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Biomedical subjects

David G Gorenstein

Publications and source records attributed to David G Gorenstein.

At least 19 recordsLinked to original sources
PubMed2006

Identification of proteins bound to a thioaptamer probe on a proteomics array.

A rapid method to screen and identify unknown bound proteins to specific nucleic acid probes anchored on ProteinChip array surfaces from crude biological samples has been developed in this paper. It was demonstrated with screening specific binding proteins from LPS-stimulated mouse 70Z/3 pre-B cell nuclear extracts by direct coupling of thioaptamer XBY-S2 to the pre-activated ProteinChip array surfaces. With pre-fractionation of crude nuclear extracts by ion exchange method, specific "on-chip" captured proteins have been obtained that were pure enough to do "on-chip" digestion and the subsequent identification of the "on-chip" bound proteins by microsequencing of the trypsin digested peptide fragments through tandem MS. Five mouse heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2/B1, A3, A/B, and D0 were identified. To verify those bound hnRNPs, a novel thioaptamer/antibody sandwich assay provides highly sensitive and selective identification of proteins on ProteinChip arrays.

Animals↗
PubMed2006

Structure of the envelope protein domain III of Omsk hemorrhagic fever virus.

We have solved the NMR solution structure of domain III from the Omsk hemorrhagic fever virus envelope protein and report the first sequencing of the Guriev strain of this virus. Important structural differences between tick-borne flaviviruses, such as OHFV and TBE, and mosquito-borne flaviviruses, such as West Nile virus, are discussed.

Amino Acid Sequence↗
PubMed2006

Delivery of double-stranded DNA thioaptamers into HIV-1 infected cells for antiviral activity.

Oligonucleotide agents (ODN) are emerging as attractive alternatives to chemical drugs. However, the clinical use of ODNs as therapeutics has been hindered by their susceptibility to degradation by cellular enzymes and their limited ability to penetrate intact cells. We have used various liposome-mediated transfection agents, for the in vitro delivery of DNA thioaptamers into U373-MAGI-CCR5 cells. Our lead thioaptamer, R12-2, targets the RNase H domain of the HIV-1 reverse transcriptase (RT) and inhibits viral infection in U373-MAGI-CCR5 cells. R12-2, a 62-base-pair, double-stranded DNA molecule with a monothio-phosphate modified backbone, was selected through a novel combinatorial selection method. We studied the use of oligofectamine (OF), TFX-20, Transmessenger (TM), and Gene Jammer (GJ) for transfection of the thio-modified DNA aptamers. OF-transfected U373-MAGI-CCR5 cells resulted in 68% inhibition of HIV infection in the treated cells compared to the untreated control. Inhibition was observed in a dose-dependent manner with maximal inhibition of 83%. In this report, we demonstrate that monothioate-modified DNA duplex oligonucleotides can be efficiently delivered into cells by liposome-based transfection agents to inhibit HIV replication.

Antiviral Agents↗
PubMed2006

Ab initio base-pairing energies of uracil and 5-hydroxyuracil with standard DNA bases at the BSSE-free DFT and MP2 theory levels.

Oxidized cytosine product 5-hydroxyuracil has been shown to be the major chemical precursor for the GC to AT transition, the most frequent substitution mutation observed in aerobic organisms. We have calculated the interaction energy of base-pair formation involving uracil or 5-hydroxyuracil, which is formed in cells by oxidative deamination of cytosine, bound to any of the natural DNA bases, A, C, G, and T, and discuss the effects of the hydroxyl group in this respect. The base-pair geometries and energies were calculated using the 6-311G(dp) basis set under four conditions: using density functional theory (DFT) without out basis set super-position error (BSSE) correction, using DFT with BSSE correction of geometries and energies, using Møller-Plesset second order perturbation theory (MP2) without BSSE correction, and using MP2 with BSSE geometry and energy correction. We find that the hydroxyl group of 5-HO-U (relative to U) has little effect on the base-pairs with A, C or one conformation of T, while making a substantial energy difference in base-pairs involving G or a different conformation of T. For most of the complexes studied, the BSSE-corrected energies at the DFT and MP2 levels of theory agreed to within 0.5 kcal.

Adenine↗
PubMed2006

Differential signaling networks induced by mild and lethal hemorrhagic fever virus infections.

The family Arenaviridae includes several National Institutes of Allergy and Infections Diseases category A select agents which cause hemorrhagic fever. There are few vaccines available, and treatment is limited to ribavirin, which varies in efficacy. Development of new antiviral compounds has been hindered by a lack of understanding of the molecular basis of pathogenesis. We used two variants of Pichinde virus, one attenuated and one virulent in the guinea pig model, to delineate the host determinants which lead to either viral clearance or lethal disease. By analyzing protein level changes using pathway analysis, we have identified key intermediates which may be targets for therapeutic intervention.

Animals↗
PubMed2005

Quantitative analysis of chemiluminescence signals using a cooled charge-coupled device camera.

Chemiluminescence has been considered an alternative to radioisotopic detection of materials in the life sciences. One application of this nonradioisotopic method is the electrophoretic mobility shift assay. The essential requirement for quantitative applications is that the chemiluminescence signal is linearly proportional to the concentration. However, the generation of chemiluminescence is a multi-step process, therefore linearity cannot be assumed. Therefore, it is important to verify linearity before applying the method as a quantitative tool. We used a commercial chemiluminescence generating system to evaluate the validity of quantitative measurements of biotin-labeled tRNA and single-stranded DNA. The results indicate that the relationship between the chemiluminescence signals and the quantity of biotin-labeled nucleic acids is hyperbolic rather than linear. However, it was found that with less than 50 fmol of biotin-labeled nucleic acid, which corresponds to 2.5 nM in 20 microl, linearity can be demonstrated within 5% error. Therefore, chemiluminescence-based quantitative measurements are a reliable method within these limitations.

Biological Assay↗
PubMed2005

Chemiluminescence-based electrophoretic mobility shift assay of RNA-protein interactions: application to binding of viral capsid proteins to RNA.

A chemiluminescence electrophoretic mobility shift assay was introduced for the study of RNA-protein interactions that include association of genomic RNA and viral capsid proteins. Binding of the capsid protein of Venezuelan equine encephalitis virus (VEEV) to several types of RNA was used as a model system to test the application of the method. The effects of RNA secondary structures and the significance of electrostatic interaction on binding were identified. This method may have wide application to the study of RNA-protein interactions.

Capsid Proteins↗
PubMed2005

The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers.

A PCR method for uniform amplification of a random sequence DNA library is described. A combination of 1 M betaine and 5% DMSO improves the PCR amplification by increasing the ratio of full-length products to shortened products, which are a consequence of nonuniform amplification due to stable secondary structures in the templates. This method is expected to be beneficial for obtaining high-affinity aptamers with stable secondary structures.

Betaine↗
PubMed2005

Combinatorial selection, inhibition, and antiviral activity of DNA thioaptamers targeting the RNase H domain of HIV-1 reverse transcriptase.

Despite the key role played by the RNase H of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5'-end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from Escherichia coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (K(d)) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 microg/mL) markedly inhibited viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 to 2.0 microg/mL (IC(50) < 100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from a multiplicity of infection (moi) of 0.0005 to 0.05, with a reduction of the level of virus production by more than 50% at high moi. Suppression of virus was comparable to that seen with AZT when moi <or= 0.005.

Anti-HIV Agents↗
PubMed2005

The effects of thiophosphate substitutions on native siRNA gene silencing.

RNA mediated interference has emerged as a powerful tool in controlling gene expression in mammalian cells. We investigated the gene silencing properties of six thiophosphate substituted siRNAs (all based on a commercial luciferase medium silencer) compared to that of unmodified siRNA. We also examined the cytotoxicity and dose-response using several thiophosphate modified siRNAs with unmodified siRNA. Our results show that two thiophosphate siRNA sequences convert from medium to high silencers with the addition of four randomly placed thiophosphates. Both thiophosphate siRNAs have a statistically significant difference in luciferase gene silencing (5% and 6% activity) relative to the unmodified native medium silencer referred to as siRNA-2 (18% activity) and four other thiophosphate siRNAs that maintain their medium silencing capability. This indicates that specific thiophosphate substitutions may alter native siRNA function. Further, this shows that thiophosphate siRNAs with the same nucleotide sequence but with different sulfur modification positions have different silencing effects. Both the native siRNA and the thio siRNAs showed a concentration dependent relationship, i.e., with concentration increase, the luciferase gene silencing effect also increased. Confirming cytotoxicity experiments showed no significant changes when HeLa cells were treated with 10nM thiophosphate siRNAs over the course of several days. These results suggest that specific placement of thiophosphates could play an important role in the development of siRNAs as therapeutics by engineering in properties such as strength of binding, nuclease sensitivity, and ultimately efficacy.

Gene Expression Regulation↗
PubMed2004

5-hydroxyuracil can form stable base pairs with all four bases in a DNA duplex.

NMR studies, UV-monitored melting experiments, and ab initio calculations show that 5-hydroxyuracil, produced by the oxidative de-amination of cytosines by reactive oxygen species, can form stable base-pairs with dA, dG, dC and dT residues in a DNA duplex, providing a basis for the in-vivo incorporation of 5-hydroxyuracil during DNA replication.

Base Pairing↗
PubMed2004

TATA box binding protein induces structure in the recombinant glucocorticoid receptor AF1 domain.

A number of transcription factor proteins contain domains that are fully or partially unstructured. The means by which such proteins acquire naturally folded conformations are not well understood. When they encounter their proper binding partner(s), several of these proteins adopt a folded conformation through an induced-fit mechanism. The glucocorticoid receptor (GR) is a ligand-activated transcription factor. Expressed independently as a recombinant peptide, the N-terminal transactivation domain (AF1) of the GR shows little structure and appears to exist as a collection of random coil configurations. The GR AF1 is known to interact with other transcription factors, including a critical component of the general transcription machinery proteins, the TATA box binding protein (TBP). We tested whether this interaction can lead to acquisition of structure in the GR AF1. Our results show that recombinant GR AF1 acquires a significant amount of helical content when it interacts with TBP. These structural changes were monitored by Fourier transform infrared and NMR spectroscopies, and by proteolytic digestions. Our results support a model in which TBP binding interaction with the GR AF1 induces significantly greater helical structure in the AF1 domain. This increased helical content in the GR AF1 appears to come mostly at the expense of random coil conformation. These results are in accordance with the hypothesis that an induced-fit mechanism gives structure to the GR AF1 when it encounters TBP.

Circular Dichroism↗
PubMed2004

Mutational and structural analysis of stem-loop IIIC of the hepatitis C virus and GB virus B internal ribosome entry sites.

Translation of the open reading frames (ORF) of the hepatitis C virus (HCV) and closely related GB virus B (GBV-B) genomes is driven by internal ribosome entry site (IRES) elements located within the 5' non-translated RNA. The functioning of these IRES elements is highly dependent on primary and higher order RNA structures. We present here the solution structures of a common, critical domain within each of these IRESs, stem-loop IIIc. These ten-nucleotide hairpins have nearly identical sequences and similar overall tertiary folds. The final refined structure of each shows a stem with three G:C base-pairs and a novel tetraloop fold. Although the bases are buckled, the first and fourth nucleotides of both tetraloops form a Watson-Crick type base-pair, while the apical nucleotides are located in the major groove where they adopt C(2)-endo sugar puckering with B-form geometry. No hydrogen bonding interactions were observed involving the two apical residues of the tetraloop. Stability of the loops appears to be derived primarily from the stacking of bases, and the hydrogen bonding between the fourth and seventh residues. Mutational analysis shows that the primary sequence of stem-loop IIIc is important for IRES function and that the stem and first and fourth nucleotides of the tetraloop contribute to the efficiency of internal ribosome entry. Base-pair formation between these two positions is essential. In contrast, the apical loop nucleotides differ between HCV and GBV-B, and substitutions in this region of the hairpin are tolerated without major loss of function.

Base Sequence↗
PubMed2004

High-resolution structure of a picornaviral internal cis-acting RNA replication element (cre).

Picornaviruses constitute a medically important family of RNA viruses in which genome replication critically depends on a small RNA element, the cis-acting replication element (cre), that templates 3D(pol) polymerase-catalyzed uridylylation of the protein primer for RNA synthesis, VPg. We report the solution structure of the 33-nt cre of human rhinovirus 14 under solution conditions optimal for uridylylation in vitro. The cre adopts a stem-loop conformation with an extended duplex stem supporting a novel 14-nt loop that derives stability from base-stacking interactions. Base-pair interactions are absent within the loop, and base substitutions within the loop that favor such interactions are detrimental to viral RNA replication. Conserved adenosines in the 5' loop sequence that participate in a slide-back mechanism of VPg-pUpU synthesis are oriented to the inside of the loop but are available for base templating during uridylation. The structure explains why substitutions of the 3' loop nucleotides have little impact on conformation of the critical 5' loop bases and accounts for wide variation in the sequences of cres from different enteroviruses and rhinoviruses.

Base Pairing↗
PubMed2004

Combinatorial selection and edited combinatorial selection of phosphorothioate aptamers targeting human nuclear factor-kappaB RelA/p50 and RelA/RelA.

Nuclear factor-kappaB (NF-kappaB) transcription factors are important in regulating the immune response and play critical roles in the pathogenesis of chronic inflammatory diseases and a variety of human cancers. Agents that target specific NF-kappaB dimers may serve as therapeutic agents for the prevention of pathogenic immune responses. We have selected monothiophosphate-modified aptamers, or "thioaptamers", to the NF-kappaB p50/RelA heterodimer using combinatorial selection techniques. We also utilized a "double sieve" or editing approach for the generation of thioaptamers with enhanced selectivity to the RelA/RelA homodimer. The thioaptamers from these selections and our previous selections on the p50/p50 and RelA/RelA homodimers all had unique sequences and bound tightly to the recombinant NF-kappaB dimers against which they were selected. The selected thioaptamers also appear to maintain their selectivity and specificity among other cellular proteins, because they have the ability to bind NF-kappaB proteins within nuclear extracts from lipopolysaccharide (LPS)-induced macrophages and B cells.

B-Lymphocytes↗
PubMed2004

Solution structure and antibody binding studies of the envelope protein domain III from the New York strain of West Nile virus.

The solution structure of domain III from the New York West Nile virus strain 385-99 (WN-rED3) has been determined by NMR methods. The West Nile domain III structure is a beta-barrel structure formed from seven anti-parallel beta-strands in two beta-sheets. One anti-parallel beta-sheet consists of beta-strands beta1 (Phe(299)-Asp(307)), beta2 (Val(313)-Tyr(319)), beta4 (Arg(354)-Leu(355)), and beta5 (Lys(370)-Glu(376)) arranged so that beta2 is flanked on either side by beta1 and beta5. The short beta4 flanks the end of the remaining side of beta5. The remaining anti-parallel beta-sheet is formed from strands beta3 (Ile(340)-Val(343)), beta6 (Gly(380)-Arg(388)), and beta7 (Gln(391)-Lys(399)) arranged with beta6 at the center. Residues implicated in antigenic differences between different West Nile virus strains (and other flaviviruses) and neutralization are located on the outer surface of the protein. Characterization of the binding of monoclonal antibodies to WN-rED3 mutants, which were identified through neutralization escape experiments, indicate that antibody neutralization directly correlates with binding affinities. These studies provide an insight into theoretical virus-receptor interaction points, structure of immunogenic determinants, and potential targets for antiviral agents against West Nile virus and highlight differences between West Nile virus and other flavivirus structures that may represent critical determinants of virulence.

Antibodies, Monoclonal↗