Due to an increasing incidence of stroke as well as a growing population of elderly individuals, the number of bedridden persons in Japan is increasing. Since their muscle and nervous system for oral functions may be lowered, many of them are suffered with difficulty in swallowing (dysphasia). In cases of severe swallowing disorders, patients may aspirate their food, in which bacteria carried with the food into the airway might cause a critical condition such as pneumonia. Tube feeding is one way to bypass the pharynx, and avoid the aspiration of food. However, tube feeding deprives patients of the pleasure associated with eating. Most patients want to take food through the mouth; thus methods need to be developed that address difficulties in swallowing. Mastication have been understood being a process to crash and mix with saliva for safe swallow; however, in addition to it humans sense the physical and chemical (i.e., taste) properties of food during mastication so that we secure security of the appetite. Swallowing is a reflex during which many muscles are activated in given timings to move food and/or liquid from the oral cavity to the stomach. Since affection system may strongly interpose in the swallowing process, a good appetite may be the most important factor for a smooth swallowing process. Properties required for the food include: clear taste, proper temperature, uniformity, agglutination, and moisture.
A century ago, an amino acid, L-glutamate (Glu), was found to be the important substance for umami (savory) taste of a Japanese soup stock cooked with sea tangle. Since that time, umami seasoning has been used to make foods palatable all over the world. Chemical analysis proved that Glu had been used for savory seasonings around the world, though its taste had been hidden behind the flavors of fat or herbs. Recently, research has shown that Glu affects the chemical senses not only in the oral cavity but also in the gastrointestinal tract, and it modulates the ingestion, digestion and metabolism of proteins. Umami taste, one of the five basic tastes along with sweet, salty, sour and bitter tastes, derived from Japanese cuisine, might be applicable for the nutritional care of elderly people, who are at risk for protein malnutrition even in developed countries.
L-Glutamate and 5'-ribonucleotides are known to elicit a unique taste, “umami,” that is distinct from the tastes of sweet, salt, sour and bitter. Recent progress in molecular biology has identified several umami receptor candidates, such as the heterodimer T1R1/T1R3, and brain-expressed and taste-expressed type 1 and 4 metabotropic glutamate receptors (brain- and taste-mGluR1 and mGluR4). This paper summarizes recent findings on the receptor system for umami taste. Most of the findings support the idea that multiple receptors exist for umami taste, at least in mice. The accumulating evidence indicates that the potential role of the signal mediated by the transduction pathway involving T1R1/T1R3 may be different from that mediated by the pathway involving mGluRs. The former signal occurs mainly in the anterior tongue, and plays a major role in preference behavior, whereas the latter occurs mainly in the posterior tongue, is active in mice lacking T1R3, Gα-gustducin, IP3R3 or TRPM5, and contributes to behavioral discrimination between umami and other taste compounds. In humans, unlike in mice, T1R1/T1R3 acts as an umami-specific receptor that can discriminate between umami and other tastes, and thus account for umami-linked preferences or discrimination.
Saliva performs many biological functions that are instrumental in maintaining oral health. Digestive enzymes, such as alpha-amylase and antimicrobial proteins, such as lysozyme and cystatin, are present at high concentrations in human saliva. It is a readily available atraumatic body fluid, useful in the diagnosis of disease, aging and oral health. Large scale analysis of salivary proteins is important for biomarker discovery. However, the abundance of some salivary proteins and contamination with proteins from food and mouth bacteria obscures identification of medium and low copy salivary proteins. In this review, we summarize current knowledge of the salivary proteome using mass spectrometry based technologies.
Aging is sometimes associated with decreased sensitivity to tastants, i.e., hypogeusia. The loss of taste sense induces not only the decreased quality of life (QOL), but also weight loss or health problems in the elderly. In our recent study, whole saliva secretion including minor salivary secretion, was found to be significantly decreased in the elderly with gustatory impairment, while it was normal in all of the elderly with normal taste thresholds, indicating that hyposalivation is closely related to hypogeusia. Moreover, clinical studies have shown that treatment for hyposalivation alleviates hypogeusia, even that due to the side effects of prescribed drugs or the effects of disease, e.g., nervous disorders or endocrine disorders. Thus, salivation is essential for maintenance of the normal taste function. Many medications for relief of dry mouth, primarily parasympathomimetic drugs, have serious adverse effects such as palpitation, sweating, nausea, diarrhea or dizziness, particularly in the elderly. To circumvent this problem, we use glutamate (umami taste) in an attempt to increase salivary secretion and to alleviate hypogeusia. An umami stimulus might be an effective method for the alleviation of hypogeusia through improvement of hyposalivation or dry mouth without side effects in aged patients. Consequently, attempts should be made to remedy hypogeusia by alleviation of hyposalivation so as to maintain and promote the health of the elderly.
The present study was designed to evaluate the nephroprotective role of methanolic extract of Achyranthes aspera (A. aspera) an important herb in the Indian system of medicine against lead acetate-induced nephrotoxicity in rats. Toxicity was induced in male albino rats (Wistar strain) by administering lead acetate (0.2%) in drinking water for 6 weeks, followed by extract of A. aspera (200 mg/kg body weight). Changes in kidney weights encountered upon lead administration improved after extract with A. aspera. Lead damage to the urine was evident from increase in the activity of γ-glutamyltranspeptidase (γ-GT), Cathespin D, alkaline phosphatase (ALP), acid phosphatase (ACP), β-glucuronidase lactate dehydrogenase (LDH) and N-acetyl-β-D-glucosaminidase (NAG) in urine along with some urinary constituents (urea, uric acid, creatinine, protein and phosphorous). The effects of lead were also studied in kidney (γ-GT, β-glucuronidase, NAG, Cathespin D and LDH) and showed a decline upon extract administration. Increased activities of urinary enzymes were accompanied by increase in the urinary constituents. Treatment with methanolic extract of A. aspera after lead induction completely ameliorated the lead-induced renal damage.
Dedifferentiation is an important biological phenomenon and is understood as a process in which cells develop in a reverse order from a more differentiated to a less differentiated state. In the present study, we observed that after UV treatment, the surviving keratinocytes underwent the reversion from differentiated state to dedifferentiated state, evidenced by the changes from three levels, including phenotype, morphology and function. First, the mature keratinocytes acquired a dedifferentiated phenotype indicated by reexpression of transit-amplifying (TA) cell markers, including CK14 and β1 integrin. Second, the cells experienced morphological changes during the process of dedifferentiation. Cells treated with UV were small and had a high nuclear to cytoplasmic ratio, whereas cells without treatment had well-developed cellular organelles and abundant tonofilaments. Third, after UV treatment, the cells regained strong proliferation capacity. These dedifferentiation-derived stem cells formed colonies with defined edges developed and presented multiple-layer growing profile under three-dimensional culture condition. The skin equivalent (SE) produced with UV-treated keratinocytes also showed a well-organized structure with four stratifications. We also report the signaling pathway involved in the dedifferentiation process. The extracellular signal-regulated kinase (ERK) pathway regulates the phenotype reversion of keratinocytes into progenitor cells. Inhibition of ERK kinase activities with a specific inhibitor (PD98059) substantially blocked phosphorylation of ERK1/2 and human keratinocyte dedifferentiation. These data collectively provide a proof-of-concept that UV treatment of HEKs is capable of inducing a phenotype reversion from an adult differentiated state to an immature-like dedifferentiated state via ERK Mitogen-Activated Protein Kinases (MAPK)-dependent pathway. It may offer the direct evidence for the existence of dedifferentiation and the underlying mechanisms involved in the process, which may bring a new insight for the regenerative medicine.
Shou-Wu-Pian, a herbal remedy formulated from Polygonum multiflorum (PM), has been extensively used for the treatment of hair-loss, constipation and vertigo in Asia, Europe and U.S.A. Although recent reports have indicated severe liver damage occurred after taking Shou-Wu-Pian in humans, studies on the hepatotoxicity of this herbal remedy have been limited, and this organ-specific adverse effect induced by PM intake remains to be confirmed. In this study, hepatotoxicity of hot-water-extracted PM was investigated in vitro and in vivo in mice. After treating primary cultured hepatocytes with PM extracts at 3 final concentrations (0.5, 1 and 5 mg/ml) with or without acetaminophen (10mM) for 24 hr, no cytotoxic effects were observed. In fact, cell viability and lactate dehydrogenase leakage were significantly improved at the highest PM extracts dose compared with controls. Mice orally administered twice daily with the PM extracts (20 or 340 mg extracts/mouse×2) for 10 days indicated no unfavorable effect on the liver function. Only when mice subchronically received the higher dose of the PM extracts (340 mg) before treatment with a single-bolus dose of acetaminophen (500 mg/kg; i.p.), attenuation of acetaminophen-induced hepatotoxicity was significantly established in vivo. The results in the present study contradicted hepatotoxic findings reported in humans; PM extracts do not induce any toxicological effects, at least on the liver, and may in fact elicit useful but limited beneficial effects on the liver in vivo.
Staphylococcus epidermidis (S. epidermidis) often cause sepsis and related diseases by transfusion of contaminated platelet concentrates (PCs). The proliferation process of this bacterium in PCs has been unclear, thus, bio-imaging system was applied for analyzing the dynamics of S. epidermidis in PCs. S. epidermidis were spiked into PCs or Luria Bertani (LB) broth. These samples were collected at each sampling time during incubation (up to 7 days), and colony-forming-units were counted. Bacterial number and their size distribution in each sample were also determined with a new bio-imaging system. The morphological characters of S. epidermidis growing in the samples were observed precisely by scanning electron microscopy (SEM). The numbers of S. epidermidis were stable for 48 hr after the spiking as lag-phase, while the bio-imaging analysis also showed that aggregates proliferated during “lag-phase.” The aggregates were also observed in LB media, however, their sizes were much smaller than those in PCs. SEM suggested that the aggregates were micro-colonies (MCs) of staphylococcal cells and cores of the MCs are composed with platelets (PLTs). Out results suggested that S. epidermidis formed floating MCs in PCs during “lag-phase.” Therefore, the term of lag phase of S. epidermidis in PCs should be called as “pseudo-lag phase.” The initial processes of forming MCs in PCs are thought to be an interaction between bacterial cells and PLTs. Floating MCs would be the source of biofilms on the inside of PC storage bags. New information obtained in this study would be useful for understanding the dynamics of growing bacteria in PCs.
Aqueous, ethanolic, hydroethanolic and methanolic extracts of Albizia sp., Cola nitida, Terminalia superba (T. superba), Cola odorata and Harungana madagascarensis were screened for phytochemical constituents. The evaluations of the antioxidant potential and α-amylase inhibitory activity of these extracts were also carried out. Tests for tannins and phenols were positive in all extracts, with the highest total phenol content observed in the ethanolic extract of T. superba. This extract, as well as the aqueous and hydroethanolic extracts of the same plant had the highest antioxidant properties as determined by the ferric reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods. For all the plants tested, at least one extract inhibited the activity of α-amylase. The most effective was the hydroethanolic extract of T. superba. The presence of active phytochemical substances with antioxidant properties may account for the above mentioned properties, and provide substantial basis for the use of these plants in ethno-medicine for the treatment of diabetes.
Mi Jung Oh, Gyu Young Chae, Kyong Hoon Ahn, So Jung Chu, Jong Min Choi, Jung Eun Ji, Seok Kyun Kim, Dong Hyuk Kang, Sung Hyun Kim, Jong Hoon Won, Yang Hui Park, Kwang Mook Jung, Sung Yun Jung, Dae Kyong Kim
Cadmium (Cd) is an environmental pollutant that produces numerous toxic effects in humans. Previous reports have demonstrated that Cd induces neurodegeneration and other diseases. To study the mechanism of Cd-induced neurodegeneration, SH-SY5Y cells were exposed to Cd. Cd-induced ceramide production was observed in the cells in a time- and dose-dependent manner. Ceramide is a well known lipid second messenger. Previous reports have shown that accumulation of ceramide, triggered by oxidative stress or anticancer drugs, induces apoptosis. To identify proteins involved in cellular responses to Cd or ceramide, we performed comparative proteome analysis using high-resolution two-dimensional gel electrophoresis (2-DE). Four different proteins were identified by combination of mass spectrometry and peptide fingerprinting. Among them, the level of oxidized peroxiredoxin-3 (Prx-3) simultaneously increased in Cd- and ceramide-exposed cells. Furthermore, we observed Cd- or ceramide-induced generation of reactive oxygen species (ROS), leading to SH-SY5Y cell apoptosis. Pretreatment with Trolox helped scavenge Cd- or ceramide-induced ROS and prevented apoptosis, suggesting that Cd- or ceramide-induced cell death can be attributed to ROS production. Here, we elucidate the link between Cd-induced apoptosis and ROS scavenger system in SH-SY5Y cells.
Cancer treatment in Japan is considered to be very progressive in the use of antineoplastic agents. Pharmacists are required to compound more antineoplastic preparations recently and thus are at risk for exposure to antineoplastic agents. In Japan, healthcare professionals have recognized the need for protection, but there have been few reports of environmental contamination or occupational exposure to antineoplastic agents. In the present study, urine samples over 24 hr from compounding pharmacists and wipe samples from the compounding room were collected to analyze levels of cyclophosphamide (CP). CP was detected in urine samples from all 4 pharmacists (mean=165.3 ng/24 hr) and for all wipe samples of the compounding room. From the results of these tests, the standard operating procedure (SOP) of the pharmacy was revised as a protective measure. The tests were then repeated and CP was detected again in the urine of all 4 compounding pharmacists, though the mean CP level was reduced from 165.3 to 47.4 ng/24 hr after the revision of the SOP. Although there was no correlation between the amount of CP compounded and the CP levels in urine initially, the 2 values were significantly correlated after the revision of the SOP (R2=0.87).
The ability of pilot-scale vertical subsurface flow constructed wetlands (VSCWs) to treat two types of high-strength [400 mg/l of COD or 80 mg/l total nitrogen (TN)] simulated wastewater under greenhouse conditions was studied during an 8-month period. Eighteen CWs units were used: six units were planted with Acorus calamus (A. calamus), another six units were planted with Lythrum salicaria (L. salicaria) and six units were unplanted. Each set of units was operated at hydraulic loading rates of 40 l/d. The treatment performance displayed that average removal rates of chemical oxygen demand (COD), TN, total phosphorous (TP) and total organic carbon (TOC) were 44-66%, 35-63%, 47-76% and 22-40%, respectively. Both species grew well under any high loading treatment. We noted that plant uptake and storage were both important factors responsible for nitrogen removal during the study period. Both planted wetlands improved pollutants removal compared with the unplanted control wetland. L. salicaria produced more shoots and biomass than the A. calamus, which can remove more P nutrients than A. calamus especially for high nitrogen (N) loading treatment. However, A. calamus was always more efficient species that improved nitrogen nutrients plant uptake because of their longer growth period. Our findings suggested that the vertical subsurface flow constructed wetlands could well treat the high-strength wastewater in greenhouse condition. If good capacity of all nutrients removal is considered, A. calamus is more appropriate than L. salicaria particularly under high N loading in the influent.
The present study investigated issues regarding cytotoxicity tests using mouse hepatocytes in primary culture. The results indicated that the cytotoxicity of acetaminophen (APAP) markedly varied depending on culture period for up to 5 days. P450 isoforms involved in the activation of APAP, such as CYP1A2, CYP2E1, and CYP3A, showed a marked decrease in expressions of respective mRNA. Their expression levels were a few percent within 24 hr after the start of the culture, and remained low throughout. Meanwhile, the mRNA expression of enzymes involved in the detoxification of APAP, UGT1A6 and SULT1A1, declined moderately and then recovered to approximately 40% of the in vivo level later in the culture. Treatment with APAP at non-cytotoxic concentrations increased expressions of CYP1A2, CYP2E1, CYP3A11 and CYP3A41 mRNA, while it decreased that of SULT1A1 mRNA. Using the lactate dehydrogenase (LDH) assay, the cells treated with 25-mM APAP for 24-48 or 48-72 hr showed higher cytotoxicity values than those for 72-96 or 96-120 hr, but the values after 50-mM treatment were completely inverted. By employing the methylthiazolyl tetrazolium (MTT) assay, culture period-dependent cytotoxicity was also observed on 25-mM APAP treatment, but this was not clear at 50 mM. Lower LDH or higher MTT activities than the control were observed in cells treated with lower concentrations of APAP, becoming the more prominent the longer the culture period. The observations suggested that, since the expression of metabolizing enzymes alters markedly during the primary culture and the range of cytotoxic concentrations of a test compound varies depending on the culture period, it is necessary to take account of these fluctuating parameters when assessing the cytotoxic character of a compound.
In Japan, crude drugs that are used as ingredients of kampo formulae are mainly imported from China. In China, cypermethrin and fenvalerate, which are pyrethroid pesticides, are detected frequently in food exported to Japan. In fact, cypermethrin and fenvalerate have been detected in crude drugs distributed in Japan. There is concern that cypermethrin and fenvalerate will move from crude drugs to kampo products. Cypermethrin and fenvalerate in kampo products are controlled by a self-imposed residual pesticide limit set by the Japan Kampo Medicine Manufacturers Association. Several methods to analyze cypermethrin and fenvalerate in kampo products have been reported, but they are time-consuming, require expensive column clean-up, and use large amounts of organic solvents. In this study, a simple, rapid, and inexpensive sample preparation method was developed to determine cypermethrin and fenvalerate levels in kampo products by gas chromatography (GC)-mass spectrometry (MS) with negative chemical ionization (NCI). Twenty-two samples of kampo products were analyzed according to the proposed method. No samples were contaminated by cypermethrin and fenvalerate levels greater than the detection limit.
Vibrio parahaemolyticus (V. parahaemolyticus), one of the most important foodborne pathogens in many maritime Asian countries, is frequently associated with the consumption of seafood. Thirty eight strains of V. parahaemolyticus were isolated from seafood in Hebei province of China. Resistance to 13 antibiotics was determined using broth microdilution methods. These strains were typed by pulsed-field gel electrophoresis (PFGE) technique with short pre-processing following SfiI digestion and a typing scheme was generated. The 38 strains were grouped into 5 types with 71% pattern similarity. All the type E were isolated from Shijiazhuang, Baoding and Langfang and simultaneously resistant to ampicillin, sulfisoxazole, streptonigrin and vancomycin, suggesting a clonal relationship between these strains. The data of antimicrobial susceptibility test and PFGE profiles in this study showed a good correlation among antimicrobial susceptibility test, PFGE profiles and geographic distribution.
Oral fluoroquinolones are widely known to form chelate complexes with metal-containing drugs, resulting in inhibition of their intestinal absorption. However, for intestinal sterilization, the concomitant regimen may be a selective and effective strategy due to decreased absorption of fluoroquinolones result in the retainment of antibiotics at the intestine if the mixture still perpetuated antibacterial activity. Therefore, to clarify whether the mixture of fluoroquinolones and sucralfate affects their antibacterial activity or not, we conducted in vitro study. According from the checkerboard study using a microdilution method with Mueller-Hinton broth, the antibacterial activity of these fluoroquinolones-sucralfate mixtures equaled to the parent fluoroquinolones even in the presence of sucralfate at the molar ratio of [sucralfate: fluoroquinolone] was less than 166, and the minimal inhibitory concentrations for clinical isolated Escherichia coli and Pseudomonas aeruginosa strains were independent of the existence of sucralfate. These data imply that the chelated forms of each fluoroquinolone retain antibacterial activity even in the presence of the recommended therapeutic doses of sucralfate in clinical practice.
Nematodes are highly abundant organisms found in soil or sedimentary habitats. The free-living nematode Caenorhabditis elegans (C. elegans) has been used as an excellent model for monitoring ecotoxicity in soil. We have previously demonstrated that the lifespan of C. elegans can be used as an endpoint for detecting the ecotoxicity of heavy metals and detergents, and have developed a novel ecotoxicity assay based on their shortened lifespan. Herein we used a daf-16(mu86) mutant CF1038 strain, which has a deficient transcription factor DAF-16 regulating a variety of genes involved in longevity and stress response, for ecotoxicity assays. We carefully examined the effects on reproduction, larval growth, and lifespan in the short-lived CF1038 strain and wild-type N2 strain in the presence of a perfluoro organic compound (pentadecafluorooctanoic acid) and an organophosphate insecticide (dichlorvos) in addition to heavy metals (CuSO4 and CdCl2) and detergents (sodium dodecyl sulfate and a commercially available household detergent). Unexpectedly, both strains exhibited comparable reductions in these endpoints including lifespan by exposure to these ecotoxicants, indicating that DAF-16 does not largely contribute to tolerance to these agents. By virtue of a shorter assay period, the lifespan-based assay using the daf-16 mutant can be useful for assessing the ecotoxicity of a variety of ecotoxic chemicals.
The hypoglycemic activity of Momordica charantia L. (Cucurbitaceae) (MC) was investigated in KK-Ay mice, an animal model of type 2 diabetes. The water extract of the fruit of MC (100 mg/kg) reduced the blood glucose of KK-Ay mice 2 and 4 hr after oral administration (p<0.05). In addition, the muscle adenosine monophosphate-activated protein kinase (AMPK) was significantly activated in the orally MC-treated mice when compared to that of the controls (p<0.01). These results suggest that the antidiabetic effect of MC is derived, at least in part, from an activation of AMPK in the muscle.
A 70% methanol extract from red ginseng (steamed and dried roots of Panax ginseng C. A. Meyer, a kind of Ginseng Radix) has been shown to have various actions on physiological functions. We investigated whether the ginseng extract (Ext.) affected the mRNA expression of cytochrome P450 (CYP) CYP1A1, 2B1, 2C11, 2D1, 3A1, and 3A2 in rat primary hepatocytes. After treatment with ginseng extract, the levels of CYP3A1 and 1A1 mRNA were significantly increased compared with those of the control. The increased protein levels of CYP3A1 were also observed after treatment with Ext. The mRNA levels of other examined CYPs exhibited little change. The mRNA levels of the pregnane X receptor, but not the constitutive androstane receptor, both transcription factors for CYPs, also significantly increased after treatment with ginseng extract. These results raise the possibility that ginseng Ext. promotes xenobiotic metabolism via an increase in CYP3A1 and 1A1 expression.
Mesoderm-specific transcript (Mest) is an imprinted gene that is predominantly expressed in the embryonic and extraembryonic mesoderm, and its gene expression is the marker of the size of adipocytes. To characterize the expression effect of Mest gene products, we transfected mouse Mest into the 3T3-L1 cell line, which is a mouse preadipocyte cell line. The proliferation of the transfected 3T3-L1 cells was assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and cell growth was assessed by counting the nuclei stained by Hoechst 33258. The 3T3-L1 cells transfected with recombinant Mest cDNA acquired the increased cell growth ability. Therefore, the gene products of Mest could be related to not only adipocyte expansion and adiposity but also mesoderm formation by enhancing the cell proliferation.
The persistence of DNA after the cell death causes a major issue in aspects of medical or biological studies. The signal from viable bacterial cells cannot be distinguished from the dead cells in the conventional DNA-based detection methods. In the present study, the loop-mediated isothermal amplification (LAMP) method combined with the ethidium monoazide (EMA) treatment was applied for specific detection of viable, but not dead, Salmonella cells. For this method (EMA-LAMP), we designed a series of primers, which recognize six distinct sequences of the target invA gene conserved in Salmonella. The invA gene of the viable cells was remarkably amplified within 1 hr when as small amounts as 100 fg of DNA was subjected to EMA-LAMP. Because EMA selectively penetrated into the dead cells and bound covalently to DNA, the gene of the dead cells could not be amplified. This study offers a novel DNA-based method to distinguish the viable bacterial cells from the dead cells.
With the aim of developing a quality control system for insulin formulations in the pharmaceutical industry, we tested the feasibility of chromatographic methods, including reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE), as tools for monitoring the conformational changes in insulin. Insulin samples were dissolved in 0.01 M HCl and incubated at 60°C for 24, 48, and 96 hr to induce aggregation and fibrillation, which were confirmed by studying their circular dichroism spectra and by assaying a fluorescent indicator of β-sheet structure in aggregate proteins [thioflavine T (ThT)]; the samples were also analyzed by RP-HPLC and CE. In RP-HPLC and CE, the distinct changes in the elution patterns, as reflected by the insulin peaks, revealed the process of conformational change in insulin. With respect to the cytotoxicity of these insulin samples against rat pheochromocytoma (PC12) cells, a significant increase in lactate dehydrogenase activity was observed in the culture medium containing insulin aggregates (50 μM) as compared to the medium containing native insulin. These results suggest that the cytotoxicity of insulin in terms of the conformational changes in its structure can be rapidly assessed by using RP-HPLC and CE.
Estrogen deficiency caused by ovariectomy (OVX) induces bone loss and increased B-lymphopoiesis in bone marrow. In OVX mice, the production of prostaglandin E (PGE) and the expression of receptor activator of nuclear factor-κB ligand (RANKL) were elevated in osteoblasts, and the cell adhesion of B cells induced RANKL expression in osteoblasts. However, the roles of PGE in RANKL expression and bone resorption are not clear. To examine the relationship between B-lymphopoiesis and PGE production by osteoblasts, B cells were purified from bone marrow, fixed, and co-cultured with mouse osteoblasts. Most of the fixed B cells adhered to cell surface of osteoblasts, and the cell-cell interaction markedly elevated the expression of cyclooxygenase (COX)-2 and membrane-bound PGE synthase (mPGES)-1 mRNAs, and PGE2 production in osteoblasts. Adding B cells also induced the expression of RANKL mRNA in osteoblasts, and the RANKL expression was suppressed by indomethacin, COX-2 inhibitor (NS398) and selective antagonist for PGE receptor EP4, suggesting that PGE production and EP4 signals are involved in RANKL-dependent bone resorption induced by cell-cell contact between B cells and osteoblasts. Therefore, the increased B-lymphopoiesis and PGE production by osteoblasts may contribute to the pathogenesis of osteoporosis due to estrogen deficiency.
In order to confirm the mechanism how postprandial elevation of triacylglycerol is suppressed by resistant maltodextrin (Fibersol-2), we conducted the following experiments. Firstly, Rats were fed a high-fat diet with resistant maltodextrin at 0% (control), 2.5% or 5% for 5 weeks to determine the lipid amount excreted in the feces of the last three days. The total lipid and triacylglycerol excreted in rat feces were significantly increased in a dose-dependent manner by the ingestion of resistant maltodextrin. Secondly, 10 healthy adult subjects were administrated a beverage containing 5 g resistant maltodextrin or placebo for 10 days, and crossed over after an 11-day washout. Total lipid excreted in feces was determined by collecting all the feces for the last three days. Fecal weight and fecal lipid amount of the subjects increased significantly with the ingestion of resistant maltodextrin compared to the placebo ingestion. Thirdly, oil emulsion prepared with resistant maltodextrin was assessed for its hydrolysis rate by lipase. The hydrolysis rate of lipid by lipase was not affected by resistant maltodextrin. Lastly, micelle emulsion was prepared with or without resistant maltodextrin, and their stability was compared. Resistant maltodextrin inhibited the decomposition of micelles and stabilized micellar structure. From these results, it was suggested that resistant maltodextrin suppresses lipid absorption and promotes the excretion of lipid into feces by delaying the release of fatty acids from micelles in the lipid absorption process. No inhibitory effect on lipase activity was observed by resistant maltodextrin.
The effect of four ortho-quinoid polycyclic aromatic hydrocarbons (PAHs) and seven para-quinoid PAHs on the viability of A549 cells were examined. The ortho-quinoid PAHs [1,2-naphthoquinone (1,2-NQ), 9,10-phenanthrenquinone (9,10-PQ), 5,6-chrysenequinone (5,6-CQ), and benzo[c]phenanthrene-5,6-quinone (B[c]P-5,6-Q)] overproduced hydrogen peroxide (H2O2) without being consumed themselves. These ortho-quinoid had strong cytotoxic effects except for 1,2-NQ, since of its tendency to covalently bind to thiol groups. The cytotoxicity appears to be due to the overproduction of H2O2 by ortho-quinoid PAHs in a redox cycle coupled with the consumption of thiol group. In contrast, the para-quinoid PAHs were not as strong cytotoxic and did not produce H2O2.
Listeria monocytogenes (L. monocytogenes) is a Gram-positive, intracellular bacterium that can cause severe infections in humans. Contamination by L. monocytogenes in foods represents a potential public health problem. Here we describe a rapid method for the detection of L. monocytogenes in milk by flow cytometry based on microfluidics (on-chip flow cytometry). This rapid identification of L. monocytogenes is based on fluorescence in situ hybridization (FISH) with a Cy5-labeled rRNA-targeting oligonucleotide probe. FISH experiments were successfully analyzed using a commercially available on-chip flow cytometry. FISH results with bacterial cultures indicated that the L. monocytogenes probe RL-2 was hybridized with 4 major strains of L. monocytogenes (serotype 1/2a, 1/2b, 1/2c and 4b) but not with other Listeria strains or milk spoilage bacteria. Specific FISH detection of L. monocytogenes was accomplished using the probe RL-2 when the milk contained a mixture of other bacterial species. The positive identification of L. monocytogenes in milk was completed within 5 hr (milk clearing: 40 min, hybridization: 3.5 hr, on-chip analysis: 30 min). The method presented in this study allows the specific and rapid identification of L. monocytogenes in milk.