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Biomedical subjects

J Yelle

Publications and source records attributed to J Yelle.

At least 19 recordsLinked to original sources
PubMed2006

Antiviral activity and cross-resistance profile of P-1946, a novel human immunodeficiency virus type 1 protease inhibitor.

The HIV protease inhibitor P-1946 is a member of a novel family of l-Lysine derivatives. The compound is a specific HIV-1 protease inhibitor that has potent and selective in vitro antiviral activity (EC50 152 nM) against a range of isolates resistant to commercially available protease inhibitors. The presence of at least four primary and four secondary drug resistance mutations is required to achieve greater than four-fold resistance to P-1946. P-1946's favorable resistance profile makes it a good lead for the development of new agents active against existing PI-resistant virus in treatment-experienced patient.

Cell Line↗
PubMed2001

Analysis of the DNA substrate structure and number of the processing sites on the activities of HIV-1 integrase in vitro.

A series of DNA substrates were synthesized to analyze the 3'-processing, integration and disintegration reactions taking place concurrently on the same DNA molecules and to evaluate the potential effects of various structural modifications of these molecules on the activities of HIV-1 integrase (IN). Our results indicate that DNA substrates containing multiple recognition sites for IN can produce efficiently the three activities of the enzyme. The 3'-processing and disintegration sites are recognized and processed by IN, both reactions being carried out in a competitive manner by the enzyme on the same DNA molecule. The presence of the gaps and unpaired nucleotides in the region surrounding the disintegration site had major deleterious effects on enzymes disintegration activity. Analysis of a different conformation at the base of the DNA hairpin has revealed a significant improvement of IN disintegration activity in the presence of double-stranded DNA on the 3' side of the disintegration site, suggesting that this region plays an important role in the stability of the enzyme-substrate complex. Interestingly, the efficiency of disintegration was strongly diminished in the presence of an unpaired nucleotide located immediately at the 3' end of the cleavage site. Overall, our results underline the extreme sensitivity of the HIV-1 IN to its substrates structure and conformation, especially for its disintegration activity, and the considerable importance of the disintegration activity in the reactions carried out in vitro by the purified enzyme.

DNA↗
PubMed2001

Midodrine prevents orthostatic intolerance associated with simulated spaceflight.

Many astronauts after being weightless in space become hypotensive and presyncopal when they assume an upright position. This phenomenon, known as orthostatic intolerance, may interfere with astronaut function during reentry and after spaceflight and may limit the ability of an astronaut to exit a landed spacecraft unaided during an emergency. Orthostatic intolerance is more pronounced after long-term spaceflight and is a major concern with respect to the extended flights expected aboard the International Space Station and for interplanetary exploration class missions, such as a human mission to Mars. Fully effective countermeasures to this problem have not yet been developed. To test the hypothesis that alpha-adrenergic stimulation might provide an effective countermeasure, we conducted a 16-day head-down-tilt bed-rest study (an analog of weightlessness) using normal human volunteers and administered the alpha(1)-agonist drug midodrine at the end of the bed-rest period. Midodrine was found to significantly ameliorate excessive decreases in blood pressure and presyncope during a provocative tilt test. We conclude that midodrine may be an effective countermeasure for the prevention of orthostatic intolerance following spaceflight.

Adrenergic alpha-Agonists↗
PubMed2001

Lipid-based delivery of combinations of antisense oligodeoxynucleotides for the in vitro inhibition of HIV-1 replication.

We evaluated a new approach to AIDS therapy by using combinations of oligodeoxynucleotides (ODNs), delivered with a lipid-based carrier system, that target different HIV viral genome sites. We identified some of the factors that seem to influence the effectiveness of a combination strategy in cell cultures including ODN concentrations, type of infection (acute vs chronic), backbone modification of the ODN, and the number of sequences. When delivered by the DLS carrier system, some advantages of using a combination of ODNs over treatment with only one ODN could be observed in acute infection assays but not in the chronic infection model. These results suggest that in the acute infection model, the 3 different antisense ODNs in the "cocktail" might block an early step of virus replication by combined inhibitory effects. Various combinations of phosphorothioate-modified (PS) and unmodified oligonucleotides delivered by the DLS system were compared for their antiviral activity in a long-term acute assay using HIV-1 (IIIB strain)-infected MOLT-3 cells. The most effective combination had 3 phosphorothioate antisense ODNs: Srev, SDIS, and SPac (>99% inhibition at 100 pM). However, the additive effect determined when using ODN combinations was rather low, revealing the high level of nonsequence specificity in HIV-1 cell culture models. Data illustrated the high sequence nonspecific activity of ODNs, especially when comparing activity of antisense ODNs with activity of random control sequence ODNs. The latter exhibited an inhibitory effect similar to that of antisense ODNs under our experimental conditions. Nevertheless, we demonstrated that it is possible to achieve high anti-HIV activity by using, in combination, picomolar range concentrations of antisense oligonucleotides complexed to a lipid-based carrier system such as the DLS system, without increasing cell toxicity.

Anti-HIV Agents↗
PubMed2000

Characterization of mutant HIV-1 integrase carrying amino acid changes in the catalytic domain.

To gain insight into the importance of conserved residues in the core domain of HIV-1 IN, we performed site-directed mutagenesis of the full-length enzyme, overexpressed the mutant proteins in E. coli, purified and analyzed their 3'-processing, integration and disintegration activities in vitro. Change of E152V in the DD(35)E motif abolished all detectable activities of IN. Alteration of two highly conserved residues, P145 and K156, by isoleucine, resulted in a substantial loss or completely abolished the three activities of the enzyme. Mutant P90D weakly reduced the 3'-processing but severely affected the two other IN activities. Results obtained from double and triple mutations, P90D/P1451 and P145I/F185K/C280S, clearly suggest a crucial role of P145 in the catalytic function of IN, whereas the mutants V150E, L158F and L172M had no detectable effect on any of the IN activities. Taken together, these results allowed us to conclude that all the conserved amino acids in the core domain of IN are not equally important for catalytic functions: like D64, D116 and E152, our data suggest that P90, P145 and K156 are also essential for all three enzymatic activities of HIV-1 IN in vitro, whereas V150, L158 and L172 appear to be less critical.

Amino Acid Sequence↗
PubMed1996

Lessons from operational cardiovascular studies in space.

The Space Shuttle program has produced a database of information on the cardiovascular responses to spaceflight, based on in-flight as well as pre- and post-flight assessments undertaken as part of the assessment of the health, safety, and efficiency of Shuttle crews. The methods used in routine cardiovascular assessments of Space Shuttle astronauts are reviewed, and the major findings of these investigations are presented.

Cardiovascular Physiological Phenomena↗
PubMed1994

Analysis of long-term viral expression in CEM cells persistently infected with non syncytium-inducing HIV-1 strains.

CEM cells were infected with three HIV-1 non syncytium-inducing (NSI) strains obtained from AIDS patients or seropositive individuals. The surviving cells were followed for several months in the persistently infected cultures designated 65870/CEM, 65871/CEM and 3929/CEM, and analyzed for virus expression using light and electron microscopy, immunofluorescence, reverse transcriptase assay, polymerase chain reaction amplification (PCR), nucleic acid hybridization and flow cytometry. The virus isolates induced relatively few syncytia and other cytopathic effects in the corresponding cell lines and the number of cells positive for virus expression never rose above 44%. Distinct peaks of antigen-positive cells were obtained, coincident with high levels of reverse transcriptase activity. The cultures were strongly resistant to superinfection by laboratory strain Lai, with the exception of 65870/CEM which expressed HIV antigens in up to 15% of the cells for a few days. However, cell lysis was minimal in all cases. After long-term cultivation of the three cultures, no antigen-positive cells were detected and no trace of virus expression could be observed. The remaining cells consisted entirely of CD4-negative cells. PCR analyses indicated that cells harboring a provirus were progressively eliminated from the cultures, leaving only virus-free cells. In this system, cells carrying a latent provirus survive for a limited period of time before virus activation induces cell lysis. These results suggest that at least three types of cells exist in the CEM cell line: CD4-positive cells which are rapidly killed by the virus, a second type harboring a latent viral genome after the infection and which grow normally until activation of the resident genome by external or internal signal(s), and a third type which represents rare CD4-negative cells present in the initial CEM population and which are selected for by the NSI isolates. This is the first study documenting specific interactions between NSI strains of HIV-1 and distinct subpopulations of CEM cells grown as a single cell culture.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Efficient recovery of cloned human cytomegalovirus DNA fragments from agarose gels.

A simple and efficient method for the recovery of DNA fragments from agarose gels is described. After electrophoresis, bands of interest are cut out of the gel and agarose slices pushed through the opening of a syringe needle. The resulting gel slurry is frozen and thawed three times and then centrifuged. DNA in the supernatant is precipitated, resuspended in a small volume of buffer and, finally, desalted. A recombinant pAT153 plasmid carrying the BamHI-P fragment of the human cytomegalovirus (HCMV) genome was detected using purified viral HindIII-E DNA fragment as a probe. Restriction endonuclease analysis was used to confirm the identity of the cloned fragment. Experiments performed with the recombinant pLCR127 plasmid indicate that our freeze/thaw method, with about 80% recovery and very little DNA degradation, is more advantageous than the well known electroelution and NaI/glass methods.

Adsorption↗
PubMed1993

SV40 DNA extracted from persistently infected Vero cells using miniprep columns for plasmids.

A recombinant pAT153 plasmid carrying the whole simian virus 40 (SV40) genome in the form of two consecutive Pst I restriction fragments was rapidly isolated using a commercially available minipreps DNA purification system. This rapid and simple method was used to extract SV40 DNA from infected Vero monkey cells. Viral DNA replication in persistently infected SVP-1 monkey cells was also conveniently followed over a period of 8 d by agarose gel electrophoresis and molecular hybridization using minipreps and the recombinant plasmid (pLCB104) as a probe. Unsatisfactory results were obtained, however, when herpes simplex virus DNA was tentatively extracted from infected Vero cells with the above method. Only covalently closed circular DNA molecules, with two strands unable to separate fully under denaturing conditions, were apparently retained after rapid neutralization by the silicone-based minipreps DNA purification resin.

Animals↗
PubMed1990

Low tumorigenicity of canine cells transformed by the human cytomegalovirus.

Dog embryo kidney cells transformed by the human cytomegalovirus (HCMV) were obtained after non-permissive infection or transfection with viral DNA digested by restriction endonuclease EcoR I. The transformed cells, growing rapidly and showing an unlimited division potential, could use medium with only 2% serum for growth, contained nuclear virus antigens, and formed small colonies (less than 0.2 mm) in agarose. From 40 mice inoculated with transformed canine cells, only one eventually developed a tumor. Results indicate that dog cells are immortalized but not tumorigenically transformed by the human cytomegalovirus.

Animals↗
PubMed1990

Physical mapping of a temperature-sensitive mutation of human cytomegalovirus by marker rescue.

Physical mapping of a temperature-sensitive (ts) mutation of human cytomegalovirus (HCMV) strain AD-169 was attempted here using cloned HindIII restriction endonuclease fragments and the mutant virus. The DNA-positive mutant tested (HCMV ts 1585) was successfully rescued by viral DNA sequences between 0.277 and 0.303 map units. The product of this gene is apparently a structural protein of molecular weight 40,000. Marker rescue could thus be used to establish the physical location of essential HCMV genes, even if the viral DNA molecule is extremely large and complex.

Chromosome Mapping↗
PubMed1990

Gel and buffer effects on the migration of DNA molecules in agarose.

The migration rate of linear and supercoiled DNA molecules in four brands of agarose from two different lots was compared using three buffer systems. Differences in DNA mobility were observed between brands and lots of agarose, in standard as well as in pulsed-field gel electrophoresis. A buffer-related effect on the migration of DNA molecules in agarose gels was also observed. Our results underline the need for an actual lot analysis of agarose, this extraction product varying from one preparation to another in several aspects.

Bacteriophage lambda↗
PubMed1986

A simplified procedure for the rapid identification of recombinant pAT153 plasmids in Escherichia coli HB101 cells.

Insertion of foreign DNA into the unique HindIII site of the high copy number plasmid pAT153 reduces but does not completely abolish the resistance of Escherichia coli HB101 cells to tetracycline. Recombinant DNA-containing colonies could then be phenotypically differentiated from non-recombinant ones by their smaller size on nutrient agar plates with ampicillin and tetracycline at a final concentration of 50 and 4 micrograms/ml, respectively. A wide variety of human cytomegalovirus DNA fragments have been found in pAT153 molecules propagated by the ampicillin-resistant tetracycline-sensitive bacteria selected.

Cytomegalovirus↗
PubMed1986

Distinct regions of the human cytomegalovirus genome are responsible for the immortalization and tumorigenicity of animal cells.

Dog embryo kidney cells were efficiently transformed by human cytomegalovirus (HCMV) particles or intact viral DNA. Negative results were obtained after transfection of the canine cells with recombinant plasmids carrying the HCMV Hind III-E subgenomic fragment or with limit Bgl II and Hind III digests of the viral genome. Immortalized dog cells with typical transformation properties appeared, however, after transfection with EcoR I fragments of the HCMV DNA. Distinct regions of the viral genome are probably responsible for the immortalization and the tumorigenicity of mammalian cells.

Cell Line↗
PubMed1986

Transformation of dog embryo kidney cells by human herpesviruses.

The infection of dog embryo kidney (DEK) cells with herpes simplex virus type 2 (HSV-2) or human cytomegalovirus (HCMV) led to the development of transformed cell lines. Rapidly dividing DEK cells with unlimited division potential exhibited growth in 2% serum, contained nuclear virus antigens, and formed small (+/- 0.2 mm) colonies in 0.3% agarose. Immortal cell lines showing the same transformation properties were also obtained after transfection with purified HSV-2 or HCMV DNA. These results confirm the transforming capacity of both herpesviruses as well as the usefulness of this different type of mammalian cells in transformation studies.

Animals↗