Alkaline extracellular conditions promote the proliferation and mineralization of a human cementoblast cell line.

AIM: To investigate the proliferation and mineralization of a human cementoblast cell line under alkaline conditions. METHODOLOGY: A human cementoblast cell line was cultured in alkaline media with several pHs (pH 7.6, 8.0 and 8.4) without CO2 . Cell numbers, phospho-p44/42 expression, alkaline phosphatase (ALP) activity and mineralization were evaluated. The significance of differences between groups was assessed using two-way analysis of variance 15 (ANOVA) followed by Bonferroni's multiple comparison test (α = 0.01). RESULTS: Cell numbers increased in a time-dependent manner in the high pH medium groups. Western blot analysis revealed the upregulated expression of phospho-p44/42 under alkaline conditions. ALP activity was also increased at pH 8.0 and 8.4. Alizarin red staining revealed increased mineralization in the high pH medium groups. The incorporation of the transient receptor potential ankyrin subfamily member 1 (TRPA1) antagonist HC030031 markedly negated the effect on proliferation and mineralization. CONCLUSIONS: Extracellular alkaline conditions promoted the proliferation and mineralization of human cementoblasts in vitro via TRPA1.

Field test of quantum key distribution in the Tokyo QKD Network.

A secure communication network with quantum key distribution in a metropolitan area is reported. Six different QKD systems are integrated into a mesh-type network. GHz-clocked QKD links enable us to demonstrate the world-first secure TV conferencing over a distance of 45km. The network includes a commercial QKD product for long-term stable operation, and application interface to secure mobile phones. Detection of an eavesdropper, rerouting into a secure path, and key relay via trusted nodes are demonstrated in this network.

Stereoselective pharmacokinetics of oxybutynin and N-desethyloxybutynin in vitro and in vivo.

In vitro studies and the multiple applications of an oxybutynin (OXY) transdermal delivery system to Japanese healthy volunteers were conducted to characterize the stereoselectivity in the pharmacokinetics of OXY and its metabolite, N-desethyloxybutynin (DEOB). In human liver microsomes, (R)-OXY and (R)-DEOB were eliminated slightly slower than the corresponding (S)-enantiomers. The production of DEOB from OXY for the (R)-enantiomer was also slower than that for the (S)-enantiomer. In human P450-expressing liver microsomes, OXY was metabolized mainly by CYP3A4 among five cytochrome P450s (CYPs) tested (CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A5) and the kinetics were slightly different for the enantiomer. The unbound fraction of (R)-OXY in plasma was almost two times higher than that of (S)-OXY, whereas (R)-DEOB was bound to plasma protein more than (S)-DEOB. No differences were observed in the blood-plasma concentration ratios for the enantiomers. After multiple applications of the transdermal delivery system, the plasma concentrations of (R)-OXY were lower than those of (S)-OXY. These data indicate that for the stereoselectivity of OXY, the unbound fraction of each OXY enantiomer was a major factor and the metabolism in liver had a minimal effect.

Pure red cell aplasia and myelofibrosis in B-cell neoplasm.

We describe an unusual case of B-cell neoplasm accompanied by pure red cell aplasia (PRCA) and myelofibrosis in a 67-year-old male presenting with severe anaemia. A few unclassified, myeloperoxidase-negative blastoid cells were seen on bone marrow aspiration, and erythroid cell hypoplasia and myelofibrosis on bone marrow biopsy. An autoimmune PRCA was suspected, as serum CH50, C3 and C4 levels were consistently low. Ciclosporin was effective in treating the anaemia, but anaemia returned when the drug was discontinued. Thirteen months later, the patient was admitted with pleural effusion and ascites that contained monoclonal CD19+ CD20+ immature blast cells with a complex karyotype, thought to be neoplastic B-cells. The unclassified blastoid cells seen earlier may therefore have been from the same origin. The patient deteriorated rapidly and died. Only one case of non-Hodgkin's lymphoma with PRCA and myelofibrosis has been reported previously. We discuss the possibility that dysregulated T-cells induced by neoplastic B-cells may have given rise to concomitant PRCA and myelofibrosis.

WT1 expression level and clinical factors in multiple myeloma.

Although Wilm's Tuomor gene (WT1) was first identified as a tumor suppressor gene for Wilm's tumor, WT1 overexpression has been detected in different malignant cell types including leukemia. Increased expression of WT1 in acute leukemia is potentially used as a marker of minimal residual disease. However, the significance of the gene for multiple myeloma is still not clear. To determine the clinical relevance of WT1 expression in multiple myeloma, we examined the association of clinical parameters and WT1 expression in bone marrow for 17 newly diagnosed multiple myeloma patients. WT1 was assessed by real-time quantitative polymerase chain reaction (RQ-PCR) and calculated standardized WT1 expression level per 100 plasma cells in the bone marrow specimen as "corrected WT1". The expression of standardized WT1 and corrected WT1 in myeloma was 59 to 1,600 copies/microg RNA and 0.05 to 406.3 copies/microg RNA/100 plasma cells, respectively, lower than in leukemia. WT1 transcripts increased when clinical factors worsen, including the stage, amount of M protein, Hb, platelet count, blood urea nitrogen (BUN), creatinine, serum alkaline phosphatase (ALP), calcium, beta2-microglobulin, thymidine kinase activity (TK), and C-reactive protein (CRP). In conclusion, the expression level of WT1 could be an additional marker to the standard parameters considered in risk assessment for multiple myeloma.

Transcriptional control of intestinal cytochrome P-4503A by 1alpha,25-dihydroxy vitamin D3.

It was previously shown that CYP3A4 is induced in the human intestinal Caco-2 cell model by treatment with 1alpha,25-dihydroxy vitamin D3 (1,25-D3). We demonstrate the vitamin D analog, 19-nor-1alpha,25-dihydroxy vitamin D2, is also an effective inducer of CYP3A4 in Caco-2 cells, but with half the potency of 1,25-D3. We report that treatment of LS180 cells, a human intestinal cell line, with 1 to 10 nM 1,25-D3 dose dependently increased CYP3A4 protein and CYP3A4 mRNA expression. CYP3A4- and CYP3A23-promoter-Luciferase reporter constructs transiently transfected into LS180 cells were transcriptionally activated in a dose-dependent manner by 1,25-D3, whereas mutation of the nuclear hormone receptor binding motif (ER6) in the CYP3A4 promoter abrogated 1,25-D3 activation of CYP3A4. Although the CYP3A4 ER6 promoter element has been shown to bind the pregnane X receptor (PXR), this receptor does not mediate 1,25-D3 induction of CYP3A4 because a) PXR is not expressed in Caco-2 cells; b) PXR mRNA expression is not induced by 1,25-D3 treatment of LS180 cells; and c) the ligand binding domain of human PXR was not activated by 1,25-D3. 1,25-D3 uses the vitamin D receptor to induce CYP3A4 because a) the vitamin D receptor (VDR)-retinoid X receptor (RXR) heterodimer binds specifically to the CYP3A4 ER6; b) selective mutation of the CYP3A4 ER6 disrupted the binding of VDR-RXR; and c) reporter constructs containing only three copies of the CYP3A4 ER6 linked to a TK-CAT reporter were activated by 1,25-D3 only in cells cotransfected with a human VDR expression plasmid. These data support the hypothesis that 1,25-D3 and VDR induce expression of intestinal CYP3A by binding of the activated VDR-RXR heterodimer to the CYP3A PXR response element and promoting gene transcription.

Trandolaprilat, an angiotensin-converting enzyme inhibitor, is not excreted in bile via an ATP-dependent active transporter (cMOAT).

Trandolapril is the prodrug of an angiotensin-converting enzyme (ACE) inhibitor. It has been proposed that its active metabolite, trandolaprilat, is mainly excreted in bile, but this has not been clearly demonstrated. Recently it has been reported that temocaprilat, an active metabolite of the ACE inhibitor temocapril, is effectively excreted in bile via an ATP-dependent active transporter (canalicular multispecific organic anion transporter: cMOAT). To investigate whether trandolaprilat has the pharmacological ability to affect the cMOAT system in a manner similar to temocaprilat. The lipophilicity of trandolaprilat and temocaprilat was measured to determine the n-octanol-water partition coefficients. The dose-dependent inhibition of the up-take of [3H]-estradiol-17beta-D-glucuronide and [3H]-2,4-dinitrophenyl-S-glutathione, which are good substrates for cMOAT, in canalicular membrane vesicles (CMVs) prepared from Sprague-Dawley rats was determined in the presence of trandolaprilat and temocaprilat. The partition coefficient of trandolaprilat (log Po/w - 1.1) was about 30 times higher than that of temocaprilat (log Po/w - 2.5). The uptake of [3H]-estradiol-17beta-D-glucuronide and [3H]-2,4-dinitrophenyl-S-glutathione was dose-dependently inhibited by the presence of temocaprilat, but trandolaprilat had no effect on the transport of [3H]-estradiol-17beta-D-glucuronide or [3H]-2,4-dinitrophenyl-S-glutathione into CMVs even at concentrations as high as 200 microM. It could be concluded that trandolaprilat has a higher lipophilicity than temocaprilat. But the hepatobiliary excretion system via cMOAT may not contribute to the excretion of trandolaprilat in bile.

Stable operation of a 300-m laser interferometer with sufficient sensitivity to detect gravitational-wave events within our galaxy.

TAMA300, an interferometric gravitational-wave detector with 300-m baseline length, has been developed and operated with sufficient sensitivity to detect gravitational-wave events within our galaxy and sufficient stability for observations; the interferometer was operated for over 10 hours stably and continuously. With a strain-equivalent noise level of h approximately 5x10(-21)/sqrt[Hz], a signal-to-noise ratio of 30 is expected for gravitational waves generated by a coalescence of 1.4M-1.4M binary neutron stars at 10 kpc distance. We evaluated the stability of the detector sensitivity with a 2-week data-taking run, collecting 160 hours of data to be analyzed in the search for gravitational waves.

A case of anaplastic large cell (Ki-1) lymphoma of B-cell phenotype, occurring in Waldenström's macroglobulinemia.

A case of anaplastic large cell (Ki-1) lymphoma of B-cell lineage occurred in a 59-year-old male with Waldenström's macroglobulinemia. Immunostaining of the lymphoma cells showed sporadic positivity for IgM and occasional positivity for kappa chain. This immunoglobulin specificity is the same as that of plasmacytoid lymphocytes in the bone marrow; therefore anaplastic transformation of Waldenström's macroglobulinemia was strongly suggested. This seems to be the first reported case of anaplastic large cell lymphoma, confirmed by CD30 expression, arising in Waldenström's macroglobulinemia.

A study on the effects of countermeasures for vibrating tool workers using an impact wrench.

The aims of this study were (1) to measure frequency-weighted vibration acceleration and (2) to study the effects of introducing a vibration-proof impact wrench on VWF in workers. The subject pool was 383 male workers who were regularly using an impact wrench and taking special medical examinations for vibration syndrome in a factory from 1982 to 1999. The prevalence of workers with VWF increased gradually after 1982, reached a peak value (4.8%) in 1986, gradually decreased after 1987, and disappeared in 1994. Sixteen subjects who had had VWF at least one time during the observation period were selected for this study. The stages of VWF were at stage I on the Stockholm Workshop scale in all subjects. After the vibration-proof impact wrench was introduced in 1986, the vibration acceleration of the impact wrench measured on the handle decreased from 8.6-11.1 m/s2 to 5.1-7.1 m/s2. The actual time per day that subjects were assumed to use the impact wrench was 108 minutes. The subjects actually used an impact wrench more than the occupational exposure limit allowed. However, VWF disappeared after the introduction of a vibration-proof impact wrench. This might have resulted from the combined effect of introducing the vibration-proof impact wrench and certain countermeasures that were taken against cold working environments.

Three-dimensional bone structure around hydroxyapatite and titanium implants in rabbits.

Long-term support of dental implants requires adequate bone thickness in the area surrounding the implant. A three-dimensional examination, including calculations of percent bone-implant contact and percent bone volume, was conducted to clarify the bone structure around pure titanium (Ti) and dense hydroxyapatite (HA) implants. The implants were installed in the tibiae of rabbits, and the bone structure was examined after 2, 4, 6 and 8 weeks. The bone formation following implantation differed in the cortical bone and cancellous bone areas. In the cortical bone area, the percent bone-implant contact and percent bone volume were comparatively consistent for both Ti and HA implants during the observation period. In the cancellous bone area, both findings were influenced by the implantation period, and the chronological bone structure in the cancellous bone area also differed between Ti and HA implants. The percent bone-implant contact and percent bone volume in the Ti implant increased over 8 weeks, whereas the dense HA implant increased for the first 4 weeks and then decreased. The implant materials, Ti and HA, affected the bone remodeling in the cancellous bone area.

Pharmacokinetic study of S-1, a novel oral fluorouracil antitumor drug.

S-1 is a novel oral fluorouracil antitumor drug that combines three pharmacological agents: tegafur (FT), which is a prodrug of 5-fluorouracil (5-FU); 5-chloro-2,4-dihydroxypyridine (CDHP), which inhibits dihydropyrimidine dehydrogenase (DPD) activity; and potassium oxonate (Oxo), which reduces gastrointestinal toxicity. Phase I and early Phase II clinical trials have already been completed. On the basis of the results of these trials, 80 mg/m2/day, given daily in two divided doses after breakfast and supper, a 28-day consecutive oral regimen is recommended. In this study, we investigated the pharmacokinetics of 5-FU, intact FT, CDHP, and Oxo, after administration of S-1, at a standard dose of 80 mg/m2/day, in advanced cancer patients. Twelve patients were recruited to the study; 5 patients with gastric cancer, 4 with colorectal cancer, and 3 with breast cancer. Among them, analysis was conducted on 12 patients for single administration and on 10 patients for consecutive administration. The initial dose of S-1 for each patient was determined according to his/her body surface area (BSA) as follows: for BSA < 1.25 m2, 80 mg/body/day; for 1.25 m2 < or = BSA < 1.5 m2, 100 mg/day; and for 1.5 m2 < or = BSA, 120 mg/day. For single administration, half of the standard dose was used. For 28-day consecutive administration, the standard dose was given daily in two divided doses. The average single dose per BSA was 35.9 mg/m2 (31.7-39.7 mg/m2). Pharmacokinetic parameters of plasma 5-FU were as follows: Cmax, 128.5 +/- 41.5 ng/ml; Tmax, 3.5 +/- 1.7 h; AUC(0-14), 723.9 +/- 272.7 ng x h/ml; and T(1/2), 1.9 +/- 0.4 h. In the 28-day consecutive regimen, there were no fluctuations in pharmacokinetics nor any drug accumulation. Because the pharmacokinetics of orally administered S-1 is almost similar to that of continuous i.v. infusion of 5-FU, we concluded that S-1 may improve patients' quality of life.

Species differences in the transport activity for organic anions across the bile canalicular membrane.

Species differences in the transport activity mediated by canalicular multispecific organic anion transporter (cMOAT) were examined using temocaprilat, an angiotensin-converting enzyme inhibitor whose biliary excretion is mediated predominantly by cMOAT, and 2,4-dinitrophenyl-S-glutathione, a typical substrate for cMOAT, in a series of in vivo and in vitro experiments. Temocaprilat was infused to examine the biliary excretion rate at steady-state. The in vivo transport clearance values across the bile canalicular membrane, defined as the biliary excretion rate divided by the hepatic unbound concentrations, were 9.8, 39.2, 9.2, 1.1, and 0.8 ml/min/kg for mouse, rat, guinea pig, rabbit, and dog, respectively. The K(m) and V(max) values for ATP-dependent uptake of 2, 4-dinitrophenyl-S-glutathione into canalicular membrane vesicles were 15.0, 29.6, 16.1, 55.8, and 30.0 microM and 0.38, 1.90, 0.15, 0. 47, and 0.23 nmol/min/mg protein, yielding the in vitro transport clearance across the bile canalicular membrane (V(max)/K(m)) of 25.5, 64.2, 9.4, 8.4, and 7.7 for mouse, rat, guinea pig, rabbit, and dog, respectively. A close in vivo and in vitro correlation was observed among animal species for the transport clearance across the bile canalicular membrane. These results suggest that the uptake experiments with canalicular membrane vesicles can be used to quantitatively predict in vivo excretion across the bile canalicular membrane.

Age-related changes in the response to thermal noxious heat and reduction of C-fibers by neonatal treatment with capsaicin.

Developmental changes of the response to nociceptive heat were examined in mice treated with capsaicin (50 mg/kg) on postnatal days 2-15. Tests of hot-plate (55 degrees C) and irradiation by infrared (IR test) were carried out after 10 days of capsaicin administration up to 120 days at intervals of 10 or 20 days. The time until forepaw (hot-plate) and hindpaw (IR test) withdrawal was assessed as the response latencies to suprathreshold and thermal threshold, respectively. Moreover, the numbers of unmyelinated C-fibers and myelinated fibers in the L4 dorsal roots of the animals treated on postnatal days 2 and 5 were counted on electron micrograph montages. Despite the marked reduction of C-fibers (60% mean decrease) in the 4 dorsal roots of the animals treated on postnatal day 2, thresholds were normal compared with those of controls. However, the animals treated with capsaicin on postnatal day 5 showed an apparent delay of thermal threshold latency only in the IR test, although the mean reduction of C-fibers was very likely the same as that of the animals pretreated on day 2. The reduction of C-fibers in mice treated on postnatal days 10 and 15 was lower than the animals treated on days 2 or 5, but their threshold latencies were significantly increased (hypoalgesia). A possible implication of these findings is discussed on the basis of the development of inhibitory systems in the intraspinal and supraspinal dorsal horn and sprouting from the surviving primary afferent neurons in the superficial dorsal horn.

FRAP-positive and capsaicin-sensitive terminals in the substantia gelatinosa of the mouse spinal trigeminal nucleus caudalis.

FRAP (fluoride-resistant acid phosphatase)-reactivity in the substantia gelatinosa of the mouse spinal trigeminal nucleus caudalis (STNC) was examined by light and electron microscopy. Degenerated figures of terminals caused by capsaicin were compared with the FRAP-positive terminals. Scalloped (fan-like) or indented, sinuous, slender, and cap-like figures with closely packed agranular synaptic vesicles of various sizes were common to both FRAP-positive and capsaicin-sensitive terminals. These terminals had glomerular or nonglomerular endings. Sometimes FRAP-positive and capsaicin-sensitive glomerular terminals made presynapses with surrounding dendrites. Frequently, both nonglomerular terminals were in direct contact with the neuronal soma. The terminal features of FRAP-positive and capsaicin-sensitive ones in the mouse STNC are the same as those seen in the superficial dorsal horn of the spinal cord. These findings suggest that some of the FRAP-positive terminals are capsaicin-sensitive, thereby indicating their nociceptive primary afferent.

Contribution of P-glycoprotein to bunitrolol efflux across blood-brain barrier.

In this study, we investigated the mechanism of the blood-brain barrier (BBB) transport of bunitrolol (BTL), as a model of beta-blocker, in vivo and in vitro. In order to define the contribution of P-glycoprotein (P-gp) to the active efflux of BTL from brain to blood, we examined the in vivo brain distribution of BTL in mdr1a(-/-) mice with a disrupted mdr1a gene. After intravenous administration of BTL to mdr1a(-/-) mice, the brain concentration and Kp value of BTL were significantly increased as compared with those in mdr1a(+/+) mice. Next, the contribution of the mdr1a P-gp to in vitro uptake of BTL was compared in LV500 cells and L cells (mouse mdr1a-expressing cells and host cells, respectively). The intracellular accumulations of [3H]vinblastine and BTL by LV500 cells were lower than those by L cells, but were significantly increased by verapamil, a P-gp inhibitor. Furthermore, the BTL uptake by KB-VJ300 cells, which express human P-gp, was also significantly lower than that by KB host cells, and was increased by verapamil. The steady-state uptake of BTL by LLC-GA5-COL300 cells, expressing human P-gp, was significantly increased in the presence of 20 microM cyclosporin A (another P-gp inhibitor), which had no effect in the LLC-PK1 host cells. On the other hand, the steady-state intracellular accumulation of BTL by MBEC4 cells, which express mdr1b P-gp instead of mdr1a P-gp, was not significantly changed in the presence of verapamil. This finding suggested that BTL is not a good substrate for mdr1b P-gp. In conclusion, our results suggest that BTL is transported from brain to blood by mdr1a P-gp in mice and by MDR1 in humans, and this presumably accounts for the low brain distribution of BTL.

Management of primary gastric lymphomas from a surgeon's viewpoint.

Primary gastric lymphoma is a relatively uncommon disease and controversy still exists over its management. In order to assess prognostic factors of lymphomas, we carried out a retrospective study of 28 surgically treated gastric lymphoma patients. The overall survival rate for all the patients in the study was 33% at 10 years. On univariate analysis, lymph node metastasis and depth of tumor infiltration proved to be a significant prognostic factor while size and location of tumor, sex, and method of resection was not. From our results we believe that gastric lymphoma can be regarded as a localized disease in the early stages and a curative resection can be attained when aggressive surgery is possible.

Prediction of the development of hepato-cellular-carcinoma in patients with liver cirrhosis by the serial determinations of serum alpha-L-fucosidase activity.

OBJECTIVE: Evaluation of the usefulness of the serial determinations of serum alpha-L-fucosidase (AFU) activity for prediction of the development of hepatocellular carcinoma (HCC) was performed. METHODS AND PATIENTS: Serum AFU activity was determined monthly for 42 months in 73 patients with liver cirrhosis (LC). RESULTS: HCC was diagnosed in 27 patients by means of ultrasonography during this observation period. In 23 (85%) of the 27 patients, serum AFU activity was found to exceed 700 nmole/ml/h during the LC stage. HCC developed within a few years in 23 (82%) of 28 LC patients with AFU activity exceeding 700 nmole/ml/h, in contrast, it developed in only 4 (9%) of 45 LC patients with AFU activity below 700 nmole/ml/h. AFU activity was already elevated in 23 (85%) of 27 patients at least 6 months before the detection of HCC by ultrasonography. CONCLUSION: It is conceivable that the development of HCC can be predicted by means of serial determinations of serum AFU activity in patients with LC.