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T Sakurai

Publications and source records attributed to T Sakurai.

At least 19 recordsLinked to original sources
PubMed2001

Biallelic inactivation of retinoic acid receptor beta2 gene by epigenetic change in breast cancer.

A growing body of evidence supports the hypotheses that retinoic acid receptor beta2 (RAR beta2) is a tumor suppressor gene. Although the loss of RAR beta2 expression has been reported in many malignant tumors, including breast cancer, the molecular mechanism is still poorly understood. We hypothesized that loss of RAR beta2 activity could result from multiple factors, including epigenetic modification and loss of heterozygosity (LOH). Using methylation-specific polymerase chain reaction and LOH analysis, we found that biallelic inactivation via epigenetic changes of both maternal and paternal alleles, or epigenetic modification of one allele combined with genetic loss of the remaining allele, could completely suppress RAR beta2 expression in breast cancer. Thus, it is possible that substantial numbers of human cancers arise through suppressor gene silencing via epigenetic mechanisms that inactivate both alleles. Because of this, chromatin-remodeling drugs may provide a novel strategy for cancer prevention and treatment.

Alleles↗
PubMed2001

Modulation of cell adhesion and viability of cultured murine bone marrow cells by arsenobetaine, a major organic arsenic compound in marine animals.

1. In this study, we investigated the biological effects of trimethyl (carboxymethyl) arsonium zwitterion, namely arsenobetaine (AsBe), which is a major organic arsenic compound in marine animals using murine bone marrow (BM) cells and compared them with those of an inorganic arsenical, sodium arsenite, in vitro. 2. Sodium arsenite showed strong cytotoxicity in BM cells, and its IC(50) was 6 microM. In contrast, AsBe significantly enhanced the viability of BM cells in a dose-dependent manner during a 72-h incubation; about a twofold increase in the viability of cells compared with that of control cells cultured with the medium alone was observed with a microM level of AsBe. 3. In morphological investigations, AsBe enhanced the numbers of large mature adherent cells, especially granulocytes, during a 72-h BM culture. When BM cells were cultured together with AsBe and a low dose (1 u ml(-1)) of recombinant murine granulocyte/macrophage colony-stimulating factor (rMu GM-CSF), significant additive-like increasing effects were observed on the numbers of both granulocytes and macrophages originated from BM cells. However, AsBe did not cause proliferation of BM cells at all as determined by colony-forming assay using a gelatinous medium. 4. These findings demonstrate the unique and potent biological effects in mammalian cells of AsBe, a major organic arsenic compound in various marine animals which are ingested daily as seafood in many countries.

Animals↗
PubMed2000

In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially localized jellyfish fluorescent protein gene.

We aimed to introduce foreign DNA into spermatogenic cells in the testis by injection of the DNA encoding jellyfish fluorescent proteins, green fluorescent protein (GFP) and yellow fluorescent protein (YFP) into the seminiferous tubules and in vivo electroporation. We obtained fluorescent spermatozoa only when using the gene of the YFP protein fused to a mitochondrial localization signal peptide. Intracytoplasmic injection into oocytes of these spermatozoa gave fluorescent fetuses and pups. Almost all of the individuals produced from fluorescent spermatozoa were transgenic. We confirmed integration of the gene into chromosomes and its transmission into offspring. This is the first report of gene transfer into germ cells and subsequent production of transgenic offspring.

Animals↗
PubMed2000

Exocytosis in the dissociated pancreatic acinar cells of the guinea pig directly visualized by VEC-DIC microscopy.

To elucidate the detailed process of exocytosis at the highest possible accuracy, we dissociated the pancreatic acinus of the guinea pig and observed zymogen granules under a video-enhanced contrast differential interference contrast (VEC-DIC) microscope. The preparation was thin enough to resolve each zymogen granule with the best clarity. When acinar cells were stimulated with ACh (20 microM), many zymogen granules near the lumen showed an abrupt light intensity change. For a period of 10 s immediately before exocytosis, zymogen granules neither shifted their position nor altered their shape within an accuracy of 38 nm. The time required for individual granules to change the light intensity (the releasing time) ranged from 0.15 to 0.70 s. After each response, the granule maintained its altered contrast for a few seconds until it was retrieved to a planar membrane. No compound exocytosis including granule-granule fusion was observed. We concluded that the exocytosis is not directly initiated by any supramolecular change but by a purely molecular event.

Acetylcholine↗
PubMed2000

Functional analysis of five endothelin-B receptor mutations found in human Hirschsprung disease patients.

Several missense mutations of the endothelin-B receptor (EDNRB) associated with Hirschsprung disease have recently been identified. Five mutated EDNRB (A183G, W276C, R319W, M374I and P383L) cDNAs were transiently expressed in several cell lines to examine the effects of these mutations. Ligand-receptor binding experiments demonstrated that all mutants examined here accept endothelins with a high affinity. Especially, the affinity of endothelins to P383L was increased. However, the number of binding sites of A183G, W276C and P383L was markedly decreased. The subcellular localization of these mutant receptors was the same as that of wild-type EDNRB, whereas the amount of protein of each mutant receptor was decreased. All mutant receptors were impaired in intracellular Ca(2+) mobilization. These findings indicate that these missense mutations result in loss of function of EDNRB, and may provide the molecular pathological basis of Hirschsprung disease in some individuals.

Animals↗
PubMed2000

Active allele loss of the androgen receptor gene contributes to loss of androgen receptor expression in female breast cancers.

Given the importance of androgen and androgen receptor (AR) in the control of female breast growth and the potential association with female breast cancer, we evaluated the AR expression in 114 female breast cancers and further analyzed why AR expression was lost. Immunohistochemical analysis revealed that 14.1% (17/114) of the tumors lost the AR expression completely. To unravel the molecular mechanism for AR expression loss, we first analyzed the somatic mutations in exons 2 through 8 of the AR gene by PCR-SSCP, but no mutation was detected. As the CAG repeats within exon 1 are also microsatellite markers, we then analyzed whether allelic loss was present. Interestingly, 11 of the 17 AR-negative tumors were heterozygous and 9 of them showed allelic loss. The lost allele was further demonstrated to be the active one by X-chromosome inactivation analysis. To confirm the immunohistochemical results, Northern and Western blot hybridization was performed and neither AR mRNA nor protein was detected in these AR-negative tumors. Loss of the active AR allele was strongly correlated with the AR expression loss (P = 0.0005). We conclude that AR expression loss is attributed to the active allele loss of AR gene in female breast cancers. Our finding may be also crucial in predicting and influencing the response of breast cancer to endocrine therapy.

Alleles↗
PubMed2000

Orexin-induced hyperlocomotion and stereotypy are mediated by the dopaminergic system.

We demonstrated involvement of the ventral tegmental area (VTA) dopaminergic system in orexin-induced hyperlocomotion and stereotypy in rats. In double-label immunohistochemical study of rat brain, we found that tyrosine hydroxylase (TH)-immunoreactive cells in the VTA received innervation from orexin immunoreactive-fibers. Orexin-A induced an increase in [Ca(2+)](i) in isolated A10 dopamine neurons in a dose-dependent manner. In behavioral studies, we found that orexin-A induced hyperlocomotion, stereotypy and grooming behavior when administered centrally in rats, and these effects were abolished by dopamine D(2) (haloperidol and sulpiride) or D(1) (SCH23390) antagonists. These results suggest that the orexin-induced hyperlocomotion, stereotypy and grooming behavior are mediated by the dopaminergic system and this pathway might be involved in orexin-induced emotional responses.

Animals↗
PubMed2000

Orexins suppress catecholamine synthesis and secretion in cultured PC12 cells.

New orexigenic peptides called orexin-A and -B have recently been described in neurons of the lateral hypothalamus and perifornical area. No orexins have been found in adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin-receptor 2 (OX2R) in the rat adrenal gland has been reported. To test the effects of orexins on peripheral organs, we investigated their effects on catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. Orexin-A and -B (100 nM) significantly reduced basal and PACAP-induced tyrosine hydroxylase (TH) (the rate-limiting enzyme in the biosynthesis of catecholamines) mRNA levels. Orexin-A and -B (100 nM) also significantly inhibited the PACAP-induced increase in the cAMP level, suggesting that the suppressive effect on TH mRNA is mediated, at least in part, by the cAMP/protein kinase A pathway. Furthermore, orexin-A and -B (100 nM) significantly suppressed basal and PACAP-induced dopamine secretion from PC12 cells. Next, we examined whether orexin receptors (OX1R, OX2R) were present in the rat adrenal gland and PC12 cells. In the adrenal glands, OX2R was as strongly expressed as in the hypothalamus, but OX1R was not detected. On the other hand, neither OX1R nor OX2R was expressed in PC12 cells. However, binding assays showed equal binding of orexin-A and -B to PC12 cells, suggesting the existence in these cells of some receptors for orexins. These results indicate that orexins suppress catecholamine release and synthesis, and that the inhibitory effect is mediated by the cAMP/protein kinase A pathway.

Adrenal Gland Neoplasms↗
PubMed2000

Living donor liver transplantation for fulminant hepatic failure.

BACKGROUND: Living donor liver transplantation (LDLT) was originally indicated only for elective cases of pediatric patients with end-stage liver disease. In Japan, however, where liver transplantation from brain-dead donor is performed very rarely, this indication has been expanded to emergency cases such as fulminant hepatic failure (FHF). METHODS: Thirty-eight patients with FHF were treated between May 1992 and April 1999. Causes of acute liver failure were non-A, non-B hepatitis in 27 patients, hepatitis B virus in seven, and hepatitis A virus, Epstein-Barr virus, herpes simplex virus, and chrome poisoning in one each. RESULTS: Four patients did not undergo LDLT because of severe brain damage or combined multiple organ failure. The remaining 34 patients underwent a total of 36 LDLTs, including two retransplantations; 16 children received transplants of 17 lateral segments, three children and eight adults transplants of 11 left lobes, and seven adults transplants of eight right lobes. A total of 15 recipients died, four of primary graft dysfunction, three of refractory acute rejection, two of pneumonia, and one each of ductopenic rejection, sepsis, aplastic anemis, recurrence of Epstein-Barr virus hepatitis, multiple organ failure by chrome poisoning, and unknown hepatic failure. Primary graft dysfunction developed in adult recipients with small-for-size graft transplants, whereas refractory acute rejection and ductopenic rejection occurred in six grafts each of children with non-A, non-B FHF. CONCLUSIONS: LDLT can be safely expanded to cases of FHF in adult patients. Primary graft dysfunction in adult recipients with small-for-size left lobe grafts can be overcome by using right lobes. However, refractory acute rejection and ductopenic rejection in children remain a major problem.

Adolescent↗
PubMed2000

Destabilization of nucleosomes by an unusual DNA conformation adopted by poly(dA) small middle dotpoly(dT) tracts in vivo.

Poly(dA) small middle dotpoly(dT) tracts are common and often found upstream of genes in eukaryotes. It has been suggested that poly(dA) small middle dotpoly(dT) promotes transcription in vivo by affecting nucleosome formation. On the other hand, in vitro studies show that poly(dA) small middle dotpoly(dT) can be easily incorporated into nucleosomes. Therefore, the roles of these tracts in nucleosome organization in vivo remain to be established. We have developed an assay system that can evaluate nucleosome formation in yeast cells, and demonstrated that relatively longer tracts such as A(15)TATA(16) and A(34) disrupt an array of positioned nucleosomes, whereas a shorter A(5)TATA(4) tract is incorporated in positioned nucleosomes of yeast minichromosomes. Thus, nucleosomes are destabilized by poly(dA) small middle dotpoly(dT) in vivo in a length-dependent manner. Furthermore, in vivo UV footprinting revealed that the longer tracts adopt an unusual DNA structure in yeast cells that corresponds to the B' conformation described in vitro. Our results support a mechanism in which a unique poly(dA) small middle dot poly(dT) conformation presets chromatin structure to which transcription factors are accessible.

Base Sequence↗
PubMed2000

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Journal Article↗
PubMed2000

Orexin-induced food intake involves neuropeptide Y pathway.

Orexins (orexin-A and -B) are recently identified neuropeptides, which are thought to be implicated in the regulation of feeding behavior. We used a NPY-Y1 receptor specific antagonist, BIBO3304, to examine whether NPY is involved in orexin-induced feeding behavior. Intracerebroventricular administration of orexin-A (10 nmol) induced food intake in rats (food intake for 3 h; vehicle 0.3+/-0.2 g vs. orexin-A 10 nmol, 4.0+/-0.5 g, n=4). Orexin-induced feeding behavior was partially inhibited by prior administration of BIBO3304 (3 h food intake: orexin-A 10 nmol, 4.0+/-0.5 g vs. BIBO3304 (60 microgram) + orexin-A 10 nmol, 2.2+/-0.2 g, n=4). A low dose of BIBO3304 (30 microgram) did not show a significant inhibitory effect. BIBO3457, an inactive enantiomer, used as a negative control, did not show any inhibitory effect on orexin-A-induced feeding behavior. Fos expression was observed in NPY-containing neurons in the arcuate nucleus 1 h after orexin-A (10 nmol) was administered intracerebroventricularly (control 0.3+/-0.08%, orexin-A 10.2+/-0.8%, n=5 rats/group). These observations suggest that NPY is involved in orexin-induced feeding behavior. However, BIBO3304 did not completely abolish the effect of orexin-A. These results suggest that orexin-A elicits feeding behavior partially via the NPY pathway. The NPY system could be the one of downstream pathways by which orexin-A induces feeding behavior. Another pathway may also be involved in orexin-A-induced feeding behavior, because BIBO3304 did not completely abolish orexin-A-induced feeding behavior.

Animals↗
PubMed2000

Purification and characterization of dissimilatory nitrate reductase from a denitrifying halophilic archaeon, Haloarcula marismortui.

Dissimilatory nitrate reductase was purified from a denitrifying halophilic archaeon, Haloarcula marismortui, to an electrophoretically homogeneous state. The purified enzyme was inferred to be a homotetramer composed of a 63 kDa polypeptide. The electron paramagnetic resonance spectrum of the purified enzyme revealed typical rhombic signals which were ascribed to Mo(V) in the Mo-molybdopterin complex. Like the bacterial membrane-bound (Nar-) enzyme, the purified enzyme supported the catalysis of chlorate. The enzyme was activated in extreme saline conditions and the values of k(cat) and K(m) toward nitrate were 145 s(-1) and 79 microM, respectively, in the presence of 2.0 M NaCl.

Bacteria↗
PubMed2000

Subtype-specific trafficking of endothelin receptors.

We investigated the subcellular localization of two endothelin receptors (ET(A)R and ET(B)R). To visualize these receptors directly, the C terminus of each receptor was fused to the N terminus of enhanced green fluorescent protein (designated as ETR-EGFP). When transiently expressed in various mammalian cell lines, ET(A)R-EGFP was predominantly localized on the plasma membrane. By contrast, ET(B)R-EGFP was, independent of ligand stimulation, predominantly localized on the intracellular vesicular structures containing Lamp-1. Immunoblot analyses revealed that at steady state ET(B)R-EGFP was highly degraded, and its degradation was inhibited by bafilomycin A(1). Antibody uptake experiments suggested that the ET(B)R-EGFP molecules were internalized from the plasma membrane. It is therefore likely that ET(B)R is first transported to the plasma membrane and then internalized, irrespective of ligand stimulation, to lysosomes where it undergoes proteolytic degradation. Exchanging the C-terminal cytoplasmic tails of the two ETRs revealed that the cytoplasmic tail is responsible for both the intracellular localization and the degradation of the receptors. Deletion of the extreme C-terminal 35 amino acids from both receptors allowed the receptor proteins to localize predominantly in the intracellular vesicles and to degrade. These observations indicate that the cytoplasmic tail of ET(A)R determines its plasma membrane localization. Stimulation with endothelin-1 increased the amount of intact ETR-EGFP fusion proteins without increasing their de novo synthesis, suggesting that binding of endothelin-1 stabilizes the ETRs.

Amino Acid Sequence↗
PubMed2000

Distribution and serum concentration of sisomicin released from fibrin glue-sealed dacron graft in the rat and human.

We investigated whether or not fibrin glue (FG) used as a sealant in vascular prostheses to prevent leakage might be useful as a carrier of antibiotics for the prevention of local graft infection. Sisomicin (SISO) was incorporated into fibrin glue (SISO-FG) and evaluated as to its safety and pharmacokinetics. SISO (1.75 mg) -FG Dacron grafts were implanted subcutaneously in the anterior abdominal region of Sprague-Dawley rats, and then the changes in SISO concentrations in the serum and in the tissue around the implantation sites were compared with those same sites in rats that had had intravenous injection of SISO (1.75 mg). The serum SISO concentrations were significantly lower in the SISO-FG Dacron graft group than they were in the intravenous injection group. However, until 4 h after implantation the SISO concentrations in the tissues around the implantation sites were significantly higher in the SISO-FG Dacron group than they were in the iv injection group, and the peak concentrations during that time were 5.8 times higher for the SISO-FG Dacron group than they were for the intravenous injection group. The ratio of the area under the tissue concentration time curve of SISO (AUC tissue) after implantation of the SISO-FG Dacron graft to that after intravenous injection of SISO was 13.08. Therefore, FG was considered to control the release of SISO into the serum and to maintain a high SISO concentration in the tissue around the implantation site. Clinically, SISO (45 mg) -FG was applied directly to the Dacron grafts implanted in 10 patients who underwent prosthetic vascular reconstruction. No graft infection was observed in any of the patients who received SISO-FG Dacron grafts. The mean serum concentration of SISO was 0.65+/-0.17 microg/mL after 1 h and 0.33+/- 0.21 microg/mL after 3 h. The results of these clinical applications are in close correlation with those of the animal experiment and suggest that FG is useful as a carrier of SISO, allowing its controlled release for the prevention of local infection.

Aged↗
PubMed2000

Factors affecting the survival of frozen-thawed mouse spermatozoa.

Mouse epididymal spermatozoa were frozen in solutions containing various compounds with different molecular weights, and the factors affecting the postthawing survival were examined. Monosaccharides (glucose, galactose) had almost no protective effect regardless of the concentration and the temperature of exposure. On the other hand, disaccharides (sucrose, trehalose) and trisaccharides (raffinose, melezitose) resulted in higher survival rates, especially at a concentration of around 0.35 mol/kg H(2)O (0.381-0.412 Osm/kg). Macromolecules, such as PVP10, Ficoll 70, bovine serum albumin, and skim milk had almost no effect, but compounds with a molecular weight of about 800, such as metrizamide and Nycodenz, had some protective effect. When a raffinose solution was supplemented with 10% metrizamide, resulting in an osmolality of approximately 0.400 Osm/kg, a high survival rate was obtained. Solutions at about 0.400 Osm/kg containing trehalose alone, trehalose + metrizamide, raffinose alone, and raffinose + metrizamide, were all effective for sperm freezing; frozen-thawed sperm could fertilize oocytes, and the resultant embryos could develop to live young after transfer. For freezing mouse spermatozoa, aqueous solutions at approximately 0.400 Osm/kg containing a disaccharide or a trisaccharide seem to be effective.

Animals↗
PubMed2000

Angiotensin II type 1 receptor blockade abolishes specific K(ATP)channel gene expression in rats with myocardial ischemia.

The cardiac ATP-sensitive potassium (K(ATP)) channel is potentially composed of an inward rectifier potassium channel (Kir6.1 and/or Kir6.2) subunit and the cardiac type of sulfonylurea receptor (SUR2A). We reported that cardiac Kir6.1 mRNA and protein are specifically upregulated in the non-ischemic as well as the ischemic regions in rats with myocardial ischemia, suggesting that humoral and/or hemodynamic factors are responsible for this regulation. In the present study, pretreatment with TCV-116, an angiotensin (Ang) II type 1 receptor antagonist, completely inhibited the upregulation of Kir6.1 mRNA and protein expression in both regions of rat hearts subjected to 60 min of coronary artery occlusion followed by 24 h of reperfusion; whereas pretreatment with lisinopril, an Ang converting enzyme (ACE) inhibitor, partly inhibited this upregulation. Except for rats pretreated with TCV-116, Kir6.1 mRNA levels were positively correlated with those for brain natriuretic peptide (BNP), a molecular indicator of regional wall stress, in both the non-ischemic and the ischemic regions. Plasma Ang II levels were not elevated in rats with control myocardial ischemia compared with sham rats. Thus, the stress-related induction of cardiac Kir6.1 mRNA and protein expression under myocardial ischemia is inhibited by pretreatment with an AT1 antagonist, but also in part by an ACE inhibitor, suggesting that activation of local renin-angiotensin system may play a role.

Angiotensin I↗