Involvement of Protein Tyrosine Phosphatases BcPtpA and BcPtpB in Regulation of Vegetative Development,Virulence and Multi-Stress Tolerance in Botrytis cinerea

Tyrosine phosphorylation and dephosphorylation have emerged as fundamentally important mechanisms of signal transduction and regulation in eukaryotic cells, governing many processes, but little has been known about their functions in filamentous fungi. In this study, we deleted two putative protein tyrosine phosphatase (PTP) genes (BcPTPA and BcPTPB) in Botrytis cinerea, encoding the orthologs of Saccharomyces cerevisiae Ptp2 and Ptp3, respectively. Although BcPtpA and BcPtpB have opposite functions in conidiation, they are essential for sclerotial formation in B. cinerea. BcPTPA and BcPTPB deletion mutants ΔBcPtpA-10 and ΔBcPtpB-4 showed significantly increased sensitivity to osmotic and oxidative stresses, and to cell wall damaging agents. Inoculation tests showed that both mutants exhibited dramatically decreased virulence on tomato leaves, apples and grapes. In S. cerevisiae, it has been shown that Ptp2 and Ptp3 negatively regulate the high-osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway. Although both BcPtpA and BcPtpB were able to inactive Hog1 and Mpk1 in S. cerevisiae, in contrast to S. cerevisiae, they positively regulate phosphorylation of BcSak1 (the homologue of Hog1) and BcBmp3 (the homologue of Mpk1) in B. cinerea under stress conditions. These results demonstrated that functions of PTPs in B. cinerea are different from those in S. cerevisiae, and BcPtpA and BcPtpB play important roles in regulation of vegetative development, virulence and in adaptation to oxidative, osmotic and cell-wall damage stresses in B. cinerea.     

DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.     

Interactions of 14N:15N stearic acid spin-label pairs: effects of host lipid alkyl chain length and unsaturation

Electron-electron double resonance (ELDOR) and saturation recovery electron paramagnetic resonance (EPR) spectroscopy have been employed to examine the interactions of 14N:15N stearic acid spin-label pairs in fluid-phase model membrane bilayers composed of a variety of phospholipids. The [14N]-16-doxylstearate:[15N]-16-doxylstearate (16:16) pair was utilized to measure lateral diffusion of the spin-labels, while the [14N]-16-doxylstearate:[15N]-5-doxylstearate (16:5) pair provided information on vertical fluctuations of the 16-doxylstearate nitroxide moiety toward the membrane surface. Three saturated host lipids of varying alkyl chain length [dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC)], an alpha-saturated, beta-unsaturated lipid [1-palmitoyl-2-oleoylphosphatidylcholine (POPC)], and phosphatidylcholine from a natural source [egg yolk phosphatidylcholine (egg PC)] were utilized as host lipids. Lateral diffusion of the stearic acid spin-labels was only slightly affected by alkyl chain length at a given reduced temperature (Tr) in the saturated host lipids but was significantly decreased in POPC at the same Tr. Lateral diffusion in DMPC, POPC, and egg PC was quite similar at 37 degrees C. A strong correlation was noted between lateral diffusion constants and rotational mobility of [14N]-16-doxylstearate. Vertical fluctuations were likewise only slightly influenced by alkyl chain length but were strongly diminished in POPC and egg PC relative to the saturated systems. This diminution of the 16:5 interaction was observed even under conditions where no differences were discernible by conventional EPR. These studies indicate that vertical fluctuation of 16-doxylstearate is quite sensitive to host lipid unsaturation and that ELDOR studies of interactions between 14N:15N spin-label pairs can provide information on spin-label motion beyond that given by conventional EPR.     

Factor interactions with the simian virus 40 early pre-mRNA influence branch site selection and alternative splicing.

To study the interaction of splicing factors with the simian virus 40 early-region pre-RNA, which can be alternatively spliced to produce large T and small t mRNAs, we used an in vitro RNase protection assay that defines the 5' boundaries of factor-RNA interactions. Protection products reflecting factor interactions with the large T and small t 5' splice sites and with the multiple lariat branch site region were characterized. All protection products were detected very early in the splicing reaction, before the appearance of spliced RNAs. However, protection of the large T 5' splice site was detected well before small t 5' splice site and branch site protection products, which appeared simultaneously. Oligonucleotide-targeted degradation of small nuclear RNAs (snRNAs) revealed that protection of the branch site region, which occurred at multiple sites, required intact U2 snRNA and was enhanced by U1 snRNA, while protection of the large T and small t 5' splice sites required both U1 and U2 snRNAs. Analysis of several pre-RNAs containing mutations in the branch site region suggests that factor interactions involving the multiple copies of the branch site consensus determine the selection of branch points, which is an important factor in the selection of alternative splicing pathways.     

Lectin bindings and diethylstilbestrol effects on the recognition of mullerian inhibiting substance (MIS) on chick mullerian ducts by MIS-antiserum

Summary A high intensity of lectin bindings was demonstrated on the epithelial cells and serosa cells of the regressing right Mullerian ducts (Mds) in the female chick embryos. The strong lectin bindings occurs on, or in the regressing Md cells along with marked surface MIS bindings at the age of day 13. However, at the age of days 5–7 1/2, bindings of lectins were weak. Neither Wheat-germ agglutinin (WGA) or Concanavalin A (Con-A) labelings before MIS-antiserum (MIS-Ab) incubation can block antibody recognitions to the antigens, including MIS and growth hormone at the age of day 13. Our previous studies indicated that after WGA labeling on the surfaces of Md epithelial cells prior to the incubation of MIS-Ab at day 10 did not prevent the recognition of MIS-Ab (Wang 1989). On the contrary, at day 7 1/2, the specific binding of MIS was eliminated after preincubations with lectins and prenatal diethylstilbestrol (DES) treatment at the age of day 5. It is suggested that DES provides a protection to the Mds from MIS-induced regression by preventing the MIS binding to its specific membrane receptors. An increase of extra- and intracellular glycoproteins or carbohydrates of regressing Md epithelial cells were suggested. Internalization of WGA but not MIS molecules was found in Md epithelial cells. The Golgi saccules were negative of lectin bindings.