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T cells bearing I³I´ antigen receptors have been investigated as potential treatments for several diseases, including malignant tumours. However, the clinical application of I³I´T cells has been hampered by their relatively low abundance in vivo and the technical difficulty of inducing their differentiation from hematopoietic stem cells (HSCs) in vitro. Here, we describe a novel method for generating mouse I³I´T cells by co-culturing HSC-enriched bone marrow cells (HSC-eBMCs) with induced thymic epithelial cells (iTECs) derived from induced pluripotent stem cells (iPSCs). We used BMCs from CD45.1 congenic C57BL/6 mice to distinguish them from iPSCs, which expressed CD45.2. We showed that HSC-eBMCs and iTECs cultured with IL-2 + IL-7 for up to 21 days induced CD45.1+ I³I´T cells that expressed a broad repertoire of VI³ and VI´ T-cell receptors. Notably, the induced lymphocytes contained few or no αsT cells, NK1.1+ natural killer cells, or B220+ B cells. Adoptive transfer of the induced I³I´T cells to leukemia-bearing mice significantly reduced tumour growth and prolonged mouse survival with no obvious side effects, such as tumorigenesis and autoimmune diseases. This new method suggests that it could also be used to produce human I³I´T cells for clinical applications.

Dietary fish intake has proven to have health benefits in humans. n-3 polyunsaturated fatty acids (PUFAs) in fish oil (FO), especially, may provide protection against age-related cognitive disorders. Owing to the unique benefits of n-3 PUFAs, other nutrients, such as fish protein (FP), have not been well studied. To clarify the effects of FO and FP on brain function, we investigated whether FO or FP feeding can prevent age-related cognitive dysfunction in senescence-accelerated mouse-prone 10 (SAMP10) mice. The FP group maintained a better working memory compared to the control and FO groups in the Y-maze test, but not episodic memory in the novel object recognition test. To evaluate demyelination levels, we measured neurofilament H (NfH) and myelin basic protein (MBP) immunoreactivity in the hippocampus (Hipp). Axon morphology was maintained in the FP group, but not in the control and FO groups. Additionally, the percentage of positive area for double-staining with NfH/MPB was significantly higher in the Hipp of FP-fed mice than in the control (p < 0.05). These results suggest that FP intake prevents age-related cognitive dysfunction by maintaining axonal morphology in the Hipp of SAMP10 mice.

Osteoporosis is a common bone disease, particularly in menopausal women. Herein, we screened four Kampo medicines (Unkeito (UKT), Kamishoyosan (KSS), Kamikihito (KKT), and Ninjinyoeito (NYT)), frequently used to treat menopausal syndromes, for their effects on receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclast differentiation in RAW 264 cells. Considering that UKT exhibited the most potent effect, we examined its effect on RANKL-induced osteoclastogenesis, the induction of osteoclast apoptosis, and the mechanisms underlying its effects. UKT inhibits RANKL-induced osteoclast differentiation in the early stage and decreases osteoclast-related genes, including tartrate-resistant acid phosphatase (Trap), dendritic cell-specific transmembrane protein (Dcstamp), matrix metalloproteinase-9 (Mmp9), and cathepsin K (Ctsk). Specifically, UKT inhibits the nuclear factor of activated T cells 1 (NFATc1), which is essential for osteoclastogenesis. UKT increases Bcl6, which antagonizes NFATc1 and Dc-stamp, thereby blocking the progression of osteoclasts to maturation. UKT also decreased nuclear translocation by downregulating the activity of p65/NF-κB. In addition, UKT enhances mononuclear osteoclast apoptosis via activation of caspase-3. Herein, we demonstrate that UKT suppresses RANKL-mediated osteoclastogenesis via the Blimp1–Bcl6 and NF-κB signaling pathways and enhances mononuclear osteoclast apoptosis. Furthermore, UKT prevents bone loss in OVX mice. Thus, UKT might be a potential therapeutic agent for postmenopausal osteoporosis.

Dietary fish oil containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been reported to affect the diversity and composition of gut microbiota and bacterial metabolites. However, few reports have focused on the effects of EPA and DHA on gut microbiota diversity and bacterial metabolites. This study evaluated the effects of dietary EPA-ethyl ester (EE) and DHA-EE on steroid metabolism, gut microbiota, and bacterial metabolites in Wistar rats. Male rats were fed the experimental diets containing 5% (w/w) soybean oil-EE (SOY diet), EPA-EE (EPA diet), and DHA-EE (DHA diet) for four weeks. The lipid contents in the serum and liver, mRNA expression levels in the liver, and the diversity, composition, and metabolites of the gut microbiota were evaluated. The EPA and DHA diets decreased serum and liver cholesterol contents compared to the SOY diet. In addition, there were no significant changes in gene expression levels related to steroid metabolism in the liver between the EPA and DHA groups. Rats fed the DHA diet had lower microbiota diversity indices, such as Simpson and Shannon indices, than rats fed the SOY and EPA diets. In addition, rats fed EPA and DHA had significant differences in the relative abundance of microbiota at the genus level, such as Phascolarctobacterium, Turicibacter, and [Eubacterium]. Therefore, it was concluded that EPA and DHA have different effects on the diversity and composition of gut microbiota under the experimental conditions employed herein.

Due to the growing demand of n-3 polyunsaturated fatty acids (PUFA) as supplements and pharmaceutical products worldwide, there are concerns about the exhaustion of n-3 PUFA supply sources. We have successfully prepared high-quality scallop oil (SCO), containing high eicosapentaenoic acid and phospholipids contents, from the internal organs of the Japanese giant scallop (Patinopecten yessoensis), which is the largest unutilized marine resource in Japan. This study compared the cholesterol-lowering effect of SCO with fish oil (menhaden oil, MO) and krill oil (KO) in obese type II diabetic KK-A

This study focuses on the host-parasite relationship of human Ascaris lumbricoides, which is a parasite of the small intestine and is also one of the commonest parasites worldwide. As part of this investigation, we examined the host-parasite relationship assuming that there is a common antigenicity, shared protein between A. lumbricoides and human small intestinal mucosa, using molecular techniques. We obtained three DNA clones from human colon cDNA library by screening for anti-A. lumbricoides polyclonal antibodies. The transmembrane mucin12 gene was identified after sequencing analysis of these clones. Specific signals of immunostaining with polyclonal anti-mucin12 antibodies were observed in the mucous secretory organs, epidermis, and intestinal canal of A. lumbricoides. These signals disappeared when immunohistochemistry was performed using pre-absorbed polyclonal antibodies with a specific peptide. These results suggest that mucin12 is localized in the mucous secretory organs in the epidermis of A. lumbricoides. Furthermore, we examined the site of mucin12 localization in the host; specific mucin12 signals were observed on the mucosal epithelia present around intestinal crypts and villi of the small intestine. Therefore, we suggest that mucin12 is a protein that shows common antigenicity in both A. lumbricoides and its host. It is presumed that adult A. lumbricoides live in their preferred environment, which is the small intestine, by secreting mucin12 to avoid being attacked by the host immune system.

Numerous studies have evaluated the composition of gut microbiota in experimental animals fed high-fat or low-fiber diets. However, few reports have focused on the effects of different fatty acid (FA) compositions on the diversity of gut microbiota and its metabolites. Therefore, the aim of this research was to investigate the effects of different dietary fats on liver mRNA expression levels of genes related to cholesterol and bile acid (BA) metabolism, as well as to investigate cecal microbiota composition and bacterial metabolites composition in rats. Four-week-old male Wistar/ST rats were fed a 15% fat diet for 30 days, including from different sources (soybean oil, lard, menhaden oil, or tuna oil). Then, the rats' cecal microbiota composition was determined by sequencing the 16S ribosomal RNA gene using next-generation sequencing. Lard diet drastically decreased the expression level of liver ATP-binding cassette subfamily G genes (Abcg5 and Abcg8 genes) compared with other diets. Menhaden oil diet increased the fecal BA excretion compared with soybean oil and lard diets. Fecal BA excretion tended to be positively correlated with the relative abundance of Firmicutes, and negatively correlated with the relative abundance of Bacteroidetes. These results have shown that dietary fats with different FA compositions have a different effect on the relative composition of cecal microbiota, and in particular, menhaden oil may have very different effects compared to other experimental fats. The effects of fish oils on the cecal microbiota may differ greatly depending on the ratio of EPA to DHA and the composition of FA other than n-3 polyunsaturated FA. Our results provided new insights on the way different dietary fat sources affect sterol metabolism and alter cecal microbiota composition in rats.

Fish protein is not only nutritional but also promotes health by improving insulin sensitivity and hypercholesterolemia. Few studies have examined the relationship between gut microbiota and the enhanced insulin sensitivity due to the intake of Alaska pollock protein (APP). Hence, we assessed the glycolytic enzyme inhibitory activity of APP in in vitro study and the alteration of blood glucose level in insulin tolerance test (ITT) and glucose tolerance test (GTT) and gut microbiota following APP intake in the in vivo study. In initial experiments, the glycolytic enzyme (α-amylase, α-glucosidase, and sucrase) inhibitory activities of APP and its digest were not drastically altered compared with that of casein and its digests. In further experiments, rats fed an AIN-93G diet containing 20% (w/w) casein or APP for 8 weeks, and the composition of fecal microbiota analyzed by 16S rRNA amplicon sequence analysis. In addition, at 6 and 7 weeks of administration of experimental diet, insulin and glucose tolerance tests were evaluated, respectively. Compared with dietary casein, dietary APP has blood glucose-lowering activity as evident in the ITT and GTT. Moreover, APP group altered the structure of fecal microbiota, and area under the curves of the ITT and GTT and the relative abundance of Blautia, which is associated with glucose metabolism, tended to be positively correlated (P = 0.08 and 0.10, respectively). This study illustrates a novel finding that APP intake could alter the composition of gut microbiota and improve insulin sensitivity. PRACTICAL APPLICATION: Studies in animals and humans have shown that Alaska pollock protein (APP) intake improves insulin sensitivity, allowing the body to utilize blood glucose more effectively, thereby keeping blood sugar levels under control. Microorganisms residing in the human gut are associated with glucose metabolism. This study shows that the relative APP intake alters the composition of these gut microorganisms, more than casein intake and therefore might prevent hyperglycemia and type 2 diabetes.

We developed a combined conventional polymerase chain reaction (PCR) and real-time PCR (qPCR)-based assay for detecting and discriminating between Opisthorchis viverrini and Haplorchis taichui parasite infections. The first PCR amplifies the mitochondrial cytochrome c oxidase subunit I (COI) genes of parasites, and differential diagnosis is achieved by performing qPCR with specific primers and SYBR Green I. The detection limit of the assay was found to be 2.0 × 102 plasmid copies in a test in which a stool sample was spiked with a single egg, which is equivalent to 5 eggs per gram (EPG). The testing of 34 clinical stool samples that had been demonstrated to contain “Opisthorchis-like” eggs by microscopy showed that the novel assay exhibited a sensitivity of 100% for “Opisthorchis-like” parasitic infections, and 71% and 91% of these samples were found to be infected with O. viverrini and H. taichui, respectively. A further four parasitic infections were diagnosed in the 16 negative samples, and the microscopic findings of these samples were confirmed to be false negatives by sequencing analysis. The assay also displayed high specificity during the testing of 10 samples containing other common parasites. The fact that our qPCR SYBR Green I–based assay detected submicroscopic traces of parasitic DNA and was able to differentiate between parasites that produce eggs with similar morphologies indicates that it has a good potential for development of diagnostic application to use in areas where multiple parasites coexist.

Phospholipids (PL) containing n-3 polyunsaturated fatty acids (PUFA) have beneficial effects of maintaining and promoting health compared with triacylglycerols (TAG) containing n-3 PUFA or general PL. This study evaluated the effects of dietary PL containing n-3 PUFA and elucidated the effects of the glycerophosphate structure and n-3 PUFA on fatty acid (FA) metabolism in rats. Rats were fed a basal diet containing soybean oil alone, TAG containing n-3 PUFA (1.8 %), soybean PL (2.7 %), PL containing n-3 PUFA (2.7 %), or TAG containing n-3 PUFA (1.8 %) + soybean PL (2.7 %). The present n-3 PUFA-supplemented diets had similar FA compositions, and the PL diets had similar PL compositions. TAG containing n-3 PUFA reduced serum TAG contents, but did not affect serum cholesterol contents compared with soybean oil alone. PL diets containing n-3 PUFA and the combination of TAG containing n-3 PUFA and soybean PL resulted in decreased serum and liver TAG contents compared with the diet containing soybean oil alone, reflecting enhanced liver FA β-oxidation. The results of this study show that TAG containing n-3 PUFA with added soybean PL affects serum and liver TAG and cholesterol contents to a similar degree as PL containing n-3 PUFA. TAG containing n-3 PUFA and soybean PL are widely used as functional food ingredients and pharmaceutical constituents and are inexpensive compared with PL containing n-3 PUFA. Therefore, the combination of TAG containing n-3 PUFA and soybean PL has potential as a useful and inexpensive component of functional foods.

Lysophospholipids have been recognized as potent biologically active lipid mediators. However, attention has not been paid to the health benefits of dietary partial hydrolysate of phospholipids (PH-PL), which is rich in docosahexaenoic acid (DHA)-bound lysophospholipids. In this study, the effects of PH-PL on serum and liver lipid profiles of rats upon administration of PH-PL are demonstrated in comparison to those of fish oil (FO), which comprises eicosapentaenoic acid (EPA) and DHA-bound triglyceride (TG). PH-PL containing EPA and DHA was prepared via enzymatic modification of squid (Todarodes pacificus) meal that is rich in phospholipids. Male Wistar rats were fed a basal diet containing soybean oil alone (7%), FO, and PH-PL. The FO and PH-PL diets had similar EPA and DHA contents. After the rats had been fed their respective diets for 28 d, their serum and liver lipid contents, fecal lipid excretion, and hepatic gene expression level were measured. The results demonstrated that compared with the soybean oil diet alone, the PH-PL diet decreased serum and liver TG contents partially because of the enhancement of liver acyl-CoA oxidase activity and suppression of liver fatty acid synthase activity. In addition, compared with the soybean oil diet, the PH-PL group exhibited lower serum cholesterol content at least in part because of the reduction of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase mRNA expression level. We found that dietary administration of EPA and DHA containing PH-PL has a hypolipidemic effect that may help prevent the development lifestyle-related diseases.

The purpose of this study was to elucidate the effects of dietary hemoglobin on serum and liver lipid contents in rats, and the ability of hemoglobin hydrolysates to disrupt lipid absorption. After rats had been fed on casein- or porcine hemoglobin-containing diets for 4 weeks, their serum and liver lipid contents and fecal cholesterol, bile acid, and nitrogen excretion were measured. To elucidate the mechanism of lipid absorption by dietary hemoglobin, we also examined lipase activity, micellar solubility of cholesterol, and bile acid binding activity in the presence of hemoglobin hydrolysates. Dietary hemoglobin decreased serum and liver triglyceride and cholesterol contents and increased fecal fatty acid, cholesterol, and bile acid excretion. In addition, hemoglobin hydrolysates inhibited lipase activity compared with casein hydrolysates in an in vitro study. These results suggested that the hypolipidemic effect of hemoglobin is mediated by increased fecal lipid excretion, and that decreased lipase activity by hemoglobin is at least partially responsible for this result. The observed effects were documented with an 8 g/kg hemoglobin diet, which is lower than in other studies; therefore. hemoglobin may be useful in the prevention of lifestyle-related diseases.

Inferior colliculus (IC) is a major center for the integration and processing of acoustic information from ascending auditory pathways. Damage to the IC as well as normal aging can impair auditory function. Novel strategies such as stem cell (SC)-based regenerative therapy are required for functional recovery because mature neural cells have a minimal regenerative capacity after an injury. However, it is not known if there are neural stem cells (NSCs) in the IC. Herein, we screened for NSCs by surface marker analysis using flow cytometry. Isolated IC cells expressing prominin-1 (CD133) exhibited the cardinal NSC properties self-renewal capacity, expression of known NSC markers (SOX2 and nestin), and multipotency. Prominin-1-expressing cells from neonatal IC generated neurospheres, and culture of these neurospheres in differentiation-conditioned medium gave rise to gamma-aminobutyric acid-ergic (GABAergic) neurons, astrocytes, and oligodendrocytes. The presence of NSC-like cells in the IC has important implications for understanding IC development and for potential regenerative therapy. Electronic supplementary material The online version of this article (doi:10.1007/s12035-017-0701-5) contains supplementary material, which is available to authorized users.

Presbycusis is the impairment of auditory function associated with aging, which stems from peripheral cochlear lesions and degeneration of the central auditory process. The effect of age-induced peripheral hearing loss on the central auditory process is not fully understood. C57Bl/6 (C57) mice present accelerated peripheral hearing loss, which is well developed by middle-age and mimics the human presbycusis pattern. The aim of this study was to elucidate the molecular effects of peripheral hearing loss in the inferior colliculus (IC) with age between young and middle-aged C57 mice using cDNA microarray. Glutamate receptor ionotropic NMDA ζ1 (GluN1) exhibited the greatest decrease in the middle-aged group as determined using cDNA microarray and by further assessment using real-time PCR (qPCR). Histological assessment with in situ hybridization of GluN1 showed significantly decreased expression in all IC subdivisions of the middle-aged group. GluN1 is a receptor for excitatory neurotransmission, and significant downregulation of this gene may be subsequent to the decline of afferent input from the cochlea in aging C57 mice. Consequently, using the combination of microarray, qPCR, and in situ hybridization, we showed that the decline of GluN1 in the IC of aging animals might have a key role in the pathogenesis of presbycusis.

Serum and tissue iodine concentration was measured in rats fed a diet supplemented with powdered kombu (Saccharina sculpera) or potassium iodide to evaluate the absorption of iodine from kombu. Eighteen male 5-wk-old Wistar rats were divided into three groups and fed a basal AIN93G diet (iodine content, 0.2 mg/kg) or the basal diet supplemented with iodine (183 mg/kg) either in the form of kombu powder or potassium iodine (KI) for 4 wk. There were no differences in weight gain or serum biochemistry tests (alanine aminotransferase and aspartate aminotransferase activity, and total serum cholesterol and triglyceride concentration) after iodine supplementation. In addition, serum levels of the thyroid hormones thyroxine and triiodothyronine, as well as thyroid-stimulating hormone, were not affected. On the other hand, serum and tissue (thyroid, liver and kidney) iodine concentrations were markedly elevated after iodine supplementation. There was no difference in thyroid iodine concentration between KI and kombu supplementation. However, there was a significant difference observed in the iodine concentrations of serum, liver and kidney between the two iodine sources; rats fed KI had iodine concentrations in these tissues 1.8 to 1.9 times higher than those in rats fed kombu powder. These results suggest that the absorption of iodine from kombu is reduced compared to that from potassium iodide.

We estimated the nutritional availability of selenium (Se) in Se-enriched Kaiware radish sprouts (SeRS) by the tissue Se deposition and glutathione peroxidase (GPX) activity of rats administered the sprouts, and examined the effect of SeRS on the formation of aberrant crypt foci (ACF) in the colon of mice administered 1,2-dimethylhydrazine (DMH) to evaluate anti-tumor activity. Male weanling Wistar rats were divided into seven groups and fed a Se-deficient basal diet or the basal diet supplemented with 0.05, 0.10, or 0.15 microg/g of Se as sodium selenite or SeRS for 28 d. Supplementation with Se dose-dependently increased serum and liver Se concentrations and GPX activities, and the selenite-supplemented groups showed a higher increase than the SeRS-supplemented groups. The nutritional availability of Se in SeRS was estimated to be 33 or 64% by slope ratio analysis. Male 4-week-old A/J mice were divided into seven groups and fed a low Se basal diet or the basal diet supplemented with selenite, SeRS, or selenite + non-Se-enriched radish sprouts (NonSeRS) at a level of 0.1 or 2.0 microg Se/g for 9 weeks. After 1 week of feeding, all mice were given six subcutaneous injections of DMH (20 mg/kg) at 1-week intervals. The average number of ACF formed in the colon of mice fed the basal diet was 4.3. At a supplementation level of 0.1 mug Se/g, only SeRS significantly inhibited ACF formation. At a supplementation level of 2.0 microg Se/g, both selenite and SeRS significantly inhibited ACF formation. The addition of NonSeRS to the selenite-supplemented diets tended to inhibit ACF formation, but this was not statistically significant. These results indicate that SeRS shows lower nutritional availability but higher anti-tumor activity than selenite.

Protamine has been used as an antiheparin drug and a natural preservative in various food products. However, limited studies have evaluated the physicochemical and functional properties of protamine. Hence, we assessed the mechanisms underlying the inhibition of lipid absorption following salmon protamine intake in in vitro and in vivo studies. In initial experiments, a salmon protamine hydrolyzate (PH) was prepared using in vitro simulated gastrointestinal digestion suppressed pancreatic lipase activity and micellar cholesterol solubility. This PH had higher bile acid-binding capacity and emulsion breakdown activity than casein hydrolyzate and l-arginine. However, the hypolipidemic functions of protamine were dramatically reduced by pancreatin digestion. In further experiments, groups of male Wistar rats were fed an AIN-93G diet containing 5% (wt/wt) salmon protamine or a protamine amino acid mixture. After 4 wk of feeding with experimental diets, reductions in serum and liver triacylglycerol (TAG) and cholesterol contents were observed in the presence of protamine, reflecting inhibition of TAG, cholesterol, and bile acid absorption. These data suggest that the formation of insoluble PH-bile acid complexes is critical before the bile acid-binding capacity is reduced. Therefore, dietary salmon protamine may ameliorate lifestyle-related diseases such as hyperlipidemia and obesity.

Organ molybdenum (Mo) concentration and the activity of hepatic sulfite oxidase and xanthine oxidase were compared in tungsten-administered rats as well as rats fed with a low Mo diet to evaluate the use of tungsten-administered rats as a model of Mo deficiency. Twenty-four male 6-week-old Wistar rats were divided into four groups according to diet (AIN93G diet (control diet) or the control diet minus ammonium molybdate (low Mo diet)) and drinking water (deionized water or deionized water containing 200 μg/mL tungsten in the form of sodium tungstate). Mo content in the control and low Mo diets were 196 and 42 ng/g, respectively. Intake of the low Mo diet significantly reduced the Mo content of several organs and serum. Decrease in hepatic sulfite oxidase activity was also induced by the low Mo diet. The administration of tungsten induced marked decreases in organ Mo content and the activity of hepatic sulfite oxidase and xanthine oxidase. These decreases induced by tungsten administration were more pronounced than those induced by just a low Mo diet. Serum uric acid was also reduced by tungsten administration irrespective of Mo intake. Although a comparatively high accumulation of tungsten (3 to 9 μg/g) was observed in the kidneys and liver, adverse effects of tungsten accumulation on liver and kidney function were not observed in serum biochemical tests. These results indicate that tungsten-administered animals may be used as a model of Mo deficiency.

To examine the selenium (Se) status of rats intermittently supplemented with Se, we measured tissue Se contents and glutathione peroxidase (GPx) activities in rats fed a Se-deficient diet intermittently supplemented with selenate. In experiment 1, four groups of male 4-wk-old Wistar rats were fed a Torula yeast-based Se-deficient diet (Se content,0.01 microg/g) for 28 d. During the experimental period, the diet of each group was supplemented with sodium selenate (0.17 microg Se/g) for 0, 1, 2 or 7 d/wk. The tissue Se contents and GPx activities both increased gradually with an increase in frequency of the selenate supplementation, and significant linear regressions were observed between the frequency and these Se indices. In particular, the correlation coefficient in the liver and plasma indices was nearly equal to a value of 1.0. In experiment 2, three groups of rats were fed the Se-deficient basal diet for 28 d. Among these, one group was daily supplemented with sodium selenate to the Se-deficient diet at a level of 0.17 microg Se/g, and another group was intermittently supplemented with the selenate at a level of 1.19 microg Se/g for 1 d/wk. The tissue Se contents and GPx activities both were increased by the selenate supplementation and no significant difference was observed between daily and weekly supplementation in the Se indices except in erythrocyte Se. These results indicate that Se status in the growth period is dependent on total Se intake in this period and that weekly intermittent supplementation with Se can maintain adequate Se status.

Sardine oil was obtained by centrifugation of surimi wastewater without heating or chemical refining. This oil (CE) showed light yellow color and the peroxide value was less than 1.0 meq/kg. The main lipid class of CE was triacylglycerol (TG) (99%). These features indicate that CE can be directly used as food materials without further purification. Commercial sardine oil (CO) is usually prepared via some kind of refining process with high temperature (250 degrees C) and chemical treatment. The comparative study on the physiological effects of these sardine oils (CE and CO) revealed that the dietary sardine oils were more effective in reducing abdominal fat pads, plasma total cholesterol, and TG levels of rats than was a soybean oil diet (control). Furthermore, these effects were greater in CE than CO, although there was little difference in the fatty acid composition of both oils. Although the main lipid class of CE was TG (99%), CE was prepared by centrifugation from surimi waste and directly used as dietary fat without further purification. Therefore, CE may contain some kinds of minor components, which could be attributed to the higher physiological activity of CE. To reveal the involvement of the minor compounds in CE, we prepared TG from CE by column chromatography and measured its effect on lipid metabolism of rats. TG from CE also showed the reducing effects on abdominal fad pads and plasma lipid levels. The effect of TG from CE was almost the same as that of original CE, suggesting that the higher nutritional activity of CE than CO may not be due to the minor compounds in CE.

An enzyme-linked immunsorbent assay (ELISA) using worm extract antigens from lung flukes of Paragonimus westermani provided good sensitivity to sera from patients with paragonimiasis westermani but high cross-reactivity with sera from most fascioliasis patients and some patients with onchocerciasis or clonorchiasis. To improve the specificity, we tested an ELISA using fluke cysteine proteinases as antigens. Cysteine proteinases were partially purified from the excretory/secretory products of P. westermani by 40-75% ammonium sulfate fractionation, hydrophobic chromatography, and arginine affinity chromatography. An ELISA using the enzyme preparation not only had increased sensitivity to paragonimiasis westermani sera but also reduced cross-reactivity with the fascioliasis, onchocerciasis, and clonorchiasis sera to negligible levels. The reactivity of the ELISA to paragonimiasis miyazakii sera was similar to that of paragonimiasis westermani sera. A proteinase preparation from P. ohirai, which can be obtained easily from infected rats, provided similar results. Therefore, the ELISA using cysteine proteinases of Paragonimus could not distinguish the parasite species with which patients were infected, but it is a valuable assay with which to immunodiagnose paragonimiasis even when the proteinases are prepared from nonhuman species.

We characterized side population (SP) cells in the cochlear nucleus (CN). Some genes of stem/progenitor markers in sorted SP cells were identified by microarray analysis and RT-PCR. Furthermore, some cells in the CN also demonstrated self-renewal and clonal expansion activities. These results suggest that tissue stem/ progenitor like cells would be identified and characterized as a slow cycling and immaturity in SP cells of CN.SP cells were sorted and characterized as regards their activity in the CN in order to identify the tissue progenitor/stem cells in the auditory nervous system.Bromodeoxyuridine (BrdU)-injected mice were prepared and the long-term BrdU-retaining cells were detected by flow cytometry. Gene expression of SP and MP cells was analyzed by microarray analysis and RT-PCR. SP cells were cultured in conditioned medium to expand stem/progenitor cells in vitro and to estimate the spheroid-forming activity of stem cells.In all, 1% of cells in the CN were detected as BrdU-positive. SP cells were detected at a frequency of 4.4% and expressed stem/progenitor markers, Abcb1b, Abcg2, Sca1, Notch1, Notch4, Hes1, and Jag1 in microarray analysis. Expression of Abcb1b, Abcg2, Sca1,Oct3/4, and Sox2 as determined by RT-PCR was supported by the microarray data. CN cells also had sphere-forming activity in young mice, but this activity was decreased by aging.

Fish consumption is well known to provide health benefits in both experimental animals and human subjects. Numerous studies have demonstrated the beneficial effects of various protein hydrolysates on lipid metabolism. In this context, this study examined the effect of fish protein hydrolysates (FPH) on cholesterol metabolism compared with the effect of casein. FPHs were prepared from Alaska pollock meat using papain as a protease. Male Wistar rats were divided into the following four dietary groups of seven rats each: either casein (20%) or FPH (10%) + casein (10%), with or without 0.5% cholesterol and 0.1% sodium cholate. Serum and liver lipid levels, fecal cholesterol and bile acid excretions, and the hepatic expression of genes encoding proteins involved in cholesterol homeostasis were examined. In rats fed the FPH diets compared with casein diets with or without cholesterol and sodium cholate, the indexes of cholesterol metabolism-namely, serum cholesterol, triglyceride, and low-density lipoprotein-cholesterol levels-were significantly lower, whereas fecal cholesterol and bile acid excretions were higher. Rats fed the FPH diets compared with casein with cholesterol exhibited a lower liver cholesterol level via an increased liver cholesterol 7α-hydroxylase (CYP7A1) expression level. This study demonstrates that the intake of FPH has hypocholesterolemic effects through the enhancement of fecal cholesterol and bile acid excretions and CYP7A1 expression levels. Therefore, fish peptides prepared by papain digestion might provide health benefits by decreasing the cholesterol content in the blood, which would contribute to the prevention of circulatory system diseases such as arteriosclerosis.

To attempt a risk assessment of the excess intake of trivalent chromium (Cr), tissue Cr accumulation and urinary Cr excretion were examined in weanling rats fed experimental diets containing graded levels of Cr chloride (CrCl3) or Cr picolinate (CrPic). Thirty-six male weanling 4-weeks-old Wistar rats were divided into six groups and fed a casein-based semi-purified diet (Cr content

Dietary proteins influence the lipid metabolism of human subjects and animals. This study evaluated the effects of fish protein on lipid metabolism in rats. Alaska pollock fillets, widely supplied as raw materials of surimi, were used as fish protein. As parameters of lipid metabolism, cholesterol and triacylglycerol concentrations in the serum and liver, the fecal excretion of bile acids, and the hepatic expression of genes encoding proteins involved in lipid homeostasis were examined. Rats fed fish protein showed decreased cholesterol concentrations in the serum and liver, and fecal bile acid and cholesterol concentrations were increased. This was caused by the increased expression of cholesterol 7alpha-hydroxylase (CYP7A1) as the digested fish protein inhibited the absorption of bile acid and cholesterol in the small intestine. In addition, it was found that dietary fish protein affects the farnesoid X receptor/small heterodimer partner-dependent pathway, which is negatively regulated by the decreased reabsorption of bile acid. Furthermore, it increased the binding to the promoter of CYP7A1 through activated liver receptor homologue-1.

Trans and conjugated fatty acids may exhibit either beneficial or detrimental bioactive effects depending on their metabolic properties. This study was conducted to elucidate if isomerization and conjugation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) demonstrate more favorable bioactivity on lipid metabolism compared to unmodified EPA and DHA. The effects of dietary intake of trans and conjugated forms of EPA and DHA on lipid metabolism were evaluated in animal trials and compared to a control group fed soybean oil. None of the experimental diets showed significant differences from the control in terms of body weight; however, the white adipose tissue weight of rodents fed trans DHA, conjugated EPA (CEPA), and conjugated DHA (CDHA) was significantly lower than the control. Triacylglycerol levels in plasma were significantly decreased in groups fed trans DHA (17.2 mg/dL) and CDHA (31.9 mg/dL) relative to the control (51.3 mg/dL). The total cholesterol concentrations were significantly lower than the control (68.0 mg/dL) in all experimental groups (47.3 to 53.7 mg/dL) except CEPA (58.3 mg/dL). Fatty acid compositions of lipids extracted from rodent livers were influenced by the dietary fatty acid profiles, with all groups showing higher concentrations of stearic acid and lower levels of linoleic acid compared to the control. Rodents fed trans DHA did not have detectable levels of these fatty acid isomers in their livers, suggesting either quick metabolism or a difficulty with bio-absorption.

Antidepressant treatments have been described to induce neurotrophic factors (NTFs) and reverse the cell loss observed in rodent stress models. Amitriptyline (AT), a tricyclic antidepressant agent, has been reported in recent studies to induce glial cell line-derived neurotrophic factor (GDNF) synthesis and release in rat C6 glioblastoma cells. GDNF has shown protection against acoustic trauma in previous studies. Therefore, we investigated whether AT could induce GDNF synthesis in the cochlea and attenuate cochlea damage against acoustic trauma. We used Hartley guinea pigs and injected AT (30 mg/kg) or saline into the peritoneum. Subjects were exposed to 117 dB SPL octave band noise centered at 4 kHz for 24 h. Noise-induced hearing loss (NIHL) was assessed with auditory brain stem response (ABR) at 4, 8 and 16 kHz measured prior to the injection, 3 days and 7 days after noise exposure. For histological assessment, we observed the sensory epithelium using a surface preparation technique and assessed the quantitative hair cell (HC) damage. We evaluated GDNF synthesis with or without intense noise exposure at 3, 12 and 24 h after the administration of AT in the cochlea using Western blot analysis. GDNF expression was shown 3 h and 12 h after the injection without noise, whereas with noise the GDNF expression lasted for 24 h. The AT-administrated group showed significantly reduced ABR threshold shift and less HC damage than the saline-administrated group. These findings suggest that the administration of AT-induced GDNF levels in the cochlea and attenuated cochlea damage from NIHL.

Mouse embryonic stem (ES) cells are useful tools for investigating differentiation into neurons and glial cells in vitro. In order to induce ES cells to differentiate into neural cells, many researchers have investigated the efficiency of induction. Embryoid body (EB) formation and retinoic acid are potent differentiation inducers known to be a trigger at the early stage of development. Basic helix-loop-helix (bHLH) is one of the important transcription factors, which is essential for premature neural formation. In NeuroD2 and Mash1-transfected cells, neural formation was observed at day 6 after the plating of embryoid bodies in culture. Nestin was detected in NeuroD2- and Mash1-transfected cells at day 10, and strong signal was detected in Mash1 transfectants by RT-PCR analysis. Map2 and Nurr1 were also detected strongly at the early stage in transfected cells compared with the wild type control, especially in the Mash1 transfectant. In immunocytochemical analysis, Tuj1-positive neurons were detected at high frequency in Mash1 transfectants and some cells were stained by tyrosine hydrogenase (TH), a marker of dopaminergic neurons. These results demonstrate that bHLH has a potential activity at an early stage for ES cells and can induce effective and rapid neural differentiation in vitro.

We compared a microcolorimetric enzyme-linked immunosorbent assay (colorimetric elisa ) and a microchemiluminescence enzyme-linked immunosorbent assay (chemiluminescence elisa ) for the detection of specific immunoglobulin G (IgG) in the serum of 9 patients with sparganosis mansoni and 9 healthy controls. The chemiluminescence elisa was able to measure serum levels of specific IgG over a far wider range than the colorimetric assay, and its detection limit was at least 10-fold lower. An additional 5 sera from sparganosis patients and 5 more from healthy controls, together with sera from 28 patients with other parasitic diseases, were also examined by the chemiluminescence elisa . All 14 patients with sparganosis mansoni showed high levels of chemiluminescence (21 302±18 907 counts per second [cps]). All sera from the 14 healthy controls (1580±569 cps) and sera from 27 of the 28 patients with other parasites (4 with taeniasis saginata [1767±501 cps], 11 with diphyllobothriasis latum [1479±501 cps], 13 with cysticercosis cellulosae [2376±1437 cps]) showed chemiluminescence levels lower than those of any of the sparganosis mansoni patients. The exception was a patient with cysticercosis (5980 cps), who may have had a dual infection with Cysticercus cellulosae and Sparganum mansoni . Thus, the chemiluminescence elisa demonstrated high sensitivity and specificity for human sparganosis mansoni.

The supernatant from normal liver cell (Clone 1469) culture enhanced polyclonal antibody production in mouse spleen cells but not in human peripheral blood lymphocytes. The supernatants from transforming growth factor beta (TGF-β)-treated C3H/HeN spleen cells augmented greatly polyclonal antibody responses compared with the untreated supernatants. IL-10 production and expression of mRNA for IL-10 in Clone 1469 cells was detected by using enzyme-linked immunosorbent assay, ELISPOT assay and PCR amplification. TGF-β led to augmentation of IL-10 secretion from Clone 1469 cells.

A 31-year-old man visited our hospital in May 1991, com-plaining of an abdominal serpiginous eruption with pruritus (Fig. 1). He had eaten raw “firefly squid” (Watasenia scintil-lans) 2 weeks before the onset of the disease, and the erup-tion had grown to about 30 cm in length in 2 weeks. The laboratory findings included a leukocyte count of 6100/mm3 with 10% eosinophils and a serum IgE level of 134 U/mL (normal < 303 U/mL). The front of the eruption was excised. Seven parasitic sections were observed in the dermis (Fig. 2). The diameter of the sections were about 0.08 mm. The mus-cle layer was of the polymyarian coelomyarian type. In the body cavity of the parasite, there was a glandular part of the esophagus composed of vacuolated cells with many nuclei and a narrow esophageal lumen. Well-developed lateral nerve chords were also visible in these sections. Two of the parasitic sections showed intestine composed of cuboidal cells with a round lumen and lateral nerve chords of both sides touching each other in the center of body cavity. These findings indicate the characteristics of larval Spiruroidea. The morphologic appearance of the sectioned worm closely resembled the type × larva of the superfamily Spiruroidea of Hasegawa.1

A 'sandwich' enzyme-linked immunosorbent assay was established to detect circulating antigens of Trichinella spiralis in human sera, its sensitivity and specificity was evaluated using 4 antigens (Trichinella spiralis, Trichuris trichiura, Dirofilaria immitis and Ascaris suum), and it was found to be sensitive and specific for T. spiralis antigen. Samples of 347 individuals with suspected trichinellosis, who had eaten incompletely cooked bear meat containing larvae of T. spiralis, were examined. Among individuals showing clinical symptoms, circulating antigens were detected in 29.9%, and the prevalence of antibodies was 18.9%. Among individuals lacking clinical symptoms, antigens were detected in 21.4% and antibodies in 5.0%. It was concluded that detection of circulating antigens was more useful for making diagnoses than measurement of specific antibodies.

Summary Naturally occurring phagocytosis of Trypanosoma gambiense by mouse eosinophils and neutrophils was reported. In vivo and in vitro experiments using monoclonal antibodies confirmed that the phagocytosis is triggered by Gl class antibodies against variable surface antigen. Ultrastructural observation revealed the mode of entry and the intracellular fate of T. gambiense: initial attachment, pseudopodia formation and complete invagination. This phagocytosis resulted in the killing of T. gambiense by mouse eosinophils and neutrophils, suggesting that eosinophils and neutrophils give at least parital protection against infection with T. gambiense in combination with the specific antibodies.

The antibody-binding sites of the muscle larva of Trichinella spiralis were investigated by immunogold staining on the ultrathin sections of LR white resin. The antibodies, which were produced in the course of T. spiralis infection in rats, specifically bound to the inner layers of the body cuticle and the cuticle of the hindgut, but not to the cuticle of the esophagus. This is the first report that reveals the antigenic nature of the inner layers of the external coverings of T. spiralis larva.