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K Horiuchi

Publications and source records attributed to K Horiuchi.

At least 163 records · Page 9Linked to original sources
PubMed1990

Replication enhancer-independent mutation increases the co-operativity with which an initiator protein binds its origin.

The plus-strand replication origin of bacteriophage fl has a bipartite structure consisting of a required core origin region and an adjacent A + T-rich enhancer sequence that potentiates replication approximately 100-fold. The core origin binds the initiator protein (gpII) and the enhancer binds the Escherichia coli integration host factor (IHF). gpII binds the core origin in two steps, forming a binding intermediate (complex I) and a functional complex for nicking (complex II). We have used a double-label gel binding assay to determine the stoichiometry of the gpII-origin interaction. The results indicate that complex I contains two gpII molecules per origin, and complex II contains four gpII molecules per origin. Enhancer-independent mutations in gpII allow wild-type levels of replication in the absence of either the enhancer or IHF. We have examined the binding of an enhancer-independent gpII mutant (mp1) protein to the replication origin. The mp1 mutation in gpII (Met40----Ile) increases the co-operativity with which the protein binds to form complex II. In addition, the mutant gpII forms both complexes with a DNA fragment containing only two (beta-gamma) of the three repeats from the core origin sequence, while the wild-type protein forms only complex I with this fragment. We discuss how a mutation that increases the co-operativity of binding of an initiator protein might stimulate DNA replication.

Base Sequence↗
PubMed1990

Development and evaluation of the TD97 measles virus vaccine.

The TD97 strain vaccine virus was prepared from the Tanabe strain measles virus by low-temperature passages in primary cell cultures and ultraviolet (UV) mutagenesis. The TD97 strain exhibited the following characteristics: highly temperature sensitive, neither multiplying nor forming any plaques at 40 degrees C in Vero cells; genetically stable, maintaining high temperature sensitivity after ten successive passages in CE cells at 30 degrees C or 35 degrees C; and M proteins of this virus about 1 KD slower in mobility in SDS-PAGE than that of the Tanabe strain. The TD97 strain was further confirmed to be attenuated by an inoculation test into primate brain. In field trials, 752 healthy children were inoculated with a live virus vaccine prepared with this strain, and the following results were obtained: the seroconversion rate was 97% (517/533), and the average HI antibody titer was 2(5.2). An antibody-increasing effect was also observed in children who were initially seropositive. In children who seroconverted, the rates of fever were 15.7% (55/351) for 37.5 degrees C or higher and 4.0% (14/351) for 39 degrees C or higher. The rash rate was 7.7% (27/351), and the incidence of local reaction was 5.4% (19/351). The TD97 strain is thus considered to be suitable in use for an attenuated measles vaccine.

Animals↗
PubMed1990

Ligandin binding phthalein complexone complex of technetium for hepatic function studies.

In our pursuit for liver functional diagnosis, development of bifunctional radiopharmaceutical containing iminodiacetic acid (IDA), as the technetium chelating site along with phthalein or fluorescein structure, the skeleton of BSP and Rose Bengal, long used for the assessment of liver function is considered. Among the various PC, PPC, TPC and calcein IDA derivatives commercially available, 99mTc-PC (PC: 3,3'-bis(N, N-dicarboxymethylaminomethyl)o-cresolphthalein) showed the highest hepatobiliary excretion. The functionality of the various technetium labeled phthalein and fluorescein IDA derivatives was evaluated by competitive BSP binding studies and by comparative binding with the hepatocyte specific protein, ligandin.

Animals↗
PubMed1990

Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli.

The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.

Alkyl and Aryl Transferases↗
PubMed1990

Stable and lipophilic technetium-99m dithiosemicarbazone complexes with 5-6-5 membered chelate ring structure.

Modification of the chelate ring structure of technetium-99m (99mTc) dithiosemicarbazone (DTS) chelate was carried out in pursuit of a more stable and lipophilic compound. A new DTS chelating molecule, pentane-2,4-dione bis(N-methylthiosemicarbazone) (PETS), with a 5-6-5 membered chelate ring structure, was synthesized and labeled with 99mTc, PETS generated two 99mTc compounds as major products. Both had much higher stability and lipophilicity than a 5-5-5 membered 99mTc DTS compound, as well as great stability in plasma. Both 99mTc-PETS compounds were rapidly extracted by the brain and heart when injected into mice. Thus, the modified chelate ring structure afforded a preferable characteristics to DTS chelate as for the chelating site for technetium radiopharmaceuticals.

Animals↗
PubMed1990

[Nephrotoxicity of cefpirome sulfate in rabbits--single and multiple intravenous administration].

Nephrotoxic potential of cefpirome sulfate (CPR) was examined by single and multiple intravenous administrations to Japanese white male rabbits. In single administration studies, no nephrotoxic symptoms were observed at the dose level of 320 mg/kg of CPR or less. At the dose level of 500 mg/kg or more, nephrotoxic findings were noted in both CPR and cefazolin sodium(CEZ) groups, such as proteinuria, glucosuria, increase in serum level of urea nitrogen, creatinine and uric acid, and necrosis and calcification in the proximal tubular epithelium of kidney. Renal phenolsulfonphthalein(PSP) excretion was suppressed at the dose level of 500 mg/kg of CPR or more, and 2000 mg/kg of CEZ. In 14 and 21 days repeated administration studies, no nephrotoxic symptoms were observed at the dose level of 100 mg/kg of CPR and CEZ or less. At the dose level of 200 mg/kg of CPR or more, urinary and serum biochemical findings mentioned above were observed, and histopathological changes in the kidney described above were added in 400 mg/kg group. The similar nephrotoxic symptoms including histopathological changes of the kidney were observed in the groups of 100 mg/kg of cefaloridine(CER) and 200 mg/kg of CEZ. In addition, renal PSP excretion was suppressed in the group of 200 mg/kg of CEZ. The results would suggest that CPR is less nephrotoxic than CER and that the nephrotoxic potential of CPR is comparable to CEZ because CPR caused more severe renal failures in single administration study and less severe renal failures in multiple administration study than CEZ.

Animals↗
PubMed1990

[Clinical trial of inactivated hepatitis A vaccine].

A clinical trial was carried out for a lypophilized inactivated hepatitis A vaccine in order to confirm its safety and antibody response. The vaccine used for the study has been developed by Denka Institute of Biological Science (Lot. D-01), Chiba Serum Institute (Lot. C-01) and The Chemo-Sero-Therapeutic Research Institute (Lot. K-02). Thirty-six volunteers were selected and employed for the present study from healthy male adults carrying no antibody against hepatitis A virus (HAV). They were divided into three dose groups; 1.0 microgram, 0.5 microgram and 0.25 microgram viral protein respectively. Each groups were injected intramuscularly 3 times with 0, 1 and 6 months interval. As the results, though, slight side effects such as local pain were found in 16 cases out of 99 total injected, but no abnormal value was observed in the blood and urine examinations. On the other hand, the mean values of serum anti-HAV at 2 months after the first injection in 1.0 microgram, 0.5 microgram and 0.25 microgram dose-groups were 562 mIU/ml, 323 mIU/ml and 134 mIU/ml, respectively, and at the 7 months, 3630 mIU/ml, 1288 mIU/ml and 1000 mIU/ml, respectively. Namely, the high immunogenicity and safety of the vaccine were confirmed in this trail.

Adult↗
PubMed1990

Morphologic studies of sickle erythrocytes by image analysis.

An automated image analysis system was used to characterize the morphology of sickle cells under hypoxic conditions. Images observed by light microscopy were transferred to the image analysis system and were processed to binary (black and white) images, and were then analyzed by area, perimeter, and shape. A circular shape factor (CSF = 4 pi X[area]/[perimeter]2) was found to be useful for elucidating the degree of deformation, but it could not differentiate elongated sickle cells from maple leaf-shaped cells. By combining an elliptical shape factor (ESF = [short axis]/[long axis]) with CSF we could separate deoxygenated homozygous sickle red cells (SS cells) into several morphologically distinct groups, including non-sickled cells, maple leaf-shaped cells, and elongated sickle cells. Using this automated image analysis system, we studied morphologic changes of SS cells exposed to deoxygenation-oxygenation (d-o) cycles between PO2 of 0 and 100 mm Hg (one cycle = 12 minutes) at pHs of 6.9 and 7.4. We found that at both pHs the morphology of sickled cells after the first deoxygenation was predominantly maple leaf-shaped. The number of elongated sickled cells increased as the number of d-o cycles increased, indicating that SS cells changed from maple leaf morphology to classic elongated sickle shape during d-o cycles. Desickling occurred less during the oxygenation phase at pH 6.9 than at pH 7.4 and as the number of d-o cycles increased. These results suggest that during d-o cycles deoxyhemoglobin S fibers may align to form large bundles that do not depolymerize completely even at the arterial oxygen pressure.

Anemia, Sickle Cell↗
PubMed1989

Double-strand cleavage and strand joining by the replication initiator protein of filamentous phage f1.

The replication initiator protein (gene II protein (gpII] of bacteriophage f1 is a multifunctional protein that plays central roles in initiation and termination of phage DNA replication. It introduces a nick at a specific site on the (+)-strand of supercoiled replicative form DNA. The 3'-hydroxyl end of the nick serves as the primer for (+)-strand rolling-circle replication. Upon completion of a round of synthesis, gpII cleaves and circulaizes the displaced single strand. When Mn2+ is included in the buffer instead of Mg2+, gpII cleaves both strands. In this paper, we investigate the mechanism of the Mn2+-dependent double-strand cleavage activity of gpII. This reaction, unlike nicking in the presence of Mg2+, does not require superhelicity. The reaction proceeds in two kinetic steps: first nicking of the (+)-strand, and then cleavage of the (-)-strand. The nucleotide sequence requirement for nicking is reduced compared to that in the presence of Mg2+. The product of the double-strand cleavage has an unusual structure. The left end is a telomere-like hairpin since the (+)- and (-)-strands are joined, as demonstrated by base sequencing. The right end has a onebase 3'-overhang. This reaction probably reflects the cleavage-joining activity of gpII in the termination event.

Base Sequence↗
PubMed1989

Hemolysis by aliphatic alcohols and saponin measured by the coil planet centrifugation method.

Using the coil planet centrifugation method, the mechanism of hemolysis by alcohols and saponin was investigated. With this technique, erythrocytes are introduced into a gradient of hemolytic agents in saline, which is prepared in a long coiled polyethylene tube. The tube is centrifuged so that the cells move from a low to a high concentration of hemolytic agent. When the cells lyse, they release hemoglobin which remains stationary, and therefore hemolytic potency can be determined spectrophotometrically by the distance the cells move before lysing. We found that alcohols caused hemolysis at a particular concentration, whereas saponin-induced hemolysis was dependent on the amount of saponin accumulated in the environment of the cell. In addition, alcohols with longer carbon chains were more potent hemolytic agents than those with shorter chains, but each additional carbon group produced less of an increase in hemolysis per mole of alcohol. This chain-length dependency is consistent with a previous study on in vivo alcohol-induced hemolysis. The coil planet centrifugation method is also adaptable to comparative studies on the mechanism of other types of hemolysis, such as immune or drug-induced lysis, and to toxicological studies.

Alcohols↗
PubMed1989

Chitin gels.

Chitin dissolved in N,N-dimethylacetamide, N-methyl-2-pyrrolidone and their mixed solvents in the presence of 5% LiCl was treated with acetic anhydride-pyridine, and the mixture solution was heated at 100 degrees C for 6 h to give a partially O-acetylated chitin gel. Chitin dissolved in these solvents in the presence of 5% LiCl was mixed with pyridine, and the mixture solution was heated at 60 degrees C for 5 h to give a chitin gel. Both the gels were rigid and transparent, and their properties and the rate of the hydrolysis of the chitin xerogel by hen-egg white lysozyme were essentially similar to those of N-acetylchitosan gel prepared by chemical N-acetylation of chitosan.

Acetylation↗
PubMed1989

Design, synthesis and 64Cu labeling of fatty acid analogs containing dithiosemicarbazone chelate.

For the development of 62Cu labeled fatty acid analogs, two fatty acid analogs, containing dithiosemicarbazone (DTS) molecule as the 62Cu coordinating site, were designed and synthesized: a fatty acid analog containing DTS molecule at the omega-position, (a) the 12,13-dioxotetradecanoic acid di(N-methyl-thiosemicarbazone) (FA-DTS), and an omega-phenyl fatty acid analog containing DTS molecule at the para-position, (b) the p-carboxyundecylphenylglyoxal-di (N-methylthiosemicarbazone] (PFA-DTS). FA-DTS was synthesized by the reaction of ethyl diethoxyacetate with ethyl 11-bromonundecanate by successive decarboxylation and hydrolysis and final condensation with N-methylthiosemicarbazide. PFA-DTS was synthesized by the Friedel-Craft acylation of ethyl 11-phenylundecanate, selenium oxidation of the acetophenone derivative, followed by the condensation with N-methylthiosemicarbazide. Radiolabeling of FA-DTS and PFA-DTS with [64Cu]copper acetate was simple, rapid and quantitative. When injected into mice, both compounds were distributed and retained in the myocardium. These results offer a good basis for further development of 62Cu labeled fatty acid analogs.

Animals↗
PubMed1989

Structure and expression of mouse apolipoprotein E gene.

The mouse apolipoprotein E gene was isolated from a genomic library by screening with a cDNA probe. DNA including apolipoprotein E gene plus segments 2.5 kilobases upstream and 0.3 kilobase downstream of the coding region was transfected into NIH3T3 cells. The cells expressed the same-size apolipoprotein E mRNA and protein as those produced by mouse endogenously. The nucleotide sequence of the gene plus 5' and 3' flanking regions (one kilobase each) was determined. The sequence of the mouse apoliprotein E gene was highly homologous to that of the rat gene, not only in the coding regions but also in the non-coding and intron regions. The mouse and the human apolipoprotein E genes were homologous in the 5' proximal flanking region up to about 200 nucleotides as well as in the four exons. This proximal region was highly conserved for the genes of mouse, rat and human; the relative positions of the "TATA box" and the two copies of "GC box" were identical.

Animals↗
PubMed1989

[Subrenal capsule assay for chemosensitivity test of urinary tract cancers].

Subrenal capsule assay as a chemosensitivity test was performed on 8 renal cell carcinomas and 7 transitional cell carcinomas using athymic mice. Twelve of the 15 trials were considered suitable for evaluable assay. In 2 of the 3 non-evaluable trials the histological examination showed fibroblasts without cancer cells in the implants of the control group while the implants grew macroscopically. No host cell infiltration was observed microscopically in any implant of the control groups of evaluable assays. Mice were treated with several anticancer agents and sensitivities were evaluated by the tumor growth inhibition rate (TGIR) on the day 10 or 12 and by histological changes. No correspondence was observed between TGIR and histological changes. The characteristic histological finding of the treated groups of transitional cell carcinomas was cystic degeneration and it seemed to be a factor of the discordance between TGIR and the histological changes.

Animals↗