Biomedical subjects
Publications and source records attributed to K Yoshimoto.
A cDNA clone encoding a human ribosomal protein L39 (hRPL39) was isolated through a random cDNA sequencing approach to a cDNA library constructed from a human colon carcinoma cell line of COLO 205. Although levels of hRPL39 mRNA were different in several cell lines including carcinoma cell lines from different tissues, they were shown not to be cell cycle-dependent in a human fibroblast cell line of TIG-1.
This article reviews some recent studies on alcohol preference, dependence, metabolism and pharmacokinetics which were mainly carried out in our department. The inbred strains of mice with genetically different alcohol drinking behavior and alcohol animal model treated with the neurotoxins, 6-hydroxydopamine and 5,7-dihydroxytryptamine, are useful for a behavioral and pharmacological approach to evaluate the contribution of specific neural systems to alcohol, drug dependence mechanism and alcohol drinking behavior. The relations between alcohol preference and some physiological conditions are reviewed. On the drug-alcohol interaction, some drugs containing the chemical group = CHONO2, antimony and methamphetamine are addressed. This article also deals with recent topics in the pharmacokinetics and pharmacodynamics of alcohol. The dose-dependency of the alcohol elimination rate, the first-pass metabolism during alcohol drinking, and the pharmacodynamic model for describing pulse rate reaction to plasma acetaldehyde are discussed.
Previous studies have established that cytokines regulate development of Th cell subsets from naive CD4 cells, suggesting promising therapeutic potential for cytokines that has not been fully realized, in part due to high dose toxicity. Here we examined effects of sustained delivery of low doses of rIL-4 on development of cytokine-secreting primary CD4 cells in vivo. Diffusion chambers injected with X63.Ag-653 plasmacytoma transfectants that constitutively produce rIL-4 were implanted s.c. in mice 24 to 48 h before immunization with keyhole limpet hemocyanin (KLH). Five days later at the peak of the response, KLH-specific CD4 cells from controls, which received chambers containing untransfected plasmacytoma cells, secreted primarily IL-2 and IL-4, with low levels of IFN-gamma and no IL-5 following Ag restimulation. Administration of rIL-4 enhanced IL-2 and IL-4 production by two- to fivefold, with a corresponding decrease in IFN-gamma. As frequencies of KLH-specific precursors that secreted IL-4 were unaltered, exogenous cytokine apparently affected the magnitude of IL-4 secretion by primed CD4 cells. Although IL-4 was undetectable in sera of mice carrying chambers, titers of KLH-specific IgG1 and IgG3 Ab were increased by rIL-4, whereas IgM, IgG2a, and IgG2b levels were unaltered. The results indicate that sustained delivery of low doses of cytokines at sites distant from Ag exposure can selectively potentiate development of CD4 subsets.
A human CAD cDNA encoding a trifunctional enzyme of carbamoylphosphate synthetase-aspartate transcarbamoylase-dihydroorotase, which catalyzes the first three steps of de novo pyrimidine nucleotide biosynthesis, was cloned from a human fibroblast cell line of TIG-1-20 by polymerase chain reaction (PCR). The predicted open reading frame encodes a protein of 2,225 amino acids with a deduced molecular weight (Mr) OF 242,913. The deduced amino acid sequence exhibits 95.3 and 76.1% identity with the CAD sequences of hamster and Squalus acanthias. The DNA fragment of 6,679 bp containing the full-length coding sequence was amplified by nested PCR using the first-strand cDNA of human cell lines of TIG-1-20 and COLO205 as a template. Southern blot analysis suggested that the CAD gene exists as a single copy in the human genome.
The quasispecies of hepatitis C virus (HCV) core region in non-cancerous and cancerous hepatocellular carcinoma (HCC) lesions, respectively, of 7 patients with hepatocellular carcinoma were studied. Multiple fluorescence based-polymerase chain reaction (PCR)-single strand conformation polymorphism exhibited a different set and a larger number of quasispecies in cancerous portions than those in non-cancerous portions. DNA sequencing of PCR-amplified core region substantiated an accumulation of nucleotide substitutions, and a greater number of quasispecies in cancerous portions than those in non-cancerous portions. The deduced amino acid sequences disclosed that at the peptide position 45, Ser is dominant in non-cancerous lesions, and Gly in cancerous lesions, respectively. Thus, HCV in hepatocellular carcinoma includes a large number of specific quasispecies presumably due to their vigorous proliferation. A different set of quasispecies with the amino acid change is presumed to be related to the hepatocarcinogenesis.
The insulin gene is specifically expressed in pancreatic islet beta cells. Various cis-acting DNA elements in the 5'-flanking region of the human insulin gene were examined for their contribution to the transcriptional activity using sensitive human growth hormone (hGH) reporter plasmids. The hGH constructs, having successively deleted human insulin promoter sequences, were transfected to a pancreatic islet beta-cell line MIN6. The deletion of two GGAAAT (GG) motifs, GG2 at -145 to -140 bp and GG1 at -134 to -129 bp, decreased the transcriptional activity to 6.5% of that of the promoter sequence from -156 to +1 bp. The selective mutations in both GG motifs also decreased the transcriptional activity to 5.5%. One-base mutations of GG2 and GG1 decreased the transcriptional activity to 82 and 11%, respectively. The two-base mutations between GG2 and GG1 affected the transcriptional activity more strongly than those just outside the GG motifs. A single set of GG motifs in the upstream of thymidine kinase promoter increased the transcriptional activity to 216% compared to that of thymidine kinase promoter alone in MIN6 cells. With an electrophoretic mobility shift assay (EMSA), a nuclear factor in MIN6 cells was shown to bind the DNA fragments containing two GG motifs. This factor did not bind to another GGAAAT-like sequence at -313 to -305 bp in the human insulin gene. These results suggested that the GG motifs contributed to the cell-specific transcription of the human insulin gene in association with the binding of the sequence-specific nuclear factor.
To examine whether long-term administration of a beta3-adrenoceptor agonist influences sympathetic nervous system (SNS) activity, norepinephrine (NE) turnover, a reliable indicator of SNS activity, in the interscapular brown adipose tissue (IBAT), the heart, and the spleen, as well as urinary excretion of NE, were measured using mice treated with CL316,243 (CL), a highly specific beta3-adrenoceptor agonist, at a dose that stimulated thermogenesis and reduced body weight. CL significantly decreased NE turnover in the IBAT, heart, and spleen and decreased urinary excretion of NE without affecting food intake over 1 to 4 weeks of treatment. These findings show that long-term administration of the beta3-adrenoceptor agonist decreases SNS activity and urinary excretion of NE.
6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4) is a coenzyme for tyrosine, tryptophan and phenylalanine hydroxylases, the former two of which are the initial and the rate-limiting enzymes in the biosynthesis of catecholamines and serotonin, respectively. The present study was conducted to determine the levels of 6R-BH4 in the inbred strains of rats BN and F344, and the inbred strains of mice C57BL/6J, C3H/HeJ and DBA/2J, with different genetically determined alcohol preferences. Previous studies have shown that BN and F344 rats exhibit a high and low alcohol preference, respectively; that C57BL/6J and DBA/2J mice show a high and low alcohol preference, respectively, and that C3H/HeJ mice exhibit a moderate alcohol preference. The levels of 6R-BH4 were measured in five regional brain areas of rats and in two regional brain areas of mice. There was about a 3-fold difference in 6R-BH4 levels across the rat brain areas assayed, ranging from a low level in the frontal cortex to a high level in the striatum and midbrain. Midbrain 6R-BH4 levels in F344 rats were higher than those of BN rats (p < 0.05). On the other hand, striatal 6R-BH4 levels in DBA/2J mice were higher than those of the other two strains of mice (p < 0.05). These results indicate that 6R-BH4 is distributed throughout the nigro- and mesostriatal dopaminergic nervous systems, and that brain 6R-BH4 levels may be involved in the genetic differences in alcohol-drinking behavior in animal models.
Sixteen sporadic pheochromocytomas, 3 pheochromocytomas in neurofibromatosis 1, and 4 pheochromocytomas in multiple endocrine neoplasia (MEN) 2A or 2B were screened for mutations at codon 768 of the RET proto-oncogene by AluI digestion of polymerase chain reaction PCR products and mutations in exon 13 by PCR-single strand conformation polymorphism (SSCP) analysis. Although mutations at codon 768 (GAG --> GAC; Glu --> Asp) of the RET proto-oncogene were recently reported to be found in 40% of sporadic medullary thyroid carcinomas (MTCs), the absence of missense mutations at codon 768 was confirmed both with PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP analysis in all examined cases of pheochromocytomas. These results suggest that mutations at codon 768 of the RET proto-oncogene do not represent a frequent mechanism of tumorigenesis for both sporadic and hereditary pheochromocytomas.
The study on the expression of the RET proto-oncogene in parathyroid tumors disclosed obvious mRNA expression by the reverse transcription (RT)-polymerase chain reaction (PCR) method and protein expression by Western blotting. To find out whether mutations in the cysteine-rich regions or tyrosine kinase domain of the RET proto-oncogene are etiological for parathyroid tumorigenesis, sporadic parathyroid adenomas and carcinomas, parathyroid tumors from multiple endocrine neoplasia 1, familial isolated hyperparathyroidism or hereditary hyperparathyroidism-jaw tumor syndrome were screened by PCR-single strand conformation polymorphism and PCR restriction fragment length polymorphism. Missense mutations in the cysteine-rich region, or codons 768 or 918 in the tyrosine kinase domain of the RET proto-oncogene, were not detected in any of the examined cases of parathyroid tumors. These results suggest that mutations of the RET proto-oncogene do not represent a frequent mechanism of tumorigenesis for parathyroid tumors.
The method to internally label PCR products with multiple colored fluorescent dyes was developed and applied to multiple fluorescence-based PCR single-stranded conformational polymorphism (MF-PCR-SSCP) analysis. PCR-amplified fluorescent DNA fragments, which were internally labeled by adding fluorescent dUTPs ([F]dUTPs) to the PCR mixture, were heat-denatured and applied to a nondenaturing polyacrylamide gel (SSCP gel) set on an automated DNA sequencer with a gel temperature-controlling system. The image data were analyzed by GENESCAN 672 software. In spite of differences in species and amount of integrated [F]dUTPs, the SSCP profiles were not significantly affected, even by the different labeling methods used-internal labeling or post-labeling at the 3' ends-in regard to the three different [F]dUTPs examined. However, the salt concentration of the solution containing the DNA samples affected the SSCP profiles. The internally labeled [F]dUTP-containing DNA fragments beyond 1000 bp in length were successfully digested with restriction endonucleases and subjected to SSCP analysis. MF-PCR-SSCP analysis with internal fluorescence labeling affords a simple and sensitive method to detect alterations in DNA sequences.
Expression of the intercellular adhesion molecule-1 (CD54) as well as the mutations of p53 gene were studied in childhood Burkitt's lymphoma (BL). Expression of CD54 was identified in 6 of 15 fresh BL cases. Mutations of p53 gene, analyzed by polymerase chain reaction-single stranded chain polymorphism followed by sequencing, were found in 5 of 14 cases examined. Interestingly, all the cases with p53 mutation were CD54 negative. This high frequency of p53 mutation in the CD54 negative group prompted us to analyze the clinical features of these cases. Six of 15 cases died within 21 months after initiation of therapy and five of these were CD54 negative. In addition, four of these had p53 mutation. These results suggest that the lack of CD54 by BL cells may provide the background for the mutation of p53 gene to occur which could result in the transformation to a more aggressive phenotype.
To establish a practical method of somatic gene therapy, we aimed to develop a regulatory system at the cellular level using a suicide vector. We introduced the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene into the human proinsulin-producing Ltk-cells and examined whether ganciclovir (GCV) administration could control proinsulin production in vivo. The cells transfected with the HSV-tk gene showed more than 100-fold increase in sensitivity to GCV compared with the parent cells. Analysis of blood glucose in diabetic nude mice with transplanted cells showed that proinsulin production by these cells was strongly suppressed by GCV treatment in vivo as reflected by the reversal to hyperglycemia. However, in the in vivo experiment, the plasmid containing the HSV-tk gene was spontaneously lost from the transplanted cells in one of six cases resulting in the resistance to GCV as reflected by the persistent hypoglycemia and increased tumor size. This system of HSV-tk and administration of GCV may be applicable to gene therapy as a suicide vector, but the system of stable expression of the HSV-tk gene must be established.
As long-term survival has become possible in patients with autosomal dominant polycystic kidney disease (ADPKD) with progress in hemodialysis (HD), complications by various extrarenal diseases has presented new problems. Recent experience of two rare cases of ADPKD ending fatally due to complications by polycystic liver is presented. Case 1: A 60-year-old female with a family history of ADPKD without a past history of liver disease, was diagnosed as ADPKD at the age of 45 years. Hemodialysis was started at the age of 58 years. From 6 months prior to her death, abdominal circumference increased (body height: 149 cm, abdominal circumference: 100 cm). Dyspnea, abdominal pain and anorexia appeared and she died of hepatic failure leading to cachexia. Case 2: A 76-year-old female with a family history of ADPKD without a past history of liver disease, was started on HD at the age of 73 years. Abdominal circumference was 84 cm (body height: 138 cm). She was repeatedly admitted to and discharged from the hospital due to febrile episodes. Infection of polycystic liver was complicated by DIC and she died of gastrointestinal hemorrhage. Autopsy revealed abscess in some of the cysts in the liver. Hepatic cysts most frequently complicating ADPKD so far have presented with scarcely any clinical problems. Recently, however, cases of infection of hepatic cysts, portal hypertension and hepatic insufficiency have been reported. The relationship between these hepatic diseases and the prognosis of ADPKD has received attention. Increase in the number of cases of complications similar to the present cases is anticipated.
The present study was performed to examine the involvement of serotonin-3 (5-HT3) receptors in the rat nucleus accumbens (ACC) in alcohol dependence. In alcohol-treated rats, perfusion of 40 mM K+ and 100 mM ethanol (EtOH) through the microdialysis probe increased the extracellular levels of ACC dopamine (DA), compared with controls. Perfusion of the serotonin (5-HT) uptake inhibitor sertlarine enhanced the extracellular levels of ACC 5-HT in both groups. Increased 5-HT availability in the synaptic clefts on the ACC further activated ACC DA release in the alcohol-treated rats, in comparison with controls. In the final experiments, perfusion of the 5.0 microM 5-HT3 receptor agonist 2-methyl-5-HT (2-Me-5-HT) through the microdialysis probe enhanced the extracellular levels of ACC DA. Magnitude of 2-Me-5-HT-induced DA release was significantly higher in alcohol-treated rats than in controls. On the other hand, 40 mM K(+)- and 100 mM EtOH-induced extracellular 5-HT release in alcohol-treated rats were markedly inhibited. These results show that (1) chronic alcohol intake increases the sensitivity of 5-HT3 receptors, (2) 5-HT3 receptors regulate DA release in the ACC, (3) the dopaminergic neuronal systems associated with 5-HT3 ionophore in the ACC were upregulated after chronic alcohol exposure, and (4) chronic alcohol intake desensitizes the serotonergic neuronal systems in rat ACC. These findings suggest that neurochemical functions of 5-HT3 receptors in regulating DA release in the ACC after alcohol exposure compensate for the dysfunction of serotonergic activity to restore the original properties in processing alcohol tolerance and that the development of alcohol dependence may be mediated by ACC 5-HT3 receptors.
We analyzed the development of cytokine-producing effector T cells from resting CD4 memory cells. Previously we showed that such memory effectors are induced in vivo upon re-exposure to Ag. Here we demonstrate that effectors arise in vitro when memory CD4 cells are restimulated with Ag in the presence cytokines. Resting splenic CD4 cells from KLH-primed mice that were depleted of naive cells by adult thymectomy and were exclusively of memory phenotype initially secreted high titers of IL-2 and low levels of IL-4 and IFN-gamma in response to Ag. When memory CD4 cells were restimulated for 3 to 4 days in cultures containing rIL-2 and Ab to block endogenous IFN-gamma and IL-4 secretion, Th0-like effectors that produced greatly increased levels of IL-2, IL-4, and IFN-gamma developed. rIL-4 together with rIL-2 and anti-IFN-gamma induced Th2-like cells that secreted primarily IL-4. In contrast, Th1-like effectors that produced IL-2 and IFN-gamma developed in the presence of rIL-2 and anti-IL-4. Addition of rIFN-gamma further enhanced priming for IFN-gamma secretion. rIL-12 also induced effectors that produced high levels of IFN-gamma, but little IL-2. Thus, cytokines direct the development of effector subsets from memory CD4 cells. Our results suggest that memory and naive CD4 cells undergo parallel development following Ag stimulation, initially secreting predominantly IL-2 and differentiating in response to IL-4, IFN-gamma, and IL-12 into polarized effector subsets.
The effect of somatostatin (SS) on adrenocorticotrophic hormone (ACTH) secretion from COR-L103 cells derived from a human small cell lung carcinoma was examined. SS at 1 microM had no effect on ACTH secretion from the cells on either short-term or long-term incubation. Studies by the reverse transcription-polymerase chain reaction (RT-PCR) showed that mRNA transcripts of the somatostatin receptor (SSTR) 2, SSTR3 and SSTR4 genes were present in COR-L103 cells. Extra bands were obtained by PCR-single strand conformation polymorphism (SSCP) analysis of the SSTR2 gene Sequence analysis of the SSTR2 gene demonstrated one point mutation in codon 188 of TGG for tryptophan to TGA for a stop codon causing loss of 182 C-terminal amino acid residues of SSTR2. The nucleotide sequences of the SSTR3 and SSTR4 genes in COR-L103 cells were normal. Binding studies using 125I-Tyr11-SS-14 showed specific affinity binding sites on COR-L103 cells and mouse pituitary tumor AtT-20 cells. Octreotide acetate suppressed the binding of 125I-Tyr11-SS-14 to these two cell lines, but the Kd of COR-L103 cells (160 nM) was 60-fold higher than that of AtT-20 cells (2.6 nM). Affinity cross-linking studies using 125I-Tyr11-SS-14 gave three bands of 72 KDa, 55 KDa and 32 KDa from AtT-20 cells, but only two bands of 55KDa and 32kDa from COR-L103 cells. These findings suggest that SSTR2 is not expressed in the plasma membranes of COR-L103 cells due to a point mutation, but that this may have no influence on the effect of SS on ACTH secretion.
Genomic structure of rat amidophosphoribosyltransferase (ATase; EC 2.4.2.14), which catalyzes the first committed step in de novo purine nucleotide synthesis, was determined by polymerase chain reaction (PCR)-based methods. There are 11 exons and all exon-intron boundaries were conserved among rat, human, and chicken ATase genes. A rat aminoimidazole ribonucleotide carboxylase (AIRC) cDNA encoding a bifunctional enzyme of AIRC (EC 4.1.1.21) at step 6 and SAICAR synthetase (EC 6.3.2.6) at step 7 in de novo purine nucleotide synthesis was cloned and sequenced. The size of the cloned rat AIRC cDNA was 1329 bp, and amino acid identity with human and chicken AIRC was 96 and 85%, respectively. The intergenic sequence using a phage clone and the PCR product disclosed that ATase and AIRC genes are physically linked with the 736 bp sequence between the translation start sites, and the determination of the transcriptional start sites by the primer extension assay for these genes disclosed that distance between the two major transcriptional start sites is 585 bp. The amount of mRNAs of both genes showed approx. 5-6-fold increase in G1/S phase of the cell cycle over those in G0 phase in synchronized rat 3Y1 fibroblasts.