p38丝裂素活化蛋白激酶介导低氧预处理诱导的内质网应激相关的心肌细胞保护

钙网蛋白（calreticulin,CRT）和caspase-12是重要的内质网（endoplasmic reticulum,ER）应激分子,本实验在心肌细胞低氧／复氧（hypoxia／reoxygenation,H／R）模型上观察低氧预处理（hypoxic preconditioning,HPC）对CRT和caspase-12表达及活化的影响,探讨内质网应激（endoplasmic reticulum stress,ERS）在HPC保护机制中的意义及其细胞信号转导机制。原代培养的Sprague-Dawley乳鼠心肌细胞随机分为6组：H／R组、HPC＋H／R组、SB203580＋HPC＋H／R组、SP600125＋HPC＋H／R组、HPC组和对照组。以细胞存活率、乳酸脱氢酶（lactate dehydrogenase,LDH）活性及流式细胞术检测细胞损伤情况：Western blot方法检测CRT和caspase-12表达、活化及p38丝裂素活化蛋白激酶（mitogen—activated protein kinases,MAPK）、cJun N-terminal kinase（JNK）磷酸化水平。结果表明：（1）HPC具有细胞保护作用,与H／R组比较,HPC＋H／R组细胞凋亡率和LDH漏出分别降低6．6％和70．0％,存活率增高6．4％：HPC前以特异性p38MAPK抑制剂SB203580预孵育消除HPC的保护作用,与HPC＋H／R组相比,细胞凋亡率和LDH漏出分别增高5．4％和2．1倍,存活率降低5．4％,JNK特异性抑制剂SP600125预孵育对HPC的保护作用无明显影响。（2）H／R明显上调CRT表达（较对照组高8．1倍）和caspase-12活性（较对照组高33．2倍）;单独HPC可诱导CRT表达增多（较对照组高2．6倍）,但上调程度较H／R组低60％。H／R前进行HPC降低CRT过表达程度（降低72．4％）及caspase-12活化水平（降低59．6％）。（3）HPC前应用p38MAPK抑制剂,抑制CRT表达上调（分别较HPC＋H／R组和HPC组低63．9％和71．9％）,并消除HPC减轻H／R上调caspase-12活性的作用（较HPC＋H／R组高7．1倍）;HPC前抑制JNK活性对CRT、caspase-12表达和活化均无明显影响。上述结果提示：HPC可激发适当的ERS,抑制H／R诱导的过度ERS,减少ER凋亡信号介导的细胞凋亡。p38MAPK信号途径在HPC诱导的ER应激分子表达、抑制ER凋亡信号分子活化等机制中发挥重要作用。     

An unusual Group 2 LEA gene family in citrus responsive to low temperature

Six cDNAs representing unique cold-induced sequences have been cloned from the hardy citrus relative Poncirus trifoliata. Among these, pBCORc115 and pBCORc119 were found to belong to the same gene family. Sequencing data indicated that pBCORc115 and pBCORc119 each contained an open reading frame, coding for a 19.8 kDa protein (COR19) and a smaller 11.4 kDa protein (COR11) respectively. Inspection of the deduced amino acid sequences revealed three large repeats in COR19, but only one was present in the COR11. Two elements: a Q-clustered tract and a K-rich motif were identified in each repeat. The K-rich motifs were similar to those of cotton D-11 and Group 2 LEA proteins. A Serine-cluster, a common feature in many Group 2 LEA-like proteins, was also found in these proteins, but it was in an unusual position at the carboxy-terminus. A bipartite motif of basic residues, similar to known nuclear targeting sequences, was also present in COR19 and COR11, suggesting that members of this protein family may have a nuclear targeting function. The expression of COR19 mRNA in response to cold acclimation, drought, flooding, and salinization was examined. COR19 expression in leaf tissue was induced in response to cold acclimation, but repressed during drought and flooding stress.     

Synergistic effect of colonization with arbuscular mycorrhizal fungi improves growth and drought tolerance of Plukenetia volubilis seedlings

The objective of this study was to investigate the effects of arbuscular mycorrhizal fungus (AMF) inoculation on plant growth and drought tolerance in seedlings of a promising oilseed crop, Sacha Inchi (Plukenetia volubilis L.), under well-watered or drought conditions. AMF inoculation was applied in four treatments: without AMF inoculation, Glomus versiforme, Paraglomus occultum, or combination of both microorganism inoculations. The results showed that AMF colonization significantly enhanced the growth of Sacha Inchi seedlings regardless of soil water conditions, and the greatest development was reached in plants dually inoculated under well-watered conditions. G. versiforme was more efficient than P. occultum. Plants inoculated with both symbionts had significantly greater specific leaf area, leaf area ratio and root volume when compared with the uninoculated control, G. versiforme, and P. occultum treatments alone, indicating a synergistic effect in the two AMF inoculation. Photosynthetic rate and water-use efficiency were stimulated by AMF, but not stomatal conductance. Inoculation with AM fungus increased antioxidant enzymes activities including guaiacol peroxidase and catalase, thus lowering hydrogen peroxide accumulation and oxidative damage, especially under drought stress conditions. However, proline content showed little change during drought stress and AMF colonization conditions, which suggested that proline accumulation might not serve as the main compound for osmotic adjustment of the studied species. These results indicate that AMF inoculation stimulated growth and enhanced drought tolerance of Sacha Inchi seedlings, through alterations in morphological, physiological and biochemical traits. This microbial symbiosis might be an effective cultivation practice in improving the performance and development for Sacha Inchi plants.     

Calcium-mediated enhancement of copper tolerance in Elodea canadensis

The alleviative effects of exogenous calcium on copper phytotoxicity were investigated in Elodea canadensis plants. There was a significant accumulation of Cu in the plants after their exposure to 0.01 mM Cu accompanied by many symptoms of toxicity. Increased uptake of Cu severely reduced content of photosynthetic pigments, soluble proteins, and free proline. The total antioxidant capacity (T-AOC), reduced glutathione (GSH), and non-protein thiol (NP-SH) were severely suppressed in Cu-stressed plants resulting in a rapid increase in content of superoxide anion (O₂ ^·?), hydrogen peroxide, lipid peroxidation, and cell death. Simultaneous application of Ca markedly increased the content of photosynthetic pigments, soluble proteins, free proline, T-AOC, GSH, and NP-SH, and reduced oxidative damage as indicated by lowered content of MDA, O₂ ^·?, and H₂O₂; and decreased cell death. Furthermore, application of Ca reduced Cu uptake and effectively reversed the Cu-induced nutrient imbalance.     

Immunosensor based on magnetic relaxation switch and biotin-streptavidin system for the detection of Kanamycin in milk

A rapid, sensitive, and simple immunosensor was developed for the detection of Kanamycin (KM) in milk. This immunosensor is based on magnetic relaxation switch (MRS) assay and biotin-streptavidin system (B-SA system). The target analyte (KM) competed with those on the surface of the superparamagnetic iron oxide (SPIO) nanoparticles and hence affected the formation of SPIO aggregates. The dispersed and aggregated states of SPIO can modulate the spin-spin relaxation time (T(2)) of the neighboring water molecule. T(2) was then changed as an effect of the target analyte. The B-SA system was used to amplify the SPIO binding, thus enhance the sensitivity. The detection working was 1.5 to 25.2ngmL(-1) and limit of detection (LOD) was determined to be 0.1ngmL(-1). The LOD of the immunosensor decreased tenfold, and its analysis time (45min) was much shorter than that of enzyme-linked immunosorbent assay (6h to 8h). The average recoveries of the KM at various spiking levels ranged from 80.2% to 85.6% with a relative standard deviation (RSD) below 4.0%. The results showed that the MRS immunosensor was a promising platform for the determination of small molecular residues because of its high sensitivity, specificity, homogeneity, and speed.     

The influence of an intramolecular hydrogen bond in differential recognition of inhibitory acceptor analogs by human ABO(H) blood group A and B glycosyltransferases

Human ABO(H) blood group glycosyltransferases GTA and GTB catalyze the final monosaccharide addition in the biosynthesis of the human A and B blood group antigens. GTA and GTB utilize a common acceptor, the H antigen disaccharide alpha-l-Fucp-(1-->2)-beta-d-Galp-OR, but different donors, where GTA transfers GalNAc from UDP-GalNAc and GTB transfers Gal from UDP-Gal. GTA and GTB are two of the most homologous enzymes known to transfer different donors and differ in only 4 amino acid residues, but one in particular (Leu/Met-266) has been shown to dominate the selection between donor sugars. The structures of the A and B glycosyltransferases have been determined to high resolution in complex with two inhibitory acceptor analogs alpha-l-Fucp(1-->2)-beta-d-(3-deoxy)-Galp-OR and alpha-l-Fucp-(1-->2)-beta-d-(3-amino)-Galp-OR, in which the 3-hydroxyl moiety of the Gal ring has been replaced by hydrogen or an amino group, respectively. Remarkably, although the 3-deoxy inhibitor occupies the same conformation and position observed for the native H antigen in GTA and GTB, the 3-amino analog is recognized differently by the two enzymes. The 3-amino substitution introduces a novel intramolecular hydrogen bond between O2' on Fuc and N3' on Gal, which alters the minimum-energy conformation of the inhibitor. In the absence of UDP, the 3-amino analog can be accommodated by either GTA or GTB with the l-Fuc residue partially occupying the vacant UDP binding site. However, in the presence of UDP, the analog is forced to abandon the intramolecular hydrogen bond, and the l-Fuc residue is shifted to a less ordered conformation. Further, the residue Leu/Met-266 that was thought important only in distinguishing between donor substrates is observed to interact differently with the 3-amino acceptor analog in GTA and GTB. These observations explain why the 3-deoxy analog acts as a competitive inhibitor of the glycosyltransferase reaction, whereas the 3-amino analog displays complex modes of inhibition.     

α<Subscript>2A</Subscript>- and α<Subscript>2C</Subscript>-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands

The poor norepinephrine innervation and high density of Gi/o-coupled α_2A- and α_2C-adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D₂-like receptor ligands, such as the D₃ receptor agonist 7-OH-PIPAT and the D₄ receptor agonist RO-105824, to α₂-adrenoceptors in cortical and striatal tissue, which express α_2A-adrenoceptors and both α_2A- and α_2C-adrenoceptors, respectively. The affinity of dopamine for α₂-adrenoceptors was found to be similar to that for D₁-like and D₂-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α_2A- and α_2C-adrenoceptors. Their ability to activate Gi/o proteins through α_2A- and α_2C-adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α₂-adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α_2A- and α_2C-adrenoceptors was nearly identical to its binding to the crystallized D₃ receptor. Therefore, we provide conclusive evidence that α_2A- and α_2C-adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D₂-like receptor ligands, which calls for revisiting previous studies with those ligands.     

Associations of HSP90AA2 gene polymorphisms with disease susceptibility,glucocorticoids efficacy and health-related quality of life in Chinese systemic lupus erythematosus patients

Although the current glucocorticoids (GCs) treatment for systemic lupus erythematosus (SLE) is effective to a certain extent, the difference in therapeutic effect between patients is still a widespread problem. Some patients can have repeated attacks that greatly diminish their quality of life. This study was conducted to investigate the relationship between HSP90AA2 polymorphisms and disease susceptibility, GCs efficacy and health-related quality of life (HRQoL) in Chinese SLE patients. A case–control study was performed in 470 SLE patients and 470 normal controls. Then, 444 patients in the case group were followed up for 12 weeks to observe efficacy of GCs and improvement of HRQoL. Two single nucleotide polymorphisms (SNPs) of HSP90AA2 were selected for genotyping: rs1826330 and rs6484340. HRQoL was assessed using the SF-36 questionnaire. The minor T allele of rs1826330 and the TT haplotype formed by rs1826330 and rs6484340 showed associations with decreased SLE risk (T allele: P_BH?=?0.022; TT haplotype: P_BH?=?0.033). A significant association between rs6484340 and improvement of HRQoL was revealed in the follow-up study. Five subscales of SF-36 were appeared to be influenced by rs6484340: total score of SF-36 (additive model: P_BH?=?0.026), physical function (additive model: P_BH?=?0.026), role-physical (recessive model: P_BH?=?0.041), mental health (dominant model: P_BH?=?0.047), and physical component summary (additive model: P_BH?=?0.026). No statistical significance was found between HSP90AA2 gene polymorphisms and GCs efficacy. These results revealed a genetic association between HSP90AA2 and SLE. Remarkably, HSP90AA2 has an impact on the improvement of HRQoL in Chinese population with SLE.