Restless legs syndrome is associated with headache-related disabilities in patients with migraine: a prospective 7-year follow-up study.

BACKGROUND AND PURPOSE: No prospective study has evaluated the impact of restless legs syndrome (RLS) on clinical factors in patients with migraine. We planned a prospective study to assess the impact of RLS comorbid status on clinical factors in patients with migraine. METHODS: A total of 101 patients with migraine who were evaluated for RLS twice at 7-year intervals in a university hospital setting were included in this study. The RLS group was defined as positive for RLS at either baseline or follow-up and the non-RLS group was defined as negative for RLS at both baseline and follow-up. The Migraine Disability Assessment (MIDAS) questionnaire, Beck Depression Inventory-II (BDI-II), Pittsburgh Sleep Quality Index (PSQI) and Epworth Sleepiness Scale were administered to all patients. RESULTS: The RLS prevalence was 16.8% at baseline and 20.8% at follow-up. Compared with the non-RLS group (n = 27), the RLS group (n = 74) showed a significantly higher rate of smoking and higher MIDAS and BDI-II scores at 7-year follow-up. A significant reduction in MIDAS and BDI-II scores at 7-year follow-up compared with those at baseline was observed in the non-RLS group, but not in the RLS group. The non-RLS group showed a significantly lower MIDAS score at 7-year follow-up than the RLS group after adjusting for confounding variables such as age, gender, smoking status, Epworth Sleepiness Scale and PSQI scores using analysis of covariance. The persistent RLS group (n = 11) (positive for RLS at both baseline and follow-up) showed a significantly higher rate of smoking and increased MIDAS, BDI-II and PSQI scores compared with the non-RLS group (n = 74) at 7-year follow-up. CONCLUSION: Our prospective study showed that RLS had a significant impact on headache-related disability in patients with migraine.

Study of MHC class II region polymorphism in the Filipino cynomolgus macaque population.

The cynomolgus macaque (Macaca fascicularis) is currently used as an animal model in various fields of immunology especially in the development of innovative vaccines for the prevention and treatment of infectious diseases. The polymorphism of the major histocompatibility complex (MHC) influences the development of adaptive immune responses and it is crucial to characterize the polymorphism of cynomolgus MHC genes. We present here a systematic study of the MHC class II haplotypes in the Filipino macaque population. By the study of a large sample of Filipino animals (N = 353), we have characterized 18 MHC class II haplotypes by means of genotyping seven microsatellites. The animals were DRB genotyped by means of PCR-SSO or DGGE-sequencing on genomic amplified fragments. We cloned and sequenced the complementary DNA (cDNA) of DQA, DQB, DPA, and DPB genes of 117 animals. Combining the microsatellite genotyping and cDNA characterized in the 117 animals, we defined genetic association between the cDNA and the microsatellites and characterized 18 MHC class II haplotypes. For 104 animals out of the 353 studied, the presence of a recombinant haplotype was highly probable. Thirty-four percent of recombination was located in 256 kb segment between D6S2876 and D6S2747 microsatellites, a region encompassing several hot spots of recombination in the human MHC.

A novel HLA-B allele, HLA-B*44:184, identified by super high-resolution single-molecule sequence-based typing in a Japanese individual.

The novel allele HLA-B*44:184 differs from HLA-B*44:77 by three non-synonymous amino acid exchanges, T94I, L95I and S97R.

HLA-DRB1, -DRB3, -DRB4 and -DRB5 genotyping at a super-high resolution level by long range PCR and high-throughput sequencing.

Super high-resolution single molecule sequence-based typing (SS-SBT) is a human leukocyte antigen (HLA) DNA typing method to the field 4 level of allelic resolution (formerly known as eight-digit typing) to efficiently detect new and null alleles without phase ambiguity by combination of long ranged polymerase chain reaction (PCR) amplification and next-generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method for the eight classical HLA loci, A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1. In this article, we describe the development of the SS-SBT method for three DRB1 linked loci, DRB3, DRB4 and DRB5 (DRB3/4/5) and characterization of DRB1-DRB3/4/5 haplotype structures to the field 4 level. Locus specific PCR primers for DRB3/4/5 were designed to amplify the gene regions from intron 1 to exon 6 [3' untranslated region (3'UTR)]. In total 20 DRB1 and 13 DRB3/4/5 allele sequences were determined by the SS-SBT to the field 4 level without phase ambiguity using 19 DR51, DR52 and DR53 positive genomic DNA samples obtained from Japanese. Moreover, 18 DRB1-DRB3/4/5 haplotypes were estimated to the field 4 level by the SS-SBT method in contrast to 10 haplotypes estimated by conventional methods to the field 1 level (formerly known as two digit typing). Therefore, DRB1-DRB3/4/5 haplotyping by SS-SBT is expected to provide informative data for improved HLA matching in medical research, transplantation procedures, HLA-related disease studies and human population diversity studies.

Characterization of swine leukocyte antigen alleles and haplotypes on a novel miniature pig line, Microminipig.

Microminipigs are extremely small-sized, novel miniature pigs that were recently developed for medical research. The inbred Microminipigs with defined swine leukocyte antigen (SLA) haplotypes are expected to be useful for allo- and xenotransplantation studies and also for association analyses between SLA haplotypes and immunological traits. To establish SLA-defined Microminipig lines, we characterized the polymorphic SLA alleles for three class I (SLA-1, SLA-2 and SLA-3) and two class II (SLA-DRB1 and SLA-DQB1) genes of 14 parental Microminipigs using a high-resolution nucleotide sequence-based typing method. Eleven class I and II haplotypes, including three recombinant haplotypes, were found in the offspring of the parental Microminipigs. Two class I and class II haplotypes, Hp-31.0 (SLA-1*1502-SLA-3*070102-SLA-2*1601) and Hp-0.37 (SLA-DRB1*0701-SLA-DQB1*0502), are novel and have not so far been reported in other pig breeds. Crossover regions were defined by the analysis of 22 microsatellite markers within the SLA class III region of three recombinant haplotypes. The SLA allele and haplotype information of Microminipigs in this study will be useful to establish SLA homozygous lines including three recombinants for transplantation and immunological studies.

A novel HLA-B allele, HLA-B*39:01:16, identified by super high-resolution single-molecule sequence-based typing in a Japanese individual.

HLA-B*39:01:16 differs from B*39:01:03 by one nucleotide 348 (codon 116) T to C.

Super high resolution for single molecule-sequence-based typing of classical HLA loci at the 8-digit level using next generation sequencers.

Current human leukocyte antigen (HLA) DNA typing methods such as the sequence-based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS-SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3' untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.

HslO ameliorates arrested ΔrecA polA cell growth and reduces DNA damage and oxidative stress responses.

Chromosome damage combined with defective recombinase activity has been widely considered to render cells inviable, owing to deficient double-strand break repair. However, temperature-sensitive recAts polA cells grow well upon induction of DNA damage and supplementation with catalase at restrictive temperatures. These treatments reduce intracellular reactive oxygen species (ROS) levels, which suggests that recAts polA cells are susceptible to ROS, but not chronic chromosome damage. Therefore, we investigated whether polA cells can tolerate a complete lack of recombinase function. We introduced a ΔrecA allele in polA cells in the presence or absence of the hslO-encoding redox molecular chaperon Hsp33 expression plasmid. Induction of the hslO gene with IPTG resulted in increased cell viability in ΔrecA polA cells with the hslO expression plasmid. ΔrecA polA cells in the absence of the hslO expression plasmid showed rich medium sensitivity with increasing ROS levels. Adding catalase to the culture medium considerably rescued growth arrest and decreased ROS. These results suggest that hslO expression manages oxidative stress to an acceptable level in cells with oxidative damage and rescues cell growth. Overall, ROS may regulate several processes, from damage response to cell division, via ROS-sensitive cell metabolism.

Polymorphic major histocompatibility complex class II Alu insertions at five loci and their association with HLA-DRB1 and -DQB1 in Japanese and Caucasians.

We investigated polymorphic Alu insertion (POALIN) frequencies at five loci in the major histocompatibility complex (MHC) class II genomic region to determine their allele and haplotype frequencies and associations with the human leukocyte antigen (HLA)-DRB1 and -DQB1 genes for 100 Japanese, 174 Australian Caucasians and 67 HLA reference cell lines obtained from different ethnic groups. The POALINs varied in frequency between 11% and 57% with significant differences between the Japanese and Caucasians at three loci. One POALIN locus deviated significantly from Hardy-Weinberg equilibrium (HWE) and four POALIN loci were in significant linkage disequilibrium and had a high percentage association with a variety of HLA-DRB1 or -DQB1 two-digit alleles. Inferred haplotype analysis among two-locus, five-locus and seven-locus haplotype structures showed maximum differences between the Japanese and Caucasians with the seven-locus haplotypes. The most common multilocus haplotype in Caucasians was DRB1*1501/DQB1*0602/AluDQ1/AluDRB1/AluORF10/AluDPB2 (6.7%), whereas the second most common allele HLA-DRB1*15 (17.5%) in Japanese was associated with three or four Alu insertions. The HLA class II POALINs also differentiated within and between HLA-DRB1 super-haplotypes DR1, DR8, DR51, DR52 and DR53. This is the first comparative population study of multilocus POALINs in the HLA class II region, which shows that POALINs whether investigated alone or together with the HLA class II alleles are informative genetic markers for the identification of allele and haplotype lineages and variations within the same and/or different populations.

Multiple principal sigma factor homologs in eubacteria: identification of the "rpoD box".

Genes for the principal sigma factor (rpoD genes) of various eubacteria were identified with a synthetic oligonucleotide probe corresponding to a conserved sequence in rpoD gene products of Escherichia coli and Bacillus subtilis. Multiple rpoD homologs were found in the strains of Micrococcus, Pseudomonas, and Streptomyces, whereas single genes were detected in E. coli, B. subtilis, and Staphylococcus aureus. The four rpoD homologs of Streptomyces coelicolor A3(2) were cloned and sequenced. A homologous portion with 13 amino acids was found in the rpoD genes of S. coelicolor A3(2), E. coli, and B. subtilis and was named the "rpoD box."

Three-dimensional SAFT imaging for anisotropic materials using photoacoustic microscopy.

A pulsed laser illuminates a target zone that causes rapid thermoelastic expansion, generating broadband high-frequency ultrasonic wave (photoacoustic wave, PA wave). We developed a PA microscopy (PAM) with a confocal area of laser and ultrasonic wave for applications in nondestructive testing (NDT). The synthetic aperture focusing technique (SAFT) is applied in the PAM for the three-dimensional (3D) imaging of interior flaws. Here, we report proof-of-concept experiments for the NDT of a subsurface flaw in a thin laminar material. Graphical abstract (a) shows a specimen of carbon-fiber-reinforced plastic (CFRP) with an artificial delamination. Here, it should be noted that the group velocity varies directionally due to the strong anisotropy of the CFRP specimen (see Graphical abstract (b)). By considering the group velocity distribution in the SAFT, the shape and location of the subsurface delamination were accurately estimated as shown in Graphical abstract (c). Coating the surface of the CFRP specimen with a light-absorbent material improved the amplitude of the PA wave. This finding showed that the signal-to-noise ratio of the waves scattered from the flaws can be improved.

Exogenous serotonin regulates colorectal motility via the 5-HT2 and 5-HT3 receptors in the spinal cord of rats.

BACKGROUND: We previously reported that intrathecal injection of noradrenaline or dopamine causes enhancement of colorectal motility. As these monoamines are neurotransmitters of descending pain inhibitory pathways in the spinal cord, we hypothesized that serotonin, which is one of the neurotransmitters involved in descending pain inhibition, also influences the lumbosacral defecation center. Therefore, we examined whether serotonin acting on the spinal defecation center enhances colorectal motility. METHODS: Colorectal intraluminal pressure and propelled liquid volume were recorded in vivo in anesthetized rats. KEY RESULTS: Intrathecal injection of serotonin into the L6-S1 spinal cord elicited periodic increases in colorectal intraluminal pressure, being associated with increases in liquid output. Pharmacological experiments revealed that the effect of serotonin is mediated by both 5-HT2 and 5-HT3 receptors. The serotonin-induced enhancement of colorectal motility was unaffected even after disconnection of the defecation center from supraspinal regions by cutting the T8 spinal cord, while transection of the parasympathetic pelvic nerves prevented the colokinetic effect of serotonin. Finally, we investigated interactions among serotonin, noradrenaline and dopamine. Simultaneous administration of sub-effective doses of these monoamine neurotransmitters into the spinal cord caused propulsive colorectal motility slightly but substantially. CONCLUSIONS AND INFERENCES: These results demonstrate that exogenous serotonin acts on 5-HT2 and 5-HT3 receptors in the lumbosacral defecation center and activates the parasympathetic nervous system to enhance colorectal motility in cooperation with noradrenaline and dopamine.

Histamine-enhanced contractile responses of gastric smooth muscle via interstitial cells of Cajal in the Syrian hamster.

BACKGROUND: Gastric motility is controlled by the autonomic and enteric nervous systems and by interstitial cells of Cajal (ICCs). Although histamine is known to be released from enterochromaffin-like cells in the gastric mucosa, its regulatory roles in gastric motility are still controversial. Therefore, we investigated the functional roles of histamine in gastric motility. METHODS: Stomach preparations from hamsters were used because the stomach of hamsters can be easily separated into the forestomach and the glandular stomach. A whole preparation of the stomach was mounted in a Magnus tube, and mechanical responses were recorded using a force transducer. KEY RESULTS: Exogenous application of histamine had little effect on contractile activity of the glandular stomach. In contrast, the monoamine evoked regular, periodic contractions in the forestomach. An H1 receptor agonist reproduced the contractile responses and an H1 receptor antagonist blocked histamine-evoked contractions. Atropine and tetrodotoxin did not affect the histamine-evoked contractions. Pretreatment with drugs that inhibit the activity of ICCs abolished the effects of histamine. CONCLUSION & INFERENCES: The findings suggest that histamine regulates gastric motility by acting on ICCs via H1 receptors in the hamster. The remarkable ability of histamine to induce rhythmic contractions would be useful for treatment of gastric dysmotility.

Purinergic control of the quail rectum: modulation of adenosine 5'-triphosphate-mediated contraction with acetylcholine.

Electrical field stimulation (EFS) induces frequency-dependent contractions of the longitudinal muscle of isolated quail rectum which were sensitive to tetrodotoxin. The aim of the present study was to investigate whether purinergic neurons are implicated in the response to nerve stimulation. The shape of the EFS-induced contractile response was different depending on stimulus frequency; low frequencies (0.5-2 Hz) induced fast monophasic contractions with a small subsequent relaxation; whereas higher frequencies (5-50 Hz) induced biphasic contractile response that comprised fast initial component (as in case of low frequency) and a slow delayed contractile component in addition to the relaxation that follows the fast contractile component. Prior application of atropine (10 microM) completely abolished the slow delayed component but significantly enhanced the fast initial contractile component. Physostigmine (1-10 microM) significantly enhanced the slow delayed component with an inhibitory effect on the initial fast component. The nonspecific purinergic receptor antagonist, suramin (100-500 microM) significantly inhibited the fast initial contractile component with no significant effect on the slow delayed one. Complete blockade of the fast component was achieved by prior application of a combination consisted of suramin (50 microM) and pyridoxicalphosphate-6-azophenyl 2',4'-disulphonic acid tetrasodium (PPADS; 10 microM). Exogenous applications of adenosine 5'-triphosphate and acetylcholine (10 microM each), produced contractile responses that mimicked those induced by EFS. These data suggest that ATP is the main noncholinergic excitatory transmitter controlling the contractile activity of the quail rectum; and that its action could be modulated by acetylcholine.

Visualization of tumor-related blood vessels in human breast by photoacoustic imaging system with a hemispherical detector array.

Noninvasive measurement of the distribution and oxygenation state of hemoglobin (Hb) inside the tissue is strongly required to analyze the tumor-associated vasculatures. We developed a photoacoustic imaging (PAI) system with a hemispherical-shaped detector array (HDA). Here, we show that PAI system with HDA revealed finer vasculature, more detailed blood-vessel branching structures, and more detailed morphological vessel characteristics compared with MRI by the use of breast shape deformation of MRI to PAI and their fused image. Morphologically abnormal peritumoral blood vessel features, including centripetal photoacoustic signals and disruption or narrowing of vessel signals, were observed and intratumoral signals were detected by PAI in breast cancer tissues as a result of the clinical study of 22 malignant cases. Interestingly, it was also possible to analyze anticancer treatment-driven changes in vascular morphological features and function, such as improvement of intratumoral blood perfusion and relevant changes in intravascular hemoglobin saturation of oxygen. This clinical study indicated that PAI appears to be a promising tool for noninvasive analysis of human blood vessels and may contribute to improve cancer diagnosis.

Tachykinins are involved in local reflex modulation of vagally mediated striated muscle contractions in the rat esophagus via tachykinin NK1 receptors.

The objective of the present study was to investigate the hypothesis of the presence of a local neural reflex modulating the vagally mediated contractions of striated muscle in the rat esophagus and to determine the possible involvement of tachykinins in such a local neural reflex. Electrical stimulation of the vagus nerve evoked twitch contractile responses that were abolished by d-tubocurarine (5 microM). Capsaicin (1-100 microM) inhibited the vagally mediated twitch contractions o f the normal rat esophageal preparations concentration-dependently but not those of the neonatally capsaicin-treated ones. NG-nitro-L-arginine methyl ester (100 microM), a nitric oxide synthase inhibitor, blocked the inhibitory effect of capsaicin and exogenous application of a nitric oxide donor (1 mM) inhibited the vagally mediated twitch contractions. Capsaicin suppressed acetylcholine release from the normal rat esophageal segments evoked by vagus nerve stimulation but not that from the neonatally capsaicin-treated ones. A selective tachykinin NK1 receptor antagonist (0.1 or 1 microM) attenuated the inhibitory effect of capsaicin. However, antagonists of tachykinin NK2, tachykinin NK3 and calcitonin gene-related peptide receptors (1 microM) did not have any effect. A tachykinin NK1 receptor agonist (1 or 5 microM) inhibited the vagally mediated twitch contractions, which was prevented by NG-nitro-L-arginine methyl ester (100 microM). These data suggest that the rat esophagus might have a local neural reflex inhibiting the vagally mediated striated muscle motility, which consists of capsaicin-sensitive sensory neurons and myenteric nitrergic neurons, and that tachykinins might be involved in the neural reflex through tachykinin NK1 receptors.

Identification of a novel HLA-C allele, HLA-C*03:313, in a Japanese individual.

HLA-C*03:313 differs from HLA-C*03:04:01:01 by one non-synonymous amino acid exchange E161G.

Correction of the HLA-DQB1*04:01:01 sequence at position 79 in exon 1.

The HLA-DQB1*04:01:01 sequence is corrected at position 79 in exon 1 (G > A, Ala > Thr).

Serotonin-induced contractile responses of esophageal smooth muscle in the house musk shrew (Suncus murinus).

BACKGROUND: Serotonin (5-hydroxytryptamine, 5-HT) is a regulatory factor in motility of the gastrointestinal tract including the esophagus. Although we proposed that vagal cholinergic and mast cell-derived non-cholinergic components including serotonin coordinately shorten the esophagus, the precise mechanism of serotonin-induced contractions in the suncus esophagus is still unclear. Therefore, the aims of this study were to determine characteristics of contractile responses induced by serotonin and to identify 5-HT receptor subtypes responsible for regulating motility in the suncus esophagus. METHODS: An isolated segment of the suncus esophagus was placed in an organ bath, and longitudinal or circular mechanical responses were recorded using a force transducer. KEY RESULTS: Serotonin evoked contractile responses of the suncus esophagus in the longitudinal direction but not in the circular direction. Tetrodotoxin did not affect the serotonin-induced contractions. Pretreatment with a non-selective 5-HT receptor antagonist or double application of 5-HT1 and 5-HT2 receptor antagonists blocked the serotonin-induced contractions. 5-HT1 and 5-HT2 receptor agonists, but not a 5-HT3 receptor agonist, evoked contractile responses in the suncus esophagus. CONCLUSION & INFERENCES: The findings suggest that serotonin induces contractile responses of the longitudinal smooth muscle in the muscularis mucosae of the suncus esophagus that are mediated via 5-HT1 and 5-HT2 receptors on muscle cells. The serotonin-induced contractions might contribute to esophageal peristalsis and emetic response.

In Vitro Analysis of Light-Induced Transcription in the Wheat psbD/C Gene Cluster Using Plastid Extracts from Dark-Grown and Short-Term-Illuminated Seedlings.

We describe a plastid in vitro transcription system that reflects characteristic features of the light-regulated transcription observed in vivo. Multiple transcripts of the wheat (Triticum aestivum) psbD/C gene cluster comprise six distinct 5[prime] ends including four transcription initiation sites designated as D/C-1 through D/C-4. Transcripts from one particular site, D/C-3, were found to be conspicuously enhanced in abundance after 4 h of illumination in vivo. The plastid extract prepared from 5-d-old dark-grown wheat seedlings was capable of transcribing from the D/C-2 and D/C-4 sites in vitro but had almost no transcription activity from the light-responsive D/C-3 site (the D/C-1 site was not examined). The plastid extract from 4-h-illuminated seedlings initiated transcription from the light-responsive site (D/C-3). Transcription from the D/C-2 and D/C-4 sites was not enhanced by using the extract from 4-h-illuminated seedlings, indicative of specific activation of the light-responsive promoter on the D/C-3 site by the extract from 4-h-illuminated seedlings. The plastid extract from 4-h-illuminated seedlings was divided into two fractions on a heparin-Sepharose column, into which the light-induced component(s) responsible for activation of the D/C-3 promoter and RNA polymerase were separated. The fraction containing the component(s) activating the D/C-3 promoter induced the transcription activity from the D/C-3 site in the plastid extract from dark-grown seedlings. It is concluded that the plastid extract from 4-h-illuminated seedlings contains some light-regulatory component(s) that activate specifically the light-responsive promoter.