Biomedical subjects

T Uchida

Publications and source records attributed to T Uchida.

At least 847 records · Page 47Linked to original sources

PubMed1990

Effect of total jejunoileal denervation on fat-stimulated release of peptide YY and cholecystokinin.

We have investigated the effect of total denervation of the jejunoileum (JDNv) on stimulated release of peptide YY (PYY) and cholecystokinin-33/39 in five dogs prepared with chronic gastric and duodenal cannulas. JDNv was performed by stripping the adventitia from the superior mesenteric artery and vein, transecting the small bowel mesentery, and division (with reanastomosis) of the small bowel at the ligament of Treitz and ileocecal junctio. Introduodenal corn oil (3 ml/kg/hr) was given before JDNv and 1 and 2 months after JDNv. Intravenous bombesin (400 pmol/kg/hr) was given (on nonconsecutive days) before JDNv and 1 month after JDNv. Plasma PYY and cholecystokinin levels were measured by specific radioimmunoassay. Release of PYY was enhanced after JDNv. The integrated release of PYY (ng.[0 to 60 min]/ml) after intraduodenal corn oil was as follows: before JDNv, 4.1 +/- 1.2; 1 month after JDNv, 16.0 +/- 2.7; and 2 months after JDNv, 10.3 +/- 2.2. Similar results were noted with intravenous bombesin (3.7 +/- 0.9 [before JDNv] vs 12.0 +/- 0.7 [1 month after JDNv]). Corn oil-stimulated release of cholecystokinin was abolished after JDNv (before JDNv 2.2 +/- 1.1; 1 month after JDNv, 0.6 +/- 0.3; and 2 months after JDNv, 0.4 +/- 0.6). Basal plasma levels of PYY and cholecystokinin were not affected by JDNv. We conclude that JDNv enhances PYY and abolished cholecystokinin release, which provides evidence for different mechanisms of neural control.

Animals↗

PubMed1990

[Antitumor activity of a novel analog of cytarabine, 4-amino-1-beta-D-arabinofuranosyl-2(1H)-pyrimidinone 5'-(sodium octadecyl phosphate) monohydrate (YNK01)].

4-Amino-1-beta-D-arabinofuranosyl-2(1H)-pyrimidinone 5'-(sodium octadecyl phosphate) monohydrate (YNK01) was an orally active depot form of 1-beta-D-arabinofuranosylcytosine (Ara-C). In the present study, antitumor activity of YNK01 was compared with it of Ara-C in vitro and in vivo. The activity of a main metabolite of YNK01, 5'-carboxypropylphosphate of Ara-C (C-C3PCA), was also studied. Growth inhibitory activity of YNK01 against various cultured tumor cells was 1/32-1/1,100 of that of Ara-C. YNK01 exhibited antitumor activity against L1210 leukemia in mice after i.v., i.p. or p.o. administration. The activity did not depend on the administration routes. Compared with Ara-C, the activity was comparable in both i.v. and i.p. administrations, but greater in p.o. administration. Oral administration of YNK01 showed similar antitumor spectrum to i.p. administration of Ara-C. Oral activity of YNK01 against L1210 leukemia did not depend on the administration schedules but depended on a total administration dose. In contrast, activity of Ara-C greatly depended on the schedules, and the frequent i.p, administration showed greatest activity. Growth inhibitory activity of C-C3PCA against cultured tumor cells was 1/2-1/7 of Ara-C. The metabolite exhibited activity against L1210 leukemia in mice after i.p. administration. These results suggest that YNK01 is a clinically useful drug with p.o. administration for cancers as well as Ara-C.

Administration, Oral↗

PubMed1989

Simplified cosmid vectors for gene transfer to cultured mammalian cells: isolation of the gene for elongation factor 2 from the mouse.

We constructed a series of cosmid vectors that carry two tandemly arranged lambda cos and mammalian selective markers. We achieved cloning efficiencies of 1-3 x 10(7) and greater than 10(6) colony-forming units per microgram of insert, using a cloned 42-kb BamHI fragment and Sau3AI fragments of 40-50 kb from mouse genomic DNA, respectively. The modified Ca.phosphate coprecipitation method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607-616] considerably improved the efficiency of gene transfer of cosmids into cultured mammalian cells: when genes encoding thymidine kinase from herpes simplex virus type 1 and aminoglycoside 3'-phosphoribosyltransferase from Tn5 were selected, the efficiencies of gene transfer into mouse L cells were about 10(-6). The mouse genome contains one copy of the functional gene for elongation factor 2 (EF2) per haploid genome and multiple copies of the EF2-related gene. We isolated a cosmid that carried functional full-length mouse EF2 from a cosmid library of L-cell genomic DNA, by colony hybridization and subsequent gene transfer of candidate cosmids into human 143B cells.

Animals↗

PubMed1989

Intrinsic properties of reverse transcriptase in reverse transcription. Associated RNase H is essentially regarded as an endonuclease.

The intrinsic properties of reverse transcriptase in reverse transcription were studied using a synthetic, partial ovalbumin mRNA with a synthetic DNA oligonucleotide annealed to the 3'-end of the RNA as a model substrate. With or without concomitant cDNA synthesis, the RNase H activity of avian myeloblastosis virus (AMV)-reverse transcriptase cleaved the substrate at a site which would leave a hybrid of between 7 and 14 base pairs between the 3' termini of the RNA and DNA oligonucleotide. Variability in the exact size of the hybrid probably reflects some weak base preference for cleavage by the enzyme. These short hybrids can be recognized as substrates by Escherichia coli RNase H and can be utilized by reverse transcriptase as sites for continuation of cDNA synthesis. Substrates with 5'-triphosphorylated termini, 3'-OH, 3'-phosphate, 3'-end hairpin structures and 20 base pair hybrids on the middle region of long RNA more than 300 bases or on circular RNA were all cleaved by AMV-reverse transcriptase-associated RNase H, indicating that the RNase H activity is essentially regarded as an endonuclease degrading RNA moiety in RNA-DNA hybrid. The modes of action of reverse transcriptase from murine leukemia virus and Rous-associated virus 2 were the same as that of AMV-reverse transcriptase, except that the size of the remaining hybrid and the specificity for cleavage depended on the reverse transcriptase. We propose a possible model to explain the mode of action of RNase H and RNA-dependent DNA polymerase activities in reverse transcription.

Avian Leukosis Virus↗

PubMed1989

Phage particle-mediated gene transfer of recombinant cosmids to cultured mammalian cells.

An efficient procedure for the introduction of recombinant cosmids into cultured mammalian cells consists of the following steps. Cosmids were packaged, in vitro, into lambda phage particles and transduced into Escherichia coli hosts lysogenized with thermo-inducible lambda c Its phage. The introduced cosmids were repackaged into phage particles in the thermo-induced hosts. The efficiency of such in vivo cosmid packaging was further improved by construction of pTC vectors that carried three cohesive end sites (cos) of phage lambda, arrayed in tandem. Two types of cosmids, in almost equal numbers (i.e., cosmids with one cos and cosmids with two cos), were obtained from a cosmid library constructed with pTC vectors. The efficiency of packaging in vivo of cosmids with two cos, was found to be 7-20 times higher than that of corresponding cosmids with only one cos. Use of a high-copy-number derivative of pTCl further improved the phage yield by 20- to 30-fold. The packaged cosmids, which carried the thymidine kinase-encoding gene of herpes simplex virus type 1 as a selective marker, were introduced into mouse Ltk- cells with an efficiency of 10(-5), by the phage transfer method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607-616].

Animals↗

PubMed1989

Structure and expression of elongation factor 2 gene during development of Dictyostelium discoideum.

A cDNA library constructed from poly(A)+ RNA isolated from Dictyostelium discoideum cells at 12 h of development was screened with the hamster elongation factor 2 (EF-2) cDNA. Several different cDNA clones which hybridized were isolated after a second screening. A cDNA clone representing the 5'-end of the mRNA was obtained by primer extension. By comparing the amino acid sequence deduced from the nucleotide sequences of these clones with that of hamster EF-2, we found enough homology between them to conclude that the isolated clones were complementary to the mRNA of D. discoideum EF-2. The N terminus which is the GTP-binding domain and the C-terminal half where it interacts with a ribosome showed a high degree of homology. The amino acid sequence of the carboxyl half includes that it contain a site of ADP-ribosylation by diphtheria toxin. From the Northern blotting analysis, the size of the mRNA was estimated to be 2.6 kilobases. The expression of the mRNA was high in vegetative cells, became maximal at the aggregation stage, and decreased thereafter through development. Upon differentiation of prespore and prestalk cells, the mRNA was highly enriched in the former over the latter. ADP-ribosylation assay of EF-2 protein by diphtheria toxin showed nearly the same developmental changes for the protein as the mRNA. However, prestalk cells were found to contain the same amount of the protein as prespore cells. The Southern blot analyses indicated that the gene encoding EF-2 is unique.

Amino Acid Sequence↗

PubMed1989

Introduction and expression of the human insulin gene in adult rat liver.

Liposomes associated with gangliosides and loaded with the human insulin gene were incubated with HVJ (Sendai virus) and red blood cell membranes containing non-histone chromosomal protein, high mobility group 1 (HMG-1), or bovine serum albumin (BSA) to form vesicle complexes. These vesicle complexes were then injected into the portal vein of adult rats. The insulin gene co-introduced with HMG-1 was transported into the nuclei of liver cells much more efficiently than the gene co-introduced with BSA. Moreover, the amount of transcript of the insulin gene co-introduced with HMG-1 was more than 10 times greater than that of the gene co-introduced with BSA. Similar amounts of DNA and RNA of human insulin were found in the liver within 7 days after injection of the gene, and then the amounts of both decreased rapidly. The level of human insulin in rat serum gradually increased to a maximum (20-30 microunits/ml) after 7 or 8 days and then decreased rapidly.

Animals↗

PubMed1989

The histidine residue of codon 715 is essential for function of elongation factor 2.

Several mutant cDNAs of elongation factor 2 (EF-2) were constructed by site-directed mutagenesis and their products expressed in mouse cells were investigated. Amino acid substitution for the histidine residue of codon 715, which is modified post-translationally to diphthamide, resulted in non-functional EF-2 and this substitution did not render EF-2 resistant to Pseudomonas aeruginosa exotoxin A, which inactivates EF-2 transferring ADP-ribose to the diphthamide residue. These non-functional EF-2s with replacements of the histidine-715 residue showed various extents of inhibition of protein synthesis by competing with functional EF-2 in vivo. These results suggest that histidine-715 is essential for the translocase activity of EF-2 and that the region around diphthamide functions in recognition of, and/or binding to ribosomes. Substitution of proline for the alanine-713 residue and substitution of glutamine for the glycine-717 residue converted EF-2 to partially toxin-resistant forms. Two-dimensional gel analysis with fragment A of diphtheria toxin of these toxin-resistant EF-2s revealed that their ADP-ribosylations by toxin were much less than that of wild-type EF-2.

Adenosine Diphosphate Ribose↗

PubMed1989

Increased expression of DNA cointroduced with nuclear protein in adult rat liver.

DNA and nuclear proteins were transferred into cells simultaneously at more than 95% efficiency by means of vesicle complexes. The DNA was rapidly transported into the nuclei of cultured cells, and its expression reached a maximum within 6 to 8 hours after its introduction. Moreover, when the plasmid DNA and nuclear protein were cointroduced into nondividing cells in rat liver by injection into the portal veins of adult rats, the plasmid DNA was carried into liver cell nuclei efficiently by nuclear protein. The expression of the DNA in adult rat liver, on introduction of the DNA with nuclear protein, was more than five times as great as with nonnuclear protein.

Animals↗

PubMed1989

Effect of 3'-azido-3'-deoxythymidine on replication of duck hepatitis B virus in vivo and in vitro.

3'-Azido-3'-deoxythymidine (AZT) inhibits the replication of the human immunodeficiency virus (HIV) by blocking the formation of the phosphodiester bond and has been used clinically for the treatment of HIV infection. To assess the effect of AZT on the replication of hepadnaviruses, which replicate through reverse transcription, both the liver tissue and primary cultured hepatocytes from ducklings previously infected with duck hepatitis B virus (DHBV) were examined for DHBV DNA before and after the treatment with AZT. We did not observe suppression of DHBV replication at any doses in our system as measured by viral DNA synthesis in infected duck hepatocytes. The data strongly suggest that AZT has no inhibitory effect on DHBV reverse transcriptase.

Animals↗

PubMed1989

Identification of cell types containing S-100b protein-like immunoreactivity in the islets of Langerhans of the guinea pig pancreas with light and electron microscopy.

Cell types containing S-100b protein-like immunoreactivity in the islets of Langerhans of the guinea pig were studied by light- and electron-microscopic immunocytochemistry using antisera to S-100b protein, insulin, glicentin, somatostatin, and pancreatic polypeptide. Two types of S-100b-immunoreactive cells were identified. The first type was stellate and characterized by thin cytoplasmic processes sheathing endocrine-type cells, especially pancreatic A-cells. It was located predominantly in the neuro-insular complex and in large islets, both of which were located near the main pancreatic duct. Intense immunoreactivity was found in the cytoplasmic matrix as well as in the nucleoplasm. Nerve fibers or endings were occasionally ensheathed by its cytoplasmic processes. The second type, whose immunoreactivity was rather weak and varied from one cell to another, was oval to polygonal in shape and located randomly throughout the islets. It was an endocrine cell-type and its immunoreactivity was located in the secretory granule. With the use of immunostained consecutive sections for demonstrating pancreatic endocrine cell-types, it was found that a portion of the pancreatic B-cell population expressed S-100b-like immunoreactivity.

Animals↗

PubMed1989

A case of primary intrapulmonary neurilemoma and review of the literature.

In this paper, we present an extremely rare case of a primary intrapulmonary neurogenic tumor, in which localization of S-100 protein, neuron specific gamma-enolase (NSE) and CEA was investigated using immunohistochemical staining. The patient, who was a 39-year-old man, experienced no symptoms; however, a routine chest X-ray revealed a round tumor-like shadow in the infrahilar area of the right lung. As the tumor appeared to be gradually increasing in size, surgery was performed and histopathological examination of the excised tumor revealed it to be a primary intrapulmonary neurilemoma. Immunohistochemical staining demonstrated the presence of S-100 protein in the tumor cells but NSE and CEA were not detected. For the 2 years following his operation, the patient has been in good health and is now under careful observation.

Adult↗

PubMed1989

Efficient simplified cosmid cloning: construction and characterization of cosmid vectors that carry the two cohesive end sites of lambda phages arrayed in tandem.

We constructed a series of cosmid vectors that carry the two cohesive end sites (cos) of lambda phage, arrayed in tandem, which enabled us to clone fragments of genomic DNA of up to 50 kb without a vector background. An equimolar mixture of the left and right vector arms of equal length was prepared from the vector DNA, simply by treating the DNA sequentially with three enzymes, restriction enzyme PvuII, alkaline phosphatase, and restriction enzyme BamHI (or BglII), without purification by agarose gel electrophoresis. After phenol extraction and ethanol precipitation, the equimolar mixture of the vector arms, which carried a single cos oriented from left to right, was directly ligated with insert DNA without further manipulation. We established conditions for cosmid cloning, using two kinds of DNA fragment of 40-50 kb, prepared from mouse L cell genomic DNA, as insert DNAs, namely, three cloned BamHI fragments and Sau3AI fragments, size-selected on a sucrose density gradient. The most important parameters affecting the cloning efficiency were the quality of the insert DNA and the molar ratio of the insert and vector arms. We achieved cloning efficiencies of 3.6 X 10(6)-1.3 X 10(7) colony forming units (cfu)/micrograms of insert DNA and 1.7 X 10(5)-1.0 X 10(6) cfu/micrograms of insert DNA, using the cloned BamHI fragments and the Sau3AI fragments, respectively. We examined more than 5000 clones and found that they all contained insert DNA.

Animals↗

PubMed1989

The effect of ammonium chloride on the multiplication of herpes simplex virus type 1 in Vero cells.

The multiplication of herpes simplex virus type 1 (HSV-1) in Vero cells is inhibited by ammonium chloride. The formation of infectious virus was inhibited immediately after the addition of the agent into the culture fluid and was restored by removal of the agent. Although neither viral DNA replication nor nucleocapsid formation were affected by the addition of ammonium chloride at 4 h postinfection, the agent markedly inhibited the formation of enveloped particles and completely the formation of infectious progeny virus. These results indicate that one of the effects of ammonium chloride on the multiplication of HSV-1 is the inhibition of envelopment of viral nucleocapsids. In addition, the envelopment of HSV-1 nucleocapsids was inhibited immediately after the addition of monensin into the culture fluid. These findings suggest the importance of acidic pH of an intracellular compartment in the envelopment of HSV-1.

Ammonium Chloride↗

PubMed1989

Microinjection of T4 endonuclease V produced by a synthetic denV gene stimulates unscheduled DNA synthesis in both xeroderma pigmentosum and normal cells.

A structural gene for T4 endonuclease V was constructed by ligating synthetic oligonucleotides. The endonuclease V was overproduced in E. coli under control of the E. coli tryptophan promoter and purified to apparent homogeneity. The product had comparable DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease activities to the natural enzyme in vitro. When this endonuclease V was microinjected into the cytoplasm of xeroderma pigmentosum (XP) cells of complementation group A, B, C, D, F, G or H, unscheduled DNA synthesis (UDS) above the residual level was detected in all the cells at a dose of about 10(3) molecules following UV irradiation. The gain numbers of UDS in these XP cells increased with increase in the dose of enzyme and reached a plateau at the normal cell level on introduction of about 10(4) molecules. Introduction of more enzyme into either XP cells or normal human cells did not increase the grain number under regular labelling conditions (2.5 h, 37 degrees C). In normal mouse cells, introduction of the enzyme increased the grain number more than 4-fold under the same conditions during at least 8.5 h following UV irradiation. Furthermore, with a labelling time of 30 min, the enzyme more than doubled the grain number even in normal human cells.

Animals↗

PubMed1989

Bronchopulmonary foregut malformation in 3 infants--with special references to cases in childhood.

We encountered three infant patients with bronchopulmonary foregut malformation. Two of them were associated with oesophageal atresia, and the remaining one with jejunal atresia. Two of the patients showed total lung sequestration, and the rest showed intralobar sequestration. 37 paediatric cases of this type were reviewed from the literature. Total lung sequestration was observed in 19 of the 37 cases, showing an increase in the incidence in comparison with that reported previously. There were also 7 cases associated with oesophageal atresia.

Abnormalities, Multiple↗

PubMed1989

Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum.

For isolation of the gene responsible for xeroderma pigmentosum (XP) complementation group A, plasmid pSV2gpt and genomic DNA from a mouse embryo were cotransfected into XP2OSSV cells, a group-A XP cell line. Two primary UV-resistant XP transfectants were isolated from about 1.6 X 10(5) pSV2gpt-transformed XP colonies. pSV2gpt and genomic DNA from the primary transfectants were again cotransfected into XP2OSSV cells and a secondary UV-resistant XP transfectant was obtained by screening about 4.8 X 10(5) pSV2gpt-transformed XP colonies. The secondary transfectant retained fewer mouse repetitive sequences. A mouse gene that complements the defect of XP2OSSV cells was cloned into an EMBL3 vector from the genome of a secondary transfectant. Transfections of the cloned DNA also conferred UV resistance on another group-A XP cell line but not on XP cell lines of group C, D, F, or G. Northern blot analysis of poly(A)+ RNA with a subfragment of cloned mouse DNA repair gene as the probe revealed that an approximately 1.0 kilobase mRNA was transcribed in the donor mouse embryo and secondary transfectant, and approximately 1.0- and approximately 1.3-kilobase mRNAs were transcribed in normal human cells, but none of these mRNAs was detected in three strains of group-A XP cells. These results suggest that the cloned DNA repair gene is specific for group-A XP and may be the mouse homologue of the group-A XP human gene.

Animals↗

PubMed1989

Human interleukin 6 is a direct promoter of maturation of megakaryocytes in vitro.

The response of cells of the murine megakaryocytic lineage to human interleukin 6 (IL-6) was assessed in serum-depleted cultures using a variety of biological assays. IL-6 alone had no influence on megakaryocytic colony formation but augmented the numbers of these colonies induced by the multipotent colony-stimulating factor interleukin 3. However, in liquid marrow cultures, IL-6 alone promoted marked increments in megakaryocytic size and the activity of acetyl-cholinesterase, a marker enzyme of the lineage. Moreover, IL-6 induced a significant shift toward higher ploidy classes when megakaryocytic DNA was quantitated by flow cytometry. To determine whether the influence of IL-6 on megakaryocytic maturation was direct, the factor was added to cultures of single megakaryocytes isolated from megakaryocytic colonies. Fifty-four percent of these cells increased in size compared with 19% of those grown without IL-6. The data show that human IL-6 is a potent direct-acting growth factor for murine megakaryocytes with activity promoting maturation of that lineage.

Animals↗