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Biomedical subjects

R Kobayashi

Publications and source records attributed to R Kobayashi.

At least 361 records · Page 20Linked to original sources
PubMed1990

Tc-99m RBC imaging for demonstrating intermittent intestinal bleeding.

A case of intestinal bleeding caused by multiple ulcerations near the terminal ileum is presented. Abdominal scanning with Tc-99m tagged red blood cells (Tc-99m RBC) demonstrated the intermittent bleeding in short intervals. Selective angiography did not demonstrate the bleeding site. Exploratory laparotomy showed 3 ulcers on the ileum at a site near the terminal ileum, with bleeding from one site. Tc-99m RBC abdominal scanning was very useful in this case for localization of the site of gastrointestinal bleeding before laparotomy.

Erythrocytes↗
PubMed1990

Experimental infection of African green monkeys and cynomolgus monkeys with a SIVAGM strain isolated from a healthy African green monkey.

An infection occurred in all African green monkeys and cynomolgus monkeys experimentally inoculated with SIVAGM [TYO-1], as demonstrated by the appearance of an antibody to SIVAGM [TYO-1] and the isolation of the virus. No monkey exhibited overt clinical disorders throughout the experimental period of 42 weeks. Thus, SIVAGM was not pathogenic to its original host or to macaques. This system is proposed as a model for HIV infection manifesting no overt disease.

Animals↗
PubMed1990

[Hyperdiploidy (greater than 50 chromosomes) has the most favorable prognosis among the major karyotypic subgroups of childhood acute lymphoblastic leukemia].

Thirty-four children, including nine relapsed cases with acute lymphoblastic leukemia (ALL) having hyperdiploidy (greater than 50 chromosomes) were studied on clinical and cytogenetic characteristics. The majority of children initially with hyperdiploidy (greater than 50 chromosomes), who showed favorable prognostic features such as lower leukocyte counts, lower serum lactic dehydrogenase levels, ages between 2 and 10 years, or the presence of common ALL antigen, had the most favorable outcome among childhood ALL (5-year survival rate was 100%). Even nine children, who showed poor prognostic features such as ages over 10 years, leukocyte counts over 2 X 10(4)/mm3 or lymphomatous signs, had also the same favorable outcome. There were no differences in clinical features between 6 patients with additional chromosomal structural abnormalities and 19 patients without them. Duplication of the long arm of chromosome 1 was frequently observed as additional chromosomal structural abnormalities. Patients with hyperdiploidy (greater than 50 chromosomes) observed at relapse, who had the same favorable clinical features as those at diagnosis, had a poorer prognosis. These findings show that initial hyperdiploidy (greater than 50 chromosomes) is an independent favorable prognostic sign in childhood ALL and additional chromosomal structural abnormalities may not indicate a poor prognosis among childhood ALL with hyperdiploidy (greater than 50 chromosomes). On the other hand, relapsed children with hyperdiploidy (greater than 50 chromosomes) have not a favorable outcome after the onset of relapse.

Child↗
PubMed1989

Visualization of antigen on nitrocellulose membrane by the oxidative coupling reaction of N,N'-dimethyl-p-phenylenediamine and 4-chloro-1-naphthol.

A method which allows the highly sensitive and simple immunodetection of antigen-antibody complexes on nitrocellulose papers has been developed. The method is a modification of the procedure known as the "Nadi reaction" (oxidative coupling of 1-naphthol and N,N'-dimethyl-p-phenylendiamine) for histochemical purposes. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. Bound proteins were first reacted with a primary antibody and then with a horseradish peroxidase-labeled second antibody. The antigen-antibody complexes on the membranes were visualized with the oxidative coupling solution containing N,N'-dimethyl-p-phenylendiamine and 4-chloro-1-naphthol. Sensitivity was enhanced 4 to 16 times by the new method relative to that of the 4-chloro-1-naphthol method or the 3,3'-diaminobenzidine method.

3,3'-Diaminobenzidine↗
PubMed1989

Alpha-actinin expression during avian myogenesis in vivo. Evidence for the existence of an embryo-specific isoform of alpha-actinin.

The isoforms of skeletal muscle alpha-actinin present during chick embryogenesis were analyzed by two-dimensional electrophoresis in combination with the immunoblot technique. Chicken embryonic muscles at 8-15 days contain an embryo-specific isoform of alpha-actinin. The embryonic alpha-actinin isoform has a molecular mass of 112 kDa and an isoelectric point of 5.8, whereas the values for the adult isoform of alpha-actinin were 100 kDa and 5.85, respectively. To characterize the two classes of alpha-actinin polypeptides we have compared the two proteins by 125I-labeled two-dimensional peptide mapping. The embryonic isoform is highly similar to, but exhibited extensive peptide differences to, the adult isoform of alpha-actinin. The developmental sequence of the expression of the alpha-actinins was also studied. In extracts of skeletal muscle from 8-10-day-old embryos, only the embryonic isoform was detected. In extracts from 15-day-old embryos, both the embryonic and the adult isoforms coexisted. However by 21 days, the embryonic isoform had disappeared and only the adult isoform was detected. These data suggested that the embryonic and the adult isoform of alpha-actinins are distinct proteins and that during skeletal myogenesis in ovo one class of alpha-actinin is replaced by a new class of alpha-actinin polypeptides, and that the latter is maintained into adulthood.

Actinin↗
PubMed1989

Isolation and characterization of a new 36-kDa microfibril-associated glycoprotein from porcine aorta.

A new connective tissue protein of 36 kDa has been purified from porcine aorta. The biochemical and immunological properties of the protein are distinct from those of microfibril-associated proteins reported previously such as lysyl oxidase, 31-kDa microfibril-associated glycoprotein, and fibrillin. It could bind to concanavalin A-Sepharose and gelatin-Sepharose. The protein contained the sequence Arg-Gly-Asp-Ala in the amino-terminal region, which is the site for the association with cell and extracellular matrix. Using specific antibody raised to the protein, we demonstrated its restricted localization in aorta adventitia. Immunoelectron microscopy specified its location to a class of extracellular structural elements described as elastin-microfibrils.

Amino Acid Sequence↗
PubMed1989

An immunological and biochemical comparison of 67 kDa calcimedin and 67 kDa calelectrin.

The 67 kDa calcimedin is a Ca2+-binding protein isolated from several muscle tissues. A recent report [Morse & Moore (1988) Biochem. J. 251, 171-174] indicated that the 67 kDa calcimedin is distinct from 67 kDa calelectrin, which is purified from various non-muscle cells. In the present study we have purified the 67 kDa protein from bovine aorta (i.e. 67 kDa calcimedin) and liver (i.e. 67 kDa calelectrin) and compared them by immunological and biochemical criteria. The aorta calcimedin is identical with the liver calelectrin by the following criteria. (1) The calcimedin co-electrophoresed with the calelectrin on SDS/5-15%-(w/v)-linear-gradient polyacrylamide gels. (2) The two proteins selectively cross-reacted with a chicken gizzard calcimedin antibody. (3) An antibody raised against the bovine aorta calcimedin also recognized the bovine liver calelectrin. (4) One-dimensional peptide maps of the two proteins revealed no significant difference. (5) The calcimedin appeared to have an amino acid composition essentially the same as that of the liver calelectrin. (6) The amino acid sequences of the calcimedin fragments were identical with those of the calelectrin fragments.

Amino Acid Sequence↗
PubMed1989

Identification and partial separation of three distinct 32-kDa calcium/phospholipid-regulated proteins from bovine spleen.

We have separated three distinct 32-kDa calcium/phospholipid-regulated proteins from bovine spleen, which we have designated as 32kDa-Ia, 32kDa-Ib and 32kDa-II. By one-dimensional peptide mapping and chromatographic behavior, the three proteins are distinct from each other. Gizzard 35kDa calcimedin antibody recognizes both 32kDa-Ia and 32kDa-II but not 32kDa-Ib. Anti-aorta endonexin II specifically reacts with 32kDa-II. Bovine lens endonexin antibody reacts with 32kDa-Ia and 32kDa-Ib, but not with 32kDa-II. These data suggest that the 32-kDa calcium/phospholipid-regulated proteins purified from various sources can be divided into three distinct classes of proteins.

Animals↗
PubMed1989

Anti-histone autoantibodies in ddY mice, an animal model for spontaneous IgA nephritis.

The ddY mice are known to develop spontaneous glomerulonephritis resembling human IgA nephritis after 40 weeks of age. A sharp rise of circulating polyclonal IgG and IgA is also observed at this stage. Since these overproduced immunoglobulins seem to be related to the development of murine glomerulopathy, antigen-antibody interactions between renal tissue proteins and serum immunoglobulins were analyzed by Western blotting in ddY mice before and after 40 weeks of age. Serum IgG at 50 weeks reacted with an 18-kDa renal tissue protein which was identified as histone H3, as well as with histone H1. Renal histones were extracted along with IgG from the murine kidney at 50 weeks in a high salt soluble fraction. Serial studies of anti-histone antibodies by enzyme-linked immunosorbent assay showed that IgG class antibodies markedly increased after 40 weeks of age. IgA class antibodies mildly increased after 56 weeks of age. Anti-DNA antibodies were not detected. These results demonstrate that ddY mice also develop mainly IgG class and partly IgA class anti-histone autoantibody after 40 weeks of age, and that histone-anti-histone complexes may contribute to the development of murine glomerulopathy. Although anti-histone antibodies have been reported in lupus mice, ddY mice differ from these mice in that no anti-DNA antibodies develop.

Animals↗
PubMed1989

Effect of selenite on glucocorticoid receptor.

Rat liver glucocorticoid receptor was partially purified and characterized for its hormone binding using selenite. Selenite at very low concentrations irreversibly inhibited the hormone binding. The concentration for half maximal inhibition was approximately 2.8 microM. The inhibition was restored by dithiothreitol. The receptor-hormone complex became considerably insensitive to the selenite inhibition. The receptor inhibited by selenite was eluted at the same position as the native receptor from DEAE ion exchange and gel filtration columns. The results suggest that at least four sulfhydryl groups are located in the hormone binding domain of the receptor making a cluster.

Animals↗
PubMed1989

Sequencing and high expression of aminopeptidase P gene from Escherichia coli HB101.

A plasmid pAPP1 with a 4 kbp insert at the PstI site of pBR322, encoding aminopeptidase P gene of Escherichia coli HB101 (Yoshimoto et al. (1988) J. Biochem. 104, 730-734), was subcloned into pUC18 and pUC19. The transformant of E. coli JM83 harboring pAPP4 with a 1.9 kbp fragment showed more than 50-fold higher enzyme activity than that of the host, after cultivation at 37 degrees C for 40 h in LB-medium containing ampicillin. When the gene DNA was inserted reversely in pAPP4, the enzyme productivity decreased markedly. The whole nucleotide sequence of the inserted fragment of plasmid pAPP4 was clarified by the dideoxy chain-terminating method. Within this sequence, the mature enzyme protein-encoding sequence was found to start just after an ATG codon, as judged by comparison with amino-terminal protein sequencing. Eleven bases upstream from the proposed initiation codon was an AGGAGA sequence which seemed to be a ribosome binding site. Thirty-four bases upstream from the proposed start codon was the 6-base sequence TACAAA, the so-called -10 region or Pribnow box. Further, the 6-base sequence TTTACT around 77 bases upstream from the start codon was deduced to be a putative -35 region consensus sequence. The inverted repeat at 1334 was tentatively assumed to be a terminator. The molecular weight of the enzyme was estimated to be 49,650 from the nucleotide sequence. The purified enzyme contained 0.2 gram atom of zinc per subunit. The enzyme activity was inhibited by EDTA and activated 5-fold by Mn2+.

Amino Acid Sequence↗
PubMed1989

Identical properties of aldosterone and corticosterone binders and their presence in rat brain and kidney.

The [3H]corticosterone binders from rat brain and kidney were characterized by binding affinity and chromatographies, and compared with the binders for [3H]aldosterone and [3H]triamicinolone acetonide. Corticosterone-binding globulin-like molecules at very high concentrations in crude extracts were completely eliminated by a DEAE-gel adsorption procedure. [3H]Aldosterone binder in the renal, DEAE-treated fraction was recovered in a single peak by gel-filtration chromatography and by ultracentrifugation in linear sucrose gradients, independent of hormone-binding and tungstate, a stabilizer of the binder. The Stokes' radius and sedimentation coefficient of the renal aldosterone binder were 6.6 nm and 9.3S, respectively, indicating an apparent molecular weight of 263,000. Corticosterone-preferring binder also existed in the DEAE-treated fraction. Both aldosterone and corticosterone binders were found in the brain and kidney preparations. Comparison among the binders showed identical values of Stokes' radius and elution pattern from DEAE-Toyopearl in a linear salt gradient regardless of the organ and the hormones. Scatchard analyses of [3H]aldosterone and [3H]corticosterone binding showed for each ligand only one group of high-affinity sites with the equivalent dissociation constants, 4-7 nM. The orders of steroids in competing for the two high-affinity sites were equivalent: corticosterone greater than or equal to aldosterone much greater than triamcinolone acetonide, and that for the triamcinolone acetonide binding was triamcinolone acetonide much greater than aldosterone greater than or equal to corticosterone. Hydroxyapatite column chromatography separated the aldosterone and corticosterone binders from the triamcinolone acetonide binder, but not the aldosterone binder from the corticosterone binder. It is concluded that aldosterone and corticosterone binders distinct from triamcinolone acetonide binder exist in rat brain and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Chemical modification of neutral protease from Bacillus subtilis var. amylosacchariticus with tetranitromethane: assignment of tyrosyl residues nitrated.

A neutral protease from Bacillus subtilis var. amylosacchariticus was modified with tetranitromethane (TNM) at pH 8.0 for 1 h at 25 degrees C, by which treatment the proteolytic activity toward casein was markedly reduced, whereas activity changes toward N-blocked peptide substrates were variable depending upon the substrate used. The modified enzyme was digested with a Staphylococcus aureus V8 protease at pH 7.9 and the resultant peptides were separated by HPLC. Two peptides which contain nitrotyrosyl residue(s) were purified. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153-159 of the neutral protease, and Tyr-158 was identified as PTH-nitrotyrosine. The other one was the amino-terminal peptide of residue Nos. 1-22, and Tyr-21 was shown to be nitrated. From a comparison with the active site structure of thermolysin, which is a zinc metalloprotease with a high sequence homology to B. subtilis neutral proteases, nitration of Tyr-158 was inferred to be closely related to the activity changes of the neutral protease from B. subtilis var. amylosacchariticus.

Amino Acid Sequence↗
PubMed1989

[Effectiveness of local delivery of ofloxacin using controlled-release strips (PT-01) in periodontal patients. Part 2. Successive delivery system and combination with scaling].

Four different sites were randomly selected in each periodontal patient and divided into four group: PT-01-treated site (A), PT-01 treatment combined with scaling (S + A), placebo-treated site (P) and placebo treatment combined with scaling (S + P). PT-01 and the placebo were applied to the periodontal pocket weekly on days 0 to 28, and clinical and microbiological evaluations were made. The results obtained were as follows: 1. Weekly changes in plaque index, plus discharge and mobility of the tooth were not observed in any group. 2. In gingival index, bleeding on probing, pocket depth and gingival crevicular fluid, significant improvement was found in A in comparison with P and in S + A in comparison with S + P. 3. In total number of subgingival bacteria and in the ratio of motile rods and spirochetes to the total number of bacteria, significant reduction was found in A in comparison with P and in S + A in comparison with S + P.

Delayed-Action Preparations↗
PubMed1989

High-yield purification of the complex-forming glycoprotein in urine from normal and abnormal subjects.

Ordinary methods for purification of complex-forming glycoprotein (protein HC) or alpha 1-microglobulin, a protein closely related to protein HC, require either large volumes of urine or special collection of urine specimens from patients with tubular proteinuria. Here we describe a fast, efficient procedure for isolating protein HC in high yield from urines from healthy and diseased subjects, with use of Cibacron blue, hydroxylapatite, and gel chromatography. Using this procedure, we also obtained a considerable amount of polymeric protein HC from the urine of a patient with chronic renal failure. The pattern of charge heterogeneity and immunoreactivity with anti-protein HC differed between the polymeric and monomeric forms of protein HC. We also observed a variability in charge heterogeneity of protein HC among patients with renal disorders. These results demonstrate that this purification method is useful for further studies to elucidate the biochemical properties of protein HC and its clinical significance in renal disorders.

Alpha-Globulins↗
PubMed1989

[Research in similarities between EEP34 and lipocortin].

EDTA-extractable protein (EEP) is a major extrinsic protein of lens fiber membrane. The EEP of calf lens separated three major bands by SDS-PAGE, and the molecular weights of these major bands were 34 kilodalton (kd.), 33 kd. and 32 kd. Combination of EEP with F-actin and some kinds of phospholipid containing liposomes depends on Ca2+. In two-dimensional gel electrophoresis, some spots of the calf-EEP partially overlapped with spots of lipocortin, which is a substrate for epidermal growth factor receptor/kinase and an inhibitor of phospholipase A2. The 34 kd. protein of the EEP cross-reacted to the antibody prepared against lipocortin of pig's aorta: These results suggest the important function of EEP in lens.

Animals↗
PubMed1989

[Biochemical and immunological properties of EEP 32 kd protein].

EDTA-extractable protein (EEP) is a major extrinsic protein of lens membrane. EEP is composed of two major polypeptides on SDS-PAGE with apparent molecular weights of 32 kilodaltons (kd) and 34 kd. Recently it has been reported that the 34 kd protein of the EEP cross-reacted with the antibody prepared against lipocortin, an inhibitor of phospholipase A2. We have purified and characterized the 32 kd protein of the EEP (EEP 32). Crude EEP of bovine lens was applied to a Q-Sepharose column and eluted with a linear gradient system composed of 0-0.7 M NaCl. The EEP 32 was discreted at salt concentrations of approximately 0.1-0.25 M (EEP 32-A) and 0.35-0.4 M (EEP 32-B). Both EEP 32-A and EEP 32-B interact with phospholipid and F-actin in a Ca2+-dependent manner as lipocortin. Furthermore, the EEP 32-A, a major component of the EEP 32 purified by hydroxyapatite chromatography and gel filtration, inhibited phospholipase A2 activity more than lipocortin. On Western blot analysis, the 32 kd lipocortin-like protein of human placenta showed cross-reactivity to EEP 32-B, but not EEP 32-A. These results suggest that EEP 32 contains two lipocortin-like proteins differing in antigenicity and isoelectric points.

Animals↗