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Biomedical subjects

R A Ogert

Publications and source records attributed to R A Ogert.

9 recordsLinked to original sources
PubMed2001

N-linked glycosylation sites adjacent to and within the V1/V2 and the V3 loops of dualtropic human immunodeficiency virus type 1 isolate DH12 gp120 affect coreceptor usage and cellular tropism.

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linked glycosylation sites on its gp120 subunit. In this study, specific glycosylation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eliminated by site-directed mutagenesis and the properties of the resulting mutant envelopes were evaluated using a recombinant vaccinia virus-based cell-to-cell fusion assay alone or in the context of viral infections. Of the glycosylation sites that were evaluated, those proximal to the V1/V2 loops (N135, N141, N156, N160) and the V3 loops (N301) of gp120 were functionally critical. The glycosylation site mutations near the V1/V2 loop compromised the use of CCR5 and CXCR4 equally. In contrast, a mutation within the V3 loop preferentially inhibited the usage of CCR5; although this mutant protein completely lost its CCR5-dependent fusion activity, it retained 50% of the wild-type fusion activity with CXCR4. The replication of a virus containing this mutation was severely compromised in peripheral blood mononuclear cells, MT-4 cells, and primary monocyte-derived macrophages. A revertant virus, which acquired second site changes in the V3 loop that resulted in an increase in net positive charge, was isolated. The revertant virus fully recovered the usage of CXCR4 but not of CCR5, thereby altering the tropism of the parental virus from dualtropic to T-tropic. These results suggest that carbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interaction with receptors. Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may be virus isolate dependent.

Base Sequence↗
PubMed2000

Rous sarcoma virus DR posttranscriptional elements use a novel RNA export pathway.

Rous sarcoma virus (RSV), a simple retrovirus, needs to export unspliced viral RNA from the nucleus to the cytoplasm, circumventing the host cell restriction on cytoplasmic expression of intron-containing RNA. The cytoplasmic accumulation of full-length viral RNA is promoted by two cis-acting direct repeat (DR) elements that flank the src gene; at least one copy of the DR sequence is necessary for viral replication. We show here that the DR mediates export of a reporter construct from the nucleus, suggesting it is a constitutive transport element (CTE). In contrast, human immunodeficiency virus type 1 (HIV-1) and other complex retroviruses encode accessory proteins, Rev or Rex, which promote export of incompletely spliced viral transcripts. This RNA export pathway is CRM1 dependent and can be blocked by the cytotoxic agent leptomycin B. We show here that DR-mediated export is CRM1 independent, suggesting that RSV uses a different export pathway from that of HIV-1 and other complex retroviruses. The simian retroviruses have a CTE which interacts with the cellular Tap export protein. However, we were unable to detect binding of the RSV DR RNA to Tap, suggesting it may use a different export pathway from that of the simian retroviruses. These data suggest that the RSV DR element uses a novel nucleocytoplasmic export pathway.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Mutational analysis of the rous sarcoma virus DR posttranscriptional control element.

The direct repeat (DR) sequences flanking the src gene in Rous sarcoma virus are essential posttranscriptional control elements; at least one copy of this sequence is necessary for cytoplasmic accumulation of unspliced viral RNA. These sequences promote Rev-independent human immunodeficiency virus type 1 expression, suggesting they act as constitutive transport elements (CTEs). To determine which regions of this sequence are critical for CTE function, mutations in the downstream DR were generated and tested in a viral deletion construct lacking src and the upstream DR. Two single-point mutations and three different clustered mutations caused substantial reductions in reverse transcriptase activity, Gag protein levels, and unspliced viral RNA in the cytoplasm. Three conserved regions of the CTE, including nucleotides 8844 to 8847, 8862 to 8864, and 8868 to 8870, were most sensitive to inactivation by mutagenesis.

Animals↗
PubMed1996

Avian retroviral RNA element promotes unspliced RNA accumulation in the cytoplasm.

All retroviruses need mechanisms for nucleocytoplasmic export of their unspliced RNA and for maintenance of this RNA in the cytoplasm, where it is either translated to produce Gag and Pol proteins or packaged into viral particles. The complex retroviruses encode Rev or Rex regulatory proteins, which interact with cis-acting viral sequences to promote cytoplasmic expression of incompletely spliced viral RNAs. Since the simple retroviruses do not encode regulatory proteins, we proposed that they might contain cis-acting sequences that could interact with cellular Rev-like proteins. To test this possibility, we initially looked for a cis-acting sequence in avian retroviruses that could substitute for Rev and the Rev response element in human immunodeficiency virus type 1 expression constructs. A cis-acting element in the 3' untranslated region of Rous sarcoma virus (RSV) RNA was found to promote Rev-independent expression of human immunodeficiency virus type 1 Gag proteins. This element was mapped between RSV nucleotides 8770 and 8925 and includes one copy of the direct repeat (DR) sequences flanking the RSV src gene; similar activity was observed for the upstream DR. To address the function of this element in RSV, both copies of the DR sequence were deleted. Subsequently, each DR sequence was inserted separately back into this deleted construct. While the viral construct lacking both DR sequences failed to replicate, constructs containing either the upstream or downstream DR replicated well. In the absence of both DRs, Gag protein levels were severely diminished and cytoplasmic levels of unspliced viral RNA were significantly reduced; replacement of either DR sequence led to normal levels of Gag protein and cytoplasmic unspliced RNA.

Animals↗
PubMed1992

Kinetics of antibody binding at solid-liquid interfaces in flow.

We have developed the theoretical framework for a displacement immunoassay conducted in flow under nonequilibrium conditions. Using a repetitive displacement technique, we determined the displacement rate and apparent dissociation rate constant at different flow rates. Our data suggest that the kinetics are best described by a first-order function. The displacement efficiency, the displacement rate, and therefore the apparent dissociation rate constant were calculated and demonstrated to be flow rate dependent. The theoretical framework developed in this study was successful in predicting the behavior of antigen displacement in flow.

Antibodies, Monoclonal↗
PubMed1992

Detection of Clostridium botulinum toxin A using a fiber optic-based biosensor.

A rapid, sensitive, analytical method for the detection of Clostridium botulinum toxin has been developed. The fiber optic-based biosensor utilizes the evanescent wave of a tapered optical fiber for signal discrimination. A 50 mW argon-ion laser, which generates laser light at 514 nm, is used in conjunction with an optical fiber probe that is tapered at the distal end. Antibodies specific for C. botulinum are covalently attached to the surface of the tapered fiber. The principle of the system is a sandwich immunoassay using rhodamine-labeled polyclonal anti-toxin A immunoglobin G (IgG) antibodies for generation of the specific fluorescent signal. Various anti-toxin antibodies were immobilized to the fibers. Affinity-purified polyclonal horse anti-toxin A antibodies performed better than the IgG fraction from the same horse serum or than the monoclonal anti-toxin A antibody BA11-3. Botulinum toxin could be detected within a minute, at concentrations as low as 5 ng/ml. The reaction was highly specific and no response was observed against tetanus toxin.

Animals↗
PubMed1990

Complete amino acid sequence of fetal bovine serum acetylcholinesterase and its comparison in various regions with other cholinesterases.

The complete amino acid sequence of a mammalian acetylcholinesterase from fetal bovine serum (FBS AChE) is presented. This enzyme has a high degree of sequence identity with other cholinesterases, liver carboxyesterases, esterase-6, lysophospholipase, and thyroglobulin. The locations of 191 amino acids in 10 regions of the FBS enzyme were compared with corresponding sequences of Torpedo, human, and Drosophila AChEs and human serum butyrylcholinesterase (BChE). In one region there is a marked difference in both the number of amino acids and their sequence between mammalian AChE and other AChEs and the human serum BChE. The amino acid sequence of FBS AChE showed overall homologies of 90% with human AChE, 60% with T. california AChE, 50% with human serum BChE, and 39% with Drosophila AChE in these regions.

Acetylcholinesterase↗
PubMed1990

Studies on the topography of the catalytic site of acetylcholinesterase using polyclonal and monoclonal antibodies.

Polyclonal and monoclonal antibodies were generated against a synthetic peptide (25 amino acid residues) corresponding to the amino acid sequence surrounding the active site serine of Torpedo californica acetylcholinesterase (AChE). Prior to immunization, the peptide was either coupled to bovine serum albumin or encapsulated into liposomes containing lipid A as an adjuvant. To determine whether this region of AChE is located on the surface of the enzyme and thus accessible for binding to antibodies, or located in a pocket and thus not accessible to antibodies, the immunoreactivity of the antibodies was determined using enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, Western blots, and competition ELISA. The polyclonal antibody and several of the monoclonal antibodies failed to react with either Torpedo or fetal bovine serum AChE in their native conformations, but showed significant cross-reactivity with the denatured enzymes. Human serum butyrylcholinesterase, which has a high degree of amino acid sequence homology with these AChEs, failed to react with the same antibodies in either native form or denatured form. Chymotrypsin also failed to react with the monoclonal antibodies in either form. Eighteen octapeptides spanning the entire sequence of this region were synthesized on polyethylene pins, and epitopes of representative monoclonal antibodies were determined by ELISA. The reactivity of peptides suggest that a portion of the 25 mer peptide in AChE containing the active site serine is the primary epitope. It is not exposed on the surface of the enzyme and is most likely sequestered in a pocket-like conformation in the native enzyme.

Acetylcholinesterase↗