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Biomedical subjects

E Arnold

Publications and source records attributed to E Arnold.

At least 91 records · Page 5Linked to original sources
PubMed1994

Subunit specificity of mutations that confer resistance to nonnucleoside inhibitors in human immunodeficiency virus type 1 reverse transcriptase.

We constructed plasmid vectors that simultaneously express both the p66 and p51 subunits of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in Escherichia coli. These vectors allow us to generate HIV-1 RT heterodimers in which either the p66 or the p51 subunit has the wild-type sequence and the other subunit has a specific amino acid substitution. We used these vectors to express HIV-1 RT heterodimers containing several different amino acid substitutions reported to confer resistance to nonnucleoside inhibitors. Most of the amino acid substitutions conferred resistance to nonnucleoside inhibitors R86183 (TIBO) and TSAO-m3T only when present in the p66 subunit of the p66-p51 heterodimer; heterodimers that contained a wild-type p66 subunit and a mutant p51 subunit remained sensitive to the inhibitors. However, there was one mutation, E138K, that conferred drug resistance when the mutation was present in the p51 subunit. The corresponding heterodimer with the E138K mutation in the p66 subunit and a wild-type p51 subunit remained sensitive to the inhibitors. Analysis of the three-dimensional structure of HIV-1 RT indicated that residue 138 of the p51 subunit is in the nonnucleoside inhibitor-binding pocket while residue 138 of the p66 subunit is not. The mutagenesis results, combined with structural data, support the idea that the nonnucleoside inhibitors exert their effects by binding to a hydrophobic pocket in the RT heterodimer and that mutations which give rise to drug resistance directly interfere with the interactions between the nonnucleoside inhibitors and HIV-1 RT.

Antiviral Agents↗
PubMed1994

Use of random systematic mutagenesis to generate viable human rhinovirus 14 chimeras displaying human immunodeficiency virus type 1 V3 loop sequences.

Random systematic mutagenesis was used to generate a library of human rhinovirus 14 chimeras that each display a segment from the V3 loop of human immunodeficiency virus type 1. The sequence XXIGPGRAXX, where X could be any of the 20 amino acids, was inserted at the neutralizing immunogenic site II of human rhinovirus 14 between VP2 residues 159 and 160. Twenty-five unique chimeric viruses were isolated, and the identity of their randomized residues was determined. A nonrandom amino acid distribution that may reflect structural requirements for viability was observed at the randomized positions. Fifteen of 25 chimeras were neutralized by one or more of a panel of four anti-human immunodeficiency virus type 1 V3 loop antibody preparations, indicating that antigenicity had been successfully transplanted. Libraries of chimeric viruses produced by using the techniques described may be a source of vaccines and other immunotherapeutic reagents. The random systematic mutagenesis methodology described should be generally useful for the rapid transplantation of foreign sequences into viral coat and other proteins to produce libraries containing members with the desired properties.

Amino Acid Sequence↗
PubMed1994

Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP.

Recent studies indicate that nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) may inhibit the proliferation of vascular smooth muscle cells (SMC) in vitro. The purpose of this study was to investigate the mechanism of NO- and cGMP-dependent inhibition of cultured rat aortic SMC. The cytokine interleukin-1 beta (IL-1 beta) inhibited serum- and platelet-derived growth factor-stimulated [3H]thymidine incorporation into DNA in subcultured rat aortic SMC. Incubation with IL-1 beta for 24 h markedly increased cGMP levels but not adenosine 3',5'-cyclic monophosphate (cAMP) levels. However, the IL-1 beta-induced increase in cGMP was correlated with an activation of the cAMP-dependent protein kinase (cAMP kinase) activity ratio. The activation of the cAMP kinase was prevented by treatments that blocked NO and cGMP production. The NO-generating vasodilator, S-nitroso-N-acetylpenicillamine (SNAP) also inhibited DNA synthesis and elevated cGMP levels. The inhibition of DNA synthesis by both IL-1 beta and SNAP was observed only when cGMP levels were elevated to high levels (10-fold or more). As was the case for IL-1 beta, SNAP increased the activity ratio of cAMP kinase. Selective inhibition of cAMP kinase using (R)-p-bromoadenosine 3',5'-cyclic monophosphorothioate prevented the inhibition of proliferation by IL-1 beta. By contrast, the inhibitor of the cGMP-dependent protein kinase, (R)-p-bromoguanosine 3',5'-cyclic monophosphorothioate, had no effect on IL-1 beta-induced inhibition of cellular proliferation. These studies suggest that cGMP-dependent activation of the cAMP kinase may be responsible in part at least for the NO-dependent inhibition of proliferation of subcultured rat aortic SMC.

Animals↗
PubMed1994

Libraries of human rhinovirus-based HIV vaccines generated using random systematic mutagenesis.

Human rhinovirus (HRV), an immunogenic and relatively nonpathogenic virus, has been engineered to display HIV-1 immunogens with the intent of developing a vaccine against AIDS. HIV immunogens from the V3 loop have been placed into the neutralizing immunogenic (NIm) sites on the surface of HRV14 naturally recognized by the immune system. To increase the likelihood of recovering viable chimeras displaying the transplanted HIV-1 V3 loop sequences in conformations that mimic that of HIV, we have used random systematic mutagenesis to produce libraries of chimeric HRV14 in which the transplanted epitope from HIV-1 is flanked by one or more randomized amino acid residues. This allows the HIV epitope to be accommodated into the HRV coat proteins in many conformations, some of which should result in the production of viable, immunogenic hybrids. Using this approach, a library containing the sequence XXIGPGRAXX, where X could be any of the 20 amino acids, was generated. A nonrandom distribution of residues was found at the randomized positions, which may be a reflection of the structural requirements for viability. A subset of chimeras was identified that reacted with neutralizing anti-HIV-1 V3 loop antibody preparations, indicating that the antigenicity of the epitopes had been transplanted. Another chimeric virus library was designed to reflect the natural diversity of the V3 loop by incorporating amino acids at frequencies similar to those found among naturally occurring isolates of HIV-1. Powerful selection techniques utilizing anti-HIV-1 V3 loop neutralizing antibodies are being employed to isolate efficiently antigenic chimeras that could serve as potential vaccine candidates.

AIDS Vaccines↗
PubMed1993

Crystal structure of Myxococcus xanthus nucleoside diphosphate kinase and its interaction with a nucleotide substrate at 2.0 A resolution.

The X-ray crystallographic structure of nucleoside diphosphate (NDP) kinase from Myxococcus xanthus has been determined using multiple isomorphous replacement techniques and refined at 2.0 A resolution to a crystallographic R-factor of 0.17. This is the first report of the structure of an enzymatically active NDP kinase and of the enzyme with a bound nucleotide. The structure has been determined in P4(3)2(1)2 and I222 crystal forms. The enzyme monomer consists of a four-stranded antiparallel beta-sheet. The surfaces of the sheet are partially covered with five helical segments. There are two protein molecules in the asymmetric unit of the tetragonal crystal form. They form a dimer with an extensive interface in which 1092 A2 per monomer is buried. The majority of the contact area in the dimer interface is between hydrophobic or aromatic residues. Two dimers are related by a crystallographic 2-fold axis to yield a tetramer. This tetramer is also present in the orthorhombic crystals; however, in this case, the 222 symmetry is entirely crystallographic. Upon tetramer formation, an additional 473 A2 of solvent-accessible surface area from each monomer becomes buried. The interface between dimers in the tetramer is stabilized by salt bridges. Equilibrium sedimentation studies are consistent with the enzyme being a tetramer in solution. The structure of a complex of adenosine diphosphate (ADP) with the enzyme was determined and reveals that most of the nucleotide interactions with the protein are with the pyrophosphate and ribose groups, while the base has no hydrogen bonds with the protein and interacts only by stacking with the side chain of Phe59. The Mg2+ interacts with the pyrophosphate of the ADP and via a solvent molecule with the side chain of the conserved Asp120 residue. The mode of interaction with the nucleotide is novel, with the nucleotide binding at the side of the beta-sheet. The structures of the nucleotide in crystals grown in the presence or absence of Mg2+ are essentially identical. In addition, the phosphotransfer reaction from adenosine triphosphate (ATP) to the enzyme can occur without Mg2+. This suggests that only the second step of the reaction in which the enzyme transfers the phosphate to a nucleoside diphosphate acceptor is significantly catalyzed by the metal.

Adenosine Diphosphate↗
PubMed1993

SCH 38057: a picornavirus capsid-binding molecule with antiviral activity after the initial stage of viral uncoating.

The activity of a new water-soluble molecule, SCH 38057, against picornaviruses is described. SCH 38057 inhibited plaque formation of selected entero- and rhinoviruses in a range of 10.2 to 29.1 microM (50% endpoint) and had a therapeutic index of 10 against poliovirus type 2 (polio 2) in HeLa cells. When administered orally or subcutaneously, SCH 38057 protected mice infected with either coxsackievirus B3 (CVB3) or echovirus-9 from mortality. The molecule provided a low level of protection against thermal inactivation of virus, indicating that SCH 38057 interacts with the picornavirus capsid. Binding studies with [3H]SCH 38057 revealed that the molecule binds to CVB3 and human rhinovirus 14 (HRV14) in a ratio of 29 and 19 molecules per viral particle, respectively. The affinity constant for SCH 38057 binding to CVB3 was 7.0 x 10(-4) M. When added to cultures of infected cells at 3 h after infection, SCH 38057 markedly inhibited viral RNA synthesis. This finding with lack of inhibition of attachment and loss of infectious virus after attachment were interpreted to indicate that, although SCH 38057 binds to the viral capsid, the molecule exerts its antiviral effect after the initial stage of picornavirus uncoating, i.e., after conversion of the 156S infectious viral particle to smaller subviral species.

Animals↗
PubMed1992

Structure of HIV-1 reverse transcriptase/DNA complex at 7 A resolution showing active site locations.

AIDS, caused by human immunodeficiency virus (HIV), is one of the world's most serious health problems, with current protocols being inadequate for either prevention or successful long-term treatment. In retroviruses such as HIV, the enzyme reverse transcriptase copies the single-stranded RNA genome into double-stranded DNA that is then integrated into the chromosomes of infected cells. Reverse transcriptase is the target of the most widely used treatments for AIDS, 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI), but resistant strains of HIV-1 arise in patients after a relatively short time. There are several nonnucleoside inhibitors of HIV-1 reverse transcriptase, but resistance to such agents also develops rapidly. We report here the structure at 7 A resolution of a ternary complex of the HIV-1 reverse transcriptase heterodimer, a monoclonal antibody Fab fragment, and a duplex DNA template-primer. The double-stranded DNA binds in a groove on the surface of the enzyme. The electron density near one end of the DNA matches well with the known structure of the HIV-1 reverse transcriptase RNase H domain. At the opposite end of the DNA, a mercurated derivative of UTP has been localized by difference Fourier methods, allowing tentative identification of the polymerase nucleoside triphosphate binding site. We also determined the structure of the reverse transcriptase/Fab complex in the absence of template-primer to compare the bound and free forms of the enzyme. The presence of DNA correlates with movement of protein electron density in the vicinity of the putative template-primer binding groove. These results have important implications for developing improved inhibitors of reverse transcriptase for the treatment of AIDS.

Base Sequence↗
PubMed1992

Crystallization and preliminary X-ray diffraction studies of coxsackievirus B1.

Preparations of coxsackievirus B1 (CVB1) derived from an infectious cDNA clone have been crystallized in multiple crystal forms. Using high intensity synchrotron radiation, an orthorhombic form of the crystals was shown to diffract X-rays to at least 2.9 A resolution. The unit cell has a primitive lattice with dimensions a = 323 A, b = 450 A, and c = 522 A. A crystallographic asymmetric unit of these CVB1 crystals probably contains an entire virus particle, implying the presence of 60-fold non-crystallographic redundancy. This CVB1 crystal form appears to be suitable for high-resolution structure determination by X-ray crystallography.

Crystallography↗
PubMed1992

Is the tyramine test for depressive illness useful in elderly patients?

There were no significant differences in tyramine sulphate excretion following tyramine ingestion between elderly depressed, demented or control patient groups, in contrast with younger subjects where this test is a trait marker for unipolar endogenous depression. There are inherent problems in urine collection studies in the elderly and the results may have been influenced by the medication that elderly patients have to take for other disorders. This study suggests that the tyramine test is unlikely to be of clinical usefulness in the over 65 age group.

Aged↗
PubMed1991

Crystals of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase with a monoclonal antibody Fab fragment and double-stranded DNA diffract x-rays to 3.5-A resolution.

Two crystal forms of complexes have been grown that contain human immunodeficiency virus type 1 reverse transcriptase and a monoclonal antibody Fab fragment. One of the crystal forms (form II, space group P3112, a = 168.7 A, c = 220.3 A) diffracts x-rays to 3.5-A resolution and appears suitable for moderate-resolution structure determination. The form II crystals have the unusual property that their maximum resolution of diffraction and resistance to radiation damage are enhanced by either crystallization in the presence of or soaking with double-stranded DNA primer-template mimics. These crystals may permit structural studies of catalytically relevant complexes and eventually enable us to experimentally observe successive steps in the reverse transcription process.

Antibodies, Monoclonal↗
PubMed1991

Crystallization and preliminary X-ray diffraction analysis of oucleoside diphosphate kinase from Myxococcus xanthus.

Nucleoside diphosphate (NDP) kinase catalyzes the transfer of the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. Human and rodent forms of this enzyme have been shown to be suppressors of metastasis. Crystals that diffract X-rays to high resolution have been obtained for the recombinant Myxococcus xanthus NDP kinase expressed in and purified from Escherichia coli. Two crystal forms have been obtained. Both forms are orthorhombic, space group I222 (or I2(1)2(1)2(1)) with a = 267.1 A, b = 74.0 A and c = 75.1 A for form I and a = 53.5 A, b = 74.0 A and c = 75.1 A for form II. Form I appears to have five molecules in the asymmetric unit approximately related to each other by a translation of 0.2 along the a axis. Diffraction data have been recorded to 1.9 A for form I and to 2.2 A for form II.

Crystallization↗
PubMed1991

HIV reverse transcriptase structure-function relationships.

HIV reverse transcriptase (RT) is the target of the most widely used treatments for AIDS. Biochemical and mutagenesis studies performed on HIV-1 RT are reviewed in light of the enzyme's structure and functions. Features described include domain arrangement, dimerization, proteolytic processing, and specific recognition of the priming tRNA. Possible regions of functional importance as determined by comparative amino acid sequence analysis and by site-directed mutagenesis are identified. Among the conclusions of the analysis is the unexpected realization that the substrate for proteolytic maturation of the HIV-1 RT p66/p66 homodimer to the p66/p51 heterodimer is most likely an unfolded RNase H domain. In addition, the current progress in crystallization and structure determination of HIV-1 RT is described. Finally, a functional-model of the active reverse transcription complex is presented.

Amino Acid Sequence↗
PubMed1990

Analysis of the structure of a common cold virus, human rhinovirus 14, refined at a resolution of 3.0 A.

Human rhinovirus 14 has a pseudo T = 3 icosahedral structure in which 60 copies of the three larger capsid proteins VP1, VP2 and VP3 are arranged in an icosahedral surface lattice, reminiscent of T = 3 viruses such as tomato bushy stunt virus and southern bean mosaic virus. The overall secondary and tertiary structures of VP1, VP2 and VP3 are very similar. The structure of human rhinovirus 14, which was refined at a resolution of 3.0 A [R = 0.16 for reflections with F greater than 3 sigma(F)], is here analyzed in detail. Quantitative analysis of the surface areas of contact (proportional to hydrophobic free energy of association) supports the previously assigned arrangement within the promoter, in which interactions between VP1 and VP3 predominate. Major contacts among VP1, VP2 and VP3 are between the beta-barrel moieties. VP4 is associated with the capsid interior by a distributed network of contacts with VP1, VP2 and VP3 within a promoter. As the virion assembly proceeds, the solvent-accessible surface area becomes increasingly hydrophilic in character. A mixed parallel and antiparallel seven-stranded sheet is composed of the beta C, beta H, beta E and beta F strands of VP3 in one pentamer and beta A1 and beta A2 of VP2 and the VP1 amino terminus in another pentamer. This association plays an essential role in holding pentamers together in the mature virion as this contact region includes more than half of the total short non-bonded contacts between pentamers. Contacts between protomers within pentamers are more extensive than the contacts between pentamers, accounting in part for the stability of pentamers. The previously identified immunogenic regions are correlated with high solvent accessibility, accessibility to large probes and also high thermal parameters. Surface residues in the canyon, the putative cellular receptor recognition site, have lower thermal parameters than other portions of the human rhinovirus 14 surface. Many of the water molecules in the ordered solvent model are located at subunit interfaces. A number of unusual crevices exist in the protein shell of human rhinovirus 14, including the hydrophobic pocket in VP1 which is the locus of binding for the WIN antiviral agents. These may be required for conformational flexibility during assembly and disassembly. The structures of the beta-barrels of human rhinovirus 14 VP1, VP2 and VP3 are compared with each other and with the southern bean mosaic virus coat protein.

Amino Acid Sequence↗