Termite repellent sesquiterpenoids from Callitris glaucophylla heartwood

Fractions of methanol and ethanol extracts from the heartwood of white cypress pine (Callitris glaucophylla Thompson et Johnson) were investigated for their repellent activity against subterranean termite Coptotermes formosanus Shiraki worker using a two-choice semicircular filter paper test at 0.5% (w/w) concentration. Fraction CY-E2 composed of (−)-citronellic acid, guaiol, α-, β-, and γ-eudesmol isomers as well as an unknown compound, showed the highest statistically significant repellency (97.8% ± 2.2 SEM) of all fractions tested. Bioactivity-guided fractionations using high-performance liquid chromatography led to the isolation of two, oxygenated eudesmane-type sesquiterpenes with α-methylene moieties, both termite-repellent compounds. These compounds were subsequently identified as ilicic acid methyl ester (IAME) and costic acid by means of spectroscopic analyses, electron impact mass spectrometry, and nuclear magnetic resonance spectroscopy. We report the isolation of both IAME and costic acid from C. glaucophylla heartwood for the first time. Part of this paper was presented at the 47th Symposium on the Chemistry of Terpenes, Essential Oils, and Aromatics, Tokyo, November 2003     

Genetic diversity of Populus euphratica populations in northwestern China determined by RAPD DNA analysis

Five Populus euphratica Oliver populations in northwestern China were analyzed using RAPD DNA markers to determine genetic diversity among and within populations. One hundred-and-five polymorphic bands were observed, ranging in size from 250 bp to 1700 bp, using 10 primers. Only one population on the north side of the Tianshern Range had a unique band common to all individuals that was not found in individuals from populations in the Tarim River valley. Intra-population genetic diversity was high in two populations along the Tarim River and low in the other three populations. There was no significant correlation between genetic distances and geographic distances. The result of correspondence analysis shows that the individuals from the three populations with low genetic diversity are isolated from each other. The result of cluster analysis based on genetic distance shows that the population in the Tianshern Range is genetically distant from the other populations. These results suggest that the Tianshern Range population was genetically isolated from the other populations.     

Detecting endotoxin activity in bovine serum using an automated testing system

The aim of the present study was to compare the ability of the commercially available portable test system (PTS^TM) to detect endotoxin activity in bovine serum, with that of the traditional LAL-kinetic turbidimetric (KT) and chromogenic (KC) assays. Prior to testing, serum samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTS^TM was not significantly different from that of the traditional LAL-based assays. The results using PTS^TM correlated with those using KT (r²=0.963, P<0.001) or KC assays (r²=0.982, P<0.001). Based on these findings, the PTS^TM could be applied as a simplified system to assess endotoxin activity in bovine serum.     

Effect of ovarian hormones on maturation of dendritic cells from peripheral blood monocytes in dogs

Previously, we reported that ovarian hormones affect the immune response against E. coli isolated from the dogs affected with pyometra. In order to investigate mechanisms underlying the immune modulation, we examined the effects of ovarian hormones on the generation of dendritic cells (DCs), the most potent antigen presenting cell. DCs were differentiated from peripheral blood monocytes (PBMOs) using a cytokine cocktail. Both estrogen receptor and progesterone receptors were expressed by the PBMOs and immature DCs. When various ovarian hormones were added to the culture for the DC differentiation, progesterone significantly decreased the expression of DC maturation markers, such as CD1a, CD80 and CD86, on mature DCs. Conversely, the addition of estrogen to the cultures increased the expression of CD86, but not other maturation makers. Furthermore, DCs differentiated in the presence of progesterone did not stimulate allogeneic mononuclear cells in PB. Taken together, these results indicate that progesterone diminishes the maturation of DCs, leading to decreased immune responses against invading pathogens.     

Accessory corpora lutea formation in pregnant Hokkaido sika deer (Cervus nippon yesoensis) investigated by examination of ovarian dynamics and steroid hormone concentrations

Generally, sika deer conceive a single fetus, but approximately 80% of pregnant females have two corpora lutea (CLs). The function of the accessory CL (ACL) is unknown; moreover, the process of ACL formation is unclear, and understanding this is necessary to know its role. To elucidate the process of ACL formation, the ovarian dynamics of six adult Hokkaido sika deer females were examined ultrasonographically together with peripheral estradiol-17β and progesterone concentrations. ACLs formed in three females that conceived at the first estrus of the breeding season, but not in those females that conceived at the second estrus. After copulation, postconception ovulation of the dominant follicle of the first wave is induced by an increase in estradiol-17β, which leads to formation of an ACL. A relatively low concentration of progesterone after the first estrus of the breeding season is considered to be responsible for the increase in estradiol-17β after copulation.     

Specific detection of potentially allergenic kiwifruit in foods using polymerase chain reaction

Kiwifruit (Actinidia deliciosa and Actinidia chinensis) is allergenic to sensitive patients, and, under Japanese regulations, it is one of the food items that are recommended to be declared on food labeling as much as possible. To develop PCR-based methods for the detection of trace amounts of kiwifruit in foods, two primer pairs targeting the ITS-1 region of the Actinidia spp. were designed using PCR simulation software. On the basis of the known distribution of a major kiwifruit allergen (actinidin) within the Actinidia spp., as well as of reports on clinical and immunological cross-reactivities, one of the primer pairs was designed to detect all Actinidia spp. and the other to detect commercially grown Actinidia spp. (i.e., kiwifruit, Actinidia arguta, and their interspecific hybrids) except for Actinidia polygama. The specificity of the developed methods using the designed primer pairs was verified by performing PCR experiments on 8 Actinidia spp. and 26 other plants including fruits. The methods were considered to be specific enough to yield target-size products only from the target Actinidia spp. and to detect no target-size products from nontarget species. The methods were sensitive enough to detect 5-50 fg of Actinidia spp. DNA spiked in 50 ng of salmon testis DNA used as a carrier (1-10 ppm of kiwifruit DNA) and 1700 ppm (w/w) of fresh kiwifruit puree spiked in a commercial plain yogurt (corresponding to ca. 10 ppm of kiwifruit protein). These methods would be expected to be useful in the detection of hidden kiwifruit and its related species in processed foods.     

Determination of theaflavins including methylated theaflavins in black tea leaves by solid-phase extraction and HPLC analysis

A quantitative method for four theaflavins and two methylated theaflavin derivatives in black tea leaves was developed by solid-phase extraction and a high-performance liquid chromatographic method with photodiode array detection. The theaflavins in black tea leaves were extracted three times with 40 vol 50% aqueous ethanol (mg dry tea powder/mL) containing 2% ascorbic acid. The ethanol extracts were diluted 4-fold with distilled water. All diluted extracts were directly applied to the solid-phase C18 cartridge column without concentration. The fraction of theaflavins was obtained by 40% ethanol extraction after rinsing with water followed with 15% ethanol extraction. An aliquot of theaflavins after concentration was injected onto an ODS C18 reversed-phase column, and four theaflavins and two methylated theaflavins were sufficiently separated by a linear gradient system using distilled water and acetonitrile with 0.5% acetic acid. This analytical method is sensitive for the determination of a small amount of methylated theaflavins, since various interfering substances produced during the fermentation process were eliminated in advance by solid-phase extraction. Using this analytical method, we also demonstrated that methylated theaflavins were easily produced during the manufacture of black tea.