Molecular Modeling Study of the Genotoxicity of the Sudan I and Sudan II Azo Dyes and Their Metabolites.

Azo dyes are defined by the presence of a characteristic N=N group. Sudan I and Sudan II are synthetic azo dyes that have been used as coloring agents. Although animal toxicity studies suggest that Sudan dyes are mutagenic, their molecular mechanism of action is unknown, thus making it challenging to establish thresholds for tolerable daily intake or to understand how these molecules could be modified to ameliorate toxicity.

Data Sharing Advances Rare and Neglected Disease Clinical Research and Treatments.

Because of the decreased cost and increased ease of whole genome analysis, the diagnosis of rare, orphan diseases has entered a new era. This new technological advance, combined with the worldwide web connections, now permits sharing, searching, and linking genotype, phenotype, and other information to facilitate diagnosis. Databases currently accessible and searchable by researchers, clinicians, and patients will be presented and discussed.

Computational methods for fragment-based ligand design: growing and linking.

Fragment-based drug design has proved itself as a powerful technique for increasing the sampling and diversity of chemical space and enabling the design of novel leads and compounds. Computational techniques for identifying fragments, binding sites and particularly for linking, growing, and evolving fragments play a significant role in the process. Information from ADME studies and clustering property information in the form of toxicophores and chemotypes can play a significant role in aiding the design of novel, selective fragments with good activity profiles.

Solvation methods for protein-ligand docking.

Hydration has a significant impact on ligand binding within protein active sites. Specific water molecules and their placement within protein active sites have been shown to make specific contributions to the energetics of protein-ligand binding and need consideration in the design of efficient binding ligands. These specific nonbulk water molecules and their interactions are different and have more significant impact in ligand design than the generalized bulk solvation of ligand-protein systems.

Association of Nrf2 polymorphism haplotypes with acute lung injury phenotypes in inbred strains of mice.

Aims: Nrf2 is a master transcription factor for antioxidant response element (ARE)-mediated cytoprotective gene induction. A protective role for pulmonary Nrf2 was determined in model oxidative disorders, including hyperoxia-induced acute lung injury (ALI). To obtain additional insights into the function and genetic regulation of Nrf2, we assessed functional single nucleotide polymorphisms (SNPs) of Nrf2 in inbred mouse strains and tested whether sequence variation is associated with hyperoxia susceptibility.

Phylogenetic analysis and evolutionary origins of DNA polymerase X-family members.

Mammalian DNA polymerase (pol) β is the founding member of a large group of DNA polymerases now termed the X-family. DNA polymerase β has been kinetically, structurally, and biologically well characterized and can serve as a phylogenetic reference. Accordingly, we have performed a phylogenetic analysis to understand the relationship between pol β and other members of the X-family of DNA polymerases.

Novel mutator mutants of E. coli nrdAB ribonucleotide reductase: insight into allosteric regulation and control of mutation rates.

Ribonucleotide reductase (RNR) is the enzyme critically responsible for the production of the 5'-deoxynucleoside-triphosphates (dNTPs), the direct precursors for DNA synthesis. The dNTP levels are tightly controlled to permit high efficiency and fidelity of DNA synthesis. Much of this control occurs at the level of the RNR by feedback processes, but a detailed understanding of these mechanisms is still lacking.

Computational drug design targeting protein-protein interactions.

Novel discoveries in molecular disease pathways within the cell, combined with increasing information regarding protein binding partners has lead to a new approach in drug discovery. There is interest in designing drugs to modulate protein-protein interactions as opposed to solely targeting the catalytic active site within a single enzyme or protein. There are many challenges in this new approach to drug discovery, particularly since the protein-protein interface has a larger surface area, can comprise a discontinuous epitope, and is more amorphous and less well defined than the typical drug design target, a small contained enzyme-binding pocket.

The H6D variant of NAG-1/GDF15 inhibits prostate xenograft growth in vivo.

Background: Non-steroidal anti-inflammatory drug-activated gene (NAG-1), a divergent member of the transforming growth factor-beta superfamily, has been implicated in many cellular processes, including inflammation, early bone formation, apoptosis, and tumorigenesis. Recent clinical studies suggests that a C to G single nucleotide polymorphism at position 6 (histidine to aspartic acid substitution, or H6D) of the NAG-1 protein is associated with lower human prostate cancer incidence. The objective of the current study is to investigate the activity of NAG-1 H6D variant in prostate cancer tumorigenesis in vivo.

Characterization of a discontinuous epitope of the HIV envelope protein gp120 recognized by a human monoclonal antibody using chemical modification and mass spectrometric analysis.

A subset of the neutralizing anti-HIV antibodies recognize epitopes on the envelope protein gp120 of the human immunodeficiency virus. These epitopes are exposed during conformational changes when gp120 binds to its primary receptor CD4. Based on chemical modification of lysine and arginine residues followed by mass spectrometric analysis, we determined the epitope on gp120 recognized by the human monoclonal antibody 559/64-D, which was previously found to be specific for the CD4 binding domain.

Progressive external ophthalmoplegia and vision and hearing loss in a patient with mutations in POLG2 and OPA1.

Objective: To describe the clinical features, muscle pathological characteristics, and molecular studies of a patient with a mutation in the gene encoding the accessory subunit (p55) of polymerase gamma (POLG2) and a mutation in the OPA1 gene.

Design: Clinical examination and morphological, biochemical, and molecular analyses.

Setting: Tertiary care university hospitals and molecular genetics and scientific computing laboratory.

The DNA polymerase gamma Y955C disease variant associated with PEO and parkinsonism mediates the incorporation and translesion synthesis opposite 7,8-dihydro-8-oxo-2'-deoxyguanosine.

Mitochondrial DNA is replicated and repaired by DNA polymerase gamma (pol gamma), encoded by the POLG gene. The Y955C substitution in POLG leads to autosomal dominant progressive external ophthalmoplegia (PEO) with other severe phenotypes. PEO patients with this mutation can further develop parkinsonism or premature ovarian failure.

Structural determinates for apolipoprotein E-derived peptide interaction with the alpha7 nicotinic acetylcholine receptor.

Neuronal nicotinic acetylcholine receptor (nAChR) signaling has been implicated in a variety of normal central nervous system (CNS) functions as well as an array of neuropathologies. Previous studies have demonstrated both neurotoxic and neuroprotective actions of peptides derived from apolipoprotein E (apoE). It has been discovered that apoE-derived peptides inhibit native and recombinant alpha7-containing nAChRs, indicating a direct interaction between apoE peptides and nAChRs.

Human glucocorticoid receptor beta binds RU-486 and is transcriptionally active.

Human glucocorticoid receptor (hGR) is expressed as two alternately spliced C-terminal isoforms, alpha and beta. In contrast to the canonical hGRalpha, hGRbeta is a nucleus-localized orphan receptor thought not to bind ligand and not to affect gene transcription other than by acting as a dominant negative to hGRalpha. Here we used confocal microscopy to examine the cellular localization of transiently expressed fluorescent protein-tagged hGRbeta in COS-1 and U-2 OS cells.

Functional analysis of the LXXLL motifs of the human glucocorticoid receptor: association with altered ligand affinity.

Although LXXLL motifs in coactivators mediate binding to liganded nuclear receptors, the roles of comparable motifs within nuclear receptors are less understood. We investigated the role of the LXXLL motifs in the human glucocorticoid receptor both in transcriptional activation and repression of gene expression. The two conserved LXXLL motifs within the ligand binding domain of the receptor, amino acids 532-536 (helix 1) and 718-722 (helix 10), were characterized by evaluating LXXLL mutant receptors as well as comparable mutants in other helices of the ligand binding domain.

The C-terminal zinc finger of UvrA does not bind DNA directly but regulates damage-specific DNA binding.

In prokaryotic nucleotide excision repair, UvrA recognizes DNA perturbations and recruits UvrB for the recognition and processing steps in the reaction. One of the most remarkable aspects of UvrA is that it can recognize a wide range of DNA lesions that differ in chemistry and structure. However, how UvrA interacts with DNA is unknown.

Antiretroviral nucleosides, deoxynucleotide carrier and mitochondrial DNA: evidence supporting the DNA pol gamma hypothesis.

Design: Nucleoside reverse transcriptase inhibitors (NRTIs) exhibit mitochondrial toxicity. The mitochondrial deoxynucleotide carrier (DNC) transports nucleotide precursors (or phosphorylated NRTIs) into mitochondria for mitochondrial (mt)DNA replication or inhibition of mtDNA replication by NRTIs. Transgenic mice (TG) expressing human DNC targeted to murine myocardium served to define mitochondrial events from NRTIs in vivo and findings were corroborated by biochemical events in vitro.

Molecular insights into NRTI inhibition and mitochondrial toxicity revealed from a structural model of the human mitochondrial DNA polymerase.

NRTI-based therapy used to treat AIDS can cause mitochondrial toxicity resulting from the incorporation of NRTIs into mitochondrial DNA by DNA polymerase gamma (pol gamma). Pol gamma has poor discrimination against many of the currently used NRTIs resulting in aborted DNA synthesis and subsequent depletion of mtDNA. Pol gamma readily incorporates ddCTP, ddITP and D4T-TP with an efficiency similar to the incorporation of normal nucleotides, whereas AZT-TP, CBV-TP, 3TC-TP and PMPApp act as moderate inhibitors to DNA synthesis.

Consequences of mutations in human DNA polymerase gamma.

DNA polymerase gamma is responsible for replication and repair of the mitochondrial genome. Human DNA polymerase gamma is composed of a 140-kDa catalytic subunit and a 55-kDa accessory subunit. Mutations in the gene for the catalytic subunit (POLG) have been shown to be a frequent cause of mitochondrial disorders.

Microglial NADPH oxidase is a novel target for femtomolar neuroprotection against oxidative stress.

Inflammation has been increasingly recognized to contribute to the pathogenesis of Parkinson's disease. Several compounds are neuroprotective at femtomolar concentrations through the inhibition of inflammation. However, the mechanisms mediating femtomolar-acting compounds are poorly understood.

Structure-function defects of human mitochondrial DNA polymerase in autosomal dominant progressive external ophthalmoplegia.

Progressive external ophthalmoplegia (PEO) is a mitochondrial disorder associated with mutations in the POLG gene encoding the mitochondrial DNA polymerase (pol gamma). Four autosomal dominant mutations that cause PEO encode the amino acid substitutions G923D, R943H, Y955C and A957S in the polymerase domain of pol gamma. A homology model of the pol gamma catalytic domain in complex with DNA was developed to investigate the effects of these mutations.

Basal transcription defect discriminates between xeroderma pigmentosum and trichothiodystrophy in XPD patients.

Mutations in the XPD gene result in xeroderma pigmentosum (XP) and trichothiodystrophy (TTD), the phenotypes of which are often intricate. To understand the genotype/phenotype relationship, we engineered recombinant TFIIHs in which XPD subunits carry amino acid changes found in XPD patients. We demonstrate that all the XPD mutations are detrimental for XPD helicase activity, thus explaining the NER defect.

Molecular origins for the dominant negative function of human glucocorticoid receptor beta.

This study molecularly elucidates the basis for the dominant negative mechanism of the glucocorticoid receptor (GR) isoform hGRbeta, whose overexpression is associated with human glucocorticoid resistance. Using a series of truncated hGRalpha mutants and sequential mutagenesis to generate a series of hGRalpha/beta hybrids, we find that the absence of helix 12 is neither necessary nor sufficient for the GR dominant negative phenotype. Moreover, we have localized the dominant negative activity of hGRbeta to two residues and found that nuclear localization, in addition to heterodimerization, is a critical feature of the dominant negative activity.

Structural and functional characterization of the human DNA repair helicase XPD by comparative molecular modeling and site-directed mutagenesis of the bacterial repair protein UvrB.

A molecular model for the human nucleotide excision repair protein, XPD, was developed based on the structural and functional relationship of the protein with a bacterial nucleotide excision repair (NER) protein, UvrB. Whereas XPD does not share significant sequence identity with UvrB, the proteins share seven highly conserved helicase motifs that define a common protein structural template. They also have similar functional roles in their ATPase activity and the ability to unwind DNA and verify damaged strands in the process of NER.