Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1Ser137 involvement in spindle formation and REC8 cleavage

Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1^Ser137 and pPlk1^Thr210) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1^Ser137 with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1^Thr210 was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1^Ser137 was colocalized with microtubule organizing center (MTOC) proteins, γ-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1^Thr210 was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1^Ser137 accumulation at MTOCs and between chromosome arms, consequently disturbed γ-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1^Ser137 and pPlk1^Thr210, as well as the subcellular distribution of pPlk1^Thr210, were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1^Ser137 is the action executor, in mouse oocytes during meiotic division.     

High-speed broadband monitoring of cell viscoelasticity in real time shows myosin-dependent oscillations

Study of the dynamic evolutions of cell viscoelasticity is important as during cell activities such as cell metastasis and invasion, the rheological behaviors of the cells also change dynamically, reflecting the biophysical and biochemical connections between the outer cortex and the intracellular structures. Although the time variations of the static modulus of cells have been investigated, few studies have been reported on the dynamic variations of the frequency-dependent viscoelasticity of cells. Measuring and monitoring such dynamic evolutions of cells at nanoscale can be challenging as the measurement needs to meet two objectives inherently contradictory to each other—the measurement must be broadband (to cover a large frequency spectrum) but also rapid (to capture the time-elapsed changes). In this study, we exploited a recently developed control-based nanomechanical protocol of atomic force microscope to monitor in real time the dynamic evolutions of the viscoelasticity of live human prostate cancer cells (PC-3 cells) and study its dependence on myosin activities. We found that the viscoelasticity of PC-3 cells, followed the power law, and oscillated at a period of about 200 s. Both the amplitude and the frequency of the oscillation strongly depended on the intracellular calcium and blebbistatin-sensitive motor proteins.     

Molecular phylogeny of the New World Dipsadidae (Serpentes: Colubroidea): a reappraisal

We present a phylogenetic analysis of the New World dipsadids based on an expanded data matrix that includes 246 terminal taxa including 196 dipsadids. The species are sampled for eight genes (12S, 16S, cytb, nd2, nd4, bdnf, c‐mos, rag2). The data are explored using two distinct optimality procedures—maximum parsimony and maximum likelihood—and two alignment strategies—dynamic homology and static homology. Two previously unsampled dipsadid genera, Sordellina and Rhachidelus, are now included in the analysis. The definitions of the genera, Erythrolamprus, Clelia, Hypsirhynchus, Philodryas and Phimophis, and the tribes Alsophiini, Echinantherini and Conophiini, are revised. In order to maintain monophyly, the genus Umbrivaga is synonymized with Erythrolamprus, and two new genera are erected to accommodate Phimophis iglesiasi and Clelia rustica, as well as their closely related species. The West Indian genera Schwartzophis, Darlingtonia, Antillophis and Ocyophis are resurrected. © The Willi Hennig Society 2012.     

Detection of quercetin based on Al-amplified phosphorescence signals of manganese-doped ZnS quantum dots

A simple phosphorescence method is proposed for quercetin detection based on Al³⁺-amplified room-temperature phosphorescence (RTP) signals of 3-mercaptopropionic acid (MPA)-capped Mn-doped ZnS quantum dots (QDs). The sensor was established based on some properties as follows. Al³⁺ can interact with carboxyl groups on the surface of MPA-capped Mn-doped ZnS QDs via chelation, which will lead to the aggregation of QDs and amplification of RTP signals, After the addition of quercetin, it can form more stable complex with Al³⁺ in alkaline aqueous solution and dissociate Al³⁺ from the surface of Mn-doped ZnS QDs, which will result in significant recovery of RTP intensity of the MPA-capped Mn-doped ZnS–Al³⁺ system. Under the optimized conditions, the change of RTP intensity was proportional to the concentration of quercetin in the range from 0.1 to 6.0 mg L⁻¹, with a high correlation coefficient of 0.996 and a detection limit of 0.047 mg L⁻¹. The proposed method is potentially suitable for detection of quercetin in real samples without complicated pretreatment.     

Targeting Hedgehog signaling pathway and autophagy overcomes drug resistance of BCR-ABL-positive chronic myeloid leukemia

The frontline tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, drug resistance is the major clinical challenge in the treatment of CML. The Hedgehog (Hh) signaling pathway and autophagy are both related to tumorigenesis, cancer therapy, and drug resistance. This study was conducted to explore whether the Hh pathway could regulate autophagy in CML cells and whether simultaneously regulating the Hh pathway and autophagy could induce cell death of drug-sensitive or -resistant BCR-ABL⁺ CML cells. Our results indicated that pharmacological or genetic inhibition of Hh pathway could markedly induce autophagy in BCR-ABL⁺ CML cells. Autophagic inhibitors or ATG5 and ATG7 silencing could significantly enhance CML cell death induced by Hh pathway suppression. Based on the above findings, our study demonstrated that simultaneously inhibiting the Hh pathway and autophagy could markedly reduce cell viability and induce apoptosis of imatinib-sensitive or -resistant BCR-ABL⁺ cells. Moreover, this combination had little cytotoxicity in human peripheral blood mononuclear cells (PBMCs). Furthermore, this combined strategy was related to PARP cleavage, CASP3 and CASP9 cleavage, and inhibition of the BCR-ABL oncoprotein. In conclusion, this study indicated that simultaneously inhibiting the Hh pathway and autophagy could potently kill imatinib-sensitive or -resistant BCR-ABL⁺ cells, providing a novel concept that simultaneously inhibiting the Hh pathway and autophagy might be a potent new strategy to overcome CML drug resistance.     

Determination of free amino acids in burley tobacco by high performance liquid chromatography

A reversed-phase high performance liquid chromatographic method was developed for determining free amino acids in burley tobacco. The test was done by OPA/3-mercaptopropionic acid as the pre-column derivatizing reagent. Chromatographic column was Elitte C¹⁸ column (4.6 mm × 250 mm i.d., 5 μm). Mobile phase A was 18 mol/l NaAc (pH7.2) including 0.002%(v/v) triethylamine and 0.3%(v/v) furanidine. Mobile phase B was 100 mol/l NaAc (pH7.2)–acetonitrile–methanol (v/v = 1:2:2). The column temperature was 40 °C and the flow rate was 1.0 ml/min. The fluorescence detector was used with 350 nm excitation wave length and 450 nm emission wave length. The average recoveries of the method ranged from 95.3–100.7% with the relative standard deviation of 2.32–9.24%. The method is simple, accurate and has good repeatability. The results of the determination of seventeen kinds of free amino acids in burley leaves were produced by the way of different ratios of cake fertilizer and inorganic fertilizer. The results show that Aspartic acid has the highest content however ratio of cake fertilizer and inorganic fertilizer. The contents of most of the free amino acids are increased and then gradually decreased with the increase in organic manure. The contents of most of the free amino acids are very close at 15:85% ratio and 30:70% ratio of cake fertilizer and inorganic fertilizer. The total amount of free amino acids is the highest at 30:70% ratio of cake fertilizer and inorganic fertilizer. Considering comprehensively, the quality of burley leaves is the best at 30:70% ratio of cake fertilizer and inorganic fertilizer.