1,722,762 research outputs found
Enzymatic deglycosylation of glycoproteins
No full textRecombinant protein expression using eukaryotic expression systems has certain advantages, such as addition of posttranslational modifications that help protein stability and activity. Asparagine-linked sugar attachment is one of the most common posttranslation modifications. However, sugar modification can impede the growth of high-quality protein crystals for structural studies using X-ray crystallography. To overcome this problem, consensus sites of N-linked attachments can be mutated into other similar residues, such as aspartic acid. Alternatively, enzymatic deglycosylation can be used to remove sugars. Peptide-N-Glycosidase F (PNGase F; EC 3.5.1.52) and Endoglycosidase H (Endo H; EC 3.2.1.96) are the most popular enzymes for this purpose.116sciescopu
SELF-MONITORING, AND INDIVIDUAL EXPECTATION OF PERFORMANCE-NORMS IN SPORT TEAMS
No full textThe main purpose of this study was to examine the relationship between self-monitoring and individuals' expectation of performance-norms, the attitudes shared among team members about how high a performance the group should achieve in team sports. A secondary purpose was to assess whether the relationship between self-monitoring and individual expectation of performance-norms would be moderated by the type of group selected. Analysis suggests that for an elite sport team there is no difference between the performance-norm for the team and individuals' expectations in terms of self-monitoring. For recreational sport teams, however, those high on self-monitoring had higher individual expectations of performance-norms than the low self-monitors.X111Nsciescopu
Design of a fuzzy logic controller with Evolutionary Q-Learning
No full textIn this paper, an Evolutionary Q-Learning(EQL) algorithm is proposed, which is based on the modified Q-learning and evolutionary algorithm. The objective of the proposed EQL algorithm is to find a fuzzy logic controller (FLC) when only a binary reinforcement signal is available from un unknown target environment. The proposed EQL algorithm utilizes and evolves a group of FLCs simultaneously to obtain more feasible solution set. By defining Q-values as functional values of states and FLCs, whole FLCs in the group experience Q-learning process together during the same generation. The Q-learning process assists the proposed EQL algorithm in finding better FLCs with good quality consequent parts. At the end of each generation, the best FLC is constructed by the unique elite construction algorithm. In usual case where evolutionary process, which is basically parallel process, is used with reinforcement learning, multiple instances of target systems are necessary to make the algorithm applied on-line. Otherwise, series experimentation for each individual should be performed in turn with a single target system. However, the proposed EQL algorithm alleviates those necessities and makes it applicable on-line with only a single target system. The feasibility of the proposed EQL algorithm is shown through simulations on the well-known cart-pole balancing problem
Two-longitudinal-mode He-Ne laser for heterodyne interferometers to measure displacement
No full textWe propose a new configuration for a high-resolution heterodyne interferometer that employs a two-longitudinal-mode He-Ne laser with an intermode beat frequency of 600-1000 MHz. The high beat frequency is downconverted to 5 MHz such that the phase change of the interferometer output is precisely measured with a displacement resolution of 0.1 nm. A thermal control scheme is adopted to stabilize the cavity length of the He-Ne plasma tube such that a frequency stability of 2 parts in 109 is obtained by suppression of frequency drifts caused by the phenomena of frequency pulling and polarization anisotropy. This two-longitudinal-mode He-Ne laser yields a high output power of 2.0 mW, which permits multiple measurements of as many as 10 machine axes simultaneously. (C) 2002 Optical Society of America
Two-way frequency-conversion phase measurement for high-speed and high-resolution heterodyne interferometry
No full textHe-Ne heterodyne laser interferometers are widely used as standard tools for displacement metrology to produce high-resolution measurements. However, such systems use specially designed complex and costly high-speed electronics. In this paper, we present a new unsophisticated method of phase measurement that achieves a resolution of 0.15 nm using simple electronics for a target speed of 2.4 ms(-1). The method adopts a frequency-conversion technique to lower the original beat frequency to 100 kHz by mixing it with a stable reference signal generated from a phase-locked loop. At the same time, to avoid the accompanying unwanted decrease in the maximum measurable speed caused by the lowered beat frequency, a two-way frequency conversion and a special fringe-counting method are combined to perform the required phase unwrapping, using widely available programmable digital gates. This allows velocity measurements up to the limit achievable using the original beat frequency
Assembly Pathway and Characterization of the RAG1/2-DNA Paired and Signal-end Complexes
No full textMammalian immune receptor diversity is established via a unique restricted set of site-specific DNA rearrangements in lymphoid cells, known as V(D)J recombination. The lymphoid-specific RAG1-RAG2 protein complex (RAG1/2) initiates this process by binding to two types of recombination signal sequences (RSS), 12RSS and 23RSS, and cleaving at the boundaries of RSS and V, D, or J gene segments, which are to be assembled into immunoglobulins and T-cell receptors. Here we dissect the ordered assembly of the RAG1/2 heterotetramer with 12RSS and 23RSS DNAs. We find that RAG1/2 binds only a single 12RSS or 23RSS and reserves the second DNA-binding site specifically for the complementary RSS, to form a paired complex that reflects the known 12/23 rule of V(D)J recombination. The assembled RAG1/2 paired complex is active in the presence of Mg2+, the physiologically relevant metal ion, in nicking and double-strand cleavage of both RSS DNAs to produce a signal-end complex. We report here the purification and initial crystallization of the RAG1/2 signal-end complex for atomic-resolution structure elucidation. Strict pairing of the 12RSS and 23RSS at the binding step, together with information from the crystal structure of RAG1/2, leads to a molecular explanation of the 12/23 rule.11sciescopu
Crystallization and preliminary X-ray crystallographic analysis of Escherichia coli RbsD, a component of the ribose-transport system with unknown biochemical function
Full text linkThe Escherichia coli high-affinity ribose-transport system consists of six proteins encoded by the rbs operon (rbsD, rbsA, rbsC, rbsB, rbsK and rbsR). Of the six components, RbsD is the only one whose function is unknown. In order to gain insights into the function of RbsD by structural analysis, we overexpressed and crystallized the protein as a first step toward this goal. RbsD was overexpressed in E. coli and crystallized using the hanging-drop vapour-diffusion method at 296 K. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 285.9, b = 92.3, c = 93.3 Angstrom, beta = 105.0 degrees. The unit cell is likely to contain 64 molecules of RbsD, with a crystal volume per protein mass (V-M) of 2.43 Angstrom (3) Da(-1) and a solvent content of about 49.3% by volume. An equilibrium centrifugation analysis demonstrated that RbsD (MW = 15 292 Da) exists as an octamer in solution, suggesting that the asymmetric unit contains two octameric assemblies of RbsD. A native data set to 2.7 Angstrom resolution was obtained from a flash-cooled crystal.11Nsciescopu
Human RAG mutations: biochemistry and clinical implications
No full textThe recombination-activating gene 1 (RAG1) and RAG2 proteins initiate the V(D)J recombination process, which ultimately enables the generation of T cells and B cells with a diversified repertoire of antigen-specific receptors. Mutations of the RAG genes in humans are associated with a broad spectrum of clinical phenotypes, ranging from severe combined immunodeficiency to autoimmunity. Recently, novel insights into the phenotypic diversity of this disease have been provided by resolving the crystal structure of the RAG complex, by developing novel assays to test recombination activity of the mutant RAG proteins and by characterizing the molecular and cellular basis of immune dysregulation in patients with RAG deficiency.114230Nsciescopu
Generating mammalian stable cell lines by electroporation
No full textExpression of functional, recombinant mammalian proteins often requires expression in mammalian cells (see Single Cell Cloning of a Stable Mammalian Cell Line). If the expressed protein needs to be made frequently, it can be best to generate a stable cell line instead of performing repeated transient transfections into mammalian cells. Here, we describe a method to generate stable cell lines via electroporation followed by selection steps. This protocol will be limited to the CHO dhfr - Urlaub et al. (1983) and LEC1 cell lines, which in our experience perform the best with this method.1143sciescopu
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