-containing cell extracts, and fosters inhibition of transcription by AP2 as measured by transient transfection in mouse and human cell lines and in in vitro transcription reactions. These results indicate that AP2 functions as a repressor of human ACHE and mouse Ache transcription.
Assessment of autonomic function in myotonic dystrophy by spectral analysis of heart-rate variability.
We analyzed by means of autoregressive spectral analysis the spontaneous beat-to-beat heart-rate variability (HRV) of 10 myotonic dystrophy (MD) patients (4 men and 6 women, aged 37-53 years) and 10 age- and sex-matched healthy, sedentary humans (control) at rest in the supine position. All MD patients had no cardiac conduction disturbances (i.e., atrioventricular or intraventricular conduction defects) on 12-lead electrocardiogram and were able to walk and perform daily activities. In the MD group, the total power, the power of the low-frequency component (a marker of sympathetic and vagal modulation of heart rate) and that of the high-frequency component (a marker of vagal modulation of heart rate) were smaller than those in the control group (P < 0.01, P < 0.05 and P < 0.05, respectively). The results of this study suggest that the cardiovascular autonomic nervous system contributing to the HRV may be disturbed even in the MD patients who can walk and perform daily activities. Therefore, one must give careful consideration to the cardiovascular autonomic dysfunction, as well as the cardiac conduction disturbance in the MD patients.
Purification and characterization of platelet-activating factor acetylhydrolase II from bovine liver cytosol.
Platelet-activating factor (PAF) acetylhydrolase, which inactivates PAF by removing the acetyl group at the sn-2 position, is distributed widely in plasma and tissues. In a previous study, we demonstrated that the PAF acetylhydrolase activity present in the soluble fraction of bovine brain cortex could be separated chromatographically into three peaks (tentatively designated isoforms Ia, Ib, and II) (Hattori, M., Arai, H., and Inoue, K. (1993) J. Biol. Chem. 268, 18748-18753). In this study, these three isoforms were also detected in kidney and liver cytosols, although their relative activity ratios in these tissues differed. In particular, isoform II was responsible for the majority of the bovine liver PAF acetylhydrolase activity. We purified isoform II from bovine liver cytosol to near homogeneity and demonstrated that it is a single 40-kDa polypeptide. This enzyme was inactivated by diisopropyl fluorophosphate and 5,5'-dithiobis(2-nitrobenzoic acid), suggesting that both serine and cysteine residues are required for the enzyme activity, and [3H]diisopropyl fluorophosphate labeled only the 40-kDa polypeptide, confirming the enzyme's identity. Isoform II showed a comparatively broader substrate specificity than isoform Ib. Isoform II hydrolyzed propionyl and butyroyl moieties at the sn-2 position approximately half as effectively as it did PAF, whereas isoform Ib hardly hydrolyzed these substrates. Taken together with previous data, the current findings indicate that tissue cytosol contains at least two types of PAF acetylhydrolase with respect to polypeptide composition, substrate specificity, and tissue distribution and suggest that these two enzymes may share distinct physiological functions in tissues.