Biomedical subjects

K Obara

Publications and source records attributed to K Obara.

At least 37 records · Page 2Linked to original sources

PubMed2001

Direct evidence of active and rapid nuclear degradation triggered by vacuole rupture during programmed cell death in Zinnia.

Differentiation into a tracheary element (TE) is a typical example of programmed cell death (PCD) in the developmental processes of vascular plants. In the PCD process the TE degrades its cellular contents and becomes a hollow corpse that serves as a water conduct. Using a zinnia (Zinnia elegans) cell culture we obtained serial observations of single living cells undergoing TE PCD by confocal laser scanning microscopy. Vital staining was performed and the relative fluorescence intensity was measured, revealing that the tonoplast of the swollen vacuole in TEs loses selective permeability of fluorescein just before its physical rupture. After the vacuole ruptured the nucleus was degraded rapidly within 10 to 20 min. No prominent chromatin condensation or nuclear fragmentation occurred in this process. Nucleoids in chloroplasts were also degraded in a similar time course to that of the nucleus. Degradations did not occur in non-TEs forced to rupture the vacuole by probenecid treatment. These results demonstrate that TE differentiation involves a unique type of PCD in which active and rapid nuclear degradation is triggered by vacuole rupture.

Apoptosis↗

PubMed2001

Transcriptional induction of matrix metalloproteinase-13 (collagenase-3) by 1alpha,25-dihydroxyvitamin D3 in mouse osteoblastic MC3T3-E1 cells.

The removal of unmineralized matrix from the bone surface is essential for the initiation of osteoclastic bone resorption because osteoclasts cannot attach to the unmineralized osteoid. Matrix metalloproteinases (MMPs) are known to digest bone matrix. We recently reported that among the MMPs expressed in mouse osteoblastic cells, MMP-13 (collagenase-3) was the one most predominantly up-regulated by bone resorbing factors including 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. In this study, we examined the mechanism of regulation of MMP-13 expression by 1alpha,25(OH)2D3 in mouse osteoblastic MC3T3-E1 cells. 1Alpha,25(OH)2D3 increased steady-state messenger RNA (mRNA) and protein levels of MMP-13. De novo protein synthesis was essential for the induction because cycloheximide (CHX) decreased the effect of 1alpha,25(OH)2D3 on the MMP-13 mRNA level. 1Alpha,25(OH)2D3 did not alter the decay of MMP-13 mRNA in transcriptionally arrested MC3T3-E1 cells; however, it increased the MMP-13 heterogeneous nuclear RNA (hnRNA) level and MMP-13 transcriptional rate. The binding activity of nuclear extracts to the AP-1 binding site, but not to the Cbfa1 binding site, in the MMP-13 promoter region was up-regulated by 1alpha,25(OH)2D3, suggesting the mediation of AP-1 in this transcriptional induction. To determine the contribution of MMPs to bone resorption by 1alpha,25(OH)2D3, the inhibitory effect of BB94, an MMP inhibitor, on resorbed pit formation by mouse crude osteoclastic cells was examined on either an uncoated or collagen-coated dentine slice. BB94 did not prevent resorbed pit formation on uncoated dentine whereas it did on collagen-coated dentine. We therefore propose that the transcriptional induction of MMP-13 in osteoblastic cells may contribute to the degradation of unmineralized matrix on the bone surface as an early step of bone resorption by 1alpha,25(OH)2D3.

3T3 Cells↗

PubMed2001

Involvement of different activator Ca(2+) in the rate-dependent stretch-induced contractions of canine basilar artery.

Stretch evoked a contraction in a rate-dependent manner in canine basilar artery; slow stretch at rates less than 3 mm/s produced no active tension, whereas quick stretch at rates over 5 mm/s did. Large conductance Ca(2+)-activated K(+) channel blockers, including charybdotoxin, iberiotoxin, and tetraethylammonium (TEA) sensitized the basilar artery to mechanical stimulation. TEA shifted the stretch rate-tension relationship toward the left. Thus, in the presence of TEA, the slow stretch (0.1-3 mm/s) could increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and active tension. The contraction in response to slow stretch (1 mm/s) was abolished by nicardipine and Gd(3+). Quick stretch (100 mm/s) increased [Ca(2+)](i) and active tension, both of which were partially inhibited by nicardipine or Gd(3+). The Gd(3+)-insensitive component of quick stretch-induced contraction was eliminated by thapsigargin, but not by nicardipine. Ryanodine, cyclopiazonic acid, thapsigargin, U-73122, and calphostin C also abolished the nicardipine-insensitive component of quick stretch-induced contraction. These results suggest that the slow stretch-induced contraction was exclusively dependent on the Ca(2+) influx through L-type voltage-dependent Ca(2+) channels (VDCs), whereas the quick stretch-induced contraction was dependent on Ca(2+) release from sarcoplasmic reticulum (SR) and Ca(2+) influx through L-type VDCs.

Animals↗

PubMed2001

Evaluation of myc and chromosome 8 copy number in colorectal cancer using interphase cytogenetics.

To reveal the significance of genetic abnormalities of the c-myc gene, 56 colorectal tumors (43 colorectal carcinomas, 5 recurrent or metastatic tumors, and 8 adenomatous polyps) were analyzed using fluorescence in situ hybridization (FISH). Two probes specific for c-myc and the chromosome 8 centromere were used for dual color FISH. In each case, 100-200 nuclei were observed for signals from the probes. The percent of nuclei with c-myc amplification (PMA) was defined as the proportion of nuclei representing the ratio of c-myc/chromosome 8 >1.0, and the percent of nuclei with the greater number of c-myc (PGNM) was defined as the proportion of nuclei representing the ratio of c-myc/chromosome 8 > or =2.0. Low level amplification was defined as a case with PMA > or =10% and PGNM <10%. High level amplification was defined as a case with PGNM > or =10%. While adenomatous polyps and in situ carcinomas showed no c-myc amplification, the low level amplification and high level amplification of c-myc were observed in 48.8% (21/43) and 20.9% (9/43) of primary colorectal carcinomas. In addition, the group including cases of stage IIIb and IV exhibited significantly higher average copy numbers of c-myc (CN-myc), PMA and PGNM than the other group of earlier stages. FISH was thought a useful cytogenetic method to detect genetic abnormalities in solid tumors. It was shown that the c-myc gene amplification identified using FISH was associated with the aggressiveness of colorectal carcinoma.

Adult↗

PubMed2001

Usefulness of endoscopic ultrasonographic analysis of variceal hemodynamics for the treatment of esophageal varices.

The correlation of between the endoscopic findings of esophageal varices and endoscopic ultrasound findings of the collaterals outside the esophageal wall in patients with portal hypertension remains unclear. We investigated the relationship between esophageal varices and the collaterals by endoscopy and endoscopic ultrasound. Moreover, we investigated the correlation between the collaterals around the esophagus and recurrence of esophageal varices in patients with portal hypertension who had undergone endoscopic injection sclerotherapy. The collaterals were divided into two groups: 1; those with peri-esophageal collateral veins (peri-ECVs) adjacent to the muscularis externa of the esophagus, and 2; those with para-esophageal collateral veins (para-ECVs) distal to the esophageal wall without contact with the muscularis externa. Peri- and para-ECVs were scored as mild or severe according to the stage of development. According to endoscopy, the varix form was significantly larger in severe peri-ECVs group than in mild peri-ECVs group. In contrast, the varix form did not differ significantly between the mild and severe para-ECVs group. The prevalence of perforating veins increased according to the varix form. With regard to variceal recurrence, in patients with variceal recurrences, EUS findings included a significantly higher incidence of severe-type peri-ECVs, a significantly larger number of perforating veins, and a significantly larger diameter of perforating veins compared with patients without recurrence. Moreover, when EUS found the abnormalities when no endoscopic recurrence was found, the results were the almost same as the findings when EUS was performed at the same time when endoscopic recurrence was found. In conclusion, the presence of severe peri-ECVs and large perforating veins in the esophageal wall strongly correlates with occurrence and recurrence of esophageal varices in patients with portal hypertension. An understanding of these EUS abnormalities on the basis of hemodynamics around the esophagus is thought to be important for management of esophageal varices in patients with portal hypertension.

Collateral Circulation↗

PubMed2001

[Induction of mucosal immunity to mycobacterial heat shock protein (hsp) 65 by colonic inoculation of plasmid DNA encoding hsp65].

Mycobacterial heat shock protein (hsp) 65 has more than 50% sequence homology with human hsp60 and immune responses against mycobacterial hsp65 may cross-react with human hsp60 and could cause autoimmune diseases including inflammatory bowel diseases (IBD). Since the colonic mucosa is a main inflammatory site in IBD, mucosal immunity to hsp65 may be more important for the mucosal inflammation than systemic immunity to hsp65. We inoculated plasmid DNA (pDNA) encoding mycobacterial hsp65 (pACB-hsp 65) into the colon of Wistar rats and evaluated the mucosal humoral immune response and the effect of these immune responses on the colonic mucosa. Four weeks after pDNA inoculation, significantly elevated titers of hsp65-specific IgA antibody were seen in fecal extracts of rats immunized intra-colonic mucosa with pACB-hsp65 (40 +/- 9 U/ml), whereas the fecal IgA antibody titers of rats inoculated intradermal with pACB-hsp65 did not arise (8 +/- 5 U/ml). Colonic inoculation of pACB-hsp65 induced systemic and mucosal immune responses to hsp65. However, macroscopic and histological examinations of the colonic mucosa inoculated with pACB-hsp65 showed no evidence of mucosal damage. These results suggested that the mucosal immunity to hsp65 on the colonic mucosa may not play a crucial role in the induction of colonic mucosal inflammation as was seen in IBD.

Animals↗

PubMed2001

[Chronological changes of lacunar infarctions on fluid-attenuated inversion recovery magnetic resonance images].

In 26 patients with lacunar syndromes, emergence of new lacunar infarctions were identified within 13 days from onset by diffusion-weighted magnetic resonance images. The identified lacunar infarctions were repeatedly imaged using fluid-attenuated inversion recovery (FLAIR) sequence up to 600 days from onset. On FLAIR images taken by 23 days from onset, lacunar infarctions showed homogeneous hyperintensity. On the later FLAIR images beyond 25 days from onset they were observed as heterogeneously hyperintense lesions in half of the patients. In the other patients, lacunar infarctions were observed as hypointense areas with a hyperintense rim beyond 41 days from onset, which indicates cystic transformation with surrounding gliosis. These FLAIR images of lacunar infarction differ from those of dilated perivascular space which is observed as an area of simple hypointensity.

Adult↗

PubMed2000

Preferential role of intracellular Ca2+ stores in regulation of isometric force in NIH 3T3 fibroblast fibres.

Fibroblast contraction plays a major role in wound repair, but the regulatory mechanisms are not well known. We investigated the relations between isometric force and intracellular calcium concentration ([Ca2+]i) in fibroblast fibres. These fibres were made with mouse NIH 3T3 fibroblasts cultured with native collagen in a three-dimensional matrix. Calf serum (CS; 30%) elicited a monotonic increase in force that attained a maximum within 15 min and could be sustained indefinitely. In contrast, [Ca2+]i increased to a peak at 3 min after CS stimulation, then returned to baseline levels by 10 min. Pretreatment with Ca2+-free medium or the Ca2+-channel antagonist nicardipine (10 microM) blocked the CS-induced [Ca2+]i increase, but force was not affected. KCl (50 mM) stimulation on the other hand, elicited a prolonged increase in [Ca2+]i but did not increase force. Inhibition of the endoplasmic reticulum Ca2+ release with Ca2+-ATPase inhibitors cyclopiazonic acid (5 microM) or thapsigargin (5 microM) nearly abolished (<20% control) the increase in [Ca2+]i and force response to CS. Treatment with ryanodine (10 microM) and caffeine (20 mM) had a similar effect. The phospholipase C inhibitor U73122 (3 microM) reduced the CS-induced increases in [Ca2+]i and force by 70 and 40%, respectively. We conclude that fibroblast isometric force is not coupled to Ca2+ arising from transmembrane influx but is correlated with the transient [Ca2+]i increase due to release from intracellular stores. Store-released Ca2+ may initiate activation pathways for fibroblast force development, but is not required for force maintenance.

3T3 Cells↗

PubMed2000

Low-temperature magnetization of submonolayer 3He adsorbed on HD preplated graphite

NMR studies of submonolayer 3He adsorbed on a bilayer of HD preplated graphite have been made down to 100 &mgr;K, which is more than 1 order of magnitude smaller than the exchange energy ( J). In the highly frustrated antiferromagnetic solid region, the magnetization obeys a Curie-Weiss law even at temperatures around J and then increases gradually down to 100 &mgr;K. Nevertheless, it does not show any anomalous behavior corresponding to a spin gap. The normalized magnetization versus the reduced temperature ( T/J) is independent of the density just after solidification. This is consistent with the result in the high-temperature region, that the main multiple-spin exchanges have a similar density dependence.

Journal Article↗

PubMed2000

Anti-tumor immunity against CT26 colon tumor in mice immunized with plasmid DNA encoding beta-galactosidase fused to an envelope protein of endogenous retrovirus.

Endogenous retroviral gene products have been recognized as being expressed in human cancerous tissues. However, these products have not been shown to be antigenic targets for T-cells, possibly due to immune tolerance. Since carcinogen-induced colon tumor CT26 expresses an envelope protein, gp70, of an endogenous ecotropic murine leukemia virus that is comparable to human tumor-associated antigens, we examined whether a DNA vaccine containing the gp70 gene induces protective immunity against CT26 cells. Injection of mice with plasmid DNA (pDNA) encoding gp70 alone failed to induce anti-gp70 antibody (Ab) or anti-CT26 cytotoxic T lymphocyte (CTL) responses. However, immunization with pDNA encoding the beta-galactosidase (beta-gal)/gp70 fusion protein induced anti-gp70 Ab and anti-CT26 CTL responses and conferred protective immunity against CT26 cells. These results indicate that beta-gal acts as an immunogenic carrier protein that helps in the induction of immune responses against the poorly immunogenic gp70. Considering these results, it is possible that potential tolerance to the endogenous retroviral gene products expressed by human tumors may be overcome by DNA vaccines that contain an endogenous retroviral gene fused to genes encoding immunogenic carrier proteins.

Animals↗

PubMed2000

Protein kinase cdelta and alpha are involved in the development of vasospasm after subarachnoid hemorrhage.

We have previously shown the enhanced activity of protein kinase C in the membrane fraction of the canine vasospastic artery after subarachnoid hemorrhage, which increased with progression of angiographic vasospasm. This study examined identification of protein kinase C isoforms in the canine basilar artery, and the changes in expression and/or translocation of each isoform during the development of vasospasm. Vasospasm was produced by using the "two-hemorrhage" canine model in the basilar artery, and angiographic progression of vasospasm was assessed consecutively. Two isoforms, protein kinase Calpha and delta were identified in basilar arteries by Western blotting. Densitometric analysis showed that the expression of protein kinase Cdelta in the membrane fraction was significantly increased in the earlier stage, and protein kinase Calpha was increased later as vasospasm progressed. These results indicate that protein kinase Cdelta and alpha isoforms may play a significant role in the development and maintenance of vasospasm.

Actins↗

PubMed2000

Injection of plasmid DNA into the gastric mucosa induces mucosal and systemic immunity.

Nearly all mucosal surfaces participate in a common mucosal immune system, and application of an antigen to one mucosal surface elicits local as well as distant mucosal immune responses. However, whether the gastric mucosa is a part of this network has not been examined directly. We show here that the injection of plasmid DNA encoding beta-galactosidase into the gastric wall caused transfection of gastric mucosal epithelial cells, induced systemic and mucosal antibody responses at both local (digestive tract) and distant (genital and respiratory tracts) sites, and induced cytotoxic T lymphocyte responses in the spleen and the mesenteric and iliac lymph nodes.

Animals↗

PubMed2000

Regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by bone resorptive factors in osteoblastic cells.

In addition to their stimulating function on osteoclastic bone resorption, bone resorptive factors may regulate proteinases and related factors in osteoblastic cells to degrade bone matrix proteins. This study investigated the regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by bone resorptive factors in the cultures of mouse osteoblastic MC3T3-E1 cells, mouse primary osteoblastic (POB) cells, and neonatal mouse calvariae. Expression of either MMP-2, -3, -9, -11, -13, and -14 or TIMP-1, -2, and -3 was detected in MC3T3-E1 cells and POB cells. When the bone resorptive factors parathyroid hormone, 1,25-dihydroxyvitamin D(3), prostaglandin E(2), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha) were added to the cell cultures, MMP-13 mRNA levels were found predominantly to increase by all resorptive factors in the three cultures. mRNA levels of either MMP-3 and -9 or TIMP-1 and -3 were found to increase mainly by the cytokines IL-1beta and TNF-alpha. BB94, a nonselective MMP inhibitor, neutralized the (45)Ca release stimulated by these resorptive factors to an extent similar to that of calcitonin, strongly suggesting that bone resorptive factors function at least partly through MMP formation. We propose that MMP-13 mRNA expression in osteoblastic cells may play an important role in stimulating matrix degradation by both systemic and local resorptive factors, whereas either MMP-3 and -9 or TIMP-1 and -3 might modulate matrix degradation by local cytokines only.

Animals↗

PubMed2000

Rapid-growing carcinosarcoma of the esophagus arising from intraepithelial squamous cell carcinoma: report of a case.

A case of carcinosarcoma arising from the intraepithelial spreading area of esophageal squamous cell carcinoma (SCC) is reported herein. A 64-year-old man was referred to our hospital for investigation of a sore throat and dysphagia. An endoscopic examination revealed a 2. 5-cm polypoid mass in the mid-esophagus. Esophagograms taken 1 month prior to consultation by our hospital and just before surgery revealed marked change within a period of less than 2 months from a 2-cm sessile elevated mass to a 4-cm polypoid mass with a lobular appearance. The resected specimen contained two lesions in the esophagus. The larger one measured 4.0 x 2.0 x 2.0 cm and had a pedunculated polypoid shape, while the smaller one, measuring 1 cm in diameter, was a plateau-type elevated lesion located 3 cm distal from the larger mass. Histologically, the distal smaller lesion was diagnosed as primary SCC associated with a high frequency of intraepithelial spread, while the larger polypoid mass was composed of spindle-shaped sarcomatous cells, arising from the intraepithelial spread of SCC. To our knowledge, this is the only reported case of esophageal carcinosarcoma arising from an area of intraepithelial spread of SCC that showed such surprisingly rapid growth.

Carcinoma in Situ↗

PubMed2000

Expression of m2 muscarinic acetylcholine receptor mRNA in primary culture of human prostate stromal cells.

The aim of this study was to investigate the expression of the muscarinic acetylcholine receptor (mAchR) subtypes mRNA in primary culture of human prostate stromal cells using the reverse transcription polymerase chain reaction (RT-PCR), RNA blotting and in situ hybridization (ISH). Using an explant method, we obtained a primary culture of prostate stromal cells from three patients with benign prostatic hypertrophy. Total RNA was extracted using the acid guanidinium method for cDNA synthesis. First-strand cDNA was then used for PCR with primers designed to amplify the fragments of each mAchR subtypes (m1-m5) cDNA sequence. The m2, m3 and m4 subtype expected bands were detected; in particular m2 transcripts was strongly detected in the stromal cell culture. Each of the PCR products were subcloned into the pGEM-T plasmid vector, sequenced and random primer labeled using 32P. Digoxigenin-labeled cRNA probes were synthesized by in vitro transcription. RNA blotting using a m2 muscarinic receptor cDNA probe revealed a 4.5 kb single transcript. However, m3 and m4 probes did not hybridize. Using in situ hybridization (ISH), m2 receptor mRNA signals were detected in several smooth muscle cells. The staining was predominantly localized to the perinuclear cytoplasm. The m3 and m4 probes did not hybridize. These results suggested that m2 receptor subtype plays a role in smooth muscle activity of the human prostate.

Blotting, Northern↗

PubMed2000

Characterization of whole-cell currents elicited by mechanical stimulation of Xenopus oocytes.

Whole-cell mechanosensitive current (I(ms)) in Xenopus oocytes was studied using the two-electrode voltage-clamp technique. I(ms) was evoked by mechanically pressing the oocyte surface with a glass micropipette. The current was found to depend on the amplitude of the stimulus, showed a time-dependent decay, and turned off immediately after the stimulus was removed. The current-voltage relationship for the peak current exhibited inward and outward rectification at negative and positive potentials, respectively, while that for the sustained current exhibited only inward rectification. I(ms) was significantly suppressed by 30 microM Gd3+. One millimolar amiloride also significantly suppressed the inward I(ms) at negative potentials, but not the outward one at positive potentials. Replacing extracellular Na+ with K+ did not change the current-voltage relationship, whereas replacing extracellular Na+ with choline+ or tetraethylammonium+ significantly decreased the inward I(ms). The outward rectifier at positive potentials was abolished by replacing extracellular Cl- with gluconate-, by intracellular injection of 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), by extracellular application of anthracene-9-carboxylic acid, and by replacing extracellular Ca2+ with Mg2+. These results suggest that mechanical stimulation activates stretch-activated cation channels and Ca2+-activated Cl- channels, the latter being secondarily activated by an increase in intracellular Ca2+ concentration by Ca2+ influx through stretch-activated cation channels.

Animals↗

PubMed2000

Anti-cathepsin G antibodies in the sera of patients with ulcerative colitis.

The presence of perinuclear anti-neutrophil cytoplasmic antibodies (P-ANCAs) and that of antibodies against cathepsin G, a target antigen for P-ANCAs, was determined in the sera of patients with ulcerative colitis (UC), relative to the endoscopic severity and disease activity. P-ANCAs were detected by indirect immunofluorescent assay (IIF) on ethanol-fixed human neutrophils. Antibodies to cathepsin G were detected by an enzyme-linked immunosorbent assay (ELISA) and Western blotting. P-ANCAs were detected by IIF in 62.5% of 32 patients with active UC. Anti-cathepsin G antibodies were detected in 40.6% of 32 patients with active UC, and their prevalence was significantly higher in patients with severe colitis, as determined by endoscopy, than in those with mild or moderate colitis (P < 0.05). The prevalence and titers of anti-cathepsin G antibodies were significantly higher during the active than the inactive phase of the disease (P < 0.05). Measurement of titers of anti-cathepsin G antibodies by ELISA in the serum is useful for evaluating the activity of UC.

Adolescent↗

PubMed2000

Specific inhibition of stretch-induced increase in L-type calcium channel currents by herbimycin A in canine basilar arterial myocytes.

The effects of protein-tyrosine kinase (PTK) and protein-tyrosine phosphatase (PTP) inhibitors on voltage-activated barium currents (I(Ba)) through L-type calcium channels increased by hypotonic solution were investigated in canine basilar arterial myocytes by the whole-cell patch-clamp technique. I(Ba) was elicited by depolarizing step from a holding potential of -80 to +10 mV and identified by using an L-type calcium channel agonist, Bay K 8644 (100 nM), and an L-type calcium channel blocker, nicardipine (1 microM). Hypotonic superfusate induced cell swelling and acted as a stretch stimulus, which reversibly increased peak I(Ba) amplitude at +10 mV. I(Ba) was also decreased by nicardipine (1 microM) under the hypotonic condition. PTK inhibitors such as herbimycin A (30 nM), genistein (10 microM), and lavendustin A (10 microM) decreased I(Ba) enhanced by hypotonic solution. Genistein also decreased I(Ba) in a concentration-dependent manner under the isotonic condition. The inactive genistein analogue daidzein (10 microM) had no effect on I(Ba) under either the isotonic or hypotonic condition. By contrast, herbimycin A did not decrease I(Ba) under the isotonic condition. Sodium orthovanadate (10 microM), a PTP inhibitor, increased I(Ba) under both conditions. The present results suggest that cell swelling by hypotonic solution increases the L-type calcium channel currents in canine basilar artery and that herbimycin-sensitive PTK activity is primarily involved in the enhancement of calcium channel currents.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗