Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli

DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases. DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.     

Fast and precise Fourier transforms

Many applications of fast Fourier transforms (FFTs), such as computer tomography, geophysical signal processing, high-resolution imaging radars, and prediction filters, require high-precision output. An error analysis reveals that the usual method of fixed-point computation of FFTs of vectors of length 2^l leads to an average loss of l/2 bits of precision. This phenomenon, often referred to as computational noise, causes major problems for arithmetic units with limited precision which are often used for real-time applications. Several researchers have noted that calculation of FFTs with algebraic integers avoids computational noise entirely. We combine a new algorithm for approximating complex numbers by cyclotomic integers with Chinese remaindering strategies to give an efficient algorithm to compute b-bit precision FFTs of length L. More precisely, we approximate complex numbers by cyclotomic integers in Z[e(2πi/2ⁿ)] whose coefficients, when expressed as polynomials in e(2πi/2ⁿ), are bounded in absolute value by some integer M. For fixed n our algorithm runs in time O(log(M)), and produces an approximation with worst case error of O(1/M(2^n-2-1)). We prove that this algorithm has optimal worst case error by proving a corresponding lower bound on the worst case error of any approximation algorithm for this task. The main tool for designing the algorithms is the use of the cyclotomic units, a subgroup of finite index in the unit group of the cyclotomic field. First implementations of our algorithms indicate that they are fast enough to be used for the design of low-cost high-speed/high-precision FFT chips     

Enzymes hydrolyzing organophosphates as potential catalytic scavengers against organophosphate poisoning

Mechanosensitive channels are ion channels whose activity is dependent on a mechanical stress on the membrane. They are believed to play a central role in mechanotransduction, the process by which mechanical energy is converted into electrical or chemical signals in biological cells. Recent progress, which has been made at the molecular level, is presented, and the two current models of activation of these channels are discussed.     

The MAT locus revisited within a 9.8 kb fragment of chromosome III containing BUD5 and two new open reading frames.

This paper reports the DNA sequence of a segment of 9.8 kb of the chromosome III. The sequenced DNA contains the MAT alpha locus. The new sequence of the MAT alpha locus differs from the previously reported sequence by six modifications in the W segment. We have found the same modifications in the HML locus. The corrected sequence contains, in HML, an open reading frame (ORF) of 190 codons which ends at the border between the W segment and the flanking DNA. In the MAT locus, this ORF extends in the flanking DNA up to 538 codons. This ORF corresponds to a gene independently identified as BUD5 (Chant et al., 1991). This gene presents homologies with the exchange factors SDC25 and CDC25. A large ORF of 1399 codons is found on the opposite side of MAT alpha (toward the telomere). This ORF corresponds to a new gene YCR724. Next to this gene is a small ORF, YCR725, of 127 codons. The localization of this fragment on chromosome III, originally supposed to be distal from the MAT locus based on genetic distance, illustrates variation in recombination frequency along the chromosome and suggests the existence of hot spots of recombination between MAT and the THR4 locus.     

Better LHe withdrawal from a liquefier

Evaporation losses during handling of liquid helium are generally substantial¹. LHe consumption is continuously growing, but the liquefier capacities are essentially limited, so there is an obvious incentive to minimize liquid losses wherever they may occur. This paper presents a system to effect better LHe withdrawal from a liquifier.     

Xenobiotics and xenoestrogens in fish: modulation of cytochrome P450 and carcinogenesis

The periaqueductal gray matter (PAG) serves as the midbrain link between forebrain emotional processing systems and motor pathways used in the defense reaction. Part of this response depends upon PAG efferent pathways that modulate cardiovascular-related sympathetic outflow systems, including those that regulate the heart. While it is known that the PAG projects to vagal preganglionic neurons, including possibly cardiovagal motoneurons, no information exists on the PAG circuits that may affect sympathetically mediated cardiac functions and, thus, the purpose of this study was to use neuroanatomical methods to identify these pathways. First, viral transneuronal retrograde tracing experiments were performed in which pseudorabies virus (PRV) was injected into the stellate ganglion of rats. After 4 days survival, five PAG regions contained transynaptically infected neurons; these included the dorsomedial, lateral and ventrolateral PAG columns as well as the Edinger-Westphal and precommissural nuclei. Second, the descending efferent PAG projections were studied with the anterograde axonal marker Phaseolus vulgaris leuco-agglutinin (PHA-L) with a particular focus on determining whether the PAG projects to the intermediolateral cell column (IML). Almost no axonal labeling was found throughout the thoracic IML suggesting that the PAG modulates sympathetic functions by indirect pathways involving synaptic relays through sympathetic premotor cell groups, especially those found in the medulla oblongata. This possibility was examined by a double tracing study. PHA-L was first injected into either the lateral or ventrolateral PAG and after 6 days, PRV was injected into the ipsilateral stellate ganglion. After an additional 4 days survival, a double immunohistochemical procedure for co-visualization of PRV and PHA-L was used to identify the sympathetic premotor regions that receive an input from the PAG. The PAG innervated specific groups of sympathetic premotor neurons in the hypothalamus, pons, and medulla as well as providing reciprocal intercolumnar connections within the PAG itself (Jansen et al., Brain Res. 784 (1998) 329-336). The major route terminates in the ventral medulla, especially within the medial region which contains sympathetic premotor neurons lying within the raphe magnus and gigantocellular reticular nucleus, pars alpha. Both serotonergic and non-serotonergic sympathetic premotor neurons in these two regions receive inputs from the PAG. Weak PAG projections to sympathetic premotor neurons were found in the rostral ventrolateral medulla (including to C1 adrenergic neurons), locus coeruleus, A5 cell group, paraventricular and lateral hypothalamic nuclei. In summary, both the lateral and ventrolateral PAG columns appear to be capable of modulating cardiac sympathetic functions via a series of indirect pathways involving sympathetic premotor neurons found in selected sites in the hypothalamus, midbrain, pons, and medulla oblongata, with the major outflow terminating in bulbospinal regions of the rostral ventromedial medulla.     

Double dynamic self‐healing polymers: supramolecular and covalent dynamic polymers based on the bis‐iminocarbohydrazide motif

Self‐healing polymers are a class of functional polymers that, by the virtue of the presence of certain dynamic chemical linkages, may undergo self‐repair at a mechanically cut surface. Herein we report the synthesis of a self‐healing polymer giving access to double dynamicity within the polymer network by making use simultaneously of reversible covalent bonds and dynamic non‐covalent hydrogen bonding interactions. These features are provided, respectively, by doubly dynamic cassettes comprising chemically reversible imine linkages and multiply hydrogen‐bonded urea groups, connected by a siloxane‐based backbone that imparts softness to the material. Such a system can be envisaged to give access to a broad spectrum of functional materials, which can be tuned by convenient modulation of the structural motifs of the polymer. © 2013 Society of Chemical Industry     

Inside an agent

When we discuss agent-based system construction with software developers or ask students to implement common agent architectures using object-oriented techniques, we find that it is not trivial for them to create an elegant system design from the standard presentation of these architectures in textbooks or research papers. To better communicate our interpretation of popular agent architectures, we draw UML (Unified Modeling Language) diagrams to guide an implementer's design. However, before we describe these diagrams, we need to review some basic features of agents. The paper considers an architecture showing a simple agent interacting with an environment. The agent senses its environment, uses what it senses to choose an action, and then performs the action through its effectors. Sensory input can include received messages, and action can be the sending of messages. To construct an agent, we need a more detailed understanding of how it functions. In particular, if we are to build one using conventional object-oriented analysis and design techniques, we should know in what ways an agent is more than just a simple object     

Humanistic psychology as an educational program.

States that since a perceptive young person's interest in psychology begins with the problem of understanding himself and his own life, this vital interest should be utilized in starting out with studies of human life as a whole in terms of life history studies. These, viewed in an integrative frame, could easily serve as starting point for studying more specific aspects of psychology, e.g., the biological substructure of life, psychophysiological functions, individual and social behavior, cognition, motivation, human development, normal and abnormal personality, and all other functions and performances. The segmented studies would be more meaningful to the student, if related to the whole of human life and of the person. (19 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Value Potential in Psychotherapy.

This is a comment on value potential in psychotherapy. The concept of "value potential" versus that of "insight potential" is discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)