Induction of CYP1A by a diterpene quinone tanshinone IIA isolated from a medicinal herb Salvia miltiorrhiza in C57BL/6J but not in DBA/2J mice

Effects of tanshinone IIA, an active diterpene quinone of the herbal medicine Salvia miltiorrhiza (Danshen), on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in the arylhydrocarbon (Ah)-responsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice. Oral treatment of tanshinone IIA caused a dose-dependent increase of liver microsomal 7-methoxyresorufin O-demethylation (MROD) activity in B6 but not in D2 mice. In B6 mice, tanshinone IIA increased hepatic benzo(a)pyrene hydroxylation (AHH), 7-ethoxyresorufin O-deethylation, MROD, and 7-ethoxycoumarin O-deethylation activities. The levels of Cyp1A2 protein and mRNA were elevated. On the contrary, in D2 mice, tanshinone IIA decreased hepatic AHH and nifedipine oxidation activities and the CYP3A protein level without affecting other activities determined. Cyp1A2 protein and mRNA levels were not affected by tanshinone IIA in D2 mice. Tanshinone IIA had no effects on UGT and GST activities in both B6 and D2 mice. These results demonstrated that induction of CYP1A2 by tanshinone IIA depended on the Ah-responsiveness and occurred at pre-translational level.     

QTL mapping of partial resistance in winter wheat to Stagonospora nodorum blotch.

Stagonospora nodorum blotch is an important foliar and glume disease in cereals. Inheritance of resistance in wheat appears to be quantitative. To date, breeding of partially resistant cultivars has been the only effective way to combat this pathogen. The partial resistance components, namely length of incubation period, disease severity, and length of latent period, were evaluated on a population of doubled haploids derived from a cross between the partially resistant Triticum aestivum 'Liwilla' and susceptible Triticum aestivum 'Begra'. Experiments were conducted in a controlled environment and the fifth leaf was examined. Molecular analyses were based on bulked segregant analyses using 240 microsatellite markers. Four QTLs were significantly associated with partial resistance components and were located on chromosomes 2B, 3B, 5B, and 5D. The percentage of phenotypic variance explained by a single QTL ranged from 14 to 21% for incubation period, from 16 to 37% for disease severity, and from 13 to 28% for latent period,     

Cellular and luminal forms of rat intestinal calcium-binding protein as studied by counter ion electrophoresis

A novel method, termed counter ion electrophoresis, has been developed to identify calcium-binding proteins. In this procedure labeled calcium (${}^{45}\text{CaCl}{}_2$) is added to the lower (anode) chamber reservoir, and the protein sample is applied to the polyacrylamide gel in the upper (cathode) chamber reservoir. As the calcium migrates toward the cathode and the proteins move toward the anode, calcium-binding sites in the gel become labeled and gel slices, containing these sites, can be identified by liquid scintillimetry after solubilization. Application of this procedure to a study of the vitamin D-dependent calcium-binding protein in growing rats has shown that calcium-binding protein occurs consistently as two protein bands in material from mucosal scrapings, but only as one band (band 1) in material from isolated intestinal cells. Bands 1 and 2 are shown to behave as charge isomers, with band 2 more negatively charged. In weanling rats, material from mucosal scrapings yielded only band 1. When calcium-binding protein from mucosal scrapings of growing rats was prepared in the presence of phenylmethylsufonyl fluoride, the amount of band 2 was reduced. Incubation for 2 h at 37 °C of partially purified calcium-binding protein from mucosal scrapings transformed band 1 to band 2, a conversion inhibited by phenylmethyl-sulfonyl fluoride. Similar treatment of partially purified calcium-binding protein from isolated cells had no effect on band 1. It is concluded that band 1 is the cellular and native form of calcium-binding protein which is transformed enzymatically to band 2 in the presence of luminal material.     

The effect of age and 1,25-dihydroxyvitamin D3 treatment on the intestinal calcium-binding protein of suckling rats.

The intestinal level of the vitamin D-dependent duodenal calcium-binding protein was assayed by an equilibrated column technique in rat embryos, neonates, and pups. Calcium-binding protein was undetectable in unborn, newborn, and 1- to 2-day-old rats i.e., the level was lower than in severely vitamin D-deficient animals. Calcium-binding protein was detected after the animals were 5-days old and thereafter rose monotonically as a function of body weight. Treatment with 1,25-dihydroxyvitamin D₃ failed to raise the calcium-binding protein levels of newborn or 1-day-old rats, but doubled the level in 11- or 12-day-old pups. Plasma calcium was raised in all treated animals. The failure to detect calcium-binding protein in vitamin D-replete suckling animals provides evidence for a dissociation between calcium absorption and calcium binding protein.     

Sequential utilization of mixed monosaccharides by yeasts.

Four yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilus, and Rhodotorula toruloides) were tested for their ability to grow and consume D-glucose, D-xylose, D-xylulose, and D-xylitol. Sequential utilization of substrates was observed when D-glucose as mixed with D-xylulose as the carbon source. Catabolite inhibition was tentatively concluded to be responsible for this regulatory mechanism. D-Glucose was also found to inhibit the utilization of D-xylose and D-xylitol in C. utilus and R. toruloides. D-Xylose, D-xylitol, and D-xylulose were consumed simultaneously by R. toruloides and C. utilus.     

Dimemorfan N-demethylation by mouse liver microsomal cytochrome P450 enzymes

Dimemorfan (d-3-methyl-N-methylmorphinan), an analogue of dextromethorphan, is commonly used as a non-opioid antitussive. To clarify the contribution of cytochrome P450 (P450) in dimemorfan N-demethylation, effects of selective inducers and inhibitors were studied in ICR mice. Phenobarbital (PB)- and dexamethasone (Dex)-treatments caused 5-fold increases of liver microsomal dimemorfan N-demethylation activity. In untreated mouse liver microsomes, demethylation activity was strongly inhibited by a CYP3A inhibitor, ketoconazole. In PB-and Dex-treated mouse liver microsomes, ketoconazole caused strong inhibition, whereas orphenadrine caused a decrease of less than 20%. Pretreatment of control mouse liver microsomes with anti-CYP3A inhibited demethylation activity, whereas pre-treatment with anti-CYP2B had no effect. In PB-and Dex-treated mouse liver microsomes, the demethylation activity was inhibited by both anti-CYP3A and anti-CYP2B. In control mice, the intrinsic clearance of dimemorfan from N-demethylation was 5.8 microl min(-1)mg protein(-1). In PB- and Dex-treated mice, the correlation coefficient of fitting using one-enzyme and two-enzyme models were similar. The intrinsic clearances of induced mouse liver microsomes were similar. These results revealed that CYP3A played a major role in hepatic demethylation in untreated mice. Both CYP3A and CYP2B were involved in this demethylation in PB- and Dex-treated mice.     

Sequential utilization of mixed monosaccharides by yeasts

Four yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilus, and Rhodotorula toruloides) were tested for their ability to grow and consume D-glucose, D-xylose, D-xylulose, and D-xylitol. Sequential utilization of substrates was observed when D-glucose as mixed with D-xylulose as the carbon source. Catabolite inhibition was tentatively concluded to be responsible for this regulatory mechanism. D-Glucose was also found to inhibit the utilization of D-xylose and D-xylitol in C. utilus and R. toruloides. D-Xylose, D-xylitol, and D-xylulose were consumed simultaneously by R. toruloides and C. utilus.     

D-Mannitol dehydrogenase from Absidia glauca. Purification, metabolic role, and subunit interactions.

When Absidia glauca was grown in minimal media with D-mannitol as the only source of carbon, an NAD+ specific D-mannitol dehydrogenase (EC 1.1.1.67) was induced. The crude extract also gave evidence of mannitol kinase, mannitol-1-phosphate dehydrogenase, phosphofructokinase, and L-iditol dehydrogenase activity. The heat labile purified preparation was judged enzymically homogeneous based on evidence derived from substrate specificity studies and activity staining, following disc gel electrophoresis. The enzymic monomer, with a weight of about 67000 daltons, slowly polymerizes when stored at -20 degrees C, giving a multiplicity of protein bands on electrophoresis distributed predominantly across a spectrum from dimer to pentamer, with enzymic activity resident predominantly in even multiples of the monomer. Depolymerization occurred rapidly (hours) when a frozen preparation was brought to and held between 4 and 20 degrees C. Aggregate fragmentation with sodium dodecyl sulfate showed a time-temperature dependence, terminating in a subunit component of 13000 daltons. pH optimum for polyol oxidation occurs at 9.6 (NaOH-glycine buffer) while ketose reduction proceeded most rapidly at pH 7.0-7.2 (phosphate buffer). A regulatory role is suggested for this enzyme based on dead-end inhibition by mannitol 1-phosphate, multiple enzyme forms, and its locus at the initiation site for mannitol utilization. The physiological relevance of low-temperature aggregation to regulatory control remains to be established.     

Establishment of two basal-like breast cancer cell lines with extremely low tumorigenicity from Taiwanese premenopausal women

The research of carcinogenetic mechanisms of breast cancer in different ethnic backgrounds is an interesting field, as clinical features of breast cancers vary among races. High premenopausal incidence is distinctive in East-Asian breast cancer. However, human cell lines derived from Asian primary breast tumor are rare. To provide alternative cell line models with a relevant genetic background, we aimed to establish breast cancer cell lines from Taiwanese patients of Han-Chinese ethnicity. Fresh tissue from mammary tumors were digested into organoids, plated and grown in basal serum-free medium of human mammary epithelial cells (HuMEC) with supplements. Cells were further enriched by positive selection with CD326 (epithelial cell adhesion molecule; EpCAM)-coated micro-magnetic beads. Two breast cancer cell lines derived from premenopausal women were successfully established by this method, and named Chang-Gung Breast Cancer 01 (CGBC 01) and 02 (CGBC 02). These two cell lines had a similar phenotype with weak expression of estrogen receptor (ER), progesterone receptor (PR), and without amplification of receptor tyrosine protein kinase erbB-2 (HER2/neu). Genome-wide Single Nucleotide Polymorphism (SNP) array showed multiple copy number alterations in both cell lines. Based on gene expression profiles, CGBC 01 and 02 were clustered into basal-like subtype with reference to the breast cancer cell line gene expression database. The tumorigenicity of both cell lines was extremely low in both anchorage-independence assay and transplantation into the mammary fat pads of nude mice. CGBC 01 and CGBC 02 are low tumorigenic breast cancer cell lines, established from Han-Chinese premenopausal breast cancer patients, which serve as in vitro models in studying the biological features of Asian breast cancer.