Growth of recombinant equine herpesvirus 1 (EHV-1) replaced with passage-induced mutant gene 1 and gene 71 derived from an attenuated EHV-1 in cell cultures and in the lungs of mice

The relationship of passage-induced mutant genes 1 and 71 of an attenuated equine herpesvirus 1 (EHV-1) with virulence was analysed by constructing nine recombinant EHV-1 viruses by homologous recombination. Gene 1 or/and gene 71 of a virulent EHV-1 strain, HH1, was replaced by a mutant gene 1 or/and 71 of an attenuated HH1 strain, BK343, respectively. The beta-galactosidase gene of Escherichia coli was inserted within the gene 1 or 71 coding sequence of HH1 to inactivate the genes. Virus replications of these recombinant viruses in cell cultures were similar, but release of the gene 71-inactivated virus from infected cells was delayed compared to that of the other viruses. Plaque sizes of the recombinant viruses were similar to those of HH1, but those of BK343 were significantly smaller, indicating an effect of some unknown factor(s) on viral cell-to-cell spread. The growth abilities of the recombinant viruses with a mutant gene 1 or/and 71 in lungs of mice were similar to those of HH1, but those of gene 71-inactivated viruses were reduced to the level of BK343 and the titers were about 100-times lower than those of the other recombinant viruses. These results indicate that the mutant genes 1 and 71 of BK343 might not confer an attenuated nature to EHV-1.     

Genomic analysis of Theileria sergenti stocks in Japan with DNA probes.

Restriction fragment length polymorphisms of Theileria sergenti DNA from 18 different infections of cattle in 14 locations in Japan were analyzed by Southern blotting using T. sergenti genomic DNA fragments as probes. Probe pTs 2 hybridized with four fragments in BamHI digested piroplasm DNA, at 8.0, 7.3, 6.0 and 3.4 kb. Probe pTs 11-D1 hybridized with multiple fragments. With each probe, polymorphisms were observed among stocks from different locations. However, there was no correlation between the patterns of hybridization bands and the locations where parasites were collected. Analysis of the hybridization patterns of stocks obtained from individual cattle in the same grazing areas showed an almost identical pattern.     

Angiotensin I-converting enzyme-inhibitory peptides obtained from chicken collagen hydrolysate

In this study, collagen extracted from chicken legs (which are the yellow keratin parts containing a nail) was hydrolyzed with various enzymes, and the angiotensin I-converting enzyme (ACE)-inhibitory activity of each hydrolysate was determined. The hydrolysate by treatment with an Aspergillus species-derived enzyme had the highest activity (IC 50 = 260 microg/mL). The fraction of this hydrolysate obtained by ultrafiltration with a molecular-weight cutoff of 3000 Da (low fraction) had a stronger activity (IC 50 = 130 microg/mL) than the fractionated one. This fraction was further fractionated by HPLC, and the peptides in the fraction with high ACE-inhibitory activity were identified. The amino acid sequences of the four peptides were identified using a protein sequencer. These peptides were synthesized to confirm their ACE-inhibitory activities; this showed that peptides with a Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro sequence had the highest activity (IC 50 = 29 microM). When the low fraction was administered to spontaneous hypertensive rats, a decrease in their blood pressure was observed after 2 h of administration, and a significant decrease in blood pressure (-50 mmHg) was observed after 6 h. Moreover, long-term administration studies indicated that the low fraction showed a significant suppression of increased blood pressure.     

Establishment of reliable methods of in vitro pollen germination and pollen preservation of Brassica rapa (syn. B. campestris)

A simple and reliable method for evaluating the viability of Brassica pollen was established in which the in vitro germination rate of pollen was adopted as the index of the viability of pollen grains. Pollen grains were preincubated in an atmosphere in which the relative humidity (RH) was fixed to 52% or 66% at 20 °C for 5 hours. They were cultured for 16 hours at 25 °C in a liquid Kwack's medium (1964) supplemented with 20% sucrose, and the pH was adjusted to 8.0. They were then observed under a microscope and the number of germinating and unchanged pollen grains were counted. The germination rate of pollen was improved and stabilized by preincubation and the use of a high pH medium. More than 90% of the freshly harvested pollen grains of Brassica rapa (syn. B. campestris) germinated constantly in these conditions Undehisced anthers were collected from flowers at anthesis and dehydrated by incubation at 20 °C for 16–24 hours in an atmosphere where the RH was fixed to 15% or 32%. They were put into a plastic vial and preserved in a freezer at -20 °C. The germination percentage of the preserved pollen was scored at intervals during preservation. The germination rate of the pollen grains preserved at -20°C for 1 year was higher than 50% and the pollen proved to be efficient for seed set. Most of the seeds germinated normally. This revised version was published online in August 2006 with corrections to the Cover Date.     

Fecal progestagens to detect and monitor pregnancy in captive female cheetahs (Acinonyx jubatus)

The purposes of the present study were to establish a noninvasive monitoring assay of fecal progestagen measurement to detect pregnancy and to identify the components of fecal progestagens in early, middle and late pregnancy in cheetahs. Feces were collected from 7 female cheetahs and analyzed from 30 days before the last copulation to parturition in 9 pregnancies. Blood was collected from one cheetah. Fecal progestagen and serum progesterone concentrations were determined by enzyme immunoassay (EIA). The profiles of the fecal progestagen concentrations were similar to the serum progesterone profile. Fecal progestagen and serum progesterone concentrations remained at the baseline until copulation. In the mean fecal progestagen profile during pregnancy (92.8 ± 0.4 days; from the last copulation to parturition), the concentrations increased 3-4 days after the last copulation and remained high until parturition. To investigate changes in the components of progestagen metabolites in the tripartite periods of gestation, fecal progestagens were analyzed by HPLC-EIA. Marked immunoreactive peaks consistent with 5α-pregnan-3α/β-ol-20-one and 5α-pregnan-3,20-dione and small peaks consistent with 5β-pregnan-3α/β-ol-20-one were detected. There were no distinct difference in the components of progestagens among the first, second and third trimesters of pregnancy. The hormone assay, as an indicator of fecal 5α-reduced pregnanes, is useful for detecting pregnancy and monitoring pregnant luteal activity in cheetahs.     

Anticancer activities of thelephantin O and vialinin A isolated from Thelephora aurantiotincta

Thelephora aurantiotincta is an edible mushroom belonging to the genus Thelephora; it grows in symbiosis with pine trees. Recently, phytochemical investigations have revealed that the genus Thelephora is an abundant source of p-terphenyl derivatives. However, their bioactivity has not yet been well characterized. In screening for natural materials with anticancer activity, a T. aurantiotincta ethanol extract (TAE) was found to decrease cell viability in human hepatocellular carcinoma cells (HepG2). In this study, a new p-terphenyl derivative, thelephantin O, and a known compound, vialinin A, were isolated as the principal bioactive components of TAE. These compounds decreased cell viability in HepG2 and human colonic carcinoma cells (Caco2), but not in noncancerous human hepatocytes. This is the first report of the isolation from T. aurantiotincta of selective cytotoxic agents against cancer cells.     

Antidiabetic and Antioxidant Effects of Polyphenols in Brown Alga <Emphasis Type="Italic">Ecklonia stolonifera</Emphasis> in Genetically Diabetic KK-A<Superscript>y</Superscript> Mice

The dietary intake and control of blood glucose levels are very important in hyperglycemic patients and α-glucosidase inhibitors are a cost-effective means to preventing the progression of diabetes. In search of a natural inhibitor from food materials, α-glucosidase inhibitory activity and the anti-hyperglycemic effects of a brown alga, Ecklonia stolonifera, were investigated using non-insulin dependent diabetic mice. Methanolic extract of E. stolonifera (MEE), which contains a high content of polyphenols, showed strong inhibition of α-glucosidase in vitro. Male KK-A^y mice, a genetically non-insulin dependent diabetic model, showed hyperglycemia with aging, but the ingestion of MEE suppressed the increase in plasma glucose and lipid peroxidation levels in unfasted KK-A^y mice dose dependently. In KK-A^y mice, which were fed the MEE diet for 4 weeks, MEE moderated the elevation of plasma glucose levels after the oral administration of maltose. The polyphenols in MEE were estimated to be phlorotannins by HPLC-PDA and LC/MS analyses. These results demonstrate that E. stolonifera, seaweed typically used as a health food, has strong antidiabetic and antioxidant effects in vivo, thus, it may have beneficial properties in the prevention of diabetes and could be useful in the development of an antidiabetic pharmaceutical and functional food.