Arginine kinase from Litopenaeus vannamei: Cloning, expression and catalytic properties

Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. in this paper, the full-length cDNA of AI( was cloned from shrimp, Litopenaeus vannamei by using RT-PCR and RACE PCR. It was 1446 bp encoding 356 amino acids, and belongs to the conserved phosphagen kinase family. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of AK with the highest expression in the muscle and the lowest in the skin. The expression of AK after challenge with LIPS was tested in hemocytes and muscle, which indicated that the two peak values were 6.2 times (at 3 h) and 10.14 times (at 24 h) in the hemocytes compared with the control values, respectively (P < 0.05), while the highest expression of AK was 41 times (at 24 h) in the muscle compared with the control (P < 0.05). In addition, AK was expressed in Eschetichia coli by prokaryotic expression plasmid pGEX-4T-2. The recombinant protein was expressed as glutathione s-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. After cleavage from GST by using a site-specific protease, the recombinant protein was identified by ESI-MS and showed AK activity. After treatment with 10 mM ATP, the enzyme activity significantly increased. However, the enzyme activity was inhibited by 10 mM alpha-ketoglutarate, 50 mM glucose and 200 mM ATP. This research suggested that AK might play an important role in the coupling of energy production and utilization and the immune response in shrimps. (C) 2009 Elsevier Ltd. All rights reserved.

Purification and partial characterization of Mn superoxide dismutase from muscle tissue of the shrimp Macrobrachium nipponense

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that protects against oxidative stress from superoxide radicals in living cells. This enzyme had been isolated, purified and partially characterized from muscle tissue of the shrimp Macrobrachium nipponense. The purification was achieved by heat treatment, ammonium sulfate fractionated precipitation and column chromatograph on DEAE-cellulose 32. Some physiological and biochemical characterization of it was tested. The molecular weight of it was about 21.7 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had an absorption peak of 278 nm in ultraviolet region, and the enzyme remained stable at 25-45 degreesC within 90 min. However, it was rapidly inactivated at higher temperature. Treatment of the enzyme with 1 mM ZnCl2, SDS and 1 mM or 10 mM mercaptoethanol showed some increasing activity. However, the enzyme activity was obviously inhibited by 10 mM CaCl2, CuSO4, ZnCl2 and 1 mM CaCl2 and 10 mM K2Cr2O7. SOD activity did not show significantly variation after incubated with 1 mM CaCl2, EDTA and 10 MM SDS. The enzyme was insensitive to cyanide and contained 1.03 +/- 0.14 atoms of manganese per subunit shown in atomic absorption spectroscopy, which revealed that purified SOD was Mn superoxide dismutase. (C) 2004 Elsevier B.V. All rights reserved.

Identification of archaeon-producing hyperthermophilic alpha-amylase and characterization of the alpha-amylase

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca2+ for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The mitochondrial manganese superoxide dismutase gene in Chinese shrimp Fenneropenaeus chinensis: Cloning, distribution and expression

Manganese superoxide dismutase (MnSOD) plays an important role in crustacean immune defense reaction by eliminating oxidative stress. Knowledge on MnSOD at molecular level allows us to understand its regulatory mechanism in crustacean immune system. A novel mitochondrial manganese superoxide dismutase (mMnSOD) was cloned from hepatopancreas of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1185 bp with a 660 bp open reading frame, encoding 220 amino acids. The deduced amino acid sequence contains a putative signal peptide of 20 amino acids. Sequence comparison showed that the mMnSOD of F. chinensis shares 88% and 82% identity with that of giant freshwater prawn Macrobrachium rosenbergii and blue crab Callinectes sapidus, respectively. mMnSOD transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill by Northern blotting. RT-PCR analysis indicated that mMnSOD showed different expression profiles in shrimp hemocytes and hepatopancreas after artificial infection with while spot syndrome virus (WSSV). In addition, a fusion protein containing mMnSOD was produced in vitro. LC-ESI-MS analysis showed that two peptide fragments (-GDVNTVISLAPALK- and -NVRPDYVNAIWK-) of the recombinant protein were identical to the corresponding sequence of M. rosenbergii mMnSOD, and the enzyme activity of the refolded recombinant protein was also measured. (c) 2006 Elsevier Ltd. All rights reserved.

中国明对虾免疫系统中抗氧化相关基因的克隆与表达分析

对虾病害在世界范围内的广泛传播，给水产养殖和沿海农村经济造成了重大损失。深入开展对虾免疫机制研究并在此基础上寻找对虾疾病防治的有效方法已成为当务之急。研究表明，当对虾等甲壳动物受到外界病原刺激时，其体内的吞噬细胞在吞噬活动中会激活磷酸己糖支路的代谢，引起呼吸爆发，产生多种活性氧分子。另外，受到病原侵染的对虾还会产生其他多种免疫反应，这些免疫反应将消耗大量的能量（ATP），产能的呼吸链会加速运转，由此也会引发大量活性氧的产生。这些活性氧分子可以杀灭入侵的病原微生物，但同时由于活性氧分子反应的非特异性，它们也会对宿主的细胞、组织和器官造成严重伤害，进而导致对虾生理机能的损伤和免疫系统的破坏。所以，消除对虾体内因过度免疫反应产生的过量氧自由基将能够增强其抵御病原侵染的能力，提高免疫力。本论文从中国明对虾体内克隆了线粒体型超氧化物歧化酶（mMnSOD）、胞质型超氧化物歧化酶（cMnSOD）、过氧化氢酶（Catalase）和过氧化物还原酶（Peroxiredoxin）等四种与免疫系统相关的抗氧化酶基因，分析了它们的分子结构特征，组织分布及应答不同病原刺激的表达变化模式，并对其中的mMnSOD基因和Peroxiredoxin基因进行了体外重组表达、分离纯化和酶活性分析。 采用RACE技术从中国明对虾血细胞中克隆了两个超氧化物歧化酶（SOD）基因，通过序列比对分析发现，其中一个为mMnSOD基因，另一个为cMnSOD基因。mMnSOD基因的cDNA全长为1185个碱基，其中开放阅读框为660个碱基，编码220个氨基酸，其中推测的信号肽为20个氨基酸。多序列比对结果显示中国明对虾mMnSOD基因的推导氨基酸序列与罗氏沼虾、蓝蟹的推导氨基酸序列同源性分别为88%和82%。Northern blot结果表明，该基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。半定量RT-PCR结果显示，对虾感染病毒3 h时，该基因在血细胞和肝胰脏中的转录水平显著升高。此外，通过构建原核表达载体，本研究对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活分析。cMnSOD基因的cDNA全长为1284个碱基，其中开放阅读框为861个碱基，编码287个氨基酸。多序列比对结果显示中国明对虾cMnSOD基因的推导氨基酸序列与斑节对虾和凡纳滨对虾的同源性高达98%和94%。组织半定量结果显示，cMnSOD基因在对虾被检测的各个组织中均有表达。 另外，半定量RT-PCR结果表明，对虾感染病毒23h时，该基因在肝胰脏中的转录上升到正常水平的3.5倍；而感染后59 h时，该基因在血细胞中的转录上升到正常水平的2.5倍。 利用根据其他生物过氧化氢酶保守氨基酸序列设计的简并引物，结合RACE技术，从中国明对虾肝胰脏中克隆到了过氧化氢酶基因的部分片段，片段长1725个碱基。多序列比对结果发现目前所得中国明对虾Catalase基因部分片段的推导氨基酸序列与罗氏沼虾和皱纹盘鲍Catalase氨基酸序列的同源性分别达到95%和73%。通过实时荧光定量PCR技术对中国明对虾Catalase基因在各个组织中的分布情况及病毒感染后该基因在血细胞和肝胰脏中的转录变化进行了研究。结果发现，该基因在肝胰脏、鳃、肠和血细胞中表达水平较高，在卵巢、淋巴器官和肌肉中的表达水平相对较弱；感染病毒23 h和37 h时，对虾血细胞和肝胰脏中该基因mRNA的表达量分别出现显著性上升。 依据中国明对虾头胸部cDNA文库提供的部分片段信息，结合SMART-RACE技术，从中国明对虾肝胰脏中克隆到了过氧化物还原酶基因（Peroxiredoxin）， 该基因的cDNA全长为942个碱基，其中开放阅读框为594个碱基，编码198个氨基酸。中国明对虾Peroxiredoxin基因的推断氨基酸序列与伊蚊、文昌鱼和果蝇等Peroxiredoxin基因的推断氨基酸序列同源性分别为77%、76%和73%。其蛋白理论分子量为22041.17 Da，pI为5.17。Northern blot结果表明，Peroxiredoxin基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。实时荧光定量PCR结果显示，弧菌感染后，该基因在对虾血细胞和肝胰脏中的转录水平都有明显变化并且表达模式不同。另外，对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活性分析。酶活性分析表明，复性后的重组蛋白能在DTT存在的条件下还原H2O2。

Protein phosphatase inhibition assay for detection of diarrhetic shellfish poison in oyster

A method based on protein phosphatase enzyme activity inhibition for the detection of diarrhetic shellfish poison (DSP) was used to analyze the DSP toxicity in three oyster samples. Based on the standard dose-effect curve developed with a series of okadaic acid (OA) standard solutions, the DSP toxicity of the three oyster samples collected were screened, and the results showed that there were no OA and dinophysis toxins ( DTXs) in the samples without hydrolization. However, the OA toxicity could be detected in two of the hydrolyzed samples, and the OA toxicity of the two samples were 1.81 and 1.21 mu g OA eq./kg oyster, respectively.

Fucoidan protects against dopaminergic neuron death in vivo and in vitro

Parkinson's disease is a neurodegenerative disorder of uncertain pathogenesis characterized by a loss of dopaminergic neurons in substantia nigra pars compacta, and can be modeled by the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Oxidative stress may contribute to MPTP- and Parkinson's disease-related neurodegeneration. Fucoidan is a sulfated polysaccharide extracted from brown seaweeds which possesses a wide variety of biological activities including potent antioxidative effects. Here we investigated the effect of fucoidan treatment on locomoter activities of animals, striatal dopamine and its metabolites and survival of nigral dopaminergic neurons in MPTP-induced animal model of Parkinsonism in C57/BL mice in vivo and on the neuronal damage induced by 1-methyl-4-phenylpyridinium (MPP+) in vitro, and to study the possible mechanisms. When administered prior to MPTP, fucoidan reduced behavioral deficits, increased striatal dopamine and its metabolites levels, reduced cell death, and led to a marked increase in tyrosine hydroxylase expression relative to mice treated with MPTP alone. Furthermore, we found that fucoidan inhibited MPTP-induced lipid peroxidation and reduction of antioxidant enzyme activity. In addition, pre-treatment with fucoidan significantly protected against MPP+-induced damage in MN9D cells. Taken together, these findings suggest that fucoidan has protective effect in MPTP-induced neurotoxicity in this model of Parkinson's disease via its antioxidative activity. (C) 2009 Elsevier B.V. All rights reserved.

Rapid protein identification using monolithic enzymatic microreactor and LC-ESI-MS/MS

A monolithic enzymatic microreactor was prepared in a fused-silica capillary by in situ polymerization of acrylamide, glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, followed by ammonia solution treatment, glutaraldehyde activation and trypsin modification. The choice of acrylamide as co-monomer was found useful to improve the efficiency of trypsin modification, thus, to increase the enzyme activity. The optimized microreactor offered very low back pressure, enabling the fast digestion of proteins flowing through the reactor. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at high flow rate. The digests were then characterized by CE and HPLC-MS/MS with the sequence coverage of 57.7%. The digestion efficiency was found over 230 times as high as that of the conventional method. in addition, for the first time, protein digestion carried out in a mixture of water and ACN was compared with the conventional aqueous reaction using MS/MS detection, and the former solution was found more compatible and more efficient for protein digestion.

Muscle signaling in exercise intolerance: Insights from the McArdle mouse model

We recently generated a knock-in mouse model (PYGM p.R50X/p.R50X) of McArdle disease (myophosphorylase deficiency). One mechanistic approach to unveil the molecular alterations caused by myophosphorylase deficiency, which is arguably the paradigm of 'exercise intolerance', is to compare the skeletal-muscle tissue of McArdle, heterozygous, and healthy (wild type (wt)) mice. We analyzed in quadriceps muscle of p.R50X/p.R50X (n=4), p.R50X/wt (n=6) and wt/wt mice (n=5) (all male, 8 wk-old) molecular markers of energy-sensing pathways, oxidative phosphorylation (OXPHOS) and autophagy/proteasome systems, oxidative damage and sarcoplamic reticulum (SR) Ca handling. We found a significant group effect for total AMPK (tAMPK) and ratio of phosphorylated (pAMPK)/tAMPK (P=0.012 and 0.033), with higher mean values in p.R50X/p.R50X mice vs. the other two groups. The absence of massive accumulation of ubiquitinated proteins, autophagosomes or lysosomes in p.R50X/p.R50X mice suggested no major alterations in autophagy/proteasome systems. Citrate synthase activity was lower in p.R50X/p.R50X mice vs. the other two groups (P=0.036) but no statistical effect existed for respiratory chain complexes. We found higher levels of 4-hydroxy-2-nonenal-modified proteins in p.R50X/p.R50X and p.R50X/wt mice compared with the wt/wt group (P=0.011). Sarco(endo)plasmic reticulum ATPase 1 (SERCA1) levels detected at 110kDa tended to be higher in p.R50X/p.R50X and p.R50X/wt mice compared with wt/wt animals (P=0.076), but their enzyme activity was normal. We also found an accumulation of phosphorylated SERCA1 in p.R50X/p.R50X animals. Myophosphorylase deficiency causes alterations in sensory energetic pathways together with some evidence of oxidative damage and alterations in Ca handling but with no major alterations in OXPHOS capacity or autophagy/ubiquitination pathways, which suggests that the muscle tissue of patients is likely to adapt overall favorably to exercise training interventions.

Rubisco small subunit, chlorophyll a/b-binding protein and sucrose : fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light

Chungui Lu, Olga A. Koroleva, John F. Farrar, Joe Gallagher, Chris J. Pollock, and A. Deri Tomos (2002). Rubisco small subunit, chlorophyll a/b-binding protein and sucrose : fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light. Plant Physiology, 130 (3) pp.1335-1348 Sponsorship: BBSRC RAE2008

Analysis of the chlorophyll catabolism pathway in leaves of an introgression senescence mutant of Lolium temulentum

Maria Roca, Caron James, Adriana Pruzinsk?, Stefan H?rtensteiner, Howard Thomas and Helen Ougham. Analysis of the chlorophyll catabolism pathway in leaves of an introgression senescence mutant of Lolium temulentum. Phytochemistry, 65 (9), 1231-1238. Sponsorship: BBSRC RAE2008

Efeitos da tetraciclina em Gambusia holbrooki

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Characterization of TRAF6 mediated ubiquitination of presenilins and γ-secretase substrates

Post-translational modification of the γ-secretase protease complexes and their substrates has an important role in controlling receptor-initiated signalling events, which are critically important in the pathogenesis of cancer, inflammatory and Alzheimer’s disease. Our lab has previously characterised an interaction between TRAF6 and presenilin-1, which lead to the identification of interleukin-1 (IL-1) receptor type 1 (IL-1R1) and Toll-like receptor-4 (TLR4) as novel γ-secretase substrates. Subsequently our group showed that TRAF6 promoted ubiquitination and γ-secretase cleavage of IL-1R1. The aim of this project is to study the association between TRAF6 and the presenilins, the critical γ-secretase complex components, and to determine the functional importance of TRAF6-mediated ubiquitination of γ-secretase substrates. Firstly, we show that the full-length presenilins are novel substrates of TRAF6-mediated Lysine-63-linked polyubiquitination. Secondly, we show that co-expression of TRAF6 and the presenilins increases the stability and alters the turnover of the presenilins. Thirdly, we reveal that TRAF6-mediated ubiquitination of presenilin does not affect γ-secretase enzyme activity, but may regulate the full-length presenilin functions such as ER Ca2+ signalling. Previously, we have reported IL-1R1 as a novel substrate of TRAF6-mediated ubiquitination. In this study, we identified five lysine residues in the IL-1R1 intracellular domain targeted by TRAF6-mediated polyubiquitination. Furthermore, mutagenesis of these five lysine residues led to decreased IL-1R1 cell surface expression, precluded the ectodomain shedding and attenuated the responsiveness to IL-1β stimulation, demonstrating the critical role of TRAF6 in IL-1R1 trafficking.

A novel mutation of the ACADM gene (c.145C>G) associated with the common c.985A>G mutation on the other ACADM allele causes mild MCAD deficiency: a case report.

A female patient, with normal familial history, developed at the age of 30 months an episode of diarrhoea, vomiting and lethargy which resolved spontaneously. At the age of 3 years, the patient re-iterated vomiting, was sub-febrile and hypoglycemic, fell into coma, developed seizures and sequels involving right hemi-body. Urinary excretion of hexanoylglycine and suberylglycine was low during this metabolic decompensation. A study of pre- and post-prandial blood glucose and ketones over a period of 24 hours showed a normal glycaemic cycle but a failure to form ketones after 12 hours fasting, suggesting a mitochondrial β-oxidation defect. Total blood carnitine was lowered with unesterified carnitine being half of the lowest control value. A diagnosis of mild MCAD deficiency (MCADD) was based on rates of 1-14C-octanoate and 9, 10-3H-myristate oxidation and of octanoyl-CoA dehydrogenase being reduced to 25% of control values. Other mitochondrial fatty acid oxidation proteins were functionally normal. De novo acylcarnitine synthesis in whole blood samples incubated with deuterated palmitate was also typical of MCADD. Genetic studies showed that the patient was compound heterozygous with a sequence variation in both of the two ACADM alleles; one had the common c.985A>G mutation and the other had a novel c.145C>G mutation. This is the first report for the ACADM gene c.145C>G mutation: it is located in exon 3 and causes a replacement of glutamine to glutamate at position 24 of the mature protein (Q24E). Associated with heterozygosity for c.985A>G mutation, this mutation is responsible for a mild MCADD phenotype along with a clinical story corroborating the emerging literature view that patients with genotypes representing mild MCADD (high residual enzyme activity and low urinary levels of glycine conjugates), similar to some of the mild MCADDs detected by MS/MS newborn screening, may be at risk for disease presentation.

Harvesting life's energy: increase in the aerotolerence of the electrogenic anaerobe geobacter sulfurreducens due to over-expression of superoxide dismutase and catalase

Gemstone Team iGEM