Search for nucleon decay via n→ν[over ¯]π0 and p→ν[over ¯]π+ in Super-Kamiokande.

We present the results of searches for nucleon decay via n→ν[over ¯]π0 and p→ν[over ¯]π+ using data from a combined 172.8 kt·yr exposure of Super-Kamiokande-I,-II, and-III. We set lower limits on the partial lifetime for each of these modes: τn→ν[over ¯]π0>1.1×10(33) years and τp→ν[over ¯]π+>3.9×10(32) years at a 90% confidence level.

Search for dinucleon decay into kaons in Super-Kamiokande.

A search for the dinucleon decay pp → K+ K+ has been performed using 91.6 kton·yr data from Super-Kamiokande-I. This decay provides a sensitive probe of the R-parity-violating parameter λ112''. A boosted decision tree analysis found no signal candidates in the data. The expected background was 0.28±0.19 atmospheric neutrino induced events and the estimated signal detection efficiency was 12.6%±3.2%. A lower limit of 1.7×10(32) years has been placed on the partial lifetime of the decay O16 → C14K+ K+ at 90% C.L. A corresponding upper limit of 7.8×10(-9) has been placed on the parameter λ112''.

Evidence for the appearance of atmospheric tau neutrinos in super-Kamiokande.

Super-Kamiokande atmospheric neutrino data were fit with an unbinned maximum likelihood method to search for the appearance of tau leptons resulting from the interactions of oscillation-generated tau neutrinos in the detector. Relative to the expectation of unity, the tau normalization is found to be 1.42 ± 0.35(stat)(-0.12)(+0.14)(syst) excluding the no-tau-appearance hypothesis, for which the normalization would be zero, at the 3.8σ level. We estimate that 180.1 ± 44.3(stat)(-15.2)(+17.8) (syst) tau leptons were produced in the 22.5 kton fiducial volume of the detector by tau neutrinos during the 2806 day running period. In future analyses, this large sample of selected tau events will allow the study of charged current tau neutrino interaction physics with oscillation produced tau neutrinos.

Search for differences in oscillation parameters for atmospheric neutrinos and antineutrinos at Super-Kamiokande.

We present a search for differences in the oscillations of antineutrinos and neutrinos in the Super-Kamiokande-I, -II, and -III atmospheric neutrino sample. Under a two-flavor disappearance model with separate mixing parameters between neutrinos and antineutrinos, we find no evidence for a difference in oscillation parameters. Best-fit antineutrino mixing is found to be at (Δm2,sin2 2θ)=(2.0×10(-3) eV2, 1.0) and is consistent with the overall Super-K measurement.

Menin interacts with ß-catenin in osteoblast differentiation.

Menin promotes the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage by interacting with the BMP-2 signaling molecules Smad1/5, and Runx2. However, the relationship between menin and the Wnt-ß-catenin pathway in bone is unclear. Reduction of menin expression by transfection of a menin antisense construct did not alter the levels of ß-catenin in mouse mesenchymal C2C12 and osteoblastic MC3T3-E1 cells. However, menin co-immunoprecipitated with ß-catenin as well as LEF-1 in C2C12 and MC3T3-E1 cells. Reduction of menin expression by antisense menin transfection antagonized ß-catenin-induced transcriptional activity of the pGL3-OT luciferase reporter construct in C2C12 and MC3T3-E1 cells. Antisense menin transfection antagonized the BMP-2 and ß-catenin-stimulated increases in Runx2 and alkaline phosphatase levels in C2C12 cells. The data show that menin interacts with ß-catenin in mouse mesenchymal and osteoblastic cells, and suggest that the interaction is important for osteoblast differentiation.

Urinary deoxypyridinoline is a BMD-independent marker for prevalent vertebral fractures in postmenopausal women treated with glucocorticoid.

SUMMARY: Urinary deoxypyridinoline (DPD) level was associated with prevalent vertebral fractures in glucocorticoid (GC)-treated postmenopausal women independently of lumbar spine bone mineral density (BMD). INTRODUCTION: Bone metabolic indices are the potential predictors of bone fragility. However, their diagnostic efficiency for identifying the risk of GC-induced vertebral fractures is still unclear. We therefore evaluated whether bone metabolic indices would assess the risk of vertebral fractures in GC-treated women. METHODS: One hundred seventy-five women treated with GC for more than 6 months were enrolled in this study. RESULTS: Both premenopausal and postmenopausal women with vertebral fractures had significantly higher urinary DPD levels than those without vertebral fractures. When multivariable logistic regression analysis was performed with the presence of vertebral fractures as a dependent variable and each of DPD or osteocalcin level adjusted for age, weight, height, current and maximum doses of GC, duration of GC treatment, as well as lumbar spine BMD as an independent variable, DPD level was identified as a factor associated with the presence of vertebral fractures in postmenopausal women but not in premenopausal women. CONCLUSION: Urinary DPD level was significantly associated with prevalent vertebral fractures in GC-treated postmenopausal women independently of lumbar spine BMD.

Effect of alendronate on bone metabolic indices and bone mineral density in patients treated with high-dose glucocorticoid: a prospective study.

SUMMARY: This prospective study, in the very early phase after initiation of glucocorticoid (GC) treatment, showed that alendronate was effective in suppressing accelerated bone resorption and subsequent decrease in bone mineral density (BMD) at the lumbar spine of patients with high-dose GC treatment. INTRODUCTION: How bisphosphonates affect bone metabolism and BMD of patients with high-dose GC in the early phase, especially within 1 month is unclear. METHODS: We examined the prospective effects of daily 5 mg alendronate on bone metabolism and BMD in 20 patients with high-dose GC (at least 40 mg prednisolone/day) and compared them to 34 high-dose GC-treated patients without alendronate. RESULTS: Serum levels of calcium decreased at day 28 in the alendronate group. Urinary calcium excretion significantly increased after day 7 in both groups. The increase in serum parathyroid hormone (PTH) level at day 7 in the control group was not observed in the alendronate group, but PTH levels increased at day 28 and month 3 in the alendronate group. As for the bone turnover markers, the serum osteocalcin level decreased in both alendronate and control groups, but serum bone-type alkaline phosphatase levels did not show significant changes. Although the urinary type I collagen cross-linked N-telopeptide (NTX) level showed significant increases on days 7 and 28 in the control group; such early increases in urinary NTX were not observed in the alendronate group. Thereafter, the urinary NTX levels fell slowly in the alendronate group significantly. BMD at the lumbar spine significantly decreased from month 1 in the control group, whereas in the alendronate group, BMD at the lumbar spine maintained almost the same level at all time points observed. CONCLUSION: Alendronate was effective in suppressing bone resorption and subsequent BMD decrease at the lumbar spine in patients with high-dose GC treatment.

Bone fragility in male glucocorticoid-induced osteoporosis is not defined by bone mineral density.

UNLABELLED: Eighty-seven male Japanese subjects taking prednisolone > or = 5 mg for more than 6 months and 132 age- and body mass index (BMI)-matched control subjects were examined. Multiple regression analysis adjusted for age and BMI showed that spinal bone mineral density (BMD) in the prednisolone group was not associated with prevalent vertebral fractures (VFs). INTRODUCTION: Glucocorticoid (GC) treatment is known to increase the risk for bone fractures. However, the association between VFs and BMD in GC-treated male patients remains unclear. METHODS: Eighty-seven male subjects taking prednisolone > or = 5 mg for more than 6 months and 132 age- and BMI-matched control subjects were examined using lateral thoracic and lumbar spine radiographs and spine dual energy X-ray absorptiometry. RESULTS: The presence of GC use was an independent risk factor for VFs adjusted for age and BMI (odds ratio 10.93, P < 0.001). By receiver operating characteristic analysis, the absolute BMD values for detecting VFs were higher and the sensitivity and specificity were lower in the GC group than in the control group (0.936 vs 0.825 g/cm(2) and 53.5% vs 74.0%, respectively). Multiple regression analysis adjusted for age and BMI showed that spinal BMD in the GC group was not associated with prevalent VFs, even after adding current and past maximum GC doses as independent variables. CONCLUSIONS: These results show that lumbar BMD values are not associated with prevalent VFs in GC-treated male patients, suggesting that bone fragility in male GC users is affected by bone quality rather than by BMD.

Measurement of the differential branching fraction and forward-backward asymmetry for B --> K(*)l+l-.

We study B --> K(*)l+l- decays (l = e, mu) based on a data sample of 657 x 10(6) BB pairs collected with the Belle detector at the KEKB e+e- collider. We report the differential branching fraction, isospin asymmetry, K* polarization, and the forward-backward asymmetry (A(FB)) as functions of q2 = M(ll)(2)c2. The fitted A(FB) spectrum exceeds the standard model expectation by 2.7 standard deviations. The measured branching fractions are B(B --> K*l+l-) = (10.7(-1.0)(+1.1) +/- 0.9) x 10(-7) and B(B --> Kl+l-) = (4.8(-0.4)(+0.5) +/- 0.3) x 10(-7), where the first errors are statistical and the second are systematic, with the muon to electron ratios R(K*) = 0.83 +/- 0.17 +/- 0.08 and R(K) = 1.03 +/- 0.19 +/- 0.06.

Radical scavenging activity of bisbenzylisoquinoline alkaloids and traditional prophylactics against chemotherapy-induced oral mucositis.

BACKGROUND AND OBJECTIVE: Oral mucositis is a major severe toxic side-effect of systemic chemotherapy and irradiation in patients with cancer. Various free radical scavengers have been shown to prevent chemotherapy-induced skin necrosis. The objective of this study was to determine the antioxidant activity of a bisbenzylisoquinoline alkaloidal compound (BIQAC) and a series of chemicals, including allopurinol, used clinically for the treatment of chemotherapy-induced mucositis. METHODS: Allopurinol, melatonin, camostat mesilate, gabexate mesilate, hydroquinone and BIQAC were tested for their radical scavenging activities on four different radical species: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) cation radical (ABTS(*+)) using standard methods, and superoxide anion radical (O(2) (-)) and hydroxyl radical (OH(*)) using electron spin resonance. RESULTS: Allopurinol had radical scavenging activity against O(2) (-) only. Melatonin had strong radical scavenging activity against ABTS(*+), and weak activity against DPPH radical and OH(*). Camostat mesilate had weak radical scavenging activity against OH(*). Gabexate mesilate had no radical scavenging activity against any of these radicals. Hydroquinone had strong radical scavenging activity against DPPH radical and ABTS(*+), and moderate activity against both O(2) (-) and OH(*). BIQAC had moderate radical scavenging activity against DPPH radical, strong radical scavenging activity against ABTS(*+) and O(2) (-), and weak activity against OH(*). CONCLUSION: The BIQAC had the most braod-spectrum radical scavenging activity, suggesting that it may be effective against chemotherapy-induced mucositis. These findings also suggest that this radical-scavenging activity screening method, against four kinds of radicals, may be useful for the screening of radical scavenging activity of new natural and synthetic chemicals.

Relationship between oral mucositis and high-dose methotrexate therapy in pediatric acute lymphoblastic leukemia.

OBJECTIVE: Oral mucositis is a major toxicity in the high-dose methotrexate (HD-MTX) treatment for children with acute lymphoblastic leukemia (ALL). The first aim of this study was to evaluate the relationship between the MTX serum concentration and occurrence of oral mucositis in pediatric ALL patients. The second aim was to clarify the relationship between MTX exposure and epidermal keratinocyte cell injury using an in vitro study. METHODS: 49 patients were treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL-HR02 protocol. This protocol involves HD-MTX treatment (3 g/m2 for 24-h i.v. infusion). The MTX serum concentrations were measured by a fluorescence polarization immunoassay. The relationship between oral mucositis and MTX serum concentrations 48 and 72 h after administration was determined. The cell toxicity of MTX for human epidermal keratinocytes was analyzed by using a cell viability assay (WST-1 assay). In addition, pharmacokinetic evaluation for clearance, AUC extrapolated from 48 h to infinity (AUC48h-inf) and elimination half-life (t1/2b) were done using the 1-compartmental models. RESULTS: Oral mucositis occurred in 24 patients (49.0%), in whom 20 patients (83.3% in oral mucositis group) showed WHO severity Grade 1 or 2. Only 4 patients (16.7% in oral mucositis group) showed Grade 3 severity. 22 patients (44.9%) had oral mucositis in the group with a concentration under 10-6 M 48 h after MTX administration. There was no significant deference among the cell viabilities in the concentrations of 10-6 M, 10-5 M and 10-4 M 48 h after the MTX exposure. However, the cell viability obtained 24 h after the MTX exposure was significantly different from the respective cell viability 48, 72 and 96 h after the MTX exposure. In the group with oral mucositis, the clearance decreased significantly (p = 0.042), and the t1/2b (p = 0.025) and AUC48h- yen (p = 0.025) increased significantly compared with the non-symptom group. CONCLUSIONS: It seems that there is no significant relationship between the serum MTX concentration and oral mucositis. This in vitro study has demonstrated that the cell injury was related to the duration of MTX exposure rather than a high MTX concentration.

Analysis using fluorescence polarization immunoassay for unbound teicoplanin concentration in serum.

OBJECTIVES: The aim of this study was to develop a simpler and more rapid analytical method for unbound teicoplanin in serum. METHODS: A new analytical method was developed by modifying an existing fluorescence polarization immunoassay (FPIA) method. The validation of the developed FPIA method was compared in the quantification of unbound teicoplanin with that of the high-performance liquid chromatography (HPLC) method reported previously. The developed FPIA method was employed for the measurement of 36 clinical samples collected from patients with methicillin-resistant Staphylococcus aureus (MRSA) infection. RESULTS: The limits of detection and quantification were 0.5 and 0.8 mug/mL, respectively. The recovery rate was 97.5-106.6%. The developed FPIA method showed better accuracy than the HPLC method. The within-run and interday reproducibility of the assay was good, with relative standard deviation values of 4.76-18.75% (within-run) and 5.68-13.95% (interday). Precision and accuracy of this method were within the acceptable limits defined in the US FDA Guidance for bioanalytical method validation. The correlation between the developed FPIA method and the HPLC method was good (r(2) = 0.87). A positive bias with the FPIA method was observed from the result of the Bland-Altman difference plot. CONCLUSION: We firmly believe that the present method is useful for the adjustment of teicoplanin dosages for patients under various conditions.

Estimation of the area under the curve for mycophenolic acid in adult renal transplant patients with concomitant tacrolimus using a limited sampling strategy.

OBJECTIVES: The aim of this study was to develop a limited sampling strategy (LSS) for monitoring the use of mycophenolic acid (MPA) in maintenance therapy with tacrolimus (TCL) in renal transplant patients. METHODS: Eighteen adult patients receiving a first transplant were investigated. All patients were treated with a combination of TCL, steroid and mycophenolate mofetil (MMF). Besides the predose trough concentration (C(0)), whole blood samples were taken for measurement of the MPA concentration at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 h for a 14-point 12-h pharmacokinetic (PK) profile. Using stepwise linear regression analysis, an abbreviated area under the concentration time curve (AUC) was calculated using all 14, and any combination of sampling points to give an estimating equation with up to three predictors. RESULTS: The equation derived from C(2), C(7) and C(12,) for AUC estimation: AUC = (2.05 x C(2)) + (8.51 xC(7)) + (2.29 x C(12)) + 4.24. was found to be optimal. Using this formula, there was an excellent correlation between the estimated 3-point AUC and AUC(0-12 h). To assess the agreement between the abbreviated methods and the full PK profile, we plotted the average AUC of the abbreviated estimates and the full PK profile. This Bland-Altman analysis indicated good agreement to within +/-2 SD and a prediction variability of 7.56 microg x h/mL. CONCLUSION: Our proposed three-sampling-point estimate of AUCs is clinically acceptable. However, the sampling times are inconvenient for outpatients, and is recommended only for monitoring MMF treatment of inpatients with suspected toxicity or at high risk of organ rejection.

Biologically inactive growth hormone caused by an amino acid substitution.

Short stature caused by biologically inactive growth hormone (GH) is characterized by lack of GH action despite high immunoassayable GH levels in serum and marked catch-up growth to exogenous GH administration. We found a heterozygous single-base substitution (A-->G) in exon 4 of the GH-1 gene of a girl with short stature, clinically suspected to indicate the presence of bioinactive GH and resulting in the substitution of glycine for aspartic acid at codon 112. We confirmed the presence of mutant GH in the serum using isoelectric focusing analysis. The locus of mutation D112G was found within site 2 of the GH molecule in binding with GH receptor (GHR)/GH binding protein (GHBP). The expressed recombinant mutant GH tended to form a 1:1 instead of the 1:2 GH-GHBP complex normally produced by wild-type GH. The formation of a 1:2 GH-GHBP complex is compatible with the dimerization of GHRs by GH, a crucial step in GH signal transduction. Mutant GH was less potent than wild-type GH not only in phosphorylation of tyrosine residues in GHR, janus kinase 2 (JAK2), and signal transducers and activators of transcription 5 (STAT5) in IM-9 cells, but also in metabolic responses of BaF/GM cells, a stable clone transfected with cDNA of the chimera of the extracellular domain of human GHR, the transmembrane and the cytoplasmic domain of the human thrombopoietin receptor. These results indicate that the D112G mutation in the GH-1 gene causes production of bioinactive GH, which prevents dimerization of GHR and is therefore responsible for the patient's short stature.

Cloning and sequencing of a c-myc oncogene in a Burkitt's lymphoma cell line that is translocated to a germ line alpha switch region.

We have cloned and sequenced the translocated c-myc gene from the Burkitt's lymphoma CA46 cell line that carries a reciprocal translocation between chromosomes 8 and 14. The breakpoint lies within the first intron of c-myc, so that the first noncoding exon of the gene remains on the 8q- chromosome. The second and third coding exons are translocated to the 14q+ chromosome into the switch region of C-alpha 1. The orientation of the c-myc gene with relationship to alpha 1 is 5' to 5', with directions of transcription in opposite orientation. DNA sequencing studies predict five changes in the amino acid sequence of the myc protein, two of which occur in a region within the second exon which is highly conserved in evolution. Southern blotting data indicate that the first exon of c-myc is rearranged 3' to 3' with the pseudo-epsilon gene. Because CA46 cells contain two rearranged mu genes, the translocation must have occurred after immunoglobulin rearrangement. The position of the breakpoint in CA46 occurs within a 20-base-pair region of the first intron of c-myc to which breakpoints have been mapped for two additional B-cell lymphomas with the t(8;14) translocation, ST486 and the Manca cell line. The region of the heavy chain locus to which c-myc has translocated is different in each case. Comparisons have been made of the levels of transcripts of the translocated c-myc gene in ST486 and CA46, where the gene is not associated with the heavy chain enhancer, with its expression in the Manca cell, in which it is. The c-myc gene is transcribed at similar levels in all three cases.

Serum soluble factors induce the proliferation, alkaline phosphatase activity and transforming growth factor-beta signal in osteoblastic cells in the patient with hepatitis C-associated osteosclerosis.

Hepatitis C-associated osteosclerosis (HCAO) is a rare syndrome characterized by severe, acquired, generalized osteosclerosis and hyperostosis in adults who are infected with the hepatitis C virus. However, the detail of the pathogenesis of HCAO is still unknown. We examined the effects of serum of the HCAO patient on the proliferation, alkaline phosphatase (ALP) activity and transforming growth factor (TGF)-beta-Smad signaling in mouse osteoblastic cells. The patient was compatible with HCAO, characterized by high bone mass, bone thickening and bone pain with normal lamelar bone. The serum from the HCAO patient increased the levels of TGF-beta and Smad3 expression in osteoblastic MC3T3-E1 cells, compared with the control subject. Moreover, the serum from the HCAO patient significantly augmented TGF-beta-induced transcriptional activity with luciferase assay using 3TP-Lux with a Smad3-specific responsive element. In addition, the serum from the HCAO patient significantly stimulated the MTT intensity, the level of proliferating cell nuclear antigen expression, a proliferation marker, and ALP activity in MC3T3-E1 cells, compared with that from the control subject. In conclusion, the present study indicated that the serum from the HCAO patient stimulated TGF-beta-Smad signaling, as well as the proliferation and ALP activity in osteoblastic cells. Some soluble factors other than parathyroid hormone might be related to the pathogenesis of HCAO.

Formation of 5-formyl-2'-deoxycytidine from 5-methyl-2'-deoxycytidine in duplex DNA by Fenton-type reactions and gamma-irradiation.

5-methyl-2'-deoxycytidine (5-Me-dC) is formed by the enzymatic methylation of dC, primarily in CpG sequences in DNA, and is involved in the regulation of gene expression. In the present study, 5-Me-dC and double-stranded DNA fragments containing 5-Me-dC were either gamma-irradiated or aerobically treated with Fenton-type reagents, Fe(II)-EDTA, Fe(II)-nitrilotriacetic acid, Fe(III)-EDTA-H(2)O(2)-catechol or ascorbic acid-H(2)O(2) under neutral conditions. The formation of 5-formyl-2'-deoxycytidine (5-CHO-dC) was observed upon treatment of both 5-Me-dC and DNA fragments containing 5-Me-dC. The yields of 5-CHO-dC from 5-Me-dC and those of 5-formyl-2'-deoxyuridine from dT were comparable. These results suggest that 5-Me-dC in DNA is as susceptible to oxidation as dT in cells, and raise the possibility that 5-CHO-dC may contribute to the high mutagenic rate observed in CpG sequences in genomic DNA.

Cell cycle regulation of menin expression.

The multiple endocrine neoplasia type 1 gene product, menin, interacts with Jun D. The physiological role of menin in cell cycle control and the manner in which its inactivation contributes to tumorigenesis remain unknown. In the present study, the expression of menin was examined at various cell cycle stages in GH4C1 cells, a rat pituitary cell line. Cells synchronized at the G1-S-phase boundary expressed menin at a lower level than G0-G1-synchronized cells. The expression of menin increased as the cells entered S phase, at which time Jun D expression also increased. In contrast, cells synchronized at the G2-M phase expressed lower levels of menin. At G0-G1, G1-S, and G2-M phases of the cell cycle, menin was found predominantly in the nucleus. In summary, we show that in pituitary cells, menin is a nuclear protein whose expression is cell-cycle regulated. The data suggest that menin has an important role in cell growth regulation.

Effect of temperature on arginine incorporation by ribosomeless extracts of cells transformed by a temperature-sensitive mutant of Rous sarcoma virus.

The effect of transformation of normal rat kidney cells by a temperature-sensitive mutant of the Prague strain of Rous sarcoma virus (ts LA 24 PR-A) on the post-translational addition of arginine to the NH2-terminus of preformed acceptor molecules has been studied. Cells maintained at the permissive (35 degrees C) temperature show a high arginine-incorporating activity in ribosome free extracts compared to that found in extracts of cells grown at the non-permissive (40 degrees C) temperature. Temperature shift experiments as well as studies with cells transformed by wild type Rous sarcoma virus suggest that the decreased activity in cells grown at 40 degrees C is not due to a high temperature per se. The lower arginine incorporation in the 40 degrees C cell extracts is partially due to a decrease in the activity of arginyl transferase which catalyses the transfer of arginine from arginyl tRNA to the acceptor protein. Polyacrylamide gel electrophoresis of the radioactive product shows that the acceptor molecules present in extracts of cells grown at 40 degrees C are larger and qualitatively different from those found in extracts of cells grown at 35 degrees C.

Effect of temperature on normal and SV40-transformed human fibroblasts.

The effect of a temperature shift from low (36 degrees C) to high (40 degrees C) temperature on human fibroblasts (IMR90) at various population doubling levels and IMR90 cells transformed by SV40 virus infection at a population doubling level of 30 (SV40/IMR90) was examined. Both IMR90 and SV40/IMR90 cells showed a decrease in cell saturation density at confluency, whereas an increase in population doubling time and protein content was noted when the cells were shifted up to 40 degrees C from 36 degrees C. The modification of IMR90 chromosomal proteins by arginyl-tRNA transferase was increased by the temperature shift, whereas NH2-terminal arginylation of SV40/IMR90 chromatin was not altered. Similarly, no appreciable change in 2-deoxyglucose uptake was noted with SV40/IMR90 cells at either temperature, although 2-deoxyglucose uptake by IMR90 cells was increased by the temperature shift. Additionally, the rate of 2-deoxyglucose uptake showed no difference between IMR90 and SV40/IMR90 cells. The above results support previous findings that environmental alterations, such as temperature shift can cause acceleration of cellular senescence. These findings also imply that cellular senescence remains fixed when viral transformation occurs and is rendered refractory to further age-associated alterations.