Significant association between nonsyndromic oral clefts and arylhydrocarbon receptor nuclear translocator (ARNT)

The etiology of nonsyndromic oral clefts (cleft lip, cleft palate, or cleft lip and palate) is still controversial, but is considered to involve both genetic and environmental factors. One of suspected environmental factors is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) found in tobacco, herbicides, contaminated soil, and food. TCDD administered during organogenesis in mice causes a high incidence of CP in fetuses. There is ample evidence that aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), and cytochrome P450 1A1 (CYP1A1) are involved in TCDD metabolism. We assessed whether there is any association in the Japanese population of nonsyndromic oral clefts with single nucleotide polymorphisms (SNPs) in the AHR, ARNT, and CYP1A1 genes using transmission disequilibrium test (TDT) and case-control study. We identified and investigated three SNPs in ARNT; 567G/C (V189V), IVS12-19T/G, and 2117C/T (P706L). Two amino acid substitutions, R554L in AHR and I462V in CYP1A1, were also investigated. In the TDT, the C allele of ARNT 567G/C was preferentially transmitted to patients (P = 0.033). When a haplotype consisting of 567G/C and IVS12-19T/G in ARNT was considered, the preferential transmission of the CT (567C-IVS12-19T) haplotype was observed (P = 0.0012). In a case-control study, a significant association of IVS12-19T/G in ARNT was observed (P = 0.021). The SNPs studied in AHR and CYP1A1 were not associated with the disease. Our results suggest that ARNT is involved in the development of nonsyndromic oral clefts in the Japanese population.     

A perfusable microfluidic device with on-chip total internal reflection fluorescence microscopy (TIRFM) for in situ and real-time monitoring of live cells

A microfluidic device integrated with a Total Internal Reflection (TIR)-based chip for cell observation and analysis was developed. This integrated device enables in situ Total Internal Reflection Fluorescence Microscopy (TIRFM) on adherent cells cultured under continuous medium perfusion. This TIR-based chip, allows TIRFM to be easily performed on cells without the assembly of complicated optical components and cell culture chambers. The integrated device was evaluated by tracking the movement of fluorescent beads and monitoring the location of insulin granules in mouse pancreatic β-cells. This system offers higher signal-to-noise (S/N) ratio than epi-fluorescence microscopy (EPIFM), and comparable image quality to commercial TIRFM systems when imaging insulin granules. We also detected repetitive changes in intracellular Ca(2+) concentration in MIN6-m9 cells stimulated with KCl, which demonstrates quick perfusion for cell analysis while maintaining high S/N ratio.     

Primary somatosensory evoked magnetic fields elicited by sacral surface electrical stimulation

To explore the brain response to sacral surface therapeutic electrical stimulation (SSTES) for the treatment of refractory urinary incontinence and frequent micturition, evoked magnetic fields were measured in six healthy males. Electrical stimuli were applied between bilateral surface electrodes over the second through fourth posterior sacral foramens with intensity just below the pain threshold. Somatosensory evoked magnetic fields (SEFs) for the bilateral median (MN) and posterior tibial nerves (PTN) were also measured for the comparison. Sources of the early SEF peaks were superimposed on individual magnetic resonance images. The first peak latency for sacral stimuli, M30, occurred at 30.2+/-0.8 ms (mean+/-standard deviation, N=6), with shorter latency than those for PTN stimulus (39.3+/-1.4 ms, N=12) and longer latency than those for MN stimulus (21.0+/-0.9 ms, N=12). The second peak latency for sacral stimuli, M50, occurred at 47.2+/-2.9 ms (N=6). Both M30 and M50 peaks showed a single dipole pattern over the vertex in the isofield maps. The equivalent current dipoles of M30 and M50 were both estimated near the medial end of the central sulcus with approximately posterior current direction. These results suggest that the sacral M30 and M50 are responses from the primary somatosensory cortex. The relatively long time lag between the onset and peak of M30 suggests that SSTES directly affects both the cauda equina and cutaneous nerve of the sacral surface.     

The application of end user computing (EUC) for detection of lipoprotein lipase gene abnormality]

Lipoprotein lipase (LPL) is an enzyme digesting lipoprotein triglyceride (TG) in peripheral blood vessels. Most patients with LPL deficiency show very high plasma TG and low HDL-C. To establish an effective computer-based screening system to identify individuals with genetic LPL disorders, we selected 50 subjects whose plasma TG was over 350mg/dl and HDL-C was lower than 35mg/dl from patients at Hamamatsu University Hospital. We applied End User Computing (EUC) of our laboratory system to select high risk subjects with LPL gene abnormalities. Polymerase chain reaction (PCR) products from LPL gene exons 2-9 were screened by single-strand conformation polymorphism (SSCP), direct DNA sequence analysis and restriction fragment length polymorphism (RFLP). We found a novel missense mutation (1223C-->G, S323C) in LPL gene exon 7 from three subjects. By PCR-mediated site-directed mutagenesis and restriction digestion, the three subjects were found to be heterozygous. In addition, we identified two other common mutations in Japanese employing the RFLP method. One was the 1595C-->G (S447X) in exon 9 from six subjects, two homozygous and four heterozygous individuals. The other was a mutation of intron 3 (C-->T transition) from four heterozygous subjects. Using EUC screening method, we detected genetic LPL abnormalities more easily. The frequency of the LPL gene mutation in the 50 high-risk subjects was 26%, and was estimated to be one out of 2,000 patients at our clinic. Using the EUC system to screen for LPL mutations was established to be an effective computer-based screening system to identify individuals with genetic abnormalities.     

Salivary secretion of highly concentrated chromogranin a in response to noradrenaline and acetylcholine in isolated and perfused rat submandibular glands

Chromogranin A (CgA) is a member of a family of highly acidic proteins, chromogranins, which are co-stored in the adrenergic neurons and paraneurons and co-released with adrenaline and noradrenaline (NAd) in response to adequate stimulation. The present study provides novel evidence that CgA-like immunoreactivity (IR) is stored in the exocrine cells in the granular convoluted tubule, and is secreted into saliva by stimulation with NAd and acetylcholine (ACh) in the isolated and perfused rat submandibular gland. NAd at 1 microM produced maximum secretion of CgA-like IR (< 0.9 mM) and a marked increase in salivary flow. Further increases in NAd concentration (10 or 100 microM) yielded concentration-dependent decreases in both responses. ACh at 1 microM produced maximum salivary flow and a slight elevation of CgA-like IR secretion (6 microM); 100 microM ACh decreased the salivary flow but increased the CgA-like IR secretion (0.6 mM). Electron microscopic examination showed vigorous compound exocytosis of secretory granules in the cells of the granular convoluted tubule when the submandibular gland was stimulated with 1 microM NAd. These results provide an experimental basis for the view that the salivary CgA-like IR secretion may be a sensitive and quantitative index of the activity of the sympathetic nervous system innervating the gland.     

cAMP-independent decrease of ATP-sensitive K+ channel activity by GLP-1 in rat pancreatic β-cells

Using the patch-clamp method, we studied the mechanism of depolarization of rat pancreatic beta-cells induced by glucagon-like peptide 1 (7-36) amide (GLP-1). GLP-1 caused depolarization in a concentration-dependent manner (0.2-100 nM). Exendin (9-39) amide, a GLP-1 receptor antagonist, prevented the GLP-1-induced depolarization. GLP-1 reduced tolbutamide-sensitive membrane currents evoked by voltage ramps from -90 to -50 mV, recorded in the perforated whole-cell configuration, suggesting that GLP-1 decreased the activity of the ATP-sensitive K+ channel (KATP). This GLP-1 effect was prevented by exendin (9-39) amide. In cells treated with Rp-cAMPS, an inhibitor of the cAMP-dependent protein kinase (PKA), GLP-1 still caused depolarization and reduced the whole-cell membrane current through KATP. Examined in the cell-attached configuration, 20 nM GLP-1, applied out of the patch, had little effect on KATP activity. In the inside-out configuration, the open time probability and the single-channel conductance of KATP in the absence of ATP inside the membrane were unaffected by the presence of 20 nM GLP-1 in the pipette. In both conditions, application of ATP to the inside of the membrane reduced KATP activity. The half-maximal concentrations (ki) of ATP were 11.6 microM without and 5.6 microM with 20 nM GLP-1 in the pipette (P<0.05). The values of the Hill coefficient (h) were 1.03 without and 1.01 with GLP-1. We conclude that GLP-1 reduces KATP activity by elevating the sensitivity of KATP to ATP, resulting in depolarization of pancreatic beta-cells. This GLP-1 action is independent of the cAMP signalling pathway.     

Seroprevalence of Kaposi's sarcoma‐associated herpesvirus among men who have sex with men in Japan

Kaposi's sarcoma‐associated herpesvirus (KSHV), the etiologic agent of Kaposi's sarcoma, causes malignancies frequently in patients with acquired immunodeficiency syndrome. In the United States and Europe, KSHV infection is common among men who have sex with men. However, the seroprevalence of KSHV among men who have sex with men in Japan is unknown. In the present study, the seroprevalence of KSHV was investigated among 230 men who have sex with men and 400 age‐ and area of residence‐matched men (controls) using a mixed‐antigen (KSHV‐encoded K8.1, open reading frame 59, 65, and 73 proteins) enzyme‐linked immunosorbent assay and an immunofluorescence assay. Among the Japanese men who have sex with men, serological assays revealed that 27 (11.7%) were seropositive for KSHV; 20 (5%) of the men in the control group were also KSHV seropositive. The seroprevalence of KSHV among men who have sex with men was significantly higher than in the control group (odds ratio = 2.52, 95% confidence intervals = 1.38–4.62, P = 0.0019, Chi‐square test). Infection with the human immunodeficiency virus, Treponema pallidum, or hepatitis B and C virus did not correlate with KSHV infection. Furthermore, the association of KSHV seropositivity with specific sexual activities was not statistically significant. In conclusion, a higher KSHV seroprevalence was found among Japanese men who have sex with men than among the controls, suggesting that the circulation of KSHV infection is more efficient among men who have sex with men in Japan than among men who do not engage in such sexual activities. J. Med. Virol. 85: 1046–1052, 2013. © 2013 Wiley Periodicals, Inc.