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Biomedical subjects

K Uchida

Publications and source records attributed to K Uchida.

At least 19 recordsLinked to original sources
PubMed1992

Oxidative fragmentation of collagen and prolyl peptide by Cu(II)/H2O2. Conversion of proline residue to 2-pyrrolidone.

Oxidative degradation of collagen and the model peptides by Cu(II)/H2O2 has been studied. The depolymerization of collagen was predominantly observed by use of gel filtration chromatography. Polyproline was used as a model for collagen, and the oxidative modification was examined by amino acid analysis. Glutamic acid and gamma-aminobutyric acid were identified in the hydrolysates of oxidized polyproline. The formation of glutamic acid was reduced by treatment with NaBH4. The model peptide, (Pro-Pro-Gly)10, was also degraded by Cu(II)/H2O2, and a new N-terminal glycine was generated in proportion to the reaction time. Hydroxyl radical scavengers show only partial inhibition of the degradation of (Pro-Pro-Gly)10. In order to estimate the fragmentation mechanism, we used N-tert-butoxycarbonyl (Boc)-L-prolylglycine as a model for collagen and (Pro-Pro-Gly)10. The degradation products were isolated and characterized. Then N-tert-Boc-2-pyrrolidone, which provides gamma-aminobutyric acid by acid hydrolysis, was identified. The formation of a 2-pyrrolidone compound from oxidized Boc-L-prolylglycine is direct evidence for the scission of the peptide bond. The time-dependent formation of N-tert-Boc-2-pyrrolidone and liberation of glycine from N-tert-Boc-L-prolylglycine exposed to Cu(II)/H2O2 was observed. These results suggest that the cleavage of the peptide bond (Pro-Gly) was caused by oxidation of the proline residue, which led to the formation of the 2-pyrrolidone compound. We confirmed that proline oxidation leads to the fragmentation of proteins, accompanied by the formation of a 2-pyrrolidone structure.

Amino Acid Sequence↗
PubMed1992

Measurement of glycated albumin by the nitroblue tetrazolium colorimetric method.

A method has been developed for the measurement of glycated albumin (albumin-fructosamine) by the nitroblue tetrazolium (NBT) colorimetric method. In this method, polyethylene glycol was added to the serum and then the mixture was centrifuged to separate globulin proteins from albumin proteins. This made it possible to measure the glycated albumin in the supernatant by the NBT colorimetric method, without the interference of globulin proteins. This measurement method correlated with the measurement of glycated albumin using boronate affinity chromatography with an r value of 0.942 (P < 0.001). Our method using polyethylene glycol permits easy measurement of albumin fructosamine and is therefore useful as an index of diabetic control and for diabetic screening.

Chromatography, Affinity↗
PubMed1992

Isolation of cDNA clone encoding rat senescence marker protein-30 (SMP30) and its tissue distribution.

We have isolated and characterized two cDNA clones encoding senescence marker protein-30 (SMP30), the amounts of which are known to decrease androgen-independently with aging in the livers of rats. Of these cDNA clones, one consisted of 1588 bp nucleotides and the other of 1195 bp nucleotides generated by alternative polyadenylation. These two cDNA clones shared the same open reading frame, but the larger species had 393 bp nucleotides of 3' untranslated region in addition to the first polyadenylation site of smaller species. Northern hybridization analysis showed that two species of mRNA (1.7 kb and 1.4 kb) located in the liver and kidney were consistent with these short and long forms of cDNA. The open reading frame, 897 bp could encode 299 amino acids. The estimated molecular weight and pI of the deduced polypeptide were 33,387 and 5.1, respectively. Furthermore, immunohistochemical analysis confirmed that SMP30 was preferentially localized in the hepatocytes and renal proximal tubular epithelium. Genomic Southern hybridization analysis demonstrated that SMP30 was widely conserved among higher animals. A computer-assisted homology analysis of nucleic acid and protein databases revealed no remarkable homology with other known proteins. Therefore, SMP30 seems to be a novel protein. In addition, the existence of putative A-U rich mRNA degradation signals and protein degradation signals (PEST sequence) in the structure of SMP30 may suggest important regulatory function of this unique protein manifested by changes in its concentrations.

Aging↗
PubMed1992

Establishment of the human BSMZ breast cancer cell line, which overexpresses the erbB-2 and c-myc genes.

A new cell line, designated BSMZ, was established from a malignant pleural effusion from a woman with breast cancer. This line has a doubling time of 27 h and has now been cultured for over 120 passages. The large, rounded BSMZ cells grow as both a monolayer and as aggregations in suspension. Intracytoplasmic lumen, a finding consistent with results from cells derived from mammary tissue, was detected on ultrastructural analysis. Injection of BSMZ cells into nude mice resulted in the growth of solid tumors 4 weeks after inoculation. The solid tumor was identical to the original BSMZ cells in microscopic and electron microscopic studies. These cells possess an average of 80 chromosomes. Expression of erbB-2 and c-myc genes was increased by 10-fold, while there was no detectable overexpression of the N-ras and c-myb genes. Southern analysis has revealed amplification of the erbB-2 and c-myc loci. The BSMZ cell line may therefore provide a useful model for the study of human breast cancer and overexpression of the erbB-2 gene.

Adult↗
PubMed1992

Selective oxidation of histidine residues in proteins or peptides through the copper(II)-catalysed autoxidation of glucosone.

Glucosone has been identified as the main intermediate sugar moiety product of the copper(II)-catalysed autoxidation of the Amadori compound [Kawakishi, Tsunehiro & Uchida (1991) Carbohydr. Res. 211, 167-171]. Oxidative fragmentation of the model protein, especially selective degradation of the histidine residue in protein or peptides mediated by the copper(II)-catalysed autoxidation of glucosone, is discussed in this paper. The oxidative damage to protein could be retarded by catalase (EC 1.11.1.16) and EDTA, while superoxide dismutase (EC 1.15.1.1) and hydroxyradical scavengers showed little effect. Through the process of the oxidative degradation of N-benzoylhistidine and other histidine-containing peptides, the oxidation of the imidazole ring in histidine caused by the glucosone-copper(II) system was the same as that by the ascorbate-copper(II) system. These facts suggest that the copper-catalysed autoxidation of glucosone could generate some active-oxygen species causing oxidative damage to protein similar to that caused by the ascorbate-copper(II) system.

Amino Acids↗
PubMed1992

Selective cleavage of thioether linkage in proteins modified with 4-hydroxynonenal.

The peroxidation of polyunsaturated fatty acids leads to numerous products, including 4-hydroxynonenal (HNE). That 4-hydroxy-2-alkenal compounds react with sulfhydryl groups of proteins to form thioether adducts possessing a carbonyl function has been established [Schauenstein, E. & Esterbauer, H. (1979) Ciba Found. Symp. 67, 225-244]. Taking advantage of the fact that Raney nickel catalyzes cleavage of thioether bonds, we have developed a procedure to quantitate the amount of HNE moiety bound to protein by means of a thioether linkage. Adducts of HNE with N-acetylcysteine and glutathione were prepared, labeled with NaB[3H]H4, and then treated with Raney nickel. The 3H-labeled product was recovered in 85-90% yield from both HNE-N-acetylcysteine and HNE-glutathione adducts in a solvent [10% (vol/vol) methanol/chloroform]-estractable form. Treatment of proteins with HNE led to the disappearance of protein sulfhydryl groups. However, less than 10% of the labeled adducts obtained after subsequent reduction with NaB[3H]H4 could be released in a solvent-extractable form upon treatment with Raney nickel. This and the observation that HNE reacts with proteins lacking a sulfhydryl group attests to the fact that HNE can react with amino acid residues other than cysteinyl residues.

Acetylcysteine↗
PubMed1992

Characteristics of substrates and inhibitors in binding to rat liver L-tryptophan 2,3-dioxygenase: a Fourier transform infrared and kinetic study.

Infrared spectroscopy and steady-state kinetics were applied to rat liver L-tryptophan 2,3-dioxygenase, in order to find relations between the structure and binding characteristics of its substrates and inhibitors. The binding characteristics were reflected by changes in the infrared CO stretch band(s) of an Fe(II)-CO complex of the enzyme upon addition of L-tryptophan and 12 analogs. The CO stretch band around 1961 cm-1 of the complex was not much affected by 1-methyl-D,L-tryptophan, a noncompetitive inhibitor, implying a binding at a site distant from the Fe(II)-CO vicinity. The spectral pattern was significantly changed by any of the other compounds which conserved an indole NH, indicative of its binding to the catalytic site. All substrates, which contained a complete CH(NH2)COOH group in addition to the NH, gave spectra similar to that of an L-tryptophan-bound complex. Spectral changes caused by six inhibitors, which lacked the complete CH(NH2)COOH, were different from one another and from those by the substrates. Hence, for an analog, the indole NH is indispensable to bind to the catalytic site, and the CH(NH2)COOH is important to take a correct configuration appropriate to the catalytic reaction. The reason why L- and D-isomers of 5-hydroxytryptohan are not substrates, in spite of their conservation of the required functional groups and correct binding to the catalytic site, has been ascribed to a possible distortion of the protein structure in the heme pocket due to a strong hydrogen bond from the hydroxyl group to an amino acid side chain.

Animals↗
PubMed1992

Modification of histidine residues in proteins by reaction with 4-hydroxynonenal.

We find that histidine residues in proteins are major targets for reaction with the lipid peroxidation product 4-hydroxynon-2-enal (HNE). Reaction of insulin (which contains no sulfhydryl groups) with HNE leads to the generation of HNE-protein adducts, which are converted to radioactive derivatives upon subsequent treatment with NaB[3H]H4. Amino acid analysis of the modified protein showed that the HNE treatment leads to the selective loss of histidine residues and the stiochiometric formation of 3H-labeled amino acid derivatives. The same labeled products were detected in acid hydrolysates of polyhistidine and N-acetylhistidine after their reactions with HNE and NaB[3H]H4. The reaction of N-acetylhistidine with HNE led to the production of two compounds. Upon acid hydrolysis, both derivatives yielded stoichiometric amounts of histidine. However, after reduction with NaBH4, acid hydrolysis led to a mixture of amino acid derivatives [presumably, isomeric forms of N pi (N tau)-1,4-dihydroxynonanylhistidine] that were indistinguishable from those obtained from insulin and polyhistidine after similar treatment. Although other possibilities are not excluded, it is suggested that the modification of histidine residues in proteins by HNE involves a Michael-type addition of the imidazole nitrogen atom of histidine to the alpha, beta-unsaturated bond of HNE, followed by secondary reaction involving the aldehyde group with the C-4 hydroxyl group of HNE. The reaction of histidine residues with HNE provides the basis for methods by which the contributions of HNE in the modification of proteins can be determined.

Aldehydes↗
PubMed1992

Purification of senescence marker protein-30 (SMP30) and its androgen-independent decrease with age in the rat liver.

Age-associated changes in the soluble proteins from rat liver were examined by a newly developed two-dimensional cellulose acetate membrane electrophoresis (2D-CAME). We detected and isolated a novel rat liver protein, the amounts of which decreased androgen-independently with aging. We designated this protein, whose molecular mass was 30 kDa and pI value was 4.9, as senescence marker protein-30 (SMP30). The expression of SMP30 was not modified by castration or treatment with testosterone propionate after castration. Age-associated decrease of SMP30 level was also recognized in aged female. We noted another protein with a pI value of 7.3 that decreased with aging. Its expression seemed to be androgen-dependent and it was markedly expressed only in young and adult male rat livers. We then purified SMP30 and prepared an anti-serum to SMP30. Immunohistochemical analysis showed that the localization of SMP30 was restricted to liver and kidney among numerous organs tested. Although the amount of SMP30 in the liver was relatively large and the tissue distribution was characteristic, no known protein corresponds to this protein.

Aging↗
PubMed1992

Excitatory amino acid receptors appear to mediate paroxysmal depolarizing shifts in rat neocortical neurons in vitro.

This study was designed to assess some of the contributions of excitatory amino acids to locally evoked responses in neurons in slices from frontal motor cortex in Sprague-Dawley rats. Intracellular recordings were obtained from 54 cortical neurons. Paroxysmal depolarization shifts (PDS) were evoked by local single pulse stimulation in cortex or in a small number of cases (n = 2) occurred spontaneously. These potentials could be abolished by application of kynurenic acid, a broad spectrum excitatory amino acid receptor antagonist. They were enhanced in Mg(2+)-free medium and could then be antagonized by application of D,L-2-amino-5-phosphonovalerate (AP5), a selective blocker of the N-methyl-D-aspartate (NMDA) receptors.

2-Amino-5-phosphonovalerate↗
PubMed1992

An androgen-dependent subclone derived from a mouse mammary tumor, Shionogi carcinoma 115, secretes a heparin-binding growth factor having an apparent molecular weight of 31,000 in response to androgen.

An androgen-dependent cell line denoted SC2G is a clone of an androgen-dependent mouse mammary tumor, Shionogi Carcinoma 115. Fibroblast growth factors (FGFs), epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) are stimulatory for the growth of SC2G cells in the absence of androgen. This clone was found to secrete an androgen-induced growth factor mostly eluting at 1.8 M NaCl on a heparin-Sepharose column. This factor was partially purified by chromatography on two consecutive heparin-Sepharose columns followed by cation-exchanging chromatography on an S-Sepharose column from the chemically defined serum-free medium conditioned by SC2G cells in the presence of androgen. The factor was a heat- and acid-labile cationic protein that was inactivated by reduction with dithiothreitol. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, most of the growth-promoting activity of this factor was found at approx. 31 kDa under non-reduced conditions. Neither neutralizing antibody against basic-FGF nor that against EGF inhibited the growth-promoting activity of this factor in cell culture, suggesting the factor was distinct from basic FGF or EGF. However, the possibility that the factor was another FGF- or EGF-like growth factor was not excluded.

Animals↗
PubMed1992

Formation of beta-galactosyl compounds of pyridoxine in growing culture of Sporobolomyces singularis.

Several pyridoxine compounds were found to be formed in a high yield in a growing culture of Sporobolomyces singularis containing lactose and pyridoxine. Three compounds, I, II and III, were isolated from cultured broth by Dowex 50W-X8 column chromatography, paper chromatography, lyophilization, and then obtained as needle crystals (m.p. (decomp.): I, 204-206 degrees C; II, 192-194 degrees C; III, 222-223 degrees C. [alpha]D16: I, -27.7 degrees (c = 3.82, H2O); II, -1.6 degrees (c = 2.52, H2O); III, +8.3 degrees (c = 3.98, H2O)). Compounds I, II and III were identified as 5'-O-(beta-D-galactopyranosyl)-pyridoxine, 4'-O-(beta-D-galactopyranosyl)-pyridoxine, and 4'-O-(beta-D-galactopyranosyl-(1----4)-beta-D-galactopyranosyl)-pyrid oxi ne, respectively, on the basis of the various experimental results, viz., ultraviolet, infra-red, 1H-NMR, and 13C-NMR spectra, products by hydrolysis with acid and with alpha- and beta-galactosidases, migration on paper electrophoresis, and Gibbs reaction in the presence and absence of boric acid. Also, the yeast produced a remarkable amount of beta-glucosyl compounds of pyridoxine in cultured broth, when grown on cellobiose and pyridoxine.

Carbohydrate Sequence↗
PubMed1992

Clinical significance of the vertebral vein in prostate cancer metastasis.

A total of 75 prostate cancer and 67 lung cancer patients with positive bone scintigrams were studied. The patterns of spread in the axial skeleton and pelvis were different between the groups. The differences in the distribution of bony metastases between prostate and lung are explained by the role of Batson's vertebral venous plexus. We developed an animal model of spinal bone metastasis to prove this route. As suspension of tumor cells was injected into the tail vein of mice with vena caval occlusion. This procedure reproducibly resulted in metastatic tumor growth in the lumbar region of the vertebral column. The prevalence of spinal bone metastasis is attributed to passage of tumor cells via the vertebral venous plexus.

Adenocarcinoma↗
PubMed1992

Enhancement of host resistance to opportunistic infections by ubenimex (bestatin).

Ubenimex is a low molecular biological response modifier (BRM) which has been demonstrated to have antitumor and immunomodulatory activities. In this study, the effect of ubenimex on infection with Candida albicans was investigated in normal and immunosuppressed mice, and it showed a prophylactic effect. In normal mice, ubenimex prolonged survival time in a dose-dependent manner. In immunosuppressed mice treated with cyclophosphamide 4 days before infection, ubenimex at 5 mg/kg for 5 days significantly increased the number of survivors. Significant improvement in peritoneal leukocyte counts and in function of neutrophils including phagocytosis and release of activated oxygen was observed in ubenimex-treated mice. These results indicate that ubenimex is a potent BRM for prevention against opportunistic infections.

Animals↗
PubMed1992

Helicobacter pylori infection induces a decrease in immunoreactive-somatostatin concentrations of human stomach.

Immunoreactive-somatostatin (ir-somatostatin) concentrations of the gastric mucosa and gastric juice with Helicobacter pylori infection were measured in the human stomach. One hundred seventy-one patients (106 males, 65 females; mean age, 52.0; range, 19-84 years) were registered. Gastric juice and mucosa were obtained with the usual endoscopy procedure. Somatostatin concentration was measured by radioimmunoassay. The ir-somatostatin concentrations in the H. pylori-negative group were significantly higher than in the positive group gastric mucosa, whereas its levels in gastric juice tended to decrease with H. pylori infection. There was an inverse correlation between luminal ammonia levels and ir-somatostatin concentrations of the gastric mucosa. On the other hand, ir-somatostatin concentrations of the gastric mucosa significantly decreased with chronic and active inflammatory change. This decrease was not correlated with the grade of active inflammation, which was in close relation to H. pylori infection, but with the grade of chronic inflammation. These results indicate that H. pylori may reduce ir-somatostatin concentrations of the human stomach and that its effect is partly mediated via luminal ammonia produced by H. pylori.

Adult↗