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Biomedical subjects

A Blanchard

Publications and source records attributed to A Blanchard.

At least 37 records · Page 2Linked to original sources
PubMed1999

Role of Mycoplasma penetrans endonuclease P40 as a potential pathogenic determinant.

Recently, we reported the purification to homogeneity and characterization of Ca(2+)- and Mg(2+)-dependent endonuclease P40 produced by Mycoplasma penetrans (M. Bendjennat, A. Blanchard, M. Loutfi, L. Montagnier, and E. Bahraoui, J. Bacteriol. 179; 2210-2220, 1997), a mycoplasma which was isolated for the first time from the urine of human immunodeficiency virus-infected patients. To evaluate how this nuclease could interact with host cells, we tested its effect on CEM and Molt-4 lymphocytic cell lines and on peripheral blood mononuclear cells. We observed that 10(-7) to 10(-9) M P40 is able to mediate a cytotoxic effect. We found that 100% of cells were killed after 24 h of incubation with 10(-7) M P40 while only 40% cytotoxicity was obtained after 72 h of incubation with 10(-9) M P40. Phase-contrast microscopy observations of P40-treated cells revealed morphological changes, including pronounced blebbing of the plasma membrane and cytoplasmic shrinkage characteristic of programmed cell death, which is in agreement with the internucleosomal fragmentation of P40-treated cell DNA as shown by agarose gel electrophoresis. We showed that (125)I-radiolabeled or fluorescein isothiocyanate-labeled P40 was able to bind specifically in a dose-dependent manner to the cell membrane of CEM cells, which suggested that the cytotoxicity of P40 endonuclease was mediated by its interaction with the cell surface receptor(s). The concentration of unlabeled P40 required to inhibit by 50% the formation of (125)I-P40-CEM complexes was about 3 x 10(-9) M, indicating a high-affinity interaction. Both P40 interaction and cytotoxicity are Ca(2+) dependent. Our results suggest that the cytotoxicity of M. penetrans observed in vitro is mediated at least partially by secreted P40, which, after interaction with host cells, can induce an apoptosis-like death. These results strongly suggest a major role of mycoplasmal nucleases as potential pathogenic determinants.

Calcium↗
PubMed1999

Pathways for HCO-3 exit across the basolateral membrane in rat thick limbs.

We studied the pathways for HCO-3 transport in basolateral membrane vesicles (BLMV) purified from rat medullary thick ascending limbs (MTAL). An inward HCO-3 gradient in the presence of an inside-positive potential stimulated the rate of 22Na uptake minimally and did not induce a 22Na overshoot, arguing against the presence of electrogenic Na+-HCO-3 cotransport in these membranes. An inside-acid pH gradient stimulated to the same degree uptake of 86Rb+ (a K+ analog) with or without HCO-3. Conversely, applying an outward K+ gradient caused a modest intracellular pH (pHi) decrease of approximately 0.38 pH units/min, as monitored by quenching of carboxyfluorescein; its rate was unaffected by HCO-3, indicating the absence of appreciable K+-HCO-3 cotransport. On the other hand, imposing an inward Cl- gradient in the presence of HCO-3 caused a marked pHi decrease of approximately 1.68 pH units/min; its rate was inhibited by a stilbene derivative. Finally, we could not demonstrate the presence of a HCO-3/lactate exchanger in BLMV. In conclusion, the presence of significant Na+-, K+-, or lactate-linked HCO-3 transport could not be demonstrated. These and other data suggest that basolateral Cl-/HCO-3 exchange could be the major pathway for HCO-3 transport in the MTAL.

Acids↗
PubMed1998

A simplified gradient solvent delivery system for capillary liquid chromatography-electrospray ionization mass spectrometry.

We describe a simple solvent delivery system for gradient capillary HPLC at nanoliter per minute flow rates. The novel aspect of the system is that solvents are delivered one at a time, using a switching valve, into a relatively large-volume mixing chamber. Efficient mixing in the chamber causes the formation of a sigmoidal gradient from the initial solvent to the subsequent solvent, which is then delivered to a capillary column. The shape of the gradients formed can be predicted from a simple theoretical model. Gradients of different slope can be formed by varying either the size of the chamber or the system flow rate. The system is robust, reproducible, and simple to operate. We provide a detailed protocol of how to construct a low-cost capillary HPLC system consisting of two syringe pumps, a capillary mixing chamber, a capillary column, and a zero dead-volume microelectrospray interface. We demonstrate that the coupling of this HPLC system to a mass spectrometer enabled us to identify proteins at the low femtomole level in solution-phase digests and at the picomole level in digests of samples separated on SDS-PAGE gels. We believe that the strategy presented will be useful as a general method for the characterization of proteins and peptides by capillary HPLC-electrospray mass spectrometry.

Chromatography, High Pressure Liquid↗
PubMed1998

NH4+ as a substrate for apical and basolateral Na(+)-H+ exchangers of thick ascending limbs of rat kidney: evidence from isolated membranes.

1. We have used highly purified right-side-out luminal and basolateral membrane vesicles (LMVs and BLMVs) isolated from rat medullary thick ascending limb (MTAL) to study directly the possible roles of the LMV and BLMV Na(+)-H+ exchangers in the transport of NH4+. 2. Extravesicular NH4+ ((NH4+)o) inhibited outward H+ gradient-stimulated 22Na+ uptake in both types of vesicles. This inhibition could not be accounted for by alteration of intravesicular pH (pHi). 3. Conversely, in both plasma membrane preparations, the imposition of outward NH4+ gradients stimulated 22Na+ uptake at the acidic pHi (6.60) of MTAL cells, under conditions in which possible alterations in pHi were prevented. All NH4+ gradient-stimulated Na+ uptake was sensitive to 0.5 mM 5-(N,N-dimethyl)-amiloride. 4. The BLMV and LMV Na(+)-H+ exchangers had a similar apparent affinity for internal H+ (Hi+), with pK (-log of dissociation constant) values of 6.58 and 6.52, respectively. 5. These findings indicate that NH4+ interacts with the external and internal transport sites of the LMV and BLMV Na(+)-H+ antiporters, and that both of these exchangers can mediate the exchange of internal NH4+ ((NH4+)i) for external Na+ (Na+o) at the prevailing pHi of MTAL cells. 6. We conclude that operation of the BLMV Na(+)-H+ exchanger on the NH4(+)-Na+ mode may represent an important pathway for mediating the final step of NH4+ absorption, whereas transport of NH4+ on the apical antiporter may provide negative feedback regulation of NH4+ absorption.

Ammonia↗
PubMed1998

Diagnosis and management of coronary artery disease.

Coronary artery disease (CAD) is a major cause of morbidity and mortality in the United States. This review considers the diagnosis and therapy of CAD in the general population free of renal disease. Diagnostic methods include clinical examination, noninvasive techniques and catheterization. Pharmacological therapy includes aspirin, beta-blockers, nitrates, calcium antagonists, lipid-lowering agents, and angiotensin-converting enzyme inhibitors. Revascularization includes percutaneous coronary angioplasty and coronary artery bypass grafting. Recent clinical trials that provide the basis for current cardiology guidelines are highlighted, however individualized care is underscored.

Cardiac Catheterization↗
PubMed1998

Molecular evidence for nosocomial transmission of human immunodeficiency virus from a surgeon to one of his patients.

We have investigated the molecular evidence in favor of the transmission of human immunodeficiency virus (HIV) from an HIV-infected surgeon to one of his patients. After PCR amplification, the env and gag sequences from the viral genome were cloned and sequenced. Phylogenetic analysis revealed that the viral sequences derived from the surgeon and his patient are closely related, which strongly suggests that nosocomial transmission occurred. In addition, these viral sequences belong to group M of HIV type 1 but are divergent from the reference sequences of the known subtypes.

Base Sequence↗
PubMed1998

Water and solute permeabilities of medullary thick ascending limb apical and basolateral membranes.

The medullary thick ascending limb (MTAL) reabsorbs solute without water and concentrates NH4+ in the interstitium without a favorable pH gradient, activities which require low water and NH3 permeabilities. The contributions of different apical and basolateral membrane structures to these low permeabilities are unclear. We isolated highly purified apical and basolateral MTAL plasma membranes and measured, by stopped-flow fluorometry, their permeabilities to water, urea, glycerol, protons, and NH3. Osmotic water permeability at 20 degrees C averaged 9.4 +/- 0.8 x 10(-4) cm/s for apical and 11.9 +/- 0.5 x 10(-4) cm/s for basolateral membranes. NH3 permeabilities at 20 degrees C averaged 0.0023 +/- 0.00035 and 0.0035 +/- 0.00080 cm/s for apical and basolateral membranes, respectively. These values are consistent with those obtained in isolated perfused tubules and can account for known aspects of MTAL function in vivo. Because the apical and basolateral membrane unit permeabilities are similar, the ability of the apical membrane to function as the site of barrier function arises from its very small surface area when compared with the highly redundant basolateral membrane.

Ammonia↗
PubMed1998

Functional and molecular characterization of luminal and basolateral Cl-/HCO-3 exchangers of rat thick limbs.

Cl-/HCO-3 exchange was measured in luminal (LMV) and basolateral (BLMV) membrane vesicles purified from rat medullary thick ascending limb (MTAL). Cl-/HCO-3 exchange in BLMV and LMV was inhibited by DIDS, with respective IC50 values of 3.2 +/- 0.9 and 15.2 +/- 5.2 microM, whereas Cl- conductances were DIDS insensitive. At constant external pH, BLMV 36Cl-/HCO-3 and 36Cl-/Cl- exchanges exhibited a sigmoidal pattern of activation as internal pH (pHi) increased from 6.1 to 8.0, whereas LMV 36Cl-/Cl- exchange was unchanged between pHi 6.7 and 7.8. The 165-kDa AE2 polypeptide and approximately 115-kDa AE1-related polypeptide were present only in BLMV. In contrast, AE1-related polypeptides of approximately 90 and 95 kDa were present not only in BLMV but also (in variable abundance) in LMV. We conclude that rat MTAL BLMV and LMV express distinct anion exchange activities and distinct sets of AE polypeptides. AE2 (and perhaps AE1) in BLMV likely contribute to HCO-3 absorption. In contrast, LMV exchangers may contribute to NaCl absorption via parallel coupling with the luminal Na+/H+ antiporters and/or may provide negative feedback regulation of HCO-3 absorption.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Influence of myopia and aging on the optimal spherical aberration of soft contact lenses.

Soft contact lenses with different levels of third-order spherical aberration were tested in two samples of subjects aged between 20 and 45 years: 18 emmetropes and 19 myopes. Contrast sensitivity was measured at 12 cycles/degree to determine the optimal lens spherical aberration required by each individual. The optimal third-order coefficient was found to be negative on average in both refractive error groups. Myopic subjects required contact lenses with more negative spherical aberration than did emmetropes. The optimal aberration was also found to become increasingly negative with aging. The rate of this age-related change was faster in the myopic group. In comparison with aberration-free soft contact lenses, an improvement in contrast detection threshold of more than 25% was observed with optimal spherical aberration in half of the myopic subjects.

Adult↗
PubMed1998

Analysis of tissue- and hormone-specific regulation of the human prolactin-inducible protein/gross cystic disease fluid protein-15 gene in transgenic mice.

The human prolactin-inducible protein/gross cystic disease fluid protein-15 (PIP/GCDFP-15) gene is expressed in more than 90% of human breast cancer biopsies but not in the normal mammary gland. However, it is expressed in several normal human apocrine glands such as the lacrimal and salivary glands. In human breast cancer cell lines, the gene is regulated by a number of hormones including androgen and prolactin. It is not known whether gene expression in normal tissues is under similar hormonal control. To understand the mechanisms by which hormone- and tissue-specific expression of the human PIP/GCDFP-15 gene are regulated in vivo, we generated transgenic mice using a 13.7 kb genomic DNA fragment containing the entire 7 kb human gene, together with 2.9 kilobases of 5' and 3.8 kilobases of 3' flanking sequences. The human PIP/GCDFP-15 transgene was found to be expressed in both the lacrimal and salivary glands but was not expressed in the mammary glands of transgenic mice. This tissue-specific pattern of the transgene expression in the mouse was very similar to that of the endogenous human PIP/GCDFP-15 gene, and to the endogenous mouse,gene. In the mouse salivary glands, the transgene expression was highest in the parotid, considerably less in the submaxillary (submandibular) and absent in the sublingual glands. In the mouse lacrimal gland, as in the human breast cancer cell lines, the human PIP/GCDFP-15 transgene was also up-regulated by androgen. These studies demonstrate that the human gene with its 6.3 kb flanking sequences is able to confer gene expression in vivo in a tissue-specific and hormone-responsive manner.

Animals↗
PubMed1997

Mycoplasmas and HIV infection, a possible interaction through immune activation.

The persistence of HIV in the host is associated with a state of chronic immune activation with deleterious effects. In addition to HIV, several infectious agents can contribute to this activation and, consequently, to an increased HIV load. We have postulated that mycoplasmas could act as cofactors of HIV, contributing to a faster progression of the disease through increased immune activation. Mycoplasma penetrans was shown to be associated with HIV infection and recent data have indicated the capacity of this mycoplasma to activate different functions of the immune system. However, its role as an HIV cofactor remains to be demonstrated.

AIDS-Related Opportunistic Infections↗
PubMed1997

P0 is constitutively expressed in the rat neural crest and embryonic nerves and is negatively and positively regulated by axons to generate non-myelin-forming and myelin-forming Schwann cells, respectively.

We show that in the rat, the major gene of PNS myelin, P0, is expressed long before myelination in the neural crest, Schwann cell precursors, and embryonic Schwann cells irrespective of whether they will myelinate or not. This myelin-independent P0 expression is constitutive and likely to serve as a specific marker for the Schwann cell lineage. The much higher P0 expression accompanying myelination is therefore not new gene expression but strong up-regulation of preexisting basal levels. We provide new evidence that the up-regulation to myelination-related levels depends on positive extrinsic signals and therefore does not represent a constitutive phenotype. P0 mRNA is not detectable in mature non-myelin-forming Schwann cells of the sympathetic trunk, but is detectable after transection, indicating that there is a P0-inhibitory signal associated with mature unmyelinated axons. Thus, the regulation of the P0 gene is complex, encompassing extrinsically signaled amplification superimposed on a highly lineage-specific and constitutive basal expression.

Animals↗
PubMed1997

Influence of microbial infections on the progression of HIV disease.

HIV infection is associated with immune activation, which in turn stimulates HIV replication. Certain other co-infections cause immune activation and may contribute to an increased viral load. The impact of co-infections by microorganisms such as Mycobacterium tuberculosis can be important for patient survival, particularly those at high risk of exposure to infection and with poor access to medical care.

AIDS-Related Opportunistic Infections↗
PubMed1997

Human immunodeficiency virus infection and AIDS in a person with negative serology.

A 38-year-old woman resident of Ivory Coast died of AIDS, while remaining human immunodeficiency virus (HIV)-seronegative. She had been regularly tested because her husband was HIV-seropositive. The subject's lack of specific antibodies was assessed using commercial tests and confirmed by a radioimmunoprecipitation assay of the patient's virus. She was unquestionably HIV-1-infected, with a high plasma virus load, and her virus could be isolated. Molecular analyses indicated this retrovirus was clade A, which is common in Africa, and it was highly homologous to the virus isolated from her husband. The subject's seronegative status was thought to be due to rapid depletion of specific CD4+ helper T cells, resulting from accelerated disease progression, and was host-related rather than due to a specific HIV strain.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Spiralin, a mycoplasmal membrane lipoprotein, induces T-cell-independent B-cell blastogenesis and secretion of proinflammatory cytokines.

Mycoplasmas are bacteria which can cause respiratory, arthritic, and urogenital diseases. During the early phase of infection, mycoplasmas usually induce an inflammatory response and a humoral response preferentially directed against their membrane-bound, surface-exposed lipoproteins. In this report, we describe the effects on immune cells of spiralin, a well-characterized mycoplasmal lipoprotein. Purified spiralin stimulated the in vitro proliferation of human peripheral blood mononuclear cells and murine splenocytes. The stimulation pathway was probably different from that followed by Escherichia coli lipopolysaccharide because the effect of spiralin was not abolished by polymyxin B. Comparison of the effects of whole, native spiralin with those induced by proteinase K-digested spiralin or by the C-terminal half of spiralin (peptide p[13.5]T) revealed that the first half of the protein, which contains the lipoylated N terminus, is responsible for the mitogenic activity. In contrast to whole spiralin, proteinase K-digested spiralin did not trigger murine B-cell differentiation and immunoglobulin G and M secretion. Stimulation of human or murine immune cells led to early secretion of proinflammatory cytokines (human tumor necrosis factor alpha and murine interleukin 1 or 6). Spiralin induced the T-cell-independent blastogenesis of murine B cells but did not stimulate T cells. Altogether, our data demonstrate that spiralin possesses potent immunostimulating activity, similar to that reported for lipoproteins of pathogenic gracilicutes (gram-negative eubacteria; e.g., Borrelia burgdorferi OspA and E. coli Braun lipoprotein), and are consistent with the fact that lipoproteins are major antigens during mycoplasma infections.

Animals↗
PubMed1997

Purification and characterization of Mycoplasma penetrans Ca2+/Mg2+-dependent endonuclease.

The major nuclease from Mycoplasma penetrans has been purified to homogeneity. The enzyme seems to be present as a membrane-associated precursor of 50 kDa and as a peripheral membrane monomeric polypeptide of 40 kDa that is easily removed by washing of cells with isotonic buffers and in the aqueous phase upon Triton partitioning of Triton X-114-solubilized protein. The 40-kDa nuclease was extracted from M. penetrans cells by Triton X-114 and phase fractionation and was further purified by chromatography on Superdex 75 and chelating Sepharose (Zn2+ form) columns. By gel filtration, the apparent molecular mass was 40 kDa. The purified enzyme exhibits both a nicking activity on superhelical and linear double-stranded DNA and a nuclease activity on RNA and single-stranded DNA. No exonuclease activity was found for this enzyme. This nuclease required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity and exhibited a pH optimum between pH 7 and 8 for DNase activity. It was inhibited by Zn2+, Mn2+, heparin, sodium dodecyl sulfate, and chelator agents such EDTA and EGTA, but no effect was observed with ATP, 2-mercaptoethanol, N-ethylmaleimide, dithiothreitol, nonionic detergents, phenylmethylsulfonyl fluoride, and iodoacetamide. Nuclease activity was inhibited by diethylpyrocarbonate at both pH 6 and 8 and by pepstatin, suggesting the involvement of a histidine and an aspartate in the active site. When added to human lymphoblast nuclei, the purified M. penetrans endonuclease induced internucleosomal fragmentation of the chomatin into oligonucleosomal fragments. On the basis of this result, and taking into account the fact that M. penetrans has the capacity to invade eucaryotic cells, one can suggest, but not assert, that produced Ca2+/Mg2+-dependent endonuclease may alter the nucleic acid metabolism of host cells by DNA and/or RNA degradation and may act as a potential pathogenic determinant.

Antigens, Bacterial↗