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At least 253 records · Page 14Linked to original sources

PubMed1999

The structure of adrenodoxin reductase of mitochondrial P450 systems: electron transfer for steroid biosynthesis.

Adrenodoxin reductase is a monomeric 51 kDa flavoenzyme that is involved in the biosynthesis of all steroid hormones. The structure of the native bovine enzyme was determined at 2.8 A resolution, and the structure of the respective recombinant enzyme at 1.7 A resolution. Adrenodoxin reductase receives a two-electron package from NADPH and converts it to two single electrons that are transferred via adrenodoxin to all mitochondrial cytochromes P 450. The structure suggests how the observed flavin semiquinone is stabilized. A striking feature is the asymmetric charge distribution, which most likely controls the approach of the electron carrier adrenodoxin. A model for the interaction is proposed. Adrenodoxin reductase shows clear sequence homology to half a dozen proteins identified in genome analysis projects, but neither sequence nor structural homology to established, functionally related electron transferases. Yet, the structure revealed a relationship to the disulfide oxidoreductases, permitting the assignment of the NADP-binding site.

Adrenodoxin↗

PubMed1980

Properties and transforming activities of two plasmids in Streptococcus pneumoniae.

Two plasmids from group B streptococcus were introduced into pneumococcus (Streptococcus pneumoniae) and examined for copy number, stability, and some features of the process by which they transform pneumococcal recipients. The 3.6 Mdal pMV158 (tet) was present at a minimum of 12 to 16 copies per chromosome and was never observed to be cured. The 20 Mdal pIP501 (cat erm) had a minimum copy number of 3 to 4 per chromosome and was lost spontaneously at a frequency near 0.03 per division. The presence of novobiocin increased this frequency 2 to 3-fold. Competence for chromosomal transformation and the membrane endonuclease needed for normal DNA entry were required for plasmid transformation. Plasmid transformants segregated transformed cells one generation ahead of chromosomal transformants. Both single and multiple hit components of the transformation reaction kinetics were observed, but the latter could not be seen in the presence of competing chromosomal DNA. The major of the transforming activity behaved as covalently closed circular DNA in dye-buoyancy gradients. Although most of the activity for both plasmids sedimented in sucrose gradients more rapidly than did monomeric closed circular DNA, a significant fraction was found at a position suggesting that it may have been due to monomeric plasmids.

Centrifugation, Density Gradient↗

PubMed1997

Deciphering protein sequence information through hydrophobic cluster analysis (HCA): current status and perspectives.

Ten years after the idea of hydrophobic cluster analysis (HCA) was conceived and first published, theoretical and practical experience has shown this unconventional method of protein sequence analysis to be particularly efficient and sensitive, especially with families of sequences sharing low levels of sequence identity. This extreme sensitivity has made it possible to predict the functions of genes whose sequence similarities are hardly if at all detectable by current one-dimensional (1D) methods alone, and offers a new way to explore the enormous amount of data generated by genome sequencing. HCA also provides original tools to understand fundamental features of protein stability and folding. Since the last review of HCA published in 1990 [1], significant improvements have been made and several new facets have been addressed. Here we wish to update and summarize this information.

Amino Acid Sequence↗

PubMed2004

Stiffness characteristics of hamstring tendon graft fixation methods at the femoral site.

In ACL reconstruction, stiffness and strength of a tendon graft complex are important features for knee stability and rehabilitation. The fixation between tendon and bone is known to be one of the weakest components of the graft complex. We examined the tensile load-displacement characteristics of looped semitendinosus tendons in a porcine femoral tunnel. Two groups of six cadaveric semitendinosus tendons and porcine femurs were tested, secured with either an aperture or non-aperture fixation method. Constructs were tested at 1 mm/s until failure in a materials testing machine, which allowed force and displacement data to be recorded. The non-aperture fixation group was significantly less stiff for the first 4 mm of displacement and had significantly higher ultimate failure loads. Provided that adequate ultimate strength can be achieved, stiffness properties of a tendon graft will be improved by using aperture fixation in femoral-site ACL reconstruction.

Animals↗

PubMed2006

Redox properties of the Fe3+/Fe2+ couple in Arthromyces ramosus class II peroxidase and its cyanide adduct.

The thermodynamics of the one-electron reduction of the ferric heme in free and cyanide-bound Arthromyces ramosus peroxidase (ARP), a class II plant peroxidase, were determined through spectro-electrochemical experiments. The data were compared with those for class III horseradish peroxidase C (HRP) and its cyanide adduct, and were interpreted in terms of ligand binding features, electrostatic effects and solvent accessible surface area of the heme group and of catalytically relevant residues in the heme distal site. The E(o)' values for free and cyanide-bound ARP (-0.183 and -0.390 V, respectively, at 25 degrees C and pH 7) are higher than those for HRP and HRP-CN. ARP features an enthalpic stabilization of the ferrous state and a remarkably negative reduction entropy, which are both unprecedented for heme peroxidases. Once the compensatory contributions of solvent reorganization are partitioned from the measured reduction enthalpy, the resulting protein-based deltaH(o)'(rc(int)) value for ARP turns out to be less positive than that for HRP by +10 kJ mol(-1). The smaller stabilization of the oxidized heme in ARP most probably results from the less pronounced anionic character of the proximal histidine, and the decreased polarity in the heme distal site as compared with HRP, as indicated by the X-ray structures. The surprisingly negative deltaS(o)'(rc) value for ARP is the result of peculiar reduction-induced solvent reorganization effects.

Crystallography, X-Ray↗

PubMed2006

Bond-coating in plasma-sprayed calcium-phosphate coatings.

The influence of bond-coating on the mechanical properties of plasma-spray coatings of hydroxyatite on Ti was investigated. Plasma-spray powder was produced from human teeth enamel and dentine. Before processing the main apatite coating, a very thin layer of Al2O3/TiO2 was applied on super clean and roughened, by Al2O3 blasting, Ti surface as bond-coating. The experimental results showed that bond-coating caused significant increase of the mechanical properties of the coating layer: In the case of the enamel powder from 6.66 MPa of the simple coating to 9.71 MPa for the bond-coating and in the case of the dentine powder from 6.27 MPa to 7.84 MPa, respectively. Both tooth derived powders feature high thermal stability likely due to their relatively high content of fluorine. Therefore, F-rich apatites, such those investigated in this study, emerge themselves as superior candidate materials for calcium phosphate coatings of producing medical devices. The methods of apatite powder production and shaping optimization of powder particles are both key factors of a successful coating. The methods used in this study can be adopted as handy, inexpensive and reliable ways to produce high quality of powders for plasma spray purposes.

Apatites↗

PubMed1983

Orthopedic coordination of dentofacial development in skeletal Class II malocclusion in conjunction with edgewise therapy. Part II.

This second of two articles describes the effects of mandibular growth enhancement in a comprehensive orthopedic approach to the correction of skeletal Class II malocclusion. Clinical and experimental evidence gives support to the concept, and case histories show the clinical mode of use of the appliance system. An initial 10- to 12-month orthopedic phase is followed by a second phase of similar duration in which full edgewise therapy is used to establish a gnathologically ideal occlusion. As the essential corrections are achieved in the orthopedic phase, the second phase is much reduced in complexity and is generally confined to alignment and final detailing. This combined approach to skeletal Class II treatment offers the possibility of better harmony of dental and facial features, with continued stability through the period of facial growth.

Cephalometry↗

PubMed1985

An evaluation of the use of a pH indicator for the detection of beta-lactamase in enzyme immunoassay.

The use of a mixed pH indicator containing bromocresol purple and bromothymol blue was evaluated for the detection of beta-lactamase activity in enzyme immunoassays (EIA) based on this enzyme. The EIA was found to correlate well with a bioassay for antibodies to tetanus toxoid in sheep sera. Results could be read spectrophotometrically at 450 nm or visually as a colour change from dark blue to yellow. The mixed pH indicator was found to have a number of desirable features including good stability, ease of preparation and a sharp colour change which makes the system suitable for visual determination of titration end points.

Animals↗

PubMed1988

Model for the three-dimensional folding of 16 S ribosomal RNA.

We have derived a model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA, using interactive computer graphic methods. It is based on (1) the secondary structure derived from comparative sequence analysis, (2) the three-dimensional co-ordinates for the centers of mass of the 30 S subunit proteins, and (3) the locations of sites in 16 S rRNA that interact with specific ribosomal proteins, from footprinting and crosslinking studies. We present a detailed description of the derivation of the model. About 75% of the RNA chain is sufficiently constrained to provide a useful model. This contains most of the universally conserved core of the molecule. In all but a few instances, protected and crosslinked sites can be placed within or very close to their cognate proteins, while obeying stereochemical rules. The overall shape of the model and locations of specific regions of the RNA correspond well to data derived from electron micrographs of 30 S subunits, although such data were not used to construct the model. Phylogenetic variations in the structure are readily accommodated; as an example, we have modeled the 950-nucleotide mammalian mitochondrial 12 S rRNA by superimposing it on the E. coli structure. The three major RNA domains, as defined by secondary structure, appear to exist as autonomous structural units in three dimensions, for the most part. There is an extensive interface between the 5' and central domains, whereas the 3' major domain has relatively little apparent contact with the rest of the structure. The 5', central and 3' major domains form structures that resemble the body, platform and head, respectively, seen in electron micrographs of 30 S subunits. We discuss possible roles for the ribosomal proteins in stabilizing specific structural features of the RNA during ribosome assembly. The decoding site, as deduced from footprinting and crosslinking studies involving the tRNA anticodon stem-loop, is well-localized. Bases protected from chemical probing by the anticodon stem-loop line the cleft of the subunit. The conserved loop at position 530, which contains some of the bases protected by A site-bound tRNA, is remote (approx. 80 A) from the decoding site. Protection of these bases by the anticodon stem-loop is thus unlikely to be due to direct contact.

Base Sequence↗

PubMed1987

Lectin-specific targeting of lysosomal enzymes to reticuloendothelial cells.

The principles and methods used for enzymatic modification of the carbohydrate portion of glucocerebrosidase are similar to those performed by Ashwell and Morell, Stahl, and others. It is difficult to explain the lack of uptake of native enzyme through binding of the high-mannose type glycopeptide to Man/GlcNAc receptors since approximately 20% of the total oligosaccharides on the native enzyme are high mannose type. Possibly a requirement for multiple sites of attachment to the receptor is not met by a single high-mannose type oligosaccharide per molecule. Alternatively, the presence of complex type oligosaccharides on this enzyme, demonstrated by structural studies, may mask the mannose site and thus account for the poor uptake of native enzyme. The ability to successfully deglycosylate any protein or enzyme in order to specifically target a selected cell type requires that there be (1) an available source of pure enzyme; (2) specific exoglycosidases of high specific activity available either commercially or relatively easily purified; (3) chemical or biochemical means available for the testing of the product, preferably at each step; and (4) a means of separating the glycosidases used from the desired enzyme product. The characteristic and unique accumulation of glucocerebroside only in cells of the monocyte- histiocyte series, makes Gaucher's disease an excellent prototype for the study of enzyme replacement therapy. The principles demonstrated for the enzymatic deglycosylation of glucocerebrosidase may be applied to the cell-specific delivery of other glycoproteins as well. Other lysosomal diseases in which storage occurs in multiple cell types may be ameliorated by administration of macrophage-directed enzymes if, by so doing, storage material can be digested during the normal phagocytic turnover of senescent cells. Consideration of the kinetics of degradation and the structural features affecting the stability of enzymes in vivo are prerequisites to improving the bioengineering of targeted lysosomal enzymes. Animal and culture models have been developed for the study of glucocerebrosidase delivery to specific cell types and substrate degradation. Other studies have progressed toward a definition not only of the receptors at the plasma membrane involved in the internalization of exogenous enzyme, but also of internal receptors or properties of the lysosome responsible for intracellular protein trafficking. A complete understanding of the forces acting to direct endogenous or exogenously supplied enzyme to a given subcellular organelle will require a synthesis of experimental results from all areas of glycoprotein research.

Animals↗

PubMed1993

Structural characterization of the 3' noncoding region of the gene encoding rat mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase.

We report the structural analysis of the 3' noncoding region of the rat mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase-encoding gene. This region, encoded by a single exon of the gene, spans 410 nucleotides. By using S1-nuclease protection analysis, we have mapped two major alternative polyadenylation signals, in close proximity. Computer analysis of the 3' noncoding region has detected the existence of four inverted repeats that could form stem-and-loop structures, with the thermodynamic free energy of formation ranging from -12.2 kcal/mol to -20.4 kcal/mol. The putative regulatory role of these structural features in mRNA stability is discussed.

Animals↗

PubMed2006

Angiotensin II induces histomorphologic features of unstable plaque in a murine model of accelerated atherosclerosis.

BACKGROUND: We explored the role of angiotensin II in determining the histomorphometric features of plaque stability in apolipoprotein E-deficient mice submitted to ligation of the carotid artery. METHODS: Six-month-old apolipoprotein E-deficient mice underwent ligation of the common left carotid artery and were immediately assigned to receive either angiotensin II (1.4 mg . kg(-1) . d(-1) subcutaneously) or vehicle (phosphate-buffered saline; control) via a subcutaneous osmotic minipump for 4 weeks. RESULTS: Ligated arteries from control animals developed intimal lesions composed of macrophage foam cell plaques, which accumulated adjacent to the internal elastic lamina and were surrounded by a fibromuscular layer. Angiotensin II-treated mice had a greater intimal area (threefold), which was accompanied by a fivefold increase in the foam cell area. Lesions from angiotensin II-treated mice also displayed complex morphology characterized by intralesional neovasculature and hemorrhage. The content of active matrix metalloproteinase 2, mainly colocalized with macrophage foam cells, and the production of the inflammatory mediators monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1 were also increased by angiotensin II treatment. Although angiotensin II induced vessel expansion and lumen loss to a similar extent, only vessel enlargement correlated with intimal area. CONCLUSIONS: Taken together, this study's results support a role of angiotensin II in plaque vulnerability by promoting intraplaque neovascularization/hemorrhage, inflammation, and expansive remodeling.

Angiotensin II↗

PubMed1996

Modeling protein folding: the beauty and power of simplicity.

It is argued that simplified models capture key features of protein stability and folding, whereas more detailed models may be more appropriate for protein structure prediction. A brief overview of experimental and theoretical results is presented that corroborates these points. I argue that statistical models capture the key principle of protein stability-cooperativity- and therefore provide a reasonable estimate of protein free energy whereas more detailed but less physically transparent calculations fail to do so. I also explain that the previously published claim that simple models give predictions that are inconsistent with experiments on polypeptide block-copolymers is based on incomplete analysis of such experiment.

Hydrogen Bonding↗

PubMed1987

Interaction of synthetic analogues of distamycin with poly(dA-dT): role of the conjugated N-methylpyrrole system.

Two synthetic analogues of distamycin (Dst), PPA and PAP, containing a saturated beta-alanine moiety substituting for an N-methylpyrrole chromophore were studied for their interactions with the double-stranded alternating copolymer poly(dA-dT).poly(dA-dt) [abbreviated as poly(dA-dT)], with UV absorption and circular dichroism (CD) spectroscopy. The distinctive feature of these analogues is the difference in the extents of extended conjugation due to contiguous pyrrole rings: it decreases in the order Dst greater than PPA greater than PAP. Both these analogues bind to poly(dA-dT) in a way similar to Dst, as suggested from the observed red shift in the UV spectra of the ligands upon complexation and the appearance of induced Cotton effects (in the 290-350-nm region) in the CD spectra of the complexes. A comparative study of (i) the spectral features of the complexes between these ligands, Dst and netrospin (Nt) and poly(dA-dT), and (ii) the binding parameters for the association with the polynucleotide suggests that the number and relative positions of the pyrrole moieties influence the spectral features and thermodynamic stabilities of the complexes, and the latter show a progressive decrease in the order Dst greater than Nt greater than PPA greater than PAP. Implications of these results vis-à-vis the molecular basis of Dst-DNA interaction are discussed.

Circular Dichroism↗

PubMed2004

Following ligand binding and ligand reactions in proteins via Raman crystallography.

Raman crystallography permits the monitoring of chemical events in single-protein crystals in real time. Using a Raman microscope, it is possible to obtain protein Raman spectroscopic data of unprecedented quality and stability. The latter features allow us to obtain the Raman spectrum for small molecules soaking into crystals under normal (nonresonance) Raman conditions. Thus, via an approach utilizing Raman difference spectroscopy, we can quantitate the amount of ligand in the crystal, determine the chemistry of inhibitor-protein interactions, and follow chemical reactions in the active site on the time scale of minutes. While providing unique chemical insights, these data also provide an invaluable guide for determining the conditions for flash-freezing crystals for X-ray crystallographic analysis. In addition, the Raman difference spectra often contain contributions from protein modes due to protein conformational changes occurring upon ligand binding. These features allow us to probe events ranging from small cooperative conformational changes to massive and unexpected secondary structure changes in the crystal. An experimental advantage of Raman crystallography is that the data can be collected from crystals in situ, in sitting or hanging drops, under the conditions used to grow the crystals.

Ligands↗

PubMedDate not supplied

Nanoparticulate system for efficient gene transfer into refractory cell targets.

A biocompatible, nanoparticulate formulation has been designed to retain, protect, and deliver adenoviral gene constructs over an extended time course. Such devices can be administered locally or systemically with low toxicity. A multipolymeric nanoparticulate system, featuring very high stability in physiologic media, was designed to allow efficient in vitro gene transfer. The efficacy of nanoparticulate delivery is effective in cell systems that are normally refractory to gene transfer, such as pancreatic islets and antigen-presenting cells. The findings suggest a nonspecific uptake system that permits adenoviral particle release within the transfected cells. A comparison with literature data revealed that our system is efficient at much lower levels (at least three orders of magnitude) of infectious viral particles.

Animals↗

PubMed2002

Molecular and stable carbon isotopic source identification of oil residues and oiled bird feathers sampled along the Atlantic Coast of France after the Erika oil spill.

The Erika tanker broke in two close to the Atlantic coast of France on December 12, 1999. On December 25th, some heavy fuel oil released by the tanker came ashore along the French Atlantic Coast. Some oil residues and oiled bird feathers were collected all along the Atlantic Shoreline of France after the wreck of the Erika tanker. The aim of this study was to differentiate oil residues and oiled bird feathers related to the Erika oil spill from the ones resulting from the numerous tar ball incidents which had occurred after the Erika oil spill. Alkane and PAH quantification of oil residues allowed differentiation of the samples collected on the north part of the Atlantic Coast from those collected on the south part of the Atlantic shoreline. All oiled birds appear to have been contaminated by the Erika oil. Samples collected on the south part of the Atlantic Coast contain a different molecular fingerprint compared to the Erika oil indicating that they are not related to the Erika oil spill. Bulk and molecular 13C/12C ratio measurements were performed in order to check the discriminative feature and the stability of the isotopic approach. Bulk stable carbon isotopic composition has been shown to be a valuable screening correlation tool as it confirms the link of samples collected in the north part of the Atlantic Coast with the Erika oil spill. All the samples collected along the south part of the Atlantic Shoreline exhibit 13C-enriched bulk isotopic compositions compared to Erika oil. Molecular isotopic composition of saturated hydrocarbons and of phenanthrene compounds also allows unambiguous differentiation of samples related to the Erika oil spill from those due to tar ball incidents. Over the long-term, when molecular distribution will have been modified by the different processes affecting oil in the marine environment, molecular isotopic composition should then be of particular help for Erika oil residues identification.

Animals↗

PubMed2004

Structural increment system for 11-vertex nido-boranes and carboranes.

An increment system forming a set of quantitative rules that govern the relative stabilities of 11-vertex nido-boranes and carboranes is presented. Density functional theory computations at the B3LYP/6-311+G//B3LYP/6-31G level with ZPE corrections were carried out for 61 different boron hydride and carborane structures from [B(11)H(14)](-) to C(4)B(7)H(11) to determine their relative stabilities. Disfavored structural features that destabilize a cluster structure relative to a hypothetical ideal situation were identified and weighted by so-called energy penalties. The latter show additive behavior and allow us to reproduce (within 5 kcal mol(-)(1)) the DFT computed relative energies. Energy penalties for four structural features, i.e., adjacent carbon atoms, CC, a hydrogen atom bridging between a carbon and a boron atom, CH-B, an endo-terminal hydrogen atom at an open face carbon atom, CH(2) and an endo-H between two carbon atoms, C(BH(2))C for the 11-vertex nido-cluster are quite similar to those reported for the 6-vertex nido-cluster, thus showing a behavior independent of the cluster size. Hydrogen structural features, however, vary strongly with the cluster size. Two unknown 11-vertex nido-carboranes were identified which are thermodynamically more stable than known positional isomers.

Journal Article↗