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.

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.

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.

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.

Phosphorylation of the AfsR protein involved in secondary metabolism in Streptomyces species by a eukaryotic-type protein kinase.

A global regulatory protein, AfsR, involved in secondary metabolism, was found to be phosphorylated by a membrane-associated phosphokinase, named AfsK, of Streptomyces coelicolor A3(2) and S. lividans. The N-terminal portion of AfsK, deduced from the nucleotide (nt) sequence of the afsK gene, which was located downstream from the afsR gene, showed significant sequence similarity to the catalytic domain of eukaryotic Ser/Thr protein kinases (PKs). Consistent with this, experiments with AfsK produced by use of an Escherichia coli host-vector system revealed a self-catalyzed phosphate incorporation into both Ser and Tyr residues of AfsK. The recombinant AfsK phosphorylated the purified AfsR at both Ser and Thr residues. Disruption of the chromosomal afsK gene with the phage vector KC515 resulted in significant, but not complete, loss of actinorhodin production. This result implies the involvement of afsK in the regulation of secondary metabolism. The presence of an additional PK able to phosphorylate AfsR is predicted, because the afsK-disrupted strain still contained an activity able to phosphorylate Ser and Thr residues of AfsR. Southern hybridization experiments showed that nt sequences homologous to afsK, as well as afsR, were distributed among many Streptomyces spp. It is thus concluded that a signal transduction system similar to that found in higher organisms is involved in the regulation of secondary metabolism in the bacterial genus Streptomyces.

Combined quantitative cytophotometric method for staining indolyl and sulfhydryl groups and protein.

Sulfenylation with 2-nitrophenylsulfenyl chloride (NPS-Cl), which is specific for tryptophyl and cysteinyl residues in protein, was applied to quantitative histochemistry. By measurement of the absorbance values at 370 nm of sections stained with NPS-Cl, Beer-Lambert's law was found to hold for NPS staining. Treatment of NPS-stained sections with 2-mercaptoethanol (ME) (NPS-ME staining) resulted in sulfenylation of tryptophyl residues only. For determination of the amounts of tryptophyl and cysteinyl residues per unit of protein, protein staining with Coomassie Brilliant Blue (CB) was combined with NPS and NPS-ME staining. CB and NPS-CBB staining also followed Beer-Lambert's law. By measuring the absorbance values at 370 and 650 nm of doubly stained sections, the relative contents of tryptophyl and cysteinyl residues in various tissue proteins were calculated. This method will be useful for the investigation of changes in both protein amount and composition.

gamma-Glutamyl transpeptidase in rat liver during 3'-Me-DAB hepatocarcinogenesis: immunohistochemical and enzyme histochemical study.

Immunohistochemical localization of gamma-glutamyl transpeptidase (gamma-GTP) in rat liver during 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) hepatocarcinogenesis was investigated and compared with sites of gamma-GTP activity. Immunohistochemically, gamma-GTP was stained in the apical border of proliferating oval cells during the early stages of azo-dye carcinogen feeding. After 7 weeks, multiple hyperplastic nodules appeared in which gamma-GTP was localized in the bile canaliculi. In hepatoma tissues, positive staining for gamma-GTP was observed in the bile canaliculi-like spaces, on the cell membrane, and sometimes in the cytoplasm of malignant cells. Enzyme histochemical staining showed gamma-GTP activity to be present in almost the same areas as the immunoreactive gamma-GTP. However, some areas adjacent to hepatoma tissue showed immunohistochemically reactive protein but no enzyme activity. Immunoreactive gamma-GTP was present in all locations at which enzyme activity was seen. The present data suggest that an altered form of gamma-GTP might be present in tissues during 3'-Me-DAB hepatocarcinogenesis.

Immunohistochemical study of alpha-fetoprotein in rat embryos during ontogenesis.

In order to clarify the dynamics of alpha-fetoprotein (AFP) during ontogenesis, the appearance and localization of AFP and albumin in rat embryos were immunohistochemically investigated. AFP began to appear in the embryos on day 9 in some of the cells constituting the embryonic ectoderm. From day 10, both AFP and albumin became strongly stained in the yolk sac. The formation of liver anlage started on day 11; here AFP alone was clearly apparent in the primitive hepatocytes and also in some other cells, such as somites, or those of the central nervous system. After further development of the embryos, AFP, albumin, and transferrin were detected by means of serial section examination in the same hepatocytes. Besides being found in the liver, AFP began to be stained in the dorsal pancreas and mesonephric vesicles on day 12, in the stomach on day 13, and in the lung anlage and metanephros on day 14. Albumin was also localized in the same sites in these tissues, but the intensity of albumin staining was not as strong as that of AFP. Therefore, we assume that AFP plays an important role in the rapid growth of rats during the early gestational stages.

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.

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.

Antitumor activity of murine monoclonal antibody NCC-ST-421 on human cancer cells by inducing apoptosis.

BACKGROUND: The murine monoclonal antibody NCC-ST-421 (ST-421) recognizes dimeric Le(a) antigen expressed on gastrointestinal cancer cells. MATERIALS AND METHODS: Direct antitumour activity of ST-421 was evaluated using dimeric Le(a) positive cell lines Colo 205, Colo 201, HT-29 and WiDr; and negative cell lines MX-1 and K562. RESULTS: While time- and concentration-dependent antitumour activity was observed against Colo 205 and Colo 201, no antitumour activity was detected against the other cell lines tested in an in vitro cytotoxicity assay. When ST-421 was administered intraperitoneally daily for 2 weeks to severe combined immunodeficient (SCID) mice transplanted with tumour xenografts, inhibition of tumour growth was observed against Colo 205, and to a lesser extent HT-29 and WiDr. Anti-asialo GM1 antibody did not block this antitumour activity, suggesting ST-421 has a direct cytotoxic effect. The degree of antitumour activity of ST-421 dependent on the grade of Le(a)-expression as detected by immunohistochemical staining. CONCLUSIONS: Flow cytometric and immunohistochemical analysis suggests the induction of apoptosis may play a key role in the direct antitumour activity of ST-421 on Colo 205 cells.

Quantitative electron-microscopic analyses of pulpal nerve fibres in the mouse lower incisor after neonatal capsaicin treatment.

A single dose of capsaicin (50 mg/kg) was injected subcutaneously into four mice on day 2 of life; four untreated mice were used as controls. Six months later, a drop of 30 microM capsaicin was instilled on to the cornea of all the mice and the number of times the eyes were wiped was counted to assess the effect of capsaicin on trigeminal sensory neurones. Ultrathin cross-sections were made of the apical pulp of the incisors on both sides of control (n = 8) and capsaicin-treated animals (n = 8). Electron micrographs of pulp nerves were taken and enlarged to a final magnification of x34,000. The numbers of unmyelinated axons in the pulps of all 16 incisors and of unmyelinated axons per Schwann cell in the pulps of four incisors each from the control and capsaicin-treated groups were counted. The short diameters of unmyelinated axons were measured with a computer-operated image analyser. The number of eye wipings was eight-fold less in the capsaicin-treated than in the normal group. This finding clearly indicated that capsaicin irreversibly affected the chemogenic nociceptive trigeminal neurones. The mean number of unmyelinated axons was 345 in controls and 217 (37.1% reduction) in capsaicin-treated animals. The number of unmyelinated axons of less than 0.6 microns dia was 41.5% less in capsaicin-treated mice than in controls. Thus, fine unmyelinated axons in the mouse incisor pulp are capsaicin sensitive, and they are assumed to be nociceptive fibres conveying pain stimuli from the tooth. Capsaicin affected Schwann cells, even those with few unmyelinated axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron microscopic study of the effect of capsaicin on the mouse chorda tympani nerves.

Studies were made to determine whether this nerve contains capsaicin-sensitive fibres. Capsaicin (50 mg/kg, subcutaneously) was injected into 2 animals on day 2 after birth and into one animal on days 2 and 3 after birth. Both chorda tympani of these and 4 control mice were later excised. The constituent fibres of 3 of the capsaicin-treated and all 8 control nerves were then analysed. The myelinated fibres in 3 chorda tympani of treated and control animals were measured, and the unmyelinated axons in Schwann cells were counted from electron-micrograph montages of the entire nerve. Normal chorda tympani contained about 600 nerve fibres, 55% myelinated and 45% unmyelinated. Capsaicin-treatment did not change the constituent fibres nor the size distribution of the myelinated fibres. Thus no capsaicin-sensitive, nociceptive fibres were found in the mouse chorda tympani. Capsaicin does not destroy the neurones of the geniculate ganglion and parasympathetic, presynaptic fibres. Therefore, gustation and secretion of saliva are not influenced by capsaicin.

Effect of 1-hydroxyethane-1,1-diphosphonic acid (HEDP) on new bone formation induced by bone matrix gelatin (BMG).

The effect of the biphosphonate 1-hdroxyethane-1,1 diphosphonic acid (HEDP) on induced ectopic osteoneogenesis was studied by light and electron microscopy. Demineralized bone matrix gelatin (BMG) was implanted into the rectus abdominis muscle of 8-week-old Sprague-Dawley rats, HEDP was administered subcutaneously with a mini-osmotic pump, and histological changes were examined 4 days, 1, 2 and 3 weeks later. Four days after implantation, "acellular mineral deposition" in the control group was suppressed in the HEDP group. Two weeks after BMG implantation, cartilage formation was more intensive in the HEDP-treated group than in the control group. Three weeks after implantation, new lamellar bone with bone marrow was observed in the control group, while immature osteoid tissue detected by type II collagen in the central area was prominent in the HEDP-treated group. HEDP-treatment also suppressed the appearance and activity of osteoclast-like cell.

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.

Vibration acceleration magnitudes of hand-held tools and workpieces.

The magnitudes of the hand-transmitted vibration, 297 hand-held tools and workpieces were measured at workplaces. The tools were used mainly in construction and manufacturing industries. The current standard for the measurement of hand-transmitted vibration has been based on the frequency-weighted acceleration of dominant axis method. According to the proposal of ISO/CD5349-1, the results of measurements (462 points) were recalculated by the method of the frequency-weighted acceleration sum. There is 32% of difference in average value between the acceleration sum and the dominant axis method.

Efficiency of respirator canisters with methyl bromide.

Two kinds of respirator canisters, an organic vapor (OV) canister containing activated carbon and a methyl bromide (MB) canister containing activated carbon impregnated with triethylenediamine were investigated for their filtration efficiency of MB gas under various conditions of MB concentration, humidity and temperature. At lower MB concentrations, the breakthrough time of the OV canister was shorter than that of the MB canister. It also decreased with an increase in temperature, whereas the breakthrough time of the MB canister did not change with temperature. Humidity affected the breakthrough times of the OV canister as much as those of the MB canister. The desorption and the remaining filtration efficiency of MB gas were examined in both canisters which had been used for fumigation in plant quarantine. More than 5 ppm of MB gas leaked from 11 out of 18 used OV canisters, whereas less than 0.1 ppm MB leaked out from each of the used MB canisters. The mean remaining breakthrough time of the used OV canisters was 9.3 min, whereas that of the used MB canisters was 74.4 min. From the present study, it was shown that the gas masks with the OV canisters were not suitable for fumigating with MB gas.

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.

[Development of syringe-type off line pre-column and simultaneous quantitation of four xanthine derivatives (caffeine, theobromine, theophylline and paraxanthine) in serum].

A new syringe-type minicolumn, called Extrashot-Silica (EXS-Silica), containing diatomaceous earth granules was described. The EXS-Silica differs from the conventional pretreatment column. Using the EXS-Silica we can execute the simultaneous extraction-injection to HPLC, column. Therefore, an analysis using the EXS-Silica is an easier and faster method than the general HPLC analysis method. In this study, we carried out the simultaneous determination of four xanthine derivatives, such as caffeine, theobromine, theophylline and paraxanthine, in serum specimens. We used dichloromethane containing 4% ethanol (v/v) for the extraction-injection and water-acetic acid-ethanol-dichloromethane (0.2:0.2:4:95.6, v/v) for the mobile phase of HPLC. The eluent was monitored with a UV detector at 275 nm. A linear relationship between the amount of drug and the peak height was confirmed in the range of 1-40 micrograms/ml for the above-mentioned four xanthine derivatives in the serum. When a 5 microliters aliquot of the serum was subjected to this method, the observed detection limits of the drug were far less than therapeutic concentrations. The analytical accuracy of our method was finally confirmed by comparing the obtained analytical data by the new method with those obtained using the fluorescense polarization immunoassay method. Serum concentrations of theophylline obtained by these two methods correlate satisfactorily. Except for minor modifications in the injector, the existing liquid-chromatographic equipment can be used.