果实成熟衰老与抗病性的调控机制研究

　　果实为开花植物所特有的发育器官，在种子的成熟和传播过程中发挥着重要作用。同时，肉质果实中含有丰富的营养物质，包括纤维素、维生素、抗氧化剂等，成为人们饮食的重要组成部分。由于果实的成熟衰老和抗病性直接影响果品的质量和市场价值，因此，研究果实成熟衰老和抗病性的调控机制具有重要的理论意义和应用前景。本文主要利用蛋白质组学的方法，探讨外源化学物质抑制果实成熟衰老和诱导抗病性的调控机制。 1. 硅对果实的抗病性诱导：用硅酸钠（1%）处理采后的甜樱桃果实，再接种褐腐病原菌（Molinilia fracticola），置于20C下，观测贮藏期间果实的发病率，并分析硅处理后诱导的主要蛋白质及调控机制。研究结果表明：硅酸钠处理可显著抑制贮藏期间褐腐病的发生，其抑病机理与硅诱导PR-蛋白的表达，提高果实的抗氧化水平，减轻由病原菌侵染造成的氧化胁迫相关。同时，硅处理还能保护细胞骨架结构，有利于增强果实对病原菌入侵的抵抗力。 2. 水杨酸对果实的抗病性诱导：用水杨酸（SA，2mM）在果园处理三种成熟度的甜樱桃果实，然后接种青霉病原菌（Penicillium expansum）观察其发病情况，并取样分析参与抗病性应答的主要蛋白质及调控机制。试验结果表明：SA处理能显著降低青霉病的发病率和抑制病斑扩展，而且SA对低成熟度甜樱桃果实的抗性诱导效果更好。在八成熟的果实中，有5个热激蛋白和4个脱氢酶蛋白被SA诱导，这些蛋白参与了糖酵解和三羧酸循环。抗氧化蛋白和PR蛋白主要参与较低成熟度果实的抗性应答，而热激蛋白和脱氢酶在较高成熟度果实的抗性应答中更明显，SA诱导的抗性与代谢途径相关。 　　3. 草酸对果实的抗性诱导：用5mM的草酸处理冬枣果实后，接种青霉菌（P. expansum），观察果实发病情况，测定果实相关的生理指标，分析参与果实抗性应答的主要蛋白质及调控机制。结果表明：草酸能明显延缓冬枣果实的衰老，提高果实对青霉菌的抗性。草酸处理能抑制果实乙烯的释放量和呼吸强度，延缓叶绿素的降解，减少乙醇积累。利用蛋白质组学的研究方法证实了在25个参与了草酸处理应答的蛋白中，胱硫醚-β-合酶结构域包含蛋白（CBB domain-containing protein）和3个与光合作用相关蛋白[二磷酸核酮糖羧化酶/加氧酶（Ribulose bisphosphate carboxylase/oxygenase activase, chloroplast precursor），二磷酸核酮糖羧化酶/加氧酶大亚基结合蛋白（RuBisCO large subunit-binding protein subunit beta, chloroplast precursor），植物光系统Ⅱ放氧复合蛋白2（PSII oxygen-evolving complex protein 2）]的表达量上调，乙醇脱氢酶的表达量出现下调。草酸处理还提高了与乙烯合成前体相关蛋白的表达，抑制了ACC合成酶的活性。草酸提高果实抗病的机制与延缓果实成熟衰老和保持果实抗性有关。 4. 果实衰老的调控机制：采用高氧（100%）和低氧（2-3%）处理苹果果实，观察果实衰老的进程，并基于蛋白质组学的研究方法，探讨苹果果实衰老与线粒体蛋白质组的关系。结果表明，在苹果衰老过程中有22个蛋白的表达量发生变化，这些蛋白主要参与了三羧酸循环，电子传递，碳代谢和胁迫应答。高氧处理能诱导氧化胁迫，加速了果实的衰老。质谱鉴定结果证明：在高氧胁迫下，超氧化物歧化酶（manganese superoxide dismutase，MnSOD）和线粒体外膜通道蛋白（porin) 的表达量降低，MnSOD的活性受到抑制，由此提高了线粒体中超氧阴离子的含量，增加了蛋白质的氧化损伤。 此外，高氧处理改变了porin的功能，导致了线粒体膜的透势发生变化，从而引起外膜损伤。由此阐明了活性氧在果实的成熟衰老调控中的重要作用。

Genomic organization and sequence analysis of the vomeronasal receptor V2R genes in mouse genome

Two multigene superfamilies, named V1R and V2R, encoding seven-transmembrane-domain G-protein coupled receptors (GPCRs) have been identified as pheromone receptors in mammals. Three V2R gene families have been described in mouse and rat. Here we screened

蛙虹彩病毒cop基因的克隆表达及亚细胞定位

从蛙虹彩病毒(Rana gryliovirus,RGV)基因组中克隆了含凋亡相关结构域的新基因-cop(Caspaserecruit ment domain only protein,COP)基因的全部编码区,成功构建了重组表达载体,进行了原核表达,并在鲤鱼上皮瘤细胞(Epithelioma papulosumcyprini,EPC)中进行了亚细胞定位.序列分析表明,RGVcop基因全长288 bp,编码一个长为95 aa,分子量为10.4×103的推定蛋白.二级结构预测表明其含有5个α螺旋.同源性比对分

Molecular cloning and characterization of crucian carp (Carassius auratus L.) interferon regulatory factor 7

Interferon (IFN) can induce an antiviral state via interferon-regulatory transcription factors (IRFs), which bind to and control genes directed by the interferon-stimulated response element (ISRE). Here we describe a fish IRF, termed CaIRF7, cloned from a subtractive cDNA library which is constructed with mRNAs obtained from crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. CaIRF7 cDNA was found to be 1816 bp in length, with a 42 bp 5' UTR and a 508 bp 3' UTR. The open reading frame translates into 421 amino acids in which a DNA-binding domain (DBD) containing the repeated tryptophan motif and IRFs association domain have been identified. Like chicken GgIRF3, CaIRF7 was most similar to mammalian IRF7 with 27 to 30% identity overall and some 37% identity in their DBDs. A single transcript of 1.9 kb was detected in virally induced CAB cells by virtual Northern blotting. RT-PCR analysis revealed a wide tissue distribution of CaIRF7 constitutive expression, with detectable transcript in non-infected CAB cells and various tissues of healthy crucian carp. In addition, CaIRF7 expression was differentially increased by stimulation of the CAB cells with active GCHV, UV-inactivated GCHV or CAB IFN, indicating that the activation of CaIRF7 was directly regulated by IFN. (C) 2003 Published by Elsevier Ltd.

The examination of the traditional femininity and women’s lives in ancient China

This thesis explores the evolution of the concept of traditional Chinese femininity in relation to women’s lives in ancient China (221 BCE – A.D.1840). It proposes that the traditional Chinese femininity had been trying to seek a balance between the permanent principles and contingency plans for the stability and development of the society, which caused women’s humiliation and freedom. In reality, politicians and thinkers in ancient China had been transforming the concept of femininity itself to make it more adaptable to the social conditions of that time. This may be discussed in terms of three aspects. Firstly, the traditional concept of Chinese human relationships, including the ethical order, always emphasised the influence of individual behaviour on others and the overall stability and linked development of family, society and nation. Thus, both men and women, must be placed within this interrelated, interacting and cooperating relationship. Secondly, the association of family and country created an overlap of family and public affairs, which, objectively, facilitated the movement of women from the inner to the public arena. Thirdly, the notions of political and ethical morality and of men’s virtues and women’s virtues were integrated because of the union of family and nation. Therefore, typically virtuous women could be a source of encouragement for men and, furthermore, men formulated their virtues in the public space by formulating women’s virtues in the private space. The shaping of the gender image and concept of women in ancient China reflected the country’s changing cultural and gender norms. Chinese femininity and lifestyles, like Chinese history, were a continuous presence in the society but were also constantly changing. Through this study, it could be noted that Chinese women were not hidden and that their subjectivity and the concepts motivating them were not merely devised by a male-dominated society and culture.

UNC-6 (netrin) stabilizes oscillatory clustering of the UNC-40 (DCC) receptor to orient polarity.

The receptor deleted in colorectal cancer (DCC) directs dynamic polarizing activities in animals toward its extracellular ligand netrin. How DCC polarizes toward netrin is poorly understood. By performing live-cell imaging of the DCC orthologue UNC-40 during anchor cell invasion in Caenorhabditis elegans, we have found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin). Time-lapse analyses revealed that UNC-40 clusters assemble, disassemble, and reform at periodic intervals in different regions of the cell membrane. This oscillatory behavior indicates that UNC-40 clusters through a mechanism involving interlinked positive (formation) and negative (disassembly) feedback. We show that endogenous UNC-6 and ectopically provided UNC-6 orient and stabilize UNC-40 clustering. Furthermore, the UNC-40-binding protein MADD-2 (a TRIM family protein) promotes ligand-independent clustering and robust UNC-40 polarization toward UNC-6. Together, our data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering. We propose that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.

Irisin and FNDC5 in retrospect: An exercise hormone or a transmembrane receptor?

FNDC5 (fibronectin domain-containing [protein] 5) was initially discovered and characterized by two groups in 2002. In 2011 FNDC5 burst into prominence as the parent of irisin, a small protein containing the fibronectin type III domain. Irisin was proposed to be secreted by skeletal muscle cells in response to exercise, and to circulate to fat tissue where it induced a transition to brown fat. Since brown fat results in dissipation of energy, this pathway is of considerable interest for metabolism and obesity. Here I review the original discoveries of FNDC5 and the more recent discovery of irisin. I note in particular three problems in the characterization of irisin: the antibodies used to detect irisin in plasma lack validity; the recombinant protein used to demonstrate activity in cell culture was severely truncated; and the degree of shedding of soluble irisin from the cell surface has not been quantitated. The original discovery proposing that FNDC5 may be a transmembrane receptor may deserve a new look.

Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus.

Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn't localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don't play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn't show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated "paradoxical growth effect" at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.

Cyclin-dependent kinase 11(p58) interacts with HBO1 and enhances its histone acetyltransferase activity.

CDK11(p58), a 58kDa protein of the PITSLRE kinase family, plays an important role in cell cycle progression, and is closely related to cell apoptosis. To gain further insight into the function of CDK11(p58), we screened a human fetal liver cDNA library for its interacting proteins using the yeast two-hybrid system. Here we report that histone acetyltransferase (HAT) HBO1, a MYST family protein, interacts with CDK11(p58) in vitro and in vivo. CDK11(p58) and HBO1 colocalize in the cell nucleus. Recombinant CDK11(p58) enhances the HAT activity of HBO1 significantly in vitro. Meanwhile, overexpression of CDK11(p58) in mammalian cells leads to the enhanced HAT activity of HBO1 towards free histones. Thus, we conclude that CDK11(p58) is a new interacting protein and a novel regulator of HBO1. Both of the proteins may be involved in the regulation of eukaryotic transcription.

Functional domains of the human epididymal protease inhibitor, eppin

Eppin has two potential protease inhibitory domains: a whey acid protein or four disulfide core domain and a Kunitz domain. The protein is also reported to have antibacterial activity against Gram-negative bacteria. Eppin and its whey acid protein and Kunitz domains were expressed in Escherichia coli and their ability to inhibit proteases and kill bacteria compared. The Kunitz domain inhibits elastase (EC 3.4.21.37) to a similar extent as intact eppin, whereas the whey acid protein domain has no such activity. None of these fragments inhibits trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1) at the concentrations tested. In a colony forming unit assay, both domains have some antibacterial activity against E. coli, but this was not to the same degree as intact eppin or the two domains together. When bacterial respiratory electron transport was measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, eppin and its domains caused an increase in the rate of respiration. This suggests that the mechanism of cell killing may be partly through the permeablization of the bacterial inner membrane, resulting in uncoupling of respiratory electron transport and consequent collapse of the proton motive force. Thus, we conclude that although both of eppin’s domains are involved in the protein’s antibacterial activity, only the Kunitz domain is required for selective protease inhibition.

RUNX3 downregulation in human lung adenocarcinoma is independent of p53, EGFR or KRAS status

RUNX3 aberrations play a pivotal role in the oncogenesis of breast, gastric, colon, skin and lung tissues. The aim of this study was to characterize further the expression of RUNX3 in lung cancers. To achieve this, a lung cancer tissue microarray (TMA), frozen lung cancer tissues and lung cell lines were examined for RUNX3 expression by immunohistochemistry, while the TMA was also examined for EGFR and p53 expression. RUNX3 promoter methylation status, and EGFR and KRAS mutation status were also investigated. Inactivation of RUNX3 was observed in 70% of the adenocarcinoma samples, and this was associated with promoter hypermethylation but not biased to EGFR/KRAS mutations. Our results suggest a central role of RUNX3 downregulation in pulmonary adenocarcinoma, which may not be dependent of other established cancer-causing pathways and may have important diagnostic and screening implications.

Constitutive expression of human keratin 14 gene in mouse lung induces premalignant lesions and squamous differentiation

Squamous cell carcinoma accounts for 20% of all human lung cancers and is strongly linked to cigarette smoking. It develops through premalignant changes that are characterized by high levels of keratin 14 (K14) expression in the airway epithelium and evolve through basal cell hyperplasia, squamous metaplasia and dysplasia to carcinoma in situ and invasive carcinoma. In order to explore the impact of K14 in the pulmonary epithelium that normally lacks both squamous differentiation and K14 expression, human keratin 14 gene hK14 was constitutively expressed in mouse airway progenitor cells using a mouse Clara cell specific 10 kDa protein (CC10) promoter. While the lungs of CC10-hK14 transgenic mice developed normally, we detected increased expression of K14 and the molecular markers of squamous differentiation program such as involucrin, loricrin, small proline-rich protein 1A, transglutaminase 1 and cholesterol sulfotransferase 2B1. In contrast, wild-type lungs were negative. Aging CC10-hK14 mice revealed multifocal airway cell hyperplasia, occasional squamous metaplasia and their lung tumors displayed evidence for multidirectional differentiation. We conclude that constitutive expression of hK14 initiates squamous differentiation program in the mouse lung, but fails to promote squamous maturation. Our study provides a novel model for assessing the mechanisms of premalignant lesions in vivo by modifying differentiation and proliferation of airway progenitor cells. © The Author 2008. Published by Oxford University Press. All rights reserved.

Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction

The recognition of microbial pathogens by the innate immune system involves Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns. Different TLRs recognize different pathogen-associated molecular patterns, with TLR-4 mediating the response to lipopolysaccharide from Gram-negative bacteria. All TLRs have a Toll/IL-1 receptor (TIR) domain, which is responsible for signal transduction. MyD88 is one such protein that contains a TIR domain. It acts as an adapter, being involved in TLR-2, TLR-4 and TLR-9 signalling; however, our understanding of how TLR-4 signals is incomplete. Here we describe a protein, Mal (MyD88-adapter-like), which joins MyD88 as a cytoplasmic TIR-domain-containing protein in the human genome. Mal activates NF-kappaB, Jun amino-terminal kinase and extracellular signal-regulated kinase-1 and -2. Mal can form homodimers and can also form heterodimers with MyD88. Activation of NF-kappaB by Mal requires IRAK-2, but not IRAK, whereas MyD88 requires both IRAKs. Mal associates with IRAK-2 by means of its TIR domain. A dominant negative form of Mal inhibits NF-kappaB, which is activated by TLR-4 or lipopolysaccharide, but it does not inhibit NF-kappaB activation by IL-1RI or IL-18R. Mal associates with TLR-4. Mal is therefore an adapter in TLR-4 signal transduction.

Nicotinamide Riboside Is a Major NAD+ Precursor Vitamin in Cow Milk

Background: Nicotinamide riboside (NR) is a recently discovered NAD+ precursor vitamin with a unique biosynthetic pathway. Although the presence of NR in cow milk has been known for more than a decade, the concentration of NR with respect to the other NAD+ precursors was unknown.

Objective: We aimed to determine NAD+ precursor vitamin concentration in raw samples of milk from individual cows and from commercially available cow milk.

Methods: LC tandem mass spectrometry and isotope dilution technologies were used to quantify NAD+ precursor vitamin concentration and to measure NR stability in raw and commercial milk. Nuclear magnetic resonance (NMR) spectroscopy was used to test for NR binding to substances in milk.

Results: Cow milk typically contained ∼12 μmol NAD+ precursor vitamins/L, of which 60% was present as nicotinamide and 40% was present as NR. Nicotinic acid and other NAD+ metabolites were below the limits of detection. Milk from samples testing positive for Staphylococcus aureus contained lower concentrations of NR (Spearman ρ = −0.58, P = 0.014), and NR was degraded by S. aureus. Conventional milk contained more NR than milk sold as organic. Nonetheless, NR was stable in organic milk and exhibited an NMR spectrum consistent with association with a protein fraction in skim milk.

Conclusions: NR is a major NAD+ precursor vitamin in cow milk. Control of S. aureus may be important to preserve the NAD+ precursor vitamin concentration of milk.

Estratégias de alteração da funcionalidade de proteínas de soja

Este trabalho teve como principal objetivo estudar e modificar as propriedades funcionais das proteínas de soja de forma a otimizar e diversificar a sua aplicação industrial. Para tal, foram propostas e estudadas quatro estratégias: i) extração do isolado de proteínas de soja (IPS) a partir de diferentes matérias-primas, ii) adição de galactomananas (GM) com graus de ramificação e massas moleculares diferentes, iii) hidrólise enzimática controlada das proteínas de soja, iv) processamento por alta pressão hidrostática. O estudo e a interpretação da influência destas estratégias sobre as propriedades funcionais das proteínas de soja, nomeadamente, na capacidade gelificante e emulsionante, foram realizados recorrendo fundamentalmente a ensaios reológicos dinâmicos a baixas deformação, espectroscopia de infravermelho, electroforeses, calorimetria diferencial de varrimento e ensaios de microscopia confocal de varrimento laser. O estudo da extração e caracterização dos isolados de proteínas de soja obtidos a partir de diferentes matérias-primas permitiu concluir que as caraterísticas físico-químicas dos isolados são dependentes da origem da matéria-prima de extração e da severidade dos tratamentos industriais prévios à extração do isolado. Contudo, as propriedades viscoelásticas dos géis obtidos por aquecimento controlado não foram significativamente distintas embora tenha sido possível relacionar o grau de agregação com a diminuição da temperatura de gelificação e com o aumento inicial dos módulos viscoelásticos. As alterações sofridas pelos isolados de origem comercial mostraram ser irreversíveis resultando em géis menos rígidos e com maior caráter viscoso. A adição de galactomanana alterou significativamente o mecanismo de gelificação induzido termicamente das proteínas de soja, bem como as propriedades viscoelásticas dos géis e a microestrutura dos géis, demonstrando-se a ocorrência de separação de fases, em virtude da incompatibilidade termodinâmica entre os biopolímeros, resultando em géis mais rígidos e no decréscimo da temperatura de gelificação. A extensão destas alterações foi dependente da massa molecular, grau de ramificação e da razão IPS/GM. O efeito da hidrólise enzimática por ação da bromelina, nas propriedades gelificantes e emulsionantes das proteínas de soja, mostrou ser dependente do grau de hidrólise (GH). Valores de GH inferiores a 15 % melhoraram as propriedades gelificantes das proteínas de soja. Por outro lado, o aumento do GH teve um efeito negativo nas propriedades emulsionantes, o qual foi atenuado por adição da goma de alfarroba, com efeito positivo na gelificação das proteínas de soja. A concentração crítica limite de compatibilidade entre os hidrolisados de proteína de soja e a goma de alfarroba aumentou com o decréscimo do GH e da massa molecular do polissacacrídeo. O efeito da AP sobre as propriedades físico-químicas e funcionais dos IPS foi influenciado pela origem do isolado e pelas condições de tratamento. O processamento até 100 MPa desencadeou um aumento da atividade emulsionante e considerável melhoria da capacidade gelificante. Contudo, valores de pressão superiores promoveram a desnaturação das proteínas constituintes dos isolados, resultando no decréscimo da temperatura de gelificação e numa re-associação das subunidades proteicas, diminuindo a elasticidade dos géis finais. Os resultados sugeriram que as alterações nas proteínas de soja promovidas durante o tratamento por AP constituem um fator limitante para o desdobramento e re-associação durante o aquecimento térmico, necessários para a formação e fortalecimento de gel formado. O processamento por AP influenciou a estrutura secundária e a microestrutura das amostras. A presença de GA teve um papel baroprotetor. Assim, com este trabalho demonstrou-se que com as estratégias seguidas para manipulação das propriedades funcionais de proteínas de soja, nomeadamente através da adição de um polissacarídeo com propriedades estruturais controladas, da adequada combinação da adição de um polissacarídeo neutro com a hidrólise controlada das proteínas ou com tratamento por alta pressão, é possível a criação de novas funcionalidades, com utilidade no desenvolvimento de novas formulações alimentares, permitindo expandir a aplicação destas proteínas vegetais.