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.

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.

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.

Genetic analysis of Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy: nucleotide-binding fold-2 mutation impairs cooperative binding of adenine nucleotides to sulfonylurea receptor 1.

To elucidate the genetic etiology of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) in the Japanese population, we conducted a polymerase chain reaction-single-strand conformation polymorphism analysis of the sulfonylurea receptor 1 (SUR1) and Kir6.2 genes in 17 Japanese PHHI patients, including a pair of siblings from a consanguineous family. We also analyzed the glutamate dehydrogenase gene for the exons encoding an allosteric regulatory domain of the enzyme. In the SUR1 gene, we identified one frameshift (I446fsdelT) and two missense (R1420C, R1436Q) mutations. None of these mutations were found in control Japanese subjects. Siblings homozygous for the R1420C mutation had a mild form, whereas two patients heterozygous for the I446fsdelT and R1436Q mutations, respectively, exhibited a severe form of PHHI. Functional consequences of these mutations on K(ATP) function were evaluated using 86Rb+ efflux studies in COS-7 cells. SUR1-446fsdelT and SUR1-1436Q did not form a functional K(ATP). Western blot analysis after transient expression in COS-7 cells revealed the expression of SUR1-1436Q protein to be markedly reduced, suggesting SUR1-1436Q to be unstable in these cells. K(ATP)(SUR1-1420C) showed reduced responses to metabolic inhibition by oligomycin and 2-deoxyglucose. K(ATP) channels are under complex regulation by intracellular ATP and ADP. ATP both inhibits and activates these channels. The inhibition is probably mediated through direct ATP interaction with a pore-forming subunit Kir6.2, whereas the activation is likely to be through a regulatory subunit SUR1. There is a cooperative regulation of ATP and ADP binding to SUR1, and this cooperativity may be involved in regulating the K(ATP) channel. In SUR1-1420C, high-affinity binding of ATP to the nucleotide-binding fold (NBF)-1 was indistinguishable from that of wild-type SUR1. However, stabilization of ATP binding to NBF-1 by MgATP or MgADP was impaired, suggesting that this defect may account for impaired K(ATP)(SUR1-1420C) function. This is the first direct biochemical evidence that the cooperativity of nucleotide binding to SUR1 is impaired in a SUR1 mutant causing PHHI. No mutations were identified in the Kir6.2 and glutamate dehydrogenase genes. The genetic etiology of PHHI appears to be heterogeneous. SUR1 mutations may account for no more than 20% of PHHI cases in Japanese patients. Mutations of Kir6.2 and glutamate dehydrogenase genes are likely to be even less common.

Cloning of three Caenorhabditis elegans genes potentially encoding novel matrix metalloproteinases.

Three genes potentially encoding novel matrix metalloproteinases (MMPs) were identified by sequence similarity searching of Caenorhabditis elegans genome database, and cDNAs for these MMPs were cloned. The predicted gene products (MMP-C31,-H19 and -Y19) display a similar domain organization to human MMPs. MMP-H19 and -Y19 are unique in that they have an RXKR motif between the propeptide and catalytic domains that is a furin-like cleavage site, and conserved only in stromelysin-3 and membrane-type MMPs. The amino acid sequence homology with MMP-1/human interstitial collagenase at the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively. Recombinant proteins of C. elegans MMPs cleaved an MMP peptide substrate with efficiency proportional to their amino acid homology with human MMPs. Digestion of gelatin was observed only with MMP-C31. Enzyme activity of MMP-C31 and -H19 was inhibited by human tissue inhibitor of MMPs (TIMP)-1, TIMP-2 and synthetic MMP inhibitors, BB94 and CT543, indicating that the catalytic sites of these C. elegans MMPs are structurally closely related with those of mammalian MMPs.

Human membrane type-4 matrix metalloproteinase (MT4-MMP) is encoded by a novel major transcript: isolation of complementary DNA clones for human and mouse mt4-mmp transcripts.

Five distinct membrane-type matrix metalloproteinases (MT-MMP) have been reported by cDNA cloning. However, the mt4-mmp gene product (MMP-17) has not been identified yet in spite of the cDNA isolation [Puente et al. (1996), Cancer Res. 56, 944-949]. In this study, we re-examined the transcripts for human mt4-mmp by 5' RACE and identified two types of transcripts. The minor one corresponded to the cDNA reported by Puente et al. and failed to express protein, and the other is the major transcript that has an extended open reading frame and expressed 67 and 71 kDa translation products. Thus, functional mt4-mmp has been identified for the first time.

Necrotizing bronchial aspergillosis in a patient receiving neoadjuvant chemotherapy for non-small cell lung carcinoma.

We describe a case of necrotizing bronchial aspergillosis which developed after lobectomy following neoadjuvant chemotherapy in a 73-year-old woman with non-small cell lung cancer. The lesion was visualized and biopsied through FBS, which played a useful role for early diagnosis of this disease. Itraconazole therapy was effective and safe.

Enhanced colonic and rectal absorption of insulin using a multiple emulsion containing eicosapentaenoic acid and docosahexaenoic acid.

The aim of this study was to test the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on insulin absorption from rat intestinal loops in situ, using a water-in-oil-in-water (W/O/W) multiple emulsion. The enhancement effect of these long-chain polyunsaturated fatty acids was compared with that of free fatty acids having a C18 alkyl chain. The emulsion (insulin dose, 50 units/kg) was administered directly into the colonic and rectal loops. Both EPA and DHA strongly enhanced insulin absorption and induced hypoglycemia after colonic and rectal dosing. Comparing the pharmacological availability, the order of effectiveness with respect to the enhanced absorption of insulin was DHA >/= EPA > C18 unsaturated fatty acids >> C18 saturated fatty acid at both sites. DHA showed greater effects upon rectal dosing than upon colonic dosing. Histological studies revealed that the emulsion incorporating DHA did not induce gross morphological changes in the structure of the intestinal mucosa. Our results indicate that a W/O/W multiple emulsion incorporating DHA is a possible means of facilitating the intestinal absorption of insulin without inducing any serious damage to the epithelial cells.

Oral delivery of insulin using pH-responsive complexation gels.

The goal of oral insulin delivery devices is to protect the sensitive drug from proteolytic degradation in the stomach and upper portion of the small intestine. In this work, we investigate the use of pH-responsive, poly(methacrylic-g-ethylene glycol) hydrogels as oral delivery vehicles for insulin. Insulin was loaded into polymeric microspheres and administered orally to healthy and diabetic Wistar rats. In the acidic environment of the stomach, the gels were unswollen due to the formation of intermolecular polymer complexes. The insulin remained in the gel and was protected from proteolytic degradation. In the basic and neutral environments of the intestine, the complexes dissociated which resulted in rapid gel swelling and insulin release. Within 2 h of administration of the insulin-containing polymers, strong dose-dependent hypoglycemic effects were observed in both healthy and diabetic rats. These effects lasted for up to 8 h following administration.

Enhanced enteral bioavailability of vancomycin using water-in-oil-in-water multiple emulsion incorporating highly purified unsaturated fatty acid.

The aim of this study was to evaluate the potential of an emulsion incorporating unsaturated fatty acids to improve the mucosal absorption of poorly absorbed drugs from rat intestinal loops in situ, using a water-in-oil-in-water (W/O/W) multiple emulsion. Vancomycin hydrochloride (VCM) was used as a model drug with low oral bioavailability. The entrapment efficiency of VCM in the emulsion was approximately 60% and remained constant over storage for 1 month at 4 degrees C. The emulsion incorporating C18 unsaturated fatty acids or docosahexaenoic acid (DHA) markedly enhanced VCM absorption after colonic and rectal dosing. The effectiveness of DHA on VCM colonic absorption improvement was the same as that of oleic acid, and less than that of linoleic and linolenic acids. For rectal dosing, bioavailability was similar among various emulsions, in the range 40-50%. The effect of the emulsion incorporating oleic acid or DHA on improving VCM enteral bioavailability was not increased proportional to the incorporated amount. The electrical resistance of membranes was not changed by the incorporation of various fatty acids in emulsions. Our results indicated that W/O/W emulsions incorporating C18 unsaturated fatty acid or DHA were useful carriers for improving the absorption of poorly absorbable drugs via the intestinal tract without gross changes to tight junction function.

Continuous infusion versus intermittent short infusion of metoclopramide for cisplatin-induced acute emesis.

Metoclopramide is an active antiemetic against cisplatin-induced acute emesis. However, the optimal administration method (continuous infusion versus intermittent short infusion) for metoclopramide has not yet been clearly defined. We have conducted a randomized crossover study to compare the antiemetic efficacy of continuous infusion of metoclopramide with that of intermittent short infusion of metoclopramide in 54 evaluable patients. Patients were stratified according to sex and were randomized to receive either a continuous-infusion regimen (regimen A) or an intermittent-short infusion regimen (regimen B). Patients were switched to the alternate therapy in the second course. In regimen A, metoclopramide at 3 mg/kg i.v. was given before cisplatin, and then metoclopramide at 4 mg/kg was infused intravenously over 7.5 hours. In regimen B, metoclopramide at 3 mg/kg i.v. was followed by 2 mg/kg i.v. for two doses. Dexamethasone and diphenhydramine were given intravenously in both regimens. There was no significant difference between two regimens in their ability to prevent emesis. Complete protection (no episode of emesis) and major protection (< or = 2 episodes of emesis), respectively, were obtained by 67% (95% confidence interval: 53-79%) and 85% (95% confidence interval: 73-93%) of all patients given regimen A and by 59% (95% confidence interval: 45-72%) and 81% (95% confidence interval: 68-91%) of those given regimen B. The two regimens were also equally effective in controlling nausea. However, male patients showed better control of nausea and vomiting than did female patients, regardless of treatment regimen. Toxicity was mild in both regimens and was well tolerated. Our findings indicate that both continuous-infusion metoclopramide and intermittent-short infusion metoclopramide are effective in controlling cisplatin-induced acute nausea and vomiting.

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.

The dose-related hypoglycemic effects of insulin emulsions incorporating highly purified EPA and DHA.

The dose-related pharmacological effects of insulin emulsion incorporating highly purified eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were investigated. Water-in-oil-in-water multiple emulsions (insulin dose, 0, 10, 25 and 50 IU/kg) incorporating 2% DHA or EPA were administered directly into the colonic and rectal loops in situ. Serum insulin levels rose and serum glucose levels decreased in an insulin dose-related fashion. The relationship of insulin dose and C(max) or AUC(insulin) was linear at the rectum, but a non-linear relationship was observed at the colon. The trend was more predominant in DHA. In the in vivo rectal absorption experiment using emulsions incorporating 2% DHA, 5 IU/kg of insulin emulsion produced a rapid, transitory increase in serum insulin levels and strong reduction of serum glucose levels. The pharmacological availability determined from the dose-response curve by s.c. administration of insulin reached 43.2+/-26.3% (mean+/-S.D.). Mucosal irritation caused by administration of emulsions incorporating 2% EPA or DHA was evaluated by a lactate dehydrogenase (LDH) release study, and compared with those of the emulsion incorporating 2% oleic or linolenic acid. Only when emulsion incorporating 2% oleic acid was applied in the intestine did significant LDH release into the mesenteric veins occur. Our results indicate that emulsion incorporating highly purified long-chain polyunsaturated fatty acid, especially DHA, has the potential of becoming the formulation for enteral delivery of insulin.

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.

Relationship between insulin and blood pressure in Japanese obese subjects.

OBJECT: The association of obesity and hypertension is well recognized. However, the nature of the relationship between increased body weight and blood pressure (BP) elevation has remained obscure. PATIENTS AND METHODS: We evaluated BP, insulin sensitivity, insulin clearance and fasting plasma insulin concentration in 19 younger (over 40 years) and in 15 older (more than 40 years) obese subjects to determine the relationships between BP and other factors. Insulin sensitivity and clearance were determined with the euglycemic clamp technique. RESULTS: BP was not associated with insulin sensitivity although most of the subjects showed insulin resistance. In the younger obese group, a positive correlation between diastolic BP and body mass index (kg/m2) was found (r=0.740; p=0.043). In the older obese group, systolic and diastolic BP were correlated with fasting plasma insulin levels (r=0.705; p=0.003; r=0.574; p=0.025, respectively), and systolic BP was inversely correlated with insulin clearance (r=-0.715, p=0.003). CONCLUSION: These results suggest that insulin is an important factor in BP elevation in older obese subjects, but not in younger obese subjects.

[Relationship between refractive elements and morphometry of corneal endothelial cells in normal eyes of young Japanese].

We analyzed corneal endothelial cells in 1,026 eyes from 12 to 29-year-old patients who had no eye disease except refractive error. Morphometric parameters were examined for their distribution and correlation with refractive elements. The distribution of cell density and hexagonal cells in percentage was normal. The logarithmic value of mean cell area and coefficient of variation (CV) also had normal distribution. There was also significant correlation between aging and morphometric parameters, i.e., positive correlation between the mean cell area and the CV, and negative correlation between the cell density and the hexagonality. There was significant correlation between corneal refraction and morphometric parameters, i.e., negative correlation with mean cell area, and positive correlation with cell density. There was also significant negative correlation between the corneal astigmatism and the hexagonality. Consequently, we suggest that the distribution of each morphometric parameter and the difference in age and refractive elements should be considered when discussing morphometry of the corneal endothelial cells.

[Corneal curvature of myopia].

We classified myopia by total refractive error, measured the corneal curvature, and analyzed the relationship between the diopter value and corneal curvature in growing teen-agers and in adults in their twenties, whose myopic progress is presumed to have stopped. We adopted as the harmonic average the central 3.0 mm of the cornea. We also examined peripheral corneal curvature in upper side, lower side, temporal side and nasal side at about 4.5 mm and 6.5 mm diameter from the center. There was very little difference in the harmonic average of corneal curvature from -1D to -6D. Similarly, there was very little difference in the peripheral corneal curvature. The corneal curvature was larger from central to periphery in all directions. The upper side was larger than lower side and the nasal side was larger than the temporal side. We concluded that the refractive power did not depend on the corneal refractive power in mild to moderate myopic patients.

[Detection of resting state of accommodation by accommodative microfluctuation].

Accommodative microfluctuations were recorded when the subjects were looking at a stable target. The waves of the accommodative microfluctuation were analyzed by fast Fourier transform. When accommodation was in a resting state, the high frequency components were minimized. This suggested that the resting state of accommodation might be measured when the subject was looking at a target. At a little distance from the resting state of accommodation, the high frequency components were maximized. This might suggest a negative accommodation.