Effects of lactoferrin and lactoperoxidase-containing food on the oral microbiota of older individuals.

The oral microbiota influences health and disease states. Some gram-negative anaerobic bacteria play important roles in tissue destruction associated with periodontal disease. Lactoferrin (LF) and lactoperoxidase (LPO) are antimicrobial proteins found in saliva; however, their influence on the whole oral microbiota currently remains unknown. In this randomized, double-blinded, placebo-controlled study, the effects of long-term ingestion of LF and LPO-containing tablets on the microbiota of supragingival plaque and tongue coating were assessed. Forty-six older individuals ingested placebo or test tablets after every meal for 8 weeks. The relative abundance of bacterial species was assessed by 16S rRNA gene high-throughput sequencing. Most of the bacterial species in supragingival plaque and tongue coating that exhibited significant decreases in the test group were gram-negative bacteria, including periodontal pathogens. Decreases in the total relative abundance of gram-negative organisms in supragingival plaque and tongue coating correlated with improvements in assessed variables related to oral health, such as oral malodor and plaque accumulation. Furthermore, there was significantly less microbiota diversity in supragingival plaque at 8 weeks in the test group than in the placebo group and low microbiota diversity correlated with improvements in assessed variables related to oral health. These results suggest that LF and LPO-containing tablets promote a shift from a highly diverse and gram-negative-dominated to a gram-positive-dominated community in the microbiota of supragingival plaque and tongue coating. This microbial shift may contribute to improvements in oral health, including oral malodor and state of the gingiva.

Factors affecting masticatory function of community-dwelling older people: Investigation of the differences in the relevant factors for subjective and objective assessment.

OBJECTIVE: This study aimed to examine relevant factors for subjective and objective assessment of masticatory functions and elucidate any differences between the two methods. BACKGROUND: Previous studies have reported that the results of subjective and objective assessment of masticatory function in older people do not necessarily agree. MATERIALS AND METHODS: This study included 487 community-dwelling Japanese older people (205 male and 282 female; mean age 74.1±6.3 years) who participated in a comprehensive geriatric health examination. Basic information (gender and age), higher level of competence in daily living, depression, subjective masticatory function (SMF) and objective masticatory function (OMF) assessments, cognitive function, skeletal muscle mass, handgrip strength, gait speed and oral status (number of remaining and functional teeth, mouth dryness and occlusal force) were recorded. RESULTS: Multiple logistic regression analysis showed that depression (odds ratio [OR]: 1.181, 95% confidence interval [CI]: 1.094-1.275), mouth dryness (OR: 2.037, CI: 1.212-3.423) and occlusal force (OR: 0.997, CI: 0.996-0.999) were significantly associated with SMF, whereas higher level of competence in daily living (OR: 0.730 CI: 0.586-0.910), skeletal muscle mass (OR: 0.521 CI: 0.283-0.960), number of functional teeth (OR: 0.862 CI: 0.775-0.959), number of remaining teeth (OR: 0.868 CI: 0.810-0.930) and occlusal force (OR: 0.994, CI: 0.991-0.998) were associated with OMF. CONCLUSION: Subjective masticatory functionSMF and OMF were associated with different factors, suggesting that both mental and physical factors should be taken into consideration when treating decreased masticatory function.

Relationship between mortality and Council of Nutrition Appetite Questionnaire scores in Japanese nursing home residents.

OBJECTIVE: This 1-y cohort study examined whether Council of Nutrition Appetite Questionnaire (CNAQ) scores predicted mortality in 316 elderly Japanese residents of five nursing homes (60 men, 256 women; mean age: 84.9 ± 8.3 y). METHODS: The baseline survey included participant characteristics (e.g., age, sex, height, weight, and medical history), and Barthel Index (BI), Clinical Dementia Rating (CDR), Mini Nutritional Assessment-Short Form (MNA®-SF), CNAQ, Simplified Nutritional Appetite Questionnaire (SNAQ; simplified CNAQ), and SNAQ for the Japanese elderly (SNAQ-JE) scores. RESULTS: Following the baseline survey, mortality data were collected for 1 y; during this time, 62 participants (19.6%) died. The deceased group's CNAQ scores (25.1 ± 4.8) were significantly lower than those of the survival group (28 ± 3.6; P < 0.001). After adjusting for age, sex, medical history, BI, CDR, and MNA®-SF scores in Cox proportional regression, CNAQ (hazard ratio [HR], 0.91; 95% confidence interval [CI], 0.85-0.97; P = 0.004), SNAQ (HR, 0.84; 95% CI, 0.75-0.93; P = 0.001), and SNAQ-JE (HR, 0.84; 95% CI, 0.76-0.92; P < 0.001) scores were related to mortality. CONCLUSIONS: This study showed that CNAQ scores were inversely associated with 1-y mortality. Furthermore, appetite assessment using the CNAQ predicted the death of Japanese nursing home residents. Similarly, the SNAQ and SNAQ-JE scores were inversely associated with 1-y mortality.

Discovery of a novel class of 1,3-dioxane-2-carboxylic acid derivatives as subtype-selective peroxisome proliferator-activated receptor alpha (PPARalpha) agonists.

A new series of 1,3-dioxane-2-carboxylic acid derivatives was synthesized and evaluated for agonist activity at human peroxisome proliferator-activated receptor (PPAR) subtypes. Structure-activity relationship studies led to the identification of 2-methyl-c-5-[4-(5-methyl-2-phenyl-1,3-oxazol-4-yl)butyl]-1,3-dioxane-r-2-carboxylic acid 4b as a potent PPARalpha agonist with high subtype selectivity at human receptor subtypes. This compound exhibited a substantial hypolipidemic effect in type 2 diabetic KK-A(y) mice.

Structure-activity studies on 1,3-dioxane-2-carboxylic acid derivatives, a novel class of subtype-selective peroxisome proliferator-activated receptor alpha (PPARalpha) agonists.

A series of 1,3-dioxane carboxylic acid derivatives was synthesized and evaluated for human PPAR transactivation activity. Structure-activity relationships on the phenyloxazole moiety of the lead compound 3 revealed that the introduction of small hydrophobic substituents at the 4-position of the terminal phenyl ring increased the PPARalpha agonist activity, and that the oxazole heterocycle was essential to the maintenance of both potency and PPARalpha subtype-selectivity. This investigation led to the identification of 14d (NS-220) and 14i as highly potent and selective human PPARalpha agonists. In KK-A(y) type 2 diabetic mice, these compounds significantly lowered plasma triglyceride and very-low-density plus low-density lipoprotein cholesterol levels while simultaneously raising HDL cholesterol levels. Our results suggest that highly potent and subtype-selective PPARalpha agonists will be promising drugs for the treatment of metabolic disorders in type 2 diabetes.

A comparison of vasodilation mode among selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide]), its active metabolite MRE-269 and various prostacyclin receptor agonists in rat, porcine and human pulmonary arteries.

Selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N- (methylsulfonyl)acetamide]) is a novel, orally available non-prostanoid prostacyclin receptor (IP receptor) agonist that has recently been approved for the treatment of pulmonary arterial hypertension (PAH). We examined the effect of the active metabolite of selexipag, MRE-269, and IP receptor agonists that are currently available as PAH therapeutic drugs on the relaxation of rat, porcine and human pulmonary artery. cAMP formation in human pulmonary artery smooth muscle cells was induced by all test compounds (MRE-269, epoprostenol, iloprost, treprostinil and beraprost sodium) and suppressed by IP receptor antagonists (CAY10441 and 2-[4-(1H-indol-4-yloxymethyl)-benzyloxycarbonylamino]-3-phenyl-propionic acid). MRE-269 induced endothelium-independent vasodilation of rat extralobar pulmonary artery (EPA). In contrast, endothelial denudation or the addition of a nitric oxide synthase inhibitor markedly attenuated the vasodilation of EPA induced by epoprostenol, treprostinil and beraprost sodium but not iloprost. The vasorelaxant effects of MRE-269 on rat small intralobar pulmonary artery (SIPA) and EPA were the same, while the other IP receptor agonists induced less vasodilation in SIPA than in EPA. Furthermore, a prostaglandin E receptor 3 antagonist enhanced the vasodilation induced by all IP receptor agonists tested except MRE-269. We also investigated the relaxation induced by IP receptor agonists in pulmonary arteries from non-rodent species and found similar vasodilation modes in porcine and human as in rat preparations. These results suggest that MRE-269, in contrast to other IP receptor agonists, works as a selective IP receptor agonist, thus leading to pronounced vasorelaxation of rat, porcine and human pulmonary artery.

Relationship between skeletal muscle mass and swallowing function in patients with Alzheimer's disease.

AIM: The present study verified the hypothesis that decreased skeletal muscle in older adults with Alzheimer's disease is related to Alzheimer's disease progression and decreased oral or swallowing function. METHODS: We investigated 232 patients with Alzheimer's disease (31 men, 201 women, average age 85.4 ± 5.9 years) in two regions in Japan. The patients provided basic information (sex and age), and were assessed for skeletal muscle index, dementia severity (clinical dementia rating), activities of daily living, nutritional status, oral status and swallowing function. RESULTS: Stratification by clinical dementia rating was as follows: clinical dementia rating 0.5:21 patients (9.0%), clinical dementia rating 1:85 patients (36.6%), clinical dementia rating 2:88 patients (37.9%) and clinical dementia rating 3:38 patients (16.3%). Alzheimer's disease severity was significantly related to skeletal muscle index. Logistic regression analysis showed that clinical dementia rating 3 (odds ratio 11.68, 95% confidence interval 4.52-30.20), body mass index < 18.5 (odds ratio 3.18, 95% confidence interval 1.27-8.00), calf circumference <30.5 cm (odds ratio 9.33, 95% confidence interval 2.01-43.27) and poor swallowing function (odds ratio 4.93, 95% confidence interval 1.10-22.04) had a significant effect on decreased skeletal muscle index. CONCLUSIONS: Therefore, decreased skeletal muscle mass in patients with Alzheimer's disease requires strategies to manage swallowing dysfunction. Geriatr Gerontol Int 2016; 17: 402-409.

DHCR24 gene knockout mice demonstrate lethal dermopathy with differentiation and maturation defects in the epidermis.

Desmosterolosis is an autosomal recessive disorder due to mutations in the 3beta-hydroxysterol-Delta24 reductase (DHCR24) gene that encodes an enzyme catalyzing the conversion of desmosterol to cholesterol. To date, only two patients have been reported with severe developmental defects including craniofacial abnormalities and limb malformations. We employed mice with targeted disruption of DHCR24 to understand the pathophysiology of desmosterolosis. All DHCR24-/- mice died within a few hours after birth. Their skin was wrinkleless and less pliant, leading to restricted movement and inability to suck (empty stomach). DHCR24 gene was expressed abundantly in the epidermis of control but not of DHCR24-/- mice. Accordingly, cholesterol was not detected whereas desmosterol was abundant in the epidermis of DHCR24-/- mice. Skin histology revealed thickened epidermis with few and smaller keratohyaline granules. Aberrant expression of keratins such as keratins 6 and 14 suggested hyperproliferative hyperkeratosis with undifferentiated keratinocytes throughout the epidermis. Altered expression of filaggrin, loricrin, and involcrin were also observed in the epidermis of DHCR24-/-. These findings suggested impaired skin barrier function. Indeed, increased trans-epidermal water loss and permeability of Lucifer yellow were observed in DHCR24-/- mice. DHCR24 thus plays crucial role for skin development and its proper function.

DHCR24-knockout embryonic fibroblasts are susceptible to serum withdrawal-induced apoptosis because of dysfunction of caveolae and insulin-Akt-Bad signaling.

The DHCR24 gene encodes an enzyme catalyzing the last step of cholesterol biosynthesis, the conversion of desmosterol to cholesterol. To elucidate the physiological significance of cholesterol biosynthesis in mammalian cells, we investigated proliferation of mouse embryonic fibroblasts (MEFs) prepared from DHCR24(-/-) mice. Both DHCR24(-/-) and wild-type MEFs proliferated in the presence of serum in culture media. However, the inhibition of external cholesterol supply by serum withdrawal induced apoptosis of DHCR24(-/-) MEFs, which was associated with a marked decrease in the intracellular and plasma membrane cholesterol levels, Akt inactivation, and Bad dephosphorylation. Insulin is an antiapoptotic factor capable of stimulating the Akt-Bad cascade, and its receptor (IR) is enriched in caveolae, cholesterol-rich microdomains of plasma membrane. We thus analyzed the association of IR and caveolae in the cholesterol-depleted MEFs. Subcellular fractionation and immunocytochemical analyses revealed that the IR and caveolin-1 contents were markedly reduced in the caveolae fraction of the MEFs, suggesting the disruption of caveolae, and that large amounts of IR were present apart from caveolin-1 on plasma membrane, indicating the uncoupling of IR with caveolae. Consistent with these findings, insulin-dependent phosphorylations of insulin receptor substrate-1, Akt, and Bad were impaired in the cholesterol-depleted MEFs. However, this impairment was partial because treatment of the MEFs with insulin restored Akt activation and prevented apoptosis. Cholesterol supply also prevented apoptosis. These results demonstrate that the cellular cholesterol biosynthesis is critical for the activation and maintenance of the Akt-Bad cell survival cascade in response to growth factors such as insulin.

Relationship between swallowing function and the skeletal muscle mass of older adults requiring long-term care.

AIM: The present study investigated the risk factors for dysphagia among older adults who require long-term care, and also examined their systemic decrease in skeletal muscle mass. METHODS: We evaluated 399 people who required long-term care and who were residing in Omori town, Yokote city, Akita prefecture, Japan. We then analyzed data from 255 participants who had complete information available regarding their sex, age, case history (stroke, Parkinson's disease and dementia), Barthel Index, Skeletal Muscle Mass Index, oral function test and modified water swallowing test results. Participants' water swallowing test results were used to create groups with good or poor swallowing function, and a univariate analysis was carried out for each parameter. Parameters with a P-value of <0.25 in the univariate analysis were subsequently included in a multiple logistic regression model as explanatory variables, and good or poor swallowing function were defined as the dependent variables. RESULTS: After adjusting for age and sex, our analysis showed that poor tongue motility (odds ratio 17.23, 95% confidence interval 5.90-50.31, P < 0.001) and decreased Skeletal Muscle Mass Index (odds ratio 3.36, 95% confidence interval 1.41-7.99, P = 0.006) were significantly correlated with decreased swallowing function. CONCLUSIONS: Decreased swallowing function was closely correlated with poor tongue motility, and this finding is similar to those of previous studies. However, the present results also show that decreased Skeletal Muscle Mass Index is a novel risk factor for dysphagia among older adults who require long-term care.

Blockade of voltage-gated calcium channel Cav1.2 and α1-adrenoceptors increases vertebral artery blood flow induced by the antivertigo agent difenidol.

Difenidol (1,1-diphenyl-4-piperidino-1-butanol hydrochloride) is an effective drug for the treatment of vertigo and dizziness. This drug is known to improve the blood flow in vertebral arteries, though the precise mechanism underlying this action remains unclear. In the present study, we investigated the effect of difenidol on voltage-gated calcium channel Ca(v)1.2 and α(1)-adrenoceptor subtypes that regulate the intracellular calcium concentration ([Ca(2+)](i)), as well as their possible involvement in the action of difenidol on vertebral artery relaxation and blood flow in dogs. In vitro binding assays demonstrated that difenidol at micromolar concentrations bound to the α(1A)-, α(1B)- and α(1D)-adrenoceptor subtypes. Difenidol inhibited the phenylephrine-induced increase in [Ca(2+)](i) in Chinese hamster ovary cells expressing human α(1A)-, α(1B)- or α(1D)-adrenoceptor subtypes with similar IC(50) values in the low micromolar range. In an electrophysiological assay, difenidol inhibited L-type calcium channel (Ca(v)1.2 subunit). In dogs, i.v. difenidol preferentially enhanced vertebral over femoral arterial blood flow. Phenylephrine and potassium induced contraction of dog vertebral arterial rings, and difenidol inhibited this action. Inhibition of phenylephrine-induced contraction by difenidol was mimicked by the α(1)-adrenoceptor antagonist phentolamine, the α(1A)-adrenoceptor antagonist RS 17,053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethanamine hydrochloride) and the α(1D)-adrenoceptor antagonist BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride). In addition, the L-type calcium channel blocker nifedipine, like difenidol, attenuated the potassium-induced contraction. These findings suggest that the difenidol-induced increase in vertebral arterial blood flow may be due to vascular relaxation mediated by mixed blocking actions at α(1)-adrenoceptors and voltage-gated calcium channel Ca(v)1.2.

Structure of asparagine-linked oligosaccharides of an aspartic proteinase from the zygomycete fungus Rhizomucor pusillus.

The zygomycete fungus Rhizomucor pusillus (previously called Mucor pusillus) secretes an aspartic proteinase containing two asparagine-linked, high-mannose type oligosaccharide chains at Asn79 and Asn188. For structural elucidation of the carbohydrate moieties, the protein was divided into two portions, an N-terminal portion containing Asn79 and a C-terminal portion containing Asn188, by a specific autocatalytic cleavage under alkaline conditions. Each of the asparagine-linked oligosaccharides was then released by peptide-N-glycosidase F digestion and pyridylaminated with a fluorescent reagent, 2-aminopyridine, at the reducing end. High-performance liquid chromatography analyses showed that the structure of the asparagine-linked oligosaccharide chain attached to residue Asn79 was Man5GlcNAc2, and that bound to residue Asn188 was Man5GlcNAc2 and Man6GlcNAc2. These observations suggest that the processing of mannose residues in asparagine-linked oligosaccharides in the Golgi apparatus of Rhizomucor resembles that in mammalian cells.

Kinetic analysis of enhanced thermal stability of an alkaline protease with engineered twin disulfide bridges and calcium-dependent stability.

The thermal stability of a cysteine-free alkaline protease (Alp) secreted by the eukaryote Aspergillus oryzae was improved both by the introduction of engineered twin disulfide bridges (Cys-69/Cys-101 and Cys-169/Cys-200), newly constructed as part of this study, and by the addition of calcium ions. We performed an extensive kinetic analysis of the increased thermal stability of the mutants as well as the role of calcium dependence. The thermodynamic activation parameters for irreversible thermal inactivation, the activation free energy (deltaG), the activation enthalpy (deltaH), and the activation entropy (deltaS) were determined from absolute reaction rate theory. The values of deltaH and deltaS were significantly and concomitantly increased as a result of introducing the twin disulfide bridges, for which the increase in the value of deltaH outweighed that of deltaS, resulting in significant increases in the value of deltaG. The enhancement of the thermal stability obtained by introducing the twin disulfide bridges is an example of the so-called low-temperature stabilization of enzymes. The stabilizing effect of calcium ions on wild-type Alp is similar to the results we obtained by introducing the engineered twin disulfide bridges.

Prognosis of breast cancer patients treated with sentinel node biopsy in Japan.

BACKGROUND: Sentinel node biopsy predicts accurate pathological nodal staging. The survival of node-negative breast cancer patients should be evaluated between the patients treated with sentinel node biopsy alone and those treated with axillary lymph node dissection. METHODS: Ninety-seven patients with negative axillary nodes underwent sentinel node biopsy immediately followed by axillary lymph node dissection between January 1998 and June 1999 (the ALND group). Since then, if sentinel lymph nodes were negative on the frozen-section diagnosis, 112 patients underwent sentinel node biopsy alone without axillary lymph node dissection between July 1999 and December 2000 (the SNB group). We retrospectively observed the outcome of the two study groups. RESULTS: Median follow-up was 52 months in all patients. Relapse-free survival rates at 3 years in the ALND and SNB groups were 94% and 93%, respectively. Five of the 112 patients in the SNB group had overt axillary metastases. Three of them with axillary metastases alone were treated with delayed axillary lymph node dissection. These three patients have been free of other events for 3 years after local salvage treatment. CONCLUSIONS: Sentinel node biopsy will emerge as a standard method to diagnose axillary nodal staging for clinically node-negative breast cancer patients.

A novel selective peroxisome proliferator-activated receptor alpha agonist, 2-methyl-c-5-[4-[5-methyl-2-(4-methylphenyl)-4-oxazolyl]butyl]-1,3-dioxane-r-2-carboxylic acid (NS-220), potently decreases plasma triglyceride and glucose levels and modifies lipoprotein profiles in KK-Ay mice.

2-Methyl-c-5-[4-[5-methyl-2-(4-methylphenyl)-4-oxazolyl]butyl]-1,3-dioxane-r-2-carboxylic acid (NS-220) was newly synthesized and demonstrated to be a novel potent peroxisome proliferator-activated receptor alpha (PPARalpha) agonist with high subtype selectivity. In cell-based reporter gene assays, the EC(50) values of NS-220 for human PPARalpha, PPARgamma, and PPARdelta were 1.9 x 10(-8), 9.6 x 10(-6), and >10(-4) M, respectively, and for mouse PPARalpha, PPARgamma, and PPARdelta were 5.5 x 10(-8), 3.3 x 10(-5), and >10(-4) M, respectively. In addition, [(3)H]NS-220 bound to the ligand-binding domain of human PPARalpha with a K(D) value of 1.85 x 10(-7) M. Fenofibric acid and bezafibrate showed weak agonist activity for PPARalpha (EC(50), 2-8 x 10(-5) M), with poor subtype selectivity. NS-220 (0.1-3 mg/kg p.o.) decreased plasma triglyceride levels in ddY mice in a dose-dependent manner, but its hypolipidemic activity was abolished in PPARalpha-deficient mice. In KK-A(y) mice, an animal model of type-2 diabetes, NS-220 (0.3-1 mg/kg p.o.; 4 days) and fenofibrate (100-300 mg/kg p.o.; 4 days) decreased plasma triglyceride and glucose levels in a dose-dependent manner. In a 2-week repeated administration test, NS-220 (0.3-1 mg/kg p.o.) decreased plasma glucose levels markedly without increasing in plasma insulin levels. Furthermore, NS-220 increased high-density lipoprotein levels and decreased triglyceride-rich lipoprotein levels. In conclusion, a newly synthesized dioxanecarboxylic acid derivative, NS-220, is a potent and highly selective PPARalpha agonist that ameliorates metabolic disorders in diabetic mice. These results strongly suggest that it will be a promising drug for the treatment of hyperlipidemia or metabolic disorders in type-2 diabetes.