Conformationally Restricted Criegee Intermediates: Evidence for Formation and Stereoelectronically Controlled Fragmentation

The Baeyer-Villiger reaction of 2-(2-oxocyclohexyl) acetic acid occurs via a bicyclic Criegee intermediate, which fragments with stereoelectronic control, as evidenced by product analysis; the reaction of the but-2-yl ester and of 2-(2-oxocyclopentyl) acetic acid also show evidence of such stereoelectronic control, but less convincingly.

Excision of uracil from the ends of double stranded DNA by uracil DNA glycosylase and its use in high efficiency cloning of PCR products

We show that uracil DNA glycosylase from E. coli excises uracil residues from the ends of double stranded oligos. This information has allowed us to develop an efficient method of cloning PCR amplified DNA. In this report, we describe use of this method in cloning of E. coli genes for lysyl- and methionyl-tRNA synthetases. Efficiency of cloning by this method was found to be the same as that of subcloning of DNA restriction fragments from one vector to the other vector. Possibilities of using other DNA glycosylases for such applications are discussed.

Electrical switching studies on Ge-Te-Tl chalcogenide glasses: Effect of thallium on the composition dependence of switching voltages

Bulk Ge(17)Te83_,JI glasses (05x.5_13), have been found to exhibit memory type electrical switching. The switching voltages (also known as threshold voltage V-th) of Ge17Te83-xTlx glasses are found to decrease with increasing thallium content. The rate of decrease of Vtry is greater at lower concentrations and \textbackslashid, falls at a slower rate for higher thallium concentrations (x 6). The addition of thallium to the Ge-Te network fragments the covalent network and introduces ionic nature to it; the reduction in network connectivity leads to the decrease in switching voltages with thallium content. The decrease in the glass transition temperatures of Ge17Te83-xTlx glasses with increasing thallium concentration supports the idea of decrease in network connectivity with TI addition. The more metallic nature of TI also contributes to the observed reduction in the switching voltages of Ge17Te83-xTlx glasses with TI content. Further, there is an interesting correlation seen between the threshold voltage V-th and the average bond energy, as a function of TI content. In addition, the switching voltages of Ge17Te83-xTlx glasses have been found to decrease with sample thickness almost linearly. The set-reset studies indicate that the Ge17Te83-xTl2 sample can be switched for more than 10 cycles, whereas other glasses could not be reset beyond two switching cycles. (C) 2010 Elsevier B.V. All rights reserved.

Thermodynamic and structural consequences of changing a sulfur atom to a methylene group in the M13Nle mutation in ribonuclease-S

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex. We have substituted the wild-type residue at position 13, methionine (Met), with norleucine (Nle), where the only covalent change is the replacement of the sulfur atom with a methylene group. The thermodynamic parameters associated with the binding of this variant to S-protein, determined by titration calorimetry in the temperature range 10-40 degrees C, are reported and compared to values previously reported [Varadarajan, R., Connelly, P. R., Sturtevant, J. M., & Richards, F. M. (1992) Biochemistry 31, 1421-1426] for other position 13 analogs. The differences in the free energy and enthalpy of binding between the Met and Nle peptides are 0.6 and 7.9 kcal/mol at 25 degrees C, respectively. These differences are slightly larger than, but comparable to, the differences in the values for the Met/Ile and Met/Leu pairs. The structure of the mutant complex was determined to 1.85 Angstrom resolution and refined to an R-factor of 17.4% The structures of mutant and wild-type complexes are practically identical although the Nle side chain has a significantly higher average B-factor than the corresponding Met side chain. In contrast, the B-factors of the atoms of the cage of residues surrounding position 13 are all somewhat lower in the Nle variant than in the Met wild-type. Thus, the large differences in the binding enthalpy appear to reside entirely in the difference in chemical properties or dynamic behavior of the -S- and -CH2- groups and not in differences in the geometry of the side chains or the internal cavity surface. In addition, a novel method of obtaining protein stability data by means of isothermal titration calorimetry is introduced.

Topology of wolfgram proteins and 2?, 3?-cyclic nucleotide 3?-phosphodiesterase in CNS myelin: Studies with proteases

The topological disposition of Wolfgram proteins (WP) and their relationship with 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in human, rat, sheep, bovine, guinea pig and chicken CNS myelin was investigated. Controlled digestion of myelin with trypsin gave a 35KDa protein band (WP-t) when electrophoresed on dodecyl sulfate-polyacrylamide gel in all species. Western blot analysis showed that the WP-t was derived from WP. WP-t was also formed when rat myelin was treated with other proteases such as kallikrein, thermolysin and leucine aminopeptidase. Staining for CNPase activity on nitrocellulose blots showed that WP-t is enzymatically active. Much of the CNPase activity remained with the membrane fraction even after treatment with high concentrations of trypsin when WP were completely hydrolysed and no protein bands with M.W > 14KDa were detected on the gels. Therefore protein fragments of WP with M.W < 14KDa may contain CNPase activity. From these results, it is suggested that the topological disposition of all the various WP is such that a 35KDa fragment is embedded in the lipid bilayer and the remaining fragment exposed at the intraperiod line in the myelin structure which may play a role in the initiation of myelinogenesis.

Characterization of the DNA-binding domain of beta protein, a component of phage lambda Red-pathway by UV catalyzed cross-linking

beta protein, a key component of Red-pathway of phage lambda is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli. To facilitate studies on structure-function relationships, we overexpressed beta protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of beta protein in solution. A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis. Stoichiometric measurements indicated a site-size of 1 monomer of beta protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that beta protein formed stable nucleoprotein complexes with 36-mer, but not with 27- or 17-mer DNA. Interestingly, the interaction of beta protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgCl2, and the binding was abolished by 250 mM NaCl. The K-d of beta protein binding to 36-mer DNA was on the order of 1.8 x 10(-6) M. Photochemical cross-linking of native beta protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain. Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end. These findings provide a basis for further understanding oi the structure and function of beta protein.

Cloning And Characterization of Mycobacteriophage-13 Promoters

Restriction fragments of mycobacteriophage 13 DNA capable of initiating transcription have been cloned into a promoter selection vector of Escherichia coli, and selected on the basis of development of resistance to chloramphenicol. The growth pattern of these 'promoter clones' on a concentration gradient of chloramphenicol and the biochemical assays of the chloramphenicol acetyl transferase have permitted the assessment of their relative promoter strengths. DNA sequence analysis revealed significant homology of these promoters to the -35 regions of the mycobacterial- and E. coli promoter consensus, but less so to the - 10 region. Based on the sequence of phage 13 promoters identified here and the reported sequences of mycobacterial promoters, a promoter consensus for mycobacteria has been generated.

The determination of spatial pattern in Dictyostelium discoideum

Free-living amoebae of the cellular slime mould Dictyostelium discoideum aggregate when starved and give rise to a long and thin multicellular structure, the slug. The slug resembles a metazoan embryo, and as with other embryos it is possible to specify a fate map. In the case of Dictyostelium discoideum the map is especially simple: cells in the anterior fifth of the slug die and form a stalk while the majority of those in the posterior differentiate into spores. The genesis of this anterior-posterior distinction is the subject of our review. In particular, we ask: what are the relative roles of individual pre-aggregative predispositions and post-aggregative position in determining cell fate? We review the literature on the subject and conclude that both factors are important. Variations in nutritional status, or in cell cycle phase at starvation, can bias the probability that an amoeba differentiates into a stalk cell or a spore. On the other hand, isolates, or slug fragments, consisting of only prestalk cells or only prespore cells can regulate so as to result in a normal range of both cell types. We identify three levels of control, each being responsible for guiding patterning in normal development: (i) 'coin tossing', whereby a cell autonomously exhibits a preference for developing along either the stalk or the spore pathway with relative probabilities that can be influenced by the environment; (ii) 'chemical kinetics', whereby prestalk and prespore cells originate from undifferentiated amoebae on a probabilistic basis but, having originated, interact (e.g. via positive and negative feedbacks), and the interaction influences the possibility of conversion of one cell type into the other, and (iii) 'positional information', in which the spatial distribution of morphogens in the slug influences the pathway of differentiation. In the case of possibilities (i) and (ii), sorting out of like cell types leads to the final spatial pattern. In the case of possibility (iii), the pattern arises in situ.

Isolation of an 800 kb contiguous DNA fragment encompassing a 3.5-cM region of chromosome 1 in Arabidopsis using YAC clones

The apetalal mutation of Arabidopsis affects floral meristem identity and the development of sepal and petal primordia of the flower. We mapped the available RFLP markers on chromosome 1 that are in the general vicinity of apetalal on a fine structure map and then chose the closest RFLP as a starting point for contiguous DNA (contig) generation. We report here a contig of about 800 kilobases (kb) that spans a 3.5 cM region of chromosome 1. We used genomic libraries of Arabidopsis prepared in yeast artificial chromosome (YAC) vectors and the detailed characterization of 19 YACs is reported. RFLPs displayed by the end fragments from the walk were mapped to align and correlate the genetic and physical maps for this region of chromosome 1. In this segment of the genome, 1 cM corresponds to a little over 200 kb of physical distance.

Primary structure of a cytotoxin-like basic protein from Naja naja naja (Indian Cobra) venom

The complete amino acid sequence of a cytotoxin-like basic protein (CLBP) from the venom of Naja naja naja (Indian Cobra) was determined by manual degradation using a 4-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. Peptide fragments obtained by chemical cleavage with cyanogen bromide and enzymic cleavages with trypsin and Staphylococcus aureus proteases for sequence analysis were purified by reversed-phase chromatography. The total number of amino acid residues was 61, with leucine as the C-terminal residue. (C) Munksgaard 1995.

Simulation and modelling of orbital debris environment by equivalent breakups

It is well known that the increasing space activities pose a serious threat to future missions. This is mainly due to the presence of spent stages, rockets spacecraft and fragments which can lead to collisions. The calculation of the collision probability of future space vehicles with the orbital debris is necessary for estimating the risk. There is lack of adequately catalogued and openly available detailed information on the explosion characteristics of trackable and untrackable debris data. Such a situation compels one to develop suitable mathematical modelling of the explosion and the resultant debris environment. Based on a study of the available information regarding the fragmentation, subsequent evolution and observation, it turns out to be possible to develop such a mathematical model connecting the dynamical features of the fragmentation with the geometrical/orbital characteristics of the debris and representing the environment through the idea of equivalent breakup. (C) 1997 COSPAR.

Molecular structure and reactions of 1,1 '-bi(acenaphthen-1-ylidene)-2,2 '-dione

The molecular structure of 1,1'-bi(acenaphthen-1-ylidene)-2,2'-dione 1, a potential building-block for the synthesis of fullerene fragments, has been investigated by X-ray crystallography and semi-empirical (AM1 and PM3) calculations. There is a good agreement between the calculated and crystal structure which is essentially planar and has E-configuration. In the solid state, molecules of 1 pack in an interesting manner as corrugated sheets sustained by a network of C-H ... O hydrogen bonds and resulting in the formation of tetrameric loops. While steric factors limit the reactivity of the carbonyl groups in 1, the ene double bond of the ene-dione moiety present in it exhibits propensity toward [4 + 2]-cycloadditions to furnish novel and highly compressed polycycles 8-10.

Nematogens with more flexible chains than aromatic rings in the core

Mesogens containing four rings in the main core can accommodate one terminal and two nearby lateral chains on each outside aromatic ring. These compounds containing six chains present an enantiotropic nematic range which is influenced by the rigidity of the links. The conformational behaviour of the first methyleneoxy group within the chains was investigated by one and two dimensional C-13 NMR. The sign of the jump in chemical shifts when entering the nematic phase indicates the folding of each lateral branch. Dipolar oscillations during cross-polarization contact provide the values of the bond order parameter. The two First lateral fragments do not behave in the same way, demonstrating the influence of the fragment along which the chain is back: folded.

PDB Goodies - a web-based GUI to manipulate the Protein Data Bank file

PDB Goodies is a web-based graphical user interface (GUI) to manipulate the Protein Data Bank file containing the three-dimensional atomic coordinates of protein structures. The program also allows users to save the manipulated three-dimensional atomic coordinate file on their local client system. These fragments are used in various stages of structure elucidation and analysis. This software is incorporated with all the three-dimensional protein structures available in the Protein Data Bank, which presently holds approximately 18 000 structures. In addition, this program works on a three-dimensional atomic coordinate file (Protein Data Bank format) uploaded from the client machine. The program is written using CGI/PERL scripts and is platform independent. The program PDB Goodies can be accessed over the World Wide Web at http:// 144.16.71.11/pdbgoodies/.

The absolute orientation of a molecule in space: Extension of the 'CIP' system

A scheme for denoting the absolute spatial orientation of molecules and molecular fragments is presented; this is based on three angular deviations - one for each spatial dimension - with respect to a reference orientation, which is itself defined with the help of certain features of the Cahn-Ingold-Prelog stereochemical notation. The new scheme helps in reconstructing the three-dimensional characteristics of molecules from purely verbal descriptors, and may thus find application in various information storage and retrieval processes (e.,-. encrypting holograms, etc.).