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At least 1,315 records · Page 73Linked to original sources
PubMed1993

Synthesis and evaluation of the antitumor activity of 4,5-diamino-substituted 1,2-benzoquinones.

A series of 4,5-diamino-substituted-1,2-benzoquinones were prepared from catechol and the corresponding secondary amines in high yield in a single step using copper complex formation to stabilize the intermediate. The cytotoxicity of the products under various conditions was evaluated using the EMT-6 mammary carcinoma cell line, and antitumor activity was tested in the L1210 murine leukemia. The 4,5-diaziridinyl-1,2-benzoquinone was a more potent cytotoxic agent than diaziquone (AZQ) and was very effective against the L1210 leukemia. The azetidine, pyrrolidine, and diethylamine derivatives were not effective antitumor agents.

Animals↗
PubMed2006

Liquid crystal nanoparticles prepared as miniemulsions.

Liquid crystal nanoparticles using the low molecular weight liquid crystal E7 are prepared by a miniemulsion approach with a droplet size between 180 and 630 nm. The sizes of liquid crystal droplets prepared in this work are one of the smallest sizes ever reported for liquid crystals, and they are considerably dependent on the type and amount of the surfactant used, as well as on the amount of hexadecane. Furthermore, the behavior of a liquid crystal confined to small droplets is investigated. DSC measurements reveal a large shift in the nematic-isotropic phase transition temperature. Further investigations on liquid crystal droplets are carried out using AFM measurements showing that these particles have an order of the liquid crystal molecules within the droplets. Light scattering measurements yield the temperature dependence of the anisotropy and the temporal stability of the droplets. An effect of the director fluctuations within the droplets on dynamic depolarized scattering was found.

Journal Article↗
PubMed2006

Kinetically controlled self-assembly of pseudorotaxanes on crystallization.

[reaction: see text] Mixing of equimolar amounts of cyclobis(paraquat-p-phenylene) (CBPQT(4+)) with a bis-4-methylphenyl ether (MPE twice) of a 1,5-dioxynaphthalene (DNP) derivative in MeCN/CH(2)Cl(2) (3:1) results in the formation of a [2]pseudorotaxane which, on crystallization, yields a [4]pseudorotaxane in the solid state that is stabilized by multiple [C-H...F] interactions: a mixture of the same components in a 1:3 ratio affords a crystalline [2]pseudorotaxane after vapor diffusion of methyl-tert-butyl ether into a solution of these components in MeCN/CH(2)Cl(2) (3:1).

Crystallization↗
PubMed2007

Differences in the subunit interface residues of alternatively spliced glutathione transferases affects catalytic and structural functions.

GSTs (glutathione transferases) are multifunctional widespread enzymes. Currently there are 13 identified classes within this family. Previously most structural characterization has been reported for mammalian Alpha, Mu and Pi class GSTs. In the present study we characterize two enzymes from the insect-specific Delta class, adGSTD3-3 and adGSTD4-4. These two proteins are alternatively spliced products from the same gene and have very similar tertiary structures. Several major contributions to the dimer interface area can be separated into three regions: conserved electrostatic interactions in region 1, hydrophobic interactions in region 2 and an ionic network in region 3. The four amino acid side chains studied in region 1 interact with each other as a planar rectangle. These interactions are highly conserved among the GST classes, Delta, Sigma and Theta. The hydrophobic residues in region 2 are not only subunit interface residues but also active site residues. Overall these three regions provide important contributions to stabilization and folding of the protein. In addition, decreases in yield as well as catalytic activity changes, suggest that the mutations in these regions can disrupt the active site conformation which decreases binding affinity, alters kinetic constants and alters substrate specificity. Several of these residues have only a slight effect on the initial folding of each subunit but have more influence on the dimerization process as well as impacting upon appropriate active site conformation. The results also suggest that even splicing products from the same gene may have specific features in the subunit interface area that would preclude heterodimerization.

Alternative Splicing↗
PubMed1999

Oxidative stabilization of C/C tow pregs studied by light microscopy

Tow pregs obtained from polyacrylonitrile-based carbon fibres and a commercial binder coal-tar pitch were stabilized by oxidation at 548, 588 and 623 K for periods ranging between 1 and 15 h. The microstructural changes involved in the stabilization of tow pregs on the carbonized laminates were studied by light microscopy. Under the conditions studied, the oxidative stabilization of tow pregs produced a significant increase in carbon yield. At the same time, the development of porosity on carbonization was avoided and the size of the optical texture was reduced. However, the extreme oxidation of pitch components at the edges of the carbonized laminate adversely affected pitch binding capability and the sinterizability of the laminates.

Journal Article↗
PubMed1999

Expression, purification and copper-binding studies of the first metal-binding domain of Menkes protein.

The cDNA, coding for the first metal-binding domain (MBD1) of Menkes protein, was cloned into the T7-system based vector, pCA. The T7 lysozyme-encoding plasmid, pLysS, is shown to be crucial for expression, suggesting that the protein is toxic to the cells. Adding copper to the growth medium did not affect the plasmid stability. MBD1 is purified in two steps with a typical yield of 12 mg.L-1. Menkes protein, a P-type ATPase, contains a sequence GMXCXSC that is repeated six times, at the N-terminus. The paired cysteine residues are involved in metal binding. MBD1 has only two cysteine residues, which can exist as free thiol groups (reduced), as a disulphide bond (oxidized) or bound to a metal ion [e.g. Cu(I)-MBD1]. These three MBD1 forms have been investigated using CD. No major spectral change was seen between the different MBD1 forms, indicating that the folding is not changed upon metal binding. A copper-bound MBD1 was also studied by EPR, and the lack of an EPR signal suggests that the oxidation state of copper bound to MBD1 is Cu(I). Cu(I) binding studies were performed by equilibrium dialysis and revealed a stoichiometry of 1 : 1 and an apparent Kd = 46 microM. Oxidized MBD1, however, is not able to bind copper. Different copper complexes were investigated for their ability to reconstitute apo-MBD1. Given the same total copper concentration CuCl43- was superior to Cu(I)-thiourea (structural analogue of metallothionein) and Cu(I)-glutathione (used at fivefold higher copper concentration) although the latter two were able to partially reconstitute apo-MBD1. Cu(II) was not able to reconstitute apo-MBD1, presumably due to Cu(II)-induced oxidation of the thiol groups. Based on our results, glutathione and/or metallothionein are likely candidates for the in vivo incorporation of copper to Menkes protein.

Adenosine Triphosphatases↗
PubMed1992

The synapsis event in the homologous pairing of DNAs: RecA recognizes and pairs less than one helical repeat of DNA.

A key step in homologous recombination is the alignment and pairing of homologous DNAs. The Escherichia coli RecA protein initiates pairing by binding to single-strand DNA, forming a helical nucleoprotein filament. We demonstrate that in the presence of the nonhydrolyzable ATP analogue adenosine 5'-[gamma-thio]triphosphate and ADP, RecA can pair a homologous oligonucleotide 15 bases long with a duplex DNA to yield synaptic complexes consisting of the oligonucleotide and duplex DNA stabilized by RecA. RecA can pair as few as eight bases of homology to form such synaptic complexes. The homologous DNAs remain paired to each other upon removal of RecA provided that the length of shared homology is at least 26 base pairs. Based on our findings and the work of others, we propose that in vitro, one helical turn of a RecA nucleoprotein filament containing approximately six RecA monomers and 15 bases of single-strand DNA is the functional unit sufficient to carry out the homology search.

Base Sequence↗
PubMed1999

Selection for improved subtiligases by phage display.

Engineering enzyme activity has been challenging because of uncertainties in structure-function relationships and difficulties in screening a large number of mutant enzymes. A product capture strategy using phage display is presented here for the selection of improved enzymes from a large library of variants (>10(9) independently derived mutants). Subtiligase, a double mutant of subtilisin BPN' that catalyzes the ligation of peptides, was displayed on phage. Twenty-five active site residues were randomly mutated in groups of four or five to yield six different libraries that were independently sorted. Variants that ligated a biotin peptide onto their own extended N termini were selectively captured. Mutant subtiligases were identified that had increased ligase activity. The selection also yielded unexpected subtiligase mutants having residues known to improve the stability and oxidative resistance of wild-type subtilisin. These studies are exemplary for the use of phage to improve enzyme function when it is closely linked to a selectable catalytic event.

Bacteriophages↗
PubMed2000

Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors.

A cellular assay system for measuring the activity of cytoplasmically expressed anti-GCN4 scFv fragments directed against the Gcn4p dimerization domain was established in the budding yeast Saccharomyces cerevisiae. The inhibitory potential of different constitutively expressed anti-GCN4 scFv intrabodies was monitored by measuring the activity of beta-galactosidase expressed from a GCN4-dependent reporter gene. The in vivo performance of these scFv intrabodies in specifically decreasing reporter gene activity was related to their in vitro stability, measured by denaturant-induced equilibrium unfolding. A framework-engineered stabilized version showed significantly improved activity, while a destabilized point mutant of the anti-GCN4 wild-type showed decreased effects in vivo. These results indicate that stability engineering can result in improved performance of scFv fragments as intrabodies. Increasing the thermodynamic stability appears to be an essential factor for improving the yield of functional scFv in the reducing environment of the cytoplasm, where the conserved intradomain disulfides of antibody fragments cannot form.

Amino Acid Sequence↗
PubMed1993

Measuring astatine-211 distributions with SPECT.

We have investigated standard SPECT techniques (rotating gamma cameras, multi-hole collimators, and filtered backprojection reconstruction) for imaging astatine-211 distributions. Since 211At emits alpha particles, this nuclide has potential for use in radiotherapy. The capability of imaging this nuclide would allow in vivo evaluation of the distribution and stability of potential 211At-labelled radiotherapeutic agents. 211At decay yields x-rays in the 77-92 keV range in addition to 500-900 keV gamma rays. This study evaluates the feasibility of SPECT imaging using the x-ray emissions of 211At. We have evaluated several collimators, with the determination that the medium-energy collimators we used are suitable, with 7% penetration (uncollimated counts versus collimated counts). Several phantoms were imaged and attenuation coefficients were measured (narrow-beam mu = 0.182 cm-1 for 77-80 keV x-rays in water). Reconstructed images demonstrate qualitative capabilities and a simple quantitative study demonstrates good correction for attenuation and scatter (approximately 10% error), at low count densities, at least for the phantom geometries used in this study.

Astatine↗
PubMed1997

Viroid processing: switch from cleavage to ligation is driven by a change from a tetraloop to a loop E conformation.

A longer-than-unit-length transcript of potato spindle tuber viroid is correctly processed in a potato nuclear extract only if the central conserved region is folded into a multi-helix junction containing at least one GNRA tetraloop-hairpin. The cleavage-ligation site between G95 and G96 was mapped with S1 nuclease and primer extension. The structural motifs involved in the processing mechanism were analysed by UV crosslinking, chemical mapping, phylogenetic comparison and thermodynamic calculations. For processing, the first cleavage occurs within the stem of the GNRA tetraloop; a local conformational change switches the tetraloop motif into a loop E motif, stabilizing a base-paired 5' end. The second cleavage yields unit-length linear intermediates, whose 3' end is also base-paired and most probably coaxially stacked in optimum juxtaposition to the 5' end. They are ligated to mature circles autocatalytically, with low efficiency, or enzymatically, with high efficiency.

Base Sequence↗
PubMed2003

Vulnerability curves from conifer sapwood sections exposed over solutions with known water potentials.

The cohesion-tension (CT) theory requires stability of liquid water in conducting elements under high tensions. This stability has been measured using different methods, some of which yielded contradictory results. In this study a method is presented to establish known tensions in the water inside conifer tracheids, to detect cavitation events under these conditions and to construct vulnerability curves. Tangential sapwood sections of Juniperus virginiana L. were placed closely over the surface of NaCl solutions with water potentials ranging from -0.91 to -7.57 MPa. Water potentials were measured with a thermocouple hygrometer in contact with the section, and ultrasound acoustic emissions (UAE) from the sections were registered with an ultrasound transducer. The emission rate of signals increased with the concentration of the solution. Exposure of 100 microm sections in the airspace over a solution provided optimal conditions for the rupture of the water column: many tracheid walls bordered on air, and water in the lumen came under high tension. Nevertheless, the water remained in the metastable liquid state for periods of many hours. The vulnerability obtained from simultaneous measurements of water potentials and ultrasound acoustic emissions on sapwood sections was substantially higher than from conventionally measured curves of detached branches. It is argued that the isolation of tracheids in a massive organ as well as the rate of potential decline will influence the probability of cavitations at a given water potential and thus the parameters of the vulnerability curve.

Acoustics↗
PubMed1988

Chemical Equivalence of Phosphoenzyme Reaction States in the Catalytic Mechanism of the Red Beet (Beta vulgaris L.) Plasma Membrane ATPase.

A comparison of two phosphoryl enzyme reaction states associated with the plasma membrane ATPase of red beet (Beta vulgaris L.) storage tissue was carried out to determine if their differences in reactivity toward ADP and K(+) was related to a structural difference in the site of phosphorylation. Using a pulse labeling method it was possible to produce preparations where either the ADP-sensitive and -insensitive phosphoenzyme forms or the ADP-insensitive phosphoenzyme form alone were trapped as trichloroacetic acid denatured protein. Following complete digestion with Pronase, both preparations yielded radioactive tripeptides with similar properties with respect to pH stability of the covalent bond linking the phosphate to the peptide, isoelectric point, and migration on cellulose thin layer plates. Since the preparation containing both intermediate reaction states behaved in a uniform manner during analysis and displayed properties similar to the preparation containing only the ADP-insensitive phosphoenzyme form, it was proposed that both phosphoenzyme forms were chemically equivalent and derived from the same region of the catalytic active site. The observation that ethyleneimine treatment of both preparations followed by trypsin digestion resulted in the production of tripeptides similar to the Pronase fragments would support this proposal since it suggests that the tripeptides from both phosphoenyzme states contain a lysine residue on the C terminal end and are adjacent to a cysteine residue on the N-terminal end. The chemical equivalence of these two phosphoenzyme reaction states suggests that their differences in reactivity toward ligands may be related to conformational changes associated with the catalytic and transport mechanism of this enzyme.

Journal Article↗
PubMed1993

Inhibition of translational initiation in Saccharomyces cerevisiae by secondary structure: the roles of the stability and position of stem-loops in the mRNA leader.

A new modular gene-expression system for use in studies of translational control in Saccharomyces cerevisiae was constructed. A GAL::PGK fusion promoter (GPF) directed the inducible synthesis of mRNAs initiated at a single major site. A series of leader sequences were tested in combination with each of two reporter genes (encoding chloramphenicol acetyl transferase (cat) and luciferase (luc)). Stem-loop structures of three different sizes and predicted stabilities were inserted into each of two different unique restriction sites in the leader. After correction for relative mRNA abundance, a stem-loop of predicted stability equivalent to approximately -18 kcal mol-1 inhibited translation by up to 89%. The degree of inhibition exerted by the other stem-loops correlated positively with their predicted stabilities. Combinations of two stem-loops at different sites yielded an inhibitory effect greater than that of either individual stem-loop alone. Similar inhibitory effects were observed with both reporter genes. However, inhibition of translation, particularly of the cat gene, was more effective when the stem-loop was positioned close to the start codon rather than at the 5' end of the leader. The observed results reflect an important form of post-transcriptional control that is expected to act on a large number of genes in yeast.

Base Sequence↗
PubMed1994

The bacterial degradation of benzoic acid and benzenoid compounds under anaerobic conditions: unifying trends and new perspectives.

Simple homocyclic aromatic compounds are extremely abundant in the environment and are derived largely from lignin. Such compounds may enter anaerobic environments and several groups of bacteria, exhibiting diverse energy-yielding mechanisms, have evolved the capacity to overcome the thermodynamic stability of the benzene nucleus and degrade aromatic compounds under these conditions. Over the last few years considerable advances have been made in our understanding of the biochemical strategies underlying the bacterial degradation of aromatic compounds in anoxic environments. The study of the biochemistry, and more recently the molecular genetics of the photosynthetic bacterium Rhodopseudomonas palustris and several strains of denitrifying pseudomonads, has provided the greatest insight into the mechanism and regulation of aromatic degradation under anaerobic conditions. Research has centred around the anaerobic degradation of benzoic acid. This involves the initial activation to form benzoyl-Coenzyme A, reduction of the aromatic nucleus--a reaction that has only recently been demonstrated in vitro--and the subsequent degradation of the alicyclic intermediates. Recently, much information regarding the exact nature of these intermediates has been obtained. Also through recent studies, it has become increasingly clear that benzoyl-CoA is a central metabolic intermediate during the anaerobic degradation of structurally diverse aromatic compounds. The initial metabolism of these compounds involves the formation of a carboxyl group on the aromatic nucleus (if necessary) and the synthesis of the respective Coenzyme A thioester; this results in the direct formation of benzoyl-Coenzyme A rather than benzoate. In many cases of anaerobic aromatic degradation studied in batch culture, aromatic intermediates are transiently excreted into the medium. It is argued that the study of this phenomenon may facilitate the understanding of the regulation and kinetics of the aromatic degradative pathways.

Acyl Coenzyme A↗
PubMed1993

Major groove accessibility of RNA.

Chemical acylation experiments showed that the RNA major groove, often assumed to be too deep and narrow to permit recognition interactions, is accessible at duplex termini. Reactivity extended further into the helix in the 5' than in the 3' direction. Asymmetric and large loops between helices uncoupled them, which yielded both enhanced reactivity at terminal base pairs and weaker stabilization enthalpy compared to that in small loops or symmetric loops of the same size. Uncoupled helices have effective helix ends with accessible major grooves; such motifs are attractive contributors to protein recognition, tertiary folding, and catalysis.

Base Sequence↗
PubMed1994

Lignin peroxidase oxidation of aromatic compounds in systems containing organic solvents.

Lignin peroxidase from Phanerochaete chrysosporium was used to study the oxidation of aromatic compounds, including polycyclic aromatic hydrocarbons and heterocyclic compounds, that are models of moieties of asphaltene molecules. The oxidations were done in systems containing water-miscible organic solvents, including methanol, isopropanol, N, N-dimethylformamide, acetonitrile, and tetrahydrofuran. Of the 20 aromatic compounds tested, 9 were oxidized by lignin peroxidase in the presence of hydrogen peroxide. These included anthracene, 1-, 2-, and 9-methylanthracenes, acenaphthene, fluoranthene, pyrene, carbazole, and dibenzothiophene. Of the compounds studied, lignin peroxidase was able to oxidize those with ionization potentials of <8 eV (measured by electron impact). The reaction products contain hydroxyl and keto groups. In one case, carbon-carbon bond cleavage, yielding anthraquinone from 9-methylanthracene, was detected. Kinetic constants and stability characteristics of lignin peroxidase were determined by using pyrene as the substrate in systems containing different amounts of organic solvent. Benzyl alkylation of lignin peroxidase improved its activity in a system containing water-miscible organic solvent but did not increase its resistance to inactivation at high solvent concentrations.

Journal Article↗
PubMed1983

Examination of immobilized fungal cells by phase-contrast and scanning electron microscopy.

Conidia of Penicillium urticae were immobilized in kappa-carrageenan beads and then shaken, in a growth-supporting medium to yield an in situ grown population of mycelia. The physical stability of these beads and the degree of mycelial growth inside the beads were significantly affected by the concentrations of kappa-carrageenan and locust bean gum (LBG) in the bead matrix and by the porous or nonporous nature of the interior. Thus 16-h-old porous and nonporous beads, prepared from 1.25% kappa-carrageenan, 0.5% LBG, and conidia, possessed a very dense mycelial mass at the surface. Only the porous beads possessed a moderately dense mycelial mass at the centre. The conidia at the centre of nonporous beads either failed to germinate or formed very small germ tubes. When washed, 36-h-old porous beads were repeatedly (i.e., 48 h) transferred into nitrogen-free medium, the density of mycelia at the centre increased to equal that at the surface after three transfers or 8 days. Mycelia at the surface exhibited signs of physical damage, while those in the centre did not. The addition of 100 micrograms/mL of cycloheximide to these replacement cultures was reflected by the distortion of interior mycelia.

Carrageenan↗