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Biomedical subjects

T Shimada

Publications and source records attributed to T Shimada.

At least 145 records · Page 8Linked to original sources

Developmental morphology of blood and lymphatic capillary networks in mammalian hearts, with special reference to three-dimensional architecture.

The development of blood and lymphatic capillaries in the cardiac ventricles was studied using the rat and monkey hearts from later gestation to adulthood. For scanning electron microscopy, the tissue was treated with ultrasonication followed by HCl in order to remove epicardial mesothelial cell, subepicardial connective tissue and basement membrane. At the end of fetal life and neonate, the blood capillaries ran in parallel between the bundles consisting of several cardiac myocytes with small size. Endothelial extensions were found frequently. Numerous pericytes with short processes were observed on the capillary wall. Blood capillaries in the pubertal myocardium ran between cardiac myocytes to form a dense network. Processes of pericytes became elongated along the capillaries. In the adult, the capillaries showed occasionally Y-shaped branching and H-shaped anastomoses. The lymphatic capillaries were identified by large, variable sizes and absence of pericytes. In early postnatal rats, they appeared in the subepicardial region. In young and adult animals, the lymphatic capillary networks with relatively dense reticular meshes surrounded the bundles of several myocytes. It was noted that numerous nerve fibers were in close to the lymphatic capillary wall.

Aging↗

[An elderly woman with breast cancer and multiple liver metastasis that responded well to combination therapy of fadrozole and tamoxifen].

Few reports suggest a clinical benefit from combination treatment of fadrozole and tamoxifen for advanced breast cancer in elderly patients. We report the case of an 82-year-old breast cancer patient with multiple liver metastasis. After mastectomy, combination treatment of fadrozole and tamoxifen was added. Two months after the start of this treatment, there was a remarkable reduction in the size of metastatic lesions that continued over 6 months. CT examination revealed the largest lesion was reduced from 8.0 cm to 5.0 cm in largest diameter. The other two lesions were reduced from 3.0 cm to 2.0 cm. The reduction rate was 36%, indicating PR in the Response Evaluation Criteria in Solid Tumors (RECIST). The tumor marker CEA was remarkably reduced from 318 to 85 (ng/ml), and CA15-3 was reduced from 430 to 150 (U/ml). Tumor marker reduction continued over the 6 months corresponding to CT findings. No adverse effect was experienced. This combination therapy was useful and safe against metastatic breast cancer in a patient over 80 years of age.

Administration, Oral↗

[Not Available].

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Journal Article↗

Hepatocyte growth factor protects gastric epithelial cells against ceramide-induced apoptosis through induction of cyclooxygenase-2.

Forced overexpression of cyclooxygenase-2 (COX-2) in intestinal cells has been shown to be associated with resistance to apoptosis. However, the role of physiologically-induced COX-2 in the regulation of apoptosis remains unclear. In the present study, we examined whether hepatocyte growth factor (HGF)-induced COX-2 affects ceramide-induced apoptosis in RGM-1 gastric epithelial cells. An externally applied cell permeable ceramide analogue, C2-ceramide, caused RGM-1 cell death in a dose-dependent manner, whereas an inactive ceramide analogue, C2-dihydroceramide, did not. TdT-mediated dUTP nick end labeling (TUNEL) assay showed that the C2-ceramide-induced cell death was apoptosis. Application of HGF rapidly induced the expression of COX-2, and HGF prevented the apoptotic cell death induced by C2-ceramide. However, the anti-apoptotic action of HGF was antagonized by coapplication of NS-398, a selective inhibitor of COX-2. Thus, these results indicate that COX-2 is involved in the survival signaling from HGF in gastric epithelial cells, and suggest a role for physiologically-induced COX-2 in the protection of the cells from apoptosis.

Animals↗

The chitinase gene of the silkworm, Bombyx mori, contains a novel Tc-like transposable element.

We have determined the cDNA sequence and the genomic organization of the chitinase gene of the silkworm, Bombyx mori. The cDNA encodes 544 amino acids having 83% amino acid homology to the chitinase of the tobacoo hornworm, Manduca sexta. The total length of the gene is larger than 25 kilobase pairs, and it is separated into 11 exons. The intron-exon boundaries are all in accordance with the GT-AG rule. Also, the TATA box sequence was found in the 5' upstream region of the gene, and the gene is mapped on the seventh chromosome. A novel DNA type transposon that shows similarity to the Tc-like element was found in the third intron in some strains of B. mori; other strains, however, lack this element in the same intron. This element has long terminal inverted repeats, presumably encodes a transposase of about 340 amino acids with a DDE motif, and has an amino-terminal domain with a strong nuclear localization function. Seven other transposable elements with homologous but distinct sequences were isolated from the B. mori genome. Together with plaque hybridization results, our findings suggest that these novel elements exist in multiple copies constituting a new Tc-like transposable element family in the silkworm genome.

Amino Acid Sequence↗

Contribution of endogenous nitric oxide to basal vasomotor tone of peripheral vessels and plasma B-type natriuretic peptide levels in patients with congestive heart failure.

OBJECTIVES: We examined whether a relationship exists between the vasoconstrictive response to endogenous nitric oxide (NO) synthesis inhibition and the severity of heart failure in patients with congestive heart failure (CHF). BACKGROUND: Controversy exists as to whether the vasoconstrictive response to NO synthesis inhibition in patients with CHF is comparable to that in normal subjects or is enhanced. METHODS: Forearm blood flow (FBF) and calculated forearm vascular conductance (FVC) were obtained using plethysmography before and after administration of the NO synthesis inhibitor L-NMMA (NG-monomethyl-L-arginine) in 40 patients with CHF due to dilated cardiomyopathy and in 16 normal control subjects. Basal plasma B-type natriuretic peptide (BNP) and nitric oxide concentrations were measured in all subjects. RESULTS: Plasma BNP and nitrite/nitrate (NOx) levels in the patients group were significantly greater and baseline FBF was significantly less. Administration of L-NMMA significantly decreased FBF and FVC in both groups. The percent changes in FBF (%FBF) and FVC (%FVC) from the baseline after L-NMMA correlated significantly with plasma BNP level (%FBF: r = 0.72; %FVC: r = 0.76; both p < 0.001). Percent changes in both FBF and FVC were greater in patients with BNP > or = 100 pg/ml than in normal subjects; however, in patients with BNP < 100 pg/ml they were comparable to those in normal subjects. CONCLUSIONS: Vasoconstrictive response to L-NMMA in patients with CHF was preserved or enhanced in proportion to the basal plasma BNP level, indicating a close relationship between the contribution of endogenous NO to basal vasomotor tone and the severity of heart failure.

Adult↗

Non-Sonogashira-type palladium-catalyzed coupling reactions of terminal alkynes assisted by silver(I) oxide or tetrabutylammonium fluoride.

Palladium-catalyzed reaction of aryl and alkenyl halides with terminal alkynes in the presence of silver(I) oxide as an activator furnishes various arylated or alkenylated alkynes in good to excellent yields. The similar coupling reaction is also found to proceed using tetrabutylammonium fluoride (TBAF) or tetrabutylammonium hydroxide (TBAOH) as an activator.

Journal Article↗

DNA vaccination of macaques by a full genome HIV-1 plasmid which produces noninfectious virus particles.

In this study, we tried a DNA vaccination regime in rhesus macaques using a full genome HIV-1 plasmid. The HIV-1 genome is under the control of its original LTR promoter, but has a mutated zinc finger motif gene in the nucleocapsid region. Due to the lack of genomic RNA packaging, the plasmid produces only noninfectious viral particles. We repeatedly injected four macaque monkeys intramuscularly with the naked DNA over a period of 40 weeks. To evaluate the humoral and cell-mediated immunity provided by this DNA vaccination, no other booster or other recombinant viral vectors were used. Immunological responses against HIV-1 were elicited in all of the vaccinated monkeys: stable anti-HIV-1 Env antibodies were raised in two monkeys and CTL activities were induced in the other monkeys. The macaques were intravenously challenged at 54 weeks with 100 TCID(50) of SHIV-NM-3rN, which possesses an envelope gene homologous to the one in the vaccinated plasmid. In all of the vaccinated macaques, the peak plasma viral loads induced by the challenge virus were two to three orders of magnitude lower than those of the naive controls. These results suggest that a DNA vaccination regime with a full genome plasmid alone is potentially efficacious and provides a new possibility for the development of an AIDS vaccine.

AIDS Vaccines↗

Targeted and stable gene delivery into muscle cells by a two-step transfer system.

We developed a muscle-specific gene delivery system based on two-step gene transfer. The first step involved adenovirus-mediated transfer of the ecotropic retrovirus receptor (EcoRec) gene driven by the muscle-specific desmin promoter. Both human primary myoblasts and fibroblasts were efficiently transduced with this adenovirus vector. However, expression of EcoRec was detected only in myoblasts. In the second step, EcoRec-expressing myoblasts could be stably transduced with the ecotropic retroviral vector with the beta-galactosidase gene. Approximately 15% of myoblasts were transduced by this two-step strategy. When the transduced myoblasts were differentiated into myotubes, extensive cell-cell fusion occurred, and the apparent number of beta-galactosidase-positive cells increased to 28%. These results indicate that our two-step gene delivery system could be used for targeted and stable gene transfer into muscle cells.

Adenoviridae↗

Decreased UV sensitivity, mismatch repair activity and abnormal cell cycle checkpoints in skin cancer cell lines derived from UVB-irradiated XPA-deficient mice.

Xeroderma pigmentosum group A gene (XPA)-deficient mice are defective in nucleotide excision repair (NER) and are therefore highly sensitive to ultraviolet (UV)-induced skin carcinogenesis. We established cell lines from skin cancers of UVB-irradiated XPA-deficient mice to investigate the phenotypic changes occurring during skin carcinogenesis. As anticipated, the skin cancer cell lines were devoid of NER activity but were less sensitive to killing by UV-irradiation than the XPA(-/-) fibroblast cell line. The lines were also more resistant to 6-thioguanine (6-TG) than XPA(-/-) and XPA(+/+) fibroblasts, which was suggestive of a mismatch repair (MMR) defect. Indeed, in vitro mismatch binding and MMR activity were impaired in several of these cell lines. Moreover, these cell lines displayed cell cycle checkpoint derangements following UV-irradiation and 6-TG exposure. The above findings suggest that MMR downregulation may help cells escape killing by UVB, as was seen previously for methylating agents and cisplatin, and thus that MMR deficient clones are selected for during the tumorigenic transformation of XPA(-/-) cells.

Animals↗

A retroviral vector capable of targeted gene transfer into cells expressing HIV envelope glycoprotein.

An expression vector encoding a chimeric envelope protein composed of CD4 and ecotropic retroviral envelope glycoprotein was constructed with the aim of accomplishing targeted gene transfer into HIV-1-infected cells. The chimeric protein was efficiently expressed and transported to the surfaces of various cell types and supported HIV-1 entry into human cells. A packaging cell line producing retroviral vectors carrying chimeric envelope proteins was then established. The vector particles produced were shown to be capable of specific gene transfer into human cells expressing HIV envelope glycoprotein. Blocking experiments confirmed that the vector particles entered the cells via an interaction between the chimeric and HIV envelope proteins. This targeting vector may thus be a useful tool with which to develop effective gene therapies against HIV infection.

Animals↗

cDNA cloning of acyl-CoA desaturase homologs in the silkworm, Bombyx mori.

We have isolated two acyl-CoA desaturase clones from a pheromone gland cDNA library by using the EST (expressed sequence tag) database of Bombyx mori. The putative acyl-CoA desaturases encoded by the clones desat 1 (2029bp) and desat 2 (2341bp) have 98% identity, and both proteins show 61% identities to Trichoplusia ni acyl-CoA Delta(11) desaturase. The deduced amino acid sequences conserve well the histidine clusters that are catalytically essential for acyl-CoA desaturase activity. Northern blot and RT-PCR analyses revealed that both transcripts of desat 1 and desat 2 were expressed predominantly in the pheromone gland. Both transcripts detected 3days before adult eclosion dramatically increased a day before adult eclosion, keeping the mRNA levels high even after eclosion. These results, combined with the fact that Delta(11) and Delta(10, 12) desaturation of palmitate is a key step to synthesize pheromone in B. mori, suggest that the desaturases encoded by desat 1 and desat 2 are involved in either or both of the desaturation steps in the pheromone biosynthetic pathway of B. mori. The mRNA levels of desat 1 and desat 2 were not affected by decapitation or injection of the pheromone biosynthesis activating neuropeptide (PBAN) into the adult female moth, suggesting that the transcription of desat 1 and desat 2 is not regulated by PBAN. In addition to the clones in the pheromone gland, eight other clones encoding the same Delta(9) desaturase homolog were found in an embryonic cDNA library by searching from the EST database of B. mori. The deduced amino acid sequence from one of the clones (desat 3) shows 79% identity to T. ni Delta(9) desaturase but only 52% identity to the desaturases in the pheromone gland of B. mori. Northern blot analysis showed that the mRNA corresponding to the desat 3 was detected in the ovary and fat body, but not in the pheromone gland. Abundance of the Delta(9) desaturase clones (eight out of the 762 randomly sequenced clones) in the library prepared from diapause-destined embryos (40h after oviposition) suggests that the Delta(9) desaturase encoded by desat 3 plays an important role in embryonic development in B. mori.

Amino Acid Sequence↗

Synthesis and Physical Properties of Sterically Congested Cycloalkenes, 1,2-Di-tert-butyl-3,3,5,5-tetramethylcyclopentene and 1,2-Di-tert-butyl-3,3,6,6-tetramethylcyclohexene.

Two sterically congested cycloalkenes (9 and 10), congeners of tetra-tert-butylethylene, were synthesized and characterized. Oxidation of the bicyclic 1,3-dithietane 8 with dimethyldioxirane (DMD) gave the endo,endo-disulfoxide 13, thermal isomerization of which to the endo,exo-disulfoxide 15 followed by oxidation with DMD gave the trioxide 18. Heating 18 in refluxing 1,3-dimethyl-2-imidazolidinone furnished 1,2-di-tert-butyl-3,3,5,5-tetramethylcyclopentene (9) in 69% yield by a 2-fold extrusion process. The reaction of the 1,6-diketone dihydrazone 23 with Se(2)Cl(2) gave the selenadiazoline 34 and the 1,3-diselenetane 35. Heating 34 at 115-130 degrees C gave 1,2-di-tert-butyl-3,3,6,6-tetramethylcyclohexene (10), a "didehydro" derivative of tetra-tert-butylethylene, in 43% yield. The C=C bond in 10 is strained in degree comparable to those of most strained alkenes reported so far.

Journal Article↗

Role of apoptosis induction in both peripheral lymph nodes and thymus in progressive loss of CD4+ cells in SHIV-infected macaques.

To investigate the role of apoptosis in the progressive loss of CD4+ lymphocytes in HIV infection, we have used macaques infected with SHIV, a hybrid virus of HIV and simian immunodeficiency virus (SIV). In the present study, we sequentially analyzed apoptosis induction in the acute phase of SHIV infection. Four macaques infected with a pathogenic SHIV, SHIV89.6P, and four macaques infected with a nonpathogenic SHIV, NM-3rN, were analyzed during the first 2 or 4 weeks postinfection. In the 89.6P-infected macaques the number of peripheral CD4+ cells sharply decreased at 2 weeks postinfection and was maintained below 50/microl until 4 weeks postinfection, while in the NM-3rN-infected macques the number of the CD4+ cells did not change significantly. Plasma viral loads peaked at 2 and 2-3 weeks postinfection, and the peak values were about 1 x 10(9) and 10(6)-10(7) copies/ml in the 89.6P- and the NM-3rN-infected macaques, respectively. In the 89.6P-infected macaques, Fas antigen expression and the extent of apoptosis in PBMCs and peripheral lymph nodes increased at 1-2 weeks postinfection. A high number of apoptotic cells was also observed in thymus sections 2 and 4 weeks postinfection. On the other hand, apoptosis was scarcely induced in the NM-3rN-infected macaques. These results suggest that the extent of apoptosis induction is closely correlated with the pathogenicity of SHIV and that the apoptosis induction in peripheral lymphoid tissues and thymus, where T cell maturation occurs, may play an important role in the depletion of CD4+ lymphocytes in 89.6P infection.

Acquired Immunodeficiency Syndrome↗

Use of genetically engineered Salmonella typhimurium OY1002/1A2 strain coexpressing human cytochrome P450 1A2 and NADPH-cytochrome P450 reductase and bacterial O-acetyltransferase in SOS/umu assay.

The major pathway of bioactivation of procarcinogenic heterocyclic aromatic amines (HCAs) is cytochrome P450 1A2 (CYP1A2)-catalyzed N-hydroxylation and subsequent esterification by O-acetyltransferase (O-AT). We have previously reported that an umu tester strain, Salmonella typhimurium OY1001/1A2, endogenously coexpressing human CYP1A2 and NADPH-P450 reductase (reductase), is able to detect the genotoxicity of some aromatic amines [Aryal et al., 1999, Mutat Res 442:113-120]. To further enhance the sensitivity of the strain toward HCAs, we developed S. typhimurium OY1002/1A2 by introducing pCW"/1A2:hNPR (a bicistronic construct coexpressing human P450 1A2 and the reductase) and pOA102 (constructed by subcloning the Salmonella O-AT gene in the pOA101-expressing umuC"lacZ gene) in S. typhimurium TA1535. In addition, as an O-AT-deficient strain, we developed the OY1003/1A2 strain by introducing pCW"/1A2:hNPR and pOA101 into O-AT-deficient S. typhimurium TA1535/1,8-DNP. Strains OY1001/1A2, OY1002/1A2, and OY1003/1A2 expressed, respectively, about 150, 120, and 140 nmol CYP1A2/l culture (in whole cells), and respective cytosolic preparations acetylated 15, 125, and > or = 0 nmol isoniazid/min/mg protein as the O-AT activities of cytosolic preparations, respectively. We compared the induction of umuC gene expression as a measure of genotoxicity and observed that the OY1002/1A2 strain was more sensitive than OY1001/1A2 strain toward the genotoxicity of 2-amino-1,4-dimethylimidazo[4,5-f]quinol ine(MeIQ), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ),2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx),2-aminoanthracene, 2-amino-6-methyldipyrido[1,2-a::3,2'-d]i midazole,3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole, and 3-amino-1-methyl-5H-pyrido[4, 3-a]indole. However, the genotoxicity of MeIQ, IQ, and MeIQx was not detected with the OY1003/1A2 strain. These results indicate that the newly developed strain OY1002/1A2 can be employed in detecting potential genotoxic aromatic amines requiring bioactivation by CYP1A2 and O-acetyltransferase.

Acetyltransferases↗

Roles of NADPH-P450 reductase in the O-deethylation of 7-ethoxycoumarin by recombinant human cytochrome P450 1B1 variants in Escherichia coli.

Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants were used to reconstitute 7-ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E. coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems. In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E. coli coexpressing CYP1B1 and the reductase proteins. Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required. However, in membranes of bicistronic constructs, there was no additional stimulation of 7-ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was <0.5. These results suggest that NADPH-P450 reductase produced in the bacterial membranes is more active in interacting with CYP1B1 proteins in the bicistronic system than the reductase added to artificial phospholipid vesicles or bacterial membranes.

Alkylation↗

Characterization of liver microsomal 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation activities in Japanese and Caucasian subjects genotyped for CYP2E1 gene.

To determine whether the CYP2E1 genetic polymorphisms cause alterations in protein expression and enzyme catalytic activities, three CYP2E1 genetic polymorphisms, namely RsaI/PstI, DraI, and MspI types, were determined in liver genomic DNA isolated from 39 Japanese and 45 Caucasians. These genotypes were compared with levels of CYP2E1 and activities of 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation in liver microsomes from these human samples. In combination of three types of CYP2E1 polymorphisms, it was classified into seven genotypes in the Japanese population and four in the Caucasian population. The incidence in the occurrence of RsaI/PstI polymorphism or DraI polymorphism was 0.24 and 0.29 for Japanese, and 0.01 and 0.02 for Caucasians. Ethnic difference was also noted in the MspI polymorphism in which frequencies in Japanese and Caucasian populations were 0.15 and 0.02, respectively. Studies with liver microsomes showed that there were no significant differences in the levels of expression of CYP2E1 protein between wild-type (group A) and other 6 genotypes (B, C, D, E, F, and G) in Japanese and other three genotypes (B, D, and F) in Caucasians. Catalytic activities for 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation by liver microsomes were also found to be less significantly affected by mutations in the CYP2E1 gene in human samples examined in this study. These results support the view that RsaI/PstI, DraI, and MspI types of CYP2E1 genetic polymorphisms may not cause significant alterations in protein expression and enzyme catalytic activities of CYP2E1 enzyme in human livers.

Asian People↗