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At least 1,531 records · Page 85Linked to original sources
PubMed2003

Molecular modeling of diastereoisomeric aggregates of l/d ser/histamine amide with 5'-TpTpdC-3'.

Novel nonpeptide serine/histamine amides (1: l-Ser-Hism,2: d-Ser-Hism) with potent DNA cleavage activity were designed. Conformational analysis and docking study were carried out in an attempt to understand the DNA cleavage mechanism of the designed enantiomeric nonpeptides. First, the most stable conformers of the designed amides were obtained from the conformational analysis by random search. Next, the three-dimensional structures of l-Ser-Hism.5'-TpTpdC-3' and d-Ser-Hism.5'-TpTpdC-3' complexes were built using molecular docking techniques. The docked diastereoisomeric aggregates show that both l-Ser-Hism and d-Ser-Hism bind to two neighboring phosphates in the 5'-TpTpdC-3' backbone through H-bonds. This binding mode suggests a possible phosphodiester bond hydrolysis mechanism. In addition, the binding energies of two constructed complexes were also calculated with the Tripos force field. It indicates that the binding ability between l-Ser-Hism and 5'-TpTpdC-3' is stronger than that of d-Ser-Hism, suggesting a stronger DNA cleavage activity of l-Ser-Hism than that of d-Ser-Hism. The results agree with our experimental DNA cleavage assays. Supplementary material is available for this article if you access the article at http://dx.doi.org/10.1007/s00894-002-0114-9. A link in the frame on the left on that page takes you directly to the supplementary material. Figure Docking structures of1 and2 binding with oligonucleotide: l-Ser-Hism.5'-TpTpdC-3'(left), d-Ser-Hism.5'-TpTpdC-3'(right). Hydrogen bonds are shown in dotted lines. Only one strand of the oligonucleotide is shown for clarity

Amides↗
PubMed2000

Rapid identification of bacteria from positive blood cultures by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

Bacteremia continues to result in significant morbidity and mortality, particularly in patients who are immunocompromised. Currently, patients with suspected bacteremia are empirically administered broad-spectrum antibiotics, as definitive diagnosis relies upon the use of blood cultures, which impose significant delays in and limitations to pathogen identification. To address the limitations of growth-based identification, the sequence variability of the 16S rRNA gene of bacteria was targeted for rapid identification of bacterial pathogens isolated directly from blood cultures using a fluorescence-based PCR-single-strand conformation polymorphism (SSCP) protocol. Species-specific SSCP patterns were determined for 25 of the most common bacterial species isolated from blood cultures; these isolates subsequently served as a reference collection for bacterial identification for new cases of bacteremia. A total of 272 blood-culture-positive patient specimens containing bacteria were tested. A previously determined SSCP pattern was observed for 251 (92%) specimens, with 21 (8%) specimens demonstrating SSCP patterns distinct from those in the reference collection. Time to identification from blood culture positivity ranged from 1 to 8 days with biochemical testing, whereas identification by fluorescence-based capillary electrophoresis was obtained as early as 7 h at a calculated cost of $10 (U.S. currency) per specimen when tested in batches of 10. Limitations encountered included the inability to consistently detect mixed cultures as well as some species demonstrating identical SSCP patterns. This method can be applied directly to blood cultures or whole-blood specimens, where early pathogen identification would result in a timely diagnosis with possible implications for patient management costs and the mortality and morbidity of infections.

Bacteremia↗
PubMed1984

[Contribution of NMR spectroscopy to the study of structure-function relations of proteins and peptides].

NMR spectroscopy provides a unique means to study molecular conformation, mechanisms of action and structure-function relationships for peptides and proteins in solution under conditions approaching those of their physiological environment. Development of NMR techniques, especially directed to the peptide and protein conformational analysis, is considered under the topics of two-level signal assignment and structural significance of homo- and heteronuclear spin-spin couplings. The results of NMR conformational analysis are presented for solution spatial structure of valinomy cin and gramicidin A antibiotics, honey-bee neurotoxin apamin, scorpion insectotoxin I5A and snake venom neurotoxins of "short" and "long" types. The structure-function relationships are discussed for these biologically active molecules.

Amino Acid Sequence↗
PubMed2000

Single strand conformation polymorphism analysis of K-ras gene mutations by capillary electrophoresis with laser-induced fluorescence (LIF) detector.

Mutations of K-ras gene play an important role in neoplastic progression. The capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) technique is available for the detection of gene mutations. Using an automated capillary electrophoresis with short-chain linear polyacrylamide, after denaturation of PCR products, injections were performed at reverse polarity of 5 kV for 15 s and the separations were carried out under a constant voltage of 8 kV. Of 16 specimens of lung cancer tissue, two specimens were found to have abnormal peaks in the electrophoretogram. CE-SSCP is rapid, automated, and has high performance.

Electrophoresis, Capillary↗
PubMed1998

Snapback SSCP analysis: engineered conformation changes for the rapid typing of known mutations.

Several approaches may be applied to detect known mutations, including restriction enzyme cleavage, allele-specific oligonucleotide (ASO) hybridization or amplification, dideoxy fingerprinting, and direct DNA sequencing. All these approaches require several extra steps after PCR and may involve radioactive isotopes, time-consuming hybridization, template purification, or digestion steps. The ease and simplicity of the SSCP test make it a popular choice for mutation detection, but a significant limitation is that some DNA changes will not alter the overall conformation of either single strand and are thus not amenable to SSCP typing. We describe Snapback SSCP to genotype normal and mdx mice (an animal model of Duchenne muscular dystrophy) that previously could not be differentiated by conventional SSCP analysis. A snapback primer was designed with additional bases at the 5' terminus, which were complementary to the normal sequence flanking the mdx mutation and used under the original amplification conditions. Each single strand of these snapback PCR products now had one terminus capable of re-annealing or "snapping back" to the normal sequence but not the mdx mutation. In this manner, a conformation change was engineered into the normal strand that could be readily distinguished from the mdx allele on a SSCP gel. This approach could be applied to the routine screening of other known mutations that are not amenable to detection by simple SSCP analysis.

Animals↗
PubMed1992

Polymerase chain reaction--single-strand conformation polymorphism analysis of polymorphism in DPA1 and DPB1 genes: a simple, economical, and rapid method for histocompatibility testing.

A new technical trial was carried out to detect polymorphism in HLA-DP genes, based on the diversity in electrophoretic mobility of single-stranded DNA (single-strand conformation polymorphism, SSCP). Genomic DNAs from 31 cell lines homozygous for 2 and 14 different DPA1 and DPB1 alleles, respectively, and from peripheral blood cells of a normal individual homozygous for another DPB1 allele were subjected to polymerase chain reaction (PCR) to amplify the polymorphic exon 2 of DPA1 or DPB1 genes. The PCR samples were denatured by heating in the presence of formamide to obtain single-stranded DNA, electrophoresed in a neutral polyacrylamide gel, and visualized by silver staining. Allelic differences were detected by the distinctive electrophoretic pattern of each single strand, depending on the sequence-specific conformation. Fifteen DPB1 alleles showed 11 distinct electrophoretic patterns, leaving four allelic combinations not distinguished. These four allelic combinations could be further distinguished by using another couple of primers in PCR, with which a part of the exon was amplified, and by subsequent SSCP analysis. The use of four pairs of primers in PCR allowed for discrimination of all the 15 DPB1 alleles tested. Two allelic differences in exon 2 of DPA1 gene could be clearly demonstrated. In addition, putative new alleles of DPA1 and DPB1 genes were detected by SSCP analyses. The PCR-SSCP analysis is simple and rapid, requires neither radioactive materials nor restriction enzymes, and is expected to be a useful tool for investigating the fine HLA-matching required for clinical transplantation of organs.

Alleles↗
PubMed2005

Analysis of conformational changes in 16 S rRNA during the course of 30 S subunit assembly.

Ribosome biogenesis involves an integrated series of binding events coupled with conformational changes that ultimately result in the formation of a functional macromolecular complex. In vitro, Escherichia coli 30 S subunit assembly occurs in a cooperative manner with the ordered addition of 20 ribosomal proteins (r-proteins) with 16 S rRNA. The assembly pathway for 30 S subunits has been dissected in vitro into three steps, where specific r-proteins associate with 16 S rRNA early in 30 S subunit assembly, followed by a mid-assembly conformational rearrangement of the complex that then enables the remaining r-proteins to associate in the final step. Although the three steps of 30 S subunit assembly have been known for some time, few details have been elucidated about changes that occur as a result of these three specific stages. Here, we present a detailed analysis of the concerted early and late stages of small ribosomal subunit assembly. Conformational changes, roles for base-pairing and r-proteins at specific stages of assembly, and a polar nature to the assembly process have been revealed. This work has allowed a more comprehensive and global view of E.coli 30 S ribosomal subunit assembly to be obtained.

Base Pairing↗
PubMed1994

Minisatellite isoalleles can be distinguished by single-stranded conformational polymorphism analysis in agarose gels.

Minisatellite isoallelism, i.e. the occurrence of minisatellite alleles with different internal sequence composition but indistinguishable length, is a common limitation of minisatellite allele length analysis. Internal sequence variation can be used to distinguish such isoalleles, provided that detailed sequence knowledge of its basis is available. We now show that minisatellite isoalleles can also be simply resolved by single-stranded conformational polymorphisms (SSCP) arising during agarose gel electrophoresis. SSCP on agarose gels can be used to distinguish minisatellite isoalleles either after PCR amplification, or by standard Southern blot analysis of genomic DNA.

Alleles↗
PubMed1988

Method of modeling protein structure by the two-dimensional nuclear magnetic resonance spectroscopy data; application to the proteinase inhibitor BUSI IIA from bull seminal plasma.

A new approach is suggested to model the spatial structure of protein molecules in solution based on combined use of the methods of theoretical conformational analysis and NMR spectroscopy data. At the first stage, special means are used to convert d connectivity information into the most probable values of dihedral angles. This allows search for possible spatial structures in the limited regions of the conformational space at further stages using the methods of the theoretical conformational analysis. The suggested approach was verified in reconstructing the spatial backbone structure of the fragment 17-57 of the proteinase inhibitor BUSI IIA from the bull seminal plasma. The structural model parameters are compared with the corresponding characteristics obtained from the X-ray analysis data for the homologic proteinase inhibitor from the Japanese quail ovomucoid. The suggested approach is shown to correctly reproduce both the general molecule topology and the conformations of individual amino acid residues.

Amino Acid Sequence↗
PubMed1981

Structure of N-acetyl-D-alanyl-D-alanine hydrate. An analogue of the COOH-terminal segment of peptidoglycan of bacterial cell walls.

The solid state conformational analysis of Ac-D-Ala-D-Ala-OH.H2O, carried out by infrared absorption and X-ray diffraction, has indicated that the molecules are not extended in a regular beta conformation, but rather that they are partially folded, the phi, psi torsional angles of the COOH-terminal residue in particular being in the region of the left-handed alpha helix of the Ramachandran map. The acetylamino and peptide groups ar found in the usual trans conformation, the latter, however, exhibiting a deviation from rigid planarity. Only intermolecular hydrogen bonds occur in the crystal state. The solution conformational analysis, performed by infrared absorption and CD, has revealed that the amount of intramolecular N--H ... O==C hydrogen-bonded folded forms, if any, should be extremely small, even in deuteriochloroform at high dilution. In water, solvated, unordered species largely predominate.

Bacteria↗
PubMed1999

Single strand conformational polymorphism analysis of human CD1 genes in different ethnic groups.

CD1 molecules are able to present unusual antigens, lipids or glycolipids from mycobacterium cell walls to T lymphocytes. Previous studies have suggested that polymorphism of these genes is very limited, in contrast with classical major histocompatibility complex (MHC) antigen-presenting molecules. Our aim was to study possible allelic variations of exons 2 and 3, encoding for the alpha1 and alpha2 domains, respectively, of human CD1A, -B, -C and -D genes. We analyzed genomic samples of unrelated, healthy individuals from different ethnic background: 70 Caucasians from Europe, 33 Black Africans (13 from Tanzania and 20 Zulus), 19 Caucasians from the Sahara and 44 Asian individuals. We have found CD1A to be a biallelic locus with a common allele which was present in the majority of the individuals studied. The second allele differed from the common one by a single-point mutation, resulting in a change of Cys to Trp at position 52 in the alpha1 domain. This second allele was found in heterozygosis in 7 out of 70 Caucasians from Europe (allelic frequencies P=0.95 and q=0.05). In the Chinese population, we found the second allele present in heterozygosis in 19 from the 44 individuals studied, and we also found 6 homozygous individuals for the second allele (allelic frequencies P=0.64 and q=0.35). In addition, we detected a synonymous mutation (C to T transition) in codon 34 of CD1C exon 2 in 4 out of 20 Zulus and in 2 of the 13 Blacks from Tanzania.

Africa↗
PubMed1995

TP53 gene mutations in gastric carcinoma detected by polymerase chain reaction/single-strand conformation polymorphism analysis of archival material.

TP53 gene mutations, one of the most common alterations described in human tumors, have also been detected in gastric carcinoma, and shown to occur rather late in disease progression. A better assessment of the prognostic value of TP53 gene mutations can be obtained by examining archival material, as this allows stored cases with well-defined histories to be monitored. We performed immunohistochemical and polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analyses of formalin-fixed paraffin-embedded material from nine selected cases of gastric carcinoma at different pathological stages. PCR-SSCP analysis of TP53 exons 5-8 detected missense point mutations in two out of five immunostain(PAb1801)-positive tumors, and a deletion (allowing for a premature stop codon) in one of the remaining four immunostain-negative tumors. Thus, PCR-SSCP analysis represents a feasible strategy for the detection of TP53 alterations in archival material of gastric carcinoma cases.

Aged↗
PubMed2004

The structure and conformational behavior of sulfonium salt glycosidase inhibitors in solution: a combined quantum mechanical NMR approach.

We present the conformational analysis of an inhibitor of alpha-mannosidase, based on a novel sulfonium salt structure (1) that mimics the mannosyl cation intermediate. Because of the number of possible isomeric structures for 1, as well as its complex molecular structure, traditional conformational analysis by NMR was not applicable. Instead, a single experimentally consistent structure was obtained from finite perturbation quantum mechanical calculations of the NMR J-couplings at the B3LYP/6-311G** level. Using a full relaxation matrix analysis, we showed that the quantum-predicted NMR structure was the only isomer that was consistent with the experimental NOE intensities. The results illustrate the potential for finite perturbation calculations to be useful in the analysis of complex charged molecules.

Enzyme Inhibitors↗