Genotypic and phenotypic variation of Lewis antigen expression in geographically diverse Helicobacter pylori isolates

Background: Helicobacter pylori are a persistent colonizer of the human gastric mucosa, which can lead to the development of peptic ulcer disease and gastric adenocarcinomas. However, H. pylori can asymptomatically colonize a host for years. One factor that has been hypothesized to contribute to such persistence is the production of Lewis (Le) antigens in the lipopolysaccharide layer of the bacterial outer membrane as a form of molecular mimicry, because humans also express these antigens on their gastric mucosa. Humans and H. pylori both are polymorphic for Le expression, which is driven in H. pylori by variation at the Le synthesis loci. In this report, we sought to characterize Le genotypic and phenotypic variation in geographically diverse H. pylori isolates. Materials and Methods: From patients undergoing endoscopy in 29 countries, we determined Le phenotypes of 78 H. pylori strains and performed genotyping of the galT and β‐(1,3)galT loci in 113 H. pylori strains. Results: Le antigen phenotyping revealed a significant (p < .0001) association between type 1 (Le^a and Le^b) expression and strains of East Asian origin. Genotyping revealed a significant correlation between strain origin and the size of the promoter region upstream of the Le synthesis gene, galT (p < .0001). Conclusion: These results indicate that the heterogeneity of human Le phenotypes is reflected in their H. pylori colonizing strains and suggest new loci that can be studied to assess the variation of Le expression.     

Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry

Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-¹³C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and ¹³C-labeling dynamics of intracellular metabolites using non-stationary ¹³C-metabolic flux analysis (¹³C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.     

Role of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes in influencing faecal bacteria and biochemical parameters in human subjects

Aims: To evaluate the positive influence of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes into the human gut with special reference to faecal bacterial balance, short‐chain fatty acid concentrations and enzyme activities in a randomized, double‐blind human trial in comparison with probiotic‐free artichokes (control). Methods: Twenty subjects were randomized into two groups, which consumed daily 180 g of the artichoke product (probiotic or control) during two 15‐day study periods (periods 1 and 2) separated by a 15‐day washout in a crossover manner. Faecal samples were subjected to microbiological and biochemical analyses, and a strain‐specific PCR was performed to monitor the probiotic strain. Results: The probiotic strain, transported by the vegetable matrix, transiently colonized the gut of 17/20 subjects (median 6·87 log CFU g^?1 faeces), antagonized Escherichia coli and Clostridium spp. and increased the genetic diversity of lactic population based on REP‐PCR profiles, mainly after period 1. Conclusions: The probiotic L. paracasei LMGP22043 successfully colonized the human gut and positively influenced faecal bacteria and biochemical parameters. Significance and Impact of the Study: The association of the probiotic L. paracasei with a food carrier rich in fibre can represent a new strategy for favouring a daily supply of probiotics and attracting more consumers to vegetable food fortified with probiotic strains.     

Forest gene diversity is correlated with the composition and function of soil microbial communities

The growing field of community and ecosystem genetics indicates that plant genotype and genotypic variation are important for structuring communities and ecosystem processes. Little is known, however, regarding the effects of stand gene diversity on soil communities and processes under field conditions. Utilizing natural genetic variation occurring in Populus spp. hybrid zones, we tested the hypothesis that stand gene diversity structures soil microbial communities and influences soil nutrient pools. We found significant unimodal patterns relating gene diversity to soil microbial community composition, microbial exoenzyme activity of a carbon-acquiring enzyme, and availability of soil nitrogen. Multivariate analyses indicate that this pattern is due to the correlation between gene diversity, plant secondary chemistry, and the composition of the microbial community that impacts the availability of soil nitrogen. Together, these data from a natural system indicate that stand gene diversity may affect soil microbial communities and soil processes in ways similar to species diversity (i.e., unimodal patterns). Our results further demonstrate that the effects of plant genetic diversity on other organisms may be mediated by plant functional trait variation.     

Efficient proteolysis using a regenerable metal-ion chelate immobilized enzyme reactor supported on organic-inorganic hybrid silica monolith

A metal‐ion chelate immobilized enzyme reactor (IMER) supported on organic–inorganic hybrid silica monolith was developed for rapid digestion of proteins. The monolithic support was in situ prepared in a fused silica capillary via the polycondensation between tetraethoxysilane hydrolytic sol and iminodiacetic acid conjugated glycidoxypropyltrimethoxysilane. After activated by Cu²⁺, trypsin was immobilized onto the monolithic support via metal chelation. Proteolytic capability of such an IMER was evaluated by the digestion of myoglobin and BSA, and the digests were further analyzed by microflow reversed‐phase liquid chromatography with ESI‐MS/MS. Similar sequence coverages of myoglobin and BSA were obtained by IMER, in comparison to those obtained by in‐solution digestion (91 versus 92% for 200 ng myoglobin, and 26 versus 26% for 200 ng BSA). However, the digestion time was shortened from 12 h to 50 s. When the enzymatic activity was decreased after seven runs, the IMER could be easily regenerated by removing Cu²⁺ via EDTA followed by trypsin immobilization with fresh Cu²⁺ introduced, yielding the equal sequence coverage (26% for 200 ng BSA). For ～5 μg rat liver extract, even more proteins were identified with the immobilized trypsin digestion within 150 s in comparison to the in‐solution digestion for 24 h (541 versus 483), demonstrating that the IMER could be a promising tool for efficient and high‐throughput proteome profiling.     

NaCl胁迫对甘草生长及抗氧化酶活性的影响

目的：通过分析不同浓度NaCl胁迫下甘草生长和抗氧化酶活性的变化探讨甘草对盐分胁迫的适应性机理;方法：采用不同浓度的NaCl溶液（配成Hoagland液）处理盆栽一年生甘草,分别于35d、70d和105d取样,测定甘草株高、地上部分鲜、干重、根鲜、干重及甘草叶片SOD、POD、CAT活性,分析各生长指标与抗氧化酶活性的相关性;结果：NaCl胁迫70d和105d,0．6％和0．9％处理组的株高、地上部分鲜、干重及根鲜、干重均显著低于CK,SOD、POD及CAT活性均显著高于CK,经相关性分析得知,SOD、POD及CAT活性与各生长指标均负相关,其中POD活性与各生长指标极显著负相关;结论：甘草对NaCl胁迫的响应有胁迫时间和浓度的依赖性,在遭遇胁迫时,通过改变自身的生长和提高抗氧化酶活性,来提高机体的抗盐能力。     

血清与血浆中HBsAg在全自动酶免分析检测结果比较

目的：探讨血浆取代血清检测乙肝标志物的检测结果以及临床意义。方法：选取我科检测乙肝标志物的血样30份,分别放在抗凝剂管和普通干燥试管,采用全自动酶免疫分析仪检测HBsAg,将检测结果进行OD及S/CO值统计处理,并进行比较分析。结果：血清与二种血浆检测结果相关系数均0.99,二种血浆与血清结果相关关系良好（P〉0.0 5）,无显著性差异。结论：血浆代替血清完全可以用于全自动酶标分析仪进行检测,既可以节省预处理时间,又可以减少标本在分离吸移血清过程中出现差错,值得临床推广和应用。     

Inhibitory role of TACE/ADAM17 cytotail in protein ectodomain shedding

AIM:To determine if the cytotail of the principal sheddase tumor necrosis factor-α converting enzyme (TACE;ADAM17) controls protein ectodomain shedding.METHODS:Site-directed mutagenesis was performed to derive TACE variants. The resulting TACE expression plasmids with amino acid substitutions in the extracel-lular,cysteine-rich disintegrin domain (CRD) and/or deleted cytotail,along with an expression vector for the enhanced green fluorescence protein were transfected into shedding-defective M1 mutants stably expressing transmembrane L-selectin or transforming growth factor (TGF)-α. The expression levels of the TACE substrates at the cell surface were determined by flow cytometry. RESULTS:Consistent with published data,a single point mutation (C600Y) in the CRD led to shedding defi-ciency. However,removal of the cytotail from the C600Y TACE variant partially restored ectodomain cleavage of TGF-α and L-selectin. Cytotail-deleted mutants with any other substituting amino acid residues in place of Cys600 displayed similar function compared with tail-less C600Y TACE.CONCLUSION:The cytotail plays an inhibitory role,which becomes evident when it is removed from an enzyme with another mutation that affects the enzyme function.     

Purification, immobilization, and characterization of a specific lipase from Staphylococcus warneri EX17 by enzyme fractionating via adsorption on different hydrophobic supports

Staphylococcus warneri strain EX17 produces three lipases with different molecular weights of 28, 30, and 45 kDa. The 45 kDa fraction (SWL-45) has been purified from crude protein extracts by one chromatographic step based on the selective adsorption of this lipase by interfacial activation on different hydrophobic supports at low ionic strength. The adsorption of SWL-45 on octyl-Sepharose increased the enzyme activity by 60%, but the other lipases were also adsorbed on this support. Using butyl-Toyopearl, which is a lesser hydrophobic support, the purification factor was close to 20, and the only protein band detected on the sodium dodecyl sulfate-polyacrylamide electrophoresis analysis gel was that corresponding to the SWL-45, which could be easily desorbed from the support by incubation with triton X-100, producing a purified enzyme. SWL-45 was immobilized under very mild conditions on cyanogen bromide Sepharose, showing similar activities and stability as for its soluble form but without intermolecular interaction. The effects of different detergents over the activity of the immobilized SWL-45 were analyzed, which was hyperactivated by factors of 1.3 and 2.5 with 0.01% Tween 80 and 0.1% Triton X-100, respectively, while ionic detergents produced detrimental effects on the enzyme activity even at very low concentrations. Optimal reaction conditions and the effect of other additives on the enzyme activity were also investigated.     

Synthesis and performance of 3D-megaporous structures for enzyme immobilization and protein capture

The preparation of megaporous bodies, with potential applications in biotechnology, was attempted by following several strategies. As a first step, naive and robust scaffolds were produced by polymerization of selected monomers in the presence of a highly soluble cross‐linker agent. Ion‐exchange function was incorporated by particle embedding, direct chemical synthesis, or radiation‐induced grafting. The total ionic capacity of such systems was 1.5 mmol H⁺/g, 1.4 mmol H⁺/g, and 17 mmol H⁺/g, respectively. These values were in agreement with the ability to bind model proteins: observed dynamic binding capacity at 50% breakthrough was ?7.2 mg bovine serum albumin/g, ?7.4 hen egg‐white lysozyme (HEWL) mg/g, and ?108 HEWL mg/g. In the later case, total (static) binding capacity reached 220 mg/g. It was observed that the structure and size of the megapores remained unaffected by the grafting procedure which, however, allowed for the highest protein binding capacity. Lysozyme supported on grafted body showed extensive clarification activity against a Micrococcus lysodekticus suspension in the flow‐through mode, i.e., 90% destruction of suspended microbial cells was obtained with a residence time ≈ 18 min. Both protein capture and biocatalysis applications are conceivable with the 3D‐megaporous materials described in this work. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011