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At least 523 records · Page 29Linked to original sources
PubMed1995

Chromatin structure analysis based on a hierarchic texture model.

The quantification of chromatin structures is an important part of nuclear grading of malignant and premalignant lesions. In order to achieve high accuracy, computerized image analysis systems have been applied in this process. Chromatin texture analysis of cell nuclei requires a suitable texture model. A hierarchic model seemed to be most compatible for this purpose. It assumes that texture consists of homogeneous regions (textons). Based on this model, two approaches to texture segmentation and feature extraction were investigated using sections of cervical tissue. We examined the reproducibility of the measurement under changing optical conditions. The coefficients of variations of the texture features ranged from 2.1% to 16.9%. The features were tested for their discriminating capability in a pilot study including 30 cases of cervical dysplasia and carcinoma. The overall classification accuracy reached 65%. This study presents an automated technique for texture analysis that is similar to human perception.

Carcinoma in Situ↗
PubMed1997

Structural analysis of N-glycans of storage glycoproteins in soybean (Glycine max. L) seed.

The structures of N-linked sugar chains (N-glycans) of storage glycoproteins in soybean seeds have been identified. Eight pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the storage glycoproteins by reverse-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were first identified by two-dimensional PA-sugar chain mapping and ion-spray mass analysis, considering the results of sugar composition analysis or sequential exoglycosidase digestion. The deduced structures were further analyzed by ion-spray tandem mass spectrometry and 500 MHz 1H-NMR spectrometry. The eight structures fell into two categories; the major class (96.6%) was a typical high mannose-type, the minor class was a xylose containing-type (Man3Xyl1GlcNac2, Man3Fuc1Xyl1GlcNac2; 3.4%).

Carbohydrate Sequence↗
PubMed2000

Methods for immunoelectron microscopic and fine structural analysis of synaptonemal complexes and nodules in yeast.

Several gene products involved in meiotic chromosome pairing and recombination in yeast have been identified in recent years. Two nuclear structures play key roles in the meiotic processes: the synaptonemal complex (SC), which is essential for the pairing of the chromosomes, and the recombination nodules (RNs), which mark the sites of recombination. Good morphological representation of the yeast SC and RNs is needed in order to show structural changes caused by specific mutations in protein-coding genes and for fine localization of proteins using immunoelectron microscopy (immuno-EM). This paper presents a newly developed preparation method for EM and immuno-EM that allows analysis of fine structural details and localization of proteins in the SC and RNs in yeast. Structural components of the SC are clearly seen and appear strikingly similar to those in the SC in other organisms. Antibodies against the SC protein Zip1, a transverse filament protein, label the central region of the SC strongly and specifically as expected. The improved method will be an important tool in high-resolution determination of the location of proteins in the meiotic yeast nucleus.

Antibodies↗
PubMed1993

Cloning of the cDNA encoding human xanthine dehydrogenase (oxidase): structural analysis of the protein and chromosomal location of the gene.

The primary structure of human xanthine dehydrogenase (hXDH) was determined by cloning and sequence analysis of the cDNAs encoding the enzyme. The nucleotide (nt) sequence has an open reading frame of 3999 nt encoding a protein of 1333 amino acids (aa) with a calculated M(r) of 146,604. The deduced aa sequence of hXDH is homologous to the previously reported rat XDH (rXDH) and Drosophila melanogaster XDH sequences with identities of 90.2 and 52.0%, respectively. The aa residues involved in both the reversible and the irreversible conversion from the dehydrogenase type to the oxidase type of rXDH are completely conserved between the rat and the human enzymes. This implies that the molecular mechanisms of the conversion of hXDH from dehydrogenase to oxidase are common to those of the well-characterized rXDH. Five sequence variations were detected in the isolated cDNA clones. Spot blot hybridization using flow-sorted human chromosome revealed that the hXDH-encoding gene (hXDH) was located on chromosome 2.

Amino Acid Sequence↗
PubMed1992

Design and synthesis of novel 6,7-imidazotetrahydroquinoline inhibitors of thymidylate synthase using iterative protein crystal structure analysis.

Antifolate inhibitors of thymidylate synthase (TS) have primarily been based on the structure of folic acid. This paper describes the identification and development of novel 6,7-imidazotetrahydroquinoline TS inhibitors by iterative ligand design, synthesis, and crystallographic analysis of protein-inhibitor complexes. Beginning with a high-resolution crystal structure of E. coli TS (TS, EC 2.1.1.45), an imidazotetrahydroquinoline inhibitor was designed de novo to occupy the folate binding pocket. Structural modifications of the initial compound 1h (Ki approximately 5 microM human/E. coli TS) were then made on the basis of feedback from additional cocrystal structures and activity data. An amino group in the 2-position of the imidazole was found to increase the potency of the series by 1-2 orders of magnitude. Other substitutions on the imidazole ring (1-CH3, 2-CH3, 2-NHCH3, 2-SCH3) generally led to weaker inhibition. Additional improvements in activity were obtained by modification of the substituents on the tetrahydroquinoline nitrogen, bringing the Ki of three of the compounds below 15 nM against the human TS enzyme. The compounds were tested for cytotoxicity and were shown to inhibit the growth of three tumor cell lines in vitro.

Adenocarcinoma↗
PubMed1996

Prediction of permanent work disability in a follow-up study of early rheumatoid arthritis: results of a tree structured analysis using RECPAM.

The objectives of this study are: (a) to determine the occurrence of permanent work disability (PWD) in early rheumatoid arthritis (RA); (b) to identify prognostic groups of patients; (c) to assess the employment rates for these groups over time. Seventy-three gainfully employed consecutive out-patients with early RA (> or = 5 ARA 1958 criteria, disease duration < or = 12 months) at time one (T1) were re-examined at time two (T2) after a mean follow-up of 6 yr (S.D. +/- 2 yr). Potential risk factors, identified at T1, for PWD at T2 were entered in a tree structured survival analysis using RECPAM (RECursive Partition and AMalgamation). Cumulative 3 yr employment rates (3-yrER +/- S.E.M.) were computed from the resulting Kaplan-Meier curves. At T2, PWD occurred in 27 of the 73 patients (37%). The fastest decline in the employment rate was found within the first 3 yr of the disease onset, with a 3-yrER reduced to 73 +/- 5%. The group with the poorest prognosis (n = 14; 3-yrER 14 +/- 9%) was defined by age > or = 50 yr with either ESR > or = 60 mm/h or the combination of modified functional class (1-7) > or = 4 with a disease duration > or = 7 months. An intermediate group (n = 38; 3-yrER 79 +/- 6%) was defined by (a) age > or = 50 yr and low or moderate disease activity, (b) age < 50 yr and more strenuous job-related physical requirements, (c) age < 50 yr and less strenuous work, but joint count > or = 15. No case of PWD occurred in 21 individuals aged < 50 yr with a joint count < 15 and less physically demanding jobs. PWD occurs early in a substantial number of patients with RA. RECPAM defines risk profiles that can readily be applied in actual clinical situations and allow an estimation of the risk of PWD at different time points using the resulting Kaplan-Meier curves.

Adult↗
PubMed2003

Structural analysis of gelsolin using synchrotron protein footprinting.

Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron "white" beam results in the formation of stable side chain oxidation products, which can be digested with proteases for mass spectrometry (MS) analysis. Liquid chromatography-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its "inactive" and Ca2+-activated state. Oxidation rate data for 81 peptides derived from the trypsin digestion of gelsolin are presented; 60 of these peptides were observed not to be oxidized, and 21 had detectable oxidation rates. We also report the Ca2+-dependent changes in oxidation for all 81 peptides. Fifty-nine remained unoxidized, five increased their oxidation rate, and two experienced protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca2+-insensitive and Ca2+-dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand-induced structural changes, and the data provide a detailed model for gelsolin activation.

Calcium↗
PubMed2000

Structural analysis of WW domains and design of a WW prototype.

Two new NMR structures of WW domains, the mouse formin binding protein and a putative 84.5 kDa protein from Saccharomyces cerevisiae, show that this domain, only 35 amino acids in length, defines the smallest monomeric triple-stranded antiparallel beta-sheet protein domain that is stable in the absence of disulfide bonds, tightly bound ions or ligands. The structural roles of conserved residues have been studied using site-directed mutagenesis of both wild type domains. Crucial interactions responsible for the stability of the WW structure have been identified. Based on a network of highly conserved long range interactions across the beta-sheet structure that supports the WW fold and on a systematic analysis of conserved residues in the WW family, we have designed a folded prototype WW sequence.

Amino Acid Sequence↗
PubMed1997

Structural analysis of the capsular antigen of Escherichia coli O8:K41:H11.

The primary structure of the acidic capsular antigen of Escherichia coli O8:K41:H11 was shown by monosaccharide analysis, methylation analysis, and by 1D and 2D (1)H amd (13)C NMR spectroscopy to be composed of branched pentasaccharide repeating units with the structure (formula: see text)

Bacterial Capsules↗
PubMed1999

The reaction center complex from the green sulfur bacterium Chlorobium tepidum: a structural analysis by scanning transmission electron microscopy.

The three-dimensional (3D) structure of the reaction center (RC) complex isolated from the green sulfur bacterium Chlorobium tepidum was determined from projections of negatively stained preparations by angular reconstitution. The purified complex contained the PscA, PscC, PscB, PscD subunits and the Fenna-Matthews-Olson (FMO) protein. Its mass was found to be 454 kDa by scanning transmission electron microscopy (STEM), indicating the presence of two copies of the PscA subunit, one copy of the PscB and PscD subunits, three FMO proteins and at least one copy of the PscC subunit. An additional mass peak at 183 kDa suggested that FMO trimers copurify with the RC complexes. Images of negatively stained RC complexes were recorded by STEM and aligned and classified by multivariate statistical analysis. Averages of the major classes indicated that different morphologies of the elongated particles (length=19 nm, width=8 nm) resulted from a rotation around the long axis. The 3D map reconstructed from these projections allowed visualization of the RC complex associated with one FMO trimer. A second FMO trimer could be correspondingly accommodated to yield a symmetric complex, a structure observed in a small number of side views and proposed to be the intact form of the RC complex.

Bacterial Proteins↗
PubMed1984

[Structural analysis of the correlations of genetic markers with physiological indices].

The structure of phenotypic and genetic correlations between ABO blood groups, MN, Rh(D), haptoglobins and concentration indications of the total cholesterol, of sugar curve, the levels of systolic and diastolic arterial pressure was studied in twins by the method for cluster analysis (121 monozygotic twin pairs, 120 dizygotic like sex twin pairs and 107 pairs of unlike sex twins). No correlation between the systems analysed and their indications on the phenotypic level was observed. By analysis of genetic correlations, the existence of correlations between the Rh system and arterial pressure, and the total cholesterol concentration, as well as between the ABO system and sugar concentration indexes, was established. Possible genetic causes for correlations obtained are being discussed.

Adult↗
PubMedDate not supplied

Use of Markush structure analysis techniques for descriptor generation and clustering of large combinatorial libraries.

This article outlines a system in which a Markush structure representation is used for analysis of a combinatorial library, giving savings in terms of storage and processing requirements when compared with an enumerated library. The Markush representation used is described, along with the way in which it can be built from a reaction- and precursor-based description of a library. The process used to generate SMILES, and structure fingerprint and calculated property descriptors for each specific molecule in the library is discussed. Comparative performance figures are given for the Markush approach and various standard fingerprint generation programs based on the enumerated members of the library; the former shows time savings of a couple of orders of magnitude. The use of these rapidly generated fingerprints for clustering of large combinatorial libraries is described.

Cluster Analysis↗
PubMed1996

Kinetic and structural analysis of mutant CD4 receptors that are defective in HIV gp120 binding.

The T-cell antigen coreceptor CD4 also serves as the receptor for the envelope glycoprotein gp120 of HIV. Extensive mutational analysis of CD4 has implicated residues from a portion of the extracellular amino-terminal domain (D1) in gp120 binding. However, none of these proteins has been fully characterized biophysically, and thus the precise effects on molecular structure and binding interactions are unknown. In the present study, we produced soluble versions of three mutant CD4 molecules (F43V, G47S, and A55F) and characterized their structural properties, thermostability, and ability to bind gp120. Crystallographic and thermodynamic analysis showed minimal structural alterations in the F43V and G47S mutant proteins, which have solvent-exposed mutant side chains. In contrast, some degree of disorder appears to exist in the folded state of A55F, as a result of mutating a buried side chain. Real time kinetic measurements of the interaction of the mutant proteins with gp120 showed affinity decreases of 5-fold for G47S, 50-fold for A55F, and 200-fold for F43V. Although both rate constants for the binding reaction were affected by these mutations, the loss in affinity was mainly due to a decrease in on rates, with less drastic changes occurring in the off rates. These observations suggest the involvement of conformational adaptation in the CD4-gp120 interaction. Together, the structural and kinetic data confirm that F43V is a critical residue in gp120 recognition site, which may also include main chain interactions at residue Gly-47.

Binding Sites↗
PubMedDate not supplied

Expression, purification and secondary structure analysis of Saccharomyces cerevisiae vacuolar membrane H+-ATPase subunit F (Vma7p).

The vacuolar H+-ATPase is an acid pump found in virtually all eukaryotic cells. It shares a common macromolecular organization with the F1F0-ATPase, and some V-ATPase subunits are structural and functional homologues of F-ATPase components. However, the vacuolar complex contains several subunits which do not resemble F-ATPase subunits at the sequence level, and which currently have no specific function assigned. One example is subunit F, the Vma7p polypeptide of Saccharomyces cerevisiae. A recombinant form of Vma7p was expressed in Escherichia coli and purified to homogeneity. Mass spectroscopy confirmed a mass of 13460 Da for Vma7p, and dynamic light scattering showed that the polypeptide was globular and monodisperse even at high concentrations. Analysis of secondary structure by circular dichroism and FTIR showed that Vma7p comprises 30% alpha-helix and 32-42% beta-sheet. The protein fold recognition programme 'Threader 2' produced highly significant matches between Vma7p and five alpha-beta sandwich folds. Relative proportions of secondary structure elements within these folds were broadly consistent with the spectroscopic data. Although Vma7p does not share sequence similarity with the F-ATPase epsilon subunit, the analysis suggests that the polypeptides not only have similar masses and assemble into homologous core complexes, but also share similar secondary structures. It is possible that the two polypeptides are homologous and perform similar functions within their respective ATPases. The production of high yields of homogeneous, folded, monodisperse protein will facilitate high resolution crystallography and NMR spectroscopy studies.

Circular Dichroism↗
PubMed1998

Structural analysis of apolipoprotein A-I: effects of amino- and carboxy-terminal deletions on the lipid-free structure.

An amino-terminal deletion mutant (residues 1-43) and a carboxy-terminal deletion mutant (residues 187-243) of human apoliprotein A-I (apo hA-I) have been produced from a bacterial expression system to explore the importance of the missing residues for the conformation of apo hA-I. Our focus has been to study the lipid-free structure of apo hA-I to understand how discrete domains influence the conformational plasticity of the protein and, by inference, the mechanism of lipid binding. All spectral and physical measurements indicate that both apo delta(1-43)A-I and apo delta(187-243)A-I have folded, tertiary structures. These structures differ in the specific arrangement of helical domains based, in part, on their relative thermodynamic stability, near- and far-UV CD, limited proteolysis, and the accessibility of tryptophans to fluorescence quenchers. In addition, all data indicate that the folded domains of apo hA-I and apo delta(187-243)A-I are very similar. Results from analytical ultracentrifugation suggest that lipid-free apo hA-I and the deletion mutants each exist in a dynamic equilibrium between a loosely folded, helical bundle and an elongated monomeric helical hairpin. The conformational heterogeneity is consistent with significant ANS binding exhibited by all three proteins and could help to explain the facile lipid binding properties of apo hA-I.

Amino Acid Sequence↗
PubMed1984

Structural analysis of heparin by methylation and g.l.c.-m.s.: preliminary results.

Heparin is a complex mixture of polysaccharides differing in biological activity and structure, and attempts to relate this activity to structure have suffered, owing to a lack of sufficiently sensitive and specific analytical methods. Application of methylation analysis to determination of the structure of heparin is described. Carboxyl-reduced heparin was converted into its pyridinium salt, this was dissolved in Me2SO, and free OH and NH groups were methylated with dimethylsulfinyl anion. Sulfate groups were removed by solvolysis, and after dialysis, the polymer was acetylated and depolymerized by acetolysis. The resulting monosaccharides were converted into alditol acetates, which were separated by capillary, gas-liquid chromatography, and identified by both electron impact and chemical ionization mass spectrometry. Seventeen different monosaccharides were identified in the hydrolyzate. All of the expected internal hexosaminyl and glycosyluronic residues were identified. Although several sugars were identified as nonreducing termini, only a hexosamine 6-sulfate was identified as a reducing-terminus sugar. The results indicate that methylation analysis of heparins and other complex, sulfated glycosaminoglycans is feasible.

Chemical Phenomena↗
PubMed1994

Structural analysis of antibody specificity. Detailed comparison of five Fab'-steroid complexes.

Structures of the Fab' fragment of the anti-progesterone antibody DB3 in complex with five cross-reactive steroids (aetiocholanolone, 5 beta-androstane-3,17-dione, 5 alpha-pregnane-20-one-3 beta-ol-hemisuccinate, progesterone-11 alpha-ol-hemisuccinate and progesterone) have been determined by X-ray crystallography to a maximum resolution of 2.7 A. These different steroids compete with progesterone binding with affinities in the nanomolar range despite substantial differences in their three-dimensional structures. Comparison of the unliganded DB3 Fab' and these five steroid-Fab' complexes reveals that all the steroid ligands bind to an "open" conformation of the Fab' as defined by the orientation of the indole side-chain of TrpH100, whereas in the unliganded or "closed" form the binding site is occluded by TrpH100. Small but significant conformational changes take place in the antibody to maximize the physical and chemical complementarity with each ligand. The various cross-reactive ligands are accommodated in the binding site in two distinct orientations. We term these binding modes syn and anti, as they are defined by the orientation of the steroid beta face relative to TrpH50. In all cases, the steroid D ring is inserted into a hydrophobic cavity formed mainly by TrpH50, TyrH97, TrpH100 and PheH100b; a hydrogen bond interaction with AsnH35 to the keto group at position C17 or C20 orients the steroid in the pocket. The AsnH35 hydrogen bond and the interaction with TrpH50 account for the restricted heavy chain response to immunization with progesterone-like steroids derivatized at the 11 alpha position. Cross-reactivity of the antibody with different steroids is explained by alternative binding pockets for the A ring, which generates different ligand orientations in the binding site. This study suggests which factors are most likely to contribute to the observed antibody specificity, such as linker position and the paucity of functional groups on the immunogenic hapten.

Amino Acid Sequence↗
PubMed2001

Structure analysis of soluble proteins using electron crystallography.

Electron crystallography as a structural determination technique has grown dramatically in use over recent years. Improvements in microscopes, equipment, practical techniques, computation facilities and image processing methods are reflected in the increasing number of near-atomic resolution structures that have been published. In this review we shall summarize the techniques involved in structure determination of soluble proteins using electron crystallography. Many soluble protein structures have been investigated in this manner over the past two decades. Here we present several examples where a variety of approaches have been used to gradually increase the information obtained.

Annexins↗