A reliable enzyme-linked immunosorbent assay for the determining of sesame proteins in raw food ingredients and in processed foods.

Sesame is a frequent cause of adverse food reactions in allergic patients. We developed a novel sandwich enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies and a unique extraction buffer for the detection and quantification of sesame proteins in processed foods and in raw food ingredients to clarify the validity of sesame labeling and for precautionary allergen labeling. The developed sandwich ELISA method is highly specific for sesame proteins. The limit of detection (LOD) and limit of quantification (LOQ) are 0.013 µg/g and 0.025 µg/g, respectively. The recoveries for incurred food samples, such as dressing, breads, sauce and pudding, ranged from 67 % to 81 %, while the repeatability and reproducibility coefficients of variation were less than 4.7 % and 4.5 %, respectively. The developed method has applicability for food products and is a reliable tool for the detection of hidden sesame proteins in raw food ingredients and in processed foods.

A disease-driver population within interstitial cells of human calcific aortic valves identified via single-cell and proteomic profiling.

Cellular heterogeneity of aortic valves complicates the mechanistic evaluation of the calcification processes in calcific aortic valve disease (CAVD), and animal disease models are lacking. In this study, we identify a disease-driver population (DDP) within valvular interstitial cells (VICs). Through stepwise single-cell analysis, phenotype-guided omic profiling, and network-based analysis, we characterize the DDP fingerprint as CD44highCD29+CD59+CD73+CD45low and discover potential key regulators of human CAVD. These DDP-VICs demonstrate multi-lineage differentiation and osteogenic properties. Temporal proteomic profiling of DDP-VICs identifies potential targets for therapy, including MAOA and CTHRC1. In vitro loss-of-function experiments confirm our targets. Such a stepwise strategy may be advantageous for therapeutic target discovery in other disease contexts.

Interaction of Nerve Growth Factor ß with Adiponectin and SPARC Oppositely Modulates its Biological Activity.

Both adiponectin and secreted protein, acidic and rich in cysteine (SPARC) inhibit platelet-derived growth factor-BB (PDGF-BB)-induced and basic fibroblast growth factor (FGF2)-induced angiogenic activities through direct and indirect interactions. Although SPARC enhances nerve growth factor (NGF)-dependent neurogenesis, the physical interaction of NGFß with adiponectin and SPARC remains obscure. Therefore, we first examined their intermolecular interaction by surface plasmon resonance method. NGFß bound to immobilized SPARC with the binding constant of 59.4 nM, comparable with that of PDGF-BB (24.5 nM) but far less than that of FGF2 (14.4 µM). NGFß bound to immobilized full length adiponectin with the binding constant of 103 nM, slightly higher than those of PDGF-BB (24.3 nM) and FGF2 (80.2 nM), respectively. Treatment of PC12 cells with SPARC did not cause mitogen-activated protein kinase (MAPK) activation and neurite outgrowth. However, simultaneous addition of SPARC with NGFß enhanced NGFß-induced MAPK phosphorylation and neurite outgrowth. Treatment of the cells with adiponectin increased AMP-activated protein kinase (AMPK) phosphorylation but failed to induce neurite outgrowth. Simultaneous treatment with NGFß and adiponectin significantly reduced cell size and the number of cells with neurite, even after silencing the adiponectin receptors by their siRNA. These results indicate that NGFß directly interacts with adiponectin and SPARC, whereas these interactions oppositely regulate NGFß functions.

Intracerebroventricular administration of sulphated cholecystokinin octapeptide induces anxiety-like behaviour in goldfish.

Sulphated cholecystokinin octapeptide (CCK-8s) is involved in feeding regulation as an anorexigenic neuropeptide in vertebrates. In rodents, i.c.v. administration of CCK-8s has been shown to affect not only feeding behaviour, but also psychomotor activity. However, there is still no information available concerning the psychophysiological effects of CCK-8s in goldfish. Therefore, in the present study, we examined the effect of synthetic goldfish (gf) CCK-8s on psychomotor activity in this species. Intracerebroventricular administration of gfCCK-8s at 0.1, 0.5 and 2.5 pmol g-1 body weight (BW) did not affect swimming distance (locomotor activity). Because goldfish prefer the lower to the upper area of a tank, we used this as a preference test (upper/lower test) to assess anxiety-like behaviour. Intracerebroventricular administration of gfCCK-8s at 2.5 pmol g-1 BW shortened the time spent in the upper area. The action of gfCCK-8s mimicked that of FG-7142 (the central-type benzodiazepine receptor inverse agonist, an anxiogenic agent) at 5 and 10 pmol g-1 BW. The anxiogenic-like effect of gfCCK-8s was abolished by treatment with the CCK receptor antagonist proglumide at 50 pmol g-1 BW. We also investigated the localisation of CCK/gastrin-like immunoreactivity in the goldfish brain. CCK/gastrin-like immunoreactivity was observed in the anxiety-related regions (the nucleus habenularis and the interpeduncular nucleus). These data indicate that gfCCK-8s potently affects psychomotor activity in goldfish, and exerts an anxiogenic-like effect via the CCK receptor-signalling pathway.

New CETP inhibitor K-312 reduces PCSK9 expression: a potential effect on LDL cholesterol metabolism.

Despite significant reduction of cardiovascular events by statin treatment, substantial residual risk persists, driving emerging needs for the development of new therapies. We identified a novel cholesteryl ester transfer protein (CETP) inhibitor, K-312, that raises HDL and lowers LDL cholesterol levels in animals. K-312 also suppresses hepatocyte expression of proprotein convertase subtilisin/kexin 9 (PCSK9), a molecule that increases LDL cholesterol. We explored the underlying mechanism for the reduction of PCSK9 expression by K-312. K-312 inhibited in vitro human plasma CETP activity (IC50; 0.06 µM). Administration of K-312 to cholesterol-fed New Zealand White rabbits for 18 wk raised HDL cholesterol, decreased LDL cholesterol, and attenuated aortic atherosclerosis. Our search for additional beneficial characteristics of this compound revealed that K-312 decreases PCSK9 expression in human primary hepatocytes and in the human hepatoma cell line HepG2. siRNA silencing of CETP in HepG2 did not compromise the suppression of PCSK9 by K-312, suggesting a mechanism independent of CETP. In HepG2 cells, K-312 treatment decreased the active forms of sterol regulatory element-binding proteins (SREBP-1 and -2) that regulate promoter activity of PCSK9. Chromatin immunoprecipitation assays demonstrated that K-312 decreased the occupancy of SREBP-1 and SREBP-2 on the sterol regulatory element of the PCSK9 promoter. PCSK9 protein levels decreased by K-312 treatment in the circulating blood of cholesterol-fed rabbits, as determined by two independent mass spectrometry approaches, including the recently developed, highly sensitive parallel reaction monitoring method. New CETP inhibitor K-312 decreases LDL cholesterol and PCSK9 levels, serving as a new therapy for dyslipidemia and cardiovascular disease.

Orexin A enhances locomotor activity and induces anxiogenic-like action in the goldfish, Carassius auratus.

Orexin acts as an orexigenic factor for the regulation of appetite and rhythmicity in rodents. In goldfish, intracerebroventricular (ICV) administration of orexin A has been shown to affect not only food intake, but also locomotor activity. However, as there is still no information regarding the effect of orexin A on emotional behavior in goldfish, we investigated the effect of orexin A on psychomotor activity in this species. Intracerebroventricular administration of synthetic orexin A at 2 and 4pmol/g body weight (BW) enhanced locomotor activity, and this enhancement by orexin A at 4pmol/g BW was attenuated by treatment with the orexin receptor 1 antagonist, SB334867, at 10pmol/g BW. Since intact goldfish prefer a black to a white background area, or the lower to the upper area of a tank, we used two types of preference tests (black/white and upper/lower tests) for measuring anxiety-like behavior in goldfish. Intracerebroventricular administration of orexin A at 4pmol/g BW shortened the time spent in the white background area, and increased the time taken to move from the lower to the upper area. This action of orexin A mimicked that of the central-type benzodiazepine receptor inverse agonist, FG-7142 (an anxiogenic agent), at 4pmol/g BW. The anxiogenic-like effect of orexin A was abolished by treatment with SB334867 at 10pmol/g BW. These results indicate that orexin A potently affects psychomotor activity in goldfish.

Direct evidence for pitavastatin induced chromatin structure change in the KLF4 gene in endothelial cells.

Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.

Ovine corticotropin-releasing hormone (oCRH) exerts an anxiogenic-like action in the goldfish, Carassius auratus.

Corticotropin-releasing hormone (CRH) is a member of the hypothalamic neuropeptide family that includes urocortins, urotensin I and sauvagine in vertebrates. CRH and urocortin act as anorexigenic factors for satiety regulation in rodents. In a goldfish model, intracerebroventricular (ICV) administration of ovine CRH (oCRH) affects not only food intake, but also locomotor activity. However, there is no information regarding the psychophysiological roles of CRH in goldfish. Therefore, we investigated the effect of oCRH on psychomotor activity in this species. ICV administration of synthetic oCRH at 20 pmol/g body weight (BW) enhanced locomotor activity. Since intact goldfish prefer the lower to the upper area of a tank, we developed a method for measuring the time taken for fish to move from the lower to the upper area. ICV administration of oCRH at 20 pmol/g BW and the central-type benzodiazepine receptor inverse agonist FG-7142 (an anxiogenic agent) at 1-4 pmol/g BW both increased the time taken to move from the lower to the upper area. This anxiogenic-like effect of oCRH was abolished by the CRH receptor antagonist α-helical CRH(9-41) (100 pmol/g BW). These results indicate that CRH can potently affect locomotor and psychomotor activities in goldfish.

A glass fiber sheet-based electroosmotic lateral flow immunoassay for point-of-care testing.

We have developed a quantitative immunoassay chip targeting point-of-care testing. To implement a lateral flow immunoassay, a glass fiber sheet was chosen as the material for the microfluidic channel in which the negative charge on the fiber surfaces efficiently generates the electroosmotic flow (EOF). The EOF, in turn, allows controllable bound/free separation of antigen/antibody interactions on the chip and enables precise determination of the antigen concentration. In addition, the defined size of the porous matrix was suitable for the filtration of undesired large particles. We confirmed the linear relationship between the concentration of analyte and the resulting fluorescence intensity from the immunoassay of two model analytes, C-reactive protein (CRP) and insulin, demonstrating that analyte concentration was quantitatively determined within the developed chip in 20 min. The limits of detection were 8.5 ng mL(-1) and 17 ng mL(-1) for CRP and insulin, respectively.

Effects of intramuscular administration of tiletamine-zolazepam with and without sedative pretreatment on plasma and serum biochemical values and glucose tolerance test results in Japanese black bears (Ursus thibetanus japonicus).

OBJECTIVE: To establish a safe anesthetic protocol with little effect on blood biochemical values and IV glucose tolerance test (IVGTT) results in Japanese black bears (Ursus thibetanus japonicus). ANIMALS: 16 captive female Japanese black bears (5 to 17 years of age). PROCEDURES: Bears were randomly assigned to 4 treatment groups (4 bears/group) in which various treatment combinations were administered via blow dart: tiletamine HCl and zolazepam HCl (9 mg/kg) alone (TZ), TZ (6 mg/kg) and acepromazine maleate (0.1 mg/kg), TZ (6 mg/kg) and butorphanol tartrate (0.3 mg/kg), or TZ (3 mg/kg) and medetomidine HCl (40 µg/kg). Glucose injection for the IVGTT was started 130 minutes after TZ administration. Blood samples were obtained before, at, and intermittently after glucose injection for measurement of biochemical variables as well as plasma glucose and serum insulin concentrations during the IVGTT. Rectal temperature, pulse rate, and respiratory rate were assessed every 15 minutes during the experiment. RESULTS: Induction and maintenance of anesthesia were safely achieved with little adverse effect on cardiopulmonary function when each of the 4 anesthetic regimens was used, although mild hypothermia was induced. No difference was evident between treatment groups in blood biochemical values. Blood glucose and insulin concentration profiles during the IVGTT were similar among the bears given TZ, with or without acepromazine or butorphanol, but hyperglycemia and hypoinsulinemia developed in bears given TZ with medetomidine. CONCLUSIONS AND CLINICAL RELEVANCE: All 4 anesthetic regimens yielded chemical restraint without affecting clinical and biochemical values in bears, but medetomidine appeared to affect IVGTT results. For this reason, medetomidine should not be used when anesthetizing bears for IVGTTs.

Changes in blood glucose and insulin responses to intravenous glucose tolerance tests and blood biochemical values in adult female Japanese black bears (Ursus thibetanus japonicus).

The metabolic mechanisms to circannual changes in body mass of bears have yet to be elucidated. We hypothesized that the Japanese black bear (Ursus thibetanus japonicus) has a metabolic mechanism that efficiently converts carbohydrates into body fat by altering insulin sensitivity during the hyperphagic stage before hibernation. To test this hypothesis, we investigated the changes in blood biochemical values and glucose and insulin responses to intravenous glucose tolerance tests (IVGTT) during the active season (August, early and late November). Four, adult, female bears (5-17 years old) were anesthetized with 6 mg/kg TZ (tiletamine HCl and zolazepam HCl) in combination with 0.1 mg/kg acepromazine maleate. The bears were injected intravenously with glucose (0.5 g/kg of body mass), and blood samples were obtained before, at, and intermittently after glucose injection. The basal triglycerides concentration decreased significantly with increase in body mass from August to November. Basal levels of plasma glucose and serum insulin concentrations were not significantly different among groups. The results of IVGTT demonstrated the increased peripheral insulin sensitivity and glucose tolerance in early November. In contrast, peripheral insulin resistance was indicated by the exaggerated insulin response in late November. Our findings suggest that bears shift their glucose and lipid metabolism from the stage of normal activity to the hyperphagic stage in which they show lipogenic-predominant metabolism and accelerate glucose uptake by increasing the peripheral insulin sensitivity.

Reliable enzyme-linked immunosorbent assay for the determination of coconut milk proteins in processed foods.

This study was designed to develop a novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of coconut milk proteins in processed foods. The developed sandwich ELISA was able to detect coconut milk proteins from various coconut milk products and did not show any cross-reactivity with 41 of 42 kinds of popularly used food ingredients, thus reflecting great specificity for coconut milk proteins. In addition, the established ELISA is highly sensitive and allowed the detection of 0.31 µg/g of coconut milk protein in complex food matrices. This proposed assay could serve as a useful tool for the detection of the presence of hidden coconut milk proteins in processed foods.

Seasonality and fasting effect in raccoon dog Nyctereutes procyonoides serum leptin levels determined by canine leptin-specific enzyme-linked immunosorbent assay.

Leptin is an adipocyte-derived peptide hormone that acts on the brain and regulates food intake and energy balance. Several previous reports have suggested that overwintering raccoon dogs Nyctereutes procyonoides are able to control their adiposity efficiently, but the contribution of leptin to weight regulation in these animals remains unclear. To study the seasonality of overwintering raccoon dogs as well as the effects of fasting on them, serum leptin levels were investigated using a newly established canine leptin-specific enzyme-linked immunosorbent assay (ELISA) kit. Of the nine animals studied, five were fed and four were fasted (deprived of food for 2 months in winter). Blood samples and body fat weights were monitored once a month throughout the experimental period (July 2007-March 2008). Leptin concentrations obtained by ELISA were significantly higher than and had a positive correlation with those obtained by previously used multispecies radioimmunoassay (RIA) kits. Moreover, ELISA showed a clearer correlation between the body fat weight and leptin levels compared with RIA, suggesting the efficacy of canine leptin-specific ELISA kit for leptin estimation in raccoon dogs. Autumnal fattening was observed in both groups of animals, but the wintertime loss of adipose tissue was more obvious in the fasted group. Serum leptin concentrations determined by ELISA showed seasonal changes without significant differences between the fed and fasted animals. Therefore, high levels of leptin may be responsible for the suppression of feeding behavior in raccoon dogs before winter.

Determination of walnut protein in processed foods by enzyme-linked immunosorbent assay: interlaboratory study.

Because food allergens from tree nuts, including walnuts, are a frequent cause of adverse food reactions for allergic patients, the labeling of foods containing ingredients derived from tree nuts is required in numerous countries. According to Japanese regulations, the labeling of food products containing walnuts is recommended. To ensure proper labeling, a novel sandwich ELISA kit for the determination of walnut protein in processed foods (Walnut Protein [2S-Albumin] Kit; Morinaga Institute of Biological Science, Inc.; "walnut kit") has been developed. We prepared seven types of incurred samples (model processed foods: biscuits, bread, sponge cake, orange juice, jelly, chicken meatballs, and rice gruel) containing 10 microg walnut soluble protein/g of food for use in interlaboratory evaluations of the walnut kit. The walnut kit displayed sufficient reproducibility relative standard deviations (interlaboratory precision: 5.8-9.9% RSDR) and a high level of recovery (81-119%) for all the incurred samples. All the repeatability relative standard deviation (RSDr) values for the incurred samples that were examined were less than 6.0%. The results of this interlaboratory evaluation suggested that the walnut kit could be used as a precise and reliable tool for determination of walnut protein in processed foods.

A selective ACAT-1 inhibitor, K-604, stimulates collagen production in cultured smooth muscle cells and alters plaque phenotype in apolipoprotein E-knockout mice.

Acyl-coenzyme A:cholesterol O-acyltransferase-1 (ACAT-1) plays an essential role in macrophage foam cell formation and progression of atherosclerosis. We developed a potent and selective ACAT-1 inhibitor, K-604, and tested its effects in apoE-knockout mice. Administration of K-604 to 8-week-old apoE-knockout mice for 12 weeks at a dose of 60 mg/kg/day significantly reduced macrophage-positive area and increased collagen-positive area in atherosclerotic plaques in the aorta without affecting plasma cholesterol levels or lesion areas, indicating direct plaque-modulating effects of K-604 on vascular walls independent of plasma cholesterol levels. Pactimibe, a nonselective inhibitor of ACAT-1 and ACAT-2, reduced plasma cholesterol levels but did not affect macrophage- or collagen-positive areas. The size of macrophages and cholesteryl ester contents in the aorta were reduced by K-604. Exposure of cultured human aortic smooth muscle cells to K-604 resulted in increased procollagen type 1 contents in the culture supernatant and increased procollagen type 1 mRNA levels. Procollagen production was unaffected by pactimibe even at a concentration that inhibited cholesterol esterification to the basal level. Thus, the plaque-modulating effects of K-604 can be explained by stimulation of procollagen production independent of ACAT inhibition in addition to potent inhibition of macrophage ACAT-1.

Plasma leptin concentration in dogs with diabetes mellitus.

The plasma leptin concentration was evaluated in dogs with diabetes mellitus. Twenty normal and sixteen diabetic dogs were divided into nonobese and obese groups based on body condition score, respectively. The obese normal dogs had significantly higher plasma leptin concentrations than the nonobese normal dogs, whereas there was no significant difference between the nonobese and obese diabetic dogs. In addition, the plasma leptin concentration in the obese diabetic dogs was significantly lower than that in the obese normal dogs. In conclusion, the plasma leptin concentrations in the diabetic dogs were affected by factors other than adiposity.

A novel sandwich enzyme-linked immunosorbent assay for feline insulin.

A novel sandwich enzyme-linked immunosorbent assay (ELISA) was established to determine the serum insulin concentrations in domestic cats. By using a solid-phase mouse anti-bovine insulin monoclonal antibody and a peroxidase-conjugated guinea pig anti-rat insulin polyclonal antibody, feline serum insulin concentrations in the range of 0.1 to 3.6 ng/ml could be measured. The intraassay CV and interassay CV were less than 6% and less than 10%, respectively. The present insulin assay will strongly help studies on feline diabetes mellitus.

A reliable enzyme linked immunosorbent assay for the determination of bovine and porcine gelatin in processed foods.

Since gelatin-containing foods pose a risk for eliciting allergic reactions in sensitized individuals, a novel sandwich enzyme linked-immunosorbent assay (ELISA) for the detection and quantification of bovine and porcine gelatin in processed foods was developed. Rabbits and goats were immunized with bovine gelatin, and three antisera (pAb1 and pAb2 from rabbits, and pAb3 from goats) were obtained. We established a sandwich ELISA method based on a combination of these antibodies. In this study, two sandwich ELISA methods, rabbit pAb2-pAb1 and goat pAb3-pAb3, were evaluated for sensitivity, specificity, cross-reactivity, and applicability. Both ELISA methods were highly specific for bovine and porcine gelatin but had little reactivity with fish gelatin. The detection and quantification limits for porcine gelatin were found to be 0.78 ng/mL and 1.56 ng/mL, respectively. The established sandwich ELISA methods produced no false-positives, except for heated meat products or false negatives when various commercial foods were analyzed for their gelatin content. The rabbit pAb2-pAb1 ELISA cross-reacted with boiled squid, while the goat pAb3-pAb3 ELISA did not. Thus, the proposed goat pAb3-pAb3 ELISA method is a reliable tool for the detection of gelatin contaminants present in processed foods.

Reliable enzyme-linked immunosorbent assay for the determination of walnut proteins in processed foods.

Among food allergens of tree nuts, walnuts are a frequent cause of adverse food reactions in allergic patients. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of walnut soluble proteins in processed foods was developed. The sandwich ELISA is highly specific for walnut soluble proteins. The recovery ranged from 83.4 to 123%, whereas the intra- and interassay coefficients of variation were less than 8.8 and 7.2%, respectively. This study showed that the proposed method is a reliable tool for detection in the presence of hidden walnut proteins in processed foods.

Annual changes in serum leptin concentration in the adult female Japanese black bear (Ursus thibetanus japonicus).

In the present study, assay of the serum leptin concentration of the Japanese black bear (Ursus thibetanus japonicus) was attempted using a canine-leptin-specific sandwich enzyme-linked immunosorbent assay (ELISA). The dose-response curve of the bear serum was linear and parallel to the canine leptin standard curve. In mated and unmated bears, the serum leptin concentration was stable at low levels from May to August or September, gradually increased from September or October, and then remarkably increased in late November. We conclude that this method may be useful for measuring bear serum leptin concentration and that the serum leptin concentration changes annually with a peak in late November.