Extraskeletal chondrosarcoma in the abdominal cavity of a cow.

A 25-month-old female crossbred cow presented with astasia, emaciation, and stunted growth. Macroscopic examination revealed a large mass in the abdominal cavity, approximately 100 × 30 × 30 cm. Microscopic examination revealed that the mass consisted of multilobular mature and immature cartilaginous matrices with chondrocytic cells, surrounded by spindle to pleomorphic mesenchymal tumor cells. The cartilaginous matrices consisted of hyaline and elastic cartilages, as confirmed with Azan stain, and Victoria Blue and Van Gieson stain. Immunohistochemistry revealed that the chondrocytic and mesenchymal cells both expressed S-100. The tumor was diagnosed as an extraskeletal chondrosarcoma in the abdominal cavity of this cow.

Immunohistochemical expression of autophagosome markers LC3 and p62 in preneoplastic liver foci in high fat diet-fed rats.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive deposition of droplets in hepatocytes. Patients with NAFLD can be at risk for nonalcoholic steatohepatitis, which can lead to hepatocellular carcinoma. Autophagy is a cellular pathway that is crucial for survival and homeostasis, and which protects against pathophysiological changes like obesity and cancer. We determined the expression of autophagy markers in preneoplastic hepatic lesions and the effects of an autophagy repressor chloroquine (CQ) or inducer amiodarone (AM) in a steatosis-related hepatocarcinogenesis model. Male F344 rats were fed a control diet or high fat diet (HFD), and subjected to initiation and promotion steps with N-nitrosodiethylamine injection at week 0 and a partial hepatectomy at week 3. Several HFD-fed rats were administered 0.1% CQ and 0.5% AM in their drinking water during week 2 and 8. CQ and AM did not improve HFD-induced obesity. AM, but not CQ, significantly decreased the number of glutathione S-transferase placental form-positive preneoplastic liver foci in the liver. Autophagosome markers LC3 and the LC3-binding protein p62 were heterogeneously expressed in the preneoplastic foci. CQ might inhibit autophagy by significantly increased p62/LC3 ratio, while AM might have a potential of inducing autophagy by showing an increased gene expression of the autophagy regulator, Atg5. These results suggest that preneoplastic lesions express autophagosome markers and that AM might decrease steatosis-related early hepatocarcinogenesis by potentially inducing autophagy in HFD-fed rats, while inhibition of autophagy by CQ did not alter the hepatocarcinogenesis. However, an immunohistochemical trial revealed a technical limitation in detecting autophagosome markers because there were variations in each preneoplastic lesion.

Lack of preventive effect of maternal exposure to α-glycosyl isoquercitrin and α-lipoic acid on developmental hypothyroidism-induced aberrations of hippocampal neurogenesis in rat offspring.

Hypothyroidism during the developmental stage induces disruption of hippocampal neurogenesis in later life, as well as inducing oxidative stress in the brain. The present study investigated the preventive effect of co-exposure to an antioxidant on disruptive neurogenesis induced by developmental exposure to anti-thyroid agent in rats. For this purpose, we used two antioxidants, α-glycosyl isoquercitrin (AGIQ) and α-lipoic acid (ALA). Mated female Sprague Dawley rats were either untreated (control) or treated with 12 ppm 6-propyl-2-thiouracil (PTU), an anti-thyroid agent, in drinking water from gestational day 6 to postnatal day (PND) 21, the latter group being subjected to feeding basal diet alone or diet containing AGIQ at 5,000 ppm or ALA at 2,000 ppm during PTU exposure. On PND 21, PTU-exposed offspring showed reductions in a broad range of granule cell lineage subpopulations and a change in the number of GABAergic interneuron subpopulations. Co-exposure of AGIQ or ALA with PTU altered the transcript levels of many genes across multiple functions, suggestive of enhancement of synaptic plasticity and neurogenesis. Nevertheless, immunohistochemical results did not support these changes. PTU exposure and co-exposure of AGIQ or ALA with PTU did not alter the hippocampal lipid peroxidation level. The obtained results suggest a possibility that thyroid hormone depletion itself primarily disrupts neurogenesis and that oxidative stress may not be involved in the disruption during development. Transcript expression changes of many genes caused by antioxidants may be the result of neuroprotective actions of antioxidants rather than their antioxidant activity. However, no preventive effect on neurogenesis suggested impairment of protein synthesis via an effect on mRNA translation due to hypothyroidism.

Establishment of a novel experimental model for muscle-invasive bladder cancer using a dog bladder cancer organoid culture.

In human and dogs, bladder cancer (BC) is the most common neoplasm affecting the urinary tract. Dog BC resembles human muscle-invasive BC in histopathological characteristics and gene expression profiles, and could be an important research model for this disease. Cancer patient-derived organoid culture can recapitulate organ structures and maintains the gene expression profiles of original tumor tissues. In a previous study, we generated dog prostate cancer organoids using urine samples, however dog BC organoids had never been produced. Therefore we aimed to generate dog BC organoids using urine samples and check their histopathological characteristics, drug sensitivity, and gene expression profiles. Organoids from individual BC dogs were successfully generated, expressed urothelial cell markers (CK7, CK20, and UPK3A) and exhibited tumorigenesis in vivo. In a cell viability assay, the response to combined treatment with a range of anticancer drugs (cisplatin, vinblastine, gemcitabine or piroxicam) was markedly different in each BC organoid. In RNA-sequencing analysis, expression levels of basal cell markers (CK5 and DSG3) and several novel genes (MMP28, CTSE, CNN3, TFPI2, COL17A1, and AGPAT4) were upregulated in BC organoids compared with normal bladder tissues or two-dimensional (2D) BC cell lines. These established dog BC organoids might be a useful tool, not only to determine suitable chemotherapy for BC diseased dogs but also to identify novel biomarkers in human muscle-invasive BC. In the present study, for the 1st time, dog BC organoids were generated and several specifically upregulated organoid genes were identified. Our data suggest that dog BC organoids might become a new tool to provide fresh insights into both dog BC therapy and diagnostic biomarkers.

Ameliorating effect of postweaning exposure to antioxidant on disruption of hippocampal neurogenesis induced by developmental hypothyroidism in rats.

Developmental hypothyroidism as a model of autism spectrum disorders disrupts hippocampal neurogenesis through the adult stage. The present study investigated the ameliorating effect of postweaning exposure to antioxidant on the hypothyroidism-induced disruptive neurogenesis. Mated female Sprague-Dawley rats were treated with 0 or 10 ppm 6-propyl-2-thiouracil (PTU) as an anti-thyroid agent in drinking water from gestational day 6 to postnatal day (PND) 21 on weaning. PTU-exposed male offspring were fed either basal diet, diet containing α-glycosyl isoquercitrin (AGIQ) at 5,000 ppm or α-lipoic acid (ALA) at 1,000 ppm as an antioxidant from PND 21 to PND 77. PTU-exposure decreased DCX+ and NeuN+ granule cell lineage subpopulations, synaptic plasticity-related FOS+ granule cells, and hilar PVALB+ and GAD67+ GABAergic interneurons, increased hilar SST+ and CALB2+ interneurons, and upregulated Gria3, Otx2, and antioxidant enzyme genes in the dentate gyrus on PND 77. These results suggest disruption of neurogenesis remained in relation with increase of oxidative stress and compensatory responses to the disruption at the adult stage. AGIQ recovered expression of some antioxidant enzyme genes and was effective for restoration of NeuN+ postmitotic granule cells and PVALB+ and SST+ interneurons. In contrast, ALA was effective for restoration of all interneuron subpopulations, as well as postmitotic granule cells, and upregulated Grin2a that may play a role for the restoration. Both antioxidants recovered expression of Otx2 and AGIQ-alone recovered Gria3, suggesting a reversal of disruptive neurogenesis by compensatory responses. Thus, postweaning antioxidant exposure may be effective for ameliorating developmental hypothyroidism-induced disruptive neurogenesis by restoring the function of regulatory system.

Characterization of the modes of binding between human sweet taste receptor and low-molecular-weight sweet compounds.

One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2-hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein-coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2-hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling-based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands.

Chimeric yeast G-protein α subunit harboring a 37-residue C-terminal gustducin-specific sequence is functional in Saccharomyces cerevisiae.

Despite many recent studies of G-protein-coupled receptor (GPCR) structures, it is not yet well understood how these receptors activate G proteins. The GPCR assay using baker's yeast, Saccharomyces cerevisiae, is an effective experimental model for the characterization of GPCR-Gα interactions. Here, using the yeast endogenous Gα protein (Gpa1p) as template, we constructed various chimeric Gα proteins with a region that is considered to be necessary for interaction with mammalian receptors. The signaling assay using the yeast pheromone receptor revealed that the chimeric Gα protein harboring 37 gustducin-specific amino acid residues at its C-terminus (GPA1/gust37) maintained functionality in yeast. In contrast, GPA1/gust44, a variant routinely used in mammalian experimental systems, was not functional.

Photoactive ligands probing the sweet taste receptor. Design and synthesis of highly potent diazirinyl D-phenylalanine derivatives.

Some D-amino acids such as d-tryptophan and D-phenylalanine are well known as naturally-occurring sweeteners. Photoreactive D-phenylalanine derivatives containing trifluoromethyldiazirinyl moiety at 3- or 4-position of phenylalanine, were designed as sweeteners for functional analysis with photoaffinity labeling. The trifluoromethyldiazirinyl D-phenylalanine derivatives were prepared effectively with chemo-enzymatic methods using L-amino acid oxidase and were found to have potent activity toward the human sweet taste receptor.

Anti-tumor activities of the antlered form of Ganoderma lucidum in allogeneic and syngeneic tumor-bearing mice.

We investigated the anti-tumor effects of a dry powder preparation of the antlered form of Ganoderma lucidum (G. lucidum AF, rokkaku-reishi in Japanese), a variant type of G. lucidum, not only in allogeneic Sarcoma 180-bearing ddY mice, but also in syngeneic MM 46-bearing C3H/He mice. G. lucidum AF inhibited tumor growth and elongated the life span when orally administered to mice by free-feeding of a 2.5% G. lucidum AF-containing diet. It also showed anti-tumor activity in spite of post-feeding after tumor inoculation. G. lucidum AF significantly countered the depression of splenic CD8+ cells and protected the decrease in interferon-gamma (IFN-gamma) production in regional lymph nodes of MM 46-bearing mice, indicating that the anti-tumor activity of G. lucidum AF might be caused by its immunostimulating action. These results suggest that the ingestion of G. lucidum AF can be useful for the prevention and curing of cancer.

Yellow flowers generated by expression of the aurone biosynthetic pathway.

Flower color is most often conferred by colored flavonoid pigments. Aurone flavonoids confer a bright yellow color on flowers such as snapdragon (Antirrhinum majus) and dahlia (Dahlia variabilis). A. majus aureusidin synthase (AmAS1) was identified as the key enzyme that catalyzes aurone biosynthesis from chalcones, but transgenic flowers overexpressing AmAS1 gene failed to produce aurones. Here, we report that chalcone 4'-O-glucosyltransferase (4'CGT) is essential for aurone biosynthesis and yellow coloration in vivo. Coexpression of the Am4'CGT and AmAS1 genes was sufficient for the accumulation of aureusidin 6-O-glucoside in transgenic flowers (Torenia hybrida). Furthermore, their coexpression combined with down-regulation of anthocyanin biosynthesis by RNA interference (RNAi) resulted in yellow flowers. An Am4'CGT-GFP chimeric protein localized in the cytoplasm, whereas the AmAS1(N1-60)-RFP chimeric protein was localized to the vacuole. We therefore conclude that chalcones are 4'-O-glucosylated in the cytoplasm, their 4'-O-glucosides transported to the vacuole, and therein enzymatically converted to aurone 6-O-glucosides. This metabolic pathway is unique among the known examples of flavonoid, including anthocyanin biosynthesis because, for all other compounds, the carbon backbone is completed before transport to the vacuole. Our findings herein not only demonstrate the biochemical basis of aurone biosynthesis but also open the way to engineering yellow flowers for major ornamental species lacking this color variant.

Formation of two methylenedioxy bridges by a Sesamum CYP81Q protein yielding a furofuran lignan, (+)-sesamin.

(+)-Sesamin, a furofuran class lignan, is widespread in vascular plants and represented by Sesamum spp. (+)-Sesamin has been of rapidly growing interest because of its beneficial biological effects in mammals, but its biosynthesis and physiological roles in plants remain to be clarified. It is speculated to be synthesized from (+)-pinoresinol by means of (+)-piperitol by formation of two methylenedioxy bridges mediated by two distinct Sesamum indicum cytochrome P450 (SiP450) proteins. Here, we report an SiP450, CYP81Q1, that alone catalyzes (+)-sesamin biosynthesis from (+)-pinoresinol by means of (+)-piperitol by forming two methylenedioxy bridges. The CYP81Q1 gene expression profile was temporally consistent with the accumulation pattern of (+)-sesamin during seed development. The CYP81Q1-GFP chimera protein was colocalized with an endoplasmic reticulum (ER)-targeting chimera protein, indicating that (+)-sesamin biosynthesis occurs on the ER cytoplasmic surface. Moreover, we isolated two CYP81Q1 homologs from other Sesamum spp. Sesamum radiatum CYP81Q2 showed dual (+)-piperitol/(+)-sesamin synthetic activity. CYP81Q2, as well as CYP81Q1, therefore, corresponds to a (+)-piperitol/(+)-sesamin synthase in lignan biosynthesis. In contrast, Sesamum alatum CYP81Q3 showed no activity, in accord with (+)-sesamin being deficient in S. alatum. Our findings not only provide insight into lignan biosynthesis but also unravel a unique mode of cytochrome P450 action.

Dietary supplementation of L-carnosine prevents ischemia/reperfusion-induced renal injury in rats.

The effects of dietary supplementation of L-carnosine (beta-alanyl-L-histidine) on ischemia/reperfusion-induced acute renal failure (ARF) in rats were examined. Ischemic ARF was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal functional parameters such as blood urea nitrogen, plasma creatinine, creatinine clearance, urine flow, urinary osmolality and fractional excretion of sodium were measured. Renal function in ARF rats markedly decreased at 1 d after reperfusion. Prior feeding of L-carnosine-containing diet (0.0001 w/w%) for 2 weeks attenuated the ischemia/reperfusion-induced renal dysfunction. Histopathological examination of the kidney of ARF rats revealed severe renal damages, such as tubular necrosis, proteinaceous casts in tubuli and medullary congestion, which were also significantly suppressed by the dietary supplementation of L-carnosine. These findings strongly suggest that L-carnosine supplementation is useful as a prophylactic treatment in the development of the ischemic ARF.

Effects of sesamin on aortic oxidative stress and endothelial dysfunction in deoxycorticosterone acetate-salt hypertensive rats.

In the present study, we evaluated the relationship between the antihypertensive effect of sesamin, a lignan from sesame oil, and its antioxidative activity in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. After a 5-week treatment period, systolic blood pressure was significantly elevated in normal diet-fed DOCA-salt animals compared with cases in sham-operated animals. Sesamin feeding, tempol (a superoxide dismutase mimetic) treatment or antihypertensive drugs combination (triple therapy; reserpine, hydralazine, hydrochlorothiazide) significantly suppressed the development of DOCA-salt-induced hypertension. Compared with sham-operated rats, the normal diet-fed DOCA-salt rats revealed marked increases in aortic superoxide (O(2)(-)) production. These increases in O(2)(-) production were significantly suppressed by sesamin feeding or tempol treatment, but not by triple therapy. Acetylcholine (Ach)-induced endothelium-dependent relaxation was markedly decreased in normal diet-fed DOCA-salt rats, compared with cases in sham-operated rats. Sesamin feeding and triple therapy significantly improved the DOCA-salt-induced impairment of endothelium-dependent relaxation. However, tempol treatment had no effect on the impaired vasodilator responses induced by DOCA-salt treatment. In DOCA-salt rats with or without sesamin feeding, systolic blood pressure significantly correlated with both aortic O(2)(-) production and endothelium-dependent vascular relaxation. These findings suggest that sesamin feeding inhibits the enhancement of aortic O(2)(-) production in DOCA-salt hypertensive rats, and this effect may contribute to the antihypertensive effect of sesamin. Sesamin feeding-induced improvement of endothelial dysfunction seems to result from the above antioxidative and antihypertensive effects.

Influence of histamine in a liver injury model induced by Propionibacterium acnes and lipopolysaccharide.

In normal mice, plasma histamine levels were 29.4+/-10.1 pmol/ml. When 0.1 microg of lipopolysaccharide (LPS) was intravenously injected into Propionibacterium acnes (P. acnes)-primed ICR mice, histamine levels increased remarkably to 61.2+/-15.9 pmol/ml (p<0.001). An increase was also observed in liver tissues. Oral administration of histidine at 200 mg/kg once daily for 5 d before intravenous LPS injection increased the plasma alanine aminotransferase (ALT) activity to 2936.5+/-356.3 IU/l, a significant change compared with the controls (2244.8+/-425.5 IU/l, p<0.05). The 24 h survival rate after LPS injection was 72.7% in the mice treated with 50 mg/kg of ranitidine, in contrast with 50% in the control group although the treatment did not significantly decrease the plasma ALT activity. On the other hand, 50 mg/kg of pyrilamine significantly reduced plasma ALT activity (p<0.001). The results suggested that histamine levels are related to hepatic damage in the P. acnes plus LPS induction of liver injury.

Preventive effect of L-carnosine on ischemia/reperfusion-induced acute renal failure in rats.

We investigated the effect of L-carnosine (beta-alanyl-L-histidine) on ischemic acute renal failure in rats. Ischemic acute renal failure was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function in untreated acute renal failure rats markedly decreased at 1 day after reperfusion. Pre-ischemic treatment with L-carnosine dose-dependently (1, 10 microg/kg, i.v.) attenuated the ischemia/reperfusion-induced renal dysfunction. Histopathological examination of the kidney of untreated acute renal failure rats revealed severe renal damage, which was significantly suppressed by pre-treatment with L-carnosine, at each dose given. In untreated acute renal failure rats, norepinephrine concentrations in renal venous plasma remarkably increased within 2 min after reperfusion and thereafter rapidly decreased. Pre-ischemic treatment with L-carnosine at a dose of 10 microg/kg significantly depressed the elevated norepinephrine level. On the other hand, although the higher dose of L-carnosine given 5 min after reperfusion tended to ameliorate the renal dysfunction after reperfusion, the improvement was moderate compared with those seen in pre-ischemic treatment. These results indicate that L-carnosine prevents the development of ischemia/reperfusion-induced renal injury, and the effect is accompanied by suppression of the enhanced norepinephrine release in the kidney immediately after reperfusion. Thus, the preventing effect of L-carnosine on ischemic acute renal failure is probably through the suppression of enhanced renal sympathetic nerve activity induced by ischemia/reperfusion.

Hypoglycemic action of Cyclocarya paliurus (Batal.) Iljinskaja in normal and diabetic mice.

This study examined the hypoglycaemic activity of Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus) in ICR mice by oral glucose tolerance testing. The blood glucose level was significantly lower in the C. paliurus extract treatment group than in the control group after animals were given sucrose. This difference was not observed following the administration of glucose. We demonstrated that the chronological change in the level of blood glucose in genetically hyperglycemic obese KK-Ay mice is significantly lower when C. paliurus extract is administered daily for three weeks. An in vitro study showed that C. paliurus inhibits alpha-glucosidase, a disaccharide-degrading enzyme in the small intestinal mucosa, leading to a decrease in the absorption of glucose into the blood and a subsequent lowering of the blood glucose level.

Inhibitory effect of oolong tea on the oxidative state of low density lipoprotein (LDL).

In the present study, we investigated the anti-oxidant activity of oolong tea in an oxidation model using human low-density lipoprotein (LDL). Oolong tea suppressed the oxidation of LDL induced by 2-2'-azobis 4-methoxy-2,4-dimethyvaleronitrile (V70) in a dose-dependent manner, that is, it prolonged the lag time to 114.3%, 138% and 199.9% as compared with the control group at 0.5 microg/ml, 1.0 microg/ml, and 2.5 microg/ml, respectively. We also determined the scavenging effect of oolong tea on active oxygen radicals using the electron spin resonance (ESR) technique with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent. The intensity of the ESR signals for the DMPO-OOH adduct formed by the hypoxanthine/xanthine oxidase reaction system with DMPO decreased in the presence of oolong tea. The IC(50) of oolong tea was 19.9 microg/ml. These findings suggested that oolong tea has beneficial effects on health related to its anti-oxidative action.

Hypolipemic effect of Cyclocarya paliurus (Batal) Iljinskaja in lipid-loaded mice.

We investigated the inhibitory effect of Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus) extract on postprandial hyperlipemia in mice. A single oral administration of C. paliurus extract (250 mg/kg) suppressed an increase in plasma triacylgycerol (TG) levels when fed with 5 ml/kg of lard and olive oil. The inhibition rates were 28.6% and 24.1%, respectively, but free fatty acid (FFA) levels in plasma were not significantly affected as compared with control group mice. In addition, C. paliurus extract showed inhibitory activity toward pancreatic lipase, a key enzyme of dietary TG absorption, with an IC(50) of 9.1 microg/ml in vitro. Our results suggested that the hypolipemic action of C. paliurus extract was probably interrelated with suppression of the activity of digestive lipase, and as a result, the blood lipid level was reduced.

Effects of oolong tea on metabolism of plasma fat in mice under restraint stress.

We investigated the effects of oolong tea on the basic metabolism of plasma lipids in mice under restraint stress. When a lipid emulsion (Intralipid 20%; a lipid emulsion containing 20% soybean oil) was injected intravenously into mice, the restraint stress prolonged the half-life (T 1/2) of elimination for plasma triglyceride (TG) from 28.7 to 55.5 min. The elimination rate per minute was 48.2% in stressed mice with the rate in starved control mice as 100%. Therefore, TG metabolism was disrupted by the stress, and the use of TG as an energy source decreased. We found that the metabolism of lipids significantly response to the restrained stress in the present study. Plasma TG was 515.9 +/- 29.9mg/dl 35min after Intralipid administration in control stressed mice, 478.7 +/- 26.7 mg/dl in the stressed group given caffeine 100 mg/kg of body weight, and 418.3 +/- 18.4 mg/dl in the stressed group given 1,000 mg/kg oolong tea, an improvement by 7.2% and 18.9%, respectively, with the value for the untreated control group. The intake of oolong tea alleviated the stress-induced decrease in the rate of blood lipid metabolism; this effect may have arisen from some non-specific stress-relieving property of the tea or from acceleration of lipid metabolism by properties of polyphenols, etc. in tea. Oolong tea had anti-stress effects on plasma TG metabolism, and the effects did not depend on caffeine.