Detection of Proteins Binding to the Promoter Region DNA Using a Nonradioactive Gel-Retardation Assay

Binding of nuclear proteins to the promoter region was studied by a nonradioactive gel-retardation assay. The procedure uses biotinylated oligonucleotides in combination with streptavidin and biotin-conjugated alkaline phosphatase. This method offers sensitivity comparable to radioactive detection, and the advantage of the high stability of probes. Moreover the hazards of usage associated with radiation are avoided.     

Detection of Proteins in Normal and Alzheimer's Disease Cerebrospinal Fluid with a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay

Abstract— Alzheimer's disease is a progressive degenerative dementia characterized by the abundant presence of neurofibrillary tangles in neurons. This study was designed to test whether the microtubule-associated protein, a major component of neurofibrillary tangles, could be detected in CSF. Additionally, we investigated whether CSF levels were abnormal in Alzheimer's disease as compared with a large group of control patients. We developed a sensitive sandwich enzyme-linked immunosorbent assay using AT120, a monoclonal antibody directed to human, as a capturing antibody. With this technique, the detection limit for was less than 5 pg/ml of CSF. Using ATS, which recognizes abnormally phosphorylated ser-ines 199–202 in, the detection limit was below 20 pg/ml of CSF. However, with AT8, we found no immunoreactiv-ity in CSF, suggesting that only a small fraction of CSF contains the abnormally phosphorylated AT8 epitope. Our results indicate that CSF levels are significantly increased in Alzheimer's disease. Also, CSF levels in a large group of patients with a diversity of neurological diseases showed overlap with CSF levels in Alzheimer's disease.     

Cell-Type Dependence of Stability Modulation of Lectin-initiated Contacts by Impairment of Multivalent Carbohydrate Binding and Intracellular Signaling

Initial ligand selection and the intermolecular spatial arrangement ofglycan-lectin complexes are assumed to be essential to induce formationof stable cell aggregates by a lectin. To distinguish effects of thesetwo processes, the tetrameric mistletoe lectin and its isolated B-chainwere used. A reduced impact of multivalency for Ehrlich ascites tumorcells in contrast to rat thymocytes was revealed. Signaling is thusinitiated in a cell-type-dependent manner. Using selective metabolicinhibitors to reduce signal transfer for aggregate stability, decreasein cellular SH-group level was shown to be a common effect accompanyingsuppression of lectin-dependent aggregate stability. The resultsunderscore an intrinsic variability in the relative importance oflectin-dependent glycan aggregation on the cell surface for triggeringpost-binding lectin effects.     

一种基于链霉素沉淀的PrPSc检测方法的建立

建立一种新的基于链霉素沉淀的PrPSc的Western blot检测方法,用终浓度为60 mmol/L的链霉素处理蛋白酶K消化的PrPSc贮存液,通过离心沉淀PrPSc,用Western blot对PrPSc的链霉素富集效果进行检测.结果显示,链霉索能够与PrPSc结合形成高分子量复合物,但不影响糖基化形式.此外,基于链霉素沉淀的Western blot,无论是在低浓度或是大容积的条件下,均可显著地提高对PrPSc检测的敏感性.基于链霉素沉淀的Western blot试验是一种敏感、特异、快速及灵活的检测方法,有潜力用于脑组织、外周组织及体液中低水平的PrPSc的检测.     

Sensitive Detection of Norovirus Using Phage Nanoparticle Reporters in Lateral-Flow Assay

Noroviruses are recognized worldwide as the principal cause of acute, non-bacterial gastroenteritis, resulting in 19-21 million cases of disease every year in the United States. Noroviruses have a very low infectious dose, a short incubation period, high resistance to traditional disinfection techniques and multiple modes of transmission, making early, point-of-care detection essential for controlling the spread of the disease. The traditional diagnostic tools, electron microscopy, RT-PCR and ELISA require sophisticated and expensive instrumentation, and are considered too laborious and slow to be useful during severe outbreaks. In this paper we describe the development of a new, rapid and sensitive lateral-flow assay using labeled phage particles for the detection of the prototypical norovirus GI.1 (Norwalk), with a limit of detection of 10⁷ virus-like particles per mL, one hundred-fold lower than a conventional gold nanoparticle lateral-flow assay using the same antibody pair.     

Sensitive In Vivo Assay for Detection of Murine Leukemia Viruses

A test is reported for the in vivo detection of the replicative ability of murine leukemia viruses (MLV) in the BALB/c mouse strain. Growth in the spleen was assayed by the complement-fixation assay for MLV group-specific antigen after injection of newborn mice. Low doses of laboratory-derived and wild-type MLV from cell-culture and animal-grown sources were detected as early as 7 to 14 days postinoculation. The sensitivity of this in vivo test compared favorably with in vitro assays for MLV. In vivo detection of MLV replication was correlated with long-term oncogenicity.     

Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold

We have engineered pH sensitive binding proteins for the Fc portion of human immunoglobulin G (hIgG) (hFc) using two different strategies – histidine scanning and random mutagenesis. We obtained an hFc-binding protein, Sso7d-hFc, through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus; Sso7d-hFc was isolated from a combinatorial library of Sso7d mutants using yeast surface display. Subsequently, we identified a pH sensitive mutant, Sso7d-his-hFc, through systematic evaluation of Sso7d-hFc mutants containing single histidine substitutions. In parallel, we also developed a yeast display screening strategy to isolate a different pH sensitive hFc binder, Sso7d-ev-hFc, from a library of mutants obtained by random mutagenesis of a pool of hFc binders. In contrast to Sso7d-hFc, both Sso7d-his-hFc and Sso7d-ev-hFc have a higher binding affinity for hFc at pH 7.4 than at pH 4.5. The Sso7d-mutant hFc binders can be recombinantly expressed at high yield in E. coli and are monomeric in solution. They bind an epitope in the CH3 domain of hFc that has high sequence homology in all four hIgG isotypes (hIgG_1–4), and recognize hIgG_1–4 as well as deglycosylated hIgG in western blotting assays. pH sensitive hFc binders are attractive candidates for use in chromatography, to achieve elution of IgG under milder pH conditions. However, the surface density of immobilized hFc binders, as well as the avidity effect arising from the multivalent interaction of dimeric hFc with the capture surface, influences the pH dependence of dissociation from the capture surface. Therefore, further studies are needed to evaluate if the Sso7d mutants identified in this study are indeed useful as affinity ligands in chromatography.     

Self-Powered Wireless Carbohydrate/Oxygen Sensitive Biodevice Based on Radio Signal Transmission

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.     

Linker Dependence of Avidity in Multivalent Interactions Between Disordered Proteins

Multidomain proteins often interact through several independent binding sites connected by disordered linkers. The architecture of such linkers affects avidity by modulating the effective concentration of intramolecular binding. The linker dependence of avidity has been estimated theoretically using simple physical models, but such models have not been tested experimentally because the effective concentrations could not be measured directly. We have developed a model system for bivalent protein interactions connected by disordered linkers, where the effective concentration can be measured using a competition experiment. We characterized the bivalent protein interactions kinetically and thermodynamically for a variety of linker lengths and interaction strengths. In total, this allowed us to critically assess the existing theoretical models of avidity in disordered, multivalent interactions. As expected, the onset of avidity occurs when the effective concentration reached the dissociation constant of the weakest interaction. Avidity decreased monotonously with linker length, but only by a third of what is predicted by theoretical models. We suggest that the length dependence of avidity is attenuated by compensating mechanisms such as linker interactions or entanglement. The direct role of linkers in avidity suggests they provide a generic mechanism for allosteric regulation of disordered, multivalent proteins.     

Sensitive Detection of Colorectal Cancer in Peripheral Blood by Septin 9 DNA Methylation Assay

Background

Colorectal cancer (CRC) is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC.

Methodology/Principal Findings

Using a PCR assay for analysis of Septin 9 (SEPT9) hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls) and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls). 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48%) and 7/102 controls (7%). In the test study 73/126 CRCs (58%) and 18/183 control samples (10%) were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected) while maintaining 90% specificity (19/183 for controls). Positive rates for plasmas from the other cancers (11/96) and non-cancerous conditions (41/315) were low. The rate of polyp detection (>1 cm) was ∼20%.

Conclusions/Significance

Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.     

Affinity Purification of Human τ Proteins and the Construction of a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay for Human τ Detection

Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity-purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml.     

植物钙结合蛋白与钙离子结合鉴定技术的研究进展

Ca2＋在植物生长发育和环境适应过程中发挥着中心调控作用,钙信号是植物生长发育和逆境响应的主要调控因子,钙结合蛋白是植物钙信号传导途径的最重要组分之一,然而植物钙结合蛋白在体内和体外与Ca2＋结合的技术体系还有待完善和发展。为了系统总结植物钙结合蛋白的鉴定方法与技术,本文从定性结合、定量结合和结合方式等角度,综述了植物钙结合蛋白在体内和体外条件下与Ca2＋结合的原理、方法、特点和应用前景,详细阐述了近年来的主要检测方法,并对其今后的发展趋势作了展望。本文将为植物钙结合蛋白的分离、功能鉴定和作用机制的研究提供技术支撑。     

检测脱氨基速率的超灵敏方法及应用

脱氨基被认为是引起细胞突变的主要因素,如果这些脱氨基的产物不被修复,将引起转换(transition)突变.为了理解DNA结构和其化学活性的关系,介绍一种新的灵敏的遗传学方法,它应用在DNA特定点的脱氨基速率的测定.这种方法基于M13mp2噬菌体内的1acZα基因中的CCC脯氨酸密码子的反转突变,即每个脱氨基事件表现为在白色菌斑背景中的一个蓝色菌斑,其灵敏度可达10⁵ M13mp2 DNA分子中检验出一个脱氨基事件.此外,该法可以计算脱氨基动力学速率常数和反应活化能.     

Development of a p38 Kinase Binding Assay for High Throughput Screening

p38 is a member of the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases, which is activated by cellular stressors and has been shown to be a critical enzyme in the synthesis and action of proinflammatory cytokines, tumor necrosis factor-a (TNF-alpha) and interleukin-1beta (IL-1beta). A group of pyridinyl imidazole compounds such as SB202190 have been identified as selective inhibitors of p38 that bind directly to the ATP pocket of the enzyme. These compounds inhibit the p38 kinase activity, block TNF-alpha and IL-1beta secretion both in vivo and in vitro and are found to be effective in animal models of arthritis, bone resorption, and endotoxin shock. We postulated that other classes of compounds capable of competing the binding of pyridinyl imidazole with p38 enzyme could have efficacy in the treatment of inflammatory diseases. Therefore, a simple and robust assay was developed to measure the ability of small molecules to inhibit the binding of tritium-labeled pyridinyl imidazole, SB202190, to recombinant p38 kinase. For assay development, the human p38 gene was cloned in baculovirus and then expressed in insect cells. Tritiated SB202190 was synthesized and used as the p38 ligand for a competitive filter binding assay. This assay has been used successfully to screen both synthetic and combinatorial chemical libraries for other classes of p38 kinase inhibitors.     

Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence

Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to a broad range of cellular functions, including cell motility, signal transduction, and virus replication. Here, we describe a dual-color (DC) extension of the fluorescence z-scan technique, which has proven successful for quantification of peripheral membrane protein binding to the PM in living cells. We demonstrate that the coexpression of a second, distinctly colored fluorescent protein provides a soluble reference species that delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM-binding measurements by an order of magnitude. DC z-scan generates an intensity profile for each detection channel that contains information on the axial distribution of the peripheral membrane and reference protein. Fit models for DC z-scan are developed and verified using simple model systems. Next, we apply the quantitative DC z-scan technique to investigate the binding of two peripheral membrane protein systems for which previous z-scan studies failed to detect binding: human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein and lipidation-deficient mutants of the fibroblast growth factor receptor substrate 2α. Our findings show that these mutations severely disrupt PM association of fibroblast growth factor receptor substrate 2α but do not eliminate it. We further detected binding of HIV-1 MA to the PM using DC z-scan. Interestingly, our data indicate that HIV-1 MA binds cooperatively to the PM with a dissociation coefficient of K_d ∼16 μM and Hill coefficient of n ∼2.     

Karyopherin-Centric Control of Nuclear Pores Based on Molecular Occupancy and Kinetic Analysis of Multivalent Binding with FG Nucleoporins

Intrinsically disordered Phe-Gly nucleoporins (FG Nups) within nuclear pore complexes exert multivalent interactions with transport receptors (Karyopherins (Kaps)) that orchestrate nucleocytoplasmic transport. Current FG-centric views reason that selective Kap translocation is promoted by alterations in the barrier-like FG Nup conformations. However, the strong binding of Kaps with the FG Nups due to avidity contradicts rapid Kap translocation in vivo. Here, using surface plasmon resonance, we innovate a means to correlate in situ mechanistic (molecular occupancy and conformational changes) with equilibrium (binding affinity) and kinetic (multivalent binding kinetics) aspects of Karyopherinβ1 (Kapβ1) binding to four different FG Nups. A general feature of the FxFG domains of Nup214, Nup62, and Nup153 is their capacity to extend and accommodate large numbers of Kapβ1 molecules at physiological Kapβ1 concentrations. A notable exception is the GLFG domain of Nup98, which forms a partially penetrable cohesive layer. Interestingly, we find that a slowly exchanging Kapβ1 phase forms an integral constituent within the FG Nups that coexists with a fast phase, which dominates transport kinetics due to limited binding with the pre-occupied FG Nups at physiological Kapβ1 concentrations. Altogether, our data reveal an emergent Kap-centric barrier mechanism that may underlie mechanistic and kinetic control in the nuclear pore complex.     

细胞分裂素结合蛋白的研究进展

迄今为止,已从多种植物中分离到细胞分裂素结合蛋白(CBPs),它们可能在细胞分裂素的信号转导、体内运输及代谢中起作用。根据现有研究结果认为,大多数CTKs受体可能位于膜上,通过与G_蛋白耦联的信号转导系统或双组分信号转导系统完成CTKs信号的跨膜转导。少数CTKs受体可能位于细胞质中,与胞内CTKs结合后进入细胞核,直接调节基因的表达。本文综述了近年来对CBPs的研究进展,分析了CTKs受体的可能类型及CBPs作用的可能机制。