Search PubMed⌕ Search

Biomedical subjects

G Kikuchi

Publications and source records attributed to G Kikuchi.

At least 19 recordsLinked to original sources
PubMed1998

Biosafety monitoring of patients receiving intracerebral injections of murine retroviral vector producer cells.

Patients with recurrent malignant brain cancer, who were receiving gene therapy by intracerebral injection of murine retroviral vector producer cells (VPCs), were monitored for the presence of replication-competent retrovirus (RCR). RCR sequences were not detected by polymerase chain reaction (PCR) in any of the 608 peripheral blood leukocyte (PBL) samples analyzed. Vector DNA sequences were detected transiently in PBL samples from a subset of 34 patients. Humoral immune responses to a retroviral core protein p30 and murine VPC were detected in some patients, most frequently in patients receiving repeated administrations of VPC. RCR was not detected in biological assays of PBLs from 41 patients who had either anti-retroviral antibodies in sera and/or vector DNA in PBLs. Our data suggest that in situ generation of RCR was not detected following intracerebral inoculation of VPCs in any of the 128 patients evaluated.

Animals↗
PubMed1993

Multiple cis-acting elements are required for proper transcription of the mouse V delta 1 T cell receptor promoter.

To gain insight into the developmentally regulated expression of the mouse TCR V delta-gene segments, we have investigated the role of the 5' promoter region of the V delta 1-gene. Transient transfection assays showed that a construct encompassing 267 nucleotides upstream from the mapped transcriptional start site was capable of driving promoter activity when transfected into V delta 1+ T cells. The inclusion of an additional 459-bp 5' segment to this construct did not affect promoter activity. However, a deletion of 222 5' nucleotides from the same construct dramatically decreased promoter activity. In vivo genomic footprinting localized several protein-DNA interactions to the stretch of DNA shown to have transcriptional activity. A computer analysis revealed that the segments of DNA participating in these protein-DNA interactions were identical to the previously described cyclic AMP response element (CRE), E box, and leukemia virus E26 cis-acting elements. Transient transfection assays performed with -267 bp constructs containing mutations at each of the localized cis-acting elements revealed that the CRE, E box, and Ets elements work together in driving promoter activity and that the CRE and Ets elements are the most important for driving transcription. Gel mobility shift analyses showed that each of these cis-acting elements is capable of binding specific nuclear factors present in V delta 1-expressing cells. These data indicate that multiple transcription factors acting in concert are responsible for V delta 1 gene expression.

Animals↗
PubMed1992

Mouse autoreactive gamma/delta T cells. I. Functional properties of autoreactive T cell hybridomas.

A minor population of dendritic epidermal T cells (DETC) express the V gamma 1.1C gamma 4V delta 6 T cell receptor and T cell clones and hybridomas derived from this subset constitutively secrete cytokines in culture secondary to recognition of an autoantigen. Activation of these autoreactive cells requires the use of the vitronection receptor (VNR) as an accessory molecule which interacts with the Arg-Gly-Asp-Ser (RGDS) sequence of extracellular matrix (ECM) proteins. We have compared the functional properties of C gamma 4+ hybridomas derived from newborn thymocytes and from adult spleen with the DETC hybridomas/lines in terms of their ability to secrete cytokines spontaneously and for the use of the VNR as an accessory molecule. Almost all the C gamma 4+ thymocyte hybridomas secreted cytokines spontaneously and in the majority of lines the most prominent cytokine secreted was granulocyte-monocyte colony-stimulating factor. In contrast, none of the four splenic C gamma 4+ hybridomas secreted cytokines spontaneously although all were capable of cytokine production following activation via the T cell receptor. Although the thymocyte hybridomas did not grow as adherent cell lines in culture, constitutive cytokine production required engagement of the VNR by its ligand in ECM proteins. In all cases, cytokine production could be inhibited by an anti-VNR monoclonal antibody as well as by soluble RGDS. The strong correlation of functional and molecular properties between thymocyte C gamma 4+ hybridomas and C gamma 4+ DETC suggests that the C gamma 4+ DETC may be of thymic origin and that cells with potential for autoreactivity residing in the thymus at birth may populate other peripheral locations in the mouse. The data also support the concept that the VNR, and possibly other integrins, play a role as accessory elements for autoreactive cells and may be essential for the regulation of such activity.

Animals↗
PubMed1988

Deoxyuridine triphosphate nucleotidohydrolase: distribution of the enzyme in various rat tissues.

The distribution of deoxyuridine triphosphate nucleotidohydrolase (dUTPase) [EC 3.6.1.23] in the cytosol of various rat tissues was investigated by measuring the enzyme activity and by immunochemical analyses. Among nine rat tissues, thymus, and spleen showed the highest activities of the enzyme per gram of tissue, while intestine, stomach, lung and liver showed very low levels. Rabbit antibodies directed against purified dUTPase of anemic rat spleen showed reactivity with partially purified dUTPases from other rat tissues such as thymus, testis, or regenerating liver. Immunotitration and immunoblot experiments also indicated that the dUTPases in various rat tissues had very similar antigenicity and apparently the same subunit molecular size (Mr = 19,500), suggesting that the enzyme lacks organ-specificity. Immunoblot analysis of dUTPase protein with crude extracts from various rat tissues showed a similar distribution to that of the enzyme activity. No immuno-reactive band corresponding to the dUTPase was detected in intestine, although intestinal mucosa has been recognized as an actively proliferating tissue.

Anemia↗
PubMed1987

The hyperosmolarity-induced response of the ocular standing potential in mature rabbits.

The hyperosmolarity-induced response of the ocular standing potential (SP) provides a method of testing the function of the retinal pigment epithelium (RPE) without using light stimulation. In this study, the changes in potential level occurring after a short-term intravenous injection of 10 ml of 20% mannitol were determined by means of a direct current amplifier for the following groups: Group 1, normal rabbit eyes; Group 2, rabbit eyes in which the RPE was damaged by sodium iodate; Group 3, rabbit eyes in which the photoreceptors were damaged by monoiodoacetic acid; Group 4, rabbit eyes with uveoretinitis experimentally induced by Arthus-type inflammation. The following results were obtained: 1. The hyperosmolarity-induced SP response consisted of a transient increase in potential level (positive wave) in Group 1. 2. For Group 2 a transient decrease in potential level (negative wave) was obtained, i.e., a reversal of the normal positive response to negative wave. 3. A positive wave and no reversal was found for Group 3. 4. For Group 4 a negative wave and a reversal of the normal response was obtained. These hyperosmolarity-induced SP responses provide additional information concerning the possibilities of the method for studying the function of the RPE.

Aging↗
PubMed1987

Deoxyuridine triphosphate nucleotidohydrolase activity and its correlation with multiplication of erythroid cells in rat spleen.

The administration of phenylhydrazine to rats brought about a marked increase in the dUTPase activity in the cytosol fractions of spleen and red blood cells; the activity began to increase with a two-day lag and reached the maximum at the 5th or 6th day of the phenylhydrazine treatment (13 and 5 times the control values in total activity in the spleen and red blood cells, respectively), and then the activity decreased. The activities of thymidine kinase and sigma-aminolevulinate synthase in the spleen and red blood cells also changed in parallel with that of dUTPase. The increases of these activities were suppressed completely by methotrexate, an inhibitor of DNA synthesis. The time courses of the enzyme activity changes in the red blood cells, however, were slightly behind those in the spleen. Thus, a close correlation was assumed between the dUTPase activity and the multiplication of erythroid cells in rat spleen.

Animals↗
PubMed1986

An immunochemical study of delta-aminolevulinate synthase and delta-aminolevulinate dehydratase in liver and erythroid cells of rat.

The relationship between erythroid delta-aminolevulinate (ALA) synthase and hepatic ALA synthase in rat was analyzed immunochemically, using antibodies directed against rat liver ALA synthase and against chicken liver ALA synthase. Rat erythroid ALA synthase showed no cross-reactivity with anti-liver ALA synthase antibodies, but hepatic ALA synthases from rat, mouse, and chicken share substantial cross-reactivity with one another. These results clearly distinguish the isozyme relationship between erythroid ALA synthase and hepatic ALA synthase in rat and suggest that there may be at least two different ALA synthase genes in rat. ALA dehydratase in rat liver, on the other hand, could not be immunochemically distinguished from ALA dehydratase in rat erythroid cells when antibody against rat erythroid ALA dehydratase was used. The finding that erythroid ALA synthase is an entity different from hepatic ALA synthase may provide a clue to understanding the different features in hepatic and erythropoietic porphyrias.

5-Aminolevulinate Synthetase↗
PubMed1984

Identification of a high affinity growth hormone receptor in rat adipocyte membranes.

Affinity cross-linking techniques have been used to identify a growth hormone (GH) receptor in rat adipocyte membranes. Adipocytes were incubated with 125I-human GH (125I-hGH) for 2 h at 37 degrees C and washed once to remove unbound hGH. The bivalent cross-linking reagent disuccinimidyl suberate (0.4 mM) was added to the cells for 15 min at 15 degrees C. A plasma membrane-enriched fraction was prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The autoradiographs of gels containing samples treated with reductant revealed an intense band of apparent Mr = 134,000 which was not present when cells were incubated with an excess of hGH or bovine GH. In contrast, the intensity of the Mr = 134,000 band was not altered by the presence of a similar excess of insulin or rat prolactin during the binding step. Multiple lower molecular weight species displaying the same hormone sensitivity as the Mr = 134,000 species were also present but at much lower levels. In the absence of reductant, the affinity-labeled GH receptor migrated as a broad band of Mr = 116,000-125,000 and as a less intense band of Mr = 230,000. At low reductant concentrations, both of the hGH-labeled complexes exhibited larger apparent molecular weights (Mr (X 10(3) ) = 135 and 270), indicating the presence of intrachain disulfide bonds. At higher reductant concentrations, the Mr = 270,000 species disappeared as the Mr = 134,000 band increased in intensity. Use of a cleavable cross-linking reagent, ethylene glycol bis(succinimidyl succinate), in conjunction with two-dimensional gel electrophoresis, revealed that the Mr = 134,000 complex is composed of iodinated monomeric hGH (Mr = 22,000) bound to a membrane protein. The molecular weight of the reduced hGH receptor protein itself was calculated to be 112,000, assuming a 1:1 stoichiometry of hormone to receptor.

Adipose Tissue↗
PubMed1984

Relation of the extra-sequence of the precursor form of chicken liver delta-aminolevulinate synthase to its quaternary structure and catalytic properties.

Precursor and mature forms of delta-aminolevulinate (ALA) synthase were purified to near homogeneity from chicken liver mitochondria and cytosol, respectively, and their properties were compared. The enzyme purified from mitochondria had apparently the same subunit molecular weight (65,000) as that of the native mitochondrial enzyme. The enzyme purified from the cytosol fraction, however, showed a subunit molecular weight of about 71,000 which was somewhat smaller than that estimated for the native cytosolic enzyme (73,000). The enzyme purified from liver cytosol seems to have been partially degraded by some endogenous protease during the purification, but may have the major part of the signal sequence. On sucrose density gradient centrifugation, the purified mitochondrial and cytosolic ALA synthases showed an apparent molecular weight of about 140,000, indicating that both enzymes exist in a dimeric form. The ALA synthase synthesized in vitro was also shown to exist as a dimer. Apparently the extra-sequence does not interfere with the formation of dimeric form of the enzyme. The purified cytosolic ALA synthase had a specific activity comparable to that of the purified mitochondrial enzyme. Kinetic properties of the two enzymes, such as the pH optimum and the apparent Km values for glycine and succinyl-CoA, were quite similar. The extra-sequence does not appear to affect the catalytic properties of ALA synthase. The isoelectric point of the cytosolic ALA synthase was 7.5, whereas that of the mitochondrial enzyme was 7.1. This suggests that the extra-sequence in the cytosolic enzyme may be relatively rich in basic amino acids.

5-Aminolevulinate Synthetase↗
PubMed1983

Inhibition by hemin of in vitro translocation of chicken liver delta-aminolevulinate synthase into mitochondria.

The precursor form of chicken liver delta-aminolevulinate synthase was synthesized in a reticulocyte lysate cell-free translation system and then incubated with the homologous liver mitochondria. The precursor enzyme was incorporated into the mitochondria with an attendant processing to the mature enzyme. In this in vitro experimental system, both the transport and processing of the enzyme were significantly inhibited by the addition of hemin as low as about 3 microM. This provides further support to the view, which had been derived from the studies in vivo, that inhibition by hemin of the translocation of delta-aminolevulinate synthase into mitochondria could be one of the regulatory mechanisms for heme biosynthesis in the liver cells.

5-Aminolevulinate Synthetase↗
PubMed1983

Translational inhibition by heme of the synthesis of hepatic delta-aminolevulinate synthase in a cell-free system.

Synthesis of delta-aminolevulinate synthase in a rabbit reticulocyte lysate system directed by total polysomes from the liver of allylisopropylacetamide-treated rats was studied with the combined use of [3H]leucine and a specific rabbit antibody. The protein synthesis observed in the cell-free system employed represented mainly the peptide chain elongation and its termination rather than the net synthesis involving initiation. Synthesis of delta-aminolevulinate synthase in this cell-free system was inhibited progressively with the increased addition of hemin; the synthesis was reduced to about 40% by about 30 microM hemin. Synthesis of total protein, however, was not significantly affected by the addition of hemin. The data obtained suggest that heme inhibits a peptide chain elongation step in the synthesis of delta-aminolevulinate synthase.

5-Aminolevulinate Synthetase↗
PubMed1983

delta-Aminolevulinate synthase isozymes in the liver and erythroid cells of chicken.

Antibodies raised against the purified chicken liver delta-aminolevulinate synthase showed a partial cross-reactivity with the chicken erythroid delta-aminolevulinate synthase. delta-Aminolevulinate synthase synthesized in vitro using polysomes from erythroid cells showed a subunit molecular weight of 55,000, whereas the enzyme synthesized in vitro using liver polysomes had a subunit molecular weight of 73,000. delta-Aminolevulinate synthase isolated from mitochondria of erythroid cells showed a molecular weight of 53,000, while the enzyme in liver mitochondria had a value of 65,000. These observations imply that the erythroid delta-aminolevulinate synthase differs from the hepatic enzyme.

5-Aminolevulinate Synthetase↗
PubMed1983

Induction of heme oxygenase in rat liver. Increase of the specific mRNA by treatment with various chemicals and immunological identity of the enzymes in various tissues as well as the induced enzymes.

A specific antibody was prepared against rat liver heme oxygenase which had been induced by bromobenzene treatment. Immunochemical studies with this antibody (IgG) revealed that heme oxygenases from livers of rats treated with hemin, Cd2+, Co2+, or bromobenzene from rat spleen and also from kidney of Sn2+-treated rats were all immunochemically identical. Cell-free synthesis of heme oxygenase was performed in a rabbit reticulocyte lysate system using polysomes isolated from livers of rats treated with either hemin, Cd2+, or bromobenzene, and it was found that translatable mRNA specific for heme oxygenase was actually increased in the liver of rats treated with any of those inducers. Also, the ability of liver polysomes to direct cell-free synthesis of heme oxygenase was apparently proportional to the activity of heme oxygenase in the liver from which polysomes were prepared. The heme oxygenase protein synthesized either in vivo or in vitro showed a molecular weight of 31,000 when examined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. This value is essentially identical with the molecular weight of heme oxygenase purified from rat liver and indicates that a precursor form of heme oxygenase may not be involved in the heme oxygenase synthesis.

Animals↗
PubMed1983

A stoichiometric study of heme degradation catalyzed by the reconstituted heme oxygenase system with special consideration of the production of hydrogen peroxide during the reaction.

In heme degradation catalyzed by the reconstituted heme oxygenase system, 8 to 9 mol of dioxygen and 11 to 12 mol of NADPH were consumed per mol of hemin lost, and about half the amount of dioxygen consumed could be accounted for by the production of hydrogen peroxide, which accumulated in the reaction mixture. Production of hydrogen peroxide in the heme oxygenase reaction did not appear to be due to the bimolecular dismutation of superoxide anions but rather seemed to be due to dissociation of a "peroxo" species formed on heme or intermediates of heme degradation. The hydrogen peroxide produced appeared to cause a considerable degree of non-specific degradation of heme (not leading to the formation of biliverdin) and also caused an inactivation of heme oxygenase. By taking into account the amount of dioxygen incorporated into hydrogen peroxide and some other factors, it could be deduced that 3 mol of dioxygen is consumed for the formation of 1 mol of biliverdin in the heme oxygenase reaction.

Animals↗
PubMed1983

Nonketotic hyperglycinemia: two patients with primary defects of P-protein and T-protein, respectively, in the glycine cleavage system.

The glycine cleavage system was investigated in the livers and brains of two patients with typical nonketotic hyperglycinemia who died in the neonatal period. The overall activity of the glycine cleavage system was found to be extremely low in both the liver and brain of each patient. In one patient, the disturbance of the glycine cleavage system was due to absence of activity of the P-protein. Immunochemical analysis indicated that this resulted from an absence of the enzyme protein. In the other patient, the activity of the T-protein was undetectable in the brain and was extremely low in the liver. Clinically classic nonketotic hyperglycinemia resulted from molecular defects in two different protein components of the glycine cleavage system.

Amino Acid Metabolism, Inborn Errors↗