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Biomedical subjects

A Blanchard

Publications and source records attributed to A Blanchard.

At least 73 records · Page 4Linked to original sources
PubMed1994

Inhibition of HIV type 1 reverse transcriptase assay by nucleases produced by contaminating mycoplasmas.

Mycoplasmal contamination of HIV-1-infected cells has been found to induce reduction of reverse transcriptase (RT) activity; however, the exact mechanism of this phenomenon was not clearly elucidated. Our results indicate that the apparent reduction in RT activity is due to a calcium-dependent nuclease(s) that is (are) produced by contaminating mycoplasmas. The interference with the RT assay was found to be due to the degradation of products of the RT activity. Addition of EGTA at a 1 mM concentration was sufficient to remove the inhibitory effect. The particular HIV-1-producing cell line that was under study was found to be contaminated with Mycoplasma fermentans and Mycoplasma pirum and the latter was isolated in pure culture. Nuclease activity was also observed with pure cultures of mycoplasmas from different species. The activity was found to be of the endonuclease type because it was active with both supercoiled and linear DNAs.

Cell Line↗
PubMed1994

Molecular characterization of the P1-like adhesin gene from Mycoplasma pirum.

A DNA fragment has been isolated from the genome of Mycoplasma pirum by use of a genetic probe derived from the conserved region within the genes for the major adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae. A gene encoding an adhesin-like polypeptide was localized, and sequence analysis indicated a G + C content of only 28%, with A- and T-rich codons being preferentially used. A total of 91% of positions 3 were either A or T. The deduced polypeptide is 1,144 amino acids long (126 kDa) and shows 26% identity with the adhesins of M. genitalium and M. pneumoniae. Other features in common with these two membrane proteins include a similar hydropathic profile and a proline-rich C terminus. Antibodies were prepared by using as an immunogen a peptide derived from the C terminus of the M. pirum adhesin-like polypeptide and were found to recognize on immunoblots a 126-kDa polypeptide from an M. pirum cellular extract. The characterization of the adhesin-like gene is a first step toward a better understanding of the mechanisms allowing this human mycoplasma to attach to host cells.

Adhesins, Bacterial↗
PubMed1994

AIDS-associated mycoplasmas.

Previously, we hypothesized that mycoplasmas could act as cofactors accelerating the progression of HIV disease. In the present paper, we review the current knowledge on three mycoplasmas (Mycoplasma fermentans, M. penetrans, and M. pirum) that have been implicated as these putative cofactors. All three mycoplasmas have been isolated from patients with HIV infection, and serological studies have suggested that the presence of M. penetrans could be associated with HIV infection. These mycoplasmas share the capacity to hydrolyze arginine and ferment glucose as well as to attach to and invade eukaryotic cells. The possible mechanisms that could allow mycoplasmas to influence HIV pathogenesis, specifically through the activation of the immune system or the production of superantigen or by contributing to the oxidative stress observed in HIV-infected subjects, are discussed. These studies have offered and will continue to offer major contributions to a better understanding of mycoplasmal flora in humans and have begun to unveil some of the mechanisms of virulence of these organisms.

AIDS-Related Opportunistic Infections↗
PubMed1993

Phylogenetic analysis of Mycoplasma penetrans, isolated from HIV-infected patients.

A novel mycoplasmal species designated as Mycoplasma penetrans has recently been isolated from patients infected with human immunodeficiency virus. The 16S rRNA gene from this mycoplasma was cloned and its nucleotide sequence determined. This sequence was aligned with previously published homologous sequences from several mycoplasmas and with related Gram-positive bacteria and a phylogenetic tree was constructed. The results indicate that M. penetrans belongs to the evolutionary group Pneumoniae.

DNA, Bacterial↗
PubMed1993

Detection of Ureaplasma urealyticum by polymerase chain reaction in the urogenital tract of adults, in amniotic fluid, and in the respiratory tract of newborns.

On the basis of the nucleotide sequence of the urease genes of Ureaplasma urealyticum serotype 8, polymerase chain reaction (PCR) primers were selected, evaluated for specificity and sensitivity, and tested for their ability to detect U. urealyticum in the adult urogenital tract, amniotic fluid, and endotracheal aspirates of newborns. All 14 reference serotypes of U. urealyticum were detected with equal sensitivity (1-10 cfu), whereas multiple strains of 12 other mycoplasma species found in humans as well as eukaryotic DNA were not detected. A total of 638 clinical specimens was evaluated. Results indicate that PCR is equal to if not more sensitive than culture for detection of U. urealyticum. Faster detection of U. urealyticum by PCR (< 24 h) compared to culture (2-5 days) will be particularly important in management of very low birth-weight infants in whom this organism has been shown to be a significant cause of meningitis, respiratory disease, and death. This method of detection will also be helpful in further determining the role of this organism in intraamniotic infection and premature birth.

Adult↗
PubMed1993

Use of the polymerase chain reaction for detection of Mycoplasma fermentans and Mycoplasma genitalium in the urogenital tract and amniotic fluid.

In an attempt to further define the natural history of Mycoplasma genitalium and Mycoplasma fermentans infections in humans, we used cultures and the polymerase chain reaction (PCR) to determine whether these organisms were present in the urethra and cervix of sexually active adults and in the amniotic fluid of women whose membranes were intact and collected at the time of cesarean delivery (to preclude cervical contamination). M. genitalium was detected by PCR but not by culture in 11% of patients with urethritis or cervicitis. It was not detected by either PCR or culture in the 232 amniotic fluid samples analyzed. In contrast, M. fermentans was not detected by either method in patients with urethritis or cervicitis but was detected by PCR in 4 of 232 amniotic fluid samples tested. These results indicate that in these four cases M. fermentans was transferred transplacentally. Histological evidence of chorioamnionitis was present in two of the four patients, a finding suggesting that M. fermentans may be a cause of chorioamnionitis. These results must be confirmed by other investigators, and further studies should be undertaken to determine the potential clinical significance of M. fermentans infection.

Adolescent↗
PubMed1993

Identification of Mycoplasma pirum genes involved in the salvage pathways for nucleosides.

Genes encoding enzymes involved in the salvage pathway for nucleosides have been cloned and sequenced from the mollicute Mycoplasma pirum. One of them, encoding deoxyriboaldolase, was functionally identified by complementation of an Escherichia coli mutant. These genes are clustered, suggesting an operon organization, and they are immediately followed by the putative gene for the triose phosphate isomerase, an enzyme used during glycolysis.

Aldehyde-Lyases↗
PubMed1993

Evaluation of intraspecies genetic variation within the 16S rRNA gene of Mycoplasma hominis and detection by polymerase chain reaction.

Mycoplasma hominis is a heterogeneous species with DNA-DNA hybridization values ranging from 51 to 100%. We report here the sequencing of the 16S rRNA gene of a strain (183) that greatly differs from the type strain (PG21) of this species. Comparison of 16S rDNA sequences from these two strains showed limited differences, indicating that the two strains belong to the same rRNA species complex. Using these nucleotide sequence data, we established a rapid method for the detection of M. hominis by using polymerase chain reaction. This method was shown to be sensitive and specific when tested with reference strains and clinical isolates.

Base Sequence↗
PubMed1993

Ritanserin, imipramine, and placebo in the treatment of dysthymic disorder.

Fifty outpatients with dysthymic disorder (DSM-III) were divided by double-blind randomized assignment into three groups given ritanserin, imipramine, and placebo, respectively. The trial was of 7 weeks' duration; by week 6, imipramine was clearly superior to placebo, whereas by week 7, both drugs caused significantly more improvement than the placebo. Although imipramine had slightly greater efficacy than ritanserin, it also had significantly more side effects. This was particularly evident in the higher dropout rate with imipramine. The efficacy and side effect profile of ritanserin makes it well tolerated and acceptable with dysthymic patients who, although they do not respond as quickly as patients with major depressive disorder, do show significant improvement, given sufficient time.

Adult↗
PubMed1993

Mycoplasmas as cofactors in infection due to the human immunodeficiency virus.

Results obtained in vitro suggest that mycoplasmas act as cofactors with the human immunodeficiency virus (HIV) in the development of AIDS. Mycoplasmas, including Mycoplasma fermentans, Mycoplasma pirum, and Mycoplasma penetrans have since been isolated from HIV-infected individuals. In addition, M. fermentans has been detected by different investigators in numerous tissues and in the blood of HIV-infected patients. Higher titers of antibodies to M. penetrans have also been found in HIV-infected patients as compared with noninfected individuals. These mycoplasmas have been shown to have the capacity to invade cells and to be potent immunomodulators. Although there is no doubt that mycoplasmas are found in HIV-infected individuals and eventually produce systemic infections, their pathogenic role in association with HIV remains to be determined.

HIV Infections↗
PubMed1993

Reduction in suicidal ideation with SSRIs: a review of 459 depressed patients.

This paper is a review of 459 outpatients treated in double-blind clinical drug trials using similar protocols which compared the clinical responses to specific serotonin reuptake inhibitors, norepinephrine reuptake inhibitors, mixed norepinephrine serotonin reuptake inhibitors, serotonin-2 antagonists and placebo. Although improvements in the total score on the Hamilton Rating Scale for Depression did not differ significantly among the groups, there were differences in the profile of response based on analysis of the items of the scale. The most striking difference was the significantly more rapid and effective improvement in depressed mood and the lessening of suicidal ideation among the patients treated with specific serotonin reuptake inhibitors.

Adolescent↗
PubMed1992

Rapid detection of tetM in Mycoplasma hominis and Ureaplasma urealyticum by PCR: tetM confers resistance to tetracycline but not necessarily to doxycycline.

Tetracycline resistance in Mycoplasma hominis and Ureaplasma urealyticum has been associated with the tetM determinant and has recently been increasing in incidence. We report here a rapid method for detection of the tetM determinant based on the use of the polymerase chain reaction (PCR) to amplify a 397-bp DNA fragment from the tetM gene and verification of specificity using the restriction enzyme TaqI. Analysis of 42 U. urealyticum and 49 M. hominis isolates indicates that the PCR method may be clinically useful for determination of tetracycline sensitivity, as tetM is presently the only known determinant associated with tetracycline resistance in these two organisms. All of the tetM-positive M. hominis isolates were sensitive to doxycycline, indicating that tetM does not necessarily confer resistance to this antibiotic.

Base Sequence↗
PubMed1992

Role of mycoplasma infection in the cytopathic effect induced by human immunodeficiency virus type 1 in infected cell lines.

In addition to previously reported tetracycline analogs, other antibiotics known for antimycoplasmal activities inhibited the cytopathic effect in CEM cl13 cells infected with human immunodeficiency virus type 1 (HIV-1) or HIV-2 but were unable to block virus replication. A contaminating mycoplasma was isolated from our CEM cl13 cells and identified as a strain of Mycoplasma fermentans. Following infection of lymphoblastoid (CEM) or promonocytic (U937 and THP1) cell lines with HIV-1, cytopathic effect was observed only in association with mycoplasmal contamination. Moreover, HIV-1 infection of U937 cells after experimental inoculation with a human isolate of M. fermentans led to pronounced cell killing. We have verified that this effect is not merely an artifact caused by arginine and/or glucose depletion in the cell culture medium. These results confirm that mollicutes, in particular M. fermentans, are able to act synergistically with HIV-1 to kill infected cells in some in vitro systems.

Anti-Bacterial Agents↗
PubMed1991

Detection and identification of mycoplasmas by amplification of rDNA.

Alignment of published 16S rRNA sequences allowed the definition of a pair of oligonucleotides suitable for polymerase chain reaction (PCR). Using this pair of PCR primers, several mycoplasmas including the four human parasites Mycoplasma genitalium, M. hominis, M. salivarium and M. orale were detected. This DNA amplification was restricted to species of the genus Mycoplasma while no cross-reaction was observed with DNA from other bacteria and eukaryotic cells. Subsequent analysis of amplified products by either specific oligonucleotide hybridization or dideoxy sequencing specified the identity of the detected mycoplasmas. This method offers a highly discriminating and sensitive assay for the direct detection and identification of these microorganisms without the need for prior cultivation.

Base Sequence↗
PubMed1991

Cyclic adenosine monophosphate-stimulating agents induce ecto-5'-nucleotidase activity and inhibit DNA synthesis in rat cultured mesangial cells.

The ecto-5'-nucleotidase activity of rat glomerular mesangial cells increases after exposure to prostaglandin E2 (PGE2) via cAMP stimulation (Savic et al., 1990, Immunology 70, 321). Therefore we examined whether other cAMP-stimulating agents had a similar effect. Forskolin (1 microM), PGE2 (10 microM), and isoproterenol (10 microM), three products stimulating rat mesangial cell adenylate cyclase activity, enhanced cAMP accumulation within 5 min and 5'-nucleotidase activity after a lag time of at least 24 h, 3-Isobutyl-1-methylxanthine (IBMX) and Ro 20-1724, two drugs inhibiting cAMP degradation, also stimulated cAMP accumulation and 5'-nucleotidase activity. The effects of these agents on 5'-nucleotidase activity were additive with those of the three products stimulating adenylate cyclase activity, except for Ro 20-1724 and forskolin which acted synergistically. Cycloheximide, a blocker of protein synthesis, suppressed the cAMP-dependent increase of 5'-nucleotidase activity. Because ecto-5'-nucleotidase activity is a marker of cell differentiation, the effect of the same cAMP-stimulating agents on cell proliferation was also studied. Forskolin, PGE2, and isoproterenol inhibited [3H]thymidine incorporation into rat mesangial cells in a dose-dependent manner. The same effect was obtained with IBMX (100 microM) and Ro 20-1724 (50 microM). Stimulation of 5'-nucleotidase activity and inhibition of [3H]thymidine incorporation occurred over the same range of concentrations for the various agonists tested. Taken together, these results indicate that expression of ecto-5'-nucleotidase in rat mesangial cells is induced by cAMP whatever the reason for its accumulation. The simultaneous inhibition of DNA synthesis may occur independently or be associated with the stimulation of 5'-nucleotidase expression.

5'-Nucleotidase↗
PubMed1990

Ureaplasma urealyticum urease genes; use of a UGA tryptophan codon.

Nucleotide sequence analysis of a Ureaplasma urealyticum DNA fragment, homologous to cloned urease genes of other prokaryotes, revealed three consecutive open reading frames. The molecular weights of the three deduced polypeptides are 11.2 kD, 13.6 kD and 66.6 kD. These values are consistent with the size of the three subunits previously reported for purified native urease. A significant sequence homology was found between the three polypeptides of the ureaplasmal urease and the single polypeptide of jack bean (Canavalia ensiformis) urease. Codon usage indicates that UGA is a tryptophan codon in this mollicute. Use of polymerase chain reactions has disclosed the existence of genetic polymorphism among the urease genes of different serotypes of U. urealyticum.

Amino Acid Sequence↗