Biomedical subjects

S Takahashi

Publications and source records attributed to S Takahashi.

At least 19 recordsLinked to original sources

PubMed1992

[Diagnostic imaging of polysplenia syndrome in the adult].

Polysplenia syndrome is a congenital disorder of situs that is characterized by the presence of multiple spleens and a variable combination of thoracic and visceral anomalies. We present three adult cases of polysplenia syndrome with emphasis on the diagnostic imaging findings. Computed tomography could best determine the exact location and shape of the anomalous organs. The visceral anomalies observed in our patients included multiple spleens, interruption of the inferior vena cava (IVC), azygos or hemiazygos continuation, left-sided IVC, symmetrical liver, anomalous fissure of the liver, anomalous lobe of the liver, median location of the gallbladder, short pancreas, inverted stomach, gallbladder and pancreas, and intestinal malrotation.

Female↗

PubMed1992

Interaction of alpha subunit of GTP-binding protein Go with a 20-kDa Triton-insoluble membrane protein in bovine brain.

Heterotrimeric Go bound to the membranes of bovine brain, but Go alpha remained bound to the membranes even after activation with GTP gamma S. Furthermore, Go alpha bound to a Triton X-100-insoluble fraction of the membranes in a saturable manner. However, the 37-kDa Go alpha eliminated by trypsin at the amino-terminus could not bind to the fraction. Using a blot overlay approach of the Triton-insoluble fraction, only a 20-kDa protein was identified that interacts with Go alpha. These results indicate that Go alpha binds to a 20-kDa Triton-insoluble protein in the bovine brain membranes.

Animals↗

PubMed1992

Tumor necrosis factor and interleukin-1 induce activin A gene expression in a human bone marrow stromal cell line.

Activin A, a homodimer of the beta A chain, regulates hematopoiesis. In a human bone marrow-derived stromal cell line, KM-102, phorbol myristate acetate, tumor necrosis factor-alpha and interleukin-1 beta induced great increases in beta A chain mRNA levels and production of activin A activities. The phorbol ester-induced beta A chain gene expression was inhibited by cycloheximide and down regulation of protein kinase C, whereas the cytokine-induced expression was little affected by these treatments. These results indicate that the inflammatory cytokines directly stimulate beta A chain gene expression via protein kinase C-independent pathways.

Activins↗

PubMed1992

In vitro proliferation and the cytotoxic specificity of a cryopreserved cytotoxic T cell clone reacting against human autologous tumor cells.

Proliferation and functional maintenance of CTL after cell cryopreservation often proves to be quite difficult. We developed an improved method for proliferating cryopreserved CTL, and for gaining their specific cytotoxic function. T cells were cryopreserved at -180 degrees C in RPMI 1640 containing 50% FCS and 10% DMSO. The cryopreserved T cells were well recovered by culturing in a medium containing the supernatant of primary cultures with TIL and autologous tumor cells, in addition to a high concentration (350 U/ml) of rIL-2. Furthermore, these cells were proliferated more efficiently when MMC-treated autologous tumor cells were used in vitro as a feeder and an antigenic stimulant. However, such a high dose IL-2 cultivation resulted in the loss of cytotoxic reactivity of CTL clone. In contrast, the withdrawal of rIL-2 from in vitro cultivation for 24 h prior to the cytotoxic assays conferred the specificity of cytotoxicity on CTL. By these methods, one can obtain a large number of CTL, and pursue the physiologic detail of the specific cytotoxic mechanism of CTL against autologous human tumor cells.

Antigens, Surface↗

PubMed1992

Rabbit very low density lipoprotein receptor: a low density lipoprotein receptor-like protein with distinct ligand specificity.

A cDNA that expresses a receptor for very low density lipoprotein (VLDL) was isolated from a rabbit heart cDNA library and characterized. The deduced amino acid sequence of the cDNA revealed that the cDNA encodes a protein with striking homology to the low density lipoprotein (LDL) receptor. Like the LDL receptor, the mature protein consists of the following five domains spanning 846 amino acids: 328 N-terminal amino acids including an 8-fold repeat of 40 amino acids homologous to the ligand binding repeat of the LDL receptor; 396 amino acid residues homologous to the epidermal growth factor precursor including three cysteine-rich repeats; a region immediately outside of the plasma membrane rich in serines and threonines; 22 amino acids traversing the plasma membrane; and 54 amino acids including the NPVY sequence that is required for clustering of the LDL receptor in coated pits and that projects into the cytoplasm. LDL-receptor-deficient Chinese hamster ovary cells transfected with the cDNA bound and internalized VLDL, beta-migrating VLDL, and intermediate density lipoprotein but did not bind LDL with high affinity. The 3.6- and 9.5-kilobase mRNAs for the VLDL receptor are highly abundant in heart, muscle, and adipose tissue. Barely detectable amounts of the mRNAs were present in liver. Based on the structural features, ligand specificity, and tissue expression of the mRNAs, we suggest that this VLDL receptor may mediate uptake of apolipoprotein E-containing lipoproteins enriched with triglyceride in nonhepatic tissues that are active in fatty acid metabolism.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of furin, a Kex2-like processing endoprotease, produced in Chinese hamster ovary cells.

Furin, a mammalian homolog of the yeast Kex2 protease, is associated with Golgi membranes and is involved in cleavage of precursor proteins at sites marked by the Arg-X-Lys/Arg-Arg (RXK/RR) motif. We have recently shown that a furin mutant lacking the transmembrane domain can be secreted from cDNA-transfected cells with proteolytic activity for the fluorogenic peptide t-butoxycarbonyl-Arg-Val-Arg-Arg-4-methylcoumarin-7- amide. In this study, we purified and characterized the recombinant furin from the conditioned medium of these cells. Furin was purified as a mixture of 83- and 81-kDa forms and a 96-kDa form. The differences in molecular mass were not due to differences in molecular mass were not due to differences in glycosylation. Moreover, all forms had the same NH2-terminal sequence beginning at the residue after the Arg-Ala-Lys-Arg sequence. These data suggest that the three different forms may be produced by differential COOH-terminal processing of a furin molecule and that mature furin may be autocatalytically produced. Both enzyme preparations showed a pH optimum at 7.0, required Ca2+ for the activity, and showed essentially the same inhibitor profile. These properties resembled those of the Kex2 protease. Both preparations efficiently cleaved fluorogenic peptides with an RXK/RR sequence and moderately cleaved a peptide with an RXXR sequence, but did not cleave dibasic peptides. The sequence requirements determined in vitro were compatible with those determined by expression studies in cultured cells. These data unequivocally demonstrate that furin is an endogenous cellular protease responsible for cleavage of precursor proteins mainly at RXK/RR sites.

Amino Acid Sequence↗

PubMed1992

A selective reduction of excitatory amino acids in cerebrospinal fluid of patients with Alzheimer type dementia compared with vascular dementia of the Binswanger type.

We determined the concentrations of the putative transmitter amino acids in the cerebrospinal fluid of patients with Alzheimer type dementia (ATD) and vascular dementia of the Binswanger type (VDBT). In ATD, aspartate and glutamate concentrations were significantly and selectively reduced, while in VDBT, concentrations of aspartate, glutamate, gamma-aminobutyric acid (GABA) and many other amino acids were decreased non-selectively. In both ATD and VDBT, we found a tendency for all amino acids to increase with progression of the disease, and this reached statistical significance for some amino acids.

Aged↗

PubMed1992

Concentrations of serotonin and its related substances in the cerebrospinal fluid in patients with Alzheimer type dementia.

We studied concentrations of free and total serotonin (5-hydroxytryptamine, 5-HT) and its related substances in the cerebrospinal fluid from patients with Alzheimer type dementia (ATD) compared with controls. In ATD patients, concentrations of total 5-HT, tryptophan, 5-hydroxytryptophan (5-HTP), melatonin, kynurenine, and 3-hydroxykynurenine decreased significantly. The rate of concentration of tryptophan metabolites to that of tryptophan was significantly reduced for total 5-HT and 3-hydroxykynurenine only. The 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio was significantly larger in ATD patients than in controls. The greater reduction in the 3-hydroxykynurenine concentration (81% vs. controls) than in the total 5-HT concentration (51% vs. controls) suggests that the metabolism of tryptophan to 5-HT and 3-hydroxykynurenine is in favor of 5-HT.

Aged↗

PubMed1992

Structure of the D-mannan of the pathogenic yeast, Candida stellatoidea ATCC 20408 (type II) strain, in comparison with that of C. stellatoidea ATCC 36232 (type I) strain.

Acid treatment of the cell-wall D-mannas of Candida stellatoidea strains ATCC 36232 (Type I, A3 strain) and ATCC 20408 (Type II, A2 strain) gave (1----2)-linked beta-D-manno-oligosaccharides (dp 2-5), whereas treatment with alkali gave the (1----2)-linked alpha-D-mannobiose. Conventional acetolysis of the acid- and alkali-treated D-mannan of the A3 strain gave oligosaccharides consisting of (1----2)- and (1----3)-linked alpha-D-mannopyranose residues, similar to those of Candida albicans serotype B strain. Mild acetolysis of the acid- and alkali-treated D-mannan of the A2 strain gave higher oligosaccharides that were digested by the Arthrobacter GJM-1 strain exo-alpha-D-mannosidase. The results of 1H- and 13C-NMR analyses indicated this D-mannan to contain branches with the following structures: beta-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp++ +-(1----2)-alpha-D-Manp- (1----2)-D-Man, beta-D-Manp-(1----2)-beta-D-Manp-(1----2)-alpha-D-Manp -(1----2)- alpha-D-Manp-(1----2)-D-Man, and beta-D-Manp-(1----2)-beta-D-Manp-(1----2)-beta- D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp-(1- ---2)-alpha-D-Manp- (1----2)-D-Man, in common with the D-mannans of C. albicans serotype A strains.

Agglutination Tests↗

PubMed1992

The human gene for renin-binding protein.

The human renin-binding protein (RnBP) gene was isolated from a human placental genomic library and characterized. The gene spans about 10 kilobases and consists of 11 exons separated by 10 introns. The 5'-flanking region and the exon-intron boundaries were sequenced. Residue G* in 5'-CGAG*TGG-3' was identified as the major transcription initiation site, and "GC" boxes were found in the vicinity of the cap site. No typical "TATA" or "CCAAT" box exists in the 5'-flanking region. The hydrophobic domain followed by a leucine-zipper motif in RnBP is encoded by the sixth exon. A fragment of the human RnBP gene (nucleotides -739 to +244) linked to the chloramphenicol acetyltransferase gene was transfected into human Wilms' tumor G401 and mouse L929 cells. The expression of this chimeric gene in G401 cells was 4-fold higher than that in L929 cells, the tissue-specific regulation of RnBP gene expression thus being suggested. The promoter for the RnBP gene was shown to be localized in nucleotides -35 to +244 on assaying of the promoter activity using deletion mutants of the chimeric constructs. The RnBP gene was found to be located in human chromosome X by means of polymerase chain reaction of hybrid DNAs from human and hamster somatic cells.

Animals↗

PubMed1992

An immunoreactive xanthine oxidase protein-possessing xanthinuria and her family.

The presence of immunoreactive xanthine oxidase protein was proven in a xanthinuric patient, using a polyclonal antibody against xanthine oxidase. The antibody was raised against purified human liver xanthine oxidase in a rabbit. Double immunodiffusion method demonstrated the existence of an immunologically reactive xanthine oxidase which did not possess xanthine oxidase activity. In addition, urinary excretion of oxypurines in the patient and her family was investigated. The results indicated that a brother and a sister had xanthinuria.

Adult↗

PubMed1992

Recognition of an antiparallel beta-sheet structure of human epidermal growth factor by its receptor. Site-directed mutagenesis studies of Ala-30 and Asn-32.

The Ala-30 and Asn-32 residues involved in the major antiparallel beta-sheet structure of human epidermal growth factor (hEGF) were substituted with various amino acid residues, and the receptor-binding affinities of the nine variant hEGFs were determined by the use of human KB cells. The Ala-30----Arg, Ala-30----His and Ala-30----Phe substitutions drastically reduced the binding affinity, suggesting that the side chain in position 30 of Ala-30 of hEGF is required to be small for the receptor binding. The Asn-32----Asp substitution significantly reduced the binding affinity, while the Asn-32----His variant could bind to the receptor as well as to the wild-type hEGF. Therefore, it seems to be important for receptor binding that the side chain in position 32 does not have a negative charge but does have an NH group. Thus, we propose that, in the ligand-receptor complex, the receptor recognizes, on one side of the antiparallel beta-sheet structure of hEGF, a wider contact area than previously suggested.

Alanine↗

PubMed1992

A site-directed mutagenesis study on the role of isoleucine-23 of human epidermal growth factor in the receptor binding.

The isoleucine-23 residue of human epidermal growth factor (hEGF) was substituted by a variety of amino acid residues and the receptor-binding activities of variant hEGFs were determined by the use of human KB cell. Tight receptor binding was found of variants with hydrophobic amino acid residues in position 23. The size of the isoleucine residue was nearly optimum for the receptor binding as compared with other hydrophobic residues. The structure analysis by two-dimensional nuclear magnetic resonance spectroscopy showed that the substitution at position 23 only slightly affected the tertiary structure of hEGF. These indicate that the side chain of isoleucine residue in position 23, which is exposed on the protein surface, directly binds to a hydrophobic pocket of the receptor.

Amino Acid Sequence↗

PubMed1992

Stable isotope aided nuclear magnetic resonance study to investigate the receptor-binding site of human interleukin 1 beta.

Resonance assignments for interleukin 1 beta at neutral pH were made by using three-dimensional NMR in combination with specific labeling and double-labeling methods with stable isotopes. On the basis of the present assignments, 15N single-quantum coherence spectra of N-terminal truncated and fusion mutants were compared with that of the wild-type. Although these mutants have reduced biological activity, they showed 15N-SQC spectra similar to that of the wild-type. However, small but significant chemical shift changes were observed for amino acid residues within a loop 86-99, in spite of the modification at the N-terminus, supporting the idea that this loop forms a biologically active part of interleukin 1 beta. Receptor-binding activity was studied for mutants (Asp-93)-, (Leu-93)- and des-(Arg-98)interleukin 1 beta's. The results show significant loss of the receptor-binding activity. The N-terminus, the C-terminus, and the loop 86-99 form a part of the open end of a beta-barrel [Finzel, B. C., Clancy, L. L., Holland, D. R., Muchmore, S. W., Watenpaugh, K. D., & Einspahr, H. M. (1989) J. Mol. Biol. 209, 779-791; Clore, G. M., Wingfield, P. T., & Gronenborn, A. M. (1991) Biochemistry 30, 2315-2323], which forms the receptor-binding site of IL-1 beta.

Amides↗

PubMed1992

pH-dependent membrane fusion and vesiculation of phospholipid large unilamellar vesicles induced by amphiphilic anionic and cationic peptides.

We studied fusion induced by a 20-amino acid peptide derived from the amino-terminal segment of hemagglutinin of influenza virus A/PR/8/34 [Murata, M., Sugahara, Y., Takahashi, S., & Ohnishi, S. (1987) J. Biochem. (Tokyo) 102, 957-962]. To extend the study, we have prepared several water-soluble amphiphilic peptides derived from the HA peptide; the anionic peptides D4, E5, and E5L contain four and five acidic residues and the cationic peptide K5 has five Lys residues in place of the five Glu residues in E5. Fusion of egg phosphatidylcholine large unilamellar vesicles induced by these peptides is assayed by two different fluorescence methods, lipid mixing and internal content mixing. Fusion is rapid in the initial stage (12-15% within 20 s) and remains nearly the same or slightly increasing afterward. The anionic peptides cause fusion at acidic pH lower than 6.0-6.5, and the cationic peptide causes fusion at alkaline pH higher than 9.0. Leakage and vesiculation of vesicles are also measured. These peptides are bound and associated with vesicles as shown by Ficoll discontinuous gradients and by the blue shift of tryptophan fluorescence. They take an alpha-helical structure in the presence of vesicles. They become more hydrophobic in the pH regions for fusion. When the suspension is made acidic or alkaline, the vesicles aggregate, as shown by the increase in light scattering. The fusion mechanism suggests that the amphiphilic peptides become more hydrophobic by neutralization due to protonation of the carboxyl groups or deprotonation of the lysyl amino groups, aggregate the vesicles together, and interact strongly with lipid bilayers to cause fusion. At higher peptide concentrations, E5 and E5L cause fusion transiently at acidic pH followed by vesiculation.

Amino Acid Sequence↗

PubMed1992

Involvement of protein kinase C in thrombin-induced translocation of Gi2 alpha from the membrane to the cytosol in mouse mastocytoma P-815 cells.

Recently, we reported that in mouse mastocytoma P-815 cells the cytosol contains some factor(s) which promotes the release of GTP-activated Gi2 alpha from the membrane, and that thrombin induces the translocation of Gi2 alpha from the membrane to the cytosol (Takahashi, S., Negishi, M. and Ichikawa, A. (1991) J. Biol. Chem. 266, 5367-5370). Here we investigated the mechanism underlying the thrombin-induced translocation of Gi2 alpha in mastocytoma cells. Thrombin induced a rapid and transient increase in the intracellular Ca2+ concentration ([Ca2+]i) within 1 min, attenuated pertussis toxin-catalyzed ADP-ribosylation of Gi2 in the membrane, and caused the subsequent translocation of Gi2 alpha. Thrombin induced the translocation of protein kinase C from the cytosol to the membrane, and a protein kinase C inhibitor, staurosporine, completely inhibited the thrombin-induced translocation of Gi2 alpha. When cells were treated with thrombin, the ability of the cytosol to release Gi2 alpha from the membrane in the presence of GTP gamma S markedly increased. This stimulatory effect of thrombin on the ability of the cytosol was mimicked by 12-O-tetradecanoylphorbol 13-acetate (TPA), but not by the Ca2+ ionophore, ionomycin. The thrombin- and TPA-induced potentiation of the ability of the cytosol to release Gi2 alpha was completely abolished by staurosporine. Furthermore, phosphorylation of the cytosol by protein kinase C markedly potentiated the ability of the cytosol to release Gi2 alpha. These results together demonstrate that the thrombin-induced translocation of Gi2 alpha is due to enhancement of the ability of the cytosol to release Gi2 alpha via activation of protein kinase C.

Adenosine Diphosphate Ribose↗

PubMed1992

Expression of activin A/erythroid differentiation factor in murine bone marrow stromal cells.

Accumulating evidence suggests that activin A/erythroid differentiation factor (EDF), a homodimer of beta A chain, is a physiologic hematopoietic factor, particularly of erythroid lineage. Media conditioned by phorbol myristate acetate (PMA)-stimulated murine bone marrow stromal cell lines, MC3T3-G2/PA6 and ST2, contained activin A/EDF, assayed as the activity inducing erythroid differentiation of an activin A/EDF-responsive murine erythroleukemia cell clone F5. Follistatin, a protein specifically binding to activin A/EDF, abolished this erythroid differentiation. Northern blot analysis showed that PMA rapidly increased beta A chain messenger RNA levels in MC3T3-G2/PA6 and ST2 cells. In a search for natural stimulators, we found that tumor necrosis factor-alpha, in itself and synergistically with interleukin-1 beta, induced activin A/EDF production in both cell lines. These results indicate that stromal cells produce activin A/EDF in bone marrow.

Activins↗

PubMed1992

Morphological and northern blot analysis of juxtaglomerular cells in experimental hydronephrotic mice.

The purpose of this study is to demonstrate the developmental changes of the experimental hydronephrotic kidney using immunohistochemical, histoplanimetrical, and Northern blot techniques. At 1 month after ligation of the ureter, a large number of renin-positive cells were detected immunohistochemically even at a dilution of 1:10,000 in this hydronephrotic kidney; however, there were few renin-positive cells in the non-ligated side. At 6 months after ligation, no difference in reactivity for renin between ligated and non-ligated kidneys was demonstrated. In the morphometrical analysis of the renin-positive region, the numerical value of the ligated side was already increased at 2 weeks, reached the highest value at 1 month, and then decreased gradually to almost the same value as the control kidney by the end of the experiment. On the other hand, the value of the non-ligated side decreased immediately after the unilateral ligation, increased later, and finally reached almost the same value as the control kidney. In the Northern blot analysis, the activity of renin mRNA in the ligated side at 1 month after ligation was markedly higher than that in the non-ligated side. However, the difference between the ligated and the non-ligated sides was not demonstrated at 6 months and the value came to be almost the same as in the non-operated kidney.

Animals↗