Quantitative profiling of drug-associated proteomic alterations by combined 2-nitrobenzenesulfenyl chloride (NBS) isotope labeling and 2DE/MS identification

The identification of drug-responsive biomarkers in complex protein mixtures is an important goal of quantitative proteomics. Here, we describe a novel approach for identifying such drug-induced protein alterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and control samples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and protein alterations are identified by MS through the differential intensity of paired NBS peptide peaks. Using NBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity. We found the NBS labeling procedure to be both technically and biologically reproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DE differential protein quantitation, with discrepancies largely due to the comigration of distinct proteins in the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78, CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4 expression can serve as a novel prognostic factor for disease-free survival in two independent breast cancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes in breast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator (SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellular protein quantitation than conventional 2DE approaches.     

拟南芥根毛衰老死亡过程的PCD检测

根毛是植物体吸收养分的重要器官, 自然条件下根毛的寿命很短, 仅能存活2–3周, 随即脱落死亡。以模式植物拟南芥(Arabidopsis thaliana)根毛为材料, 对根毛死亡的细胞学特征进行了报道。结果发现, 根毛衰老死亡后细胞内的原生质体发生了收缩, 并在胞质中观察到凝集物的出现; 通过原位末端标记(TUNEL)检测, 发现幼根上的根毛细胞核DNA发生了片段化。上述结果表明, 拟南芥根毛的衰老死亡很可能是植物体自主调控的程序性细胞死亡(PCD)。另外, 当根毛衰老死亡后,细胞核大多会迁移到靠近根毛基部的位置, 且正常的长管状根毛发生旋转扭曲。     

Comparative evaluation of different cell disruption methods for the release of recombinant hepatitis B core antigen from <Emphasis Type="Italic">Escherichia coli</Emphasis>

A comparative evaluation of five different cell-disruption methods for the release of recombinant hepatitis B core antigen (HBcAg) from Escherichia coli was investigated. The cell disruption techniques evaluated in this study were high-pressure homogenization, batch-mode bead milling, continuous-recycling bead milling, ultrasonication, and enzymatic lysis. Continuous-recycling bead milling was found to be the most effective method in terms of operating cost and time. However, the highest degree of cell disruption and amounts of HBcAg were obtained from the high-pressure homogenization process. The direct purification of HBcAg from the unclarified cell disruptate derived from high-pressure homogenization and bead milling techniques, using batch anion-exchange adsorption methods, showed that the conditions of cell disruption have a substantial effect on subsequent protein recovery steps.     

农药残留检测用植物酯酶的筛选

基于酶法分析中的酶抑制原理,通过对8种植物种子及2种植物加工材料总酯酶活力及其比活力进行比较,筛选农药残留检测用适宜的植物酯酶。结果显示,花豇豆总酯酶活力和比活力分别达7.325 U和0.617 U/mg,显著高于其它9个实验材料。用花豇豆酯酶对6种有机磷及氨基甲酸酯类农药——敌敌畏、辛硫磷、敌百虫、速灭威、克百威、灭多威进行敏感性分析,最低检测限均比国家规定的最大残留量小,符合检测要求。结果表明,花豇豆的酯酶总活力、比活力及其敏感性最好,是一种农药残留检测的理想酶源植物。     

A cDNA clone encoding Brassica calmodulin

A 834 bp cDNA encoding calmodulin (CaM) has been isolated from Brassica juncea. On Northern analysis this cDNA hybridises this cDNA to mRNAs of about 0.9 kb in leaf, silique and peduncle. Genomic Southern analysis indicates the presence of a CaM multigene family in Brassica juncea. Comparison of the predicted amino acid sequence of Brassica CaM with that of Arabidopsis CaM ACaM-2 and ACaM-3 showed 100% homology, which is not unusual, since both plants belong to the family Cruciferae. In situ hybridisation studies on Brassica seedlings using a digoxigenin-labelled RNA probe showed that high levels of CaM mRNA were detected in the leaf primordia and the shoot apical meristem, and to a lesser degree, in the zone of root elongation of the root tip. The occurrence of a higher rate of cell division and growth in these regions than its surrounding tissue may possibly be related to higher levels of CaM mRNA.     

A histological study of the hypophysial-gonadal system during sexual maturation and spawning in the milkfish, Chanos chanos (Forskal)

The pituitary gland of the milkfish, Chanos chanos , was studied at different stages of sexual maturation and spawning. Consecutive median sagittal sections were treated with a range of stains to demonstrate the different cell types and regions. The milkfish pituitary consists of a neural component, the neurohypophysis, and an epithelial component, the adenohypophysis, which in turn consists of three regions: the rostral pars distalis (RPD), proximal pars distalis (PPD), and pars intermedia (PI). However, unlike most teleosts, the pituitary gland of the milkfish is encased in a bony chamber, has dorsal and ventral lobes and extends anteriorly from its point of origin at the base of the brain. PAS (+) basophils are found in all regions of the adenohypophysis, but mostly in the proximal pars distalis. These cells undergo hypertrophy and hyperplasia during sexual maturation, shrinkage and degranulation during spawning.     

Cardioprotective effect of isorhamnetin against myocardial ischemia reperfusion (I/R) injury in isolated rat heart through attenuation of apoptosis

In this study, we investigated the effects of isorhamnetin on myocardial ischaemia reperfusion (I/R) injury in Langendorff-perfused rat hearts. Isorhamnetin treatment (5, 10 and 20 μg/mL) significantly alleviated cardiac morphological injury, reduced myocardial infarct size, decreased the levels of marker enzymes (LDH and CK) and improved the haemodynamic parameters, reflected by the elevated levels of the left ventricular developed pressure (LVDP), coronary flow (CF) and the maximum up/down velocity of left ventricular pressure (+dp/dt_max). Moreover, isorhamnetin reperfusion inhibited apoptosis of cardiomyocytes in the rats subjected to cardiac I/R in a dose-dependent manner concomitant with decreased protein expression of Bax and cleaved-caspase-3, as well as increased protein expression of Bcl-2. In addition, I/R-induced oxidative stress was manifestly mitigated by isorhamnetin treatment, as showed by the decreased malondialdehyde (MDA) level and increased antioxidant enzymes activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). These results indicated that isorhamnetin exerts a protective effect against I/R-induced myocardial injury through the attenuation of apoptosis and oxidative stress.     

氧合血红蛋白Fe-O2键合态 ab initio 研究

本文报道用自旋非限制Hartree-Fock方法(spin unrestricted Hartree-Fock method简写UHF)对氧合血红蛋白的Fe-O₂键合态进行ab initio研究。结果表明Fe^(Ⅱ)、Oc和Or的Mulliken布居值分别为24.18、8.19和7.64。这说明氧合血红蛋白的Fe-O₂键合态不发生电子转移。研究模型所得到的频率与实验频率基本一致。研究结果不支持Weiss提出的Fe³⁺O₂^-模型,为解释血红蛋白传输氧的作用机理提供了新的理论依据。     

Cloning, sequencing and function ofsanA, a gene involved in nikkomycin biosynthesis ofStreptomyces ansochromogenes

Several genetically stable mutants blocked in nikkomycin biosynthesis were obtained after the slightly germinated spores of Streptomyces ansochromogenes, a nikkomycin producer, were treated with ultra violet radiation. One of the mutants is the same in morpholotical differentiation as the wild type strain and is designated as NBB19. A DMA library was constructed using plasmid plJ702 as cloning vector, NBB19 as cloning recipient. A 6 kb DNA fragment which can genetically complement NBB19 was cloned when screening the library for antifungal activity. Sequence analysis showed that the 3 kb Bgl II-Sal I fragment contains one complete ORF (ORF1) and one partial ORF (ORF2). ORF1 is designated as sanA. sanA is 1 365 bp, encoding a protein consisting of 454 amino acid residues. Database searching indicated that sanA is homologous to the hypothetical methyltransferase in Pyrococcus horikoshli with 25% identities and 41% positives. Disruptant of sanA lost the ability to synthesize nikkomycin. It indicated that sa     

Gene therapy with iNOS provides long-term protection against myocardial infarction without adverse functional consequences

Previous studies have shown that gene therapy with inducible nitric oxide synthase (iNOS) protects against myocardial infarction at 3 days after gene transfer. However, the long-term effects of iNOS gene therapy on myocardial ischemic injury and cardiac function are unknown. To address this issue, we used a recombinant adenovirus 5 (Ad5) vector (Av3) with deletions of the E1, E2a, and E3 regions, which enables long-lasting recombinant gene expression for at least 2 mo due to lack of inflammation. Mice received intramyocardial injections in the left ventricular (LV) anterior wall of Av3/LacZ (LacZ group) or Av3/iNOS (iNOS group); 1 or 2 mo later, they were subjected to myocardial infarction (30-min coronary occlusion followed by 4 h of reperfusion). Cardiac iNOS gene expression was confirmed by immunoblotting and activity assays at 1 and 2 mo after gene transfer. In the iNOS group, infarct size (percentage of risk region) was significantly reduced (P < 0.05) both at 1 mo (24.2 +/- 3.4%, n = 6, vs. 48.0 +/- 3.6%, n = 8, in the LacZ group) and at 2 mo (23.4 +/- 3.1%, n = 8, vs. 36.6 +/- 2.4%, n = 7). The infarct-sparing effects of iNOS gene therapy were as powerful as those observed 24 h after ischemic preconditioning (23.1 +/- 3.4%, n = 10). iNOS gene transfer had no effect on LV function or dimensions up to 8 wk later (echocardiography). These data demonstrate that iNOS gene therapy mediated by the Av3 vector affords long-term (2 mo) cardioprotection without inflammation or adverse functional consequences, a finding that provides a rationale for further preclinical testing of this therapy.