Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking

The phox homology (PX) domain is a phosphoinositide-binding module that typically binds phosphatidylinositol 3-phosphate. Out of 47 mammalian proteins containing PX domains, more than 30 are denoted sorting nexins and several of these have been implicated in internalization of cell surface proteins to the endosome, where phosphatidylinositol-3-phosphate is concentrated. Here we investigated a multimodular protein termed PXK, composed of a PX domain, a protein kinase-like domain, and a WASP homology 2 domain. We show that the PX domain of PXK localizes this protein to the endosomal membrane via binding to phosphatidylinositol 3-phosphate. PXK expression in COS7 cells accelerated the ligand-induced internalization and degradation of epidermal growth factor receptors by a mechanism requiring phosphatidylinositol 3-phosphate binding but not involving the WASP homology 2 domain. Conversely, depletion of PXK using RNA interference decreased the rate of epidermal growth factor receptor internalization and degradation. Ubiquitination of epidermal growth factor receptor by the ligand stimulation was enhanced in PXK-expressing cells. These results indicate that PXK plays a critical role in epidermal growth factor receptor trafficking through modulating ligand-induced ubiquitination of the receptor.Both constitutive endocytosis and activated endocytosis are highly regulated events by which cells take up nutrients and internalize receptors for recycling or degradation (47). Endocytosed molecules are delivered to early endosomes, where the components are sorted to the cell surface for recycling back to the plasma membrane, or to late endosomes to be degraded in lysosomes (17). The molecular mechanisms regulating these events are not fully understood.One of the major protein families involved in the trafficking of membrane compartments is sorting nexins (SNXs), which are characterized by the presence of phox homology (PX) domains (8, 65). The PX domain is a protein module which consists of approximately 130 amino acids with three β-strands followed by three α-helices forming a helical subdomain, and the general function of this module is to interact with the head groups of inositol phospholipids through which parental proteins are targeted to specific cellular compartments. Most of the SNXs examined to date specifically recognize phosphatidylinositol 3-phosphate [PtdIns(3)P], which is found predominately in early endosomes (11). The founding member of the SNX family, SNX1, was initially identified as an interaction partner of epidermal growth factor receptor (EGFR), and the expression of SNX1 enhanced lysosomal degradation of EGFR (38); therefore, SNXs are most likely to be involved in the trafficking of many different families of receptors which are recycled to the cell surface or sent to the lysosome for degradation (19). On the other hand, PX domain-containing proteins have also been reported to bind to phosphoinositides other than PtdIns(3)P and to have functions independent of receptor trafficking (54). For example, phospholipase D is a PX domain-containing protein that hydrolyzes phosphatidylcholine to produce a second-messenger molecule, phosphatidic acid. Interestingly, phospholipase D has been recently shown to accelerate EGFR endocytosis by activating dynamin GTPase through its PX domain but independently of lipase activity (39). Cytokine-independent survival kinase (CISK) is a PX domain-containing protein kinase that has also been shown to regulate sorting of a chemokine receptor CXCR4 through AIP4, the CXCR4 ubiquitin ligase (60). RGS-PX1, a GTPase-activating protein for Gαs of heterotrimeric GTP-binding proteins, and KIF16B, a PX domain-containing kinesin superfamily member, have been shown to regulate EGFR trafficking (27, 72) and are now grouped into the SNX family as SNX13 and SNX26, respectively.Another feature of the PX domain is a well-conserved polyproline sequence (PXXP) in the variable loop between α1 and α2 helices, which led to the original identification of the PX domain as a SH3 domain-binding partner (53). The physiological importance of both intermolecular and intramolecular interactions mediated by polyproline sequences has been shown in various molecules, including phospholipase D2 (33) and p47^phox (1). In mammals, there are currently 47 proteins harboring PX domains, and 30 proteins are termed SNXs (59). The functions of these proteins have just begun to be revealed.Actin cytoskeletal dynamics have been implicated not only in cell motility and cytokinesis but also in endocytic processes, although the necessity and role in endocytosis in higher eukaryotic cells remain ambiguous (12, 34, 35, 55). The WASP homology 2 (WH2) domain is known as an actin-binding motif found in regulators of the actin cytoskeleton, including Wiskott-Aldrich syndrome protein (WASP), Scar/WASP-family verprolin-homologous protein (WAVE), verprolin/WASP-interacting protein (WIP), missing in metastasis (MIM), and β-thymosins (52). Some proteins with WH2 domains, such as β-thymosin, prevent actin filament assembly by sequestering actin monomers, while others, such as N-WASP and the Drosophila protein Ciboulot participate in barbed-end actin assembly (52). Recently, the structural basis for these opposite functions of WH2 domains was demonstrated; the interaction of the C-terminal region of β-thymosin/WH2 domain with the pointed end of the actin monomer accounts for the switch in function from inhibition to promotion of actin assembly (26). WH2 domains exist in almost 20 proteins, whose functions remain to be clarified.In the present study, we isolated a new multimodular protein (termed PXK), conserved in multicellular organisms including humans through flies, which possesses a PX domain, a protein kinase-like domain, and a WH2 domain. We show that the PX and WH2 domains function as PtdIns(3)P and actin-binding domains, respectively. PXK expression in COS cells accelerated ligand-induced EGFR endocytosis and degradation that was dependent on a functional PX domain but independent of the WH2 domain. PXK also enhanced ubiquitination of EGFR induced by EGF stimulation in these cells. Based on these results, we propose that PXK is a functional sorting nexin that may play an additional role in cellular function via its interaction with the actin cytoskeleton.     

Remediation of Nitrobenzene Contaminated Soil by Combining Surfactant Enhanced Soil Washing and Effluent Oxidation with Persulfate

The combination of surfactant enhanced soil washing and degradation of nitrobenzene (NB) in effluent with persulfate was investigated to remediate NB contaminated soil. Aqueous solution of sodium dodecylbenzenesulfonate (SDBS, 24.0 mmol L^-1) was used at a given mass ratio of solution to soil (20:1) to extract NB contaminated soil (47.3 mg kg^-1), resulting in NB desorption removal efficient of 76.8%. The washing effluent was treated in Fe²⁺/persulfate and Fe²⁺/H₂O₂ systems successively. The degradation removal of NB was 97.9%, being much higher than that of SDBS (51.6%) with addition of 40.0 mmol L^-1 Fe²⁺ and 40.0 mmol L^-1 persulfate after 15 min reaction. The preferential degradation was related to the lone pair electron of generated SO₄•⁻, which preferably removes electrons from aromatic parts of NB over long alkyl chains of SDBS through hydrogen abstraction reactions. No preferential degradation was observed in •OH based oxidation because of its hydrogen abstraction or addition mechanism. The sustained SDBS could be reused for washing the contaminated soil. The combination of the effective surfactant-enhanced washing and the preferential degradation of NB with Fe²⁺/persulfate provide a useful option to remediate NB contaminated soil.     

毒品快速定量测试方法的探讨

阐述了毒品金免疫层析试条的定量测试机理,推导出浓度值与吸光度之间的测试模型,并对主要干扰因素及消除方法进行分析。最后验证了毒品快速定量测试方法的可行性。     

Design,synthesis and binding affinity of 3'-fluoro analogues of Cl-IB-MECA as adenosine A3 receptor ligands

Several 3'-fluoro analogues, 1a, 1b, and 1c of selective and potent adenosine A(3) receptor agonist, Cl-IB-MECA were synthesized from D-xylose via highly regioselective opening of lyxo-epoxides, 8a and 8b with fluoride anion. Compared to the high binding affinity of Cl-IB-MECA to the A(3) adenosine receptor, the corresponding 3'-fluoro derivative showed remarkably decreased binding affinity, indicating that 3'-hydroxyl group acts as hydrogen bonding acceptor, not hydrogen bonding donor like fluorine atom in binding to the A(3) adenosine receptor.     

Regulation of gamete release in the economic brown seaweed <Emphasis Type="Italic">Hizikia fusiforme</Emphasis> (Phaeophyta)

Gamete release is an essential event in artificial seeding of the economic brown seaweed, Hizikia fusiforme. Mass egg release occurred in the dark, with few eggs being discharged in the light. Release of eggs was elicited with eight practical salinity units (one PSU ≡ 1 g sea salts l⁻¹) and was inhibited by salinity levels >32 PSU. Egg release was optimal at 23 °C, and was decreased by 72% in agitated seawater compared to unstirred seawater. Inhibitors of photosynthesis and ions channels suppressed egg release, indicating that this process was physiologically associated with photosynthetic activity and ion transport.     

Iodine Nutrition and Thyroid Function in Pregnant Women Exposed to Different Iodine Sources

Determination of the public health concern about magnesium (Mg) in health and disease has been confounded by the lack of a practical measure of status. This has resulted in a lack of consistency in associating Mg deficiency with specific pathological conditions. Some attempts at associating Mg with a chronic disease have used the Dietary Reference Intakes (DRIs) as a status assessment measure. Use of current DRIs for Mg is problematic because recent evidence suggests that they should be updated and based on body weight. An evidence-based suggested Estimated Average Requirement (EAR) and Recommended Dietary Allowance (RDA) for a 70-kg individual is 175 and 250 mg/day, respectively. However, numerous dietary and physiological factors can affect the need for Mg and thus affect the use of the current or suggested new DRIs to assess Mg status. Calcium intakes above normal requirements can decrease Mg balance and exacerbate signs of Mg deficiency. Mg deficiency apparently occurs often in obesity because of increased need to counteract the inflammatory stress induced by adipose tissue dysfunction. Deficiency in anti-oxidant nutrients such as vitamin E and selenium can exacerbate a response to low dietary Mg indicated by increased oxidative stress which can lead to chronic disease. Dietary modifiers of Mg absorption and excretion affect balance and thus the need for Mg. Factors decreasing Mg balance include low dietary protein and non-fermentable fiber, while factors that can increase balance include fructose and fermentable fiber and fructose-containing oligosaccharides. Use of the DRIs to assess the Mg status of a population or group needs to consider their physiological characteristics and dietary habits and be aware that the DRIs may need updating. The DRIs only can be considered a component of a toolbox that presently includes serum Mg concentration and the daily urinary Mg excretion to assess the Mg status of an individual.

     

ε-聚赖氨酸高产菌选育与发酵性能评价

【目的】选育ε-聚赖氨酸(ε-PL)高产菌,并探究不同碳源对其发酵性能的影响。【方法】借助基因组重排和核糖体工程两种育种手段强化ε-PL产生菌的合成能力,并利用p H冲击工艺评价不同碳源对ε-PL发酵的影响。【结果】经过4轮基因组重排和4轮核糖体工程连续选育,获得1株高产突变株Streptomyces albulus GS114,其摇瓶ε-PL产量达到3.0 g/L,较出发菌提高了1.7倍。该改造菌株在5 L发酵罐中分别以葡萄糖和甘油为碳源进行192 h的补料-分批发酵时,ε-PL发酵产量分别达到了43.4 g/L和45.7 g/L,较出发菌提高了11.0%和14.9%,而菌体量分别减少了24.0%和33.2%,ε-PL得率提高了34.2%和30.7%。【结论】基因组重排结合核糖体工程育种是一种有效的ε-PL高产菌选育手段,研究结果将为ε-PL高产菌改造和工业生产碳源选择提供直接指导。