Application of methyl parathion hydrolase (MPH) as a labeling enzyme

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 +/- 13.19 s(-1), which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)(5)-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 mu L hemolymph from an infected shrimp and differentiated it from a healthy shrimp.

缺磷胁迫导致高等植物叶片中磷脂酰甘油（PG）含量降低的机理研究

磷脂酰甘油（PG）是植物类囊体膜中唯一的磷脂，在它的sn-2位上总是连着一个棕榈酸（16：0）或反式十六碳烯酸（16：1 trans）。由于PG的分子结构独特，对它的功能已有了很多研究，目前认为PG在维持类囊体膜的结构与功能方面具有非常重要的作用。缺磷胁迫下，蓝藻、衣藻及拟南芥、大麦等物种中均检测到了PG含量的下降。对这一现象的常见解释是缺磷导致了PG生物合成受阻，从而引起了其含量的降低。但迄今为止尚没有试验证据支持。本研究比较了缺磷对不同叶龄的小麦与烟草叶片中PG含量与PG水解酶的活性的影响，同时对缺磷叶片酶粗提液水解外源PG后的主要产物、几种磷脂酶抑制剂对上述酶反应的影响等进行了研究，以阐明缺磷条件下叶片中PG含量下降的主要原 因。 缺磷小麦第一叶完全展开时，PG含量与PG水解酶活性均与对照相似;而第三叶完全展开时，尽管缺磷第三叶中PG水解酶活性也与对照相似，但其PG含量低于对照。这一结果表明，在小麦叶片完全展开之前，缺磷条件未影响叶片中的PG水解酶活性，第三叶中较低的PG含量应由PG的生物合成受阻引起。并且，由于缺磷植株第一叶完全展开时PG含量未受影响而第三叶中却表现出了轻微降低，可以推测叶片萌发越晚，PG生物合成受到的抑制就会越严重。 为了研究叶片衰老过程中PG含量下降的原因，我们比较了6，10，14与18日龄时缺磷与对照小麦植株第一叶中PG的相对含量与PG水解酶活性。研究发现：6日龄时，刚刚完全展开的缺磷和对照小麦第一叶中无论是PG含量还是PG水解酶活性都较为相似;而随着叶片的逐渐衰老，缺磷植株第一叶中PG含量大幅度下降，同时伴随着PG水解酶活性的急剧上升。18日龄时，缺磷小麦第一叶中的PG含量较对照降低了69.1%，其PG水解酶活性也远高于对照，37ºC下温育30min后，缺磷叶片的酶粗提液使外源PG含量降低了74.16%，而对照中只降低了13.7%。上述结果表明，缺磷条件下，小麦叶片中PG含量降低的程度与PG水解酶活性的强弱密切相关，PG水解加剧是导致老叶中PG含量降低的一个重要原因。 磷脂酶是水解磷脂的主要酶类。目前在植物体中发现的磷脂酶种类主要有磷脂酶D（PLD）、磷脂酶C（PLC）与磷脂酶A（PLA）。通过薄层层析（TLC），我们发现缺磷小麦叶片的酶粗提液水解外源PG后的主要产物是磷脂酸（PA）、二脂酰甘油（DAG）与游离脂肪酸（FFA）。将n-丁醇加入到缺磷小麦叶片的体外酶反应体系中后，观察到PA、DAG与FFA的生成量均表现出一定程度的降低。由于n-丁醇是PA经PLD途径生成的抑制剂，因此，上述结果表明PLD参与了缺磷条件下小麦叶片中PG的水解。硫酸新霉素是PLC的非特异性抑制剂，低浓度的硫酸新霉素（100μM 和 200μM ）加入到缺磷小麦叶片的体外酶反应体系后，三种产物的生成受到了严重抑制，表明PLC也与缺磷叶片中PG的降解密切相关。 为了进一步分析缺磷导致PG含量降低的原因，我们以烟草为试验材料，检测了缺磷胁迫对烟草嫩叶和老叶中的PG含量、PG水解酶活性、与PG降解相关的酶的种类及PLC、PLDα、PLDβ与PAT-1基因在mRNA上表达水平的的影响。结果表明，缺磷烟草叶片中PG含量的降低由PG生物合成受阻与PG降解加剧共同导致，PLC和PLD活性与烟草叶片中PG的降解有关。缺磷植株老叶中PG水解酶活性及PLC、PLDα、PLDβ基因在mRNA水平上的表达量均高于对照，表明在磷胁迫条件下，老叶中PG水解酶活性可能受到转录水平上的调节， PLC、PLDα、PLDβ转录活性的增强导致了PLC、PLD活性加强，从而引起PG降解的加剧，最终导致了PG含量的降低。与PLC、PLDα和PLDβ不同，缺磷胁迫对patatin蛋白（表现PLA2活性）的编码基因PAT-1在转录水平上的表达无影响，TLC分析PG的水解产物也未检测到溶血磷脂酰甘油（LPG）的生成。由此可见，PLA活性可能与缺磷条件下PG的降解无关。

小麦杂交坏死蛋白质组学研究

小麦杂交坏死是某些小麦杂交种表现出的叶片提前逐渐死亡的现象。它是由两个坏死基因Ne1和Ne2在杂交种中相遇后发生显性互补引起的。坏死从叶片尖端逐渐过渡到叶片基部，从成熟叶片发展到幼嫩叶片。一些严重坏死的F1完成它的生活周期前就在不同的生长阶段死去，无法获得F1种子，这就限制了携带优良性状的亲本的选择和优良基因的交流。另外，小麦杂交坏死是一个独特的研究植物程序性死亡的遗传系统。虽然小麦杂交坏死这种现象已经发现很多年，但其详细的分子机理却仍然未知。对小麦杂交坏死的分子机理进行深入研究将有助于克服小麦杂交利用中杂交坏死的遗传障碍，此外，也为深入研究植物的PCD机理提供可操作靶分子。 本论文采用高通量蛋白质组研究技术对小麦杂交坏死进行了研究。携带坏死基因Ne2的小麦品种Pan555（P）和携带坏死基因Ne2的小麦品种Zheng891（Z）生长发育完全正常，将两个亲本杂交，所得杂交F1代PZF1表现杂交坏死。在小麦生长阶段8，旗叶（Flag leaf）刚刚出现，PZF1的旗叶下第一片叶子（FL-1）还是完全绿色，FL-2叶尖开始有坏死斑出现。在这个阶段，分别将PZF1，P，Z的FL-2叶剪成相等的尖，中，基三段。我们选择的PZF1的FL-2叶，其叶尖段已经有成片的坏死斑出现;中间段零星出现少量坏死斑点;基部段和亲本一样还是完全的绿色，代表坏死进程中的不同阶段。又选PZF1的FL-1和FL-2分别代表杂交坏死启动前和杂交坏死启动后。两个亲本P和Z的FL-2叶的三段及FL-1叶正常，都是完全绿色。 首先分别分析了PZF1，P和Z的FL-2叶的尖、中、基三段的蛋白表达情况。在PZF1的尖、中、基三段共检测到23个差异表达蛋白点。这23个点在两个亲本的尖、中、基三段中的表达丰度没有显著差异（p<0.05），说明这23个蛋白的差异表达不是由于叶段的不同引起，确与杂交坏死相关。对这23个蛋白进行了MALDI-TOF质谱鉴定，其中18个得到成功鉴定。然后对PZF1，P和Z的FL-1叶和FL-2叶的蛋白表达情况进行了分析。与PZF1的FL-1叶比较，在FL-2叶中检测到19个蛋白上调，20个蛋白下调。这39个蛋白的丰度在两个亲本的FL-1和FL-2叶之间没有显著差异，说明这39个蛋白的差异表达不是由于叶位的不同引起，确与杂交坏死相关。对这39个蛋白进行质谱鉴定其中26个得到成功鉴定。 根据被鉴定蛋白的功能及其表达丰度的变化，对这些蛋白在小麦杂交坏死中可能的作用进行了讨论。与PZF1的FL-2叶基部相比，S-腺苷同型半胱氨酸水解酶（S-adenosyl homocysteine hydrolase）在中部极显著（p<0.01）下调，而在中部和尖段之间没有显著差异，保持低丰度不变。腺苷甲硫氨酸3（AdoMet synthase 3）和甲硫氨酸合成酶1（Methionine synthase 1）都在PZF1的FL-2叶尖段上调。甲基化循环中的这3个酶比例的不协调可能会以不同的方式加速细胞老化。 与PZF1的FL-1叶比较，尿卟啉环脱羧酶（Uroporphyrinogen decarboxylase）在FL-2叶中下调，这将引起尿卟啉环III的积累。脂加氧酶（Lipoxygenases）在FL-2叶中上调。尿卟啉环III的积累和脂加氧酶的上调都会引起细胞内活性氧的增加。另外活性氧和脂加氧酶都会使脂发生过氧化作用，进而导致细胞膜完整性受到破坏，最终可能导致细胞死亡。 与基部段比较，在PZF1的FL-2叶的尖段和/或中间段;以及与PZF1的FL-1叶比较，在FL-2叶中，都有很多防御性蛋白的上调，这暗示应对活性氧、脂过氧化、甲基化循环中三个酶比例的不协调等引起的对细胞的破坏作用，细胞可能启动了抗细胞死亡系统来应对这种细胞内部的胁迫。 然而，与基部段比较，一些能量相关蛋白在PZF1的FL-2叶的尖段和/或中间段;以及与PZF1的FL-1叶比较，在FL-2叶中的异常表达可能会以干扰能量循环的方式加速细胞死亡。另外，与FL-2基部段比较，在尖段和/或中间段，以及与PZF1的FL-1比较，在FL-2中，都有一些防御性蛋白、蛋白合成相关的蛋白以及单链DNA结合蛋白下调，它们的变化可能会降低细胞的抵抗力，蛋白合成能力以及DNA修复能力。细胞正常代谢的很多方面都受到干扰从而使PZF1叶细胞最终走向死亡。 本研究中发现了三个甲基化循环中的酶变化，而且S-腺苷同型半胱氨酸水解酶是在坏死进程的较早阶段发生下调，它的变化可能是小麦杂交坏死的一个诱因，这暗示小麦杂交坏死可能是一个表观遗传学事件。另外本研究还发现一些和活性氧，脂氧化等相关的蛋白的变化，而活性氧增加和脂氧化都是细胞凋亡的典型特征。所以本研究为表观遗传细胞凋亡和氧化胁迫细胞凋亡的研究提供了很有价值的信息。

Relationship between COPD and polymorphisms of HOX-1 and mEPH in a Chinese population

Recent studies have proposed that susceptibility to chronic obstructive pulmonary disease (COPD) might be related with the polymorphisms of some genes encoding antioxidant enzymes, such as heme oxygenase-1 (HOX-1) and microsomal epoxide hydrolase (mEPH).

Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to hypoxic stress

Hypoxia, as one suboptimal environmental condition, can affect the physiological state of shrimp during pond aquaculture. To better understand the mechanism of response to hypoxic stress in Chinese shrimp Fenneropenaeus chinensis, proteome research approach was utilized. Differentially expressed proteins of hepatopancreas in adult Chinese shrimp between the control and hypoxia-stressed groups were screened. By 2-DE analysis, 67 spots showed obvious changes after hypoxia. Using LC-ESI-MS/MS, 51 spots representing 33 proteins were identified including preamylase, arginine kinase, phosphopyruvate hydratase, citrate synthase, ATP synthase alpha subunit, chymotrypsin BI, chitinase, ferritin, C-type lectin receptors, transketolase, formylglutathione hydrolase, formyltetrahydrofolate dehydrogenase, aldehyde dehydrogenase, glutathione peroxidase, cytosolic manganese superoxide dismutase, protein disulfide isomerase, beta-actin, oncoprotem nm23, crustacyanin-Cl and so on. These proteins could be functionally classified into several groups such as proteins related to energy production, metabolism-related proteins, immune-related proteins, antioxidant proteins, chaperones, cytoskeleton proteins and ungrouped proteins. The transcription levels of ten selected genes encode the identified proteins were analyzed by real-time PCR at different sampling times of hypoxia. This study is the first analysis of differentially expressed proteins in the hepatopancreas of shrimp after hypoxia and provides a new insight for further study in hypoxic stress response of shrimp at the protein level.

Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein

Lysozyme is a widely distributed hydrolase possessing lytic activity against bacterial peptidoglycan, which enables it to protect the host against pathogenic infection. In the present study, the cDNA of an invertebrate goose-type lysozyme (designated CFLysG) was cloned from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of CFLysG consisted of 829 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 603 bp encoding a polypeptide of 200 amino acid residues with a predicted molecular weight of 21.92 kDa and theoretical isoelectric point of 7.76. The high similarity of CFLysG with goose-type (g-type) lysozymes in vertebrate indicated that CFLysG should be an invertebrate counterpart of g-type lysozyme family, which suggested that the origin of g-type lysozyme preceded the emergence of urochordates and even preceded the emergence of deuterostomes. Similar to most g-type lysozymes, CFLysG possessed all conserved features critical for the fundamental structure and function of g-type lysozymes, such as three catalytic residues (Glu 82, Asp 97, Asp 108). By Northern blot analysis, mRNA transcript of CFLysG was found to be most abundantly expressed in the tissues of gills, hepatopancreas and gonad, weakly expressed in the tissues of haemocytes and mantle, while undetectable in the adductor muscle. These results suggested that CFLysG could possess combined features of both the immune and digestive adaptive lysozymes. To gain insight into the in vitro lytic activities of CFLysG, the mature peptide coding region was cloned into Pichia pastoris for heterogeneous expression. Recombinant CFLysG showed inhibitive effect on the growth of both Gram-positive and Gram-negative bacteria with more potent activities against Gram-positive bacteria, which indicated the involvement of CFLysG in the innate immunity of C. farreri. (c) 2006 Elsevier Ltd. All rights reserved.