Biomedical subjects

T Tsuchida

Publications and source records attributed to T Tsuchida.

At least 109 records · Page 6Linked to original sources

PubMed1998

Hammerhead ribozymes against gamma-glutamylcysteine synthetase mRNA down-regulate intracellular glutathione concentration of mouse islet cells.

gamma-Glutamylcysteine synthetase (gamma-GCS) is a key enzyme in glutathione synthesis and is thought to play a significant role in intracellular detoxification systems. To specifically suppress gamma-GCS gene expression, we constructed two different hammerhead ribozymes against gamma-GCS mRNA transcripts. Two cleavage sites were targeted as follows: site 1 for anti-gamma-GCS ribozyme (H), a GUU triplet located from +348 to +350 of the gamma-GCS heavy chain, and site 2 for anti-gamma-GCS ribozyme (L), a GUU triplet located from +235 to +237 of the gamma-GCS light chain. The anti-gamma-GCS ribozymes effectively cleaved gamma-GCS mRNA in a cell-free system. When transfected into a Min-6 mouse islet cell line, these anti-gamma-GCS ribozymes not only suppressed gamma-GCS gene expression, but also reduced intracellular glutathione concentration. These results suggest that the ribozyme-mediated down-regulation of gamma-GCS gene expression may be useful for analyzing the glutathione-associated cellular defense systems of pancreatic islet cells.

Animals↗

PubMed1998

Control of expression by the cellulose synthase (bcsA) promoter region from Acetobacter xylinum BPR 2001.

The 5' upstream region (about 3.1kb) of the cellulose synthase operon (bcs operon) has been isolated by cloning from Acetobacter xylinum strain BPR 2001. The expression level of the upstream region was determined using sucrose synthase cDNA as a reporter gene in the shuttle vector pSA19. The expression occurred with the 1.1-kb upstream sequence from the ATG start codon of the bcs operon but not with the 241-bp upstream sequence in A. xylinum, although neither the 1.1-kb nor the 241-bp upstream sequence caused any expression as a promoter in Escherichia coli. The level of expression with the 1. 1-kb upstream sequence in A. aceti was 75% of that in A. xylinum. These results suggest that the upstream region functions as a specific promoter for the Acetobacter genus. The expression was reduced by the introduction of the 241-bp upstream region between the lac promoter and the reporter gene in E. coli and was not detected in A. xylinum. This suggests that the short upstream region composed of 241bp contains the site(s) which causes a negative regulation on the transcription for bcs operon. The production of recombinant protein with the ribosome-binding site (RBS) of A. xylinum obtained from the bcs operon, was reduced to about half in E. coli, and that with the site of the lac promoter was also reduced to about half in A. xylinum. This shows that a species-specific predominance occurs during interaction between mRNA and 16S rRNA in the RBS between A. xylinum and E. coli.

Amino Acid Sequence↗

PubMed1998

Telomerase activity in lung cancer cells obtained from bronchial washings.

BACKGROUND: Telomerase, a ribonucleoprotein enzyme that functions in the maintenance of telomeres (specialized structures at the ends of chromosomes), has been reported to be a novel diagnostic marker for malignant diseases. We sought to determine whether measurement of telomerase activity in bronchial washings is of value in the diagnosis of lung cancer. METHODS: Extracts of cells in bronchial washings were analyzed for telomerase activity by use of a telomeric repeat amplification protocol (TRAP) assay. Telomerase activity inside cells was evaluated by use of an in situ TRAP assay. The results of both TRAP assays were compared with those obtained from cytologic examination, which employed standard Papanicolaou staining. RESULTS: When results from the two TRAP assays were combined, telomerase activity was detected in bronchial washings from 18 (82%; 95% confidence interval [CI] = 60%-95%) of 22 patients with lung cancer. In contrast, cancer cells were detected by cytologic examination in the bronchial washings of nine (41%; 95% CI = 21%-64%) of the same 22 patients, a statistically significant difference (two-sided P = .0061). In patients with lung cancer, telomerase-positive cells could be detected in bronchial washings irrespective of tumor location--11 of 14 (79%; 95% CI = 49%-95%) peripheral cancerous lesions and seven of eight (88%; 95% CI = 47%-100%) central cancerous lesions were detected by use of TRAP assays (for comparison, two-sided P = .5349). CONCLUSIONS: A high percentage of patients with lung cancers had detectable telomerase activity in bronchial washings. Thus, the use of a cell extract-based or an in situ TRAP assay in addition to cytologic examination may make the diagnosis of lung cancer more reliable.

Adult↗

PubMed1998

Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor gene expression.

BACKGROUND: Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene is frequently associated with malignant gliomas. One type of EGFR mutation in primary gliomas results in overexpression of an aberrant EGFR messenger RNA (mRNA) that lacks sequences of exons II through VI of the human EGFR gene. We observed that the aberrantly spliced EGFR mRNA contains a ribozyme cleavable sequence (5'-AAG GUA AUU-3') created by the joining of EGFR exon I to exon VII. We hypothesized that an appropriately designed ribozyme RNA could mediate site-specific cleavage of the aberrant EGFR mRNA and reduce the growth of aberrant EGFR-producing tumor cells. METHODS: We synthesized aberrant EGFR mRNA substrates and a sequence-specific hammerhead ribozyme (abEGFR-rib) to examine the ribozyme's activity in vitro. We also constructed an abEGFR-rib plasmid and introduced it into ERM5-1 cells, which are murine NIH3T3 cells transfected to express an aberrant EGFR complementary DNA. We measured the growth potential of the cotransfected cells in culture and in nude mice. RESULTS: The synthesized abEGFR-rib efficiently and specifically cleaved aberrant EGFR mRNA substrates in vitro. Expression of the transfected abEGFR-rib suppressed expression of aberrant EGFR mRNA in ERM5-1 cells and reduced the growth of tumors formed by the cotransfected cells in nude mice. Finally, the incorporation of bromodeoxyuridine, a measure of mitotic activity, was also decreased in abEGFR-rib-producing ERM5-1 cells in vivo. CONCLUSION: Ribozymes targeted to aberrant EGFR mRNA can inhibit the growth of tumors formed by cells that express this mRNA.

Animals↗

PubMed1998

Colcemid-induced apoptosis of cultured human glioma: electron microscopic and confocal laser microscopic observation of cells sorted in different phases of cell cycle.

The effect of the antitubulin agent colcemid on human glioma cells was investigated by sorting cells with different DNA content and subjecting them to confocal laser microscopy and transmission electron microscopy. The human glioma cell line U251MG was exposed to colcemid at a concentration of 0.05 microg for 16 h. Flow cytometric analysis revealed the accumulation of cells in S/G2M phase. Cells harvested from each of G0/G1 and S/G2M peaks were then analyzed by confocal laser microscopy and transmission electron microscopy. Confocal laser microscopy revealed that colcemid-treated cells harvested from the G0/G1 peak contained mitotic and apoptotic cells in addition to interphase cells. Electron microscopy confirmed that colcemid-treated cells in the G0/G1 peak had fragmented nuclei typical of apoptotic cells and mitotic cells with altered chromatin structure. Some mitotic cells obtained by mitotic shake-off after treatment with colcemid showed DNA strand breaks defined by in situ nick end labeling. The present study indicates that mitotic as well as interphase apoptosis occurs in U251MG cells following colcemid treatment.

Apoptosis↗

PubMed1998

Non-invasive estimation of the net influx constant using the standardized uptake value for quantification of FDG uptake of tumours.

To reduce the variability of the standardized uptake value (SUV) which is widely used to evaluate 2-[18F]fluoro-2-deoxy-d-glucose (FDG) uptake by neoplasms, net influx constant (Ki) was derived from SUV. The relationship Ki=SUV.kp.V0, where kp is the plasma clearance rate and V0 is the initial distribution volume of FDG, was utilized. A total of 71 plasma input functions were measured up to 60 min after intravenous injection of FDG in 55 patients and were analysed to obtain kp and V0. SUV and V0 were calculated based on either body weight or body surface area. To validate the Ki estimation, another group of eight patients with squamous cell carcinoma of the head and neck was included. Parametric images of the net influx constant were obtained by Patlak graphical analysis of dynamic positron emission tomography (PET) data and measured plasma input function. V0 based on body weight was 0.1627+/-0.0329 (ml/g) and showed a weak negative correlation with body weight (y=0.23356-0.00138x, r=0.591). V0 based on body surface area was 5540+/-871 (ml/m2) and had no significant correlation with body weight. kp at 50 min post injection was 0. 03272+/-0.00243 (1/min), and had no correlation with the plasma glucose concentration. A highly significant positive correlation was noted between true Ki and estimated Ki based on both body weight (y=0.0033+1.0371x, r2=0.897), and body surface area (y=0.0033+1. 0351x, r2=0.926). Ki, a better indicator of FDG uptake by tumour than SUV, is derivable non-invasively. Quantification of FDG uptake by Ki will aid standardization of diagnostic criteria of FDG PET oncology.

Carcinoma, Squamous Cell↗

PubMed1998

Alterations in tumour suppressor gene p53 correlate with inhibition of thrombospondin-1 gene expression in colon cancer cells.

If activation of the p53 gene is involved in the progression or metastasis of colon cancer, it may affect the angiogenic phenotype in vivo. To verify this hypothesis, we studied the correlation between p53 accumulation and expression of thrombospondin-1 (TSP1) in colon cancer specimens. Levels of TSP1 gene expression were estimated by Northern blotting in 65 colon cancers. Accumulation of p53 and the distribution of TSP1 protein were evaluated immunohistochemically. Various levels of TSP1 gene expression were seen in colon cancers, while p53 accumulation was confirmed in 42 of the 65 colon cancers. The level of TSP1 gene expression demonstrated a significant inverse correlation with p53 accumulation in colon cancer. Colon cancer cells expressed TSP1 protein and p53 accumulation reciprocally in the same nests. These results suggest that alterations in the tumour suppressor gene p53 may inhibit TSP1 expression in colon cancer.

Adenocarcinoma↗

PubMed1998

Tibial deformities and failures of anterior cruciate ligament reconstruction in immature rabbits.

We evaluated the effects of anterior cruciate ligament (ACL) reconstruction on growth and development of the knee in immature New Zealand white rabbits (8 weeks old). In 25 rabbits, the ACL was reconstructed with an autogenous iliotibial band (ITB), and in 31 rabbits an autogenous patellar tendon bone (PTB) graft was used. Reconstructed ACLs failed in 20 of the 25 animals in the ITB group and in 26 of 31 in the PTB group. Gross and histological examinations in the animals with successfully reconstructed ACLs (5 in the ITB group and 5 in the PTB group) showed significant tibial deformity and tibial shortening, with progressive thinning of the epiphyseal plate and disorders of the cartilage column on the medial side of the tibia. These abnormalities were not observed in rabbits whose ACLs were torn. This experiment showed that successful ACL reconstruction in young animals with an open physis may result in deformities and/or shortening of the leg. Accordingly, we believe that special care should be taken when surgically reconstructing an ACL in a young patient whose physes are still open.

Animals↗

PubMed1998

Vascular endothelial growth factor (VEGF) mRNA isoform expression pattern is correlated with liver metastasis and poor prognosis in colon cancer.

Vascular endothelial growth factor (VEGF) is a well known factor that induces angiogenesis. Four isoforms, i.e. VEGF206, 189, 165, and 121, have been identified. We examined the isoform patterns of VEGF mRNA using reverse transcription polymerase chain reaction (RT-PCR) analysis in 61 colon cancers. All the colon cancers examined expressed VEGF121. The isoform patterns were classified into three groups: type 1, VEGF121; type 2, VEGF121 + VEGF165; type 3, VEGF121 + VEGF165 + VEGF189. Three of the 61 colon cancers examined showed type 1 expression, 26 showed type 2 expression and 32 showed the type 3 pattern. The patients with liver metastases showed the type 3 isoform expression pattern at a significantly higher incidence (12 of 16, 75%) than those without liver metastasis (20 of 45, 44%) (P=0.036). The type 3 isoform pattern was significantly associated with M1 stage (P=0.019). The patients with colon cancer and the type 3 isoform pattern showed significantly poor prognosis (P < 0.01, Cox-Mantel). The colon cancers with the type 3 pattern showed a significantly higher involvement of veins (P=0.006). These observations suggest that the aberrant type 3 expression pattern of VEGF189 mRNA isoforms is correlated with liver metastasis, M stage, and poor prognosis in colon cancer.

Adenocarcinoma↗

PubMed1998

An increase in apparent affinity for sucrose of mung bean sucrose synthase is caused by in vitro phosphorylation or directed mutagenesis of Ser11.

A mutational analysis of mung bean (Vigna radiata Wilczek) sucrose synthase was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli, in which two different acidic amino acid residues (Asp or Glu) were introduced at Ser11 (S11D, S11E). Only the wild-type enzyme (Ser11) was phosphorylated in vitro by a Ca(2+)-dependent protein kinase from soybean root nodules, suggesting that this is the specific target residue in mung bean sucrose synthase. The apparent affinity for sucrose was increased in this phosphorylated enzyme and also in the S11D and S11E mutant enzymes, although the affinities for UDP-glucose and fructose were similar in the wild-type, phosphorylated wild-type, and mutant enzymes. These results suggest that a monoanionic (1-) side chain at position 11 mimics the Ser11-P2- residue to bind and cleave sucrose for the synthesis of UDP-glucose. Since the S11E mutant enzyme showed the lowest K(m) (sucrose) and the highest catalytic efficiency of the recombinant proteins, the enzymic properties of this S11E mutant were further characterized. The results showed that replacement of Ser11 with Glu11 modestly protected the sucrose synthesis activity against phenolic glycosides and altered the enzyme nucleotide specificity. We postulate that the introduction of negative charge at Ser11 is possibly involved in the enzymatic perturbation of sucrose synthase.

Amino Acid Sequence↗

PubMed1998

Alterations of p53 gene and Ha-ras gene are independent events in solar keratosis and squamous cell carcinoma.

In order to clarity the multiple-step progression from solar keratosis to squamous cell carcinoma, aberrations of the p53 gene (exons 2-11) and ras genes (exons 1 and 2) in solar keratosis and squamous cell carcinoma were investigated by polymerase chain reaction and single-strand conformation polymorphism analysis. In a series of Japanese patients, eight of 27 (30%) samples of solar keratosis and three of six (50%) samples of squamous cell carcinoma showed structural abnormalities in the p53 gene. Only one solar keratosis (4%) showed a point mutation in the Ha-ras gene but not in the p53 gene. Among these cases, no mutation of ras genes could be detected in squamous cell carcinoma. Simultaneous mutation of ras genes and the p53 gene was not detected in any cases of either solar keratosis or squamous cell carcinoma. It is concluded that aberrations of the p53 gene and ras genes are induced through independent processes of ultraviolet irradiation in the course of carcinogenic change from solar keratosis to squamous cell carcinoma.

Carcinoma, Squamous Cell↗

PubMed1998

Effects of homologous atrial natriuretic peptide on drinking and plasma ANG II level in eels.

The effects of eel atrial natriuretic peptide (ANP) on drinking were investigated in eels adapted to freshwater (FW) or seawater (SW) or in FW eels whose drinking was stimulated by a 2-ml hemorrhage. An intra-arterial infusion of ANP (0.3-3.0 pmol . kg-1 . min-1), which increased plasma ANP level 1.5- to 20-fold, inhibited drinking dose dependently in all groups of eels. The drinking rate recovered to the level before ANP infusion within 2 h after infusate was replaced by saline. The inhibition at 3.0 pmol . kg-1 . min-1 was profound in FW eels and hemorrhaged FW eels, whereas significant drinking still remained after inhibition in SW eels. Plasma ANG II concentration also decreased dose dependently during ANP infusion and recovered to the initial level after saline infusion in all groups of eels. The decrease at 3.0 pmol . kg-1 . min-1 was large in FW eels and hemorrhaged FW eels compared with that of SW eels. Thus the changes in drinking rate and plasma ANG II level were parallel during ANP infusion. Plasma sodium concentration and osmolality decreased during ANP infusion in SW and FW eels, and they were restored after saline infusion. In hemorrhaged FW eels, however, ANP infusion did not alter plasma sodium concentration and osmolality. Hematocrit did not change during ANP infusion in any group of eels. Collectively, ANP infusion at physiological doses decreased drinking rate and plasma ANG II concentration in parallel in both FW and SW eels. It remains undetermined whether the inhibition of drinking is caused by direct action of ANP or through inhibition of ANG II, which is known as a potent dipsogen in all vertebrate species, including eels.

Angiotensin II↗

PubMed1998

Increased cellulose production from sucrose with reduced levan accumulation by an Acetobacter strain harboring a recombinant plasmid.

Cellulose production from sucrose by Acetobacter strains is accompanied by the accumulation of a water-soluble polysaccharide, called levan. To improve cellulose productivity, a levansucrase-deficient mutant, LD-2, was derived from Acetobacter strain 757 and used as a host for the construction of recombinant strains. An LD-2 mutant harboring a plasmid containing the sucrase gene, sucZE3, from Zymomonas mobilis together with zliS, a gene that encodes a secretion-activating factor under the control of the Escherichia coli lac promoter, had sucrase activity and produced much cellulose and little levan in a medium containing sucrose. In addition, a mutant levansucrase gene, mutant sacB, from Bacillus subtilis, which encodes a protein with little levan-forming activity, was generated by site-directed mutagenesis and introduced into the LD-2 mutant. This introduction also resulted in the higher cellulose productivity and little levan.

Acetobacter↗

PubMed1998

Brain-specific angiogenesis inhibitor 1 expression is inversely correlated with vascularity and distant metastasis of colorectal cancer.

Brain-specific angiogenesis inhibitor (BAI) 1 was recently isolated as a novel p53 inducible gene. BAI1 has been suggested to play a significant role in angiostasis. We studied the expression of BAI1 in 49 colorectal cancer specimens by RT-PCR. BAI1 expression was significantly reduced in colorectal cancers as compared to the extraneoplastic tissues (X(2) test, p=0.041). BAI1 expression was inversely correlated with vascular invasion and metastasis (Fisher's exact test, p 0.045). Moreover, vascularity in the colorectal cancer was inversely correlated with BAI1 gene expression (Mann-Whitney U-test, p=0.0003). These observations suggested that BAI1 expression might inhibit angiogenesis and metastasis of colorectal cancer.

Angiogenesis Inhibitors↗

PubMed1998

Cremimycin, a novel 19-membered macrocyclic lactam antibiotic, from Streptomyces sp.

A novel 19-membered macrocyclic lactam antibiotic, cremimycin, was isolated from the culture broth of an actinomycete strain. The producing organism, designated MJ635-86F5, was identified as a member of Streptomyces. Cremimycin was isolated from the mycelial cake by extraction with CHCl3-MeOH and precipitation with hexane-MeOH. The structure of cremimycin was determined by spectroscopic study. Cremimycin showed broad antimicrobial activities against Gram-positive bacteria including MRSA.

Animals↗

PubMed1998

Glucose transporter protein-independent tumor cell accumulation of fluorine-18-AFDG, a lipophilic fluorine-18-FDG analog.

UNLABELLED: Fluorine-18-fluorodeoxyglucose (FDG) is used clinically for tumor diagnosis, but its mechanism of accumulation in tumor cells is complicated because two factors, glucose transporter protein (GLUT) and hexokinase, govern [18F]FDG uptake directly. We selected a lipophilic [18F]FDG analog, 1,3,4,6-tetra-acetyl-2-[18F]-2-deoxy-D-glucose ([18F]AFDG), to regulate the effects of hexokinase and evaluated its characteristics in an in vitro cell culture system. METHODS: Fluorine-18-AFDG was synthesized by the method used to produce [18F]FDG, as an intermediate of [18F]FDG. Fluorine-18-AFDG uptake study was performed with LS180 tumor cells, and its metabolites were also investigated by thin-layer chromatography. To evaluate the relationship between [18F]AFDG and GLUT, we also examined [18F]AFDG uptake in the presence of cytochalasin B or with increased medium glucose concentration. The effects of lowered temperature (4 degrees C) on [18F]AFDG uptake were also investigated. RESULTS: Fluorine-18-AFDG (lipophilicity: octanol/water = 3.5) uptake was 3.3-fold higher than that of [18F]FDG. Metabolic analysis showed that [18F]AFDG was extremely stable in the incubation medium but was quickly hydrolyzed and metabolized to 2-fluoro-[18F]-2-deoxy-D-glucose-6-phosphate ([18F]FDG-6P) in tumor cells. Fluorine-18-FDG-6P accounted for approximately 45% of the total radioactivity after a 60-min incubation of [18F]AFDG. Incubation with 50 microM cytochalasin B did not affect [18F]AFDG uptake. In medium with double the control glucose level, [18F]FDG uptake was decreased by about 50%, but [18F]AFDG uptake was not affected. Fluorine-18-AFDG uptake and [18F]FDG-6P production did not show saturation and increased linearly with addition of a 10-fold higher concentration of [18F]AFDG. Lowered incubation temperature caused decreased [18F]AFDG uptake due to reduced [18F]FDG-6P production. CONCLUSION: Fluorine-18-AFDG rapidly penetrated the cell membrane as a result of its high lipophilicity and was metabolized to [18F]FDG-6P within cells. Fluorine-18-AFDG was thus characterized as "GLUT-independent [18F]FDG."

Cytochalasin B↗

PubMed1998

Fluorine-18-fluorodeoxyglucose PET versus thallium-201 scintigraphy evaluation of thyroid tumors.

UNLABELLED: To determine whether PET could help differentiate malignant from benign thyroid tumors, 18F-fluorodeoxyglucose (FDG) accumulation and 201Tl scintigraphy were examined relative to histological diagnosis. METHODS: Nodular thyroid lesions in 11 patients were evaluated before surgical resection. Static PET scanning with 370 MBq FDG was done for 20 min (from 40 to 60 min postinjection) in all patients, and standardized uptake values (SUVs) in these lesions were calculated. In addition, eight patients were evaluated with dynamic PET scan up to 60 min postinjection, and the lesions were further evaluated using graphical analysis. Thallium-201 delayed images were visually evaluated in 10 patients. RESULTS: Four of 11 nodules were well-differentiated papillary carcinoma, another five were benign follicular adenomas, one was a multinodular goiter and another a case of chronic thyroiditis that was proved not to contain a nodule. Time-activity curves of FDG uptake showed different patterns in malignant and benign tumors. In the malignant tumors, FDG uptake increased with time after the tracer injection. By contrast, FDG uptake in benign tumors gradually decreased. With use of a cutoff value of 5.0 mg/ml for SUV and 10 microl x min(-1) x ml(-1) for Kc (K complex value determined using the linear fitting of the time-activity curve of FDG accumulation), all of the four malignant nodules and the six benign nodules were separated correctly. Chronic thyroiditis had high SUV in the malignant range. Of the four patients with thyroid carcinoma, the delayed 201Tl images revealed a slightly higher or equal uptake to background activity. In a patient with chronic thyroiditis, the delayed 201Tl images revealed diffuse accumulation higher than background activity. CONCLUSION: FDG-PET is superior to 201Tl in differentiating malignant from benign tumors. Both SUVs and Kc values were useful indexes for this discrimination. Although careful evaluation is needed for chronic inflammatory lesions, this technique appears to be useful in evaluating thyroid nodules.

Adenoma↗

PubMed1998

Tissue plasminogen activator (T-PA) and plasminogen activator inhibitor (PAI-1) levels in the conditioned medium of cultured porcine brain microvascular endothelial cells.

Vascular endothelial cells are known to produce tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1). However, the release kinetics of these substances by brain microvascular endothelial cells (BMEC) is less well known. BMEC prepared from porcine brains using enzymatic digestion and percoll gradient separation was exposed to culture medium containing thrombin (1.0IU/ml), histamine (10 mumol/ml), and TN-alpha (500, 1000, 2000U/ml). The supernatant obtained at respective time points was subjected to ELISA for t-PA, PAI-1 and t-PA-PAI-1 complex. The value of these substances were significantly higher than those of the controls at respective time points. It also proved that t-PA and PAI-1 production by BMEC increases in a parallel fashion. The present study suggests that t-PA production by BMEC cultured under given stimulation increases, however, it is accompanied by concomitant PAI-1 production.

Animals↗