Focal adhesion kinase is negatively regulated by phosphorylation at tyrosine 407

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs via tyrosine phosphorylation at specific residues. Although several tyrosine phosphorylation events have been linked to FAK activation and downstream signal transduction, the function of FAK phosphorylation at Tyr(407) was previously unknown. Here, we show for the first time that phosphorylation of FAK Tyr(407) increases during serum starvation, contact inhibition, and cell cycle arrest, all conditions under which activating FAK Tyr(397) phosphorylation decreases. Transfection of NIH3T3 cells with a phosphorylation-mimicking FAK 407E mutant decreased autophosphorylation at Tyr(397) and inhibited both FAK kinase activity in vitro and FAK-mediated functions such as cell adhesion, spreading, proliferation, and migration. The opposite effects were observed in cells transfected with nonphosphorylatable mutant FAK 407F. Taken together, these data suggest the novel concept that FAK Tyr(407) phosphorylation negatively regulates the enzymatic and biological activities of FAK.     

EAG and behavioral responses of the wingless tea aphid Toxoptera aurantii (Homoptera: Aphididae) to tea plant volatiles

Electrophysiological and behavioral responses of the wingless tea aphid, Toxoptera aurantii (Boyer), to 14 synthetic volatiles identified from tea shoots, their partial (GLV mixture) and full (ACB mixture) blends, and fresh young tea leaves, buds, tender stems, adult tea leaves and tea aphid-damaged young leaves (ADYL) were studied by using an electroantennography (EAG) and a four-arm olfactometer. ACB elicited the largest EAG responses. Major volatile components, Z-3-hexen-1-ol, E-2-hexenal, n-hexanol, methyl salicylate and benzylalcohol, from the tea shoots were strongly EAG active. All the 4 tested tea shoot tissues also elicited significant EAG responses, with the young tea leaves being the strongest, followed by buds, tender stems and adult tea leaves. Surprisingly, ADYL elicited a weakly negative EAG response. In the olfactory assays, the fresh and tender tea leaves, as well as the individual major volatile components, e.g. Z-3-hexenyl acetate, methyl salicylate, E-2-hexen-1-ol and Z-3-hexen-1-ol, from the tender shoots (EAG-active) were all attractive. This result might indicate that the wingless tea aphids may use tea shoot volatiles as kairomone to find their optimal feeding sites, e.g. fresh tender tea shoots.     

Plant regeneration via direct somatic embryogenesis in Panax japonicus

Panax japonicus is one of the important medicinal plants. Here, we established the protocol for plant regeneration of P. japonicus via direct somatic embryogenesis. Somatic embryos were directly obtained from the segments of zygotic embryos on MS medium with 4.4 μM 2,4-D. Thereafter, somatic embryos were produced by repetitive secondary somatic embryogenesis. The secondary somatic embryo formation was enhanced by plasmolyzing pretreatment (1.0 M mannitol for 10 h). Frequency of secondary somatic embryo formation from cotyledon segments was lowered by plasmolyzing pretreatment, but the number of somatic embryos per explants was greatly increased. Plasmolyzing pretreatment resulted in retardation of embryo growth and required subculture to fresh medium for further growth of embryos into cotyledonary stage. Without plasmolyzing pretreatment, cotyledonary embryos were obtained after 8 weeks of culture. All the cotyledonary somatic embryos germinated by 5 μM GA₃ treatment, but only 15.3% were germinated on hormone-free medium. After 2 months of culture on 1/2 strength WPM medium, plantlets produced flowers spontaneously. In the anthers of in vitro flowers, microsporogenesis occurred normally with low number of pollen grains.     

Shotgun lipidomics identifies a paired rule for the presence of isomeric ether phospholipid molecular species

Background

Ether phospholipids are abundant membrane constituents present in electrically active tissues (e.g., heart and the brain) that play important roles in cellular function. Alterations of ether phospholipid molecular species contents are associated with a number of genetic disorders and human diseases.

Methodology/Principal Findings

Herein, the power of shotgun lipidomics, in combination with high mass accuracy/high resolution mass spectrometry, was explored to identify a paired rule for the presence of isomeric ether phospholipid molecular species in cellular lipidomes. The rule predicts that if an ether phospholipid A′-B is present in a lipidome, its isomeric counterpart B′-A is also present (where the ′ represents an ether linkage). The biochemical basis of this rule results from the fact that the enzymes which participate in either the sequential oxidation of aliphatic alcohols to fatty acids, or the reduction of long chain fatty acids to aliphatic alcohols (metabolic precursors of ether lipid synthesis), are not entirely selective with respect to acyl chain length or degree of unsaturation. Moreover, the enzymatic selectivity for the incorporation of different aliphatic chains into the obligatory precursor of ether lipids (i.e., 1-O-alkyl-glycero-3-phosphate) is also limited.

Conclusions/Significance

This intrinsic amplification of the number of lipid molecular species present in biological membranes predicted by this rule and demonstrated in this study greatly expands the number of ether lipid molecular species present in cellular lipidomes. Application of this rule to mass spectrometric analyses provides predictive clues to the presence of specific molecular species and greatly expands the number of identifiable and quantifiable ether lipid species present in biological samples. Through appropriate alterations in the database, use of the paired rule increases the number of identifiable metabolites in metabolic networks, thereby facilitating identification of biomarkers presaging disease states.     

Nitric oxide destabilizes Pias3 and regulates sumoylation

Small ubiquitin-related protein modifiers (SUMO) modification is an important mechanism for posttranslational regulation of protein function. However, it is largely unknown how the sumoylation pathway is regulated. Here, we report that nitric oxide (NO) causes global hyposumoylation in mammalian cells. Both SUMO E2 conjugating enzyme Ubc9 and E3 ligase protein inhibitor of activated STAT3 (Pias3) were targets for S-nitrosation. S-nitrosation did not interfere with the SUMO conjugating activity of Ubc9, but promoted Pias3 degradation by facilitating its interaction with tripartite motif-containing 32 (Trim32), a ubiquitin E3 ligase. On the one hand, NO promoted Trim32-mediated Pias3 ubiquitination. On the other hand, NO enhanced the stimulatory effect of Pias3 on Trim32 autoubiquitination. The residue Cys459 of Pias3 was identified as a target site for S-nitrosation. Mutation of Cys459 abolished the stimulatory effect of NO on the Pias3-Trim32 interaction, indicating a requirement of S-nitrosation at Cys459 for positive regulation of the Pias3-Trim32 interplay. This study reveals a novel crosstalk between S-nitrosation, ubiquitination, and sumoylation, which may be crucial for NO-related physiological and pathological processes.     

高血糖加重脑缺血损伤机制的研究现状

脑缺血是引起人类死亡的一个重要原因,由于其发病的分子机制十分复杂,各种因子作用相互影响,且多数因子的作用同时存在损伤和保护两种机制,使得脑缺血的研究充满了困难。目前众多研究都证实高血糖对缺血脑组织有损害作用,并可能导致局部或广泛缺血后预后更差。本文依据近几年的实验,重点阐述了五种最新的高血糖加重脑缺血过程和预后损伤的机制假说,包括高血糖通过引起过量谷氨酸释放导致的Ca2^＋大量内流造成损伤、高血糖状态下造成氧化应激从而产生各种自由基对神经元造成损伤、炎症因子相关的损伤、高血糖相关的血液灌流的减少以及高血糖造成脑内酸中毒从而引起损伤。期望这些对机制的探讨能够上加深广大医药研究人员对高血糖加重脑缺血损伤的认识,帮助找到新的药物作用靶点和治疗手段,启发新的研究思路。     

雌激素受体β基因CA重复序列多态性与绝经后骨质疏松症的关联性研究

绝经后骨质疏松症(PMO)是一种多基因调控的遗传性疾病。雌激素受体β亚型基因是骨质疏松症的重要侯选基因。此文采用病例对照设计(78名股骨颈PMO病人和122名对照以及108名腰椎PMO病人和92名对照)研究中国人(汉族)雌激素受体β基因(ESR2)第5内含子CA重复序列多态性与PMO的相关性。以CA重复序列平均数22次为界将重复序列基因分为短基因(<22)和长基因(≥22),分别以S和L表示。股骨颈及腰椎(L2-4)部位,病例组中LL基因型和L等位基因者频率显著高于对照组(P<0.01),SL、LL及SL LL基因型者较SS基因型者患PMO风险显著增高(P<0.05);调整年龄、绝经时间、绝经年龄及体质指数后,Logistic回归分析显示ESR2(CA)n多态性仍然与股骨颈(OR4.923,95%CI1.986～12.203,P=0.001)及L2-4(OR2.267,95%CI1.121～4.598,P=0.023)PMO显著相关。结果显示:ESR2基因CA重复序列多态性与股骨颈和L2-4部位PMO独立关联,L等位基因显性影响PMO的发病风险。     

Characterization of flavonoids in the traditional Chinese herbal medicine-Huangqin by liquid chromatography coupled with electrospray ionization mass spectrometry

The root of Scutellaria baicalensis, called Huangqin in Chinese, is one of the most commonly used traditional Chinese medicines for the treatment of hepatitis, tumors, diarrhea, and inflammatory diseases. The major chemical constituents of Huangqin are flavonoids. In the present paper, HPLC-DAD-ESI-MS(n) was used to analyze flavonoids in the roots of S. baicalensis. A total of 26 flavonoids were identified or tentatively characterized, including 5 C-glycosides, 12 O-glycosides, and 9 free aglycones. Two C-glycosides, apigenin-6-C-glucyl-8-C-arabinoside and chrysin-6,8-di-C-glucoside, together with some O-glycosides, are reported from S. baicalensis for the first time. This method is simple, reliable and sensitive, and could be used for the quality control of Huangqin and its related preparations.     

Quantitative determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cellular environment of a human colon adenocarcinoma cell line for pharmacokinetic studies

A simple, accurate, precise, specific and reproducible high-performance liquid chromatography (HPLC) method was developed for determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cell suspension. The assay procedure involved simple liquid-liquid extraction, the supernatant liquid was added an equal volume of water to avoid solvent effect. The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 303 nm. The analysis used a Hypersil ODS2 C18 column (5 microm, 4.6 mm x 250 mm) and methanol/distilled water as the mobile phase (flow rate=1 mL/min). A total analytical run was achieved within 6.0 min and calibration curve was linear over a wide concentration range of 0.25-40 microg/mL for plasma sample and 1.0-500 microM for cell suspension, the coefficients of correlation were 0.9997 and 0.9999 or better, respectively. There was 80.7+/-7.86%, 96.8+/-3.20% and 102.7+/-9.72% recovery from 0.5, 10, and 40 microg/mL plasma samples, respectively. Intra- and inter-batch accuracy and precision were acceptable for the both matrices. The RSD of intra- and inter-day assay variations were all less than 10%. Both analyte and IS were stable in the battery of stability studies, freeze-thaw cycles. The described assay method was applied to pharmacokinetic studies in rats and a human colon adenocarcinoma cell line (Caco-2) successfully. The application of the assay to determine the pharmacokinetic is described.