Characterisation of the interaction between syndecan-2, neurofibromin and CASK: Dependence of interaction on syndecan dimerization

Neurofibromin and calcium/calmodulin-dependent serine protein kinase (CASK) are membrane-associated signalling and scaffolding proteins which are mutated in human genetic neurological disorders. Syndecan-2 is a highly glycosylated transmembrane protein whose intracellular C-terminus has previously been shown to interact with the post-synaptic density 95/discs large/zonula occludens-1 (PDZ) domain of CASK and with two separate regions of neurofibromin. These three proteins collaborate to orchestrate the induction of filopodia and dendritic spines. We have used systematic mutagenesis of the intracellular region of syndecan-2 and a quantitative yeast two-hybrid (Y2H) assay to study the determinants of their interactions. We show that syndecan’s interactions with both CASK and neurofibromin are dependent on syndecan homodimerization and that neurofibromin largely interacts with the membrane-proximal part of the dimeric syndecan intracellular domain, leaving the membrane-distal C-terminus free to interact with CASK. We conducted a phylogenetic study of syndecan sequences, finding correspondence between conserved residues and mutations affecting both dimerization and interactions; we also find that fish have a very different syndecan repertoire from tetrapods. Further Y2H screens reveal that syndecan-2 interacts with a third distinct region of neurofibromin, and that the multiple neurofibromin regions bind competitively, rather than co-operatively, to syndecan. We combine these results to propose a model for the ternary syndecan-neurofibromin-CASK complex.     

aroA and aroD mutations influence biofilm formation in Salmonella Enteritidis

aro mutants of Salmonella enterica are frequently used as live vaccines for the oral vaccination of domestic animals. Interestingly, besides their auxotrophy, they appear to be of reduced resistance to the components of innate immune response due to a defect in outer membrane and/or cell wall integrity. Because different extracellular structures associated with the cell wall or outer membrane are involved in biofilm formation, we were interested in the ability of aroA and aroD mutants of S . Enteritidis to adhere to solid surfaces. We found that aroA and aroD mutants did not adhere to solid surfaces although they bind Congo red and produced d -mannose and d -glucose capsular polysaccharides in the same amounts as the wild-type strain. However, the aro mutants exhibited a decreased production of cellulose, N -acetyl- d -glucosamine or N -acetylneuraminic acid containing capsular polysaccharide and fimbriae which explains their inability to form biofilms. aroA and aroD containing plasmids complemented all the defects of the aro mutants. Beside its attenuation for different hosts, the loss of ability to form biofilm is an additional interesting characteristic of aro mutants.     

Molecular investigation of the distribution, abundance and diversity of the genus Pseudoalteromonas in marine samples

The genus Pseudoalteromonas has attracted interest because it has frequently been found in association with eukaryotic hosts, and because many Pseudoalteromonas species produce biologically active compounds. One distinct group of Pseudoalteromonas species is the antifouling subgroup containing Pseudoalteromonas tunicata and Ps. ulvae, which both produce extracellular compounds that inhibit growth and colonization by different marine organisms. PCR primers targeting the 16S rRNA gene of the genus Pseudoalteromonas and the antifouling subgroup were developed and applied in this study. Real-time quantitative PCR (qPCR) was applied to determine the relative bacterial abundance of the genus and the antifouling subgroup, and denaturing gradient gel electrophoresis (DGGE) was applied to study the diversity of the genus in 11 different types of marine samples from Danish coastal waters. The detection of Ps. tunicata that contain the antifouling subgroup was achieved through specific PCR amplification of the antibacterial protein gene (alpP). The Pseudoalteromonas species accounted for 1.6% of the total bacterial abundance across all samples. The Pseudoalteromonas diversity on the three unfouled marine organisms Ciona intestinalis, Ulva lactuca and Ulvaria fusca was found to be low, and Ps. tunicata was only detected on these three hosts, which all contain accessible cellulose polymers in their cell walls.     

四川2种丛生竹木质素和纤维含量的研究

以四川4个地区2种丛生竹(慈竹和硬头黄)为研究对象,对其木质素和综纤维含量进行研究.结果表明,不同地区竹种间综纤维素含量差异显著;除青神地区外,其它几个地区木质素含量差异不大.纬度对木质素积累的作用因竹种而异,对慈竹木质素含量的作用呈极明显的正效应,对硬头黄的作用则呈不明显的正效应,对综纤维含量的影响不大.竹龄影响木质素和综纤维素含量的积累,对慈竹木质素和综纤维的积累呈显著的正效应,但对硬头黄的作用却不明显.研究认为,青神、长宁和绵阳地区的硬头黄以及长宁地区的慈竹是造纸的较好原料.     

生物反应器规模化培养重组CHO-C28细胞表达HBsAg的研究

应用新型聚酯纤维盘片,采用连续灌注培养方式,分别试验了细胞接种量、pH、DO、罐流速度等因素对CHO-C28细胞生长分泌HBsAg的影响,初步建立了5L生物反应器生产重组乙型肝炎疫苗的生产工艺。经3次试验培养,每次培养60d,较适宜的培养条件确定为:pH6.80-7.10,DO 20%-30%,温度36-37℃,灌流速度138ml/h,接种浓度1.9×106cell/ml。收获液的HBsAg平均滴度是1∶256,最高滴度可达1∶512,纯化后的HBsAg产率为0.912mg/L。最后对反应器培养工艺与现行的转瓶培养工艺进行了比较,生物反应器培养具有可控制培养条件、不易污染和可使HBsAg产率提高等优点。     

Electrostatic analysis of bacterial expansins

Expansins are a family of proteins with plant cell wall remodeling‐activity, which bind cell wall components through hydrophobic and electrostatic interactions. A shallow area on the surface of the protein serves as the polysaccharide binding site (PBS) and it is composed of conserved residues. However, electric charge differences on the opposite face of the PBS produce basic, neutral, or acidic proteins. An analysis of forty‐four bacterial expansins, homologues of BsEXLX1, revealed two main groups defined by: (a) the presence or absence of disulfide bonds; and (b) by the proteins isoelectric point (pI). We determined the location of the residues responsible for the pI on the structure of representative expansins. Our results suggest that the electric charge at the opposite site of the PBS may help in substrate differentiation among expansins from different species; in addition, electrostatic polarization between the front and the back of the molecule could affect expansin activity on cellulose. Proteins 2015; 83:215–223. © 2014 Wiley Periodicals, Inc.     

Structural analysis of CPF_2247, a novel α-amylase from Clostridium perfringens

CPF_2247 from Clostridium perfringens ATCC 13124 was identified as a putative carbohydrate‐active enzyme by its low sequence identity to endo‐β‐1,4‐glucanases belonging to family 8 of the glycoside hydrolase classification. The X‐ray crystal structure of CPF_2247 determined to 2.0 Å resolution by single‐wavelength anomalous dispersion using seleno‐methionine‐substituted protein revealed an (α/α)₆ barrel fold. A large cleft on the surface of the protein contains residues that are structurally conserved with key elements of the catalytic machinery in clan GH‐M glycoside hydrolases. Assessment of CPF_2247 as a carbohydrate‐active enzyme disclosed α‐glucanase activity on amylose, glycogen, and malto‐oligosaccharides. Proteins 2011;. © 2011 Wiley‐Liss, Inc.