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PubMed2004

Mutation detection in patients with familial hypercholesterolaemia using heteroduplex and single strand conformation polymorphism analysis by capillary electrophoresis.

Mutations in the low-density lipoprotein receptor (LDLR) gene give rise to familial hypercholesterolaemia (FH). In this study we have used a 96-well capillary machine (MegaBACE, Amersham) to develop a single strand conformation polymorphism (SSCP) and heteroduplex method for the detection of mutations in the LDLR gene. We have applied this technique to 101 different mutations including single nucleotide polymorphisms in different exons of the LDLR gene. Hundred percent of these nucleotide alterations were distinguished by this method. We suggest this fast, reliable and safe method for diagnosis of FH in large patient groups.

Electrophoresis, Capillary↗
PubMed2001

Cold single-strand conformation polymorphism analysis: optimization for detection of APC gene mutations in patients with familial adenomatous polyposis.

Over 200 adenomatous polyposis coli (APC) gene mutations have been described in familial adenomatous polyposis (FAP) patients. Recent single-strand conformation polymorphism (SSCP) screening methods have introduced minigel runs, simple ethidium bromide staining and external temperature control without any loss of sensitivity (cold-SSCP). In order to test the effectiveness in APC mutation detection, cold-SSCP was employed following polymerase chain reaction (PCR) amplification in three patients with FAP. Different running parameter combinations were compared. The three mutations already known were all diagnosed by cold-SSCP. The gel concentration was found to be essential in detecting the single-base substitution in fragments of different lengths. The observation of deletions was not affected by gel concentrations and heteroduplex bands were always produced. The temperature or glycerol addition did not significantly affect sensitivity. This modified cold-SSCP method provides a simple and effective way for detecting several known Apc gene mutations without any loss of sensitivity and could be useful for large-scale molecular diagnosis of FAP.

Adenomatous Polyposis Coli↗
PubMed2001

Experimental and conformational analyses of interactions between butenafine and lipids.

Butenafine (N-4-tert-butylbenzyl-N-methyl-1-naphtalenemethylamine hydrochloride) is an antifungal agent of the benzylamine class that has excellent therapeutic efficacy and a remarkably long duration of action when applied topically to treat various mycoses. Given the lipophilic nature of the molecule, efficacy may be related to an interaction with cell membrane phospholipids and permeabilization of the fungal cell wall. Similarly, high lipophilicity could account for the long duration of action, since fixation to lipids in cutaneous tissues might allow them to act as local depots for slow release of the drug. We have therefore used computer-assisted conformational analysis to investigate the interaction of butenafine with lipids and extended these observations with experimental studies in vitro using liposomes. Conformational analysis of mixed monolayers of phospholipids with the neutral and protonated forms of butenafine highlighted a possible interaction with both the hydrophilic and hydrophobic domains of membrane phospholipids. Studies using liposomes demonstrated that butenafine increases membrane fluidity [assessed by fluorescence polarization of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene and 1,6-diphenylhexatriene] and membrane permeability (studied by release of calcein from liposomes). The results show, therefore, that butenafine readily interacts with lipids and is incorporated into membrane phospholipids. These findings may help explain the excellent antifungal efficacy and long duration of action of this drug when it is used as a topical antifungal agent in humans.

Antifungal Agents↗
PubMed1998

Polymerase chain reaction-single-strand conformational polymorphism analysis of antigen receptor rearrangements in monitoring therapeutic effect in childhood ALL.

The rearrangement of the immunoglobulin heavy chain (IgH) genes can be used as a marker of cell lineage and clonality. The polymerase chain reaction (PCR) technique using consensus primers for the IgH gene was used for remission and minimal residual disease (MRD) analysis in the follow-up of childhood acute lymphoblastic leukemia (ALL) of B-cell lineage. Single-strand conformational polymorphism (SSCP) was used to distinguish the specific clonal amplicons from the background. The Authors found that, in a series of 22 patients followed-up for 5.3 to 11.1 years, the PCR-SSCP technique could detect at least one rearrangement at initial diagnosis in 21 (95%). All patients who remained in continuous complete remission were PCR-SSCP negative at remission controls. Ten of the 22 patients had one or more bone marrow relapses. The PCR-SSCP method demonstrated MRD in three of them. In 6 of the 7 (86%) of patients with disease recurrence from whom samples were taken within 6 months before a clinically overt relapse, PCR-SSCP became positive. The Authors conclude that PCR-SSCP of a rearrangement marker might have a role as a convenient technique for monitoring emerging relapse. It may also detect unrelated clones or ongoing secondary recombination events during progression. However, PCR-SSCP is not sensitive enough to detect MRD in all patients in whom disease will later recur.

Bone Marrow Transplantation↗
PubMed2000

Lincomycin and clindamycin conformations. A fragment shared by macrolides, ketolides and lincosamides determined from TRNOE ribosome-bound conformations.

Two important lincosamide antibiotics, lincomycin and clindamycin were studied in the complex state with the bacterial ribosome after a conformational analysis by 1H and 13C NMR spectroscopy and molecular modelling of the unbound molecules. Lincosamide-ribosome interactions were investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY), resulting in a bound structure compatible with the experimental NMR data. The results compared with the conformational analysis of the substrates in solution indicate that specific conformations are preferred in the bound state. Clindamycin, the more bioactive antibiotic studied, displayed a stronger NMR response than lincomycin showing that in lincosamide-ribosome interactions, a low affinity binding level is associated to the tight binding one and is related to biological activity. This study shows that conformation plays an essential role for the low affinity binding site. Superimposition of lincosamide, macrolide and ketolide bound structures exhibited conformational similarities in a particular fragment which is in agreement with a hypothesis of partial overlapping lincosamide and macrolide binding sites.

Anti-Bacterial Agents↗
PubMed1994

Conformation of tetragastrin in DMSO. Monte Carlo simulation taking account of solvent effects.

A conformation analysis of tetrapeptide in DMSO was carried out by the Monte Carlo simulation including the solvent effects in the energy function. The lowest energy conformations in the zwitterionic and cationic states are a similar compact form, in which the two aromatic rings are close to each other. The distance distributions between the specified atoms in the molecule show that most conformations in the local energy minima are similar to this folded conformation, although there is another type of conformation of interest, which has an extended form with the two separate aromatic rings. Such results are consistent with the NMR and CD experiments, and suggest that the compact form of tetragastrin is essential as a biological active conformation. The folded structure is stabilized by the solute-solvent interaction. In particular, the nonbonding interactions between the solute and solvent molecules cause a folding effect on the molecular conformation in total. This study is a first case taking account of the solvent effect into the conformation analysis of tetragastrin.

Amino Acid Sequence↗
PubMed2004

Quantitative analysis of conformational exchange contributions to 1H-15N multiple-quantum relaxation using field-dependent measurements. Time scale and structural characterization of exchange in a calmodulin C-terminal domain mutant.

Multiple-quantum spin relaxation is a sensitive probe for correlated conformational exchange dynamics on microsecond to millisecond time scales in biomolecules. We measured differential 1H-15N multiple-quantum relaxation rates for the backbone amide groups of the E140Q mutant of the C-terminal domain of calmodulin at three static magnetic field strengths. The differential multiple-quantum relaxation rates range between -88.7 and 92.7 s(-1), and the mean and standard deviation are 7.0 +/- 24 s(-1), at a static magnetic field strength of 14.1 T. Together with values of the 1H and 15N chemical shift anisotropies (CSA) determined separately, the field-dependent data enable separation of the different contributions from dipolar-dipolar, CSA-CSA, and conformational exchange cross-correlated relaxation mechanisms to the differential multiple-quantum relaxation rates. The procedure yields precise quantitative information on the dominant conformational exchange contributions observed in this protein. The field-dependent differences between double- and zero-quantum relaxation rates directly benchmark the rates of conformational exchange, showing that these are fast on the chemical shift time scale for the large majority of residues in the protein. Further analysis of the differential 1H-15N multiple-quantum relaxation rates using previously determined exchange rate constants and populations, obtained from 15N off-resonance rotating-frame relaxation data, enables extraction of the product of the chemical shift differences between the resonance frequencies of the 1H and 15N spins in the exchanging conformations, deltasigma(H)deltasigma(N). Thus, information on the 1H chemical shift differences is obtained, while circumventing complications associated with direct measurements of conformational exchange effects on 1H single-quantum coherences in nondeuterated proteins. The method significantly increases the information content available for structural interpretation of the conformational exchange process, partly because deltasigma(H)deltasigma(N) is a signed quantity, and partly because two chemical shifts are probed simultaneously. The present results support the hypothesis that the exchange in the calcium-loaded state of the E140Q mutant involves conformations similar to those of the wild-type apo (closed) and calcium-loaded (open) states.

Calmodulin↗
PubMed1996

A proposed common spatial pharmacophore and the corresponding active conformations of some peptide leukotriene receptor antagonists.

Molecular modeling studies were carried out by a combined use of conformational analysis and 3D-QSAR methods of identify molecular features common to a series of hydroxyacetophenone (HAP) and non-hydroxyacetophenone (non-HAP) peptide leukotriene (pLT) receptor antagonists. In attempts to develop a ligand-binding model for the pLT receptor, the Apex-3D program was used to identify biophoric structural patterns that are common to 13 diverse sets of compounds showing different levels of biological activity. A systematic conformational analysis was carried out to obtain sterically accessible conformations for these flexible compounds. Apex-3D was then utilized to propose common biophoric regions based on the selection of one of several conformations (MOPAC-minimized AM1) from each compound's data set that best fits the biophoric pattern and the resulting superimposition with all the other data-set compounds. Apex-3D identified three common biophoric features important for activity: one as the hydroxyl, acetyl, carbonyl and carboxyl groups, which mimic the acid-binding region of an agonist, the other as the hydrogen-bond donating site, and the third part is represented by a plane in which lipophilic aromatic groups align. The structure-activity relationships were then assessed by using the 3D-QSAR model. A common biophore model is proposed from the Apex-3D analysis which may be useful in designing new pLT antagonists. Molecular volumes and electrostatic potential similarities were also calculated in order to obtain the important structural requirements for the activity.

Acetophenones↗
PubMedDate not supplied

[Approach to a priori calculation of stable forms of proteins].

Basing on semi-empirical conformational analysis an approach is suggested to the calculation of stable forms of protein molecules. The physical model proposed implies: 1) that the most stable conformation of a protein corresponds to its native state, 2) that interactions of three types which determine conformational states of every residue should be treated separately, 3) that the protein molecule is highly compact structure with interactions of different kinds compensating each other, 4) that interactions of the three types contribute differently to the formation of the spatial structure of a globule. These interactions are: 1) those of peptide chain atoms within a particular residue, 2) interactions of the residue with the nearest neighbours in the chain, 3) interactions with residues which are remote in the chain but close in the globule. The approach proposed allows to separate the conformational analysis of a protein into a number of subsequently solved problems. The scheme of the calculation of stable forms is presented.

Drug Stability↗
PubMed1992

Detection of heterozygous mutations in the RB1 gene in retinoblastoma patients using single-strand conformation polymorphism analysis and polymerase chain reaction sequencing.

Several families segregating the autosomal dominant form of the hereditary retinoblastoma predisposition gene have been analysed for the causative mutation. We have used the single-strand conformation polymorphism (SSCP) technique to screen for mutations, exon by exon, in the RB1 gene in affected patients from these families. The SSCP technique has proved a rapid and simple technique which relies on the sequence-dependent migration of single-stranded DNA in a non-denaturing polyacrylamide gel. Oligonucleotide primers flanking all 27 exons and the promoter region of the RB1 gene are reported here. The polymerase chain reaction (PCR)-amplified products range in size from 212 to 625 bp and include a flanking intron sequence which allows detection of mutations in these regions. The sensitivity of SSCP is optimal when DNA fragments are approximately 200 bp long. Consequently, restriction enzyme sites for each amplified region were identified, reducing the size of the PCR products analysed to less than 250 bp. Bands with aberrant migration patterns were observed on SSCP gels in the lymphocyte DNA from two patients with bilateral, familial retinoblastoma. Sequence analysis of these DNA fragments revealed the causative mutations. These consisted of a 1-bp insertion of a T in the coding strand of exon 20 and a G----A mutation in the coding strand of exon 14. This approach has proved to be a powerful method for the rapid detection of germline mutations in the RB1 gene, a programme which can be extended to individuals with new mutations.

Base Sequence↗
PubMed1997

Computational studies of sialyllactones: methods and uses.

N-Acetylneuraminic acid (1) is a common sugar in many biological recognition processes. Neuraminidase enzymes recognize and cleave terminal sialic acids from cell surfaces. Viral entry into host cells requires neuraminidase activity, thus inhibition of neuraminidase is a useful strategy for development of drugs for viral infections. A recent crystal structure for influenza viral neuraminidase with sialic acid bound shows that the sialic acid is in a boat conformation [Prot Struct Funct Genet 14: 327 (1992)]. Our studies seek to determine if structural pre-organization can be achieved through the use of sialyllactones. Determination of whether siallylactones are pre-organized in a binding conformation requires conformational analysis. Our inability to find a systematic study comparing the results obtained by various computational methods for carbohydrate modeling led us to compare two different conformational analysis techniques, four different force fields, and three different solvent models. The computational models were compared based on their ability to reproduce experimental coupling constants for sialic acid, sialyl-1,4-lactone, and sialyl-1,7-lactone derivatives. This study has shown that the MM3 forcefield using the implicit solvent model for water implemented in Macromodel best reproduces the experimental coupling constants. The low-energy conformations generated by this combination of computational methods are pre-organized toward conformations which fit well into the active site of neuraminidase.

Computer Simulation↗
PubMed1994

Human SP-A: genotype and single-strand conformation polymorphism analysis.

We have previously characterized two surfactant protein A (SP-A) cDNAs termed 1A and 6A, as well as a 6A allelic variant termed 6A1. These sequences are quite heterogeneous at the 3' untranslated region (3'UT). Differences between 6A and 6A1 alleles include an 11-bp insertion/deletion 407 bases downstream from the start of the translation termination codon and a base pair polymorphism (C or G) in exon 1 (position 1,193; White, Damm, Miller, Spratt, Schilling, Hawgood, Benson, and Cordell. Nature Lond. 317: 361-363, 1985). The 11-bp (GCCCACTGCCT) segment is present in 6A1 and absent in 6A. The 6A/6A genotype, in a small number of specimens, showed a trend toward a higher frequency in the black Nigerian population compared with Caucasians. In this report, we examine the frequency of the 6A genotype in a larger number of samples from Caucasians and black Nigerians as well as the meiotic stability of the 3'UT heterogeneity. Slot-blot analysis and allele-specific oligonucleotide probes have confirmed that the 6A/6A genotype is more frequent in the Nigerian population. Single-strand conformation polymorphisms in the 3'UT appear to be stably inherited by members of a three-generation family, suggesting that these nucleotide variants represent natural polymorphisms in the population.

Base Sequence↗
PubMed1994

Antitumor agents. 152. In vitro inhibitory activity of etoposide derivative NPF against human tumor cell lines and a study of its conformation by X-ray crystallography, molecular modeling, and NMR spectroscopy.

NPF, the title compound, was studied for its in vitro antitumor activity against 56 human tumor cell lines derived from seven cancer types. In general, NPF is about 100 times more active as compared to its parent compound, etoposide, toward all the tumor cell lines and can be considered as a lead structure for further development of anticancer agents. In order to facilitate future computer-assisted design of NPF analogs, NPF was characterized by X-ray crystallography. This crystal structure was used as the starting point for conformational analysis of this compound using several commercially available software packages, including SYBYL (Tripos Associates; Tripos force field), INSIGHT/DISCOVER (Biosym Technologies; CVFF force field), and semiempirical package MOPAC as implemented in SYBYL. The lowest energy conformation generated with the Tripos force field disagreed with the X-ray structure. On the other hand, semiempirical MOPAC/AM1 calculations showed that the X-ray structure had a lower energy than the Tripos lowest energy conformation. Subsequent NMR studies agreed well with the X-ray structure. Furthermore, conformational analysis of NPF using the DISCOVER force field identified the X-ray structure as the lowest energy conformation. Thus, the latter force field is adequate for future molecular modeling of NPF and its analogs.

Antineoplastic Agents↗
PubMed1992

[Intracomplex alkylation of octanucleotide pd(TGTTTGGC) by a 5'-(4-N-methyl-N-(2-chloroethyl)-amino)benzylphosphamide derivative of heptanucleotide pd(CCAAACA). Preparation of a covalent adduct and study of its spatial structure in an aqueous solution by two-dimensional (1)H-NMR spectroscopy].

Covalent adduct--the product of intracomplex alkylation at N-3-position of dC-8-nucleoside residue of target octanucleotide pd[TGTTTGGC] was completely synthesized by means of 4-[N-methyl-N-(2-chloroethyl)amino]benzyl-5'-phosphamido derivative of heptanucleotide pd[CCAAACA]. Its melting temperature was shown to be 70 degrees C. Tm did not depend on covalent adduct concentration and was by 40 degrees C higher than that for unmodified duplex pd[TGTTTGGC].pd[CCAAACA] at concentration of 0.5 x 10(-4) M. The spatial structure of the covalent adduct in aqueous solution was investigated by two-dimensional 3H-NMR spectroscopy. The assignment of oligonucleotide protons as well as protons of a modifying group was carried out using COSY, COSY-DQF and NOESY experiments. Conformational analysis of proton-proton coupling constants for H1', H2'a, H2'b and H3' protons showed the sugar residues to be in 2'-endo conformation. Analysis of NOE connectivities observed between the protons of the alkylating group and oligonucleotide protons yielded conclusion, regarding the 4-[N-methyl-N-(2-chloroethyl)amino]benzylamido 5'-residue being localized in the region of the lacked nucleoside residue of the heptanucleotide chain about 5 A apart from the dC-1 residue and from cytosine base of the alkylated dC-8 residue.

Alkylation↗
PubMed1998

Conformational analyses of somatostatin-related cyclic hexapeptides containing peptoid residues.

We report the conformational analysis by 1H NMR in DMSO and computer simulations involving distance geometry and molecular dynamics simulations of a series of peptoid analogues of the cyclic hexapeptide c[Phe11-Pro6-Phe7-d-Trp8-Lys9-Thr10] (1). The proline residue in compound 1 is replaced with the peptoid residues N-benzylglycine (Nphe) (compound 2), N-(S)-alpha-methylbenzylglycine [(S)-beta-MeNphe] (compound 3), and N-(R)-alpha-methylbenzylglycine [(R)-beta-MeNphe] (compound 4). The peptoid analogues 2 and 4 exhibit potent binding activities to the hsst2 receptor, while the binding affinities to the hsst5 and to the hsst3 receptors are reduced compared to that of the parent compound 1. Compound 3 shows reduced binding activities to the hsst2, hsst3, and hsst5 receptors compared to compound 1. The results of in vivo assays indicate that these compounds inhibit the growth hormone release but do not affect the insulin release. These peptoid-containing analogues show two sets of NMR signals corresponding to cis and trans conformations of the peptide bond between Phe11 and Nxaa6. We demonstrate that the backbone conformation and the orientation of the relevant side chains of compound 1 are maintained in the cis isomers of the peptoid analogues which adopt a type VI beta-turn centered around residues 11 and 6 and a type II' beta-turn with d-Trp in the i+1 position. The enhanced selectivity of the peptoid-containing analogues compared to compound 1 and the results of the conformational analysis suggest that the presence of a conformationally constrained hydrophobic group in position 6 in complementary topology to the Phe11 side chain enhances selective binding to the hsst2 receptor.

Computer Simulation↗