Solid Phase Synthesis of Phosphopeptides Incorporating 2,2-Dimethyloxazolidine Pseudoproline Analogs: Evidence for <Emphasis Type="Italic">trans</Emphasis> Leu-Pro Peptide Bonds in Stat3 Inhibitors

To answer the question of whether the conformation of the Leu-Pro bond is cis or trans in Ac-pTyr-Leu-Pro-Gln-Thr-Val-NH₂ when complexed with the SH2 domain of Stat3, we substituted 2,2-dimethyloxazolidines derived from serine (Ser(Ψ^Me,Mepro)) and threonine (Thr(Ψ^Me,Mepro)) for proline. The 2,2-dimethyloxazolidine and 2,2-dimethylthiazolidine pseudoproline (ΨPro) analogs induce predominantly cis Xxx-ΨPro peptide bonds. As these ΨPro analogs are acid-labile, the phosphopeptides were synthesized using Fmoc-based SPPS using unprotected phosphotyrosine and 4-hydroxybenzoate as the linker that allowed release from the support by alkaline ammonolysis, conditions that kept the oxazolidine rings intact. Incorporation of Ser(Ψ^Me,Mepro) resulted in 69% cis Leu-ΨPro bond content in aqueous solution whereas that for Thr(Ψ^Me,Mepro) analog was 63%. Affinities for Stat3 were 3–5 fold lower than the lead compound and were inversely correlated with cis content. Thus we conclude that the Leu-Pro peptide bond is trans when the peptide is bound to Stat3.     

Cortical and cytoplasmic flows driven by actin microfilaments polarize the cortical ER-mRNA domain along the a-v axis in ascidian oocytes

Cellular mechanisms generating the polarized redistribution of maternal Type I postplasmic/PEM mRNAs in ascidian oocytes remain unknown. We have previously shown that PEM-1 mRNA is associated with a network of rough cortical Endoplasmic Reticulum (cER) polarized along the animal-vegetal (a-v) axis forming a cER-mRNA domain in mature oocytes. We now investigate the a-v polarization of this cER-mRNA domain during meiotic maturation using H. roretzi and C. intestinalis. We show that the cER and Hr-PEM-1 aggregate as interconnected cortical patches at the cell periphery before maturation, which uniformly spread out during maturation and form a reticulated organization enriched in the vegetal hemisphere at the end of maturation. Time-lapse video recordings coupled with micromanipulations reveal that stereotyped surface, cortical and cytoplasmic flows accompany the vegetal shift of the cER-mRNA domain and mitochondria-rich myoplasm. Treatments with cytochalasin B and nocodazole indicate that both polarization of the cER-mRNA domain and mitochondria-rich myoplasm and cortical and cytoplasmic flows depend on actin cytoskeleton, but not microtubules. Using cortical fragments prepared from maturing oocytes coupled with high resolution immuno/in situ localization, we have further analyzed the effects of these inhibitors on the reorganizations the cER network and Hr-PEM-1 mRNA. We show that before maturation starts, Hr-PEM-1 mRNAs are already associated with the cER, and actin cytoskeleton inhibitors disturb their association. Finally, we hypothesize that Germinal Vesicle Break Down (GVBD) triggers an actomyosin-dependant cortical flow which directs the a-v polarization of ascidian oocytes.     

应用TDI-FP技术分析宫颈癌组织HPV16 E7基因A647G点突变

模板指导的末端碱基掺入反应结合荧光偏振检测技术(template direct dye-terminator incorporation with fluorescence- polarization,TDI-FP) 是SNP检测新技术. 应用TDI-FP方法分析中国陕西HPV16阳性宫颈组织HPV16 E7基因第647位核苷酸A→G热点突变(即A647G),首先在HPV16阳性的91例宫颈癌及49例正常/宫颈炎妇女宫颈DNA标本中,PCR扩增含647位点在内的HPV16 E7部分基因, 然后将紧邻647位点5′端的寡核苷酸探针与PCR产物内的模板杂交,并延伸一个与647位点碱基互补的荧光标记碱基：TAMRA-ddTTP或R110-ddCTP. 用荧光偏振仪读取荧光偏振 (FP) 值,根据升高的相应FP值判断647位点碱基. 结果表明,宫颈组织HPV16 E7 A647G的总体检出率为35.71% (50/140). 宫颈癌组的A→G突变率为42.86% (39/91),显著高于正常/宫颈炎组22.45% (11/49) 的突变率 (x2 = 5.778, P = 0.016),两组间的OR值为2.59 (95% CI = 1.17~5.71). 提示TDI-FP 可用于HPV有意义点突变的分析;我国陕西地区妇女HPV 16 A647G突变率及其对宫颈癌的警示性与其他地区相比有明显差异,该地区携带此突变病毒株的妇女患宫颈癌的风险可能较高     

A high-throughput assay based on fluorescence polarization for inhibitors of the polo-box domain of polo-like kinase 1

The serine/threonine kinase polo-like kinase 1 (Plk1) is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its substrates and its intracellular anchoring sites via its polo-box domain (PBD), which is unique to the family of polo-like kinases. Therefore, inhibition of the Plk1 PBD has been suggested as an approach to the inhibition of Plk1 that circumvents specificity problems associated with the inhibition of the conserved adenosine triphosphate (ATP) binding pocket. Here we report on the development of a high-throughput assay based on fluorescence polarization that allows the discovery of small-molecule inhibitors of the Plk1 PBD. The assay is based on binding of the Plk1 PBD to a phosphothreonine-containing peptide comprising its optimal binding motif with a K_d of 26 ± 2 nM. It is stable with regard to dimethyl sulfoxide (DMSO) and time, and it has a Z′ value of 0.73 ± 0.06 in a 384-well format.     

Direct evidence for excitonically coupled chlorophylls a and b in LHC II of higher plants by nonlinear polarization spectroscopy in the frequency domain

Occurrence of excitonic interactions in light-harvesting complex II (LHC II) was investigated by nonlinear polarization spectroscopy in the frequency domain (NLPF) at room temperature. NLPF spectra were obtained upon probing in the chlorophyll (Chl) a/b Soret region and pumping in the Q_y region. The lowest energy Chl a absorbing at 678 nm is strongly excitonically coupled to Chl b.     

The extremely rapid oligonucleotide hybridization and high throughput detection of microbial gene sequences using fluorescence polarization

The hybridization of oligonucleotide sequences complementary to the genes of Shiga toxins (verotoxins) types 1 and 2 of enterohaemorrhagic Escherichia coli (EHEC) and human hepatitis C virus (HCV) was monitored using fluorescence polarization under the reaction condition of high salt concentration (0.8 M NaCl), which was optimized to obtain a higher rate of hybridization. The time courses of hybridization of fluorescently labeled oligomers (probe DNAs) with the amplified DNA or RNA of the genes were recorded. Two methods, the asymmetric PCR and NASBA, were used to amplify the genetic DNA of Shiga toxins and that of RNA in HCV, respectively. Probe DNA sequences were designed which hybridized extremely rapidly with amplicons of the genes of Shiga toxins types 1 and 2 and that of HCV. In the cases using the three different DNA probes, the hybridization was 90% complete in about 1 min, considerably faster than that of the 3 min reported previously. The rapidity of this hybridization could not be explained by the melting temperature or the G+C content of the probe sequences but its relationship with high order structure of the single stranded DNA or RNA of the amplicons in the solution was strongly suggested.     

A conserved sequence in caveolin-1 is both necessary and sufficient for caveolin polarity and cell directional migration

Caveolin-1 (Cav-1) plays an important role in the organization of signaling molecules involved in a variety of signaling pathways, including those mediating cell motility. Here we show that amino acids K47-K57 of Cav-1 are a highly conserved sequence in Cav-1 and Cav-3 proteins, and that expression of either K47-K57 deletion Cav-1 mutant or wild-type Cav-2 that lacks this sequence exhibits a non-polarized distribution pattern. Expression of K47-K57 in Cav-2 leads to Cav-2 polarity, suggesting that expression of K47-K57 is sufficient to direct caveolin polarity. Importantly, we show that expression of this sequence is both necessary and sufficient to promote cell directional migration. Thus, our results support the conclusion that Cav-1 polarity is critical for cell directional migration.     

Identification of an atypical interaction site in the BTB domain of the MYC-interacting zinc-finger protein 1

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