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Biomedical subjects

A Takeda

Publications and source records attributed to A Takeda.

At least 433 records · Page 24Linked to original sources
PubMed1989

Intensification of tumor affinity of 99mTc-DL-homocysteine by cooperative use of SH-containing compounds.

The enhancement of tumor-specific distribution was investigated on 99mTc-DL-homocysteine (99mTc-Hcy), a possible tumor-imaging agent. In vitro HPLC analysis showed that 99mTc-Hcy did not bind to nonmercaptalbumin but to mercaptalbumin in blood. When glutathione, homocysteine or cysteine, which converts albumin from the mercapto- to nonmercapto-form, was intravenously injected into 99mTc-Hcy predosed mice bearing Ehrlich solid tumor, the SH-containing compounds did not affect the radioactivity distribution in tumor, but decreased that in blood to less than half that of untreated animals. In vitro gel filtration analysis showed that glutathione released 99mTc-Hcy from albumin. Free 99mTc-Hcy produced in blood by the treatment was rapidly excreted into urine. These results suggest that the combination of 99mTc-Hcy and SH-containing compounds is useful for tumor-imaging.

Animals↗
PubMed1989

Studies on chemical synthesis of human cystatin A gene and its expression in Escherichia coli.

A synthetic gene containing the coding sequence for the human cysteine proteinase inhibitor, cystatin A, was obtained by enzymatic assembly of 20 oligodeoxyribonucleotides which had been chemically synthesized by the solid phase phosphoramidite method. It was cloned into an Escherichia coli plasmid. The expression plasmid for cystatin A was constructed by introducing the synthetic gene downstream of the tac promoter of an E. coli plasmid which is a derivative of pKK223-3 with high copy number. The gene was expressed in E. coli JM109 without IPTG-induction. The expression of cystatin A was detected by SDS-polyacrylamide gel electrophoresis of the E. coli JM109 lysate, followed by immunoblotting using rabbit antiserum raised with human epidermal cystatin A and alkaline phosphatase-conjugated goat anti-rabbit IgG. The result showed that the molecular weight of the expression product is identical with that of the authentic protein and the antigenic properties are also the same. Furthermore, the expression product purified with a CM-papain Sepharose affinity column and FPLC system with a Mono-Q column showed the same inhibitory activity for various cysteine proteinases. Also, purified recombinant cystatin A was found to have identical amino acid composition, NH2-terminal amino acid sequence, and peptide-map on reverse phase HPLC with those of the authentic inhibitor.

Base Sequence↗
PubMed1989

Comparative studies on the primary structure of human cystatin as from epidermis, liver, spleen, and leukocytes.

We have studied the primary structure of human cystatin As from epidermis, liver, spleen, and leukocytes. These molecules were indistinguishable on PAGE in the presence and absence of SDS, by fast protein liquid chromatography (FPLC) chromatofocusing on a Mono P column, and in amino acid composition. The NH2- and COOH-terminal amino acid sequences of human cystatin As from epidermis, liver, and spleen were identical with those of human leukocyte cystatin A previously reported except for the lack of the NH2-terminal methionine residue in human epidermal cystatin A. The peptides obtained upon digestion of four human cystatin As with Achromobacter protease I (AP) showed identical peptide maps on HPLC except for different retention times of the NH2-terminal peptides. Furthermore, the amino acid compositions of corresponding separated peptide quartets were identical. We also determined the complete amino acid sequence of human epidermal cystatin A by sequencing peptides obtained from AP digestion and cyanogen bromide (CNBr) cleavage. It consisted of 97 amino acid residues, and was identical with those of human cystatin As from liver, spleen, and leukocytes except for the lack of the NH2-terminal methionine residue.

Amino Acid Oxidoreductases↗
PubMed1989

Characterization of copper atoms in bilirubin oxidase by spectroscopic analyses.

Bilirubin oxidase [EC 1.3.3.5], purified from the culture medium of Myrothecium verrucaria, was found to contain two blue copper atoms per protein molecule with a molecular weight of ca. 52 kDa. The two copper atoms were estimated to be in the all cupric state by the cuproine colorimetric method and also atomic absorption analysis. We could remove the reduce cuprous ions from the holo enzyme by adding ascorbate, followed by a KCN solution, yielding an apo-enzyme with no activity. The apo-enzyme can be reconstituted with Cu or other divalent cations such as Co, Fe, and Cd, with accompanying recovery of the enzyme activity. The activity recovery depended upon the species of cation employed; Cu being most effective, an almost 100% recovery, and Cd the least, only a 25% recovery. We could obtain information on the copper ions and their coordination structure by spectroscopic analyses of the apo- and reconstituted enzymes, obtaining such as absorption, CD, MCD, and XPS spectra. The bilirubin oxidase catalyzed-reaction was a second order reaction with respect to copper bound with protein. The donor set was of the CuSS*N2 (S = Cys, S* = Met, N = His) type, i.e., the same as in the case of blue copper proteins. On studying the Co-substituted enzyme, it was revealed that the copper site of the enzyme had a 4-coordinated structure.

Apoproteins↗
PubMed1989

Phosphorylation of the 27-kDa gap junction protein by protein kinase C in vitro and in rat hepatocytes.

We previously demonstrated that the 27-kDa major component protein in rat liver gap junctions was phosphorylated by protein kinase C in vitro (Takeda, A. et al. (1987) FEBS Lett. 210, 169-172). In this study, we examined this further and examined the phosphorylation of the 27-kDa gap junction protein in rat hepatocytes by metabolically labeling cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. The in vitro phosphorylation was inhibited by monoclonal antibodies recognizing the carboxyl- (C-)terminal domain of the 27-kDa protein. Protease digestion analysis revealed that phosphorylation occurred at the C-terminal domain. In rat hepatocytes, the phorbol esters, 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-dibutyrate, stimulated the 27-kDa protein phosphorylation, whereas 4 alpha-phorbol-12,13-didecanoate did not. 1-Oleoyl-2-acetyl-sn-glycerol also stimulated the 27-kDa protein phosphorylation. In addition, norepinephrine stimulated the phosphorylation and pretreatment of hepatocytes with staurosporine, a potent inhibitor of protein kinase C, inhibited this stimulatory effect of norepinephrine. Both in vitro and in hepatocytes, analysis of chemical cleavage of the 27-kDa phosphoprotein revealed that phosphorylation occurred mainly at a 10-kDa fragment which the antibodies recognized. These results indicate that protein kinase C phosphorylates the 27-kDa gap junction protein, not only in vitro but also in hepatocytes, at the C-terminal domain of the protein.

Amino Acids↗
PubMed1989

Radiation protection by Shigoka extract on split-dose irradiation in mice.

Radioprotective effect of a Shigoka extract was studied on split-dose irradiation. Mice were exposed to 4 Gy X 4 or 3 Gy X 4 for examining survival ratio or hemogram, respectively. The extract was i.p. injected immediately after each irradiation. The extract significantly (p less than 0.001) increased the survival ratio, and it increased leukocyte count within the period of the death (days 2-12 after the final exposure). Cerebral hemorrhage after 4 Gy X 4 was diminished by the extract. Recovery of leukocytogenesis seems much more important for survival at split-dose irradiation than at single acute irradiation.

Animals↗
PubMed1989

Molecular analysis of the DNA segments cross-hybridizable to the tyrosinase gene in patients affected with oculocutaneous albinism.

The human tyrosinase gene is greater than 35 kb and is organized in four introns and five exons [Tomita et al. (1989) Biochem. Biophys. Res. Commun., 164, 990-996]. Using the full-length cDNA encoding human tyrosinase and its exon-specific fragments as hybridization probes, we show that overall structural organization of the tyrosinase gene is unchanged in three patients affected with tyrosinase-negative oculocutaneous albinism (OCA). Moreover, we are able to show the presence of additional DNA segments cross-hybridizable to exon 4 or exon 5 of the tyrosinase gene in the genome of three OCA patients and a healthy individual. Namely, the exon 4 specific probe detected two bands in the DNA digested with EcoRI or HindIII and the exon 5-specific probe detected two bands in the Bgl II-digested DNA, in spite of the facts that no recognition sites for these enzymes are present in each exon. We have then isolated two phage clones harboring the distinct DNA segments, hybridizable to the exon 5 probe. Southern blotting analysis of each cloned DNA digested with Bgl II showed two different hybridization patterns as those detected in genomic DNA digested with Bgl II, confirming that there are at least two DNA segments hybridizing to the exon 5 sequence in human genome.

Albinism↗
PubMed1989

[A case report of IgD myeloma with acute renal failure and deposition of lambda light chain along the tubular basement membrane].

A case of IgD lambda type multiple myeloma that developed acute renal failure was presented. Histopathologic studies revealed apparent myeloma cast nephropathy with typical giant cells and light chain deposition along tubular basement membrane and inter-peritubular interstitium. Glomerular deposits of light chain were absent by electron and immunofluorescent microgram. The presented case showed a very uncommon histopathologic findings of unusual deposits of light chain, to tubulo-interstitium except for glomeruli, in addition to vertically rare IgD myeloma. A sixty-year old female who was suffered from acute renal failure and treated by hemodialysis for one month was admitted to our institute. Physical examinations revealed ill-apparent and emaciated uremic patient on admission. Laboratory and radiological studies demonstrated IgD lambda type monoclonal protein and numerous punched out appearance of skull bone. The diagnosis of IgD lambda myeloma was confirmed with aspiration bone marrow test. Anti-myeloma therapy of melphalan and prednisolone have started, however, fatal arrhythmia suddenly suffered the patient and resuscitation attempts failed to get any success one week after admission. Renal necropsy was performed and specimens were studied by light, immunofluorescent and electron microscope.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Kidney Injury↗
PubMed1989

Identification of irradiated pepper with the level of hydrogen gas as a probe.

A novel method to detect whether or not a particular pepper has been irradiated has been developed which is based on the fact that H2 is formed in organic substances irradiated with ionizing radiation. Following gamma irradiation, black and white peppers were ground to powder in a gastight ceramic mill. By gas-chromatographic analysis of the gas in the mill, we observed that H2 had been released from the irradiated pepper grains. Curves plotting the H2 content vs storage time at storage temperatures of 7, 22, and 30 degrees C showed that the higher the temperatures, the smaller the H2 content, and that identification of irradiated pepper was possible for 2-4 months after 10 kGy irradiation.

Chromatography, Gas↗
PubMed1989

[Loss of spinal anterior horn cells in X-linked recessive bulbospinal muscular atrophy--a morphometric study of lower motoneuron loss].

A morphometric analysis was performed to study three-dimensional distribution of the anterior horn cells in the L1 segmens from four cases of X-linked bulbospinal muscular atrophy (X-BSMA) and three age-matched controls. At autopsy, the L4 spinal segment was removed and fixed in 4% paraformaldehyde in PBS at pH 7.4, then embedded in paraffin. Serial sections of 10 microns in thickness from the rostral end of L4 segment were obtained. Every tenth section was stained with Klüver-Barrera method. The anterior spinal horn in this study was designated as the gray matter anterior to the line from the central spinal canal perpendicular to the ventral spinal fissure. The diameter of the remaining neurons with obvious nucleolus in the anterior horn was measured with TGZ-3 particle size analyzer (Zeiss) on the 205 time-magnified picture, and their location was schematically plotted on a montage of the ventral horn. Neuronal loss was more marked in X-BSMA than in amyotrophic lateral sclerosis (ALS) and Shy-Drager syndrome (SDS). In X-BSMA, the loss was most prominent in cells with large-size, and in those located in the lateral and medial vental nuclei (lamina IX after Rexed's classification). Small or intermediate cells located in the area of inner-medial portion of the ventral horn (lamina VII & VIII after Rexed's classification) were also significantly depleted. In some cases, the loss of small neuron was more marked than SDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1988

Antibody-enhanced infection by HIV-1 via Fc receptor-mediated entry.

Monocytes and macrophages, which may play a central role in the pathogenesis of infection with human immunodeficiency virus type 1 (HIV-1), express the CD4 molecule and Fc receptors (FcR) for immunoglobulin G (IgG). To explore the possibility that FcR mediate HIV-1 infection of monocytes, studies were conducted with the human monocytic cell line U937. These cells were exposed to HIV-1 complexed with various concentrations of serum from HIV-1 antibody-positive individuals and monitored for HIV-1 replication. Serum samples from antibody-negative normal individuals did not affect virus yields. High concentrations of antibody-positive sera showed virus-neutralizing activity; however, cells infected with HIV-1 in the presence of antibody-positive sera at subneutralizing concentrations significantly enhanced virus replication. This infection enhancement was blocked by heat-aggregated gamma-globulin. Moreover, the IgG fraction from an HIV-1 antibody-positive serum enhanced HIV-1 infection at the same serum dilution equivalents. In contrast, IgG-F(ab')2 did not enhance HIV-1 infection but showed neutralizing activity with HIV-1. These results are compatible with the concept of FcR-mediated infection enhancement and suggest that this immunological response to HIV-1, instead of protecting the host, potentially facilitates the infection.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Chemical properties of technetium-99m-DL-homocysteine, a possible tumor-imaging agent.

The chemical properties of 99mTc-DL-homocysteine (99mTc-Hcy) showing high accumulation in several experimental tumors were investigated. The form of tumor-tropic 99mTc-Hcy was a polymeric complex which appeared at void volume on Sephadex G-15 by eluting with 5 mM Hcy. This complex changed into smaller complexes of ca. 600 molecular weight in the presence of 150 mM NaCl and 5 mM Hcy, suggesting that 99mTc-Hcy was a complex composed of smaller polymers which are weakly bound together by an ionic bond. The complex showed a negative charge. The Hcy/Tc molar ratio in the complex was approximately 2 and no Sn was detected.

Animals↗
PubMed1988

Chemical modifications of histidyl and tyrosyl residues of inorganic pyrophosphatase from Escherichia coli.

Chemical modifications by photooxidation in the presence of rose bengal (RB) and with tetranitromethane (TNM) were carried out to elucidate the amino acid residues involved in the active site of inorganic pyrophosphatase (pyrophosphate phosphohydrolase) [EC 3.6.1.1] from Escherichia coli Q13. The photooxidation caused almost complete inactivation, which followed pseudo-first-order kinetics depending on pH and concentration of RB. The presence of Mg2+ or complex between Mg2+ and substrate or substrate analogues, imidodiphosphate and sodium methylenediphosphate, gave partial protection against the photoinactivation, whereas the substrate alone showed no protective effect. The enzyme was almost completely inactivated by chemical modification with TNM, depending upon the concentration of TNM. The amino acid analyses and enzyme activity measurements revealed that 2 histidyl residues among 5 photooxidized residues and 2 tyrosyl residues per subunit were essential for the enzyme activity. The circular dichroism (CD) spectra in the far ultraviolet region showed no significant alteration during these two modifications, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modifications altered considerably the CD bands in the near ultraviolet region and the fluorescence spectra, indicating that subtle change in conformation had occurred in the vicinity of the active site in the enzyme molecule. These results strongly suggest that histidyl and tyrosyl residues may be involved in the active site or be located in the vicinity of the active site and seem to participate in the mechanism of stability against heat inactivation.

Binding Sites↗
PubMed1988

Monoclonal antibodies recognizing different epitopes of the 27-kDa gap junction protein from rat liver.

Monoclonal antibodies (2-3E2, 6-3G11, and 7-3H6) against gap junction plaques purified from rat liver were prepared and characterized. Immunoblot analysis of liver gap junctions revealed that all three antibodies reacted with the 27-kDa protein, but not with the 22-kDa one. The 2-3E2 and 6-3G11 antibodies both reacted with the 27-kDa protein in gap junctions purified from livers of the rat, mouse, rabbit, and guinea pig; the 7-3H6 antibody, however, failed to react with the 27-kDa protein from guinea pig liver. The 7-3H6 antibody reacted strongly with the 24- to 26-kDa degradation products of the 27-kDa protein. Indirect immunofluorescence showed that the 6-3G11 and 7-3H6 antibodies both gave the same specific fluorescence labeling on rat liver cryosections, suggesting that these two antibodies recognized the cytoplasmic sites of the 27-kDa protein. Immunoblot analysis of protease-digested fragments from the 27-kDa protein revealed that the 7-3H6 antibody reacted with the 24- and 17-kDa fragments (including portions of the carboxyl-terminal domain of the 27-kDa protein) produced with endoproteinases Arg-C and Lys-C, respectively. Immunoblot analysis of CNBr fragments of the 27-kDa protein revealed that all three antibodies reacted with the 10-kDa fragment, which is thought to be the carboxyl-terminal domain of the 27-kDa protein. These results demonstrate that three monoclonal antibodies recognize different epitopes of the cytoplasmic sites (probably the carboxyl-terminal domain) of the 27-kDa liver gap junction protein.

Animals↗