Salivary Alpha-Amylase Activity and Mild Cognitive Impairment among Japanese Older Adults: The Toon Health Study.

BACKGROUND: There is growing interest in examining objective markers for early identification and behavioral intervention to prevent dementia and mild cognitive impairment in clinical and community settings. OBJECTIVE: To investigate the association between salivary alpha-amylase as an objective measure of psychological stress response and mild cognitive impairment for the implication of psychological stress in the development of mild cognitive impairment. DESIGN, SETTING, AND PARTICIPANTS: This cross-sectional study involved 865 participants aged ≥ 65 years. A saliva sample was collected in the morning, and the levels of salivary alpha-amylase were assayed. Mild cognitive impairment was evaluated using the Japanese version of the Montreal Cognitive Assessment; a score < 26 was indicative of mild cognitive impairment. A multivariable logistic regression model was used to examine the association of salivary alpha-amylase and mild cognitive impairment after adjusting for age, sex, current drinking status, current smoking status, body mass index, hypertension, diabetes mellitus, physical activity, education, social support, social network, and heart rate variability. RESULTS: Salivary alpha-amylase was associated with mild cognitive impairment (the multivariable-adjusted odds ratio [95% confidence interval] for the 1-standard deviation increment of log-transformed salivary alpha-amylase was 1.24 [1.07-1.44]). This significant association persisted after adjusting for various confounding factors. CONCLUSION: Elevation of salivary alpha-amylase was associated with mild cognitive impairment among Japanese community-dwelling older adults. This suggests that salivary alpha-amylase is a useful objective marker of psychological stress responses associated with mild cognitive impairment.

Messenger RNA expression and localization of xenin in the gastrointestinal tract in sheep.

The present study aimed to determine the primary sequence of ovine xenin and clarify the mRNA expression and peptide localization of xenin in the gastrointestinal tract in sheep. The colocalization of xenin and glucose-dependent insulinotropic polypeptide was also compared in the antrum and duodenum. Analysis of the nucleotide sequence of ovine xenin revealed a high degree (97.9%) of sequence homology of the sequence between sheep and cattle, and the amino acids sequence determined for ovine xenin coincided (100%) with that of other mammalian species. Real-time quantitative PCR for ovine xenin did not show regional difference in the mRNA expression ratio of xenin. In contrast to the real-time quantitative PCR results, anti-xenin positive cells were abundantly localized in the abomasal antrum (P < 0.01) and at a lesser amount in the duodenum, but no antixenin positive cells were observed in the other regions. Anti-xenin single-positive cells were in a majority in the abomasal antrum, whereas anti-xenin single-positive cells, and anti-GIP single-positive cells, and double-positive cells were even colocalized in the duodenum. These results suggest that abomasal antrum is a major source of xenin in the ovine gastrointestinal tract.

Microwave oscillator using piezoelectric thin-film resonator aiming for ultraminiaturization of atomic clock.

We developed a microwave oscillator and a micro electromechanical systems-based rubidium cell for the miniaturization of atomic clocks. A thin-film bulk acoustic resonator (FBAR) having a resonant frequency of the fundamental mode in the 3.5 GHz band was employed instead of a crystal resonator. It delivers a clock transition frequency of Rb atoms of 3.417 GHz without the need for a complicated frequency multiplication using a phase-locked loop. This topology considerably reduces the system scale and power consumption. For downsizing the atomic clock system toward the chip level as well as mass production, a microfabricated gas cell containing Rb and N2 gases was also developed. These microcomponents were incorporated into an atomic clock test bench, resulting in a clock operation with a short-term frequency instability of 2.1 × 10-11 at 1 s. To the best of our knowledge, this is the first report of a coherent population trapping clock operation using an FBAR-based microwave oscillator as well as a microfabricated gas cell.

Responses of plasma glucose metabolism to exogenous insulin infusion in sheep-fed forage herb plantain and exposed to heat.

The use of herbal plants as traditional medicines has a century long history. Plantain (Plantago lanceolata L.) is a perennial herb containing bioactive components with free radical scavenging activities. An isotope dilution technique using [U-13C]glucose was conducted to determine the effect of plantain on the responses of plasma glucose metabolism to exogenous insulin infusion in sheep. Six crossbred sheep (three wethers and three ewes; mean initial BW=40±2 kg) were fed either a mixed hay of orchardgrass (Dactylis glomerata) and reed canarygrass (Phalaris arundinacea) (MH-diet) or mixed hay and fresh plantain (1 : 1 ratio, dry matter basis, PL-diet) and exposed to a thermoneutral (TN, 20°C; 70% relative humidity (RH)) environment or a heat exposure (HE, 30°C; 70% RH) for 5 days using a crossover design for two 23-day periods. The isotope dilution was conducted on days 18 and 23 of the experimental period during TN and HE, respectively. Plasma concentration of α-tocopherol was greater (P<0.0001) for the PL-diet than the MH-diet and remained comparable between environmental treatments. Plasma glucose concentration before isotope dilution technique was reduced for sheep (P=0.05) during HE compared with TN and remained comparable between diets. Plasma glucose turnover rate during the preinfusion period of insulin did not differ (P=0.10) between dietary treatments and between environments (P=0.65). The response of plasma glucose utilization to exogenous insulin administration was lower (P=0.04) for the PL-diet than the MH-diet. Under present experimental conditions, the plantain group was found to be resistant to the effects of insulin infusion.

Canine distemper virus induces apoptosis through caspase-3 and -8 activation in vero cells.

We investigated the signal-transduction pathway of canine distemper virus-Onderstepoort (CDV-Ond) vaccine strain-mediated apoptosis in Vero cells. Canine distemper virus-Onderstepoort at a multiplicity of infection (MOI) of 0.1 induced DNA fragmentation 48 h after infection. Immunofluorescence staining revealed that 57% +/- 4% of the CDV-N-protein-positive cells were terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive, and all TUNEL-positive cells were CDV-N-protein-positive, indicating that CDV-Ond induced apoptosis only in the infected cells. We also found that CDV-Ond infection induced activation of caspase-3 and caspase-8. In the semi-quantitative reverse transcription-polymerase chain reaction assay for apoptosis-related genes, the expression of mRNA of the death receptor, Fas, was also increased in CDV-Ond-infected cells. In contrast, the expressions of Bcl-2 and Bax, regulators for intrinsic apoptotic signaling through the mitochondria, did not change. These results suggest that CDV-Ond induced apoptosis by activating caspase-3, possibly through caspase-8 signaling rather than through p53/Bax-mediated, mitochondrial signaling in the infected cells.

Effect of dietary protein intake on plasma leucine flux, protein synthesis, and degradation in sheep.

Combined experiments of an isotope dilution method of [1-(13)C]leucine with open circuit calorimetry and a nitrogen (N) balance test were applied to determine the effect of dietary crude protein (CP) intake on plasma leucine flux and protein synthesis and degradation in four sheep. The experiment was conducted in a 3 x 4 Latin rectangle design of three 3-week periods. Dietary CP intake was 5.6, 7.7, and 10.8 g/(kg(0.75) x d). Metabolizable energy intake was 120% of requirement for all dietary treatments. [1-(13)C]Leucine was intravenously infused for 8 h and blood and breath samples were collected during the latter 2-h period of infusion. Isotopic enrichments of plasma [1-(13)C]leucine, alpha-[1-(13)C]ketoisocaproic acid, and exhaled (13)CO(2) were determined. For the N balance test, N digestibility, N excretion in urine, and protein balance (N x 6.25) increased with increasing dietary CP intake. Rates of plasma leucine turnover, protein synthesis, and degradation changed toward reduction with increased dietary CP intake. It is likely that in sheep, high CP intake enhances protein deposition with reduced protein degradation rather than increased protein synthesis.

Three TNFalpha single nucleotide polymorphisms in the Japanese population.

BACKGROUND: Tumour necrosis factor-alpha (TNFalpha) is an essential regulator of immune responses and is implicated to relate to several types of disease susceptibilities. Population information on polymorphisms is essential for the study of genetic diseases. AIM: To obtain accurate information about single nucleotide polymorphisms (SNPs) in the TNFalpha gene in the Japanese population. SUBJECTS AND METHODS: The entire TNFalpha gene was screened for SNPs by directly sequencing 48 chromosomes derived from 24 unrelated Japanese individuals. Allele frequencies of each polymorphism were determined and compared with those previously reported in other populations. RESULTS: Three SNPs, -308G/A at nt -308, IVS1 + 125G/A at nt 492 and IVS3 + 104G/A at nt 1359 were observed, of which one (IVS3 + 104G/A at nt 1359) was novel. In addition, allele frequencies of -308G/A were remarkably different from those presented in the NCBI dbSNP, indicating a significant ethnic difference. CONCLUSIONS: The polymorphisms and allele frequencies obtained in this study will be useful for genetic studies of common diseases such as osteoporosis and rheumatoid arthritis in the Japanese population.

Membrane-type 5 matrix metalloproteinase is expressed in differentiated neurons and regulates axonal growth.

Expression of membrane-type (MT) 5 matrix metalloproteinase (MMP) in the mouse brain was examined. MT5-MMP was expressed in the cerebrum in embryos, but it declined after birth. In contrast, expression in the cerebellum started to increase postnatally and continued thereafter. The cells expressing MT5-MMP were postmitotic neurons that showed gelatinolytic activities. Specific expression of MT5-MMP was observed in the neurons but not in the glial cells when embryonal mouse carcinoma P19 cells were differentiated in vitro by retinoic acid treatment. Neurons isolated from dorsal root ganglia also expressed MT5-MMP, and it was localized at the edge of growth cone. Proteoglycans inhibit neurite extension and regulate synaptogenesis. The inhibitory effect of the proteoglycans on neurite extension of dorsal root ganglia neurons was effectively eliminated by recombinant MT5-MMP. Thus, MT5-MMP expressed in neurons may play a role in axonal growth that contributes to the regulation of neural network formation.

Linkage disequilibrium and haplotype analysis among four novel single-nucleotide polymorphisms in the human leukemia inhibitory factor (LIF) gene.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine implicated in various pathological conditions, such as rheumatoid arthritis and osteoporosis. Despite the possible importance of LIF as a therapeutic target, little is known about the bioregulation of the human LIF gene. We here sequenced the entire structure of the LIF gene of 48 alleles in the Japanese population. These experiments identified four single-nucleotide polymorphisms (SNPs) and determined their allelic frequencies from a 48-allele sequence in the Japanese population. All four SNPs found in the LIFgene were located within exon 3, that is, a C/T at nucleotide (nt) position 3951, a C/G at nt position 4376, an A/C at nt position 4442, and a G/A at nt position 5961 (nucleotide numbering starts from the ATG start codon). Based on the genotypic data, we constructed four major haplotypes in the tested population. Two-way comparisons of SNPs revealed complete linkage disequilibrium between SNPs at positions 3951, 4376, and 4442. These results may prove to be useful as genetic markers for population-based disease-association studies in osteoporosis.

Carbamoyl phosphate synthetase I deficiency: molecular genetic findings and prenatal diagnosis.

We report a Japanese boy who died at Day 28 of life because of severe carbamoyl phosphate synthetase I (CPS1) deficiency that was proven by enzyme assay. By analysis of cDNA and genomic DNA, he was shown to be a compound heterozygote with two point mutations of the CPS1 gene, 840G>C leading to an aberrant splicing and 1123C>T (predicting Q375X). The 840G>C was a mutation described in another Japanese family. Since his parents carried each mutation heterozygously, we performed prenatal diagnosis at 16 weeks of his mother's next gestation by multiplex PCR and melting curve analysis in a single capillary containing two-color fluorescent (LC-Red 640 and LC-Red 705) probes on LightCycler. We analyzed genomic DNA extracted from amniotic cells and found that the fetus was homozygous for the wild-type alleles. At term a healthy girl was born without hyperammonemia.

Thirteen single-nucleotide polymorphisms in the human osteopontin gene identified by sequencing of the entire gene in Japanese individuals.

Osteopontin (OPN) is one of the major noncollagenous bone matrix proteins produced by osteoblasts and osteoclasts. We systematically surveyed the entire structure of the OPN gene for single-nucleotide polymorphisms (SNPs) by directly sequencing 48 alleles derived from 24 unrelated Japanese individuals. We identified 13 SNPs in the OPN gene. Ten polymorphisms were identified in introns 1, 3, and 5; 2 in the coding region of exons 6 and 7; and 1 in the 3' untranslated region of exon 7. Allele frequencies for some of the polymorphisms were significantly different from those reported in the United States National Center for Biotechnology Information (NCBI) dbSNP database. These polymorphisms will be useful in genetic studies to evaluate the role of OPN proteins in bone metabolism.

Linkage disequilibrium and haplotype analysis among ten single-nucleotide polymorphisms of interleukin 11 identified by sequencing of the gene.

Interleukin (IL11) is a member of the interleukin 6 (IL6)-related cytokine subfamily, which stimulates T cell-dependent development of immunoglobulin-producing B cells. IL11 is also an important paracrine regulator of bone metabolism that induces formation of osteoclasts. In the work reported here, we sequenced the entire IL11 structural gene of 48 alleles in a Japanese test population. These experiments identified ten single-nucleotide polymorphisms (SNPs) and determined their allelic frequencies. One polymorphism was identified upstream of exon 1, one in exon 3, four in intron 4 and four in the 3' untranslated region (3'UTR) of exon 5. Based on the genotype data, we constructed six haplotypes in the tested population. Two-way comparisons of SNPs revealed two combinations in complete linkage disequilibrium, one with SNPs at nucleotide positions 2753, 3644, 5154, and 5568, and another with SNPs at positions 3686, 5141, and 5734. These results will be useful in disease-association studies where a contribution of the human IL11 gene has been suspected, especially in disorders affecting immune response and bone metabolism.

Membrane-type 1 matrix metalloproteinase cleaves CD44 and promotes cell migration.

Migratory cells including invasive tumor cells frequently express CD44, a major receptor for hyaluronan and membrane-type 1 matrix metalloproteinase (MT1-MMP) that degrades extracellular matrix at the pericellular region. In this study, we demonstrate that MT1-MMP acts as a processing enzyme for CD44H, releasing it into the medium as a soluble 70-kD fragment. Furthermore, this processing event stimulates cell motility; however, expression of either CD44H or MT1-MMP alone did not stimulate cell motility. Coexpression of MT1-MMP and mutant CD44H lacking the MT1-MMP-processing site did not result in shedding and did not promote cell migration, suggesting that the processing of CD44H by MT1-MMP is critical in the migratory stimulation. Moreover, expression of the mutant CD44H inhibited the cell migration promoted by CD44H and MT1-MMP in a dominant-negative manner. The pancreatic tumor cell line, MIA PaCa-2, was found to shed the 70-kD CD44H fragment in a MT1-MMP-dependent manner. Expression of the mutant CD44H in the cells as well as MMP inhibitor treatment effectively inhibited the migration, suggesting that MIA PaCa-2 cells indeed use the CD44H and MT1-MMP as migratory devices. These findings revealed a novel interaction of the two molecules that have each been implicated in tumor cell migration and invasion.

Five novel single-nucleotide polymorphisms of human interferon gamma identified by sequencing the entire gene.

Interferon gamma (IFNG) plays important roles in the regulation of bone remodelling. We describe here six single-nucleotide polymorphisms (SNPs) in the IFNG gene, five of which are novel, and their allelic frequencies in the Japanese population, as determined by sequencing 48 alleles of the entire gene. Four of these polymorphisms were identified inside the third intron, at nucleotide (nt) positions 2459 (A/G), 2671 (T/C), 3177 (T/G), and 3273 (G/A). In exon 4, SNPs were identified at nt positions 5199 (A/T) and 5272 (A/G). These polymorphic sites will be useful for genetic studies of disorders that affect the inflammatory process or calcium metabolism.

Novel single nucleotide polymorphisms of the human colony-stimulating factor 2 (CSF2) gene identified by sequencing the entire gene.

We describe three single nucleotide polymorphisms (SNPs) of the human colony-stimulating factor 2 (CSF2) gene and their allelic frequencies, as determined by direct sequencing of 48 alleles of the entire CSF2 gene. Three polymorphisms were identified, at nucleotide positions 1816 (T/C), 2284 (C/T), and 3079 (G/A). These polymorphisms will be useful in genetic studies not only of hematologic disorders but also of disorders of bone metabolism.

Novel single nucleotide polymorphisms of the human nuclear factor kappa-B 2 gene identified by sequencing the entire gene.

The nuclear factor kappa-B 2 (NFKB2) gene is a member of the NFKB/Rel gene family, which is known to be a pivotal regulator of the acute phase of the inflammatory response and of immune responses. We identified three novel single nucleotide polymorphisms (SNPs) and determined their allelic frequencies, as determined by the sequencing of 48 alleles of the entire gene in a Japanese population sample. Two of the three polymorphisms were identified at nucleotide (nt) position 1837 (T/C) and nt position, 1867 (GG/G) in the upstream region of the gene. The other polymorphism was identified at nt position 2584 (G/T) within intron 1. These polymorphisms will be useful in genetic studies of the processes involved in inflammatory responses and in bone differentiation.

Overestimated frequency of a possible emphysema-susceptibility allele when microsomal epoxide hydrolase is genotyped by the conventional polymerase chain reaction-based method.

A recent association study suggested that the His113 variant of microsomal epoxide hydrolase (mEPHX) may confer a risk for development of emphysema, presumably by increasing susceptibility to smoking injury. Before considering a possible role of this enzyme in pulmonary disease, we attempted to characterize the genetic polymorphism further. The Tyr/His113 polymorphism within exon 3 of mEPHX was initially examined in 62 healthy individuals by conventional methods involving polymerase chain reaction (PCR)-based determination of a restriction fragment length polymorphism (RFLP). Genomic nucleotide sequences, including the polymorphic site and the downstream primer sequence, were further analyzed in 95 unrelated, healthy Japanese volunteers by single-stranded conformation polymorphism (SSCP) analysis and direct sequencing. Genotyping by the first method (PCR-RFLP) revealed that the allelic distribution in our test population apparently deviated from Hardy-Weinberg equilibrium. Sequence analysis showed that a synonymous nucleotide substitution, AAG to AAA (Lys119), was located just within the published primer site. The AAA at codon 119 was present only in alleles with Tyr113, and its frequency reached 0.31 in our panel of 190 Japanese alleles. This substitution potentially hampered PCR amplification because of the nucleotide mismatch, with the result that the frequency of the Tyr113 variation was underestimated. The frequency of His113, a possible emphysema susceptibility allele of the mEPHX gene, was thus overestimated when human DNA samples were genotyped in the conventional way. Depending on the population(s) tested, this anomaly could represent a pitfall for PCR-based association studies.

The first case of 4-hydroxybutyric aciduria in Japan.

We report a boy with 4-hydroxybutyric aciduria resulting from a deficiency of succinic semialdehyde dehydrogenase (SSADH). A boy, 1 year 5 months, showed delayed walk with hypotonia and could not speak meaningful words. The blood levels of lactate, pyruvate and amino acids were not elevated. Head magnetic resonance imaging (MRI) and electroenchephalography (EEG) were normal. Urinary organic acid analysis with gas chromatography-mass spectrometry (GCMS) revealed increased levels of 4-hydroxybutyric acid, glutaric acid, adipic acid and suberic acid. The concentrations of 4-hydroxybutyric acid and gamma-aminobutyric acid (GABA) were elevated in the serum and cerebrospinal fluid (CSF). SSADH activity in cultured lymphoblasts was 4.5% of the normal level. So far as we know this is the first Japanese patient diagnosed as 4-hydroxybutyric acid. Urinary organic acid analysis is necessary for the diagnosis of patients with unexplained psychomotor retardation.

Accommodative microfluctuation in asthenopia caused by accommodative spasm.

BACKGROUND: Although many patients complain of eye fatigue caused by accommodative spasm, there have been no reports of a good objective examination method to diagnose it. PURPOSE: The spectral power of the high frequency component of the accommodative microfluctuation (spectral power of HFC) differs according to the constrictive degree of the accommodation. In this paper, we expatiated upon our previously reported analyzing processes of the spectral power of HFC, and we investigated the relationship between normal subjects and subjects with asthenopia. METHOD: The accommodative microfluctuation were recorded when the subjects were looking at a stable target. The waves of the accommodative microfluctuation were analyzed by FFT. RESULTS: The spectral power of HFC for the distant target was 50-60 in the subjects with normal vision, but it was higher in the subjects with asthenopia. CONCLUSION: Our results suggested that the ciliary muscle was also actively working in asthenopia caused by accommodative spasm even if the patient was looking at a distant target.