A modeling study of anaerobic biofilm systems: II. Reactor modeling

A rigorous steady-state model of anaerobic biofilm reactors taking into account acid-base and gas-phase equilibria in the reactor in conjunction with detailed chemical equilibria and mass transfer in acetate-utilizing methanogenic biofilms is presented. The performances of ideal completely stirred tank reactors (CSTRs) and plug-flow reactors, as well as reactors with nonideal hydraulic conditions, are simulated. Decreasing the surface loading rate increases the acetate removal efficiency, while decreasing the influent pH and increasing the buffering capacity improves the removal efficiency only if the bulk pH of the reactor shifts toward more optimal values between 6.8 to 7.0. The reactor can have negative or positive removal efficiencies depending on the start-up conditions. The respiration coefficient plays a critical role in determining the minimum influent pH required for reactor recovery after failure. Having multiple CSTRs-in-series generally increases the overall removal efficiency for the influent conditions investigated. Monitoring of the influent feed quality is critical for plug-flow reactors, becasue failure of the initial sections of the reactor may cause a cascading effect that may lead to a rapid reactor failure. (c) 1995 John Wiley & Sons, Inc.     

The interactions between nucleic acids and polyamines. III. Microscopic protonation constants of spermidine

Changes in the NMR chemical shifts of protons adjacent to the nitrogen atoms of Spermidine which are undergoing protonation have been measured by two-dimensional heteronuclear coupled NMR. Data thus obtained measure the dependence of the state of protonation of individual nitrogens on the pH, and permit calculation of the microprotonation constants of Spermidine and the concentrations of all of the variously protonated Spermidine species present at any pH.     

Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

Atorvastatin modifies the protein profile of circulating human monocytes after an acute coronary syndrome

Aggressive treatment with high‐dose atorvastatin reduces more effectively the incidence of cardiovascular events than moderate statin therapy. The mechanism of this benefit has not been fully elucidated. In order to know the potential effects of statin treatment on the protein expression of circulating monocytes in acute coronary syndrome (ACS) patients, a proteomic analysis of these cells was carried out by 2‐DE and MS. Twenty‐five patients with non‐ST‐elevation acute coronary syndrome (NSTEACS) were randomized, the fourth day after admission, to receive ATV 80 mg/dL (n = 14) or conventional treatment (CT) (n = 11), for two months. Blood was withdrawn at the end of the treatment, and monocytes were extracted for proteomic analysis and their protein expression patterns determined. Age, sex, total cholesterol, LDL, HDL, triglycerides, body mass index, presence of hypertension, diabetes, and smoking status were not significantly different between the two groups of patients. The expression of 20 proteins was modified by intensive ATV. Among the most relevant results stand out the normalization by intensive ATV treatment of the expression of proteins that modulate inflammation and thrombosis such as protein disulfide isomerase ER60 (PDI), Annexin I, and prohibitin, or that have other protective effects as HSP‐70. Thus, this approach shed light at the molecular level of the beneficial mechanisms of anti‐atherothrombotic drugs.     

Proteome analysis of secreted proteins during osteoclast differentiation using two different methods: two-dimensional electrophoresis and isotope-coded affinity tags analysis with two-dimensional chromatography

Bone is maintained by two cell types, bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts express two factors, osteoprotegerin and receptor activator of NF-kappaB ligand (RANKL), inhibiting and promoting osteoclast differentiation, respectively. In contrast, modulators of bone resorption expressed by osteoclasts have not been so well studied enough. In the present study, we demonstrate proteome analysis of secreted proteins during osteoclast differentiation to elucidate the molecular mechanism of bone resorption and bone remodeling. To achieve this objective, we chose RAW264.7 cells with RANKL as a homogeneous osteoclast differentiation model and used two methods, two-dimensional gel electrophoresis (2-DE) and isotope-coded affinity tags (ICAT) analysis with two-dimensional liquid chromatography. We found 23 spots in 2-DE and 19 proteins in ICAT analysis which were expressed differently during osteoclast differentiation. These two methods gave us closely related but different information about proteins, suggesting they are complementary or at least supplementary methods at present. Cathepsins, osteopontin, legumain, macrophage inflammatory protein-1alpha, and other proteins were observed as up- or down-regulated proteins and are discussed in the context of osteoclast differentiation and bone resorption. In addition to confirming previous observations, this study indicates novel proteins related to osteoclast differentiation which are potential therapeutic targets for the treatment of bone diseases, such as osteoporosis.     

Proteomic analysis of the wing imaginal discs of Drosophila melanogaster

We have combined high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of the wing imaginal discs of Drosophila melanogaster. First, we obtained a high-resolution 2-D gel pattern of [35S]methionine + [35S]cysteine-labeled polypeptides of Schneider cells, a permanent cell line of Drosophila embryonic origin, and compared it with the standard pattern of polypeptides of the wing imaginal disc. These studies reveal qualitative and quantitative differences between the two samples, but have more than 600 polypeptides in common. Second, we carried out preparative 2-D polyacrylamide gel electrophoresis using Schneider cells mixed with radioactively labeled wing imaginal discs in order to isolate some of the shared polypeptides and characterize them by matrix-assisted laser desorption/ionization-time of flight MALDI-TOF analysis. Using this strategy we identified 100 shared proteins represented in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis.     

日本血吸虫虫卵、童虫和雌雄成虫膜蛋白的双向电泳

血吸虫寄生生活复杂,中间宿主和终末宿主转换,有性繁殖和无性繁殖交替,由其感染引起的血吸虫病仍严重危害人畜健康(陈贤义等,2002).研究表明,血吸虫不仅能利用宿主的免疫信号分子伪装自己获得宿主的免疫兼容(Salzet et al.,2000),而且还在血吸虫中克隆到宿主信号分子的受体(Ahmed et al.,2001).     

Cytoskeletal scaffolding proteins interact with Lynch‐Syndrome associated mismatch repair protein MLH1

The involvement of MLH1 in several mismatch repair‐independent cellular processes has been reported. In an attempt to gain further insight into the protein's cellular functions, we screened for novel interacting partners of MLH1 utilizing a bacterial two‐hybrid system. Numerous unknown interacting proteins were identified, suggesting novel biological roles of MLH1. The network of MLH1 and its partner proteins involves a multitude of cellular processes. Integration of our data with the “General Repository for Interaction Datasets” highlighted that MLH1 exhibits relationships to three interacting pairs of proteins involved in cytoskeletal and filament organization: Thymosin β 4 and Actin γ, Cathepsin B and Annexin A2 as well as Spectrin α and Desmin. Coimmunoprecipitation and colocalization experiments validated the interaction of MLH1 with these proteins. Differential mRNA levels of many of the identified proteins, detected by microarray analysis comparing MLH1‐deficient and ‐proficient cell lines, support the assumed interplay of MLH1 and the identified candidate proteins. By siRNA knock down of MLH1, we demonstrated the functional impact of MLH1–Actin interaction on filament organization and propose that dysregulation of MLH1 plays an essential role in cytoskeleton dynamics. Our data suggest novel roles of MLH1 in cellular organization and colorectal cancerogenesis.     

人血清蛋白质组学方法的比较和优化

目的:对人未做处理的血清以及去除白蛋白和免疫球蛋白G(IgG)血清的蛋白质组学方法进行比较和优化。方法:应用双向电泳(2-DE)方法分离了未做处理的以及去除白蛋白和免疫球蛋白G(IgG)的血清,比较优化了高温变性、水化液成份组成及泡胀方式等影响血清2-DE分离效果的因素,并用质谱分析鉴定未做处理和已处理血清的2-DE谱图中部分差异蛋白点。结果:得到了分辨率和重复性较好的2-DE谱图,未做处理的血清、去除白蛋白及IgG血清的平均蛋白质点分别为(482±18)个和(523±29)个,质谱分析了9个差异蛋白点,鉴定为8种蛋白质,其中7种为功能蛋白质。仅出现在未做处理血清中的蛋白有4种,分别是维生素A结合蛋白、可溶性尿激酶血纤维蛋白溶酶原激活剂受体、蛋白激酶1抗原、血清白蛋白。4种蛋白仅出现在去除白蛋白和IgG的血清中,分别是NADH脱氢酶辅酶β亚基、肌动蛋白结合蛋白M1、T细胞活性受体β链、血小板生长因子C。结论:去除高丰度蛋白可增加一些低丰度蛋白质的检出,但非特异性吸附会导致部分功能蛋白质的丢失。     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.