Biomedical subjects

R Lavery

Publications and source records attributed to R Lavery.

At least 19 recordsLinked to original sources

PubMed2006

ARTIST: an activated method in internal coordinate space for sampling protein energy landscapes.

We present the first applications of an activated method in internal coordinate space for sampling all-atom protein conformations, the activation-relaxation technique for internal coordinate space trajectories (ARTIST). This method differs from all previous internal coordinate-based studies aimed at folding or refining protein structures in that conformational changes result from identifying and crossing well-defined saddle points connecting energy minima. Our simulations of four model proteins containing between 4 and 47 amino acids indicate that this method is efficient for exploring conformational space in both sparsely and densely packed environments, and offers new perspectives for applications ranging from computer-aided drug design to supramolecular assembly.

Alanine↗

PubMed2005

Non-additivity in protein-DNA binding.

MOTIVATION: Localizing protein binding sites within genomic DNA is of considerable importance, but remains difficult for protein families, such as transcription factors, which have loosely defined target sequences. It is generally assumed that protein affinity for DNA involves additive contributions from successive nucleotide pairs within the target sequence. This is not necessarily true, and non-additive effects have already been experimentally demonstrated in a small number of cases. The principal origin of non-additivity involves the so-called indirect component of protein-DNA recognition which is related to the sequence dependence of DNA deformation induced during complex formation. Non-additive effects are difficult to study because they require the identification of many more binding sequences than are normally necessary for describing additive specificity (typically via the construction of weight matrices). RESULTS: In the present work we will use theoretically estimated binding energies as a basis for overcoming this problem. Our approach enables us to study the full combinatorial set of sequences for a variety of DNA-binding proteins, make a detailed analysis of non-additive effects and exploit this information to improve binding site predictions using either weight matrices or support vector machines. The results underline the fact that, even in the presence of significant deformation, non-additive effects may involve only a limited number of dinucleotide steps. This information helps to reduce the number of binding sites which need to be identified for successful predictions and to avoid problems of over-fitting. AVAILABILITY: The SVM software is available upon request from the authors.

Algorithms↗

PubMed2005

CAG repeat length in an infertile male population of Irish origin.

The androgen receptor (AR) gene, located on the X chromosome, is an important regulator of human spermatogenesis. In the past decade, the link between the CAG polyglutamine tract, situated on exon one of the AR gene, and reduced spermatogenesis has become a controversial one. Alterations in the length of the CAG polyglutamine tract have been associated with prostate cancer at a reduced intrinsic length and neuromuscular diseases at a CAG repeat length of > or = 40. Minimal intermediate increases have been linked with depressed spermatogenesis in infertile males. Asian and Australian groups have published an association between increased CAG repeat length and reduced spermatogenesis while many European studies have found no such association. The aim of this study was to document the association between increased CAG repeat length and reduced spermatogenesis in a group of Irish infertile males and controls known to have fathered at least one child. The study employed the ABI 377 DNA sequencer to size the CAG repeat region of exon one of the AR gene in each group. Statistical analysis revealed no actual link between the length of the CAG tract and a reduction of spermatogenesis in a cohort of infertile patients (n = 66) of Irish ethnic origin when compared to a fertile control group (n = 77) (p = 0.599).

Electrophoresis, Polyacrylamide Gel↗

PubMed2002

A preliminary report on the implication of RT-PCR detection of DAZ, RBMY1, USP9Y and Protamine-2 mRNA in testicular biopsy samples from azoospermic men.

In this study, reverse transcription-polymerase chain reaction (RT-PCR) was optimized to analyse the presence of DAZ, RBMY1, USP9Y, protamine-2, SRY and actin messenger RNA (mRNA) in testicular cells of men suffering from idiopathic azoospermia. All samples (n=28), including five controls, showed normal expression of actin, SRY and USP9Y. Sperm was not recovered from eight patients after testicular biopsy. Of these, four patients showed altered mRNA levels for the fertility genes, DAZ, RBMY1 and protamine-2. One patient, who was previously shown to be azoospermia factor region (AZF)b deleted, lacked RBM mRNA and presented with reduced amplification of protamine-2 mRNA. This correlated with previous studies, which proposed that RBM expression is exclusive to AZFb and that the lack of testicular RBMY1 mRNA results in suppressed spermatogenesis. Two patients were each lacking DAZ mRNA but did show expression of RBMY1 mRNA at a reduced level, suggesting that there might be residual spermatogenesis in the absence of DAZ expression. Protamine-2 mRNA was detected in one patient and was absent in the second patient. Finally, one patient lacked DAZ, RBMY1 and protamine-2 mRNA. The 19 remaining azoospermic patients presented with normal expression patterns for each of the fertility genes studied. This study demonstrates that the expression of spermatogenesis-specific genes varies in azoospermia. The study of the expression of such genes in a larger number of patients might be useful in characterizing and identifying subpopulations of azoospermic men.

Biopsy↗

PubMed2001

Magnesium sulfate in the treatment of refractory ventricular fibrillation in the prehospital setting.

OBJECTIVE: To determine if magnesium sulfate (MgSO(4)) improves outcome in cardiac arrest patients initially in ventricular fibrillation (VF). METHODS: Randomized, prospective, double blind, placebo-controlled, multicenter prehospital trial using 2 g of MgSO(4). Eligible patients were non-traumatic cardiac arrest patients (> or =18 years of age) presenting in VF. The protocol included those patients refractory to three electroshocks. Epinephrine and either 2 g of MgSO(4) or placebo (normal saline) were then administered. The primary outcome variable was return of spontaneous circulation (ROSC) in the field and a perfusing pulse on arrival at the ED. Secondary endpoints included admission to the hospital (ADMT) and hospital discharge (DISC). IRB approval was obtained at all participating centers. RESULTS: Total 116 patients (58 MgSO(4), 58 placebo) were enrolled during the period from 4/1992 to 10/96 with 109 available. There were no significant differences between the groups in baseline characteristics and times to cardio pulmonary resuscitation (CPR), advanced life support (ALS), and first defibrillation, except for time to study drug administration. There was no significant differences in ROSC (placebo, 18.5%, and MgSO(4), 25.5%, P=0.38), ADMT (placebo rate=16.7%, MgSO(4)=16.4%, P=1.0) or DISC (placebo rate=3.7%, MgSO(4)=3.6%, P=1.0). CONCLUSIONS: We failed to demonstrate that the administration of 2 g of MgSO(4) to prehospital cardiac arrest patients presenting in VF improves short or long term survival.

Adolescent↗

PubMed2001

Modeling multi-component protein-DNA complexes: the role of bending and dimerization in the complex of p53 dimers with DNA.

We used molecular modeling to study the optimal conformation of the complex between two p53 DNA-binding domain monomers and a 12 base-pair target DNA sequence. The complex was constructed using experimental data on the monomer binding conformation and a new approach to deform the target DNA sequence. Combined with an internal/helicoidal coordinate model of DNA, this approach enables us to bend the target sequence in a controlled way while respecting the contacts formed with each p53 monomer. The results show that the dimeric complex favors DNA bending towards the major groove at the dimer junction by a value close to experimental findings. In contrast to inferences from earlier models, the calculation of key contributions to the free energy of the complexes indicates a determinant role for DNA in the formation of the complex with the dimer of the p53 DNA-binding domains.

Binding Sites↗

PubMed2001

High-speed molecular mechanics searches for optimal DNA interaction sites.

We have recently developed a theoretical means of studying the mechanical and interaction properties of nucleic acids as a function of their base sequence. This approach, termed ADAPT, can be used to obtain the physical properties of millions of base sequences with only modest computational expense. ADAPT is based on a multi-copy algorithm using special nucleotides ("lexides") containing all four standard bases whose contribution to the energy of the molecule can be varied. We present here a deeper study of the energy minima which occur in the multi-dimensional space defined by these variable sequences. We also present an extension of the approach termed "gene threading" which enables us to scan genomic sequence data in an attempt to locate preferential binding sites. This technique is illustrated for the case of TATA-box protein binding. ADAPT enables us to demonstrate that, for this protein, DNA deformation alone explains a large part of the experimentally observed consensus binding sequence.

Algorithms↗

PubMed2000

Optimization of nucleic acid sequences.

Base sequence influences the structure, mechanics, dynamics, and interactions of nucleic acids. However, studying all possible sequences for a given fragment leads to a number of base combinations that increases exponentially with length. We present here a novel methodology based on a multi-copy approach enabling us to determine which base sequence favors a given structural change or interaction via a single energy minimization. This methodology, termed ADAPT, has been implemented starting from the JUMNA molecular mechanics program by adding special nucleotides, "lexides," containing all four bases, whose contribution to the energy of the system is weighted by continuously variable coefficients. We illustrate the application of this approach in the case of double-stranded DNA by determining the optimal sequences satisfying structural (B-Z transition), mechanical (intrinsic curvature), and interaction (ligand-binding) properties.

Base Composition↗

PubMed2000

The impact of abasic sites on DNA flexibility.

We use internal coordinate molecular mechanics calculations to study the impact of abasic sites on the conformation and the mechanics of the DNA double helix. Abasic sites, which are common mutagenic lesions, are shown to locally modify both the groove geometry and the curvature of DNA in a sequence dependent manner. By controlled twisting and bending, it is also shown that these lesions modify the deformability of the duplex, generally increasing its flexibility, but again to an extent which depends on the nature of the abasic site and on the surrounding base sequence. Both the conformational and mechanical influence of this type of DNA damage may be significant for recognition and repair mechanisms.

Computer Simulation↗

PubMed1999

Modeling DNA deformations induced by minor groove binding proteins.

Molecular modeling is used to demonstrate that the major structural deformations of DNA caused by four different minor groove binding proteins, TBP, SRY, LEF-1, and PurR, can all be mimicked by stretching the double helix between two 3'-phosphate groups flanking the binding region. This deformation reproduces the widening of the minor groove and the overall bending and unwinding of DNA caused by protein binding. It also reproduces the principal kinks associated with partially intercalated amino acid side chains, observed with such interactions. In addition, when protein binding involves a local transition to an A-like conformation, phosphate neutralization, via the formation of protein-DNA salt bridges, appears to favor the resulting deformation.

Base Sequence↗

PubMed1999

Role of granulocyte-macrophage colony-stimulating factor and its receptor in the genesis of acute respiratory distress syndrome through an effect on neutrophil apoptosis.

HYPOTHESIS: That granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor modulate the suppression of apoptosis (Ao) of normal neutrophils incubated in the plasma of patients with postraumatic acute respiratory distress syndrome (ARDS). DESIGN: Experimental study using cultured human neutrophils. SETTING: University hospital, level I trauma center. PARTICIPANTS: Plasma was obtained from 14 patients with early, fulminant posttraumatic ARDS (mean Injury Severity Score, 22). All samples were drawn within 24 hours after injury. Plasma was also taken from up to 21 healthy control subjects. These volunteers were also used as sources of polymorphonuclear leukocytes (PMNs). MAIN OUTCOME MEASURES: (1) Effect of early, fulminant ARDS and normal plasma on spontaneous Ao and GM-CSF receptor expression in PMNs in vitro. (2) Effect of ligation of either GM-CSF or its receptor with a neutralizing monoclonal antibody (mAb) on PMN Ao in ARDS and normal plasma. (3) Correlation of extracellular GM-CSF concentration with rate of PMN Ao. (4) Levels of GM-CSF in ARDS and normal plasma and in culture supernatant of normal PMNs incubated in early, fulminant ARDS and normal plasma. RESULTS: Plasma from patients with ARDS enhanced PMN viability at 24 hours (data are given as mean +/- SEM) 52% +/- 3% control vs 60% +/- 3% ARDS, P<.05). Binding of the GM-CSF receptor with a neutralizing mAb significantly reduced PMN viability in ARDS plasma, but not in normal plasma (60% +/- 3% ARDS vs 53% +/- 3% ARDS + mAb, P<.05). Ligation of GM-CSF with mAb had no significant effect on PMN viability in either plasma. Only 1% of PMNs expressed detectable levels of the GM-CSF receptor when incubated for 24 hours in either ARDS or normal plasma. The GM-CSF levels were undetectable (>7 pg/mL) in both ARDS and normal plasma and in culture supernatants taken after 24 hours of incubation in both plasma types. Levels of GM-CSF ranging from 0 to 50000 pg/mL had no effect on PMN Ao in plasma-free medium. CONCLUSIONS: The antiapoptotic effect of ARDS plasma appears to be mediated by the GM-CSF receptor. This effect occurs at both low levels of plasma GM-CSF and surface expression of its PMN receptor. Ligation of GM-CSF had no effect of PMN Ao, suggesting that Ao is triggered by Fc portion-mediated receptor cross-linking. These results provide the theoretical basis for alphaGM-CSF receptor mAb therapy as a novel modality of treatment for ARDS.

Acute Disease↗

PubMed1999

A molecular model for RecA-promoted strand exchange via parallel triple-stranded helices.

A number of studies have concluded that strand exchange between a RecA-complexed DNA single strand and a homologous DNA duplex occurs via a single-strand invasion of the minor groove of the duplex. Using molecular modeling, we have previously demonstrated the possibility of forming a parallel triple helix in which the single strand interacts with the intact duplex in the minor groove, via novel base interactions (Bertucat et al., J. Biomol. Struct. Dynam. 16:535-546). This triplex is stabilized by the stretching and unwinding imposed by RecA. In the present study, we show that the bases within this triplex are appropriately placed to undergo strand exchange. Strand exchange is found to be exothermic and to result in a triple helix in which the new single strand occupies the major groove. This structure, which can be equated to so-called R-form DNA, can be further stabilized by compression and rewinding. We are consequently able to propose a detailed, atomic-scale model of RecA-promoted strand exchange. This model, which is supported by a variety of experimental data, suggests that the role of RecA is principally to prepare the single strand for its future interactions, to guide a minor groove attack on duplex DNA, and to stabilize the resulting, stretched triplex, which intrinsically favors strand exchange. We also discuss how this mechanism can incorporate homologous recognition.

Base Pairing↗

PubMed1999

From atomic to mesoscopic descriptions of the internal dynamics of DNA.

An analysis of four 1-ns molecular dynamics trajectories for two different 15-bp oligonucleotides is presented. Our aim is to show which groups of atoms can be treated as rigid bodies within a bead representation of DNA, independently of the base sequence and for any conformations belonging to the A/B family. Five models with moderate intragroup deformations are proposed in which the groups are formed of atoms belonging to a single nucleotide or to a complementary nucleotide pair. The influence of group deformation in two of these models is studied using canonical correlation analysis, and it is shown that the internal DNA dynamics is indeed dominated by the rigid motion of the defined atom groups. Finally, using one of the models within a bead representation of duplex DNA makes it possible to obtain stretching, torsional, and bending rigidities in reasonable agreement with experiment but points to strongly correlated stretching motions.

Base Sequence↗

PubMed1999

Abasic sites in duplex DNA: molecular modeling of sequence-dependent effects on conformation.

Molecular modeling calculations using JUnction Minimization of Nucleic Acids (JUMNA) have been used to study sequence effects on the conformation of abasic sites within duplex DNA. We have considered lesions leading to all possible unpaired bases (X), adenine, guanine, cytosine, or thymine contained within two distinct sequence contexts, CXC and GXG. Calculations were carried out on DNA 11-mers using extensive conformational search techniques to locate the most stable abasic conformations and using Poisson-Boltzmann corrected electrostatics to account for solvation effects. The results, which are in very good agreement with available experimental data, point to strong sequence effects on both the position of the unpaired base (intra or extrahelical) and on the overall curvature induced by the abasic lesion. For CXC, unpaired purines are found to lie within the helix, while unpaired pyrimidines are either extrahelical or in equilibrium between the intra and extrahelical forms. For GXG, all unpaired bases lead to intrahelical forms, but with marked, sequence-dependent differences in induced curvature.

Base Pairing↗

PubMed1999

Unraveling proteins: a molecular mechanics study.

An internal coordinate molecular mechanics study of unfolding peptide chains by external stretching has been carried out to predict the type of force spectra that may be expected from single-molecule manipulation experiments currently being prepared. Rather than modeling the stretching of a given protein, we have looked at the behavior of simple secondary structure elements (alpha-helix, beta-ribbon, and interacting alpha-helices) to estimate the magnitude of the forces involved in their unfolding or separation and the dependence of these forces on the way pulling is carried out as well as on the length of the structural elements. The results point to a hierarchy of forces covering a surprisingly large range and to important orientational effects in the response to external stress.

Biophysical Phenomena↗

PubMed1999

Collective variable modelling of nucleic acids.

Collective variable models continue to contribute to our knowledge of nucleic acids. The past year has seen considerable progress both in modelling sequence-dependent effects on nucleic acid conformation and in understanding how proteins or external stresses influence nucleic acid structure. Algorithmic developments have also allowed collective models to be applied to studies of thermal fluctuations and dynamics. For larger systems, models with varying degrees of resolution are being refined and applied to nucleic acids containing hundreds or thousands of nucleotides.

Algorithms↗

PubMed1999

Modelling DNA stretching for physics and biology.

We have used internal coordinate molecular mechanics calculations to study how the DNA double helix deforms upon stretching. Results obtained for polymeric DNA under helical symmetry constraints suggest that two distinct forms, an unwound ribbon and a narrow fibre, can be formed as a function of which ends of the duplex are pulled. Similar results are also obtained with DNA oligomers. These experiments lead to force curves which exhibit a plateau as the conformational transition occurs. This behaviour is confirmed by applying an increasing force to DNA and observing a sudden length increase at a critical force value. It is finally shown some DNA binding proteins can also stretch DNA locally, to conformations related to those created by nanomanipulation.

Bacterial Proteins↗

PubMed1999

Experimental and theoretical studies of the conformational perturbations induced by an abasic site.

The three-dimensional structure of the natural undecamer duplex d(CGCACACACGC). d(GCGTGTGTGCG) has been determined by the combined use of NMR spectroscopy and restrained molecular dynamics (rMD) and also by molecular mechanics calculations using the JUMNA program without experimental distance constraints. Both procedures have also been used to model the abasic structure d(CGCACOCACGC).d(GCGTGTGTGCG), where 'O' indicates a modified abasic site: 3-hydroxy-2-(hydroxymethyl) tetrahydrofuran. For the natural duplex, 134 interproton distances have been obtained by complete relaxation matrix analysis of the NOESY cross-peaks intensities, using MARDIGRAS software. These distances along with 100 torsion angles for sugar ring and additional data derived from canonical A and B-DNA, have been used for structures refinement by restrained molecular dynamics. Comparison of the natural oligomer with the abasic structure obtained earlier by NMR/rMD (Y. Coppel, N. Berthet, C. Coulombeau, Ce. Coulombeau, J. Garcia and J. Lhomme, Biochemistry 36, 4817-4830, 1997) confirms that the creation of an abasic site, in this sequence context, leads to marked helix kinking. It is also shown that the JUMNA procedure is capable of reproducing the overall structural features of the natural and damaged DNA conformations without the use of experimental constraints.

Base Sequence↗