Purification and partial characterization of an iron regulated outer membrane protein of B. fragilis under non-denaturing conditions

The CHAPS-PAGE gelsystem we applied gave a good separation of the proteins of Bacteroides fragilis under non-denaturing conditions. We succeeded with preparative CHAPS-PAGE in purifying an iron regulated outer membrane protein (a 44 kDa polypeptide on SDS-PAGE) of B. fragilis. This integral membrane protein proved to be a lipopolysaccharide binding protein with an isoelectric point of approximately pH 5.5. This method of purifying membrane proteins could be an important step in research into the function of membrane proteins.     

一种新型免疫层析技术在临床乳腺癌Her-2蛋白定量检测中的应用

建立一种靶点蛋白质快速定量检测方法。在原有侧向流动免疫层析技术的基础上,通过优化层析材料和纳米微球的均一性、改进检测区的检测方法,经逐点扫描技术,建立标准浓度曲线,以达到对临床靶点蛋白质的定量检测。以乳腺癌组织中的Her2表达为例,通过对已知浓度样品的检测,验证本技术方法的准确度大于96%。另外,以蛋白质免疫印迹作为组织中特定蛋白质检测金标准,分析临床肿瘤组织中Her2蛋白的含量,其准确率也达到95.5%,而免疫组织化学方法检测准确率仅为69.58%。新型免疫层析法检测结果与靶向治疗患者的愈后密切相关(P<0.01)。改进后的新型免疫层析方法能够准确地对临床靶点蛋白质进行定量检测,而且结合侧向流动技术的简单、快速和易用性,这种新型检测方法可以广泛应用于临床组织标本、血液标本和体液标本中靶点蛋白质的临场定量检测,在一定程度上可以替代免疫组化技术。     

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.     

Glyceraldehyde-3-phosphate dehydrogenase is required for efficient repair of cytotoxic DNA lesions in Escherichia coli

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with diverse biological functions in human cells. In bacteria, moonlighting GAPDH functions have only been described for the secreted protein in pathogens or probiotics. At the intracellular level, we previously reported the interaction of Escherichia coli GAPDH with phosphoglycolate phosphatase, a protein involved in the metabolism of the DNA repair product 2-phosphoglycolate, thus suggesting a putative role of GAPDH in DNA repair processes. Here, we provide evidence that GAPDH is required for the efficient repair of DNA lesions in E. coli. We show that GAPDH-deficient cells are more sensitive to bleomycin or methyl methanesulfonate. In cells challenged with these genotoxic agents, GAPDH deficiency results in reduced cell viability and filamentous growth. In addition, the gapA knockout mutant accumulates a higher number of spontaneous abasic sites and displays higher spontaneous mutation frequencies than the parental strain. Pull-down experiments in different genetic backgrounds show interaction between GAPDH and enzymes of the base excision repair pathway, namely the AP-endonuclease Endo IV and uracil DNA glycosylase. This finding suggests that GAPDH is a component of a protein complex dedicated to the maintenance of genomic DNA integrity. Our results also show interaction of GAPDH with the single-stranded DNA binding protein. This interaction may recruit GAPDH to the repair sites and implicates GAPDH in DNA repair pathways activated by profuse DNA damage, such as homologous recombination or the SOS response.     

Identification of a 13-kDa protein associated with the polyhydroxyalkanoic acid granules from Acinetobacter spp.

Abstract Transferrin-binding proteins from Neisseria meningitidis vary among different isolates. We have identified and studied a hypervariable region adjacent to the carboxyl-end of the transferrin-binding domain of the Tbp2 molecule. The tbp2 genes from six strains of N. meningitidis were cloned and sequenced in this particular region. Sequence analysis of these regions along with five other sequences available from pathogenic Neisseria showed a common organisation of seven highly variable nucleotide stretches interspersed with six conserved nucleotide stretches. The variable regions correlated with the location of immunoreactive epitopes in polyclonal antisera raised to transferrin-binding proteins identified by peptide pin technology. Sequence analysis suggested a mosaic-like organisation of the tbp2 genes. Taken together, these data suggest that the antigenic variation in this part of the protein may result from a strong host immune pressure.     

Anti-oxidative effects of silkworm storage protein 1 in HeLa cell

Silkworm hemolymph contains unique proteins that exhibit anti-apoptotic activity in mammalian cells. Among them, 30 K protein, which is one of the major anti-apoptotic molecules in silkworm hemolymph, has been well investigated. However, little is known about the biological functions of storage protein 1 (SP1), another main protein in silkworm hemolymph. In this study, the anti-apoptotic and anti-oxidative activities of SP1 were analyzed. A stable cell line expressing SP1 was constructed, which showed strong anti-apoptotic effect induced by staurosporine treatment. In addition, the cell line exhibited resistance to oxidative stress caused by hydrogen peroxide. For practical applications of SP1, recombinant SP1 was produced in Escherichia coli, and the supplementation of recombinant SP1 into culture medium exhibited anti-apoptotic and anti-oxidative activities. In addition, SP1 was found to be a cell-penetrating protein and localized in the cytosol as well as on the plasma membrane. The findings showed that SP1 itself is not an anti-oxidant; rather, it mediates intracellular anti-oxidative activity. In conclusion, the cellular resistance of SP1 to apoptosis and oxidative stress will provide a new strategy that could be utilized in the bio-industry for the production of biologics as well as for the development of anti-aging cosmetics.     

PACAP and VIP increase the expression of myelin-related proteins in rat schwannoma cells: Involvement of PAC1/VPAC2 receptor-mediated activation of PI3K/Akt signaling pathways

PACAP and its cognate peptide VIP participate in various biological functions, including myelin maturation and synthesis. However, defining whether these peptides affect peripheral expression of myelin proteins still remains unanswered. To address this issue, we assessed whether PACAP or VIP contribute to regulate the expression of three myelin proteins (MAG, MBP and MPZ, respectively) using the rat schwannoma cell line (RT4-P6D2T), a well-established model to study myelin gene expression. In addition, we endeavored to partly unravel the underlying molecular mechanisms involved. Expression of myelin-specific proteins was assessed in cells grown either in normal serum (10% FBS) or serum starved and treated with or without 100 nM PACAP or VIP. Furthermore, through pharmacological approach using the PACAP/VIP receptor antagonist (PACAP6-38) or specific pathway (MAPK or PI3K) inhibitors we defined the relative contribution of receptors and/or signaling pathways on the expression of myelin proteins. Our data show that serum starvation (24 h) significantly increased both MAG, MBP and MPZ expression. Concurrently, we observed increased expression of endogenous PACAP and related receptors. Treatment with PACAP or VIP further exacerbated starvation-induced expression of myelin markers, suggesting that serum withdrawal might sensitize cells to peptide activity. Stimulation with either peptides increased phosphorylation of Akt at Ser473 residue but had no effect on phosphorylated Erk-1/2. PACAP6-38 (10 μM) impeded starvation- or peptide-induced expression of myelin markers. Similar effects were obtained after pretreatment with the PI3K inhibitor (wortmannin, 10 μM) but not the MAPKK inhibitor (PD98059, 50 μM). Together, the present finding corroborate the hypothesis that PACAP and VIP might contribute to the myelinating process preferentially via the canonical PI3K/Akt signaling pathway, providing the basis for future studies on the role of these peptides in demyelinating diseases.