Isolation,structural elucidation,and inhibitory effects of terpenoid and lipid constituents from sunflower pollen on Epstein-Barr virus early antigen induced by tumor promoter,TPA

Eight fatty acid esters of triterpene alcohols (1-8), four free triterpene alcohols (9, 12, 17, and 18), four diterpene acids (19-22), two tocopherol-related compounds (23 and 24), four estolides (25-28), three syn-alkane-4,6-diols (29-31), one 1,3-dioxoalkanoic acid (32), and one aliphatic ketone (33), along with the mixture of free fatty acids, were isolated from the diethyl ether extract of the pollen grains of sunflower (Helianthus annuus). Among these compounds, 14 (2-8, 12, 23, 25-28, and 33) were new naturally occurring compounds, and their structures were determined on the basis of spectroscopic methods. Twenty-four terpenoids and lipids (1-4, 6-9, 12, and 19-33) and six free triterpene triols (10, 11, and 13-16), derived from their fatty acid esters (2, 3, and 5-8) by alkaline hydrolysis, were evaluated with respect to their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), in Raji cells, which is known to be a primary screening test for antitumor promoters. Among the 30 compounds tested, 21 compounds possessing a di- or a polycyclic ring system in the molecule (1-4, 6-16, and 19-24) showed potent inhibitory effects on EBV-EA induction (91-100% inhibition at 1 x 10(3) mol ratio/TPA).     

Chemical Characterization of Acid Fog and Rain in Northern Japan Using Back Trajectory and Oblique Rotational Factor Analysis

Fog/cloud and rain water were collected at the mountainside of Hachimantai range in northern Japan and rain water was also collected at Akita City in order to investigate the air pollutant scavenging mechanism. The concentrations of various ions in these samples were analyzed, and the fog drop size and the wind direction were measured at each fog event. The fog at Hachimantai range had a very high total ion concentration, and was considerably acidified by non sea salt (nss-) SO₄ ^2? and NO₃ ^?, compared with the rain at Akita and all sites in Hachimantai range. Using the oblique rotational factor analysis, three factors were extracted as the air pollutants; A: (NH₄)₂SO₄+H₂SO₄, B: sea salts+HNO₃+H₂SO₄, C: NH₄NO₃+OH^?. These salts are well-known as the cloud condensation nuclei (CCN). Combining the factor analysis with the 72h back trajectory at 850hPa level, the contribution of Factor A was closely connected to the long-range transportation of anthropogenic or natural aerosol in air masses of continental origin.     

Ca Depletion in the Soil Column on a Granite Substrate on the Island of Yakushima, a World Natural Heritage Site

We investigated the vertical profile of element distributions in the soil column on a granite substrate at three sites on the island of Yakushima, a world natural heritage site in southwestern Japan, where stream acidification due to acid rain is of increasing concern. Statistical analysis shows that the elements in the granitic soil can be classified into three groups according to their mineralogical derivations: Group 1 (K, Mg, Mn, Rb, and Nb), group 2 (Fe, Ti, Zr, Cr, and Pb), and group 3 (Ca, Na, Sr, and P), which are related to the presence of K-feldspar and K-mica, Fe-Ti oxides, and plagioclase and apatite, respectively. The pattern of distribution of group-1 elements in the soil column differs slightly among the sites because of differences in the proportions of K-feldspar and K-mica, The organic surface soil is enriched in group-2 elements and shows a strong magnetic susceptibility, indicating the presence of biogenetic magnetite. At all sites, Ca and Sr are intensely depleted due to the selective dissolution of plagioclase. Ca depletion compared to the original granite reaches more than 90 % in the C horizon, resulting in a decreasing capacity for the neutralization of acid rain and an increasing contribution of atmospheric Ca to the surface soil and vegetation.     

Effect of the addition of <Emphasis Type="Italic">Acacia mangium</Emphasis> bark on thermosetting of phenol–formaldehyde resin

The effect of addition of Acacia mangium bark powder on the thermosetting processes of two commercial phenol resins, PF-A and PF-B, was examined by bond strength test, torsional braid analysis, and differential scanning calorimetry. When the bark powder was added to PF-A, the bond strength of plywood pressed at 110°C increased and was comparable to that of plywood pressed at 120 and 130°C. However, when the bark powder was added to PF-B, the bond strength of plywood pressed at 110°C was still lower than that of plywood pressed at 120 and 130°C. The relative rigidity and loss tangent of PF-A cured with the bark powder obtained by heating at 100°C were comparable to those at 120 and 140°C, and the reaction enthalpy was increased by bark powder addition. In contrast, chemical reactions for cured PF-B were not enhanced by bark powder addition.     

Excess polyspermy reduces the ability of porcine oocytes to promote male pronuclear formation after in vitro fertilization

Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = −0.560, p < 0.05) and in the sperm lot of moderate polyspermy (R = −0.405, p < 0.05) which suggests that penetration of excessive spermatozoa disables the oocyte cytoplasm to promote MPN formation.     

Bilateral cataract in a cynomolgus monkey

Severe bilateral cataract was found in a 7 year-old naïve female cynomolgus monkey (Macaca fascicularis) 3 months before necropsy. During macroscopic examination, severe opacity and thinning of the lens were observed in both eyes. Histopathology revealed that the lens nuclei and majority of cortex lens fibers had disappeared and become excavated, while the lens fibers in the subcapsular area were swollen and distorted. Other observations included atrophy and vacuolation in the lens epithelial cells and proliferation of spindle cells and collagen fiber beneath the anterior capsule of the right eye. Immunohistochemical staining of these spindle cells revealed the presence of vimentin, cytokeratin and α-smooth muscle actin (α-SMA), which were considered to be derived from lens epithelial cells. This is a rare case of spontaneous, bilateral, hypermature cataract in a cynomolgus monkey.     

Effects of the estrous cycle on the efficacy of oocyte collection and in vitro embryo production in Duroc‐breed

Collection efficacy and in vitro embryo developmental ability of oocytes obtained from Duroc‐breed ovary donors at different stages of the estrous cycle (days 6, 12 and 16 after estrus) were performed. The numbers of collected oocytes did not differ significantly among the different estrous cycle groups (total 72–90 oocytes per gilt). However, the blastocyst rates of oocytes collected on days 12 and 16 (9.2% and 19.4%, respectively) were significantly higher than those on day 6 (1.1%). More oocytes were obtained on day 16 from small follicles (<2 mm in diameter; 85.3 oocytes per gilt) than from medium‐sized (≥2–<6 mm) and large (≥6 mm) follicles (17.5 and 12.8 oocytes, respectively). The blastocyst rates in both the medium‐sized and large follicle groups (20.0% and 19.2%, respectively) were significantly higher than that in the small follicle group (6.3%). The blastocyst cell numbers in both the medium‐sized and large follicle groups (39.4 and 43.3 cells, respectively) were significantly higher than that in the small follicle group (30.6 cells). The results suggest that oocyte collection from cycling Duroc pigs can be carried out efficiently from the late luteal to follicular stage. Those oocytes collected from medium‐sized and large follicles show better embryo development.     

Corneal Mineralization in Wistar Hannover Rats

We have recently started using Wistar Hannover rats in Japan and are now collecting background data. We have been frequently observing corneal mineralization in Wistar Hannover rats of both the RccHan^TM:WIST and Crl:WI (Han) strains. In this study, details of corneal mineralization in Wistar Hannover rats were histopathologically and ultrastructurally investigated. According to the results, Wistar Hannover rats had a much higher incidence of corneal mineralization compared with Sprague-Dawley rats. The incidence of corneal mineralization was higher in males than females. According to the histological examination, mineral deposits were positive for calcium by von Kossa’s method. Furthermore, in response to mineralization, keratocytes probably become active to play an important role against the mineralized substance.     

Fertilization Ability of Porcine Oocytes Reconstructed from Ooplasmic Fragments Produced and Characterized after Serial Centrifugations

Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos^® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.