First Report of Dimethachlon Resistance in Field Isolates of Sclerotinia sclerotiorum on Oilseed Rape in Shaanxi Province of Northwestern China.

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic fungal pathogen causing diseases in a wide range of plants, including oilseed rape (3). Substantial economic losses caused by S. sclerotiorum have been reported in the United States, Canada, Brazil, South Africa, Hungary, India, Nepal, and Japan (1). Application of fungicides is the principal tool for controlling S. sclerotiorum because of lack of high level of host resistance. Dicarboximide fungicides such as dimethachlon have been widely used to control S. sclerotiorum in recent years in China and field isolates with reduced sensitivity to dimethachlon have been reported in Jiangsu Province of eastern China (2). In order to understand the current status of dimethachlon resistance in S. sclerotiorum isolates of northwestern China, 196 and 344 isolates of S. sclerotiorum collected from oilseed rape fields in 10 counties throughout Shaanxi Province in 2011 and 2012, respectively, were assayed for sensitivity to dimethachlon using 5 µg ml-1 dimethachlon as a discriminatory dose. Mycelial plugs (6 mm in diameter) cut from the margin of a 48-h-old colony were placed in the center of petri dishes containing potato dextrose agar (PDA) amended with 5 µg ml-1 dimethachlon; PDA without fungicide served as the control. Cultures were incubated at 26°C and colony growth was measured after 72 h of incubation. Isolates that showed growth on PDA amended with fungicide were tentatively considered resistant to dimethachlon, whereas the completely inhibited isolates were considered sensitive. Results showed that 1.02% or 2 isolates of the 196 isolates collected in 2011 and 3.78% or 13 isolates of the 344 isolates collected in 2012 were resistant to dimethachlon. For all the isolates considered resistant and 42 randomly selected sensitive isolates, 50% effective concentrations (EC50) were determined on PDA amended with a series of dimethachlon concentrations. The average EC50 value of dimethachlon for sensitive isolates was 0.29 ± 0.02 µg ml-1 Resistance ratios (EC50 of resistant isolate / average EC50 of sensitive isolates) for the two resistant isolates detected in 2011 were 10.28 and 23.83, respectively, whereas resistance ratios for the 13 resistant isolates detected in 2012 ranged from 24.90 to 101.97. The average EC50 value of dimethachlon for the 13 resistant isolates detected in 2012 was 19.05 µg ml-1, and EC50 values for the two resistant isolates detected in 2011 were 2.98 and 6.91 µg ml-1, respectively. These results indicated that both resistance frequency and resistance level increased from 2011 to 2012. Bioassay results of three resistant isolates indicated that there was positive cross-resistance between dimethachlon and other dicarboximide fungicides such as iprodione and procymidone. To our knowledge, this is the first report of dimethachlon resistance in S. sclerotiorum in Shaanxi Province of northwestern China. The molecular mechanism of dimethachlon resistance in field isolates of S. sclerotiorum remains to be studied. Although resistance frequency is low at present, dimethachlon resistance should be kept in mind and fungicide resistance management tactics such as use of biological control agents, fungicide tank-mixing, or alternating dimethachlon with other fungicides having different modes of action is recommended in controlling S. sclerotiorum. References: (1) M. D. Bolton et al. Mol Plant Pathol. 7:1, 2006. (2) H. X. Ma et al. Plant Dis. 93:36, 2009. (3) L. H. Prudy. Phytopathology 69:875, 1979.

In vitro and in vivo metabolism of a novel cannabinoid-1 receptor inverse agonist, taranabant, in rats and monkeys.

The metabolism and excretion of taranabant (MK-0364, N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2{[5-(trifluoromethyl)pyridine-2-yl]oxy}propanamide), a potent cannabinoid-1 receptor inverse agonist, were evaluated in rats and rhesus monkeys. Following administration of [¹4C]taranabant, the majority of the radioactivity was excreted within 72 h. In both rats and rhesus monkeys, taranabant was eliminated primarily via oxidative metabolism, followed by excretion of metabolites into bile. Major pathways of metabolism that were common to rats and rhesus monkeys included hydroxylation at the benzylic carbon adjacent to the cyanophenyl ring to form a biologically active circulating metabolite M1, and oxidation of one of the two geminal methyl groups of taranabant or M1 to the corresponding diastereomeric carboxylic acids. Oxidation of the cyanophenyl ring, followed by conjugation with glutathione or glucuronic acid, was a major pathway of metabolism only in the rat and was not detected in the rhesus monkey. Metabolism profiles of taranabant in liver microsomes in vitro were qualitatively similar in rats, rhesus monkeys and humans and included formation of M1 and oxidation of taranabant or M1 to the corresponding carboxylic acids via oxidation of a geminal methyl group. In human liver microsomes, metabolism of taranabant was mediated primarily by CYP3A4.

Iron targeting to mitochondria in erythroid cells.

Immature erythroid cells have an exceptionally high capacity to synthesize haem that is, at least in part, the result of the unique control of iron metabolism in these cells. In erythroid cells the vast majority of Fe released from endosomes must cross both the outer and the inner mitochondrial membranes to reach ferrochelatase, which inserts Fe into protoporphyrin IX. Based on the fact that Fe is specifically targeted into erythroid mitochondria, we have proposed that a transient mitochondria-endosome interaction is involved in Fe transfer to ferrochelatase [Ponka (1997) Blood 89, 1-25]. In this study, we examined whether the inhibition of endosome mobility within erythroid cells would decrease the rate of (59)Fe incorporation into haem. We found that, in reticulocytes, the myosin light-chain kinase inhibitor, wortmannin, and the calmodulin antagonist, W-7, caused significant inhibition of (59)Fe incorporation from (59)Fe-transferrin-labelled endosomes into haem. These results, together with confocal microscopy studies using transferrin and mitochondria labelled by distinct fluorescent markers, suggest that, in erythroid cells, endosome mobility, and perhaps their contact with mitochondria, plays an important role in a highly efficient utilization of iron for haem synthesis.

Characterization of the iron transporter DMT1 (NRAMP2/DCT1) in red blood cells of normal and anemic mk/mk mice.

Divalent metal transporter 1 (DMT1) is the major transferrin-independent iron uptake system at the apical pole of intestinal cells, but it may also transport iron across the membrane of acidified endosomes in peripheral tissues. Iron transport and expression of the 2 isoforms of DMT1 was studied in erythroid cells that consume large quantities of iron for biosynthesis of hemoglobin. In mk/mk mice that express a loss-of-function mutant variant of DMT1, reticulocytes have a decreased cellular iron uptake and iron incorporation into heme. Interestingly, iron release from transferrin inside the endosome is normal in mk/mk reticulocytes, suggesting a subsequent defect in Fe(++) transport across the endosomal membrane. Studies by immunoblotting using membrane fractions from peripheral blood or spleen from normal mice where reticulocytosis was induced by erythropoietin (EPO) or phenylhydrazine (PHZ) treatment suggest that DMT1 is coexpressed with transferrin receptor (TfR) in erythroid cells. Coexpression of DMT1 and TfR in reticulocytes was also detected by double immunofluorescence and confocal microscopy. Experiments with isoform-specific anti-DMT1 antiserum strongly suggest that it is the non-iron-response element containing isoform II of DMT1 that is predominantly expressed by the erythroid cells. As opposed to wild-type reticulocytes, mk/mk reticulocytes express little if any DMT1, despite robust expression of TfR, suggesting a possible effect of the mutation on stability and targeting of DMT1 isoform II in these cells. Together, these results provide further evidence that DMT1 plays a central role in iron acquisition via the transferrin cycle in erythroid cells.

Adhesion of thymocytes to bone marrow stromal cells: regulation by bFGF and IFN-gamma.

We recently reported on selective interactions between immature T cell subpopulations and bone marrow (BM) stromal cells. To further study this process, we first examined the efficacy of methods estimating cell-cell adhesion and then investigating the effects of cytokines on thymocyte-stroma associations. Techniques based on the use of the fluorochromes calcein-acetomethylester (calcein-AM) and fluorescein diacetate (FDA) were studied and compared to regular cell counting methods. With calcein-AM labeling, the retention time was relatively long, while with FDA labeling, there was a rapid cellular efflux. Using calcein-AM, we developed an accurate quantitative fluorometric assay for determining the adherence of thymocytes to a BM stromal cell line (MBA-13). A maximal fraction of about 29% thymocytes was found to adhere to confluent MBA-13 cell layers after four to six h of coculture. Whereas interleukin 1 did not change the rate of adhesion of thymocytes to the stroma, interferon-gamma (IFN-gamma) significantly increased adhesion. Basic fibroblast growth factor (bFGF) had a dose-dependent biphasic effect on thymocyte adhesion, and a greater fraction of double negative thymocytes adhered to stroma pretreated with bFGF. Taken together, these results suggest that IFN-gamma and bFGF modulate T cells-BM stromal cell adhesion.

Selective adhesion of immature thymocytes to bone marrow stromal cells: relevance to T cell lymphopoiesis.

We investigated the interactions between the bone marrow microenvironment and T cell populations at different stages of maturation. Thymocytes were seeded onto confluent layers of bone marrow stromal cell lines (MBA-13 or 14F1.1). Within a few hours two main thymocyte populations were observed; one remained in the liquid phase and the other adhered to the stromal cells. After 24 hours of culture, most of the adhering cells expressed the phenotype of the precursors, double negative (DN) CD4-CD8-, or of immature thymocytes, double positive (DP) CD4+CD8+. The number of adhering DN cells did not change during the time of the culture, whereas that of the DP declined. The CD4+CD8- or CD4-CD8+ cells did not adhere to any significant extent. The expression of CD3 antigen on adherent thymocytes was lower than that on nonadherent ones. Sorted thymocytes at a high level of purification (>96%) were cultured over stromal layer and, after 24 hours, 60% of the DN or 22% of the DP cells were found to adhere to the stroma. The culture medium was replaced every 24 hours or after 48 hours; no significant change was noted in the number of adhering DN and DP cells. The reappearance of immature T cells in the liquid phase suggested proliferation of this cell type. Thus, early thymocytes, phenotypically characterized as DN and DP, preferentially adhere to bone marrow stromal cells. This in vitro phenomenon may represent the function of the BM stroma as an extrathymic site of T cell lymphopoiesis.

[Long-term culture of normal and leukemia bone marrow cells].

Using long-term bone marrow culture (LTBMC), we studied the in vitro growth status of bone marrow nucleated cells from 5 normal subjects and 14 patients with acute leukemia. The results showed that LTBMC could support normal hematopoiesis selectively, and in the meantime, inhibit the leukemic progenitor growth. Our preliminary clinical application indicated that this culture system could be used to purge the autologous bone marrow graft in vitro for allogeneic bone marrow transplantation.