Improved Measurements of Muonic Helium Ground-State Hyperfine Structure at a Near-Zero Magnetic Field.

Muonic helium atom hyperfine structure (HFS) measurements are a sensitive tool to test the three-body atomic system and bound-state quantum electrodynamics theory, and determine fundamental constants of the negative muon magnetic moment and mass. The world's most intense pulsed negative muon beam at the Muon Science Facility of the Japan Proton Accelerator Research Complex allows improvement of previous measurements and testing further CPT invariance by comparing the magnetic moments and masses of positive and negative muons (second-generation leptons). We report new ground-state HFS measurements of muonic helium-4 atoms at a near-zero magnetic field, performed for the first time using a small admixture of CH_{4} as an electron donor to form neutral muonic helium atoms efficiently. Our analysis gives Δν=4464.980(20) MHz (4.5 ppm), which is more precise than both previous measurements at weak and high fields. The muonium ground-state HFS was also measured under the same conditions to investigate the isotopic effect on the frequency shift due to the gas density dependence in He with CH_{4} admixture and compared with previous studies. Muonium and muonic helium can be regarded as light and heavy hydrogen isotopes with an isotopic mass ratio of 36. No isotopic effect was observed within the current experimental precision.

Proof-of-Principle Experiment for Testing Strong-Field Quantum Electrodynamics with Exotic Atoms: High Precision X-Ray Spectroscopy of Muonic Neon.

To test bound-state quantum electrodynamics (BSQED) in the strong-field regime, we have performed high precision x-ray spectroscopy of the 5g-4f and 5f- 4d transitions (BSQED contribution of 2.4 and 5.2 eV, respectively) of muonic neon atoms in the low-pressure gas phase without bound electrons. Muonic atoms have been recently proposed as an alternative to few-electron high-Z ions for BSQED tests by focusing on circular Rydberg states where nuclear contributions are negligibly small. We determined the 5g_{9/2}- 4f_{7/2} transition energy to be 6297.08±0.04(stat)±0.13(syst) eV using superconducting transition-edge sensor microcalorimeters (5.2-5.5 eV FWHM resolution), which agrees well with the most advanced BSQED theoretical prediction of 6297.26 eV.

Effect of proton pump inhibitors on the development of hypomagnesemia induced by panitumumab.

Panitumumab, a therapeutic agent for unresectable advanced/recurrent colorectal cancer, is a human IgG2 monoclonal antibody that binds to and inhibits the activity of the epidermal growth factor receptor (EGFR). The onset of hypomagnesemia is a known side effect of anti-EGFR inhibitors, including panitumumab, and it is thought that inhibition of reabsorption of Mg in renal tubules is one of the causes. In addition, recent reports have shown that long-term administration of proton pump inhibitors (PPIs) reduces serum magnesium levels. Therefore, in this study, 102 patients who received oral PPIs treated with panitumumab were classified into a PPI combination group and a PPI non-combination group, and the effect of PPIs on the development of grade 2 or higher hypomagnesemia was investigated. The incidence of hypomagnesemia in the PPI combination group (46.9%, 15/32) was higher than that in the PPI non-combination group (25.7%, 18/70). A comparison of the backgrounds of the two groups of patients showed a significant difference in serum albumin levels. PPI administration was significantly associated with panitumumab-induced hypomagnesemia development when adjusted for known risk factors, serum albumin level, renal function, and oral magnesium oxide tablets in Cox proportional hazards regression analysis (hazard ratio 2.09; 95% confidence interval 1.03-4.22; P =0.040). These results indicate that detailed monitoring of serum magnesium levels is recommended for patients treated with panitumumab and co-administration of PPIs.

Deexcitation Dynamics of Muonic Atoms Revealed by High-Precision Spectroscopy of Electronic K X Rays.

We observed electronic K x rays emitted from muonic iron atoms using superconducting transition-edge sensor microcalorimeters. The energy resolution of 5.2 eV in FWHM allowed us to observe the asymmetric broad profile of the electronic characteristic Kα and Kß x rays together with the hypersatellite K^{h}α x rays around 6 keV. This signature reflects the time-dependent screening of the nuclear charge by the negative muon and the L-shell electrons, accompanied by electron side feeding. Assisted by a simulation, these data clearly reveal the electronic K- and L-shell hole production and their temporal evolution on the 10-20 fs scale during the muon cascade process.

Temporal mapping of photochemical reactions and molecular excited states with carbon specificity.

Photochemical reactions are essential to a large number of important industrial and biological processes. A method for monitoring photochemical reaction kinetics and the dynamics of molecular excitations with spatial resolution within the active molecule would allow a rigorous exploration of the pathway and mechanism of photophysical and photochemical processes. Here we demonstrate that laser-excited muon pump-probe spin spectroscopy (photo-µSR) can temporally and spatially map these processes with a spatial resolution at the single-carbon level in a molecule with a pentacene backbone. The observed time-dependent light-induced changes of an avoided level crossing resonance demonstrate that the photochemical reactivity of a specific carbon atom is modified as a result of the presence of the excited state wavefunction. This demonstrates the sensitivity and potential of this technique in probing molecular excitations and photochemistry.

A mutation in KCNJ11 causing human hyperinsulinism (Y12X) results in a glucose-intolerant phenotype in the mouse.

AIMS/HYPOTHESIS: We identified a mouse with a point mutation (Y12STOP) in the Kcnj11 subunit of the K(ATP) channel. This point mutation is identical to that found in a patient with congenital hyperinsulinism of infancy (HI). We aimed to characterise the phenotype arising from this loss-of-function mutation and to compare it with that of other mouse models and patients with HI. METHODS: We phenotyped an N-ethyl-N-nitrosourea-induced mutation on a C3H/HeH background (Kcnj11 ( Y12STOP )) using intraperitoneal glucose tolerance testing to measure glucose and insulin plasma concentrations. Insulin secretion and response to incretins were measured on isolated islets. RESULTS: Homozygous male and female adult Kcnj11 ( Y12STOP ) mice exhibited impaired glucose tolerance and a defect in insulin secretion as measured in vivo and in vitro. Islets had an impaired incretin response and reduced insulin content. CONCLUSIONS/INTERPRETATION: The phenotype of homozygous Kcnj11 ( Y12STOP ) mice is consistent with that of other Kcnj11-knockout mouse models. In contrast to the patient carrying this mutation homozygously, the mice studied did not have hyperinsulinaemia or hypoglycaemia. It has been reported that HI patients may develop diabetes and our mouse model may reflect this clinical feature. The Kcnj11 ( Y12STOP ) model may thus be useful in further studies of K(ATP) channel function in various cell types and in investigation of the development of hyperglycaemia in HI patients.

The first clinical case of a mutation at residue K185 of Kir6.2 (KCNJ11): a major ATP-binding residue.

BACKGROUND: Closure of the adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channel plays a key role in insulin secretion from the pancreatic beta-cells. Many mutations in KCNJ11 and ABCC8, which respectively encode the pore-forming (Kir6.2) and regulatory (SUR1) subunits of the K(ATP) channel, cause neonatal diabetes. All such mutations impair the ability of metabolically generated ATP to close the channel. Although lysine 185 is predicted to be a major contributor to the ATP-binding site of Kir6.2, no mutations at this residue have been found to cause neonatal diabetes to date. METHODS: We report a 3-year-old girl with permanent neonatal diabetes (PNDM) caused by a novel heterozygous mutation (K185Q) at residue K185 of KCNJ11. The patient presented with marked hyperglycaemia and ketoacidosis at 70 days after birth, and insulin therapy was commenced. RESULTS: Wild-type and mutant K(ATP) channels were expressed in Xenopus oocytes and the effects of intracellular ATP on macroscopic K(ATP) currents in inside-out membrane patches were measured. In the simulated heterozygous state, the K185Q mutation caused a substantial reduction in the ability of MgATP to inhibit the channel. Heterozygous K185Q channels were still blocked effectively by the sulphonylurea tolbutamide. CONCLUSIONS: We report the first clinical case of a PNDM caused by a mutation at K185. Functional studies indicate that the K185Q mutation causes PNDM by reducing the ATP sensitivity of the K(ATP) channel, probably via a reduction in ATP binding to Kir6.2. Based on the experimental data, the patient was successfully transferred to sulphonylurea therapy.

Mechanism of disopyramide-induced hypoglycaemia in a patient with Type 2 diabetes.

BACKGROUND: Disopyramide, an antiarrhythmia drug, has been reported to cause hypoglycaemia. Pre-existing factors that increase the concentration of the drug in the blood increase the risk of hypoglycaemia. Furthermore, other factors can also increase the risk of hypoglycaemia even when disopyramide levels are in the therapeutic range. It has been proposed that disopyramide-induced hypoglycaemia is caused by inhibition of the pancreatic B-cell K(ATP) channels. CASE REPORT: We report a case of severe disopyramide-induced hypoglycaemia in a 62-year-old woman with Type 2 diabetes taking low-dose glimepiride treatment. She had not experienced hypoglycaemia prior to the start of disopyramide therapy. No further hypoglycaemic episodes occurred following withdrawal of disopyramide therapy. FUNCTIONAL STUDY: Current recordings of K(ATP) channels expressed in Xenopus oocytes showed that at their estimated therapeutic concentrations, disopyramide and glimepiride inhibited K(ATP) channels by about 50-60%. However, when both drugs were applied together, K(ATP) channels were almost completely closed (approximately 95%). Such dramatic inhibition of K(ATP) channels is sufficient to cause B-cell membrane depolarization and stimulate insulin secretion. CONCLUSIONS: Disopyramide therapy is not recommended for patients treated with K(ATP) channel inhibitors.

An unusual glucocerebroside in the crustacean nervous system.

High concentrations of glucocerebroside (glucosylceramide) were found in the ventral nerve cord, brain, optic nerve, and antenna, but not in the nonneural tissue, of the brown shrimp Penaeus aztecus aztecus. This lipid contained unusual sphingoid bases consisting of 14-, 15-, and 16-carbon sphinganines and sphingenines. The fatty acids were mainly nonhydroxylated homologs 22 carbons long and longer, similar to those found in galactocerebroside but differing from those in glucocerebroside in mammalian nervous systems.

Bromine residue at hydrophilic region influences biological activity of aplysiatoxin, a tumor promoter.

Aplysiatoxin and debromoaplysiatoxin, which are isolated from the seaweed, Lyngbya gracilis, differ in their chemical structure only by the presence or absence of a bromine residue in the hydrophilic region. The function and the structure-activity relation of the hydrophilic region are not known. Aplysiatoxin increased malignant transformation, stimulated DNA synthesis, and inhibited the binding of phorbol-12,13-dibutyrate and epidermal growth factor to cell receptors. Debromoaplysiatoxin inhibited the binding of these two substances as strongly as aplysiatoxin but did not increase malignant transformation or stimulate DNA synthesis. These results indicate that a slight change in the chemical structure of the hydrophilic region of aplysiatoxin affects its abilities to increase cell transformation and stimulate DNA synthesis and that the abilities of the tumor promoters to inhibit the binding of phorbol-12,13-dibutyrate and epidermal growth factor are dissociable from their abilities to increase cell transformation and stimulate DNA synthesis under some circumstances.

Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau.

The tau mutation is a semidominant autosomal allele that dramatically shortens period length of circadian rhythms in Syrian hamsters. We report the molecular identification of the tau locus using genetically directed representational difference analysis to define a region of conserved synteny in hamsters with both the mouse and human genomes. The tau locus is encoded by casein kinase I epsilon (CKIepsilon), a homolog of the Drosophila circadian gene double-time. In vitro expression and functional studies of wild-type and tau mutant CKIepsilon enzyme reveal that the mutant enzyme has a markedly reduced maximal velocity and autophosphorylation state. In addition, in vitro CKIepsilon can interact with mammalian PERIOD proteins, and the mutant enzyme is deficient in its ability to phosphorylate PERIOD. We conclude that tau is an allele of hamster CKIepsilon and propose a mechanism by which the mutation leads to the observed aberrant circadian phenotype in mutant animals.

Mammalian circadian autoregulatory loop: a timeless ortholog and mPer1 interact and negatively regulate CLOCK-BMAL1-induced transcription.

We report the cloning and mapping of mouse (mTim) and human (hTIM) orthologs of the Drosophila timeless (dtim) gene. The mammalian Tim genes are widely expressed in a variety of tissues; however, unlike Drosophila, mTim mRNA levels do not oscillate in the suprachiasmatic nucleus (SCN) or retina. Importantly, hTIM interacts with the Drosophila PERIOD (dPER) protein as well as the mouse PER1 and PER2 proteins in vitro. In Drosophila (S2) cells, hTIM and dPER interact and translocate into the nucleus. Finally, hTIM and mPER1 specifically inhibit CLOCK-BMAL1-induced transactivation of the mPer1 promoter. Taken together, these results demonstrate that mTim and hTIM are mammalian orthologs of timeless and provide a framework for a basic circadian autoregulatory loop in mammals.

Overexpression of RegIV in peritoneal dissemination of gastric cancer and its potential as A novel marker for the detection of peritoneal micrometastasis.

BACKGROUND: Regenerating gene type IV (RegIV) is a candidate marker for cancer and inflammatory bowel disease. In this study, its potential as a novel marker for the detection of gastric cancer peritoneal micrometastases was examined. PATIENTS AND METHODS: RegIV mRNA levels in the peritoneal washes of 95 gastric cancer patients and 22 with benign disease were quantified by real-time RT-PCR. To examine whether expression of RegIV enhance tumorigenicity or not, thirty two mice were injected intraperitoneally or subcutaneously with RegIV transfectants of TMK-1 cells, parental TMK-1 cells, or neomycin control transfectants. RESULTS: RegIV expression was markedly higher in patients with peritoneal metastases compared to those without. The level of RegIV mRNA in gastric cancer patients was related to the extent of wall penetration. A cut-off value for RegIV-positive expression was based on an analysis of negative control patients with benign disease, and gastric cancer patients above the cut-off value constituted the micrometastasis (MM+) group. Based on this criteria, 3 out of 43 T1 or T2 cases were MM+ (93% specificity). Among 15 patients with peritoneal dissemination (7 out of 15 cases were positive by cytology), 14 cases were positive for RegIV expression (93% sensitivity), while analysis of carcinoembryonic antigen (CEA) mRNA failed to detect micrometastases in 4 cases (73% sensitivity). Combined analysis of CEA and RegIV improved the accuracy of diagnosis to 100%. The prognosis of RegIV-positive cases was significantly worse than that of RegIV-negative cases. Multivariate analysis using the Cox proportional hazards model suggested that RegIV may be an independent prognostic factor. Stable expression of RegIV significantly enhanced peritoneal metastasis in an animal model of gastric cancer. CONCLUSION: These findings suggest that RegIV mRNA expression has the potential to serve as a novel marker for detecting peritoneal dissemination in gastric cancer.

Non-destructive elemental analysis of a carbonaceous chondrite with direct current Muon beam at MuSIC.

Electron- or X-ray-induced characteristic X-ray analysis has been widely used to determine chemical compositions of materials in vast research fields. In recent years, analysis of characteristic X-rays from muonic atoms, in which a muon is captured, has attracted attention because both a muon beam and a muon-induced characteristic X-ray have high transmission abilities. Here we report the first non-destructive elemental analysis of a carbonaceous chondrite using one of the world-leading intense direct current muon beam source (MuSIC; MUon Science Innovative Channel). We successfully detected characteristic muonic X-rays of Mg, Si, Fe, O, S and C from Jbilet Winselwan CM chondrite, of which carbon content is about 2 wt%, and the obtained elemental abundance pattern was consistent with that of CM chondrites. Because of its high sensitivity to carbon, non-destructive elemental analysis with a muon beam can be a novel powerful tool to characterize future retuned samples from carbonaceous asteroids.

Effect of peroxisome proliferator-activated receptor alpha ligand fenofibrate on K(v) channels in the insulin-secreting cell line HIT-T15.

Ligands for peroxisome proliferator-activated receptors alpha (PPARalpha) are clinically used for the treatment of patients with hyperlipidemia. As we have previously shown, a synthetic ligand of PPARalpha, fenofibrate, has a stimulatory effect on insulin secretion in clonal hamster insulinoma beta-cell line HIT-T15 cells. We have also demonstrated that fenofibrate directly inhibits ATP-sensitive potassium (K(ATP)) channels, an effect independent of PPARalpha. In this study, fenofibrate was shown to be able to reduce voltage-dependent K(+) (K(v)) channel currents in voltage-independent manner. Therefore, fenofibrate may modulate insulin secretion not only via inhibition of K(ATP) channels but also via reduction of the K(v) channel current.

Important role of serotonin in the antitumor effects of recombinant human tumor necrosis factor-alpha in mice.

The possible involvement of chemical mediator(s) in the induction of the antitumor effects of recombinant human tumor necrosis factor-alpha (rTNF-alpha) on Meth A fibrosarcoma (Meth A) in mice was studied. On day 7 after intradermal implantation of Meth A in mice, rTNF-alpha caused tumor necrosis and inhibited the tumor growth. Ketanserin, cyproheptadine, and spiperone [serotonin (5-HT) receptor blockers] inhibited or attenuated the antitumor effects of rTNF-alpha, but the other types of receptor blockers tested (histamine H1 and H2, adrenaline alpha and beta, dopamine, and acetylcholine receptor blockers) did not. The large i.v. doses of 5-HT caused tumor necrosis and inhibited tumor growth in mice when given i.v. on day 7 but not when given on day 3 after Meth A implantation, which effects closely resemble those of rTNF-alpha. Its anti-tumor effects were completely inhibited by the 5-HT receptor blockers. 5-HT, like rTNF-alpha, showed no cytotoxicity against in vitro cultured Meth A cells. The results suggest that 5-HT is, at least in part, important for the induction of antitumor effects of rTNF-alpha on Meth A in mice.

Interstrand DNA-DNA and DNA-protein cross-links by a new antitumor antibiotic, FK973, in L1210 cells.

In our previous study FK973, a novel, substituted dihydrobenzoxazine (11-acetyl-8-carbamoyloxymethyl-4-formyl-14-oxa-1,11-diazatetracyclo+ ++ [7.4.1.0(2,7).0(10,12)]tetradeca-2,4,6-trien-6,9-diyl diacetate), had potent cytotoxic and antitumor effects on murine tumors and human tumors in in vitro and in vivo experiments. In the present study the mechanism(s) of the in vitro cytotoxic effects of the drug on tumor cells were studied. After 1-h exposure of L1210 murine leukemia cells to the drug, the concentration of FK973 required to inhibit cell growth by 50% was approximately 1 microM, which was threefold more potent than the concentration of mitomycin C required. DNA synthesis was selectively inhibited in the cells treated with FK973. Alkaline elution analyses showed that FK973 formed concentration- and time-dependent interstrand DNA-DNA and DNA-protein cross-links in the cells. On the other hand, no DNA single-strand breaks were observed in the cells treated with FK973. When isolated nuclei of L1210 were exposed to FK973 for 1 h, FK973 did not form detectable interstrand DNA-DNA cross-links. We propose that FK973 is activated in the cytoplasm of cells, and forms interstrand DNA-DNA and DNA-protein cross-links which may be important for the induction of its cytotoxicity.

Recombinant human tumor necrosis factor-alpha: evidence of an indirect mode of antitumor activity.

The antitumor activity of recombinant human tumor necrosis factor (rTNF-alpha) was examined on murine tumors in mice and in cultured cells in vitro. Mice were implanted intradermally with Meth A fibrosarcoma (Meth A) on day 0. rTNF-alpha caused tumor necrosis and inhibited the tumor growth when given i.v. on day 7 or 10, but not when given on day 3. When rTNF-alpha was given i.v. in doses of 0.1-3.2 micrograms/mouse twice a week for 3 weeks beginning on day 7 or 11, the growth of solid Meth A, Colon 26 adenocarcinoma, Colon 38 carcinoma, Sarcoma-180, and M5076 reticulum cell sarcoma tumors implanted s.c. or intradermally was markedly inhibited, and the life of the mice bearing these tumors, except M5076 reticulum cell sarcoma, was prolonged. The growth of Meth A implanted i.m. was also markedly inhibited by rTNF-alpha given i.v. However, the life of mice bearing i.p. Colon 26 adenocarcinoma, MH134 hepatoma, Sarcoma-180, and Ehrlich carcinoma was not prolonged by rTNF-alpha given i.p. nine times (days 1-9) in doses up to 1.0 or 3.2 micrograms/mouse. Only in the case of mice bearing i.p. Meth A, the life was slightly prolonged by i.p. treatment with rTNF-alpha but not by i.v. treatment. In experiments against in vitro cultured cells, rTNF-alpha did not show any direct cytotoxicity against mouse tumor cells: Meth A, Colon 26 adenocarcinoma, Colon 38 carcinoma, and Sarcoma-180, but had a cytotoxic effect against L929 mouse fibroblast. The results suggest that rTNF-alpha is a unique antitumor drug with potent necrotizing activity against solid tumors in mice, and that this activity may derive from indirect mechanisms related to the growth of tumors and not to the direct cytotoxicity of the drug.

Antitumor activity and hematotoxicity of a new, substituted dihydrobenzoxazine, FK973, in mice.

FK973, a new, substituted dihydrobenzoxazine (11-acetyl-8-carbamoyloxymethyl-4-formyl-14-oxa-1,11- diazatetracyclo[7.4.1.0.0]tetra-deca-2,4,6-trien-6,9-diyl diacetate), was obtained by chemical modification of a novel antibiotic which was isolated from the fermentation products of Streptomyces sandaensis No. 6897. FK973 had cytotoxic effects against in vitro cultured human and murine tumor cells. FK973 in doses of 0.032-5.6 mg/kg (i.p.) had stronger antitumor activities and higher chemotherapeutic ratio than mitomycin C against such murine ascitic tumors as P388 and L1210 leukemia, B16 melanoma, M5076 reticulum cell sarcoma of ovarian origin, Colon 26 carcinoma, Ehrlich carcinoma, and MH134 hepatoma. In tests against murine and human solid tumors implanted s.c. in normal mice and nude mice, respectively, FK973 (i.v.) inhibited growth of murine tumors (M5076 sarcoma, Colon 38 carcinoma, B16 melanoma, and Lewis lung carcinoma) by 66-100% and human tumors (LX-1 lung, MX-1 mammary, and SC-6 stomach carcinoma) by 84-99%. In studies with drug-resistant P388 leukemia, FK973 was also effective against vincristine-resistant P388, moderately effective against mitomycin C (MMC)- and adriamycin-resistant P388, and partially effective against cyclophosphamide-resistant P388 cells in mice. Leukopenic effects of FK973 and MMC in mice were comparable at doses which gave antitumor activity almost equally. FK973 had no effect on the numbers of platelets and red blood cells, whereas MMC markedly decreased both. FK973 decreased the numbers of colony forming units in spleen and in culture and the effect was less than that of MMC. Therefore, FK973 may give weaker myelosuppression than MMC. The results suggest that FK973 will be a beneficial drug for the treatment of cancer.