Monitoring and classification of PAH toxicity using an immobilized bioluminescent bacteria

An immobilized recombinant bioluminescent Escherichia coli strain, harboring a lac::luxCDABE fused plasmid, which shows lower bioluminescence levels when cellular metabolism is inhibited, was used to monitor the cellular toxicity of polycyclic aromatic hydrocarbons (PAHs). PAHs, classified as pericondensed (PCPAHs) or catacondensed (CCPAHs) according to their molecular structures, were differentiable according to the response of this biosensor. Only CCPAHs were found to cause cellular toxicity, resulting in a dose-dependent decrease in the bioluminescent output. The induction of cellular toxicity by CCPAHs and PCPAHs was compared with acute toxicity predictions obtained using the quantitative structure-activity relationship (QSAR) model. A good relationship was obtained between the toxicities determined with the bioluminescent response of the immobilized bacterium GC2 and the QSAR model. It was also found that the present study offers a new method of predicting the cellular toxicities of CCPAHs or PCPAHs using this biosensor.     

乳酸杆菌发酵滤液中核酸抗肿瘤效应及对荷瘤鼠免疫功能的影响

目的探讨乳酸杆菌DM9811培养基滤液中核酸在体外对肿瘤细胞生长及对荷瘤鼠免疫功能的影响。方法抽提乳酸杆菌DM9811对数生长期培养基滤液中核酸,进行琼脂糖电泳分析;MTT法体外观察其对于结肠癌HT-29细胞系生长的影响;动物实验观察不同组别小鼠生存期、瘤重体重比、NK细胞活性、T细胞亚群指标。结果乳酸杆菌DM9811滤液中核酸组分为RNA;在细胞浓度为1×106时,每孔加入RNA 100μg能够明显抑制结肠癌HT-29细胞系的生长;预防组荷瘤鼠除CD8＋T细胞比例之外各项指标与阴性对照组相比差异都有统计学意义（P〈0.05）,其中NK细胞杀伤活性（58.97±3.62）、CD4＋T细胞比例（27.77±5.40）和生存期（15.1±4.48）均高于阴性对照组（43.87±3.92）、（19.68±3.00）、（10.2±3.08）,瘤重体重比（0.029±0.017）低于阴性对照组（0.066±0.024）;治疗组生存期（11.8±3.12）与阴性对照组（10.2±3.08）相比差异有统计学意义（P〈0.05）,治疗组生存期长于阴性对照组。结论乳酸杆菌DM9811培养基滤液中存在核酸组分,为100200 bp的RNA片段。100μg/mL乳酸杆菌DM9811培养基滤液中RNA组分对结肠癌HT-29细胞系生长有明显抑制作用。RNA组分可以上调荷瘤鼠的细胞免疫水平,延长荷瘤鼠的生存期。     

Quantitative profiling of the detergent-resistant membrane proteome of iota-b toxin induced vero cells

Enzyme-mediated 18O/16O differential labeling of proteome samples often suffers from incomplete exchange of the carboxy-terminus oxygen atoms, resulting in ambiguity in the measurable abundance differences. In this study, an 18O/16O labeling strategy was optimized for and applied to the solution-based comparative analysis of the detergent-resistant membrane proteome (DRMP) of untreated and Iota-b (Ib)-induced Vero cells. Solubilization and tryptic digestion of the DRMP was conducted in a buffer containing 60% methanol. Unfortunately, the activity of trypsin is attenuated at this methanol concentration hampering the ability to obtain complete oxygen atom turnover. Therefore, the incorporation of the 18O atoms was decoupled from the protein digestion step by carrying out the trypsin-mediated heavy atom incorporation in a buffer containing 20% methanol; a concentration at which trypsin activity is enhanced compared to purely aqueous conditions. After isotopic labeling, the samples were combined, fractionated by strong cation exchange and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. In total, over 1400 unique peptides, corresponding to almost 600 proteins, were identified and quantitated, including all known caveolar and lipid raft marker proteins. The quantitative profiling of Ib-induced DRMP from Vero cells revealed several proteins with altered expression levels suggesting their possible role in Ib binding/uptake.     

Integration and expression of theEscherichia coli xylose isomerase gene inSchizosaccharomyces pombe

Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively.     

Interictal Neurocognitive Processing of Visual Stimuli in Migraine: Evidence from Event-Related Potentials

Research has established decreased sensory habituation as a defining feature in migraine, while decreased cognitive habituation has only been found with regard to cognitive assessment of the relative probability of the occurrence of a stimulus event. Our study extended the investigation of interictal habituation in migraine to include cognitive processing when viewing of a series of visually-complex images, similar to those we encounter on the internet everyday. We examined interictal neurocognitive function in migraine from a habituation perspective, using a novel paradigm designed to assess how the response to a series of images changes over time. Two groups of participants--migraineurs (N = 25) and non-migraine controls (N = 25)--were asked to view a set of 232 unfamiliar logos in the context of a target identification task as their brain electrical responses were recorded via event-related potentials (ERPs). The set of logos was viewed serially in each of 10 separate trial blocks, with data analysis focusing on how the ERP responses to the logos in frontal electrodes from 200-600 ms changed across time within each group. For the controls, we found that the amplitude of the late positive potential (LPP) ERP component elicited by the logos had no significant change across trial blocks. In contrast, in migraineurs we found that the LPP significantly increased in amplitude across trial blocks, an effect consistent with a lack of habituation to visual stimuli seen in previous research. Our findings provide empirical support abnormal cognitive processing of complex visual images across time in migraineurs that goes beyond the sensory-level habituation found in previous research.     

Interaction of the lantibiotic nisin with mixed lipid bilayers: a 31P and 2H NMR study

Nisin is a positively charged antibacterial peptide which binds to the negatively charged membranes of Gram-positive bacteria. The initial interaction of the peptide with model membranes of neutral (phosphatidylcholine) and negatively charged (phosphatidylcholine/phosphatidylglycerol) model lipid membranes was studied using nonperturbing solid state magic angle spinning (MAS) (31)P NMR and (2)H wide-line NMR. In the presence of nisin, the coexistence of two bilayer lipid environments was observed both in charged and in neutral membranes. One lipid environment was found to be associated with lipid directly interacting with nisin and one with noninteracting lipid. Solid state (31)P MAS NMR results show that the acidic membrane lipid component partitions preferentially into the nisin-associated environment. Deuterium NMR ((2)H NMR) of the selectively headgroup-labeled acidic lipid provides further evidence of a strong interaction between the charged lipid component and the peptide. The segregation of acidic lipid into the nisin-bound environment was quantified from (2)H NMR measurements of selectively headgroup-deuterated neutral lipid. It is suggested that the observed lipid partitioning in the presence of nisin is driven, at least initially, by electrostatic interactions. (2)H NMR measurements from chain-perdeuterated neutral lipids indicate that nisin perturbs the hydrophobic region of both charged and neutral bilayers.     

CD164 monoclonal antibodies that block hemopoietic progenitor cell adhesion and proliferation interact with the first mucin domain of the CD164 receptor

The novel sialomucin, CD164, functions as both an adhesion receptor on human CD34+ cell subsets in bone marrow and as a potent negative regulator of CD34+ hemopoietic progenitor cell proliferation. These diverse effects are mediated by at least two functional epitopes defined by the mAbs, 103B2/9E10 and 105A5. We report here the precise epitope mapping of these mAbs together with that of two other CD164 mAbs, N6B6 and 67D2. Using newly defined CD164 splice variants and a set of soluble recombinant chimeric proteins encoded by exons 1-6 of the CD164 gene, we demonstrate that the 105A5 and 103B2/9E10 functional epitopes map to distinct glycosylated regions within the first mucin domain of CD164. The N6B6 and 67D2 mAbs, in contrast, recognize closely associated and complex epitopes that rely on the conformational integrity of the CD164 molecule and encompass the cysteine-rich regions encoded by exons 2 and 3. On the basis of their sensitivities to reducing agents and to sialidase, O-sialoglycoprotease, and N-glycanase treatments, we have characterized CD164 epitopes and grouped them into three classes by analogy with CD34 epitope classification. The class I 105A5 epitope is sialidase, O-glycosidase, and O-sialoglycoprotease sensitive; the class II 103B2/9E10 epitope is N-glycanase, O-glycosidase, and O-sialoglycoprotease sensitive; and the class III N6B6 and 67D2 epitopes are not removed by such enzyme treatments. Collectively, this study indicates that the previously observed differential expression of CD164 epitopes in adult tissues is linked with cell type specific post-translational modifications and suggests a role for epitope-associated carbohydrate structures in CD164 function.