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Biomedical subjects

T Sakai

Publications and source records attributed to T Sakai.

At least 199 records · Page 11Linked to original sources
PubMed2001

The ability of hyperoxia to limit the effects of experimental detachment in cone-dominated retina.

PURPOSE: To determine the ability of oxygen supplementation to ameliorate the effects of retinal detachment in a cone-dominated retina. METHODS: Retinal detachments were created in the right eyes of ground squirrels and the animals immediately placed in normoxic (room air) or hyperoxic (70% oxygen) conditions for 3 days. The retinas were sampled from different regions and investigated morphologically or immunocytochemically by light or confocal microscopy. Agarose embedded sections were immunostained with antibody probes to cytochrome oxidase, synaptophysin, medium-to-long wavelength-sensitive (M/L) cone opsin, rod opsin, excitatory amino acid transporter 1 (EAAT1), glutamate synthetase (GS), cellular retinaldehyde-binding protein (CRALBP), and peanut agglutinin (PNA) lectin. Retinal wholemounts were labeled with PNA and antibodies to short (S)-wavelength-sensitive cone opsin and rod opsin. Cell death was examined using a TUNEL assay on agarose sections or using toluidine blue staining on semithin sections. RESULTS: The percentage of dying cells relative to the total nuclei in the photoreceptor layer was significantly reduced, and the total number of nuclei was greater in hyperoxic animals. Triple labeling using TUNEL, anti-M/L cone opsin and anti-rod opsin showed that hyperoxia had a remarkable effect both on the reduction of cone cell death and the maintenance of the overall structure of cone photoreceptors. Analysis of the retinal wholemounts demonstrated the preservation of PNA, S-cone, and rod opsin antibody labeling in the detachments maintained in hyperoxic conditions. Although the disruption of cytochrome oxidase and synaptophysin was seen in normoxic animals, there was minimal disruption in hyperoxic animals. Labeling with anti-EAAT1, anti-GS, and anti-CRALBP was increased in the Müller cells of normoxic animals with detachments, but was decreased in the hyperoxic animals. CONCLUSIONS: Hyperoxia prevents the degeneration of both rods and cones in retinas heavily dominated by cones and mitigates the effect of detachment on Müller cell reactivity. The current results suggest that the rescue of cones is not secondary to that of rods.

Animals↗
PubMed2001

[A case of chronic methyl bromide intoxication showing symmetrical lesions in the basal ganglia and brain stem on magnetic resonance imaging].

A 30-year-old man developed paresthesia in the feet, unstable gait and blepharoptosis subsequent to pharyngeal pain while working in a warehouse of imported fruit and vegetable fumigated with methyl bromide and cyanate. Neurological examination showed bilateral blepharoptosis, diplopia, ataxic gait and paresthesia below the inguinal region. The serum concentration of thiociane, a metabolite of cyanide, was within normal limits, but that of bromide was elevated (43.7 mg/l, normal: 3.7-8.6). Thus we diagnosed the patient as intoxication of methyl bromide. Magnetic resonance imaging of the brain (MRI) revealed bilateral symmetrical lesions of high signal intensity in the posterior region of the putamen, the subthalamic nuclei, the dorsal medulla oblongata corresponding to inferior cerebellar peduncles and vestibular nuclei, the inferior colliculi and the periaqueductal gray matter surrounding the oculomotor nucleus of the midbrain. As far as we know, this is the first report of MRI detected cerebral lesions in a case with intoxication of methyl bromide.

Adult↗
PubMed2000

Importance of GlcUAbeta1-3GalNAc(4S,6S) in chondroitin sulfate E for t-PA- and u-PA-mediated Glu-plasminogen activation.

Chondroitin sulfate E (CSE) markedly enhanced plasminogen activation by tissue plasminogen activators (t-PAs) and urinary plasminogen activator (u-PA) in vitro; in the presence of 10 microg/ml of CSE, the potentiation factors of single chain of t-PA, two chain of t-PA and u-PA were 400, 140 and 130, respectively. Though the potentiation activity of CSE gradually decreased when it was depolymerized by chondroitinase ABC, the specific disaccharide from CSE still showed significant activity. Glycosaminoglycan (GAG) from sea cucumber, which possesses a very similar core structure to CSE, but has additional sulfated fucose branches exhibit very weak activity. These results suggested that the minimal structural requirement in CSE to enhance plasminogen activation by plasminogen activators is GlcUAbeta1-3GalNAc(4S,6S) and that additional branching sugars abolish the activity.

Animals↗
PubMed2000

Characterization of the promoter of the murine mac25 gene.

It is important to know the regulation of the expression of the mac25 gene because of its reduced expression in several cancer cells and of its induction by some hormonal factors. We cloned the promoter region of the murine mac25 gene and found five repeats of CCAAT sequences, four Sp1 sites, a TATA-like sequence, and an initiator (INR) sequence. Analysis using luciferase reporter plasmids indicated that CCAAT repeats have a strong enhancer activity and the second to fourth Sp1 sites are essential for basal activity of the expression of the mac25 gene. The 1 kb region that contains the promoter and exon 1 of the mac25 gene was in a typical CpG island. As hypermethylation and reduced expression of the mac25 gene were reported in murine liver tumors, methylation of this CpG island may be directly associated with the expression of the mac25 gene and tumorigenesis.

Animals↗
PubMed2000

TNF-alpha-dependent activation of NF-kappa B in human osteoblastic HOS-TE85 cells is repressed in vector-averaged gravity using clinostat rotation.

Effects of vector-averaged gravity on tumor necrosis factor (TNF)-alpha-dependent activation of nuclear factor kappa B (NF-kappa B) in human osteoblastic HOS-TE85 cells were investigated by culturing the cells using clinostat rotation (clinorotation). Cell cultures were rotated for 72 h at 40 rpm in a clinostat. At the end of clinorotation, the cells were treated with TNF-alpha for 30 min under stationary conditions. Electrophoretic mobility shift assays revealed that TNF-alpha-dependent activation of NF-kappa B was markedly reduced in the clinorotated cells when compared with the cells in control stationary cultures or after horizontal rotation (motional controls). The NF-kappa B-dependent transactivation was also impaired in the clinorotated cells, as evidenced by a transient transfection assay with a reporter plasmid containing multimerized NF-kappa B sites. Consistent with these findings, the TNF-alpha-dependent induction of endogenous NF-kappa B-responsive genes p105, I kappa B-alpha, and IL-8, was significantly attenuate in clinorotated cells. These results demonstrate that vector-averaged gravity inhibits the responsiveness of osteoblasts to TNF-alpha by repressing NF-kappa B activation.

Base Sequence↗
PubMed2000

Activation of the p21WAF1/CIP1 promoter independent of p53 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through the Sp1 sites.

Suberoylanilide hydroxamic acid (SAHA) is a novel histone deacetylase inhibitor with high potency in inducing differentiation of cultured murine erythroleukemia cells. We have recently demonstrated that SAHA induces cell cycle arrest and apoptosis in human breast cancer cells, accompanied by up-regulation of the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, via a p53-independent mechanism. In this study, we used p21 gene expression as a model system to elucidate the molecular mechanism(s) underlying SAHA-mediated gene activation. Treatment of human breast cancer cell line MCF7 cells with SAHA induced p21 mRNA as a consequence of an immediate-early gene activation. Moreover, SAHA activated the p21 promoter primarily through two Spl sites located at -82 and -69 relative to the transcription start site. Furthermore, Sp1 and Sp3 proteins were the major factors binding to the Spl site of the p21 promoter. However, SAHA did not alter their DNA binding activities, suggesting that SAHA mediates p21 promoter activity by a mechanism other than altering the DNA binding activities of Sp1 and Sp3. Further studies using the GAL4 luciferase assay system demonstrated that both GAL4-Sp1 and GAL4-Sp3 fusion proteins supported SAHA-mediated gene activation from a promoter driven by five GAL4 DNA binding sites, and that GAL4-Sp3 fusion protein was suppressive in the absence of SAHA treatment. Collectively, our results suggest that SAHA activates the p21 promoter through the Spl sites, and that both Spl and Sp3 proteins can mediate SAHA-induced gene activation.

Binding Sites↗
PubMed2000

Involvement of the Ets-1 gene in overexpression of matrilysin in human hepatocellular carcinoma.

Although matrix metalloproteinases (MMPs) are thought to be involved in the invasion and metastasis of a variety of malignant tumors, including human hepatocellular carcinoma (HCC), the mechanisms for the expression of MMPs in HCC are not known. To understand the mechanism(s) of MMP expression, the expression of matrilysin (MMP-7) and several genes of the Ets transcription factor family was investigated in human HCC and hepatoma-derived cell lines. The role of Ets-1 gene expression in HCC was also studied. Analysis by semiquantitative reverse transcription-PCR revealed that MMP-7 and Ets-1 are overexpressed and closely associated in HCC. To clarify the role of Ets-1, hepatoma cells were transduced with human Ets-1 or targeted with the Ets-1-specific antisense oligonucleotides. Cells stably transduced with the Ets-1 gene showed increased MMP-7 expression compared to parental and mock-transfected cells. Cells targeted with Ets-1-specific antisense oligonucleotides showed reduced expression of MMP-7. Cotransfection of cells with a MMP-7 promoter-reporter gene plasmid and an Ets-1 expression vector yielded an increase in MMP-7 promoter activity in an Ets-1-responsive element-dependent manner. Taken together, these data suggested that the Ets-1 oncogene is up-regulated and involved in the overexpression of MMP-7 in human HCC and may contribute to the progression of HCC.

Aged↗
PubMed2000

Dual control of the brainstem on the spindle oscillation in humans.

In human subjects, the excitability change of the brainstem was investigated over the course of the spindle oscillation. The investigation was carried out by a sequential analysis of brainstem auditory evoked potentials (BAEPs) with reference to one sequence of spindle oscillation. The method was based on the characteristics of BAEPs, i.e. far-field evoked potential. The brainstem revealed two types of excitability change: one in the lower ventral brainstem (wave-III components), and the other in the upper dorsal brainstem (wave-V components). The excitability in the dorsal brainstem showed an oscillation with one cycle period of about 1.5 s, whereas in the ventral brainstem, the excitability showed a long-range biphasic (decaying-growing) fluctuation. Both excitability changes in the brainstem preceded the spindle oscillation, and the phase was reversed during the emerging period of spindle oscillation. The results suggest a primary triggering mechanism of the brainstem for the spindle oscillation, which is independent of preceding cortical drives (K-complexes) upon the thalamus. The difference of the excitability change between the spindle oscillation and the paroxysmal discharge (spike-and-wave complex) was also discussed.

Adult↗
PubMed2000

Pepstatin A-sensitive aspartic proteases in lysosome are involved in degradation of the invariant chain and antigen-processing in antigen presenting cells of mice infected with Leishmania major.

We previously reported that CA074, a specific inhibitor of cathepsin B, significantly deviated immune responses from the disease-promoting Th2 type to the protective Th1 type in BALB/c mice infected with Leishmania major. Herein, we found that pepstatin A-sensitive aspartic proteases (PSAP) in lysosomes seem to play a different role from that of cathepsin B in antigen-processing and Ii-degradation. That is, cathepsin B appears to digest 16-, 28-, and 31-kDa peptides of soluble leishmania antigen (SLA), whereas PSAP seems to process mainly 28-kDa peptides. Furthermore, the latter protease contributed to the degradation of Ii but cathepsin B did not. Following treatment with pepstatin A, both Th1 and Th2 responses were profoundly suppressed in resistant DBA/2 mice (H-2(d)) and in susceptible BALB/c mice (H-2(d)), and both strains of mice became markedly susceptible compared with the untreated groups, probably owing to failure in degradation of Ii and partly to failure in digestion of 28-kDa peptide.

Animals↗
PubMed2000

Molecular cloning and identification of bottle-nosed dolphin flavocytochrome b gp91(phox) and p22(phox) subunits.

The bottle-nosed dolphin (Tursiops truncatus) gp91(phox) and p22(phox) cDNA were cloned from mitogen stimulated leukocytes RNA utilizing the reverse transcription-polymerase chain reaction. The sequences of these cDNAs showed that dolphin gp91(phox) and p22(phox) clones contained open reading frames encoding 569 and 192 amino acids, respectively. Analysis of the gp91(phox) amino acids sequence showed three potential N-linked glycosylation sites. Comparison of the deduced amino acid showed that dolphin gp91(phox) sequence shared 95.4, 93.8, 91.4 and 89.5% similarity with the bovine, porcine, human and mouse gp91(phox) sequences, respectively. Similarly, the amino acid sequence showed that dolphin p22(phox) shared 89.7, 84.6, 84.1, 83.6 and 83.6% similarity with the bovine, mouse, porcine, human and rattus p22(phox) sequences, respectively. Western blotting analysis with anti-peptide antibodies supported the molecular weights of the dolphin gp91(phox) and p22(phox) homologous proteins predicted from the cDNAs and amino acids sequence data.

Amino Acid Sequence↗
PubMed2000

Lipase-catalyzed transesterification of 2-hydroxy-2-(pentafluorophenyl)acetonitrile leading to (1R,2R)- and (1S,2S)-bis(pentafluorophenyl)ethane-1,2-diol

Optically pure (1R,2R)- and (1S,2S)-1,2-bis(pentafluorophenyl)ethane-1,2-diol (1) were synthesized from key intermediates (R)- and (S)-2-hydroxy-2-(pentafluorophenyl)acetonitrile (2), both of which were prepared by the lipase LIP-catalyzed transesterification (E = 465). The absolute configuration of (S)-2 was determined by X-ray structural analysis after transformation into (S)-alpha-cyano-2,3,4,5,6-pentafluorobenzyl (S)-6-methoxy-alpha-methyl-2-naphthaleneacetate (S,S)-9. In addition, the crystal structure of (S,S)-9 has an interesting well-ordered packing pattern which shows face-to-face stacking interactions and end-to-end parallel contacts between the pentafluorophenyl and 6-methoxynaphthyl groups of the adjacent molecules.

Journal Article↗
PubMed2000

Expression and role of p27(kip1) in neuronal differentiation of embryonal carcinoma cells.

We examined the expression and the regulation of p21(waf1) and p27(kip1) cdk inhibitors in P19 mouse embryonal carcinoma (EC) cells following treatment with all-trans retinoic acid (ATRA) to induce neuronal differentiation. The levels of p27 mRNA and protein increased within 24 h of treatment with ATRA, reaching a plateau 4-5 days later prior to neurite formation. In contrast, levels of p21 expression remained low until after neurites were extensively formed. Induction of muscle differentiation from P19 cells by treatment with dimethyl sulfoxide caused only transient increases in p27 levels. In a mutant P19 cell line, RAC65, treatment with ATRA induced neither p27 accumulation nor neuronal differentiation, but p21 mRNA expression increased markedly. In contrast, treatment of RAC65 cells with 9-cis retinoic acid induced both p27 expression and neuronal differentiation. Correlation between p27 expression and neuronal differentiation was also observed in NT2/D1 human EC cells. Luciferase reporter assays showed that p27 promoter activity increased in ATRA-treated cells, consistent with the elevation of p27 mRNA levels. Arrest of neuronal differentiation of P19 cells by okadaic acid resulted in inhibition of p27 expression, whereas p21 mRNA expression was greatly enhanced. Conversely, inhibition of p27 expression by antisense p27 oligonucleotides resulted in blockade of neuronal differentiation. Taken together, these results strongly suggest that the expression of p27 is indispensable for neuronal differentiation of EC cells.

Animals↗
PubMed2000

Isolation and characterization of a novel zinc finger gene, ZNF219, and mapping to the human chromosome 14q11 region.

We have isolated and characterized a novel zinc finger gene by screening a human testis cDNA library. The isolated cDNA, termed ZNF219, contains an open reading frame of 2169 nucleotides encoding 723 amino acids. Sequence analysis revealed 9 sets of Kruppel-related zinc finger structures and proline-rich regions in several parts. ZNF219 exhibited ubiquitous expression in all fetal and adult tissues examined. The transcript size was 5.5 kb in adult tissues, while the main transcript size in the embryo stage was 3.5 kb. The transcript size is developmentally regulated. When the plasmid cloned with green fluorescent protein (GFP)-tagged ZNF219 was expressed in HeLa and COS7 cells, strong fluorescence intensities were observed only in the nucleus of both cells by fluorescence confocal microscopy. These data suggest that ZNF219 may be related to the regulation of transcription and developmental regulation. Genomic structure analysis mapped ZNF219 to chromosome 14q11 between markers D14S72 and D14S990, because a sequence tagged site mapped to the locus was found in the intron region of the ZNF219 gene.

Adult↗
PubMed2000

Demonstration of MDR1 P-glycoprotein isoform expression in benign and malignant human prostate cells by isoform-specific monoclonal antibodies.

Prostate cancers are resistant to many anticancer agents at the time of presentation. P-glycoprotein (P-gp) is believed to mediate multidrug resistance phenotype. To elucidate the possible role of P-gp in such an intrinsic drug resistance of prostate cancers, its expression was examined immunohisochemically using two P-gp isoform-specific monoclonal antibodies (mAbs) with the paraffin embedded prostate samples derived from five nonmalignant and 30 untreated prostate cancer patients. In all of five normal prostate tissues, P-gp was consistently detected with both mAbs in the epithelial cells, especially at their apical site, and the level of expression was higher in the inner zone than in outer zone. On the other hand, tumor cells expressed P-gp heterogeneously in distribution and intensity; in 25 of 30 malignant cases P-gp expression was clearly demonstrated, whereas its expression was only faintly detected in other cases. However, the staining intensities for P-gp in prostate cancer cells were generally lower than in normal prostate epithelial cells. Thus, not only normal prostate epithelial cells but prostate cancer cells express at least MDR1 P-gp isoform. These results suggest that P-gp expression might play some role in intrinsic drug resistance of prostate cancer cells to many cytotoxic drugs as well as in relative resistance of the inner zone cells to the prostate carcinogenesis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Molecular characterization reveals identity of microtubule-associated proteins MAP3 and MAP4.

The identity of two microtubule-associated proteins, MAP3 and MAP4, was verified both immunologically and biochemically. MAP3 was enriched from the heat-stable fraction of rat brain extracts by reverse-phase HPLC and preparative two-dimensional gel electrophoresis. Both MAP3 and MAP4 antibodies reacted with the corresponding spots on two-dimensional Western blots. Amino acid sequences of internal peptides derived from rat MAP3 matched with corresponding sequence stretches of mouse MAP4. In the kidney cortex, the MAP3 antibody stained not only glomerular podocytes but also interstitial cells. This distribution pattern of MAP3 is identical to that of MAP4 reported previously. These results indicate that MAP3 and MAP4 are identical.

Amino Acid Sequence↗
PubMed2000

Increasing methylation of the CDKN2A gene is associated with the progression of adult T-cell leukemia.

In this study, we examined the methylation status of the CDKN2A gene in patients with different forms of adult T-cell leukemia (ATL) using Southern blot analysis, methylation-specific PCR (MSPCR), and nucleotide sequencing. We found that the CDKN2A gene was more frequently methylated in fresh tumor cells isolated from patients with acute ATL (47%) or lymphoma-type ATL (73%) than in those with less malignant chronic (17%) and smoldering (17%) ATL. In addition, deletions of the CDKN2A gene were found in 24% of acute ATL patients; thus, abnormalities of the CDKN2A gene totaled 71% in acute ATL patients. In contrast, no CDKN2A gene methylation was found in asymptomatic carriers or uninfected individuals. Methylation of the p15 gene was not found in any samples from 36 ATL patients. Direct sequencing of the CDKN2A gene after sodium bisulfite treatment of genomic DNA revealed that the methylation of CpG sites had occurred in 24 of 32 ATL cases (75%) including chronic and smoldering ATL, even when MSPCR and the Southern blot had failed to detect CDKN2A gene methylation. Among fresh ATL samples with methylation, methylation was detected in the promoter region and exon in 17 of 24 cases, and methylation in the exon without promoter region was detected in 7 of 24 cases. In one case, the pattern of methylation proved to be different between peripheral blood cells and lymph node cells, suggesting the presence of multiple subclones with regard to methylation patterns, despite the same HTLV-I integration site. Quantitative PCR showed a marked decrease in CDKN2A mRNA expression in the cells with a methylated CDKN2A gene, especially if the promoter region was methylated. These findings suggest that CpG methylation decreases CDKN2A expression and represents a critical factor in the disease progression of ATL.

Blotting, Southern↗
PubMed2000

Analysis of blood plasma proteins in patients with Alzheimer's disease by two-dimensional electrophoresis, sequence homology and immunodetection.

Blood plasma proteins of patients with Alzheimer's disease (AD; senile dementia) and non-AD-type dementia were resolved by two-dimensional electrophoresis and identified by migration position in the electrophoresis pattern, sequence homology, and immunodetection by using antibodies. For the control experiments, blood plasma proteins of a healthy young individual and non-dementia patients were examined in a manner similar to that of the plasma samples of AD patients. In the plasma sample of the healthy young individual, more than 350 spots of silver-stained proteins were observed and among these spots, 73 spots were identified. Blood plasma proteins of the AD and non-AD-type dementia patients were compared with those of the control and non-dementia patients. In the blood plasma samples of five AD patients, three patients had apolipoprotein E4, and another patient showed apolipoprotein L and complement factor H. For the AD-related proteins apolipoprotein E, tau-1, and presenilin 2, proteins were examined by immunostaining with antibodies, in both AD and non-AD patients. Among the three samples of non-AD-type dementia patients, one was distinguishable by amyloid A proteins, and the other by haptoglobin isoforms.

Adult↗