Off-pathway aggregation can inhibit fibrillation at high protein concentrations

Ribosomal protein S6 fibrillates readily at slightly elevated temperatures and acidic pH. We find that S6 fibrillation is retarded rather than favored when the protein concentration is increased above a threshold concentration of around 3.5 mg/mL. We name this threshold concentration C_FR, the concentration at which fibrillation is retarded. Our data are consistent with a model in which this inhibition is due to the formation of an off-pathway oligomeric species with native-like secondary structure. The oligomeric species dominates at high protein concentrations but exists in dynamic equilibrium with the monomer so that seeding with fibrils can overrule oligomer formation and favors fibrillation under C_FR conditions. Thus, fibrillation competes with formation of off-pathway oligomers, probably due to a monomeric conversion step that is required to commit the protein to the fibrillation pathway. The S6 oligomer is resistant to pepsin digestion. We also report that S6 forms different types of fibrils dependent on protein concentration. Our observations highlight the multitude of conformational states available to proteins under destabilizing conditions.     

OmpA can form folded and unfolded oligomers

The monomeric outer membrane protein OmpA from Escherichia coli has long served as a model protein for studying the folding and membrane insertion of β-barrel membrane proteins. Here we report that when OmpA is refolded in limiting amounts of surfactant (close to the cmc), it has a high propensity to form folded and unfolded oligomers. The oligomers exist both in a folded and (partially) unfolded form which both dissociate under denaturing conditions. Oligomerization does not require the involvement of the periplasmic domain and is not strongly affected by ionic strength. The folded dimers can be isolated and show native-like secondary structure; they are resistant to proteolytic attack and do not dissociate in high surfactant concentrations, indicating high kinetic stability once formed. Remarkably, OmpA also forms significant amounts of higher order structures when refolding in the presence of lipid vesicles. We suggest that oligomerization occurs by domain swapping favored by the high local concentration of OmpA molecules congregating on the same micelle or vesicle. In this model, the unfolded oligomer is stabilized by a small number of intermolecular β-strand contacts and subsequently folds to a more stable state where these intermolecular contacts are consolidated in a native-like fashion by contacts between complementary β-strands from different molecules. Our model is supported by the ability of complementary fragments to associate with each other in vitro. Oligomerization is probably avoided in the cell by the presence of cellular chaperones which maintain the protein in a monomeric state.     

60例食管癌手术治疗诊治分析

目的：探讨胸段食管癌三种常用术式方法的选择原则、手术疗效及术后并发症的比较。方法：本研究选择临床确诊无广泛转移胸段食管癌60例,分别采用常规左胸入路手术20例,右胸、上腹部二切口手术18例和右胸、腹、颈部三切口手术22例,均为根治性手术,术中清扫淋巴结,并送病理检查。分析不同手术径路对食管癌治疗效果及术后并发症的影响。结果：本组研究病例全部顺利完成根治性手术,术中淋巴结活检检出有淋巴结转移25例,淋巴结转移率41.67%（25/60）。本研究组发生并发症患者13例,其中吻合口瘘4例,胃排空障碍4例,肺部并发症5例。其中通过右胸、腹、颈部三切口入路手术并发症最高,达41.25%,与其它组别相比均有明显差异,P〈0.05。结论：胸段食管癌手术方式的选择应因人而异,正确的选择有助于提高肿瘤切除率、淋巴结清扫率及降低肿瘤复发率,此外不同手术方式的个体化选择是降低术后并发症和提高生存质量的有效手段。     

The influence of flume length and group size on swimming performance in shortnose sturgeon Acipenser brevirostrum

The main objectives of this study were to determine optimal methodologies to assess the general swimming performance of juvenile shortnose sturgeon Acipenser brevirostrum. Swimming densities (group v. individual swimming) and flume length (2 v. 1 m) were altered to verify if any of those variables affected performance (i.e. time to fatigue) during critical swimming (U(crit)) and endurance tests. Results for both U(crit) and endurance swimming were not significantly different between fish swum in groups of five or fish swum individually. The U(crit) values, however, were c. 22% higher for fish swum in a longer flume. Although swimming fish in groups did not improve swimming performance, group swimming lowered the variance of the data. Results also reveal that juvenile A. brevirostrum may not possess an ability to swim at high speeds (i.e. burst phase) for long periods.     

Cell-free expression and assembly of ATP synthase

Cell-free (CF) expression technologies have emerged as promising methods for the production of individual membrane proteins of different types and origin. However, many membrane proteins need to be integrated in complex assemblies by interaction with soluble and membrane-integrated subunits in order to adopt stable and functionally folded structures. The production of complete molecular machines by CF expression as advancement of the production of only individual subunits would open a variety of new possibilities to study their assembly mechanisms, function, or composition. We demonstrate the successful CF formation of large molecular complexes consisting of both membrane-integrated and soluble subunits by expression of the atp operon from Caldalkalibacillus thermarum strain TA2.A1 using Escherichia coli extracts. The operon comprises nine open reading frames, and the 542-kDa F₁F_o-ATP synthase complex is composed of 9 soluble and 16 membrane-embedded proteins in the stoichiometry α₃β₃γδ?ab₂c₁₃. Complete assembly into the functional complex was accomplished in all three typically used CF expression modes by (i) solubilizing initial precipitates, (ii) cotranslational insertion into detergent micelles or (iii) cotranslational insertion into preformed liposomes. The presence of all eight subunits, as well as specific enzyme activity and inhibition of the complex, was confirmed by biochemical analyses, freeze-fracture electron microscopy, and immunogold labeling. Further, single-particle analysis demonstrates that the structure and subunit organization of the CF and the reference in vivo expressed ATP synthase complexes are identical. This work establishes the production of highly complex molecular machines in defined environments either as proteomicelles or as proteoliposomes as a new application of CF expression systems.     

Limited expression of reticulocalbin-1 in lymphatic endothelial cells in lung tumor but not in normal lung

Lymphatic endothelial cells in tumors (T-LECs) are considered to have different characteristics from LECs in non-tumor tissues (N-LECs). However, differences between the two types have not been well analyzed at molecular level. In this report, we performed differential proteome analysis of T-LEC and N-LEC models prepared by cultivation of LECs in tumor conditioned medium. By expression profiling of identified proteins using tissue microarrays, reticulocalbin-1 was found to be expressed in clinical specimen-derived T-LECs and lung cancer cells but not N-LECs. It is suggested that reticulocalbin-1 may be an important molecule in understanding T-LEC function and control of lymphatic metastasis.     

Project Prakash: Challenging the critical period: Association of Research in Vision and Ophthalmology National Meeting

Project Prakash is an organization that reverses congenital blindness in children and adolescents in rural India with the hypothesis that these children will be able to recover some of their vision even though their visual system did not develop normally. This hypothesis challenges the scientific dogma established by the Nobel-prize winning research of Hubel and Wiesel that the brain cannot adapt to visual input after being completely deprived of vision during the critical first few months and years of life. Dr. Pawan Sinha presented his work at the largest and most respected ophthalmological research meeting, the Association for Research in Vision and Ophthalmology (ARVO), in Fort Lauderdale, Florida, on May 4, 2011.     

暗培养对黄瓜子叶节再生频率及其相关酶活性和可溶性蛋白含量的影响

以黄瓜品种新泰密刺子叶节为材料,观测了暗培养1~5 d黄瓜子叶节植株再生频率及其诱导过程中POD、SOD活性和可溶性蛋白含量的变化,以探究暗培养条件下黄瓜子叶节POD、SOD活性、可溶性蛋白含量与再生频率间的关系.结果表明,暗培养不仅可以使黄瓜子叶节POD活性持续增长,还有助于其活性保持在较高水平,D4处理在培养的第6天POD值高达243 U?g-1FW,对应的再生频率为87.20%,而对照的最高峰值仅为187.7 U?g-1FW,再生频率只有36.60%;POD的变化体现了各处理生理状态的差异,且各处理暗培养结束时的POD活性水平和POD峰值分别与植株再生频率之间存在显著正相关性,相关系数分别达到了0.921和0.839;而SOD活性水平与再生频率间无显著相关性;可溶性蛋白含量虽然可以体现子叶节生理状态的变化,但无法反映各暗处理对再生频率的影响差别.可见,暗培养有助于提高诱导过程中黄瓜子叶节POD活性的增速,使其活性保持在较高水平,且以POD活性为指标可以反映暗培养处理的有效性.     

A survey of detergents for the purification of stable,active human cystic fibrosis transmembrane conductance regulator (CFTR)

Structural knowledge of the cystic fibrosis transmembrane conductance regulator (CFTR) requires developing methods to purify and stabilize this aggregation-prone membrane protein above 1 mg/ml. Starting with green fluorescent protein- and epitope-tagged human CFTR produced in mammalian cells known to properly fold and process CFTR, we devised a rapid tandem affinity purification scheme to minimize CFTR exposure to detergent in order to preserve its ATPase function. We compared a panel of detergents, including widely used detergents (maltosides, neopentyl glycols (MNG), C₁₂E₈, lysolipids, Chaps) and innovative detergents (branched alkylmaltosides, facial amphiphiles) for CFTR purification, function, monodispersity and stability. ATPase activity after reconstitution into proteoliposomes was 2–3 times higher when CFTR was purified using facial amphiphiles. ATPase activity was also demonstrated in purified CFTR samples without detergent removal using a novel lipid supplementation assay. By electron microscopy, negatively stained CFTR samples were monodisperse at low concentration, and size exclusion chromatography showed a predominance of monomer even after CFTR concentration above 1 mg/ml. Rates of CFTR aggregation quantified in an electrophoretic mobility shift assay showed that detergents which best preserved reconstituted ATPase activity also supported the greatest stability, with CFTR monomer half-lives of 6–9 days in MNG or Chaps, and 12–17 days in facial amphiphile. Cryoelectron microscopy of concentrated CFTR in MNG or facial amphiphile confirmed mostly monomeric protein, producing low resolution reconstructions in conformity with similar proteins. These protocols can be used to generate samples of pure, functional, stable CFTR at concentrations amenable to biophysical characterization.     

Frequent mutations in NOTCH1 ligand-binding regions in Japanese oral squamous cell carcinoma

Recent studies showed that head and neck squamous cell carcinoma (HNSCC) including oral squamous cell carcinoma (OSCC) of Caucasian, Chinese and Indian patients frequently have NOTCH1 mutations. We found eight of 84 OSCC in Japanese patients have point mutations (9.5%) correspond to the ligand binding region of NOTCH1 protein. Two set of them are the same mutations and all mutations are non-synonymous G > A transitions. In addition, median disease-free survival is significantly longer in patients with NOTCH1-mutated tumors as compared to those without the mutation (P < 0.05). The protein structure simulation based on X-ray crystallography indicated that new p.A465T mutation leads to a conformational change of NOTCH1 ligand binding domain as well as the p.G481S mutant NOTCH1 with a loss of flexibility around this residue. These results suggest that NOTCH1 mutation occurs frequently in Japanese OSCC in the vicinity of the ligand binding region and, these mutations cause downregulation of the NOTCH1 function.