α-Amylase expressed in human small intestinal epithelial cells is essential for cell proliferation and differentiation.

α-Amylase, which plays an essential role in starch degradation, is expressed mainly in the pancreas and salivary glands. Human α-amylase is also detected in other tissues, but it is unclear whether the α-amylase is endogenously expressed in each tissue or mixed exogenously with one expressed by the pancreas or salivary glands. Furthermore, the biological significance of these α-amylases detected in tissues other than the pancreas and salivary glands has not been elucidated. We discovered that human α-amylase is expressed in intestinal epithelial cells and analyzed the effects of suppressing α-amylase expression. α-Amylase was found to be expressed at the second-highest messenger RNA level in the duodenum in human normal tissues after the pancreas. α-Amylase was detected in the cell extract of Caco-2 intestinal epithelial cells but not secreted into the culture medium. The amount of α-amylase expressed increased depending on the length of the culture of Caco-2 cells, suggesting that α-amylase is expressed in small intestine epithelial cells rather than the colon because the cells differentiate spontaneously upon reaching confluence in culture to exhibit the characteristics of small intestinal epithelial cells rather than colon cells. The α-amylase expressed in Caco-2 cells had enzymatic activity and was identified as AMY2B, one of the two isoforms of pancreatic α-amylase. The suppression of α-amylase expression by small interfering RNA inhibited cell differentiation and proliferation. These results demonstrate for the first time that α-amylase is expressed in human intestinal epithelial cells and affects cell proliferation and differentiation. This α-amylase may induce the proliferation and differentiation of small intestine epithelial cells, supporting a rapid turnover of cells to maintain a healthy intestinal lumen.

Towards identification of bioactive compounds in cold vacuum extracted double cherry blossom (Gosen-Sakura) leaves.

The present research examines the possibility of finding bio-molecular compounds from the double cherry blossom (termed as 'Gosen-Sakura' of Gosen-city, Niigata-prefecture, Japan) leaves, which have been long used in the preparation of the traditional Japanese sweet (wagashi) - 'sakura-mochi'. Based on its indicated anti-microbial properties historically, our study provides a new low temperature vacuum extraction method for extracting 'near natural form of water soluble leaf (cell) extracts from the Gosen-Sakura, and demonstrates the presence of some 'novel' compound(s) with anti-tumor cell lines proliferation inhibitory affects through the MTT assay. To our knowledge, no reports exist on the sakura tree 'leaf (cell) extracts' inhibiting tumor cell line growth. We further examined and compared the effects of known compounds with anti-tumor activity, coumarin and benzyl alcohol with Gosen-Sakura leaf extract; results lead us to hypothesize that the Gosen-Sakura leaf extract contains substance(s) other than the above two known compounds, with antitumor effect. Additionally, we speculate on the underlying mechanism of action of the Gosen-Sakura leaf extract by targeting cell division at the point of DNA synthesis and causing apoptosis. In conclusion, we present scientific evidence on the presence of certain 'novel' biomolecule(s), with anti-tumor activity, in the Gosen-Sakura leaf which has been long used in Japanese sweet - the 'sakura-mochi'.

Biodistribution/biostability assessment of siRNA after intravenous and intratracheal administration to mice, based on comprehensive analysis of in vivo/ex vivo/polyacrylamide gel electrophoresis fluorescence imaging.

We performed in vivo/ex vivo/polyacrylamide gel electrophoresis (PAGE) fluorescence imaging of near-infrared fluorescence (NIRF)-labeled siRNA (Cy5.5-siGL3) in mice to investigate the validity of each fluorescence imaging result as the biodistribution/biostability assessment of siRNA. Statistically significant correlations could be obtained between the in vivo and ex vivo fluorescence intensities of Cy5.5 in the relevant regions/tissues, except the lung region/tissue after intravenous administration. On PAGE fluorescence images with the naked formulation, there was no band corresponding to intact Cy5.5-siGL3 from all the tissues evaluated after intravenous administration, indicating that the fluorescence detected by in vivo and ex vivo fluorescence imaging was derived from degraded Cy5.5-siGL3 or free Cy5.5 cleaved from Cy5.5-siGL3. However, the band was detected from the lungs after intratracheal administration of the naked formulation, confirming higher stability of siRNA on the respiratory epithelium than in the blood. Regarding the polyethyleneimine formulation, the band was detected from all the tissues evaluated after intravenous administration and from the lungs after intratracheal administration, verifying the enhanced stability of siRNA in the body. These results clearly indicated the necessity of comprehensive analysis from in vivo/ex vivo/PAGE fluorescence imaging to precisely assess the distribution and stability of NIRF-labeled oligonucleotides including siRNA in the body.

Long term dietary supplementation with zeaxanthin reduces photoreceptor death in light-damaged Japanese quail.

The purpose of these studies was to evaluate the effects of light damage on Japanese quail whose retinal carotenoids had been experimentally manipulated through altered diets. The birds were raised 6 months on a commercial turkey diet (T), on a custom carotenoid-deficient diet (C-) containing 90% less carotenoid than the T diet, or on Z+ diet [the C- diet supplemented with natural zeaxanthin (35mgkg(-1) food)]. Equal numbers of males and females on each diet were exposed to nine intervals (1hr on, 2hr off) of 3200lux cool white light, then placed in the dark for 14hr before tissue collection. One retina was immediately frozen for HPLC analysis; the other eye was immediately fixed and processed for microscopy. There were no significant differences in the retinal carotenoid concentrations in hatch-mates that were and were not exposed to light. Supplementation resulted in three- to four-fold increases in retinal zeaxanthin and no change in retinal lutein or alpha-tocopherol, but the C- diet did not reduce the retinal carotenoid concentration in C- birds below that in T birds. The light-exposed retinas had significant numbers of apoptotic photoreceptors and photoreceptor ghosts. The number of ghosts was negatively correlated with the number of dying photoreceptors (P<0.05), and with retinal concentrations of zeaxanthin, alpha-tocopherol or gamma-tocopherol (P<0.04, 0.02, 0.04, respectively), but not with lutein. The number of dying photoreceptors was positively correlated with alpha-tocopherol and the sum alpha-tocopherol plus zeaxanthin (P<0.1; P0.04). Photoreceptor death was semi-quantitatively scored, assuming that ghosts were formed by removal of apoptotic photoreceptors with nuclear condensation. Stepwise regression produced a good model (r(2)=0.67;P <0.0001) for predicting death scores from retinal concentrations of zeaxanthin (Standard Coefficient=-0.76) and lutein (Standard Coefficients=+0.43). Absence of lutein in gender-specific analyses suggests lutein served as surrogate marker for gender. Combined analysis of the C- and T birds also demonstrated that dying photoreceptors were negatively correlated with retinal zeaxanthin. These data confirm our previous report that retinal carotenoids prevent photoreceptor cell death, and provide the first direct evidence that retinal zeaxanthin protects photoreceptors from light-induced death.

Elevated retinal zeaxanthin and prevention of light-induced photoreceptor cell death in quail.

PURPOSE: Inferential evidence indicates that macular pigments (lutein and zeaxanthin) protect photoreceptors and/or retard age-related macular degeneration. These experiments tested the hypothesis that retinal zeaxanthin prevents light-induced photoreceptor cell death. METHODS: Retinal damage was assessed in quail fed a carotenoid-deficient (C-) diet for 6 months. Groups of 16 birds (8 male, 8 female) were fed a C- diet supplemented with 35 mg 3R,3'R-zeaxanthin for 1, 3, or 7 days; one group was continued on C- diets. Half of each group was exposed to intermittent 3200-lux white light (10 1-hour intervals separated by 2 hours in dark). After 14 additional hours in the dark, one retina of each quail was collected for HPLC analysis, and the contralateral retina was embedded in paraffin for counts of apoptotic nuclei. RESULTS: After 7 days' supplementation, concentrations of zeaxanthin in serum, liver, and fat had increased by factors of 50.8, 43.2, and 6.5, respectively (all P < 0.001). In contrast, retinal zeaxanthin fluctuated significantly upward on day 3, but there was no net change on day 7. The number of apoptotic rods and cones in light-damaged eyes correlated significantly and inversely with zeaxanthin concentration in the contralateral retina (r = -0.61; P < 0.0001 and r = -0.54; P < 0.002), but not with serum zeaxanthin. Similar correlations were observed with retinal lutein, which correlated strongly with retinal zeaxanthin (r = 0.95; P < 0.0001). CONCLUSIONS: Retinal zeaxanthin dose dependently reduced light-induced photoreceptor apoptosis; elevated serum levels did not. These data provide the first experimental evidence that xanthophyll carotenoids protect photoreceptors in vivo.

Effect of dietary zeaxanthin on tissue distribution of zeaxanthin and lutein in quail.

PURPOSE: The xanthophyll carotenoids (lutein and zeaxanthin) are hypothesized to delay progression of age-related macular degeneration. The quail has a cone-dominant retina that accumulates carotenoids. The purpose of these experiments was to characterize the carotenoid composition of retina, serum, liver, and fat in quail and to determine whether dietary enrichment with zeaxanthin alters zeaxanthin or lutein concentrations in these tissues. METHODS: Quail were fed for 6 months with a commercial turkey diet (T group; n = 8), carotenoid-deficient diet (C- group; n = 8), or a carotenoid-deficient diet supplemented with 35 mg 3R,3'R-zeaxanthin per kilogram of food, (Z+ group; n = 8). Zeaxanthin was derived from Sphingobacterium multivorum (basonym Flavobacterium). Carotenoids in serum, retina, liver, and fat were analyzed by HPLC. RESULTS: As in the primate fovea, the retina accumulated zeaxanthin, lutein, and cryptoxanthin, and preferentially absorbed zeaxanthin (P < 0.005). In contrast, lutein was preferentially absorbed by liver (P < 0.01) and fat (P < 0.0001). In supplemented females, zeaxanthin increased approximately 4-fold in retina, and 74-, 63- and 22-fold in serum, liver, and fat, respectively. In males, zeaxanthin was elevated approximately 3-fold in retina, and 42-, 17-, and 12-fold in serum, liver, and fat, respectively. Birds fed the Z+ diet absorbed a higher fraction of dietary lutein into serum, but lutein was reduced in the retina (P < 0.05). CONCLUSIONS: Xanthophyll profiles in quail mimic those in primates. Dietary supplements of zeaxanthin effectively increased zeaxanthin concentrations in serum, retina, liver, and fat. The robust response to zeaxanthin supplementation identifies the quail as an animal model for exploration of factors regulating delivery of dietary carotenoids to the retina.