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Biomedical subjects

M Wada

Publications and source records attributed to M Wada.

At least 199 records · Page 11Linked to original sources
PubMed1999

Expression of vascular endothelial growth factor and its receptor (KDR/flk-1) mRNA in experimental choroidal neovascularization.

PURPOSE: Vascular endothelial growth factor (VEGF) is an angiogenic peptide that has been suggested to be important in the pathogenesis of choroidal neovascularization. We investigated the transcription of VEGF and its receptor KDR/flk-1 genes during the development of experimentally induced choroidal neovascularization. METHODS: Rat VEGF or KDR cDNA was inserted in PGEM or pBluescript to prepare antisense or sense riboprobes. Multiple krypton laser burns were applied to the posterior pole of pigmented rat eyes according to a previously described protocol which produces choroidal neovascularization. At intervals of up to 4 weeks after photocoagulation, the eyes were removed and cut into thin sections. The sections were subjected to in situ hybridization with digoxigenin (DIG)-labeled single-strand rat VEGF and KDR cDNA riboprobes. RESULTS: In normal adult rat retinas, VEGF and KDR mRNA expression was mainly observed in the ganglion cell and the inner nuclear layers. During the development of neovascularization, VEGF and KDR mRNAs were detected in retinal pigment epithelial-like cells, fibroblast-like cells and endothelial cells in neovascular lesions. The level of expression was strongest at 1 week after photocoagulation in lasered lesions, and decreased by 4 weeks after photocoagulation. CONCLUSIONS: Our findings demonstrate that expression of VEGF and its receptor KDR may play a role in the formation of experimentally induced choroidal neovascularization. In this study, VEGF and its receptor were co-localized, suggesting that an autocrine and/or paracrine mechanism may be operative.

Animals↗
PubMed1999

Phosphorothioate oligonucleotides induction into experimental choroidal neovascularization by HVJ-liposome system.

PURPOSE: The purpose of this study was to determine whether the inactivated hemagglutinating virus of Japan (HVJ)-liposome method can induce phosphorothioate oligonucleotides effectively into an experimentally-induced choroidal neovascularization of rats. We also examined whether antisense phosphorothioate oligonucleotides against VEGF could be induced into choroidal neovascularization as a therapeutic agent by the HVJ-liposome method. METHODS: The experiments were conducted on a rat model of choroidal neovascularization. FITC-labeled phosphorothioate oligonucleotides were coencapsulated in liposomes. The liposomes were coated with the envelope of inactivated HVJ and injected into the vitreous cavity following photocoagulation of pigmented rat eyes. The eyes were removed following injection, fixed, frozen and cut into thin sections. Induction of oligonucleotides was observed under a laser confocal scanning microscope for fluorescence and the development of choroidal neovascularization was evaluated histopathologically. RESULTS: Phosphorothioate oligonucleotides were effectively induced into ganglion cells and into the cells of the choroidal neovascularization induced by laser photocoagulation. Highly effective induction of oligos was observed 3 to 14 days after intravitreal injection of HVJ-liposomes after which the level decreased. Antisense oligonucleotides against VEGF were induced specifically into cells in the choroidal neovascularization, however neovascularization was still observed. CONCLUSIONS: Phosphorothioate oligonucleotides can be effectively induced into ganglion cells, and specifically into cells in choroidal neovascularization. Although antisense oligonucleotides against VEGF failed to prevent choroidal neovascularization, the HVJ-liposome method provided a highly effective means of inducing antisense oligos for in vivo antisense therapy.

Animals↗
PubMed1999

Genomic structure of the canalicular multispecific organic anion-transporter gene (MRP2/cMOAT) and mutations in the ATP-binding-cassette region in Dubin-Johnson syndrome.

Dubin-Johnson syndrome (DJS) is an autosomal recessive disease characterized by conjugated hyperbilirubinemia. Previous studies of the defects in the human canalicular multispecific organic anion transporter gene (MRP2/cMOAT) in patients with DJS have suggested that the gene defects are responsible for DJS. In this study, we determined the exon/intron structure of the human MRP2/cMOAT gene and further characterized mutations in patients with DJS. The human MRP2/cMOAT gene contains 32 exons, and it has a structure that is highly conserved with that of another ATP-binding-cassette gene, that for a multidrug resistance-associated protein. We then identified three mutations, including two novel ones. All mutations identified to date are in the cytoplasmic domain, which includes the two ATP-binding cassettes and the linker region, or adjacent putative transmembrane domain. Our results confirm that MRP2/cMOAT is the gene responsible for DJS. The finding that mutations are concentrated in the first ATP-binding-cassette domain strongly suggests that a disruption of this region is a critical route to loss of function.

ATP-Binding Cassette Transporters↗
PubMed1999

Serum levels of thyroid-stimulating hormone receptor antibodies during IFN-alpha2a treatment of chronic hepatitis C.

Twenty chronic hepatitis C patients with baseline levels of thyroid hormones, antithyroglobulin, and antimicrosomal antibodies within the normal range were monitored by thyroid testing during a 26-week treatment with interferon-alpha2a (IFN-alpha2a). The present study was aimed at retrospectively analyzing thyroid-stimulating hormone (TSH) receptor antibodies, employing stored serum samples obtained from these patients at baseline, at 12 weeks, and at the end of IFN-alpha2a treatment. Ten patients (group A) received IFN-alpha2a at a total dose of 474 million units (MU), and 10 patients (group B) at a total dose of 774 MU. None of the patients produced antithyroglobulin and antimicrosomal antibodies. Two patients in group A exhibited elevated TSH levels without free thyroid hormone alterations while on the treatment. At baseline, 1 patient in group A had a positive TSH binding inhibitory immunoglobulin, and 1 patient in group B was positive for thyroid-stimulating antibody at the end of treatment. Both patients remained euthyroid throughout the treatment. Treatment with IFN-alpha2a may infrequently induce the production of TSH receptor antibodies in chronic hepatitis C patients provided preexisting autoimmune thyroid disease is precluded.

Adult↗
PubMed1999

Stroke incidence and case fatality in Shiga, Japan 1989-1993.

BACKGROUND: This paper describes incidence rates and case-fatality for sub-types of stroke using data collected in Takashima, Shiga, Japan, from 1989 to 1993 and compares these with similar registers in other parts of Japan. METHODS: Registered patients included all residents of the county who experienced a first-ever stroke. Stroke was defined as sudden onset of neurological symptoms which continued for a minimum of 24 hours or led to death. Almost all such patients are hospitalized in this country. Early case fatality was defined as patients who died within 28 days of stroke onset. Diagnosis of stroke type was based on clinical symptoms as well as computed tomography (CT) scans. RESULTS: Age-adjusted incidence rates for stroke per 100,000 population aged 35 years and older were 268.7 for men and 167.5 for women. The age-specific incidence rate of both cerebral infarction and cerebral haemorrhage increased with advancing age. The occurrence of cerebral infarction in men was twice as high as in women. The 28-day case fatality for all sub-types of stroke was 16.1% in men and 15.8% in women. Case fatality for cerebral infarction, cerebral haemorrhage, and subarachnoid haemorrhage was 10.7%, 22.4% and 28.6% respectively. CONCLUSIONS: Takashima County has a moderately high stroke incidence rate and case fatality compared with other similar studies in Japan. The incidence rate of cerebral infarction in men is twice that in women, while other sub-types of stroke showed smaller differences. In order to decrease the incidence of stroke in Japan, greater efforts at primary prevention will be necessary, in particular, it is important to prevent cerebral infarction in men.

Adult↗
PubMed1999

Femoral intercondylar notch measurements in osteoarthritic knees.

METHODS: We measured the dimensions of the intercondylar notch of the femur in 32 patients with primary severe osteoarthrosis (OA) of the knee and 54 embalmed cadaveric knees. RESULTS: There were 56 knees with morphologically normal anterior cruciate ligament (ACL), 11 knees with lax or partially ruptured ACL and 19 knees with missing ACL. The average width of the intercondylar notch in knees with lax and missing ACL was significantly narrower than that of knees with normal ACL. In addition, knees with missing ACL had a significantly smaller notch depth than knees with normal ACL. In medial compartment OA (56 knees), the notch width and depth in knees with severe OA (37 knees) were significantly smaller than those in normal (19 knees) and mild to moderate OA groups (19 knees). CONCLUSION: Our results indicate that osteophyte growth in the femoral intercondylar notch seems to correlate with the progression of medial compartment OA of the knee.

Aged↗
PubMed1999

The calcium receptor and calcimimetics.

Parathyroid cells can sense small changes in plasma Ca2+ levels by virtue of a cell surface Ca2+ receptor. Calcimimetics are newly synthesized compounds that act as agonists or positive allosteric modulators at the Ca2+ receptor and can suppress parathyroid hormone secretion. The first-generation calcimimetic, NPS R-568, has undergone clinical trials in primary hyperparathyroidism and in hyperparathyroidism secondary to chronic renal insufficiency. The data accumulated so far demonstrate that calcimimetics have potential as therapeutic agents for hyperparathyroidism and related bone diseases such as osteitis fibrosa.

Aniline Compounds↗
PubMed1999

Chloroplast-avoidance response induced by high-fluence blue light in prothallial cells of the fern adiantum capillus-veneris as analyzed by microbeam irradiation

Chloroplast movement was induced by partial cell illumination using a high-fluence blue microbeam in light-grown and dark-adapted prothallial cells of the fern Adiantum capillus-veneris. Chloroplasts inside the illuminated area moved out (high-fluence response [HFR]), whereas those outside moved toward the irradiated area (low-fluence response [LFR]), although they stopped moving when they reached the border. These results indicate that both HFR and LFR signals are generated by high-fluence blue light of the same area, and that an LFR signal can be transferred long-distance from the beam spot, although an HFR signal cannot. The lifetime of the HFR signal was calculated from the traces of chloroplast movement induced by a brief pulse from a high-fluence blue microbeam to be about 6 min. This is very short compared with that of the LFR (30-40 min; T. Kagawa, M. Wada [1994] J Plant Res 107: 389-398). These data indicate that the signal transduction pathways of the HFR and the LFR must be distinct.

Journal Article↗
PubMed1999

Mechanically induced avoidance response of chloroplasts in fern protonemal cells.

Cell response to mechanical stimulation was investigated at a subcellular level in protonemal cells of the fern Adiantum capillus-veneris L. by pressing a small part of the cell with a microcapillary. In cells receiving local stimulation, the chloroplasts moved away from the site of stimulation, whereas the nuclei failed to show such avoidance movement. Mechanical stimulation for a period as short as 0.3 min was enough to induce the avoidance response to a maximal level. The avoidance movement of chloroplasts started within 30 min and the plateau level of avoidance was attained around 2 h after stimulation. By tracing the movement of chloroplasts during the response, it was shown that the mobility of chloroplasts near the stimulation site increased transiently within 1 h after the stimulation. After 2 to 3 h, it slowed down to the control level without stimulation. The avoidance response was inhibited by 0.1 mM cytochalasin B and 25 mM 2, 3-butanedione monoxime but not by 3.3 microM amiprophosmethyl or 5 mM colchicine. These findings indicate that the protonemal cells were very sensitive to mechanical stimulation and that chloroplasts moved away from the mechanically stimulated site through the actomyosin motile system.

Actomyosin↗
PubMed1999

Effect of multidrug resistance-reversing agents on transporting activity of human canalicular multispecific organic anion transporter.

The canalicular multispecific organic anion transporter (cMOAT), also termed MRP2, is a recently identified ATP-binding cassette transporter. We previously established stable human cMOAT cDNA-transfected cells, LLC/cMOAT-1 from LLC-PK1 cells, and LLC/CMV cells that were transfected with an empty vector. We found that LLC/cMOAT-1 cells have increased resistance to vincristine (VCR), 7-ethyl-10-hydroxy-camptothecin, and cisplatin but not to etoposide. The multidrug resistance-reversing agents cyclosporin A (CsA) and 2-[4-(diphenylmethyl)-1-piperazinyl]-5-(trans-4,6-dimethyl-1,3, 2-dioxaphosphorinan-2-yl)-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P) almost completely reversed the resistance to VCR, 7-ethyl-10-hydroxy-camptothecin, and cisplatin of LLC/cMOAT-1 cells; and DL-buthionine-(S,R)-sulfoximine, (3'-oxo-4-butenyl-4-methyl-threonine(1), (valine(2)) cyclosporin (PSC833), and 3-([(3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl)-((3-dimethylamino-3- oxopropyl)-thio)-methyl]thio)propanoic acid (MK571) partially reversed the resistance to these drugs. CsA and PAK-104P at 10 microM enhanced the accumulation of VCR in LLC/cMOAT-1 cells almost to the level in LLC/CMV cells without the agents. The efflux of VCR from LLC/cMOAT-1 cells was enhanced compared with LLC/CMV cells and inhibited by CsA and PAK-104P. Transport of leukotriene C(4) (LTC(4)) and S-(2, 4-dinitrophenyl)glutathione also was studied with membrane vesicles prepared from these cells. LTC(4) and S-(2, 4-dinitrophenyl)glutathione were actively transported into membrane vesicles prepared from LLC/cMOAT-1 cells. The K(m) and V(max) values for the uptake of LTC(4) by the LLC/cMOAT-1 membrane vesicles were 0. 26 +/- 0.05 microM and 7.48 +/- 0.67 pmol/min/mg protein, respectively. LTC(4) transport was competitively inhibited by PAK-104P, CsA, MK571, and PSC833, with K(i) values of 3.7, 4.7, 13.1, and 28.9 microM, respectively. These findings demonstrate that cMOAT confers a novel drug-resistance phenotype. CsA and PAK-104P may be useful for reversing cMOAT-mediated drug resistance in tumors.

Adenosine Triphosphate↗
PubMed1999

Purification and characterization of monovalent cation-activated levodione reductase from Corynebacterium aquaticum M-13.

(6R)-2,2,6-Trimethyl-1,4-cyclohexanedione (levodione) reductase was isolated from a cell extract of the soil isolate Corynebacterium aquaticum M-13. This enzyme catalyzed regio- and stereoselective reduction of levodione to (4R,6R)-4-hydroxy-2,2, 6-trimethylcyclohexanone (actinol). The relative molecular mass of the enzyme was estimated to be 142,000 Da by high-performance gel permeation chromatography and 36,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required NAD(+) or NADH as a cofactor, and it catalyzed reversible oxidoreduction between actinol and levodione. The enzyme was highly activated by monovalent cations, such as K(+), Na(+), and NH(4)(+). The NH(2)-terminal and partial amino acid sequences of the enzyme showed that it belongs to the short-chain alcohol dehydrogenase/reductase family. This is the first report of levodione reductase.

Amino Acid Sequence↗
PubMed1999

Age effect on expression of myosin heavy and light chain isoforms in suspended rat soleus muscle.

This study was designed to test the hypothesis that myosin heavy (MHC) and light chain (MLC) plasticity resulting from hindlimb suspension (HS) is an age-dependent process. By using an electrophoretic technique, the distribution of MHC and MLC isoforms was quantitatively evaluated in the soleus muscles from 3- or 12-wk-old rats after 1-3 wk of HS treatment was maintained. In normal 12- and 15-wk-old rats, the soleus muscles contained a predominance of MHCI ( approximately 94%) with small amounts of MHCIIa, but not MHCIId or MHCIIb. The suspended muscles of adult rats were characterized by the appearance of MHCIIb and MHCIId, the latter reaching approximately 6% after 3 wk of HS treatment. In contrast to changes in MHC, HS did not induce a transition in the MLC pattern in the soleus muscles from adult rats. Compared with adult rats, in juveniles HS had a much more pronounced effect on the shift toward faster MHC and MLC isoform expression. The soleus muscles of 6-wk-old rats after 3 wk of HS were composed of 37.0% MHCI, 19.1% MHCIIa, 23.7% MHCIId, and 20.2% MHCIIb. Changes in MLC isoforms consisted of an increase in MLC1f and MLC2f concomitant with a decrease in MLC2s. These results indicate the existence of a differential effect of HS on MHC and MLC transitions that appears to be age dependent. They also suggest that the suspended soleus muscles from young rats may acquire the intrinsic contractile properties that are intermediate between those in the normal soleus and typical fast-twitch skeletal muscles.

Aging↗
PubMed1999

Immunohistochemical analysis of Bcl-2, Bax, Bcl-X, and Mcl-1 expression in pancreatic cancers.

Expression of several members of the Bcl-2 family proteins was investigated by means of both immunohistochemical analysis in 30 invasive ductal adenocarcinomas and 23 intraductal papillary-mucinous tumors (IPMTs) and immunoblot analysis in 6 cancer tissues and 7 pancreatic cancer cell lines. We found that Bcl-2 was expressed in 23%, Bax in 53%, Bcl-X in 90%, and Mcl-1 in 90% of the invasive ductal adenocarcinomas. In intraductal papillary-mucinous adenocarcinomas, the expression rate of Bax was 44% and those of Bcl-XL and Mcl-1 were 88%; these values were higher than those for intraductal papillary-mucinous adenomas. Immunoblot analysis identified Bcl-XL as the predominant form of the Bcl-X protein in both pancreatic cancer tissues and cell lines, and demonstrated that both Bcl-XL and Mcl-1 protein levels were uniformly high in all cell lines. These results suggest that an imbalance between antiapoptosis proteins (such as Bcl-2, Bcl-XL, and Mcl-1) and proapoptotic proteins (such as Bax and Bcl-Xs) is involved in the distinctive biologic features of adenocarcinomas of the pancreas. Furthermore, predominantly high expressions of Bcl-XL and Mcl-1 in intraductal papillary-mucinous adenocarcinomas might be involved in the carcinogenesis in IPMT of the pancreas.

Adenocarcinoma↗
PubMed1999

Dietary intake and sources of isoflavones among Japanese.

We examined the dietary intake and sources of isoflavones (daidzein and genistein) among Japanese subjects based on dietary records (DRs). The subjects comprised two groups: 1,232 who completed one-day DRs (Group 1) and 88 men and women who kept four four-day (16-day) DRs. For quantitative data on the level of daidzein and genistein in soy foods, we extensively reviewed the literature, particularly for Japanese soy foods, and adopted the median value for each food. The median intake of daidzein was 12.1 and 9.5 mg/day among Groups 1 and 2, respectively, while the corresponding values for genistein were 19.6 and 14.9 mg/day. The top four foods (tofu, miso, natto, and fried tofu) covered about 90% of the population intake of daidzein and genistein. It did not seem feasible to estimate one's intake of isoflavones by using dietary recording/recall in epidemiological studies, since the day-to-day variation in intake was too large, the within-person coefficient of variation being 89.1% for daidzein and genistein. Therefore, we should use other methods, such as food-frequency questionnaires, focusing on the four major sources of isoflavones, to assess individual isoflavone intake.

Diet↗
PubMed1999

The 20-kilodalton (kDa) human growth hormone (hGH) differs from the 22-kDa hGH in the effect on the human prolactin receptor.

Previously we have demonstrated that 20-kDa human GH (20K-hGH) is a full agonist for hGH receptor (hGHR) even though its complex formation with hGHR and hGH-binding protein differs from that of 22-kDa human GH (22K-hGH). In this study, we focused on the effect of 20K-hGH on human PRL receptor (hPRLR). To elucidate the effects of 20K-hGH on hPRLR and compare them with those of 22K-hGH, we prepared two cells stably expressing full-length hPRLR, Ba/F3-hPRLR and CHO-hPRLR. In the proliferation of Ba/F3-hPRLR cells, which can grow in a dose-response to lactogenic hormones, both 20K- and 22K-hGH exhibited bell-shaped curves in the absence of exogenous zinc ion (Zn2+); however, the curve of 20K-hGH was shifted to a 10-fold higher concentration than that of 22K-hGH in view of EC50 value (the EC50 of 20K- and 22K-hGH were 15 nM and 1.5 nM, respectively). Addition of Zn2+ up to 25 microM increased the activities of both 20K- and 22K-hGH; however, the enhancement by Zn2+ was greater in 20K-hGH than in 22K-hGH, thereby the activities of both hGH isoforms reached the same level at 25 microM Zn2+. Nevertheless, in the presence of 0.25-1 microM free Zn2+, which is equal in human serum, the activity of 20K-hGH was still lower than that of 22K-hGH. The modest effect of 20K-hGH on activating hPRLR in the absence of Zn2+ was confirmed in the rat serine protease inhibitor 2.1 (Spi2.1) gene promoter activation and JAK2/Stat5 tyrosine phosphorylation in CHO-hPRLR. In addition, in human breast cancer cell T-47D, 20K-hGH was proved to stimulate Stat5 tyrosine phosphorylation to much lower degree than 22K-hGH via not hGHR but hPRLR. Taken together, our data suggest that 20K-hGH may be a weaker agonist for hPRLR than 22K-hGH in the human body; therefore 20K-hGH may alleviate the hPRLR-mediated side-effects such as breast cancer when administered to human body.

Animals↗
PubMed1999

Primary structure of rat spermidine synthase: an example of refining the cDNA-derived amino acid sequence.

The primary structure of rat spermidine synthase having the N-terminal acetylated methionine and 98.7% homology with that of the mouse enzyme is presented using a limited amount of the homogeneous enzyme. The study strategy was principally to compare the molecular masses of liberated peptides determined by three specific cleavage methods with those expected from known cDNA-derived amino acid sequences of mouse and human enzymes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The cleavage methods involved two enzymatic methods using lysylendopeptidase and arginylendopeptidase, and a chemical method for cleaving at the cysteine residue using 2-nitro-5-thiocyanobenzoic acid. Their usefulness was clearly demonstrated. Column-switching semimicro reversed-phase HPLC, which permits application of the entire reaction mixture, was useful for collecting a small amount of peptides containing the N-terminal amino acid, to confirm acetylation of the N-terminal methionine by MALDI TOF-MS. It was necessary in this approach to examine the amino acid sequence of certain peptides. The Edman method was used for the sequence analysis, and this will be replaced by an improved MALDI TOF-MS now available in a few laboratories.

Acetylation↗
PubMed1999

Effects of dietary protein type on the response of lipid metabolism to orotic acid in rats.

The effects of orotic acid supplementation to casein, egg protein, soy protein and wheat gluten diets on the lipids of liver and serum were compared. When orotic acid was added, the contents of total lipids and triacylglycerol in the liver of the casein group were significantly higher or tended to be higher than those of the other three dietary groups. Dietary orotic acid had no effect on the food intake. The liver weight, and liver total lipids, triacylglycerol, cholesterol and phospholipids were increased or tended to be increased by the addition of orotic acid. The serum triacylglycerol level was decreased by the addition of orotic acid to either the casein or soy protein diet. Thus, the response to liver lipid accumulation induced by orotic acid feeding depended on the dietary protein type.

Animals↗