Changes in biosynthesis of exopolysaccharide in Lactococcus lactis subspecies cremoris treated by moderate pulsed electric field treatment

Metabolome analysis and physicochemical analyses were executed with cell extracts of a Lactococcus lactis subspecies cremoris strain treated by moderate pulsed electric field (PEF) to elucidate the mechanism of enhanced production of exopolysaccharide (EPS) by the treatment. Metabolome analysis by capillary electrophoresis time of flight mass spectrometry annotated 224 metabolites from the cytoplasmic extract of the strain, which, however, showed no significant changes in metabolites related to the EPS production. Electron microscopic observation and chemical analysis of undecaprenoids as carrier of EPS biosynthetic intermediates suggested that PEF treatment dissociated immature EPSs from the intermediates due to the focal electro-condensation of hydrogen ions at the cell surface. Thus, liberated undecaprenyl phosphates were recycled efficiently, which resulted in mass increase of EPS with smaller molecular weight. The study suggested the feasibility of moderate PEF treatment as a food processing technique and revealed the mechanism of enhanced production of EPS by the treatment.     

Advances in the development of human protein microarrays

Introduction: High-content protein microarrays in principle enable the functional interrogation of the human proteome in a broad range of applications, including biomarker discovery, profiling of immune responses, identification of enzyme substrates, and quantifying protein-small molecule, protein-protein and protein-DNA/RNA interactions. As with other microarrays, the underlying proteomic platforms are under active technological development and a range of different protein microarrays are now commercially available. However, deciphering the differences between these platforms to identify the most suitable protein microarray for the specific research question is not always straightforward.

Areas covered: This review provides an overview of the technological basis, applications and limitations of some of the most commonly used full-length, recombinant protein and protein fragment microarray platforms, including ProtoArray Human Protein Microarrays, HuProt Human Proteome Microarrays, Human Protein Atlas Protein Fragment Arrays, Nucleic Acid Programmable Arrays and Immunome Protein Arrays.

Expert commentary: The choice of appropriate protein microarray platform depends on the specific biological application in hand, with both more focused, lower density and higher density arrays having distinct advantages. Full-length protein arrays offer advantages in biomarker discovery profiling applications, although care is required in ensuring that the protein production and array fabrication methodology is compatible with the required downstream functionality.     

Gene expression changes associated with cytotoxicity identified using cDNA arrays

In order to investigate gene expression changes associated with cytotoxicity, we used cDNA arrays to monitor the expression of over 5,000 genes in response to toxic stress in the HepG2 liver cell line. Cells were treated with cytotoxic doses of acetaminophen, caffeine or thioacetamide for nine time points ranging from 1 to 24 h. Samples of mRNA from each time point were used to prepare radiolabeled cDNA, which was hybridized to nylon-membrane-based cDNA arrays. High-stringency washes were applied to reduce cross-hybridization. Analysis of spot intensities revealed that each compound led to approximately 150-250 gene expression changes that were sustained over at least three adjacent time points. The affected genes could be classified into clusters based on their temporal patterns of differential expression. A common set of 44 genes showed similar expression changes in response to all three compounds. Of these changes, 90% could be confirmed by quantitative RT-PCR analysis. The results indicate that detailed array-based time-course studies, coupled with a sensitive and highly specific confirmation assay, provide a powerful means of identifying cytotoxicity-associated gene expression changes. Electronic Publication     

Genomic characterization of metastatic patterns from prospective clinical sequencing of 25,000 patients

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Target identification of covalently binding drugs by activity-based protein profiling (ABPP)

The characterization of the target proteins of drug molecules has become an important goal in understanding its mode of action and origin of side effects due to off-target binding. This is especially important for covalently binding drugs usually containing electrophilic moieties, which potentially can react with nucleophilic residues found in many proteins. This review gives a comprehensive overview of the use of activity-based protein profiling (ABPP) as an efficient tool for the target identification of covalently binding drugs.     

Development of new Malt1 inhibitors and probes

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) is a promising therapeutic target for the treatment of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL). Several research groups have reported on the development of Malt1 inhibitors and activity-based probes for in vitro and in situ monitoring and modulating Malt1 activity. In this paper, we report on two activity-based Malt1 probes (6 and 7) and a focused library of 19 new Malt1 inhibitors. Our peptide-based probe 6 labels Malt1 in an activity-based manner. In contrast, probe 7, derived from the known covalent inhibitor MI-2, labels both wild type and catalytically inactive Cys to Ala mutant Malt1, suggesting that MI-2 inhibits Malt1 by reacting with a nucleophilic residue other than the active site cysteine. Furthermore, two of our inhibitors (9, apparent IC₅₀ 3.0 μM, and 13, apparent IC₅₀ 2.1 μM) show good inhibitory activity against Malt1 and outperform MI-2 (apparent IC₅₀ 7.8 μM) in our competitive activity-based protein profiling assay.     

转人端粒酶基因骨髓间充质干细胞的蛋白表达谱分析

通过外源表达人端粒酶基因，构建了具有长期传代能力的骨髓间充质干细胞系（hTERT-hMSC），并在传代过程中尚未发现有丧失接触性生长抑制和转型现象.本文主要目的是采用双向凝胶电泳技术和MALDI-TOF-MS蛋白质谱技术分析hTERT-hMSC 细胞的蛋白差异表达图谱，研究表达外源端粒酶基因对骨髓间充质干细胞生物学特性影响的可能机制.通过分析原代第12代hMSC、第95代和275代hTERT-hMSC的蛋白凝胶图，获得原代第12代hMSC总共1543±145个蛋白点，第95代hTERT-hMSC 1 611±186个蛋白点、275代hTERT-hMSC 1451±126 个蛋白点.质谱分析鉴定100种蛋白质，其中有20种有显著差异表达.结果表明，膜联蛋白（ANX）和GSTP1表达的下调以及内质网钙结合蛋白1（RCN1）、伴侣素CCT、TUBA1B和ACTG1表达的上调可能提升了人骨髓间充质干细胞扩增的能力，而prohibitin 蛋白和p53 蛋白维持正常表达可能对hTERT-hMSC 维持细胞接触性生长抑制起着重要作用.     

Recent advances in quantitative and chemical proteomics for autophagy studies

Macroautophagy/autophagy is an evolutionarily well-conserved cellular degradative process with important biological functions that is closely implicated in health and disease. In recent years, quantitative mass spectrometry-based proteomics and chemical proteomics have emerged as important tools for the study of autophagy, through large-scale unbiased analysis of the proteome or through highly specific and accurate analysis of individual proteins of interest. At present, a variety of approaches have been successfully applied, including (i) expression and interaction proteomics for the study of protein post-translational modifications, (ii) investigating spatio-temporal dynamics of protein synthesis and degradation, and (iii) direct determination of protein activity and profiling molecular targets in the autophagic process. In this review, we attempted to provide an overview of principles and techniques relevant to the application of quantitative and chemical proteomics methods to autophagy, and outline the current landscape as well as future outlook of these methods in autophagy research.