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.

Search for a light pseudoscalar particle in the decay K_{L};{0}-->pi;{0}pi;{0}X.

We performed a search for a light pseudoscalar particle X in the decay K_{L};{0}-->pi;{0}pi;{0}X, X-->gammagamma with the E391a detector at KEK. Such a particle with a mass of 214.3 MeV/c;{2} was suggested by the HyperCP experiment. We found no evidence for X and set an upper limit on the product branching ratio for K_{L};{0}-->pi;{0}pi;{0}X, X-->gammagamma of 2.4x10;{-7} at the 90% confidence level. Upper limits on the branching ratios in the mass region of X from 194.3 to 219.3 MeV/c;{2} are also presented.

Search for the Decay K L0-->pi0nu nu[over].

We performed a search for the K L0-->pi0nu nu[over] decay at the KEK 12-GeV proton synchrotron. No candidate events were observed. An upper limit on the branching ratio for the decay was set to be 6.7 x 10(-8) at the 90% confidence level.

Function of surfactants in hair dyeing by oxidation dyes 2. Effect on formation of oxidation dyes by p-aminophenol and 5-amino-o-cresol in dye bath(1).

The effect of surfactants on an oxidation-hair-dye-formation reaction in a dye bath was studied in order to learn the mechanism of the effect of surfactants on the dyeability of hair by the oxidation dye. The dye-formation behaviours for the p-aminophenol and 5-amino-o-cresol system with the surfactants, of which the hydrophilic parts have different charges, were compared changing the concentration of surfactants. It was found that the same dyes are produced, regardless of the charge of surfactants added, and the rate of dye produced in the dyebath is increased in the presence of surfactants. The order of the production rate is, with an anionic surfactant > with non-ionic surfactant > with cationic surfactant > without surfactant. The relation between the dyeability of hair and the rate of dye produced in the dyebath in the presence of surfactants is not found. The major factor governing the dyeability of hair is different from the mechanism of the increased dye in the solution. It was also found that the dye-formation rate is increased by immersing hair into the reaction solution, and hair works as an accelerator for the dye-formation reaction.

Further evidence for the decay K+ -->pi+nu(nu).

Additional evidence for the rare kaon decay K+-->pi+nu(nu) has been found in a new data set with comparable sensitivity to the previously reported result. One new event was observed in the pion momentum region examined, 211pi+nu(nu)) = 1.57(+1.75)(-0.82)x10(-10).

Molecular phylogeny of butterflies Parnassius glacialis and P. stubbendorfii at various localities in East Asia.

The phylogeny of butterflies, Parnassius stubbendorfii and P. glacialis, collected at various localities in the Japan archipelago and the eastern part of the Asian continent was analyzed using mitochondrial DNA sequences coding for NADH dehydrogenase subunit 5 (805 bp). The molecular phylogenetic trees revealed that P. glacialis and P. stubbendorfii diverged from a common ancestor, and then the populations inhabiting the Japan archipelago and the Asian continent diverged in each species. The reliability of these divergences was supported by high bootstrap values. The divergences within the Japan archipelago and within the Asian continent in each species were unclear because of low bootstrap values. The genetic distance and a rough time-estimation in the UPGMA tree suggest that the both populations of P. glacialis and P. stubbendorfii may have been isolated in the Japan archipelago at the early time (about 1.7-2.0 Mya) of the glacial period in the Pleistocene. The genetic distance between the Japanese and the continental subspecies may be large enough that they can be classified as different species, in comparison with the genetic distances among some other parnassian species.

[Reconstruction of the pulmonary outflow tract without external conduit].

Between October 1987 and December 2000, 50 patients underwent reconstruction of the pulmonary outflow tract without external conduit. The primary malformation was tetralogy of Fallot with pulmonary atresia in 37, double outlet of right ventricle in 4, corrected transposition of the great arteries in 4, transposition of the great arteries with ventricular septal defect and pulmonary stenosis in 4, and double outlet of left ventricle in 2. Mean age at operation was 7.2 years, and mean body weight was 18.3 kg. To reconstruct posterior wall of the pulmonary outflow tract, interposition of autologous pericardium was performed in 24, direct anastomosis between pulmonary trunk and ventriculotomy in 13, longitudinal incision from ventriculotomy through pulmonary trunk in 12, and interposition of left atrial appendage in 1. Anterior wall was reconstructed with monocusp valved outflow patch (MVOP). There was one hospital death and no late death. At 10 years, the freedom from reoperation for pulmonary outflow tract obstruction was 100%, and freedom from reoperation for any cause was 86.6%. Transcatheter stenting for peripheral pulmonary stenosis was performed in 6 patients 2 to 10 months after operation.

Inhibition of platelet aggregation and the release of P-selectin from platelets by cilostazol.

To evaluate the in vitro effects of cilostazol, a phosphodiesterase III inhibitor, on platelet responses, we measured platelet aggregation and the levels of soluble P-selectin, a glycoprotein present on the alpha-granule membrane in resting platelets, and cAMP. Platelet-rich plasma and washed platelets from healthy human volunteers were treated with cilostazol (5, 25 and 50 microM). Platelet-rich plasma was stimulated by ADP (1 and 5 microM) or collagen (5 microg/ml). Washed platelets were stimulated by thrombin (4 U/ml) in the presence or absence of 1 microM forskolin. In vehicle-treated samples, soluble P-selectin levels in response to 1 microM ADP-induced primary aggregation were similar to those of circulating levels of healthy volunteers but the levels in response to 5 microM ADP-induced secondary aggregation and collagen-induced aggregation increased markedly compared to those in response to primary aggregation. This result suggests that P-selectin is released from platelets according to the extent of platelet aggregation. Cilostazol inhibited platelet aggregation as well as P-selectin release in a concentration-dependent manner. Cilostazol inhibited completely thrombin-induced aggregation in the presence of 1 microM forskolin, when cAMP levels were two-fold higher than those in the absence of forskolin. Cilostazol, which increases intracellular cAMP in platelets, may be useful in the treatment of arterial occlusive diseases.

Crystallization and preliminary X-ray crystallographic analysis of S-allelic glycoprotein S(F11)-RNase from Nicotiana alata.

Nicotiana alata S(F11)-RNase is an S-glycoprotein associated with gametophytic self-incompatibility. Crystals of S(F11)-RNase have been grown at room temperature using polyethylene glycol as a precipitant. A crystal diffracted to better than 1.4 A resolution at 100 K at the SPring-8 synchrotron-radiation source, indicating that it is very suitable for high-resolution structure analysis. The crystal belongs to the space group P2(1), with unit-cell parameters a = 65.86 (11), b = 44.73 (5), c = 64.36 (7) A, beta = 90.27 (9) degrees. The asymmetric unit contains two monomers, giving a crystal volume per protein mass (V(M)) of 2.05 A(3) Da(-1) and a solvent content of 39.6% by volume. A full set of X-ray diffraction data was collected to 1.55 A resolution with a completeness of 97.4%. A heavy-atom derivative has been successfully prepared with ethylmercury thiosalicylate (EMTS) and structure analysis is in progress.

Further search for the decay K+-->pi(+)nunu;

A search for additional evidence for the rare kaon decay K+-->pi(+)nunu; has been made with a new data set comparable in sensitivity to the previous exposure that produced a single event. No new events were found in the pion momentum region examined, 211pi(+)nunu;) = 1.5(+3.4)(-1.2)x10(-10).

Measurement of structure-dependent K+ --> &mgr;(+)nu(&mgr;)gamma decay

We report the first measurement of a structure-dependent component in the decay K+-->&mgr;(+)nu(&mgr;)gamma. Using the kinematic region where the muon kinetic energy is greater than 137 MeV and the photon energy is greater than 90 MeV, we find that the absolute value of the sum of the vector and axial-vector form factors is |F(V)+F(A)| = 0.165+/-0.007+/-0.011. This corresponds to a branching ratio of B(SD+) = (1.33+/-0.12+/-0.18)x10(-5). We also set the limit -0. 04

Presence of asparagine-linked N-acetylglucosamine and chitobiose in Pyrus pyrifolia S-RNases associated with gametophytic self-incompatibility.

S-RNases encoded by the S-locus of rosaceous and solanaceous plants discriminate between the S-alleles of pollen in gametophytic self-incompatibility reactions, but it is not clear how. We report the structures of N-glycans attached to each of the N-glycosylation sites of seven S-RNases in Pyrus pyrifolia of the Rosaceae. The structures were identified by chromatographic analysis of pyridylaminated sugar chains prepared from S4-RNase and by liquid chromatography/electrospray ionization-mass spectrometric analysis of the protease digests of reduced and S-carboxymethylated S-RNases. S4-RNase carries various types of sugar chains, including plant-specific ones with beta1-->2-linked xylose and alpha1-->3-linked fucose residues. More than 70% of the total N-glycans of S4-RNase are, however, an N-acetylglucosamine or a chitobiose (GlcNAcbeta1-->4GlcNAc), which has not been found naturally. The N-acetylglucosamine and chitobiose are mainly present at the N-glycosylation sites within the putative recognition sites of the S-RNase, suggesting that these sugar chains may interact with pollen S-product(s).

Inhibitory effects of H2-receptor antagonists on platelet function in vitro.

To evaluate in vitro inhibitory effects of four types of histamine H2-receptor antagonist (H2-receptor antagonists), famotidine, roxatidine, cimetidine and ranitidine, on platelet function, we examined aggregating potency and P-selectin levels with agonist-induced aggregation. Ranitidine and cimetidine inhibited, in concentration of 0.35 mM, the secondary aggregation induced by 5 microM adenosine diphosphate (ADP), the aggregation induced by 1 microg/mL collagen and 3 microM arachidonic acid. All of H2-receptor antagonists inhibited, in concentration of 1.4 mM, the aggregation induced by ADP, collagen and arachidonic acid. Ranitidine and cimetidine reduced markedly, in same concentration, P-selectin levels after induction of aggregation by 5 microm ADP, 1 microg/mL collagen and 3 microM arachidonic acid. When classified by the strength of inhibitory action, ranitidine and cimetidine were strong, followed by famotidine and roxatidine. It is considered that inhibitory effects of H2-receptor antagonists on platelet function are weaker than those of acetylsalicylic acid (ASA), since ASA inhibited platelet aggregation in concentration of 100 microM. No relationship was observed between inhibitory effects of H2-receptor antagonists on platelet aggregation induced by above agonists and the presence or absence of imidazole ring in the chemical structure.

Cytoprotective effect of ulinastatin, a Kunitz-type protease inhibitor, on hypoxic injury in L2 cells treated with antimycin A via stabilization of lysosomal fragility.

The cytoprotective effect of ulinastatin, a Kunitz-type protease inhibitor, was studied in L2 cells treated with antimycin A, which induces depletion of cellular adenosine triphosphate (ATP), so-called <>. Antimycin A treatment with 2 microM significantly elevated the release of N-acetyl-beta-D-glucosaminidase (NAG), a lysosomal enzyme, to 51.99+/-7.36%. In this condition, ulinastatin tended to inhibit the release of NAG at a concentration of 1000 U/ml (39.74+/-3.80%) and significantly ameliorated it at a concentration of 3000 U/ml (32.35+/-4.17%). Furthermore, ulinastatin at 10 U/ml showed a suppression on the fragility of lysosomal membrane isolated from L2 cells. These results indicate that ulinastatin has a prominent protective effect on hypoxic injury in L2 cells, and the lysosomal stabilizing effect is possibly involved as a mechanism of this action.

Compound heterozygosity for an apolipoprotein A1 gene promoter mutation and a structural nonsense mutation with apolipoprotein A1 deficiency.

Apolipoprotein (apo) A1 plays a central role in the metabolism of HDL. We describe a novel genetic variant of the apoA1 gene identified in a patient with low concentrations of plasma HDL cholesterol. The proband, a 12-year-old Japanese boy, exhibited markedly low levels of both plasma apoA1 and HDL cholesterol. Genomic DNA sequencing of apoA1 genes of the patient showed a compound heterozygosity for an A to C substitution at 27 bp upstream of the transcription start site of 1 apoA1 allele, and a C to T substitution in another allele at residue 84 resulting in aberrant termination. The point mutation at nucleotide position -27 changed ATAAATA of the putative TATA box signal sequence to ATACATA. In addition to this mutation, the patient was heterozygous for a G to A substitution at position -75. Immunoblotting of an isoelectric focusing electrophoresis gel of the proband's plasma showed a trace amount of normal apoA1. No measurable plasma apoA1 and HDL cholesterol in a patient with homozygosity for nonsense mutation at residue 84 has been reported previously. To determine the effects of substitution either at position -27 or -75, plasmids containing the 5'-flanking region of the human apoA1 promoter fused to the CAT reporter gene were constructed and transfected in HepG2 cells. A construct with the A to C substitution at position -27 showed 41. 8+/-4.2%, and G to A substitution at position -75 showed 72.8+/-15. 2% (means+/-SD, n=3) of CAT activities, compared with the wild-type promoter sequence. A construct with the double substitutions at positions -27 and -75 showed only 22.8+/-1.3% (mean+/-SD, n=3) activity relative to the wild type. Our patient is the first case with a TATA box mutation etiologically related to lipoprotein disorders.

Primary structural features of rosaceous S-RNases associated with gametophytic self-incompatibility.

We isolated cDNA clones encoding five S-RNases (S1-, S3-, S5-, S6-, S7-RNases) from pistils of Pyrus pyrifolia (Japanese pear), a member of the Rosaceae. Their amino acid sequences were aligned with those of other rosaceous S-RNases sequenced so far. A total of 76 conserved amino acid residues were stretched throughout the sequence, but were absent from the 51-66 region which was designated the hypervariable (HV) region. The phylogenetic tree of rosaceous S-RNases showed that S-RNase polymorphism predated the divergence of Pyrus and Malus. Pairwise comparison of these S-RNases detected two highly homologous pairs, P. pyrifolia S1- and S4-RNases (90.0%) and P. pyrifolia S3- and S5-RNases (95.5%). The positions of amino acid substitutions between S1- and S4-RNases were spread over the entire region, but in the pair of S3- and S5-RNases, amino acid substitutions were found in the 21-90 region including the HV region. The substitutions in this restricted region appear to be sufficient to discriminate between S3 and S5 pollen and to trigger the self-incompatible reaction.

Role of O-linked carbohydrate of human urinary trypsin inhibitor on its lysosomal membrane-stabilizing property.

Human urinary trypsin inhibitor (UTI) was digested with various enzymes to obtain O-glycoside linked N-terminal glycopeptide (UTIm1), N-glycoside linked C-terminal tandem Kunitz-domains (domain I and II, UTIm2), UTI lacking O-glycoside (UTIc), asialo UTI (UTIa) and UTI lacking N-glycoside (UTIn). We investigated the membrane stabilizing effect of these UTI derivatives on rat renal lysosome by measurement of lysosomal enzyme N-acetyl-beta-D-glucosaminidase (NAG) release after hypotonic treatment. Intact UTI suppressed NAG release, but aprotinin, gabexate mesilate (FOY), nafamostat mesilate (FUT) and recombinant domain II of UTI (R-020) had no effect, indicating that inhibition of serine proteases was not involved and the carbohydrate moiety of UTI might be necessary for this property. Among UTI derivatives, UTIm1, UTIm2, UTIm1+ UTIm2, and UTIc had no effect. In contrast, UTIa or UTIn suppressed NAG release. From these results, we conclude that O-glycoside linked core protein without N-glycoside is essential to the lysosomal membrane-stabilizing property of UTI.

Cytoprotective effect of ulinastatin on LLC-PK1 cells treated with antimycin A, gentamicin, and cisplatin.

The cytoprotective effect of ulinastatin was studied in LLC-PK1 cells treated with antimycin A, gentamicin, or cisplatin. All of the three agents induced a concentration-dependent increase in the release of lactate dehydrogenase and a decrease in the amount of remaining protein. In the cell injury models treated with 1.5 microM antimycin A, 10 mM gentamicin, and 0.3 mM cisplatin, ulinastatin tended to show a cytoprotective effect at a concentration of 3,000 U/ml and provided a significant protective effect at 10,000 U/ml. LLC-PK1 cells treated with 0.3 mM cisplatin, bovine serum albumin, and alpha1-acid glycoprotein at a concentration of 3.54 mg/ml, which is a comparable protein concentration to that of 10,000 U/ml ulinastatin, showed no protective effect but rather enhanced cell injury. These results suggest that ulinastatin exerts a direct protective effect on LLC-PK1 cells against various renal toxicities.

Inhibitory effects of antibiotics on platelet aggregation in vitro.

1. We evaluated in vitro inhibitory effects of six types of antibiotic, aztreonam (AZT), cefamandole (CMD), cefmetazole (CMZ), cefotiam (CTM), flomoxef (FMOX) and latamoxef (LMOX), on platelet aggregation, using healthy volunteers' blood. Four types--FMOX, LMOX, CTM and CMD--inhibited, in concentration of 2500 micrograms/ml, the secondary aggregation induced by 3.0 microM adenosine diphosphate (ADP), and also inhibited the aggregation induced by 0.5 micrograms/mi collagen. AZT in the same concentration, did not inhibit the aggregation induced by collagen, and it inhibited only ADP-induced aggregation. CMZ, in the same concentration, inhibited neither of the two aggregations. 2. The inhibitory effects of the antibiotics on collagen-induced aggregation were dependent on the concentration of respective antibiotics. When classified by the strength of inhibitory action, LMOX and FMOX were strong, followed by CTM and CMD. The action of AZT and CMZ was weak. In particular, LMOX showed a 32% inhibitory effect at concentration of 50 micrograms/ml, a level near the blood concentration obtained with clinical usual dose. 3. No relationship was observed between inhibitory effects of antibiotics on ADP- or collagen-induced aggregation and the presence or absence of carboxyl group and/or N-methyltetrazolethiol group in the chemical structure.

Molecular effects of M17055, furosemide and thiazide on cardiac hypertrophy of spontaneously hypertensive rats.

Although diuretics have been clinically shown to reduce cardiovascular morbidity and mortality, the effects of diuretics on cardiac hypertrophy are poorly understood. In this study, we examined the molecular effects of diuretics on hypertensive cardiac hypertrophy. Spontaneously hypertensive rats (SHR) were given p.o. M17055 (a novel "high ceiling" diuretic) 1.25, 2.5 or 5 mg/kg/day, furosemide 50 mg/kg/day or trichlormethiazide 30 mg/kg/day for 5 weeks. After the treatment, cardiac myosin isoforms were analyzed by gel electrophoresis, and cardiac hypertrophy-related gene expressions were examined by Northern blot analysis. These three diuretics significantly reduced cardiac hypertrophy of SHR. M17055 and furosemide, but not trichlormethiazide, significantly increased the proportion of cardiac V3 myosin of SHR by enhancing the gene expression of beta-myosin heavy chain. On the other hand, trichlormethiazide, but not M17055 or furosemide, suppressed the increased cardiac gene expression of skeletal alpha-actin in SHR. Cardiac collagen type III expression of SHR was decreased only by treatment with M17055. Plasma thyroid hormone levels of SHR were slightly decreased by M17055 and by furosemide and were negatively correlated with cardiac V3 myosin contents. Thus the effects on the gene expression of cardiac contractile proteins and collagen are significantly different among these three types of diuretics, which suggests that these diuretics may have different cardiac actions independent of their diuretic and antihypertensive actions. The increased cardiac V3 myosin induced by M17055 and by furosemide may be partially due to the decreased plasma thyroid hormone.