Large scale replication analysis of loci associated with lipid concentrations in a Japanese population.

BACKGROUND: Recent genome wide association studies discovered seven novel loci that influence plasma concentrations of triglycerides, high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol in Europeans. To date, large scale replication studies using populations with known differences in genome-wide linkage disequilibrium (LD) pattern have not been undertaken. METHODS: To address this issue, we tested associations between single nucleotide polymorphisms (SNPs) within the seven novel loci and plasma lipid profiles in 21 010 Japanese individuals. RESULTS: Multiple linear regression analyses showed that the rs3812316 in MLXIPL was strongly associated with triglyceride concentrations (p approximately 3.0x10(-11), 7.1 mg/dl decrease per minor C allele) and that rs599839 in CELSR2/PSRC1/SORT1 was strongly associated with LDL cholesterol concentrations (p approximately 3.1x10(-11), 4.7 mg/dl decrease per minor G allele) in the Japanese population. SNPs near ANGPTL3, TRIB1 and GALNT2 showed evidence for associations with triglyceride concentrations (3.6x10(-6)

Blockade of Angiotensin II type-1 receptor reduces oxidative stress in adipose tissue and ameliorates adipocytokine dysregulation.

Dysregulated production of adipocytokines may be involved in the development of atherosclerotic cardiovascular disease in metabolic syndrome and chronic kidney disease (CKD) associated with metabolic syndrome. The aim of this study was to determine the effects of treatment with angiotensin II (Ang II) type-1 receptor blocker (ARB) on the regulation of adipocytokines. Olmesartan, an ARB, significantly blunted the age- and body weight-associated falls in plasma adiponectin both in genetically and diet-induced obese mice, without affecting body weight, but had no effect on plasma adiponectin levels in lean mice. Olmesartan also ameliorated dysregulation of adipocytokines in obesity, such as tumor necrosis factor-alpha, plasminogen activator inhibitor-1, monocyte chemotactic protein-1, and serum amyloid A3. Olmesartan significantly reduced reactive oxygen species originating from accumulated fat and attenuated the expression of nicotinamide adenine dinucleotide phospho hydrogenase oxidase subunits in adipose tissue. In cultured adipocytes, olmesartan acted as an antioxidant and improved adipocytokine dysregulation. Our results indicate that blockade of Ang II receptor ameliorates adipocytokine dysregulation and that such action is mediated, at least in part, by targeting oxidative stress in obese adipose tissue. Ang II signaling and subsequent oxidative stress in adipose tissue may be potential targets for the prevention of atherosclerotic cardiovascular disease in metabolic syndrome and also in metabolic syndrome-based CKD.

Renal damage and salt-dependent hypertension in aged transgenic mice overexpressing endothelin-1.

The recent development of endothelin-1 (ET-1) antagonists and their potential use in the treatment of human disease raises questions as to the role of ET-1 in the pathophysiology of such cardiovascular ailments as hypertension, heart failure, renal failure and atherosclerosis. It is still unclear, for example, whether activation of an endogenous ET-1 system is itself the primary cause of any of these ailments. In that context, the phenotypic manifestations of chronic ET-1 overproduction may provide clues about the tissues and systems affected by ET-1. We therefore established two lines of transgenic mice overexpressing the ET-1 gene under the direction of its own promoter. These mice exhibited low body weight, diminished fur density and two- to fourfold increases in the ET-1 levels measured in plasma, heart, kidney and aorta. There were no apparent histological abnormalities in the visceral organs of young (8 weeks old) transgenic mice, nor was their blood pressure elevated. In aged (12 months old) transgenic mice, however, renal manifestations, including prominent interstitial fibrosis, renal cysts, glomerulosclerosis and narrowing of arterioles, were detected. These pathological changes were accompanied by decreased creatinine clearance, elevated urinary protein excretion and salt-dependent hypertension. It thus appears that mild, chronic overproduction of ET-1 does not primarily cause hypertension but triggers damaging changes in the kidney which lead to the susceptibility to salt-induced hypertension.

Reactivity of autoantibodies of autoimmune hemolytic anemia with recombinant rhesus blood group antigens or anion transporter band3.

The specificity of autoantibodies in autoimmune hemolytic anemia (AIHA) has been studied using the serological procedure and immunoprecipitation technique with rare phenotype red cells. We attempted to analyze specificity using recombinant rhesus (Rh) blood group and band3 antigens expressed on erythroleukemic cell lines, KU812E. The autoantibody eluates were isolated by the acid elution procedure from the red cells of 20 AIHA patients. The recombinant Rh antigens, RhD, cE, ce, CE, and chimera antigens CE-D and D-CE, were obtained by retroviral cDNA transduction into KU812E cells, and the cell line expressing the antigens was cloned. Band3 cDNA was also obtained and introduced into KU812E and cloned KU812 expressing RhcE. The reactivities of AIHA eluates with recombinant Rh and band3 antigens were studied by flow cytometry. Fifteen eluates reacted with at least one of the RhcE, ce, or CE antigens, and four eluates reacted with RhD. Seven eluates with strong Rh specificity were studied further using chimera antigen. Five eluates showed reduced or lost reactivity, although two eluates reacted identically with the chimera antigens as wild type. These results indicated that conformational epitopes constituted by RhD or CE specific exofacial peptide loops are important for autoantibodies in most cases. Seven eluates reacted with band3, five exclusively. The coexpression study of RhcE and band3 did not enhance the expression of either antigen nor the reactivity with patient eluates, indicating that association of Rh and band3 was not involved in the appearance of autoantigen.

Bamboo node: primary vocal fold lesion as evidence of autoimmune disease.

Descriptions of vocal fold lesions related to autoimmune diseases are rare in the literature, and focus mainly on rheumatoid nodules. This is the first report in which autoimmune diseases were promptly suspected by the observation of a unique white transverse submucosal lesion in the vocal fold during clinical examination. This lesion, reported only in autoimmune disease, has been called the bamboo node and its features are different from those of rheumatoid nodules. We report here on two patients who did not have a diagnosis of systemic disease before investigation of their main complaint of hoarseness. At the patients' first visit, vocal fold bamboo nodes were seen in the vocal fold and the otolaryngologist suspected the presence of an autoimmune disease. We requested clinical investigation to clarify our suspicion that there was an underlying systemic disease. After the investigation, both patients were shown to have autoimmune disease, Sjögren's syndrome and systemic lupus erythematous, respectively. This paper emphasizes the important role of the otolaryngologist in the detection of these unique lesions in the vocal folds through the conventional laryngeal methods. These methods consisted of direct observation with a rigid laryngeal endoscope and investigation of the patient's distinctive vibratory pattern by means of laryngeal stroboscopy. The method of treatment we used to obtain the best outcome in terms of voice improvement is also discussed.

Morphological and histochemical studies of the genioglossus muscle.

The purpose of this preliminary study was to assess the histo-anatomic composition of the genioglossus muscle fibers. The genioglossus muscles were obtained from 4 cadavers and 1 autopsy specimen. On morphological study, the average diameters of the muscle fibers were seen to gradually increase, from the fibers that ran anteriorly to the dorsum of the tongue, to the fibers that ran posteriorly to the root of the tongue. Histochemical study revealed that type II fibers were significantly predominant in the anterior portion; there was no dominant fiber type in the posterior portion. Gradual changes in diameter were independent of fiber type. These findings may suggest that the fibers of the anterior portion are suitable for phasic action, and that the posterior is relatively tonic; and the posterior has larger absolute muscle strength than the anterior. It is thought that the fibers of the posterior portion might contribute to the maintenance of the mesopharyngeal airway and to vowel production, and that the anterior fibers might contribute to some fine movements and to consonant production.

Design of synchrotron light source and its beamline dedicated to dual-energy x-ray computed tomography.

A synchrotron light source dedicated to medical applications has been designed at National Institute of Radiological Sciences. The storage ring, with circumference of 80 m, is designed for acceleration of 2.3 GeV and a stored current of 420 mA. It is equipped with two multipole wigglers to produce sufficient photon flux in a hard x-ray region required for medical applications. The purposes of the synchrotron light source are clinical performance of medical diagnoses clinically and research and development relating with medical applications. One of the most interesting applications for us is dual-energy x-ray computed tomography (CT). It gives the information about electron density of human tissue. The information plays an important role in advancing heavy-ion radiotherapy of cancers. Electron density can be derived from attenuation coefficients measured by different energy x rays. In this paper, a practical method of the dual-energy x-ray CT with synchrotron radiation is proposed with the theoretical consideration. The primitive experiment using monochromatic x rays emitted from radioisotopes proved the procedure of analysis mentioned here effective to derive electron densities from linear attenuation coefficients for two x rays of a different energy. The beamline dedicated to dual-energy x-ray CT is also proposed. It has a multipole wiggler as a light source and it mainly consists of a dual crystal monochromator and a rotating filter for attenuating photon flux of x rays and two-dimensional detector.

Heat transfer and flow characteristics on a gas turbine shroud.

The work described in this paper is an experimental investigation of the heat transfer from the main flow to a turbine shroud surface, which may be applicable to ceramic gas turbines. Three kinds of turbine shrouds are considered with a flat surface, a taper surface and a spiral groove surface opposite to the blades in an axial flow turbine of actual turbo-charger. Heat transfer measurements were performed for the experimental conditions of a uniform heat flux or a uniform wall temperature. The effects of the inlet flow angle, rotational speed, and tip clearance on the heat transfer coefficient were clarified under on- and off-design flow conditions. The mean heat transfer coefficient was correlated to the blade Reynolds number and tip clearance, and compared with an experimental correlation and measurements of a flat surface. A comparison was also made for the measurement of static pressure distributions.

The syllable method: proportion of impaired syllables as an indicator of spasmodic dysphonia severity.

We have previously proposed the 'mora method' to evaluate the degree of impairment in spasmodic dysphonia (SD) in Japanese-speaking patients. With this method, impairment is judged as the proportion of impaired morae in a 25-mora sentence in a longer passage read aloud. As the mora is the phonologically isochronic unit in Japanese, the proportion of impaired morae in speech can be used to represent the temporal proportion of impaired Japanese speech. This proportional measure of impairment reflects the perceived severity of the impairment, and is a more reliable measure than a 4-point perceptual rating scale completed either by patients or by voice professionals. In this paper, we propose a 'syllable method' to characterize the severity of impairment in English-speaking SD patients. Instead of morae, the severity of SD was represented by the proportion of impaired syllables. It should be possible to characterize SD impairments more reliably and precisely with this method than using a 4-point rating scale completed by voice professionals. Ultimately, the syllable method should become a simple and effective method for evaluating the severity of SD. Furthermore, such methods can be applied to other languages, using the appropriate phonological unit (either mora or syllable).

Molecular characterization of weak D phenotypes by site-directed mutagenesis and expression of mutant Rh-green fluorescence protein fusions in K562 cells.

BACKGROUND AND OBJECTIVES: Mutations detected in 161 weak D samples from Caucasians have been classified into 16 types. Because flow cytometry using monoclonal anti-D antibodies (mAbs) has shown that weak D red cells display type-specific antigen density, these mutations in transmembranous regions have been assigned weak D phenotypes. The present study attempts to confirm or refute this assignment. MATERIALS AND METHODS: We amplified DNA from four Japanese weak D samples using the polymerase chain reaction (PCR), and directly sequenced the amplified DNA. Using site-directed mutagenesis, we constructed three vectors expressing mutant RHDs-- G212C, V270G (weak D type 1) and G358A (type 2)--in K562 cells. The expression of RhD antigens was examined by flow cytometry using mAbs. RESULTS: A new mutation resulting in a conversion at amino acid residue 212 (Gly to Cys) was detected in a Japanese weak D sample. K562 cells transduced with mutant RhD cDNA reacted weakly in a type-specific manner with mAbs. CONCLUSIONS: The mutations--G212C (new weak D type), V270G (weak D type 1) and G358A (type 2)-- in transmembranous regions had obvious effects on the D epitopes recognized by mAbs. The results of this study provide direct evidence that these mutations can account for weak D phenotypes.

The analysis of nucleotide substitutions, gaps, and recombination events between RHD and RHCE genes through complete sequencing.

We determined the entire nucleotide sequences of all introns within the RHD and RHCE genes by amplifying genomic DNA using long PCR methods. The RHD and RHCE genes were 57,295 and 57,831 bp in length, respectively. Aligning both genes revealed 138 gaps (insertions and deletions) below 100 bp, 1116 substitutions in all introns and all exons (coding region), and 5 gaps of over 100 bp. Homologies (%) between the RH genes were 93.8% over all introns and coding exons and 91.7% over all exons and introns. Various short tandem repeats (STRs) and many interspersed nuclear elements were identified in both genes. The proportions of Alu sequences in the RHD and RHCE genes were 25.9 and 25.7%, respectively and these Alu sequences were concentrated in several regions. We confirmed multiple recombinations in introns 1 and 2. Such multiple recombination, which probably arose due to the concentrations of Alu sequences and the high level of the homology (%), is one of most important factors in the formation and evolution of RH gene. The variability of the Rh system may be generated because of these features of RH genes. Apparent mutational hotspots and regions with low of K values (the numbers of substitutions per nucleotide site) caused by recombinations as well as true mutational hotspots may be found in human genome. Accordingly, in searching for and identifying single nucleotide polymorphisms (SNPs) especially in noncoding regions, apparent mutational hotspots and areas of low K values by recombination should be noted since the unequal distribution of SNPs will reduce the power of SNPs as genetic maker. Combining the complete sequences' data of both RH genes with serological findings will provide beneficial information with which to elucidate the mechanism of recombination, mutation, polymorphism, and evolution of other genes containing the RH gene as well as to analyze Rh variants and develop new methods of Rh genotyping.

Role of endothelin-1 in stress response in the central nervous system.

Endothelin (ET)-1 is a 21-amino acid peptide that induces a variety of biological activities, including vasoconstriction and cell proliferation, and its likely involvement in cardiovascular and other diseases has recently led to broad clinical trials of ET receptor antagonists. ET-1 is widely distributed in the central nervous system (CNS), where it is thought to regulate hormone and neurotransmitter release. Here we show that CNS responses to emotional and physical stressors are differentially affected in heterozygous ET-1-knockout mice, which exhibited diminished aggressive and autonomic responses toward intruders (emotional stressors) but responded to restraint-induced (physical) stress more intensely than wild-type mice. This suggests differing roles of ET-1 in the central pathways mediating responses to different types of stress. Hypothalamic levels of ET-1 and the catecholamine metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) were both increased in wild-type mice subjected to intruder stress, whereas MHPG levels were not significantly affected in ET-1-knockout mice. Furthermore, immunohistochemical analysis showed that ET-1 and tyrosine hydroxylase, an enzyme in the catecholamine synthesis pathway, were colocalized within certain neurons of the hypothalamus and amygdala. Our findings suggest that ET-1 modulates central coordination of stress responses in close association with catecholamine metabolism.

A new device for evaluating autonomic nervous activity of cardiovascular system.

The purposes of present study are to evaluate a new measurement device with a piezo sensor to obtain fluctuation of finger blood pressure signal in comparison with the conventional tonometry system. We simultaneously measured continuous blood pressure by tonomery system and finger blood pressure using our device in 12 elderly subjects. Two time series of pulse interval variability (PIV) corresponded to RR interval were estimated respectively as the time between two successive upstrokes of these two devices and systolic blood pressure variability (SPV) was also estimated as the upstroke. In time domain the relative relation of PIV estimated by two systems was high, however, that of SPV was low. On the contrary, in frequency domain, we could estimate autonomic nervous activity of vasomotor activity from our new device. The developed device in our study may be a substitutable device for conventional method as limited to estimate the autonomic nervous activity of cardiovascular system.

Hypotension and resistance to lipopolysaccharide-induced shock in transgenic mice overexpressing adrenomedullin in their vasculature.

BACKGROUND: Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. To determine the extent to which chronic AM overproduction affects circulatory physiology under normal and pathological conditions, we used a preproendothelin-1 promoter to establish transgenic mouse lines overexpressing AM in their vasculature. METHODS AND RESULTS: Transgenic mice overexpressing AM mainly in vascular endothelial and smooth muscle cells exhibited significantly lower blood pressure (BP) and higher plasma cGMP levels than their wild-type littermates. Blockade of NO synthase with N(G)-monomethyl-L-arginine elevated BP to a greater degree in AM transgenic mice, offsetting the BP difference between the 2 groups. Despite their lower basal BP, administration of bacterial lipopolysaccharide elicited smaller declines in BP and less severe organ damage in AM transgenic mice than in wild-type mice. Furthermore, the 24-hour survival rate after induction of lipopolysaccharide shock was significantly higher in the transgenic mice. CONCLUSIONS: A chronic increase in vascular AM production reduces BP at least in part via an NO-dependent pathway. In addition, smaller responses to LPS in transgenic mice suggest that AM is protective against the circulatory collapse, organ damage, and mortality characteristic of endotoxic shock.

[Mitomycin C dissolved in a reversible thermo-setting gel: effect on aqueous flare in the rabbit eye].

PURPOSE: To determine the effects of mitomycin C (MMC) dissolved in a gel, which is as effective as a higher dose of aqueous MMC solution, on aqueous flare in rabbits. METHOD: We injected subconjunctivally a 0.1 ml solution of MMC containing 30, 10, 3.0, or 0.3 micrograms of MMC dissolved in a reversible thermo-setting gel in pigmented rabbits. As a control, a 0.1 ml aqueous solution of 30 micrograms MMC was injected subconjunctivally. Aqueous flare was measured for 28 days after the injection. RESULTS: The aqueous flare in the 30 micrograms gel group was also level similar to that in the 30 micrograms aqueous solution group, but the aqueous flare in the 10, 3.0, and 0.3 micrograms gel groups was significantly lower. The increase in the aqueous flare following the administration of MMC gel was dose-dependent. CONCLUSION: The administration of MMC dissolved in a gel can diminish the ocular inflammation by reducing the dose of MMC needed for the antiproliferative effect.

Responses of blood pressure and catecholamine metabolism to high salt loading in endothelin-1 knockout mice.

The molecular mechanism responsible for salt sensitivity is poorly understood. Mice heterozygous for the null mutation of the endothelin-1 (ET-1) gene, Edn1, may be a potential tool for studying this mechanism, because they have elevated blood pressure and disturbances in central sympathetic nerve regulation. In the present study, we used this mouse model to examine the degree to which ET-1 contributes to the responses of blood pressure and catecholamine metabolism to high salt loading. Male Edn1+/- heterozygous mice and Edn1+/+ wild-type littermates were given either a high salt (8%) or a normal salt (0.7%) diet for 4 wk. During the normal diet, renal ET-1 levels in Edn1+/- mice were approximately 50% lower than ET-1 levels in wild-type mice, whereas the high salt diet decreased renal ET-1 levels by about 50% in both Edn1+/- and wild-type mice. The high salt diet significantly increased urinary sodium excretion and fractional excretion of sodium (FENa) but did not affect circulating plasma volume, serum electrolytes, creatinine clearance, or systemic blood pressure. In addition, urinary norepinephrine and normetanephrine excretion were significantly increased, indicating that salt loading can increase sympathetic nerve activity in normal mice. These responses to salt loading did not differ between Edn1+/- mice and their wild-type littermates. We conclude that physiological changes in ET-1 production do not affect the responses of blood pressure and catecholamine metabolism to salt loading, although the renal ET-1 content is decreased by salt loading.

Elevation of blood pressure by genetic and pharmacological disruption of the ETB receptor in mice.

Exogenously administered endothelin (ET) elicits both pressor and depressor responses through the ETA and/or the ETB receptor on vascular smooth muscle cells and ETB on endothelial cells. To test whether ETB has pressor or depressor effects under basal physiological conditions, we determined arterial blood pressure (BP) in ETB-deficient mice obtained by crossing inbred mice heterozygous for targeted disruption of the ETB gene with mice homozygous for the piebald (s) mutation of the ETB gene (ETBs/s). F1 ETB-/s and ETB+/s progeny share an identical genetic background but have ETB levels that are approximately (1)/(8) and (5)/(8), respectively, of wild-type mice (ETB+/+). BP in ETB-/s mice was significantly higher, by approximately 20 mmHg, than that in ETB+/s or ETB+/+ mice. Immunoreactive ET-1 concentration in plasma as well as respiratory parameters was not different between ETB-/s and ETB+/s mice. A selective ETB antagonist, BQ-788, increased BP in ETB+/s and ETB+/+ but not in ETB-/s mice. Pretreatment with indomethacin, but not with NG-monomethyl-L-arginine, can attenuate the observed pressor response to BQ-788. The selective ETA antagonist BQ-123 did not ameliorate the increased BP in ETB-/s mice. Moreover, BP in mice heterozygous for targeted disruption of the ETA gene was not different from that in wild-type controls. These results suggest that endogenous ET elicits a depressor effect through ETB under basal conditions, in part through tonic production of prostaglandins, and not through secondary mechanisms involving respiratory control or clearance of circulating ET.