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991.
992.
Potassium channels are controlling K+ transport across plasma membrane and thus playing a central role in all aspects of osmolarity as well as numerous other functions in plants, including in sexual reproduction. We have used whole-cell and single-channel patch-clamp recording techniques investigated the regulation of intracellular free Ca2+-activated outward K+ channels in Pyrus pyrifolia pollen tube protoplasts. We have also showed the channels could be inhibited by heme and activated carbon monoxide (CO). In the presence of oxygen and NADPH, hemoxygenases catalyzes heme degradation, producing biliverdin, iron and CO. Considered the oxygen concentration approaching zero in the ovary, the heme will inhibit the K+ outward flux from the intracellular of pollen tube, increasing the pollen tubes osmolarity, inducing pollen tube burst. Here we discuss the putative role of K+ channels in plant sexual reproduction.Key words: pear, pollen, K+ channels, heme, carbon monoxideIon channels in the pollen tube play critical roles in mediation pollen germination and pollen tube growth.1–3 Early studies were focus on the plasma membrane calcium channel regulation and cytosolic free calcium concentration variation in the pollen tube reason by which was one of the most important second messengers in plants.3–7 However, reports have also showed that the potassium channels in the pollen tubes were also involved in several important steps of plant sexual reproduction.8–19 Recently, more reports further demonstrated this phenomena.20–24 In the report by Lu et al. they demonstrated that two cation/proton exchangers (CHX), CHX21 and CHX23, are essential for pollen tube growth guidance in Arabidopsis.22 chx21 chx23 double mutant induces the fertility impaired, but which is unchanged in both single chx21 or chx23 mutants. They have also found that the double mutant pollen grains germination and pollen tube growth in the transmitting tract were not difference with the wild-type, however, the double mutant pollen tubes fail to turn toward ovules.22 Protein localization experiments show CHX23 is expressed in the endoplasmic reticulum of pollen tubes; functional analysis results showed that CHX23 as a K+ transporter mediates K+ uptake in a pH-dependent manner. So, these protein affect the signal transduction pathway of pollen tube growth toward to the ovule by controlling the cation balance and pH in the pollen tube.22 Amien et al. identified a signaling ligand of defensin-like (DEFL) protein, ZmES4, which expressed in maize synergid. ZmES4 activates the maize pollen tube tip plasma membrane K+ Shaker channel KZM1.20 This finding is also very interesting. Pollen tube bursting suggested to be based on the osmotic stress; the influx of K+ mediated by ZmES4-activated KZM1 will trigger rapid plasma membrane depolarization, which induced the pollen tube tip burst.20 Furthermore, the osmotic increasing induced by too much K+ in the cytosolic of pollen tube was not only resulted by inward K+ channel activation, but also resulted by outward K+ channel inhibition in the pollen tube plasma membrane. In our report, we find a intracellular Ca2+-sensitive outward K+ channel in pear pollen tube plasma membrane, which could be inhibited by heme and activated by heme oxidative production, carbon monoxide (CO), may play a functional role in the pollen tube brusting.23In the presence of oxygen and NADPH, hemoxygenases catalyzes heme degradation, producing biliverdin, iron and CO.25 Early reports showed that oxygen plays an important role in plant sexual reproduction. Pollen tubes grow through the style toward the ovary with high speed, a process that consumes tremendous amounts of energy and requires rapid oxygen uptake by pollen tubes.26 Pollen grains have roughly 20 times the level of mitochondria and respire 10 times faster than vegetative tissue.12,27–29 Furthermore, oxygen has been proposed as a possible cue for pollen-tube guidance.30 Indeed, the existence of an oxygen gradient in the unpollinated style has been shown in some species such as Hipeastrum hybridum. Oxygen pressure is high in the stigma and style but suddenly decreases at the base of the style, approaching zero in the ovary. Moreover, pollen-tube growth itself creates hypoxic regions within the style.31 Therefore, we suggest that the outward K+ channel inhibited by heme is dominant compared with which activated by CO when pollen tubes reach the ovary, based on where the hypoxic condition (Fig. 1). However, the gene encode the outward K+ channel in the pear pollen tube remains to be determined in the further study.Open in a separate windowFigure 1Reciprocal regulation of heme and carbon monoxide in putative Ca2+-activated outward K+ channel. Under normal condition, in the presence of NADPH, heme is metabolized by hemeoxygenase to generate carbon monoxide (CO), which activates outward K+ channel. However, without the oxygen, heme cannot be metabolized. The accumulated heme acts as an inhibitor of outward K+ channel, even in the presence of NADPH. The accumulated K+ in the cytosolic of pollen will induced the pollen tube depolarized, then burst. 相似文献
993.
Ding C Zhang Y Peng X Wang Y Zhang L Cong X Ding QW Xiang RL Wu LL Yu GY 《Journal of proteome research》2011,10(5):2206-2215
Submandibular gland autotransplantation is effective for treating severe dry eye syndrome. However, more than 40% of patients show epiphora within 3-6 months after treatment. The mechanism underlying the hypersecretion in epiphora remains to be elucidated for developing novel interventions. Since salivary gland secretion is dependent on a variety of proteins, we analyzed the changes in protein expression in transplanted glands of epiphora patients with 2-D gel electrophoresis and electrospray ionization quadrupole/time-of-flight mass spectrometry and evaluated their possible roles in epiphora. There were 23 proteins that showed altered expression in the glands of epiphora patients, 15 being up-expressed and 8 being down-expressed. The expression of secretory proteins was decreased in these glands, including alpha-amylase, cystatin S, SA, and SN. In contrast, cytoskeletal proteins were all up-regulated, including actin and vimentin. Immunofluorescence revealed that the intensity ratio of F-actin in apical and lateral cytoplasm to total F-actin in acini was decreased in the glands of epiphora patients. Carbachol stimulation induced a similar redistribution of F-actin in the control glands. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was increased in both carbachol-stimulated and epiphora glands. Preincubation of submandibular glands with ERK1/2 inhibitors PD98059 or U0126 inhibited carbachol-induced F-actin redistribution. These results indicated that differentially expressed proteins participated in the hypersecretion of transplanted submandibular glands and the redistribution of F-actin might be involved in this hypersecretion in an ERK1/2-dependent manner. 相似文献
994.
Lei Y Huang K Gao C Lau QC Pan H Xie K Li J Liu R Zhang T Xie N Nai HS Wu H Dong Q Zhao X Nice EC Huang C Wei Y 《Molecular & cellular proteomics : MCP》2011,10(10):M110.005397
Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and second in females worldwide. Unfortunately 40-50% of patients already have metastatic disease at presentation when prognosis is poor with a 5-year survival of <10%. Reactive oxygen species (ROS) have been proposed to play a crucial role in tumor metastasis. We now show that higher levels of ROS accumulation are found in a colorectal cancer-derived metastatic cell line (SW620) compared with a cell line (SW480) derived from the primary lesion from the same patient. In addition, ROS accumulation can affect both the migratory and invasive capacity of SW480 and SW620 cells. To explore the molecular mechanism underlying ROS-induced migration and invasion in CRC, we have compared protein expression patterns between SW480 and SW620 cells using a two-dimensional electrophoresis-based proteomics strategy. A total of 63 altered proteins were identified from tandem MS analysis. Cluster analysis revealed dysregulated expression of multiple redox regulative or ROS responsive proteins, implicating their functional roles in colorectal cancer metastasis. Molecular and pathological validation demonstrated that altered expression of PGAM1, GRB2, DJ-1, ITGB3, SOD-1, and STMN1 was closely correlated with the metastatic potential of CRC. Functional studies showed that ROS markedly up-regulated expression of ITGB3, which in turn promoted an aggressive phenotype in SW480 cells, with concomitant up-regulated expression of STMN1. In contrast, knockdown of ITGB3 expression could mitigate the migratory and invasive potential of SW620 or H(2)O(2)-treated SW480 cells, accompanied by down-regulated expression of STMN1. The function of ITGB3 was dependent on the surface expression of integrin αvβ3 heterodimer. Furthermore, STMN1 expression and the PI3K-Akt-mTOR pathway were found to be involved in ROS-induced and ITGB3-mediated migration and invasion of colorectal cancer cells. Taken together, these studies suggest that ITGB3 plays an important role in ROS-induced migration and invasion in CRC. 相似文献
995.
Cummings J Zhou C Dive C 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2011,879(13-14):887-893
Method validation should focus on demonstrating that an assay is fit for its intended purpose. We have applied the β-expectation tolerance interval - a statistical approach that predicts the accuracy of assay measurements in the future - to the validation of two different cell death biomarker assays, the M30 and M65 ELISAs. A meta-analysis was conducted on a total of 57 different M30 and M65 assays run over a 2 year period. All code utilised in calculations was developed using MATLAB. The optimal fit to the calibration curve for the M30 assay was shown to be a quartic curve which yielded a β-expectation tolerance interval of +20.5% and -23.6% at β=95% over a wide range of QC standards (88-810 U/L). However, such a fit required at least 7 points to avoid problems with over fitting. A linear fit to the M65 calibration curve normally produced a tolerance interval of less than ±20%, however, marked inter-batch variations were evident. Amelioration of batch to batch variations was accomplished by fitting M65 calibration data preferably to a 4-parameter logistic function or a cubic spline. The minimum number of QC replicates and different assays required to produce reliable accuracy profiles was determined. The β-expectation tolerance interval approach has resulted in further optimisation of the M30 and M65 ELISAs as biomarker assays that should translate into greater accuracy in results generated from clinical trials samples. 相似文献
996.
Xiaoping Wu Xinglong Jia Yuhua Ji Cong Wang Yonglin Yu Susu Gao Quchou Li Xiaokun Li 《International journal of peptide research and therapeutics》2011,17(1):53-59
It has been known that basic fibroblast growth factor (bFGF) plays an important role in tumor progression mainly due to its
strong mitogenic activity. Antagonists targeting bFGF have been considered a potential strategy for cancer therapy via inhibiting
cell proliferation induced by bFGF. We have previously obtained a high-affinity bFGF-binding peptide (named as P7) with strong
inhibitory activity against bFGF-induced cell proliferation from the phage display random heptapeptide library. The aim of
the present investigation was to synthesize the peptide P7 by solid phase method and explore the mechanisms of the inhibitory
effect of P7-targeting bFGF stimulation on Balb/c 3T3 cells via proteomic analysis. Seven differentially expressed protein
were identified, among which four were decreased by bFGF stimulation alone and increased by addition of P7, the other three
were up-regulated by bFGF treatment alone and down-regulated by addition of P7. Among the identified proteins, epidermal fatty
acid-binding protein (E-FABP) and low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) take part in the regulation
of cell proliferation. The results suggested P7 inhibited the bioactivities of bFGF possibly via influencing the expression
of cellular proteins related to cell proliferation. 相似文献
997.
依照血管钠肽氨基酸序列,根据毕赤酵母密码子偏爱性,人工合成了血管钠肽的编码基因,通过PCR技术,扩增出5'端含有组氨酸标签的血管钠肽基因片段,构建的分泌型表达质粒pPIC9K-VNP,pPICZαB -VNP分别经SalⅠ和SacⅠ线性化后,双质粒转化毕赤酵母GS115宿主菌后,G418/Zeocin双抗筛选出整合型His+Muts表达菌株,Tricine-SDS-PAGE和Western blot分析,证明His-VNP在毕赤酵母中可有效分泌表达,摇瓶产量可达70mg/L左右,双质粒整合较单质粒整合重组子目的多肽表达量提高了75%。生物学活性研究表明,表达产物在体外具有对巨噬细胞iNOS和TNFα mRNA的抑制作用。 相似文献
998.
999.
罗布麻是中国药典收录的传统中药,本研究提取它的主要有效成分罗布麻总黄酮,采用经典抗抑郁评价模型小鼠强迫游泳实验对罗布麻叶总黄酮进行抗抑郁活性评价.试验中给小鼠罗布麻叶总黄酮提取物25 mg/kg、50 mg/kg和100 mg/kg后,观察罗布麻叶总黄酮对小鼠强迫游泳不动时间的影响,结果显示罗布麻总黄酮提取物能显著缩短小鼠强迫游泳不动时间(P<0.05),药效与氟西汀相似,表明罗布麻具有明显的抗抑郁活性.在此基础上,应用皮质酮损伤的PC12细胞模型,采用荧光相对定量RT-PCR法检测罗布麻对皮质酮损伤的PC12细胞中脑内脑源性神经营养因子(BDNF)和环磷酸腺苷反应元件结合蛋白(CREB)表达水平的影响.结果表明,皮质酮处理后PC12细胞中BDNF、CREB基因的表达量最低,罗布麻处理后,表达量显著增加,较处理前增加了近十倍,且呈剂量依赖性.本研究结果提示罗布麻抗抑郁机制可能是通过(AC-cAMP-CREB)信号通路促进BDNF、CREB的基因表达而发挥的. 相似文献
1000.
Neuronal ion channels of different types often do not function independently but will inhibit or potentiate the activity of
other types of channels, a process called cross-talk. The N-methyl-D-aspartate receptor (NMDA receptor) and the γ-aminobutyric acid type A receptor (GABAA receptor) are important excitatory and inhibitory receptors in the central nervous system, respectively. Currently, cross-talk
between the NMDA receptor and the GABAA receptor, particularly in the central auditory system, is not well understood. In the present study, we investigated functional
interactions between the NMDA receptor and the GABAA receptor using whole-cell patch-clamp techniques in cultured neurons from the inferior colliculus, which is an important
nucleus in the central auditory system. We found that the currents induced by aspartate at 100 μmol L−1 were suppressed by the pre-perfusion of GABA at 100 μmol L−1, indicating cross-inhibition of NMDA receptors by activation of GABAA receptors. Moreover, we found that the currents induced by GABA at 100 μmol L−1 (I
GABA) were not suppressed by the pre-perfusion of 100 μmol L−1 aspartate, but those induced by GABA at 3 μmol L−1 were suppressed, indicating concentration-dependent cross-inhibition of GABAA receptors by activation of NMDA receptors. In addition, inhibition of IGABA by aspartate was not affected by blockade of voltage-dependent Ca2+ channels with CdCl2 in a solution that contained Ca2+, however, CdCl2 effectively attenuated the inhibition of I
GABA by aspartate when it was perfused in a solution that contained Ba2+ instead of Ca2+ or a solution that contained Ca2+ and 10 mmol L−1 BAPTA, a membrane-permeable Ca2+ chelator, suggesting that this inhibition is mediated by Ca2+ influx through NMDA receptors, rather than voltage-dependent Ca2+ channels. Finally, KN-62, a potent inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), reduced the inhibition of I
GABA by aspartate, indicating the involvement of CaMKII in this cross-inhibition. Our study demonstrates a functional interaction
between NMDA and GABAA receptors in the inferior colliculus of rats. The presence of cross-talk between these receptors suggests that the mechanisms
underlying information processing in the central auditory system may be more complex than previously believed. 相似文献