The therapeutic effects of SET/I2PP2A inhibitors on canine melanoma

Canine melanoma is one of the most important diseases in small animal medicine. Protein phosphatase 2A (PP2A), a well conserved serine/threonine phosphatase, plays a critical role as a tumor suppressor. SET/I2PP2A is an endogenous inhibitor for PP2A, which directly binds to PP2A and suppresses its phosphatase activity. Elevated SET protein levels have been reported to exacerbate human tumor progression. The role of SET in canine melanoma, however, has not been understood. Here, we investigated the potential therapeutic role for SET inhibitors in canine melanoma. The expression of SET protein was observed in 6 canine melanoma cell lines. We used CMeC-1 cells (primary origin) and CMeC-2 cells (metastatic origin) to generate cell lines stably expressing SET-targeting shRNAs. Knockdown of SET expression in CMeC-2, but not in CMeC-1, leads to decreased cell proliferation, invasion and colony formation. Phosphorylation level of p70 S6 kinase was decreased by SET knockdown in CMeC-2, suggesting the involvement of mTOR (mammalian target of rapamycin)/p70 S6 kinase signaling. The SET inhibitors, OP449 and FTY720, more effectively killed CMeC-2 than CMeC-1. We observed PP2A activation in CMeC-2 treated with OP449 and FTY720. These results demonstrated the potential therapeutic application of SET inhibitors for canine melanoma.     

Effect of intermittent heat treatment on crystallinity in wood cellulose

The effect of intermittent heat treatment on cellulose crystallites in wood was studied to evaluate quantitatively the changes of crystallinity induced by intermittent and continuous heating. The changes in crystallinity were found to be strongly affected by the intermittent heat treatment. The increased crystallinity, the width of the crystals, and the piezoelectric properties were the same for the first intermittent heating as for continuous heating. Further intermittent heating for the same time duration and temperature had no effect on the above properties, probably due to the stopping of the thermal reaction during the interval. Our results suggested that intermittent heat treatment has certain critical cooling temperatures that stop the thermal reaction and are closely related to the duration of the interval. Samples once exposed to a certain duration of heat treatment and then cooled need more time, about twice that of the first duration of intermittent heating compared with continuous heating, to reach maximum crystallinity in wood cellulose.     

Three new algorithms to calculate the irreplaceability index for presence/absence data

The irreplaceability index is used for determining the priority of sites for conservation. We propose three new and simple algorithms for calculating it from presence/absence data without any statistical assumptions when the representative combination size is the minimum. The rare species algorithm gives accurate results but takes the longest calculation time. The species richness algorithm gives approximated results and takes the shortest calculation time. The combined algorithm is intermediate in accuracy and calculation time. We applied the combined algorithm to real data of butterflies in the Kanto district, central Japan, and derived irreplaceability values.     

Analysis of the contents of pungent compounds in fresh Korean red peppers and in pepper-containing foods

An HPLC method has been developed for the analysis of extracts of fresh peppers containing capsaicinoids and of both capsaicinoids and piperines in pepper-containing foods produced and sold in Korea. The HPLC method was optimized by defining how composition of the mobile phase affected retention times. Both identification and quantification were based on retention times and the following criteria: linearity of the UV response at 280 nm in HPLC, recoveries from spiked samples, and observed individual molecular ions in the mass spectra of the extracts determined by liquid chromatography-mass spectrometry. This method, with a limit of detection of approximately 15-30 ng, was used to quantify the distribution of capsaicinoids in 11 Korean whole peppers and in 12 commercial pepper-containing foods. Total capsaicinoid levels of whole peppers ranged from 1.21 microg/g for the PR Gang ja variety to 121.1 microg/g for the Chung yang variety. The levels in food extracts, four of which also included two piperines, ranged from 11.0 microg/g for radish kimuchi to 3752 microg/g for capsaicin sauce. The results demonstrate (a) the usefulness of the HPLC method for the simultaneous analysis of capsaicinoids derived from red peppers and piperines derived from black and white peppers extracted from complex food matrices and (b) the wide-ranging spread of levels of pungent pepper compounds in fresh peppers and in pepper-containing foods consumed in Korea.     

Effects of protein engineering of canola procruciferin on its physicochemical and functional properties

The primary structure of Brassica napus procruciferin 2/3a was engineered to elucidate structure-function relationships and to improve the functionality of cruciferin. The following mutants were constructed: (1) C287T, (2) DeltaII, variable region II was deleted; (3) C287T/DeltaII, mutation involving (1) and (2); (4) DeltaIV + A1aIV; and (5) DeltaIV + A3IV, variable region IV was replaced with variable region IV containing many charged residues from soybean glycinin A1aB1b and A3B4 subunits. Differential scanning calorimetry analysis revealed that the A1aIV region has a more favorable interaction with the procruciferin molecule than does A3IV as well as the original regions. On the basis of heat-induced precipitation analysis, it was concluded that replacement of the free cysteine residue with threonine (C287T) and insertion of charged regions (DeltaIV + A1aIV and DeltaIV + A3IV) could lead procruciferin to form soluble aggregates after heating. Low solubility was observed in mutants DeltaIV + A3IV, DeltaII, and C287T/DeltaII, especially between pH 4 and 6 at mu = 0.08, but not in DeltaIV + A1aIV, indicating that the number of acidic amino acid residues and the high number of glutamine residues are important factors for solubility at mu = 0.08. None of the mutants showed any improvements in emulsifying ability, indicating that destabilization and addition of the hydrophilic region are not effective for emulsification. The insertion of the A1aIV region in procruciferin made the molecule more susceptible to alpha-chymotrypsin.     

Antioxidative, antimutagenic, and anticarcinogenic activities of rice bran extracts in chemical and cell assays

Ethanol-water (70:30 v/v) extracts from rice brans removed from seeds of two blackish-purple pigmented (Sanhaehyanghyulla and Suwon 415) and one nonpigmented (Chuchung) brown rice cultivars were evaluated for antioxidative, anti-tumor-promoting, and anticarcinogenic activities in chemical assays and in mammalian cells (human leukemia HL-60, marmoset B lymphoblastoid B95-8, and Chinese hamster V79 lung cells) by the following tests: inhibition of xanthine oxidase activity; chelation of ferrous ions; reduction of potassium ferricyanide; scavenging of superoxide anions, hydroxyl radicals, and intracellular peroxides; inhibition of 4-nitroquinoline N-oxide-induced mutagenesis; and inhibition of phorbol ester-induced tumor promotion. The extracts from the pigmented rice seeds had generally higher activities in all tests than did the extract from the nonpigmented variety. The results suggest that brans from pigmented rice varieties may provide a source of new natural antioxidants and anticarcinogens and that such rice cultivars with high antioxidative potential also provide a genetic resource for the development of new, improved rice cultivars that may make it possible to enhance both the nutritional and medical value of rice-based diets.     

Glycoalkaloids and metabolites inhibit the growth of human colon (HT29) and liver (HepG2) cancer cells

As part of an effort to improve plant-derived foods such as potatoes, eggplants, and tomatoes, the antiproliferative activities against human colon (HT29) and liver (HepG2) cancer cells of a series of structurally related individual compounds were examined using a microculture tetrazolium (MTT) assay. The objective was to assess the roles of the carbohydrate side chain and aglycon part of Solanum glycosides in influencing inhibitory activities of these compounds. Evaluations were carried out with four concentrations each (0.1, 1, 10, and 100 microg/mL) of the the potato trisaccharide glycoalkaloids alpha-chaconine and alpha-solanine; the disaccharides beta(1)-chaconine, beta(2)-chaconine, and beta(2)-solanine; the monosaccharide gamma-chaconine and their common aglycon solanidine; the tetrasaccharide potato glycoalkaloid dehydrocommersonine; the potato aglycon demissidine; the tetrasaccharide tomato glycoalkaloid alpha-tomatine, the trisaccharide beta(1)-tomatine, the disaccharide gamma-tomatine, the monosaccharide delta-tomatine, and their common aglycon tomatidine; the eggplant glycoalkaloids solamargine and solasonine and their common aglycon solasodine; and the nonsteroidal alkaloid jervine. All compounds were active in the assay, with the glycoalkaloids being the most active and the hydrolysis products less so. The effectiveness against the liver cells was greater than against the colon cells. Potencies of alpha-tomatine and alpha-chaconine at a concentration of 1 microg/mL against the liver carcinoma cells were higher than those observed with the anticancer drugs doxorubicin and camptothecin. Because alpha-chaconine, alpha-solanine, and alpha-tomatine also inhibited normal human liver HeLa (Chang) cells, safety considerations should guide the use of these compounds as preventative or therapeutic treatments against carcinomas.     

Structure-physicochemical function relationships of soybean beta-conglycinin heterotrimers

We purified four single molecular species of beta-conglycinin heterotrimers consisting of the alpha and beta subunits or the alpha' and beta subunits from mutant soybean cultivars lacking the alpha or alpha' subunit, respectively, and examined their structural features and physicochemical functions. The extent of the hydrophobicities of the heterotrimers was related to the number of the alpha or alpha' subunit. The thermal stabilities of the heterotrimers were mainly conferred by the subunit which had lower thermal stability. Solubilities at low ionic strength (mu = 0.08) of the heterotrimers containing the alpha or alpha' subunit were very similar to those of the alpha and alpha' homotrimers, respectively. Emulsifying abilities and heat-induced associations of the heterotrimers containing one beta subunit were similar to those of the alpha or alpha' homotrimer, whereas those of the heterotrimers containing two beta subunits were similar to those of the beta homotrimer.     

Development of Expressed Sequence Tag (EST)-based Cleaved Amplified Polymorphic Sequence (CAPS) markers of tea plant and their application to cultivar identification

To develop cleaved amplified polymorphic sequence (CAPS) markers for cultivar identification of the tea leaf, 5 primer pairs designed on the basis of genes that encode proteins related to nitrogen assimilation and 26 primer pairs based on expressed sequence tag (EST) sequences of the root of tea plant were screened. From combinations of primer pair and restriction enzyme that showed polymorphism among tea plants, 16 markers were selected and applied to DNA fingerprinting of Japanese tea cultivars. Sixty-three cultivars, except for a bud sport (Kiraka) and its original cultivar (Yabukita) and a pair that was the progeny of the same crossing parent (Harumoegi and Sakimidori), were distinguished from one another. By combining the 16 markers with previously developed CAPS markers and observing the physical appearance, 67 cultivars were distinguishable. The cultivars involve approximately 95% of total tea cultivating area in Japan; therefore, about 95% of tea leaves produced in Japan can be authenticated by labeling their cultivars.