Flavonol Caffeoylglycosides as alpha-Glucosidase Inhibitors from Spiraea cantoniensis Flower

In the screening experiments for rat intestinal alpha-glucosidase inhibitors in 218 plants cultivated in the Japanese temperate region, potent maltase-inhibiting activity was found in the extract of flowers of Spiraea cantoniensis. The enzyme assay guided fractionation of the extract led to the isolation of three flavonol caffeoylglycosides, quercetin 3- O-(6- O-caffeoyl)-beta-galactoside ( 1), kaempferol 3- O-(6- O-caffeoyl)-beta-galactoside ( 2), and kaempferol 3- O-(6- O-caffeoyl)-beta-glucoside ( 3), as rat intestinal maltase inhibitors. This is the first report on the alpha-glucosidase-inhibitory activity of those flavonol caffeoylglycosides. Comparison in the activity of the isolates indicated the importance of caffeoyl substructures in the molecule for the alpha-glucosidase-inhibiting activity. The relatively high contents of the active isolates in the plant suggest that S. cantoniensis could be physiologically useful for treatment of diabetes.     

Validation of the sperm quality analyzer and the hypo-osmotic swelling test for frozen-thawed ram and minke whale (Balaenoptera bonarensis) spermatozoa

The object of the present study was to investigate the validation of the sperm quality analyzer (SQA) and the hypo-osmotic swelling (HOS) test with standard sperm analysis methods in frozen-thawed ram and minke whale spermatozoa. In rams, highly significant correlations were observed in the percentage of motile spermatozoa (P<0.01) and sperm concentration (P<0.01) between the standard and SQA methods. But, the percentage of morphologically normal spermatozoa did not significantly correlate between the standard and SQA methods. The percentages of swollen spermatozoa at 15 minutes by the HOS test were significantly correlated with the motility by the standard (P<0.05) and by the SQA (P<0.05) methods. For minke whale spermatozoa, the SVI (sperm viability index) values by the standard method were significantly (P<0.001) correlated with the sperm motility index (SMI) values by SQA. The percentage of motile spermatozoa was also significantly correlated (P<0.01) with the motility measured by SQA. Using different hypo-osmotic solutions and incubation times, the HOS test with 25, 100 and 150 mOsM did not show significant variations. Motility observed by the standard method and the percentage of swollen spermatozoa were significantly correlated (P<0.05). These results indicate that the SQA and HOS test can be utilized to assess the post-thawing motility of ram and minke whale spermatozoa, and that the SQA and HOS test values are significantly correlated in ram spermatozoa. However, sperm concentration and morphologically normal spermatozoa are not assessed accurately by SQA in minke whales.     

Expression levels of taste‐related genes in palate and tongue tip,and involvement of transient receptor potential subfamily M member 5 (TRPM5) in taste sense in chickens

The elucidation of the mechanisms underlying the taste sense of chickens will contribute to improvements in poultry feeding, because the molecular mechanism of chickens’ taste sense defines the feeding behavior of chickens. Here we focused on the gene expressions in two different oral tissues of chickens – the palate, which contains many taste buds, and the tongue tip, which contains few taste buds. Using the quantitative real‐time polymerase chain reaction method, we found that the molecular markers for taste buds of chickens, that is α‐gustducin and vimentin, were expressed significantly highly in the palate compared to the tongue tip. Our analyses also revealed that transient receptor potential subfamily M member 5 (TRPM5), a cation channel involved in taste transduction in mammals, was also highly expressed in the palate compared to the tongue tip. Our findings demonstrated that the expression patterns of these genes were significantly correlated. We showed that the aversion to bitter solution was alleviated by a TRPM5 inhibitor in behavior of chickens. Taken together, our findings enabled us to develop a simple method for screening taste‐related genes in chickens. The use of this method demonstrated that TRPM5 was involved in chickens’ taste transduction, and that a TRPM5 inhibitor can alleviate chickens’ bitter taste perception of feed ingredients.     

Effects of a meal rich in 1,3-diacylglycerol on postprandial cardiovascular risk factors and the glucose-dependent insulinotropic polypeptide in subjects with high fasting triacylglycerol concentrations

It was previously reported that compared to triacylglycerol (TAG) oil, diacylglycerol (DAG) oil improves postprandial lipid response. However, the effects of DAG oil on postprandial hyperglycemia and incretin response have not yet been determined. In this study, the effects of DAG oil on both postprandial hyperlipidemia and hyperglycemia and the response to the glucose-dependent insulinotropic polypeptide (GIP) were studied. This randomized, double-blind, crossover study analyzed data for 41 individuals with high fasting triacylglycerol concentrations. The subjects ingested test meals (30.3 g of protein, 18.6 g of fat, and 50.1 g of carbohydrate) containing 10 g of DAG oil (DAG meal) or TAG oil (TAG meal) after fasting for at least 12 h. Blood samples were collected prior to and 0.5, 2, 3, 4, and 6 h after ingestion of the test meal. Postprandial TAG concentrations were significantly lower after the DAG meal compared with the TAG meal. Postprandial TAG, insulin, and GIP concentrations were significantly lower after the DAG meal compared with the TAG meal in 26 subjects with fasting serum TAG levels between 1.36 and 2.83 mmol/L. DAG-oil-based meals, as a replacement for TAG oil, may provide cardiovascular benefits in high-risk individuals by limiting lipid and insulin excursions.     

Effect of echo-guided high-intensity focused ultrasound ablation on localized experimental tumors

We fabricated a prototype 3.25-MHz split-focus therapeutic transducer combined with a small 6.5-MHz imaging ultrasonic probe for transrectal treatment of prostate cancer and evaluated the feasibility of using split-focus high-intensity focused ultrasound (HIFU) to ablate localized tumor tissue without injuring the surrounding organs. We therefore established a localized tumor model by inoculating VX2 tumor into rabbit livers. The localized VX2 tumors of nine rabbits were transdermally treated with split-focus ablation at a peak intensity in water of 6 kW/cm2 for 4 s (6 shots) under the guidance of ultrasonic B-mode imaging. Necropsy a day after treatment found the surface of the livers and gastrointestinal tracts to be grossly normal. The VX2 tumors were completely coagulated and were surrounded by ablated liver tissue. The six shots of split-focus HIFU destroyed the VX2 tumors without injuring the liver surfaces or the surrounding organs. These results suggest that split-focus HIFU ablation could be an effective treatment of localized tumors.     

Expression and localization of alpha-tubulin N-acetyltransferase 1 in the reproductive system of male mice

The structure of microtubules is essential for the fertilizing ability of spermatozoa. Acetylation of α-tubulin plays an important role in flagellar elongation and spermatozoa motility. Previous reports have suggested that alpha-tubulin N-acetyltransferase 1 (ATAT1) is the main acetyltransferase involved in the acetylation of α-tubulin. Although ATAT1 is reported to express in the testis, no information is available regarding its expression in elongated spermatids, epididymis, and mature spermatozoa. Hence, it remains unclear whether ATAT1 is involved in spermatozoa maturation and capacitation. Therefore, we evaluated the expression of ATAT1 in the mouse male reproductive system using immunostaining and western blotting. Our results showed that ATAT1 was expressed in spermatids during spermiogenesis in mouse testes, but its expression varied according to the seminiferous tubule stage. We observed ATAT1 in the cytoplasm of round spermatids, the flagella of elongated spermatids, and in the cytoplasm of step 16 spermatids, just before its release into the lumen. In addition, ATAT1 was expressed in epithelial cells of the epididymis. In spermatozoa of the cauda epididymis, ATAT1 expression was primarily observed in the midpiece of the spermatozoa. The localization of ATAT1 protein in the male germline was observed during spermiogenesis as well as during spermatozoa maturation. Our results suggest that ATAT1 may be involved in the formation of flagella and in the acetylation process, which has attracted attention in recent years regarding male infertility.     

PDE4 and PDE5 regulate cyclic nucleotide contents and relaxing effects on carbachol-induced contraction in the bovine abomasum

The effects of various selective phosphodiesterase (PDE) inhibitors on carbachol (CCh)-induced contraction in the bovine abomasum were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4), vardenafil (type 5), BRL-50481 (type 7) and BAY73-6691 (type 9), inhibited CCh-induced contractions in a concentration-dependent manner. Among the PDE inhibitors, Ro20-1724 and vardenafil induced more relaxation than the other inhibitors based on the data for the IC₅₀ or maximum relaxation. In smooth muscle of the bovine abomasum, we showed the expression of PDE4B, 4C, 4D and 5 by RT-PCR analysis. In the presence of CCh, Ro20-1724 increased the cAMP content, but not the cGMP content. By contrast, vardenafil increased the cGMP content, but not the cAMP content. These results suggest that Ro20-1724-induced relaxation was correlated with cAMP and that vardenafil-induced relaxation was correlated with cGMP in the bovine abomasum. In conclusion, PDE4 and PDE5 are the enzymes involved in regulation of the relaxation associated with cAMP and cGMP, respectively, in the bovine abomasum.     

Canine mdr1 gene mutation in Japan

Frequency of the 4-bp deletion mutant in canine mdr1 gene was examined in 193 dogs of eight breeds in Japan. The mutant allele was found in Collies, Australian Shepherds, and Shetland Sheepdogs, where its respective frequencies were 58.3%, 33.3%, and 1.2%. The MDR1 protein was detected on peripheral blood mononuclear cells (PBMC) from a MDR1/MDR1 dog, but not on PBMC from a mdr1-1Delta/mdr1-1Delta Collie. Rhodamine 123 was extruded from MDR1/MDR1 lymphocytes. That excretion was inhibited by a MDR1 inhibitor, verapamil. On the other hand, Rh123 excretion was not observed from lymphocytes derived from a mdr1-1Delta/mdr1-1Delta Collie. These results indicated that the mutant mdr1 allele also existed in Collie-breed dogs in Japan at high rates and that mdr1-1Delta /mdr1-1Delta dogs have no functional MDR1.     

Effect of Feeding Frequency, Water Temperature, and Stocking Density on the Growth of Tiger Puffer, Takifugu rubripes

Effects of daily feeding frequency, water temperature, and stocking density on the growth of tiger puffer, Takifugu rubripes, fry were examined to develop effective techniques to produce tiger puffer in a closed recirculation system. Fish of 4, 14, and 180 g in initial body weight were fed commercial pellet diets once to five times a day to apparent satiation each by hand for 8 or 12 wk at 20 C. Daily feeding frequency did not affect the growth of 14‐ and 180‐g‐size fish. However, the daily feed consumption and weight gain of the 4‐g‐size fish fed three and five times daily were significantly higher than those of fish fed once daily (P < 0.05). Fish of 4 and 50 g in initial body weight were reared with the pellet diet at 15–30 C for 8 wk. The weight gain of fish increased with increasing water temperature up to 25 C and decreased drastically at 30 C for both sizes. Similar trends were observed for feed efficiency, although 4‐g fish had highest efficiency at 20 C. Effects of stocking density on growth were examined with fish of 8, 13, and 100 g in initial body weight. Fish were reared with the pellet diet for 8 or 16 wk at 20 C. Fish were placed in floating net cages in the culture tank, and the stocking density was determined based on the total weight of fish and volume of the net cage. Fish of 8 g in body weight grew up to 35–36 g during the 8‐wk rearing period independent of the stocking density of 8, 15, and 31 kg/m³ at the end of rearing. Final biomass per cage reached 32, 60, and 115 kg/m³ for 13‐g‐size fish, and 10, 18, and 35 kg/m³ for 100‐g‐size fish, and the growth of the fish tended to decrease with increasing stocking density for both sizes.