Biomedical subjects

T Tokunaga

Publications and source records attributed to T Tokunaga.

At least 55 records · Page 3Linked to original sources

PubMed2000

Hammerhead ribozyme specifically inhibits vascular endothelial growth factor gene expression in a human hepatocellular carcinoma cell line.

Hammerhead-type ribozymes are often utilized to suppress the expression of target genes. We evaluated the efficacy of an anti-vascular endothelial growth factor (VEGF) hammerhead-type ribozyme against GUC at exon 1 of the VEGF gene in a cell-free system (in vitro) as well as in the hepatocellular carcinoma cell line HLF (in vivo). The anti-VEGF ribozyme (alphaVRz) specifically cleaved synthetic VEGF RNA substrate, but not other triplet sequences of VEGF RNA substrate in vitro. When the alphaVRz was introduced into HLF cells, the ribozyme suppressed not only VEGF mRNA level but also that of VEGF protein. These results suggest that this ribozyme selectively inhibits VEGF gene expression in human hepatocellular carcinoma cells.

Base Sequence↗

PubMed2000

Quinolactacins A, B and C: novel quinolone compounds from Penicillium sp. EPF-6. II. Physico-chemical properties and structure elucidation.

Three novel quinolone compounds, quinolactacins A (1), B (2) and C (3), have been found from the fermentation broth of Penicillium sp. EPF-6, a fungus isolated from the larvae of mulberry pyralid (Margaronia pyloalis Welker). The molecular formulas of 1, 2 and 3 were determined to be C16H18N2O2, C15H16N2O2 and C16H18N2O3, respectively by FAB-MS and NMR spectral analyses. The structures of these compounds have a novel quinolone skeleton with a gamma-lactam ring consisting of C12H8N2O2 as the common chromophore.

Anti-Bacterial Agents↗

PubMed1999

Mouse ATF-2 null mutants display features of a severe type of meconium aspiration syndrome.

Mouse null mutants of transcription factor ATF-2 were generated by the gene targeting method. They died shortly after birth and displayed symptoms of severe respiratory distress with lungs filled with meconium. These features are similar to those of a severe type of human meconium aspiration syndrome. The increased expression of the hypoxia inducible genes suggests that hypoxia occurs in the mutant embryos and that it may lead to strong gasping respiration with consequent aspiration of the amniotic fluid containing meconium. A reduced number of cytotrophoblast cells in the mutant placenta was found and may be responsible for an insufficient supply of oxygen prior to birth. Using the cDNA subtraction and microarray-based expression monitoring method, the expression level of the platelet-derived growth factor receptor alpha gene, which plays an important role in the proliferation of trophoblasts, was found to be low in the cytotrophoblasts of the mutant placenta. In addition, ATF-2 can trans-activate the PDGF receptor alpha gene promoter in the co-transfection assay. These results indicate the important role of ATF-2 in the formation of the placenta and the relationship between placental anomalies and neonatal respiratory distress. The ATF-2 null mutants should enhance our understanding of the mechanism of severe neonatal respiratory distress.

Activating Transcription Factor 2↗

PubMed1999

Expression pattern of vascular endothelial growth factor isoform is closely correlated with tumour stage and vascularisation in renal cell carcinoma.

Vascular endothelial growth factor (VEGF) has five isoforms (VEGF206, 189, 165, 145 and 121). Increased VEGF expression in renal cell carcinoma (RCC) is associated with angiogenesis, but, it is not apparent which isoform is involved in this effect. We examined the isoform patterns of VEGF by reverse transcription-polymerase chain reaction (RT-PCR) in 47 RCCs. All showed increased VEGF expression as compared with extraneoplastic renal tissue. Four of the 47 RCCs showed VEGF121 alone, 10 showed VEGF121 + 165, and 33 showed the VEGF121 + 165 + 189 pattern. Patients with pathological stage pT3-4 RCC showed the VEGF121 + 165 + 189 isoform pattern at a significantly higher incidence (10/10, 100%) than those with pT0-2 (23/37, 62%) (P < 0.022). The VEGF121 + 165 + 189 isoform pattern was also significantly associated with high vessel counts and density (P = 0.0002, Mann-Whitney U test). These observations suggested that the VEGF189 mRNA isoform is closely associated with angiogenesis and results in the growth of RCC.

Blotting, Northern↗

PubMed1999

Thrombospondin-2 (TSP2) expression is inversely correlated with vascularity in glioma.

Thrombospondins (TSPs) are angiostatic factors in various cancers. However, the significance of TSPs has not been well characterised in glioma. We examined TSP1, TSP2 and vascular endothelial growth factor (VEGF) gene expression by reverse transcription-polymerase chain reaction (RT-PCR) in 37 gliomas. Thirty of the 37 glioma specimens showed VEGF gene expression. Eighteen of the 37 gliomas expressed the TSP1 gene. Seven gliomas lacked TSP2 gene expression, while the other 30 expressed TSP2. The lack of TSP2 gene expression was significantly associated with higher histological grade (Fisher's test, P = 0.0019) and increased vessel counts and density (Student's t-test, P < 0.0001), while there were no correlations between TSP1 and VEGF gene expression and clinicopathological features. These results indicate that the lack of TSP2 gene expression is a potent factor for enhancement of angiogenesis in glioma.

Adult↗

PubMed1999

Multidrug resistance-associated protein (MRP) expression is correlated with expression of aberrant p53 protein in colorectal cancer.

Multidrug resistance-associated protein (MRP) is one of the major factors responsible for non-P-glycoprotein (Pgp)-mediated multidrug resistance of human tumour cells. In this study, we examined MRP and aberrant p53 expression in 54 colorectal cancers (CRC), 35 carcinoma in adenomas (CIA) and 40 adenomatous polyps by immunohistochemical procedures. 38 of 54 (70%) CRCs, 16 of 35 (46%) CIAs and 3 of 40 (8%) adenomatous polyps were MRP positive (chi 2 test, P < 0.0001). 36/54 (67%) CRCs, 10/35 (29%) CIAs and 0/40 adenomatous polyps were p53 positive. 30 of the 36 p53-positive CRCs were also MRP positive and 8/10 CIAs were both p53 and MRP positive. MRP overexpression correlated with aberrant p53 accumulation in CRCs and CIAs (chi 2 test, P < = or 0.01). Coexpression of MRP and p53 in the same cells was confirmed in the CRCs and CIAs by double staining procedures. These results suggested that MRP overexpression is related to aberrant p53 expression in CRC.

ATP-Binding Cassette Transporters↗

PubMed1999

Cell-retained isoforms of vascular endothelial growth factor (VEGF) are correlated with poor prognosis in osteosarcoma.

Vascular endothelial growth factor (VEGF) is a major angiogenic factor. Osteosarcoma is characterised by hypervascularity and metastatic potential. We examined VEGF mRNA expression, VEGF isoform pattern and VEGF receptor (flt-1 and KDR) by RT-PCR analysis in 30 osteosarcomas. All 30 osteosarcomas expressed VEGF mRNA. 17 osteosarcomas (57%) expressed flt-1 mRNA, whilst 20 (67%) expressed KDR mRNA. 6/30 (20%) osteosarcomas were positive for VEGF121 only, 8 (27%) for VEGF121 + VEGF165, and 16 (53%) for VEGF121 + VEGF165 + VEGF189. Patients with osteosarcomas with VEGF165 (n = 24) had significantly poorer prognosis in comparison with those without VEGF165 (P = 0.022, Wilcoxon's test). The osteosarcomas with VEGF165 had significantly increased vascularity assessed on sections immunostained for CD34 (P < 0.001, Mann-Whitney U test). Although VEGF165 is a soluble isoform, it is also retained on the cellular surface. These results suggest that cell-retained VEGF isoforms (VEGF165, VEGF189) might be essential for neovascularisation in osteosarcoma, whilst the soluble VEGF121 isoform is not sufficient to stimulate neovascularisation in this type of neoplasm.

Adult↗

PubMed1999

Dietary heme iron does not prevent postgastrectomy anemia but fructooligosaccharides improve bioavailability of heme iron in rats.

Gastrectomized rats exhibit iron deficiency anemia. We observed the effects of dietary heme-iron and short chain frucooligosaccharides (Sc-FOS) in relation to prevention of postgastrectomy anemia in rats. Twelve laparotomized (sham-operated) rats were fed iron-citrate (control) as iron source diet without or with Sc-FOS (75 g/kg of diet) and twenty four totally gastrectomized (Bilroth II) rats, were fed a iron-citrate (control) or heme-iron (heme) as iron source diet without or with Sc-FOS (75 g/kg of diet) for 4 weeks. All rats received an intramuscular injection of vitamin B-12 every two weeks. Tail blood was collected every other week for determination of hematocrit and hemoglobin concentration. At the end of the experiment, the rats were killed and whole blood was collected. The total gastrectomy induced the postgastrectomy anemia. Dietary Sc-FOS increase iron absorption and thereby prevented completely this anemia in gastrectomized rats fed the control diet but this effect of Sc-FOS in rats fed heme diet was not complete. Dietary heme iron could not prevent postgastrectomy anemia itself, but fructooligosaccharides improve bioavailability of not only non-heme iron such as iron-citrate, but also heme-iron in rats.

Anemia↗

PubMed1999

Thrombospondin 2 expression is correlated with inhibition of angiogenesis and metastasis of colon cancer.

Two subtypes of thrombospondin (TSP-1 and TSP-2) have inhibitory roles in angiogenesis in vitro, although the biological significance of these TSP isoforms has not been determined in vivo. We examined TSP-1 and TSP-2 gene expression by reverse transcription polymerase chain reaction (RT-PCR) analysis in 61 colon cancers. Thirty-eight of these 61 colon cancers were positive for TSP-2 expression and showed hepatic metastasis at a significantly lower incidence than those without TSP-2 expression (P = 0.02). TSP-2 expression was significantly associated with M0 stage in these colon cancers (P = 0.03), whereas TSP-1 expression showed no apparent correlation with these factors. The colon cancer patients with TSP-2 expression showed a significantly low frequency of liver metastasis correlated with the cell-associated isoform of vascular endothelial growth factor (VEGF-189) (P = 0.0006). Vascularity was estimated by CD34 staining, and TSP-2(-)/VEGF-189(+) colon cancers showed significantly increased vessel counts and density in the stroma (P < 0.0001). TSP-2(-)/VEGF-189(+) colon cancer patients also showed significantly poorer prognosis compared with those with TSP-2(+)/VEGF-189(-) (P = 0.0014). These results suggest that colon cancer metastasis is critically determined by angiogenesis resulting from the balance between the angioinhibitory factor TSP-2 and angiogenic factor VEGF-189.

Analysis of Variance↗

PubMed1999

Anti-obesity and anti-diabetic activities of a new beta3 adrenergic receptor agonist, (S)-(Z)-[4-[[1-[2-[(2-hydroxy-3-phenoxypropyl)]amino]ethyl]-1-propenyl] phenoxy] acetic acid ethanedioic acid (SWR-0342SA), in KK-Ay mice.

We investigated the beta-adrenergic receptor (AR) agonistic activities in rats and humans, and the anti-obesity and anti-diabetic activities in KK-Ay mice, of a new beta3-AR agonist, SWR-0342SA ((S)-(Z)-[4-[[1-[2-[(2-hydroxy-3-phenoxypropyl)]amino]ethyl]-1-pro penyl]phenoxy] acetic acid ethanedioic acid). With regards to its beta-AR agonistic activity in rats, SWR-0342SA stimulated the atrial beating rate (beta1-AR activity) and white adipocyte lipolysis (beta3-AR activity), but did not induce uterine muscle relaxation (beta2-AR activity). The beta3-AR agonistic activity of SWR-0342SA was about 20 times stronger than its beta1-AR agonistic activity. Similarly, SWR-0342SA enhanced the accumulation of cAMP in Chinese hamster ovary (CHO) cells expressing human beta1- and beta3-ARs, while having no effect in CHO cells expressing beta2-ARs. Adenylyl cyclase stimulation by SWR-0342SA in CHO cells expressing beta3-ARs was about 35 times higher than that in CHO cells expressing beta1-ARs. With regards to anti-obesity and anti-diabetic activities, SWR-0342SA had no effect on body weight or food intake, but slightly decreased the fat pads weight in KK-Ay mice, an animal model of obesity and non-insulin-dependent diabetes mellitus (NIDDM). On the other hand, SWR-0342SA significantly decreased both blood glucose (to about 46% of control) and serum insulin levels (to about 40% of control) in KK-Ay mice. These results indicated that SWR-0342SA is a selective beta3-AR agonist, and possesses potent anti-diabetic activity, and that the anti-obesity activity is inferior to the anti-diabetic activity.

Acetates↗

PubMed1999

cAMP-independent chloride secretion activated by a vasoactive intestinal peptide in a monolayer culture of human bronchial epithelial cells.

To investigate the effects of vasoactive intestinal peptide (VIP) on Cl- transport across normal human bronchial epithelial (NHBE) cells grown in a monolayer, changes in short-circuit current (Isc) were measured in Ussing chamber systems. In the presence of 10(-4) M amiloride, the addition of VIP to the serosal solution led to an increase in the Isc in a concentration-dependent manner, the 50% effective concentration (EC50) being 2.6 x 10(-11) M. However, the addition of 10(-5) M forskolin had little effect on the increase in Isc. On the other hand, in the intracellular cAMP measurement, 10(-5) M forskolin remarkably increased the cAMP levels, but 10(-7) M VIP did not. This result suggests that Cl- secretion by VIP is not related to the raised intracellular cAMP levels in NHBE cells.

Bronchi↗

PubMed1999

Frequent expression of inducible nitric oxide synthase in esophageal squamous cell carcinomas.

Nitric oxide (NO) plays important biological roles in cardiovascular, nervous and immune systems, and is synthesized by nitric oxide synthase (NOS). Intracellular NO is known to cause DNA damage as a mutagen. We examined the expression of cytokine-inducible NOS (iNOS) in human esophageal squamous cell carcinomas. Weak iNOS immunoreactivity was seen in the basal and parabasal layers of non-neoplastic esophageal stratified squamous epithelium. iNOS expression was detected in 50 (87.7%) of the 57 esophageal squamous cell carcinomas, regardless of the depth of tumor invasion, histological differentiation and lymph node status. Early-stage cancers, i.e. mucosal squamous cell carcinomas, also showed significant iNOS expression. We speculate that increased iNOS expression is associated with the carcinogenesis of human esophageal cancer.

Adult↗

PubMed1999

Stromal expression of thrombospondin-1 is correlated with growth and metastasis of human gallbladder carcinoma.

Thrombospondin-1 (TSP1) is one of the extracellular matrix glycoproteins that affect cell adhesion, motility and growth. Based on its effects on tumors, TSP1 is thought to be a potential regulator of tumor growth and metastasis. In this study, we examined TSP1 expression in human gallbladder adenocarcinoma and its clinicopathological significance. TSP1 immunoreactivity was detected mainly in the cancer stroma and was observed infrequently in cancer cells. According to the TNM classification, 74.5% (29/39) of the T2 and T3 gallbladder cancers were TSP1-positive, while none (0/14) of the T1 cancers showed TSP1 expression (p<0.001). Lymph node metastasis and venous involvement were frequently found in the TSP1-positive cases (90.0% and 87.1%, respectively) of gallbladder adenocarcinoma (p<0.001). These observations suggested that TSP1 expression plays an important role in cancer cell growth and metastasis of human gallbladder adenocarcinomas, and that stromal TSP1 immunoreactivity is a good predictor of vascular involvement and lymph node metastasis.

Adenocarcinoma↗

PubMed1999

Expression of angiostatic factors in colorectal cancer.

Angiogenesis plays an important role in growth and proliferation of cancer. Various angiogenic and angiostatic factors regulate angiogenesis. We examined expression of genes encoding various angiostatic factors: thrombospondin 1 (TSP1), thrombospondin 2 (TSP2), brain-specific angiogenesis inhibitor 1 (BAI1) and angiopoietin 2 (AGP2) in 62 colorectal cancers and 40 samples of extraneoplastic colon mucosa. The expression of the angiostatic factors TSP2 and AGP2 were significantly increased in the cancerous mucosa as compared to these in extraneoplastic mucosa (o2 test; p<0. 0001, and Fisher's exact test; p<0.0001), while the increase in TSP1 expression was not significant. BAI1 expression was slightly decreased in the cancer tissue. These results suggested that specific types of angiostatic factors might have protective roles against cancer cell proliferation via dormancy due to hyponutrition caused by decreased vascularity.

Angiogenesis Inhibitors↗

PubMed1999

How BCG led to the discovery of immunostimulatory DNA.

The concept of immunostimulatory DNA was borne in a long series of studies on BCG-mediated tumor resistance. DNA purified from BCG inhibited the growth of various syngeneic animal tumors, augmented NK cell activity and induced IFN-alpha/beta and -gamma from mouse spleen cells and human PBL. Extending the lines of study, we found two biologically remarkable facts that (i) DNAs from invertebrates, but not from vertebrates and plants, showed the above-mentioned biologic activities, and (ii) the activities were completely dependent on particular base sequences having CpG motifs but in a senseless manner. Details of those early studies carried out mainly in the 1980's have been reviewed in the first part of this paper. In the middle part of this review, the results of toxicity and pharmacology studies and clinical trials of BCG- DNA, performed by other groups in Japan in the late 1980's, were introduced. Since a large amount of DNA had never been administered repeatedly into experimental animals or human, those experiences obtained seem to be worthwhile to introduce. Research interests of immunostimulatory DNA were galvanized in 1995 by the report of Krieg et al. showing murine B cell activation with bacterial DNA containing CpG motifs. Within a short period of time, a huge number of papers have been published in this field, and the study has expanded rapidly and largely. Now, it includes a number of research fields, for example, host-defense mechanisms against infection, allergy, autoimmune diseases, cytokine networks, plasmid vaccination, and therapeutic application of certain diseases. This paper reviewed briefly recent advances of immunostimulatory DNA research. The response of higher animals against immunostimulatory DNA must be the most primitive but important mechanism for self-nonself discrimination against foreign DNA. By utilizing immunostimulatory DNA or controlling this primitive response, it seems possible to offer many beneficial means to human health. For instance, more potent peptide- or plasmid- vaccines could be developed by the use of immunostimulatory DNA. On the other hand, many study results suggest that immunostimulatory DNA works either beneficially or harmfully for the hosts. We assume that further extensive and careful studies are required.

Adjuvants, Immunologic↗

PubMed1999

Expression of the thrombospondin 1 receptor CD36 is correlated with decreased stromal vascularisation in colon cancer.

Vascularisation is determined by the balance between various angiogenic and angio-inhibitory factors in cancer stroma. CD36 is a cell adhesion receptor shown to interact with the ligand thrombospondin 1 (TSP1). It has not been determined whether the interaction of TSP1 and its receptor CD36 is correlated with vascularisation or clinical outcome of malignant tumours in vivo. We studied the correlations between expression of TSP1, CD36 and vascularisation and prognosis in 65 colon cancers. Various levels of TSP1 gene expression were observed in 27 of 65 colon cancers. CD36 expression was detected in 33 of 65 cancers, and was significantly correlated with decreased stromal vascularisation (P<0.001). The colon cancers expressing CD36 showed better prognosis (P<0.01). TSP1 expression levels did not affect CD36 expression. These results suggested that CD36 expression which decreased stromal vascularisation is correlated with better prognosis of colon cancer.

CD36 Antigens↗

PubMed1998

Hammerhead ribozyme specifically inhibits mutant K-ras mRNA of human pancreatic cancer cells.

We have evaluated the efficacy of an anti-K-ras hammerhead ribozyme targeted against GUU-mutated codon 12 of the K-ras gene in a cell-free system as well as in cultured pancreatic carcinoma cell lines. In the cell-free system, the anti-K-ras ribozyme specifically cleaved K-ras RNA with GUU-mutation at codon 12, but not other triplet sequences at codon 12 of K-ras RNA. In the cell culture system, the anti-K-ras ribozyme significantly reduced K-ras mRNA level (GUU-mutated codon 12) in Capan-1 pancreatic carcinoma cells, but less significantly suppressed K-ras mRNA in Capan-2 (GUU/GGU heterozygous-mutation at codon 12) or MIA PaCa-2 (UGU-mutated codon 12) pancreatic carcinoma cells. The ribozyme inhibited proliferation of transfected Capan-1 cells. These results suggest that this ribozyme selectively recognizes single-base mutation of K-ras mRNA and is able to reverse the malignant phenotype in human pancreatic carcinoma cells.

Adenocarcinoma↗

PubMed1998

Novel angucycline compound with both antigastrin- and gastric mucosal protective-activities.

An angucycline series compound P371 A1 (1) from Streptomyces sp. P371 was established to have a novel structure comprising an ureido group at one of four sugar units on the basis of 2D NMR techniques. 1 exhibited an inhibitory activity against the pentagastrin-stimulated acid secretion as well as protective activities against HCl/ethanol- and indomethacin-induced gastric lesions.

Aminoglycosides↗