果树组织中总RNA提取的新方法

本研究设计了一种新的RNA提取方法 ,解决了RNA提取时容易被降解和污染这一关键问题。通过加入Rnase抑制剂 ,消除了同外源RNase对RNA的降解 ,结合DNA难呈低盐溶液(140mmol·L -1NaCl)的原理 ,去除了DNA对RNA提取液的污染 ;先后使用酚和氯仿 ,有效地去除了蛋白质和酚类物的污染 ,利用抗氧化剂PVP和巯基乙醇 ,消除了内源酚类物质氧化变色对病毒RNA逆转录的影响。采用上述方法可以在4～5h内得到纯度高、含量大、完整性好的果树总RNA ,并获得了逆转录活性较强的病毒RNA ,同时使提取RNA的成本降低。这些方法对苹果、葡萄、桃、樱桃等果树总RNA的提取均适用。

Studies of non-covalent protein complexes by MALDI methods

Several specific non-covalent protein complexes were successfully observed by matrix assisted desorption ionization mass spectrometry(MALDI MS). The methods described in this paper include the matrixes use of sinapinic acid(SA) and 6-aza-2-thiothymine (ATT) in neutral pH solution, as well as the improvement of two-layer sample preparation method to achieve a high sensitivity detection of stable non-covalent complexes, Myoglobin-heme complex was found simultaneously with the sinapinic acid matrix in the various pH solution(pH=2 or pH=5), The RNase S complex showed a striking intensity at the first shot, which was decreased with more laser shots. Most importantly, the observation of specific non-covalent complex in the brome mosaic virus(BMV) coat proteins would open up a new possibility to investigate the assembly and disassembly of viral capsids.

栉孔扇贝大规模死亡病原学研究

用平板画线法从患病栉孔扇贝（Chlamys farreri）体内分离到了一种原核生物（简称QDP）。QDP可以在改进的液体培养基MEM（含2.2%NaCl，5%小牛血清）和脑心浸液（含2.2% NaCl）中生长；菌落在显微镜下（150×）为无色、透明的小点状；革兰氏染色阴性；菌体为圆形或近似圆形。QDP在发育过程中有两种状态，一种为未成熟阶段，直径小于100nm；另一种为成熟阶段，直径变化很大，最小约60nm，最大可达4µm以上。较小的个体有拟核、核糖体和新月状的空泡，未见细胞壁；较大的个体有细胞壁，胞内大部分被空泡充满，未见拟核和核糖体。栉孔扇贝组织超簿切片电镜观查证实QDP的存在。QDP的密度随着生长发育时间的不同而有所变化，繁殖高峰期密度较大。 建立了密度梯度离心结合滤膜过滤分离技术，优化人工培养条件。最适生长温度为23℃，最适生长pH值为7.4，最适生长盐度相当于细胞培养液所需的盐浓度（0.85%NaCl）。 提取的QDP核酸能被RNase A 降解，且没有检测到DNA。以PCR、RT-PCR扩增其16SrRNA基因序列片段，PCR反应没有扩增出扩增子，而RT-PCR则扩增出了16S rRNA基因序列片段，经测定其序列全长度为1430bp，经与GENEBANK中的16S rRNA片段比较分析，与6种不同科的微生物的同源率最高的为95%-95.47%。 采用温度梯度和病原浓度梯度回归感染实验方法，较为系统地研究了QDP的致病性。研究结果表明：QDP对栉孔扇贝有强烈的致病作用，高温（23℃以上）是其致病的必要条件，证实DQP是栉孔扇贝大规模死亡的病原体之一。

Base pairing interaction between 5′- and 3′-UTRs controls icaR mRNA translation in Staphylococcus aureus

UPNa. Instituto de Agrobiotecnología. Laboratorio de Biofilms Microbianos.

Mecanismos virais no Cancro da Próstata

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

Osmolyte-induced folding of an intrinsically disordered protein: folding mechanism in the absence of ligand.

Understanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of the bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in this study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations exhibited multiphasic kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea during the titration to produce a urea-TMAO titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated, and the folding rate constants are relatively slow compared to those of intrinsically folded proteins similar in size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.

Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine pKa Measurements.

The pKa values of ionizable groups in proteins report the free energy of site-specific proton binding and provide a direct means of studying pH-dependent stability. We measured histidine pKa values (H3, H22, and H105) in the unfolded (U), intermediate (I), and sulfate-bound folded (F) states of RNase P protein, using an efficient and accurate nuclear magnetic resonance-monitored titration approach that utilizes internal reference compounds and a parametric fitting method. The three histidines in the sulfate-bound folded protein have pKa values depressed by 0.21 ± 0.01, 0.49 ± 0.01, and 1.00 ± 0.01 units, respectively, relative to that of the model compound N-acetyl-l-histidine methylamide. In the unliganded and unfolded protein, the pKa values are depressed relative to that of the model compound by 0.73 ± 0.02, 0.45 ± 0.02, and 0.68 ± 0.02 units, respectively. Above pH 5.5, H22 displays a separate resonance, which we have assigned to I, whose apparent pKa value is depressed by 1.03 ± 0.25 units, which is ∼0.5 units more than in either U or F. The depressed pKa values we observe are consistent with repulsive interactions between protonated histidine side chains and the net positive charge of the protein. However, the pKa differences between F and U are small for all three histidines, and they have little ionic strength dependence in F. Taken together, these observations suggest that unfavorable electrostatics alone do not account for the fact that RNase P protein is intrinsically unfolded in the absence of ligand. Multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function.

ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture

Replication of the ~30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

Formation of methionine sulfoxide during glycoxidation and lipoxidation of ribonuclease A

Chemical modification of proteins by reactive oxygen species affects protein structure, function and turnover during aging and chronic disease. Some of this damage is direct, for example by oxidation of amino acids in protein by peroxide or other reactive oxygen species, but autoxidation of ambient carbohydrates and lipids amplifies both the oxidative and chemical damage to protein and leads to formation of advanced glycoxidation and lipoxidation end-products (AGE/ALEs). In previous work, we have observed the oxidation of methionine during glycoxidation and lipoxidation reactions, and in the present work we set out to determine if methionine sulfoxide (MetSO) in protein was a more sensitive indicator of glycoxidative and lipoxidative damage than AGE/ALEs. We also investigated the sites of methionine oxidation in a model protein, ribonuclease A (RNase), in order to determine whether analysis of the site specificity of methionine oxidation in proteins could be used to indicate the source of the oxidative damage, i.e. carbohydrate or lipid. We describe here the development of an LC/MS/MS for quantification of methionine oxidation at specific sites in RNase during glycoxidation or lipoxidation by glucose or arachidonate, respectively. Glycoxidized and lipoxidized RNase were analyzed by tryptic digestion, followed by reversed phase HPLC and mass spectrometric analysis to quantify methionine and methionine sulfoxide containing peptides. We observed that: (1) compared to AGE/ALEs, methionine sulfoxide was a more sensitive biomarker of glycoxidative or lipoxidative damage to proteins; (2) regardless of oxidizable substrate, the relative rate of oxidation of methionine residues in RNase was Met(29) > Met(30) > Met(13), with Met(79) being resistant to oxidation; and (3) arachidonate produced a significantly greater yield of MetSO, compared to glucose. The methods developed here should be useful for assessing a protein's overall exposure to oxidative stress from a variety of sources in vivo. (c) 2006 Elsevier Inc. All rights reserved.

Proteomic method for the quantification of methionine sulfoxide

Glycoxidation and lipoxidation reactions contribute to the chemical modification of proteins during the Maillard reaction. Reactive oxygen species, produced during the oxidation of sugars and lipids in these processes, irreversibly oxidize proteins. Methionine is particularly susceptible to oxidation, yielding the oxidation product methionine sulfoxide (MetSO). Here we describe a method for the analysis of MetSO using proteomic techniques. Using these techniques, we measured MetSO formation on the model protein RNase during aerobic incubations with glucose and arachidonate. We also evaluated the susceptibility of MetSO to reduction by NaBH4, a commonly used reductant in the analysis of Maillard reaction products.

Mass spectrometric analysis of glucose-modified ribonuclease

RNase A (1 mM) was incubated with glucose (0.4 M) at 37degreesC for up to 14 days in phosphate buffer (0.2 M, pH 7.4), digested with trypsin and analysed by LC-MS. The major sites of fructoselysine formation were Lys(1), Lys(7), Lys(37) and Lys(41). Three of these sites (Lys(7), Lys(37) and Lys(41)) were also the major sites of N-epsilon-(carboxymethyl)lysine formation.

Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality

Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control On silencing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (sORNA-soaked M incognita eggs Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH2)-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhibition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually leading to embryonic lethality To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cytosolic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abundance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberration of the J2s. which recovered within 24 h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48 h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-en-I transcript which encodes an RNAi-inhibiting siRNA exonuclease We observe a marked up-regulation of MI-en-I transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RN/NI-based reverse genetic screens in plant parasitic nematodes. (C) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Promoter region of the Escherichia coli O7-specific lipopolysaccharide gene cluster:structural and functional characterization of an upstream untranslated mRNA sequence

We report the identification of the promoter region of the Escherichia coli O7-specific lipopolysaccharide (LPS) gene cluster (wbEcO7). Typical -10 and -35 sequences were found to be located in the intervening region between galF and rlmB, the first gene of the wbEcO7 cluster. Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two ops sequences. We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless lac operon. Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element. We have also found that one of the ops sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster. A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the waaQGPSBIJYZK genes. We observed another predicted stem-loop, located immediately downstream from the wbEcO7 transcription initiation site, which appeared to be involved in premature termination of transcription. This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes. wbEcO7-lac transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis. No effects were detected with temperature, osmolarity, Mg2+ concentration, and drugs inducing changes in DNA supercoiling. We therefore conclude that the wbEcO7 promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner.

Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of <1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.

The advanced glycation end product, Nepsilon-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions

Nepsilon-(Carboxymethyl)lysine (CML) is an advanced glycation end product formed on protein by combined nonenzymatic glycation and oxidation (glycoxidation) reactions. We now report that CML is also formed during metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of protein. During copper-catalyzed oxidation in vitro, the CML content of low density lipoprotein increased in concert with conjugated dienes but was independent of the presence of the Amadori compound, fructoselysine, on the protein. CML was also formed in a time-dependent manner in RNase incubated under aerobic conditions in phosphate buffer containing arachidonate or linoleate; only trace amounts of CML were formed from oleate. After 6 days of incubation the yield of CML in RNase from arachidonate was approximately 0.7 mmol/mol lysine compared with only 0.03 mmol/mol lysine for protein incubated under the same conditions with glucose. Glyoxal, a known precursor of CML, was also formed during incubation of RNase with arachidonate. These results suggest that lipid peroxidation, as well as glycoxidation, may be an important source of CML in tissue proteins in vivo and that CML may be a general marker of oxidative stress and long term damage to protein in aging, atherosclerosis, and diabetes.