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Biomedical subjects

C Ishida

Publications and source records attributed to C Ishida.

At least 37 records · Page 2Linked to original sources

All-trans retinoic acid modulates Fas antigen expression and affects cell proliferation and apoptosis in combination with anti-Fas monoclonal antibody in the human myeloma cell line, U266B1.

All-trans retinoic acid (ATRA) is a vitamin A derivative that induces the differentiation of myeloid leukemia cells in vitro and in vivo. Several investigators have recently reported that ATRA downregulates the production of interleukin-6 (IL-6) and the expression of IL-6 receptor (IL-6R) and also inhibits the proliferation of myeloma cells. It has also been reported that myeloma cells express Fas antigen, and in some of these cells apoptosis was induced by treatment with anti-Fas monoclonal antibody (mAb). In the present study, we demonstrated that ATRA increased Fas expression in the human myeloma cell line, U266B1. We observed that both apoptosis induction and growth inhibition were enhanced in cells exposed to a combination of anti-Fas mAb and ATRA compared with cells exposed to either treatment alone. We also examined whether ATRA modulated bcl-2, an anti-apoptosis protein, in U266B1 cells. Flow cytometry analysis revealed that the mean fluorescence intensity of bcl-2 protein was slightly decreased in cells treated with ATRA. These results indicate that in U266B1 cells, combined treatment with anti-Fas mAb and ATRA enhances the induction of apoptosis by modulating the expression of Fas and bcl-2 by ATRA.

Antibodies, Monoclonal↗

Functional cloning of a cDNA encoding Mei2-like protein from Arabidopsis thaliana using a fission yeast pheromone receptor deficient mutant.

To isolate Arabidopsis cDNAs that encode signal transducers and components involved in the regulation of meiosis, a trans-complementation analysis was performed using a Schizosaccharomyces pombe meiosis-defective mutant in which the genes for pheromone receptors were disabled. One cDNA obtained in this screening encodes a polypeptide, named AML1, that shows significant similarity to S. pombe Mei2 protein and has three putative RNA-recognition motifs like as Mei2. Mei2 is involved in the regulation of meiosis in fission yeast. Northern blot analysis showed that the AML1 gene is expressed in each organ. The possible functions of AML1 are discussed.

Amino Acid Sequence↗

Trypsin-sensitive and -resistant components in human T-cell membranes required for syncytium formation by human T-cell lymphotropic virus type 1-bearing cells.

Human T-cell lymphotropic virus type 1 (HTLV-1) envelope proteins play an important role in viral entry into target cells. In a syncytium formation assay consisting of a coculture of HTLV-1-bearing cells and target cells, mature gp46 and gp21 proteins each inhibited syncytium formation induced by HTLV-1-bearing cells. Experiments with 125I-labeled proteins showed that 125I-gp46 bound specifically with MOLT-4 target cells even in the presence of large amounts of gp21, whereas 125I-gp21 binding to target cells was completely blocked in the presence of large amounts of gp46. These observations suggest that HTLV-1 envelope proteins in syncytium formation interact with at least two components, which are located close to each other on the cell membrane. We isolated two components from MOLT-4 cell lysate, using Sepharose 4B columns coupled with peptides corresponding to amino acids 197 to 216 and 400 to 429, respectively, of the envelope protein. One is a trypsin digestion-sensitive component of approximately 34 to 35 kDa, which interacts specifically with gp46. The other is a nonprotein component, which interacts with gp21. This component was destroyed by sodium periodate oxidation and was partitioned into the methanol-chloroform phase. These observations suggest that these two components play an important role in HTLV-1 entry into target cells via membrane fusion.

Cell Membrane↗

[Small hand muscle atrophy as an initial sign in HTLV-I associated myelopathy].

A 68-year-old female had been well until the age of 52 when she noticed weakness and atrophy of her right small hand muscles. She began to have gait disturbance at the age of 63. On admission, neurological examinations revealed atrophy and weakness of the small hand muscles on both sides as well as spastic paraparesis. As the antibody titer for human T-lymphotropic virus type I (HTLV-I) was elevated both in the serum and CSF, a diagnosis of HTLV-I associated myelopathy (HAM) was made. Electrophysiological studies showed neurogenic changes and suggested anterior horn cell involvement. After oral administration of prednisolone, her spasticity and gait disturbance improved slightly. Small hand muscle atrophy can be one of the initial signs of HAM, and may mimic amyotrophic lateral sclerosis and cervical spondylosis.

Aged↗

A role of the amino acid residue located on the fifth position before the first aspartate-rich motif of farnesyl diphosphate synthase on determination of the final product.

Farnesyl diphosphate (FPP) synthase catalyzes consecutive condensations of isopentenyl diphosphate with allylic substrates to give FPP, C-15 compound, as a final product and does not catalyze a condensation beyond FPP. Recently, it was observed that, in Bacillus stearothermophilus FPP synthase, a replacement of tyrosine with histidine at position 81, which is located on the fifth amino acid before the first aspartate-rich motif, caused the mutated FPP synthase to catalyze geranylgeranyl diphosphate (C-20) synthesis (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095). Thus, we constructed 20 FPP synthases, each of which has a different amino acid at position 81, and analyzed them. All enzymes except for Y81P can catalyze the condensations of isopentenyl diphosphate. The final products and the product distributions are different from each other. Y81A, Y81G, and Y81S can produce hexaprenyl diphosphate (C-30) as their final product. The final product of Y81C, Y81H, Y81I, Y81L, Y81N, Y81T, and Y81V are geranylfarnesyl diphosphate (C-25), and Y81D, Y81E, Y81F, Y81K, Y81M, Y81Q, and Y81R cannot produce polyprenyl diphosphates more than geranylgeranyl diphosphate. Substitution of tryptophan does not affect the product specificity of FPP synthase. The average chain length of products is inversely proportional to the accessible surface area of substituted amino acid. However, no significant relation between the final chain length and the kinetic constants Km and Vmax are observed. These observations strongly indicate that the amino acid does not come into contact with the substrates but directly contacts the omega-terminal of an elongating allylic product. This interaction must prevent further condensation of isopentenyl diphosphate.

Alkyl and Aryl Transferases↗

Conversion of product specificity of archaebacterial geranylgeranyl-diphosphate synthase. Identification of essential amino acid residues for chain length determination of prenyltransferase reaction.

Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. To investigate the mechanism of the consecutive reaction and the determination of the ultimate chain length, a random mutational approach was planned. A geranylgeranyl-diphosphate synthase gene from Sulfolobus acidocaldarius was randomly mutagenized by NaNO2 treatment to construct a library of mutated geranylgeranyl-diphosphate synthase genes on a yeast expression vector. The library was screened for suppression of a pet phenotype of yeast C296-LH3, which is deficient in hexaprenyl-diphosphate synthase. Five mutants that could grow on a YEPG plate, which contained only glycerol as an energy source instead of glucose, were selected from approximately 1,400 mutants. All selected mutated enzymes catalyzed the formation of polyprenyl diphosphates with prenyl chains longer than geranylgeranyl diphosphate. Especially mutants 1, 3, and 5 showed the strongest elongation activity to produce large amounts of geranylfarnesyl diphosphate with a concomitant amount of hexaprenyl diphosphate. Sequence analysis revealed that each mutant contained a few amino acid substitutions and that the mutation of Phe-77, which is located on the fifth amino acid upstream from the first aspartate-rich consensus motif, is the most effective for elongating the ultimate product. Amino acid alignment of known prenyltransferases around this position and our previous observations on farnesyl-diphosphate synthase (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T.(1996) J. Biol. Chem. 271, 10087-10095) clearly indicate that the amino acid at the position of all prenyltransferases must regulate the chain elongation.

Alcohols↗

Interaction of immobilized recombinant mouse C-type macrophage lectin with glycopeptides and oligosaccharides.

Inflammatory and tumoricidal macrophages express galactose- and N-acetylgalactosamine-specific Ca(2+)-dependent lectins on their surfaces. This lectin is a family member of membrane-bound C-type animal lectins and consists of 304 amino acid residues (molecular weight 34,595). In the present study, expression vectors containing a nucleotide sequence corresponding to the carbohydrate-binding domain of mouse macrophage lectin cDNA have been prepared. The carbohydrate-binding specificity of the recombinant macrophage lectin expressed in Escherichia coli was investigated by comparing elution profiles of various glycopeptides having defined carbohydrate structures on immobilized lectins. When elution profiles of high mannose-type and complex-type Asn-linked carbohydrate chains were compared, the degree of retardation from immobilized macrophage lectin column was in the order tetraantennary complex-type with terminal galactosyl residues > triantennary complex-type with terminal galactosyl residues > biantennary complex-type with terminal galactosyl residues > high mannose-type glycopeptides. N-Terminal octapeptides from human glycophorin A that bore three NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc serine/threonine-linked tetrasaccharide chains and their sequentially deglycosylated derivatives were also applied to this column. Glycopeptides carrying three constitutive GalNAc-Ser/Thr(Tn-antigen) had the strongest affinity, whereas those with fully sialylated carbohydrate tetrasaccharide chains showed weak interaction. The association kinetics of Asn-linked glycopeptides from bovine asialofetuin to recombinant macrophage lectin was determined by surface plasmon resonance spectroscopy. The results indicate k(assoc) value of 1.63 x 10(4) M-1 s-1. The calculated value for Ka was 6.20 x 10(7) M.

Amino Acid Sequence↗

Cloning and sequence analysis of the Maackia amurensis haemagglutinin cDNA.

Maackia amurensis haemagglutinin (MAH) is a leguminous lectin which preferentially binds to a cluster of sialylated O-linked carbohydrate chains (Konami Y, Yamamoto K. Osawa T, Irimura T (1994) FEBS Lett 342:334-38). In the present study a 950 bp cDNA clone encoding MAH was isolated from a cDNA library constructed from germinated Maackia amurensis seeds. From the nucleotide sequence, MAH was predicted to consist of 285 amino acid residues containing a signal peptide of 29 amino acids. The results also confirmed our previous findings from the amino acid sequence analysis, which indicated that two highly conserved amino acid residues in all other well-known leguminous lectins were replaced in MAH. These residues were lysine-105 and aspartic acid-135. The corresponding amino acid residues in other leguminous lectins were glycine and asparagine, respectively. These differences were due to the presence of nucleotides AAA and GAT in place of AAT/C and GGA/T.

Amino Acid Sequence↗

A unique amino acid sequence involved in the putative carbohydrate-binding domain of a legume lectin specific for sialylated carbohydrate chains: primary sequence determination of Maackia amurensis hemagglutinin (MAH).

The primary sequence of 247 amino acids of Maackia amurensis hemagglutinin (MAH) was determined using a protein sequencer. After digestion with endoproteinase Lys-C, Asp-N, Arg-C, or Glu-C of MAH, the resulting peptides were purified by reversed phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. The primary sequence of MAH was compared with those of several legume lectins, and it was found that the amino acid sequence of the putative carbohydrate-binding domain of MAH exhibited a high degree of homology with those of di-N-acetylchitobiose-binding Cytisus sessilifolius lectin I (CSA-I), Laburnum alpinum lectin I (LAA-I), and Ulex europaeus lectin II (UEA-II). In the legume lectins whose primary sequences have already been determined several amino acid residues involved in carbohydrate-binding were found to be conserved. Very interestingly, in the primary sequence of MAH, one amino acid residue corresponding to the conserved amino acid, asparagine, in the primary sequences of all other legume lectins was shown to be substituted by aspartic acid. This is the first report of the occurrence of an exceptional amino acid residue among the conserved amino acid residues in the carbohydrate-binding domain of the legume lectins.

Amino Acid Sequence↗

Detection of antibodies to hepatitis C virus (HCV) structural proteins in anti-HCV-positive sera by an enzyme-linked immunosorbent assay using synthetic peptides as antigens.

We have defined 10 linear immunogenic regions encoded by the putative hepatitis C virus (HCV) structural proteins (core and envelope) by employing an enzyme-linked immunosorbent assay (ELISA) and by using 17 sequential synthetic peptides covering the N-terminal 330 amino acids of the structural polyproteins as antigens. These peptides correspond to amino acids 1 to 24, 21 to 44, 42 to 68, 64 to 91, and 100 to 120 of the putative core protein and amino acids 192 to 212, 223 to 238, 236 to 258, 250 to 266, and 307 to 330 of the putative envelope protein. In particular, the peptide covering amino acids 21 to 44 of the core protein was reactive with all but one (40 of 41) of the serum samples giving a positive signal in the passive hemagglutination assay (PHA) using the core and nonstructural proteins (NS 3/4) of the virus as antigens. We detected the HCV genome in 25 (61%) of 41 PHA-positive serum samples by the polymerase chain reaction (PCR) test. Of 25 PCR-positive serum samples, 17 serum samples had reactivity to the peptides derived from the envelope protein. On the other hand, only 1 of the 16 PCR-negative serum samples had reactivity to the peptides derived from the envelope protein. Interestingly, we often observed high serum alanine aminotransferase levels in PCR-positive individuals bearing antibodies to the envelope protein.

Alanine Transaminase↗

A new metabolite of 2,4,3',4'-tetrachlorobiphenyl in rat feces.

Metabolism in vivo of 2,4,3',4'-tetrachlorobiphenyl (TCB) was further studied using male Wistar rats. When the extract of feces of rats given TCB with chloroform was methylated and applied to gas chromatography (GC)-mass spectrometry (MS), a new metabolite was detected. The structure of this new metabolite was 4-hydroxy-2,5,3'4'-TCB based on both its retention time in GC and comparison of the mass spectrum with that of the synthetic sample. 4-Hydroxy-2,5,3',4'-TCB was assumed to be formed via a 4,5-oxide intermediate followed by NIH-shift of a chlorine atom at 4-position.

Animals↗

[A case of recurrent esophageal cancer maintaining complete long-term response after 5'-DFUR and ftorafur administrations].

The authors report a case with esophageal cancer who had undergone a non-curative resection and showed a recurrence. Treatment with only oral 5'-DFUR resulted in complete response. This was followed by oral ftorafur, and the case has remained complete response for 40 months. It is rare to obtain such an effect in esophageal cancers by oral and sole administration of a drug in the 5-FU class. Our experience with this case is described.

Administration, Oral↗

Metabolism in vitro of 3,4,3',4'- and 2,5,2',5'-tetrachlorobiphenyl by rat liver microsomes and highly purified cytochrome P-450.

Metabolism of two polychlorinated biphenyls, 3,4,3',4'- and 2,5,2',5'-tetrachlorobiphenyl (TCB), was studied using rat liver microsomes and the four forms of cytochrome P-450 (P-450), P-450b, P-450e, P-450c and P-450d. At first, effects of various inducers of P-450 such as phenobarbital (PB), 3-methylcholanthrene (MC), isosafrole (ISF) and pregnenolone 16 alpha-carbonitrile on the formation of metabolites of these TCBs by liver microsomes were compared. 3,4,3',4'-TCB was significantly metabolized by liver microsomes from MC-treated rats to form two previously reported metabolites, 4-hydroxy-3,5,3',4'-TCB and 5-hydroxy-3,4,3',4'-TCB with a relative ratio of 2.5:1. Incubation with microsomes from untreated or PB-treated rats produced none of the metabolites. On the other hand, 2,5,2',5'-TCB was metabolized to 3-hydroxy-2,5,2',5'-TCB most easily by liver microsomes from PB-treated rats and at a moderate rate by liver microsomes from ISF-treated rats. Activities of microsomes from untreated or MC-treated rats to hydroxylate 2,5,2',5'-TCB were low or undetectable. When these TCB hydroxylase activities were examined with a reconstituted system consisting of each P-450, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase, dilauroylphosphatidylcholine and NADPH-generating system, only P-450c catalyzed both the 4- and 5-hydroxylations of 3,4,3',4'-TCB at a ratio of 2.2:1. On the contrary, the hydroxylation of 2,5,2',5'-TCB proceeded efficiently with P-450b and P-450e, being more efficient with the former. P-450d did not show any catalytic activity toward 3,4,3',4'-TCB and 2,5,2',5'-TCB.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[Toxicological assessment of 2,5,2',5'-tetrachlorobiphenyl and its major metabolite, 3-hydroxy-2,5,2',5'-tetrachlorobiphenyl in rats].

We observed previously that polychlorinated biphenyl (PCB) could be classified to two groups, 3-methylcholanthrene (MC)-type and phenobarbital (PB)-type, in term of inducibility of the hepatic enzymes. MC-type PCBs such as 3,4,3',4'-tetrachlorobiphenyl (TCB), 3,4,5,3',4'-pentachlorobiphenyl (PenCB) and 3,4,5,3',4',5'-hexachlorobiphenyl (HexCB) exhibited high acute toxicity in parallel with their induction ability of microsomal benzo[a]pyrene 3-hydroxylase and cytosolic DT-diaphorase. On the contrary, PB-type PCBs such as 2,5,2',5'-TCB and 2,4,5,2',4',5'-HexCB which induce microsomal benzphetamine N-demethylase and NADPH-cytochrome P-450 reductase activities showed virtually no or very low toxicity. In the present study, we examined effects of 2,5,2',5'-TCB and its major metabolite 3-hydroxy-2,5,2',5'-TCB on body weight gain, organ weights and activities of hepatic enzymes in rats and assessed acute toxicity of these compounds. As the results, in both 2,5,2',5'-TCB and 3-hydroxy-2,5,2',5'-TCB groups, the body weights were increased during the experiment, but the rate of growth was significantly suppressed after 3 days. Significant hypertrophy of the liver and decrease of total liver lipid content were observed in 2,5,2',5'-TCB group, but the atrophy of spleen and thymus was not affected in both groups. On the other hand, in 2,5,2',5'-TCB group, benzo[a]pyrene 3-hydroxylase and benzphetamine N-demethylase activities were increased to 2. 4-fold and 1.5-fold, respectively, but were not increased in 3-hydroxy-2,5,2',5'-TCB group. After injection of 2,5,2',5'-TCB, 45% of the dose was excreted as 3-hydroxy-2,5,2',5'-TCB in feces for 5 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Respiratory function in handicapped children.

The aim of this study was to evaluate respiratory function of severely handicapped children. Tidal volumes and respiratory rates were determined in a total of 130 children with different clinical motor abilities. Tidal volume of non-sitters (n = 39) was significantly lower than ambulators (n = 49) or sitters (n = 42) (p less than 0.01). There was no difference in respiratory rate among the three groups. Among 45 children whose vital capacity could be determined, the tidal volumes showed a significant correlation with vital capacity (r = 0.56, p less than 0.001). Among four children whose tidal volume was less than 200 ml and respiratory rate was more than 30 cpm, blood gas analysis revealed hypoxia in three of them. The tidal volumes, therefore, would be a useful guide to estimate respiratory functions. It was concluded that the respiratory function in a non-sitter with reduced tidal volume is impaired, and that preventive measures must be taken against respiratory infection.

Adolescent↗