Search for the Pair Production of Dark Particles X with K_{L}

We present the first search for the pair production of dark particles X via K_{L}^{0}→XX with X decaying into two photons using the data collected by the KOTO experiment. No signal was observed in the mass range of 40-110 MeV/c^{2} and 210-240 MeV/c^{2}. This sets upper limits on the branching fractions as B(K_{L}^{0}→XX)<(1-4)×10^{-7} and B(K_{L}^{0}→XX)<(1-2)×10^{-6} at the 90% confidence level for the two mass regions, respectively.

CDK4 and cyclin D1 allow human myogenic cells to recapture growth property without compromising differentiation potential.

In vitro culture systems of human myogenic cells contribute greatly to elucidation of the molecular mechanisms underlying terminal myogenic differentiation and symptoms of neuromuscular diseases. However, human myogenic cells have limited ability to proliferate in culture. We have established an improved immortalization protocol for human myogenic cells derived from healthy and diseased muscles; constitutive expression of mutated cyclin-dependent kinase 4, cyclin D1 and telomerase immortalized human myogenic cells. Normal diploid chromosomes were preserved after immortalization. The immortalized human myogenic cells divided as rapidly as primary human myogenic cells during the early passages, and underwent myogenic, osteogenic and adipogenic differentiation under appropriate culture conditions. The immortalized cells contributed to muscle differentiation upon xenotransplantation to immunodeficient mice under conditions of regeneration following muscle injury. We also succeeded in immortalizing cryopreserved human myogenic cells derived from Leigh disease patients following primary culture. Forced expression of the three genes shortened their cell cycle to < 30 h, which is similar to the doubling time of primary cultured human myogenic cells during early passages. The immortalization protocol described here allowed human myogenic cells to recapture high proliferation activity without compromising their differentiation potential and normal diploidy.

A quinol peroxidase inhibitor prevents secretion of a leukotoxin from Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Quinol peroxidase (QPO) catalyzes peroxidase activity using quinol in the respiratory chain as a substrate. Quinol peroxidase is essential for the secretion of leukotoxin (LtxA), which destroys leukocytes and erythrocytes in humans and is one of the major virulence factors of Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis. In the present study, we aimed to find a highly potent QPO inhibitor to attenuate the virulence of A. actinomycetemcomitans. MATERIAL AND METHODS: For screening of QPO inhibitors, QPO activity was measured kinetically by SpectraMax Plus with 96-well UV plates. Three hundred compounds in the Kitasato Institute for Life Sciences Chemical Library were screened. Secretion of LtxA in the culture supernatant was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cytotoxicity against human promyelocytic leukemia cell line (HL-60) cells from the culture supernatant was measured by Trypan Blue exclusion test. RESULTS: The present study characterized ascofuranone as a highly potent inhibitor of QPO (K(i) = 9.557 +/- 0.865 nm). Ascofuranone inhibited secretion of LtxA by A. actinomycetemcomitans in a dose-dependent manner, making A. actinomycetemcomitans less pathogenic to HL-60 cells. CONCLUSION: Quinol peroxidase inhibitors are promising candidates as alternative drugs for the treatment and prevention of the onset of localized aggressive periodontitis. Using ascofuranone as a seed compound, further study of QPO inhibitors could provide novel chemotherapeutic strategies for controlling localized aggressive periodontitis.

Toward High Precision XCO2 Retrievals From TanSat Observations: Retrieval Improvement and Validation Against TCCON Measurements.

TanSat is the 1st Chinese carbon dioxide (CO2) measurement satellite, launched in 2016. In this study, the University of Leicester Full Physics (UoL-FP) algorithm is implemented for TanSat nadir mode XCO2 retrievals. We develop a spectrum correction method to reduce the retrieval errors by the online fitting of an 8th order Fourier series. The spectrum-correction model and its a priori parameters are developed by analyzing the solar calibration measurement. This correction provides a significant improvement to the O2 A band retrieval. Accordingly, we extend the previous TanSat single CO2 weak band retrieval to a combined O2 A and CO2 weak band retrieval. A Genetic Algorithm (GA) has been applied to determine the threshold values of post-screening filters. In total, 18.3% of the retrieved data is identified as high quality compared to the original measurements. The same quality control parameters have been used in a footprint independent multiple linear regression bias correction due to the strong correlation with the XCO2 retrieval error. Twenty sites of the Total Column Carbon Observing Network (TCCON) have been selected to validate our new approach for the TanSat XCO2 retrieval. We show that our new approach produces a significant improvement on the XCO2 retrieval accuracy and precision when compared to TCCON with an average bias and RMSE of -0.08 ppm and 1.47 ppm, respectively. The methods used in this study can help to improve the XCO2 retrieval from TanSat and subsequently the Level-2 data production, and hence will be applied in the TanSat operational XCO2 processing.

Identification of a Drosophila homologue of alpha-catenin and its association with the armadillo protein.

The cadherin cell adhesion system plays a central role in cell-cell adhesion in vertebrates, but its homologues are not identified in the invertebrate. alpha-Catenins are a group of proteins associated with cadherins, and this association is crucial for the cadherins' function. Here, we report the cloning of a Drosophila alpha-catenin gene by low stringent hybridization with a mouse alpha E-catenin probe. Isolated cDNAs encoded a 110-kD protein with 60% identity to mouse alpha E-catenin, and this protein was termed D alpha-catenin. The gene of this protein was located at the chromosome band 80B. Immunostaining analysis using a mAb to D alpha-catenin revealed that it was localized to cell-cell contact sites, expressed throughout development and present in a wide variety of tissues. When this protein was immunoprecipitated from detergent extracts of Drosophila embryos or cell lines, several proteins co-precipitated. These included the armadillo product which was known to be a Drosophila homologue of beta-catenin, another cadherin-associated protein in vertebrates, and a 150-kD glycoprotein. These results strongly suggest that Drosophila has a cell adhesion machinery homologous to the vertebrate cadherin-catenin system.

Cephalopod tropomyosins: identification as major allergens and molecular cloning.

Heated extracts prepared from the mantle muscles (for decapods) or leg muscles (for octapods) of nine species of cephalopods were shown to be all reactive with serum IgE in crustacean-allergic patients. No marked difference in the reactivity with IgE was recognized among the cephalopods, suggesting that they are almost equally allergenic. Immunoblotting and inhibition immunoblotting data revealed that the major allergen is tropomyosin in common with the nine species of cephalopods and that the cephalopod tropomyosins are cross-reactive with one another and also with crustacean tropomyosins. Molecular cloning experiments first elucidated the primary structures of tropomyosins from five species of cephalopods. The cephalopod tropomyosins show high sequence identity (more than 92% identity) with one another, being the molecular basis for their cross-reactivity. Although the sequence identity between cephalopod and crustacean topomyosins is only about 63-64%, some of the IgE-binding epitopes proposed for brown shrimp Penaeus aztecus tropomyosin (Pen a 1) are well conserved in the cephalopod tropomyosins, supporting the cross-reactivity between cephalopod and crustacean tropomyosins.

Purification and characterization of an agglutinin in the red alga Agardhiella tenera.

The red alga, Agardhiella tenera was found to contain a glycoprotein which agglutinates mouse leukemia cells, L5178Y but not L1210. It also agglutinates guinea pig and rabbit erythrocytes, and has weak activity against human A, B and O, mouse, horse and sheep erythrocytes and hamster and mouse lymphocytes. The agglutination was not inhibited by simple sugars. The major active component was purified and determined to be a beta-structure protein containing 2.7% glucose as sugar moiety. The molecular weight was estimated to be 12,000 by gel filtration and 13,000 by polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate. Its isoelectric point was 6.1, and it contained high amounts of glycine, serine and threonine, but no half cystine or histidine. It had no subunit structure, and the C- and N-terminal amino acids were threonine and arginine, respectively.

Novel polypeptide toxins with crab lethality from the sea anemone Anemonia erythraea.

The sea anemone Anemonia erythraea was found to contain polypeptide toxins with crab lethality as well as hemolysins. Three polypeptide toxins (AETX I, II and III) were isolated by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. A geographic variation in toxin composition was suggested. The LD50 against crabs of AETX I, II and III were estimated to be 2.2, 0.53 and 0.28 microg/kg, respectively, but none of the toxins showed lethality in mice. The amino acid sequences of the three toxins were deduced from sequencings of the whole molecules and their enzymatic fragments. Amino acid analyses and molecular mass determinations supported the accuracy of the deduced sequences. AETX I, comprising 47 amino acid residues including 6 half-Cys residues, is an analog of sea anemone type I toxins. On the other hand, AETX II and III, which are highly homologous with each other, are quite distinct from the known sea anemone polypeptide toxins in that they are composed of 59 residues including 10 half-Cys residues. Interestingly, both toxins have sequence similarities with neurotoxins isolated from the Brazilian 'armed' spider Phoneutria nigriventer.

[Emergent coronary artery bypass grafting using percutaneous cardiopulmonary support in a patient with a quadricuspid aortic valve; report of a case].

A 63-year-old man was admitted to our hospital for acute myocardial infarction. A cardiac catheter study showed 3 vessels coronary disease. He was treated by percutaneous coronary intervention for a left anterior descending arterial (LAD) lesion. Unfortunately, cardiac tamponade following stenting for LAD was complicated. A percutaneous cardiopulmonary support system was commenced along with an emergent coronary artery bypass grafting to the LAD and obtuse marginal branch. A quadricuspid aortic valve was discovered by an aortotomy and identified as Hurwitz-Roberts classification type b. Blood from the left coronary main trunk had already stopped. Intraaortic balloon pumping was instituted while weaning from the cardiopulmonary bypass. The patient's postoperative course was uneventful and all bypass grafts were sufficient. He was well 1 year after the operation.

Crustal anisotropy across northern Japan from receiver functions.

Northern Japan is a tectonically active area, with the presence of several volcanoes, and with frequent earthquakes among which the destructive Mw = 8.9-9.0 Tohoku-oki occurred on 11 March 2011. Tectonic activity leaves an imprint on the crustal structures, on both the upper and the lower layers. To investigate the crust in northern Japan, we construct a receiver function data set using teleseismic events recorded at 58 seismic stations belonging to the Japanese National (Hi-net) network. We isolate the signals, in the receiver function wavelet, that witness the presence of anisotropic structures at depth, with the aim of mapping the variation of anisotropy across the northern part of the island. This study focuses on the relation among anisotropy detected in the crust, stresses induced by plate convergence across the subduction zone, and the intrinsic characteristics of the rocks. Our results show how a simple velocity model with two anisotropic layers reproduces the observed data at the stations. We observe a negligible or small amount of signal related to anisotropy in the eastern part of the study area (i.e., the outer arc) for both upper and lower crust. Distinct anisotropic features are observed at the stations on the western part of the study area (i.e., the inner arc) for both upper and lower crust. The symmetry axes are mostly E-W oriented. Deviation from the E-W orientation is observed close to the volcanic areas, where the higher geothermal gradient might influence the deformation processes.

On the transforming growth factor beta-like activity of synthetic polypeptides comprising the amino-terminal sequence of human parathyroid hormone-related peptide.

Purified native forms of human parathyroid hormone-related peptide (PTHrp) have recently been reported to display biological activities characteristic of transforming growth factor beta (TGF-beta). The TGF-beta-like property of PTHrp may reside within the amino N-terminal PTH-receptor binding region of the polypeptide, since a synthetic analog corresponding to amino acids 1-36 of human PTHrp is as active as purified native PTHrp in bioassays specific to TGF-beta. Complete lack of structural similarity between PTHrp and TGF-beta prompted us to address the question whether copresence of the TGF-beta-like and PTH-like biological activities in the N-terminal sequence of the PTHrp molecule is a general phenomenon observable with different N-terminal PTHrp peptides of varying amino acid chain length in a variety of target cells that respond in defined ways to TGF-beta in vitro. Two forms of synthetic N-terminal human PTHrp, PTHrp-(1-34) and [Tyr40]PTHrp-(1-40), which are fully active in conventional assays for PTH/PTHrp, were tested for effects in three in vitro bioassay systems for TGF-beta: 1) stimulation, and 2) inhibition, respectively, of epidermal growth factor-dependent soft-agar colony formation of either normal rat kidney-derived fibroblasts (NRK 49F) or human lung carcinoma cells (A549); and 3) biosynthesis of metabolically labeled fibronectin in both NRK 49F cells and clonal osteoblastic rat osteosarcoma cells (ROS 17/2.8). Human TGF-beta over the dose range of 2.5-80 pM significantly stimulated or inhibited soft-agar colony formation of either NRK 49F or A549 cells, respectively, and caused a severalfold increase in biosynthetically labeled [35S]fibronectin in NRK 49F and ROS 17/2.8 cells. In contrast, none of PTHrp-(1-34), [Tyr40]PTHrp-(1-40), and synthetic human PTH-(1-34), each tested at 0.1-10 nM, displayed detectable biological activity in any of the three assay systems. In addition, covalent cross-linking of intact NRK 49F and ROS 17/2.8 cells with either [125I]TGF-beta or 125I-[Tyr40] PTHrp-(1-40) revealed the presence of several distinct affinity-labeled receptor species for TGF-beta in both cell types and the 80K PTH/PTHrp receptors in ROS 17/2.8 cells. The affinity-labeled TGF-beta receptor species were insensitive to excess PTHrp and PTH peptides, and the 80K PTH/PTHrp receptors were insensitive to excess TGF-beta, indicating that PTHrp and TGF-beta do not cross-react with respect to receptor binding for interaction with these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Episodic fluctuation in serum intact parathyroid hormone concentration in men.

To evaluate the temporal features of physiological fluctuation in serum PTH concentration, we sampled peripheral blood at 4-min intervals for 24 h from five normal men (32.8 yr; range, 26-40 yr) and measured serum PTH levels using a two-site immunoradiometric assay with the exquisite sensitivity and specificity for human PTH-(1-84) (intact PTH). The resultant 24-h time series of serum intact PTH levels were assessed by contemporary techniques in chronophysiology for rhythmic and episodic peak detection. Cosinor analysis disclosed a significant circadian rhythm in serum intact PTH concentrations in all five men, with the mean circadian amplitude and acrophase of 7.2 +/- 4.4 ng/L and 2305 +/- 401 h, respectively (mean +/- SD; n = 5). No apparent fixed ultradian periodicity was found by autocorrelation and spectral analyses. Evaluation of episodic intact PTH pulsatility by Cluster analysis revealed 23.0 +/- 4.4 discrete PTH pulses/24 h (P less than 0.01 vs. signal-free noise), which occurred at an interpulse interval of 61.6 +/- 11.1 min. The average duration of a serum intact PTH peak was 42.8 +/- 7.3 min, and its mean incremental amplitude was 12.6 +/- 1.3 ng/L, which corresponded to a 31.8 +/- 5.2% increase above the preceding nadir. Discrete PTH peaks were separated by nonpulsatile valleys which lasted for 17.9 +/- 4.4 min. Cross-correlation between the time series of serum intact PTH and whole blood ionized calcium (Ca2+) was at its maximum (-0.5) at concurrent time points in three subjects, while significant positive correlation between serum intact PTH and simultaneous serum inorganic phosphorus concentrations was observed in four of five subjects. There was no apparent correlation between the levels of serum intact PTH and serum magnesium. Our data show that serum levels of intact PTH, the only biologically active form of PTH in the blood, is characterized by a significant circadian periodicity, spontaneous episodic pulsatility with distinct peak properties, and a significant temporal coupling with Ca2+ and inorganic phosphorus concentrations. We conclude that PTH secretion, as judged by the temporal pattern of serum intact PTH levels, is pulsatile in normal men.

Primary structure of a potassium channel toxin from the sea anemone Actinia equina.

A potassium channel toxin (AeK) was isolated from the sea anemone Actinia equina by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. AeK and alpha-dendrotoxin inhibited the binding of 125I-alpha-dendrotoxin to rat synaptosomal membranes with IC50 of 22 and 0.34 nM, respectively, indicating that AeK is about sixty-five times less toxic than alpha-dendrotoxin. The complete amino acid sequence of AeK was elucidated; it is composed of 36 amino acid residues including six half-Cys residues. The determined sequence showed that AeK is analogous to the three potassium channel toxins from sea anemones (BgK from Bunodosoma granulifera, ShK from Stichodactyla helianthus and AsKS from Anemonia sulcata), with an especially high sequence homology (86%) with AsKS.

Identification of a novel transthyretin variant (Val30----Leu) associated with familial amyloidotic polyneuropathy.

A novel variant transthyretin which contains a leucine-for-valine substitution at position 30 was isolated and identified in the serum of a patient with familial amyloidotic polyneuropathy (FAP). The amino acid substitution was proven to result from a guanine-to-cytosine change at the first base of codon 30 located in exon 2 in the mutated transthyretin gene by restriction fragment length analysis on the amplified transthyretin gene using Cfr13 I. The study indicates that the point mutation of the transthyretin gene is a cause of the disorder.

Effects of sublingual nitrate in patients receiving sustained therapy of isosorbide dinitrate for coronary artery disease.

To examine the effects of sublingual isosorbide dinitrate (ISDN) in patients receiving sustained ISDN therapy, 24 patients with coronary artery disease were divided into 2 groups. Group C comprised 12 patients without sustained ISDN therapy and group N included 12 patients with sustained ISDN therapy. Before and during administration of sublingual ISDN in both groups, aortic systolic pressure, left ventricular end-diastolic pressure and coronary artery diameter were examined at cardiac catheterization. During sublingual ISDN, the aortic systolic pressure decreased by 20 +/- 6% (138 +/- 26 to 112 +/- 27 mm Hg, p less than 0.01) in group C and 10 +/- 6% (127 +/- 26 to 113 +/- 23 mm Hg, p less than 0.01) in group N (p less than 0.01, group C vs group N). The left ventricular end-diastolic pressure decreased by 65 +/- 16% (11 +/- 5 to 4 +/- 3 mm Hg, p less than 0.01) in group C and 43 +/- 14% (12 +/- 5 to 7 +/- 3 mm Hg, p less than 0.01) in group N (p less than 0.01, group C vs group N). During sublingual ISDN, the diameters of the proximal and distal segments of the left anterior descending and circumflex coronary arteries increased more significantly in group C than in group N (p less than 0.01, group C vs group N). Thus, sublingual ISDN produced less reduction of aortic systolic pressure and left ventricular end-diastolic pressure, and less dilation of coronary artery diameter in patients receiving sustained therapy with ISDN than in those without sustained therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of the VAP-peptide (BmACP-6.7) gene in the silkworm, Bombyx mori and a possible regulation of its expression by BmFTZ-F1.

The VAP-peptide (BmACP-6.7) is a hydrophobic peptide localized in adult cuticle of the silkworm, Bombyx mori. We isolated and characterized the VAP-peptide gene as a useful marker gene to analyze molecular mechanisms of terminal differentiation processes in the adult. The gene is composed of two exons interrupted by one intron. The 5' upstream promoter region is shown to bear a nucleotide sequence similar to the cis-element that is recognized and bound by the Bombyx mori FTZ-F1 protein (BmFTZ-F1). Expression of the BmFTZ-F1 gene preceded expression of the VAP-peptide gene and injection of 20-hydroxyecdysone suppressed the expression of both genes. An in vitro binding assay indicated direct interaction of BmFTZ-F1 with the VAP-peptide gene promoter sequence. Therefore, BmFTZ-F1 is proposed to be a possible factor regulating the stage-specific expression of the VAP-peptide gene towards adult life.

A hydrophobic peptide (VAP-peptide) of the silkworm, Bombyx mori: structure, expression and an enhancing function of diapause hormone activity.

We have recently identified a unique lipophilic peptide (VAP-peptide) with diapause egg inducing activity in the silkworm, Bombyx mori (Imai et al., 1996). The cloning and sequencing of cDNA encoding VAP-peptide have demonstrated that the deduced amino acid sequence consisted of 84 amino acid residues, from which the mature VAP-peptide of 68 amino acid residues was released by cleaving a signal sequence. Searches of the GenBank data base revealed no significant sequence similarity to other proteins including diapause hormone (DH). VAP-peptide gene was selectively expressed just before and at adult eclosion in the head and the thorax not in the abdomen. By a Western blot analysis, VAP-peptide was also localized in the head and the thorax of adults. The purified recombinant VAP-peptide could not induce diapause eggs even when injected at a high dose of 10 nmol/pupa. Whereas, injection of a mixture of VAP-peptide and DH clearly decreased a half-maximum dose (ED50 value) and a threshold dose (TD value) of DH, and these values decreased according to increasing molar ratios of VAP-peptide to DH. Thus, the VAP-peptide is concluded to be an endogenous protein acting as a potent enhancer of DH activity through interaction with DH.

A hydrophobic peptide (VAP-peptide) of the silkworm, Bombyx mori: a unique role for adult activity proposed from gene expression and production at the terminal phase of metamorphosis.

A unique hydrophobic peptide (VAP-peptide) isolated from male adult heads of the silkworm, Bombyx mori, has been shown to act as a synergist to the diapause hormone when administered exogenously. Here, we investigated the true role of the endogenous VAP-peptide on differentiation and development of adult organs in the silkworm. By northern blot analyses, the VAP-peptide gene was shown to be exclusively expressed at the terminal phase of adult development in epithelial tissues, especially in the wing and the thoracic integument. In situ hybridization analysis revealed that the gene was highly expressed in the epidermal cells of the wing vein and the thoracic integument. The stage- and tissue-dependent gene expression were clearly correlated to the accumulation profile of VAP-peptide. In the adult thoracic integument, VAP-peptide was predominantly deposited in the cuticle layer. Affinity chromatography indicated the ability of VAP-peptide to bind to chitin. Based on its expression patterns, localization, and chemical properties, VAP-peptide is conceived to be a structural protein that participates in mechanical strengthening of specific cuticle structures, supporting their physical requirements in the adult life of the silkworm.

Total synthesis of nafuredin, a selective NADH-fumarate reductase inhibitor.

[structure: see text] Total synthesis of nafuredin, a selective NADH-fumarate reductase inhibitor, has been accomplished by a convergent approach. The C1-C8 and C9-C18 segments were derived efficiently from D-glucose and (S)-(-)-2-methyl-1-butanol, respectively, coupled by stereoselective Julia olefination, and converted to nafuredin.

Isolation and sequence determination of two N-terminal fragments of islet amyloid polypeptide in rat pancreas.

Using a highly sensitive and specific radioimmunoassay (RIA) for the N-terminal hexadecapeptide of islet amyloid polypeptide (IAPP), we isolated two N-terminal fragments of IAPP from rat pancreas. They were identified as IAPP(1-16) and IAPP(1-17) by amino acid sequencing. The two fragments were also found in rat plasma. IAPP(1-37) was the major molecular form of rat IAPP, IAPP(1-16) and IAPP(1-17) accounting for 6.0% and 32.3% of the immunoreactivity for the N-terminal region of the peptide in pancreata of normally fed rats. In human pancreas, the N-terminal fragments of IAPP were not present, indicating that the processing of IAPP in the pancreas differs between human and rat. Food deprivation increased the molar ratios of IAPP(1-16) and IAPP(1-17) to IAPP(1-37) in comparison to values for fed rats. Identification of novel fragments of IAPP, in addition to IAPP(1-37), offers a promise for the elucidation of the physiological function of IAPP and the identification of factors that regulate the biosynthesis and catabolism of the peptide.