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P Constant

Publications and source records attributed to P Constant.

At least 19 recordsLinked to original sources
PubMed2001

Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis. Evidence that this lipid is involved in the cell wall permeability barrier.

Among the few characterized genes that have products involved in the pathogenicity of Mycobacterium tuberculosis, the etiological agent of tuberculosis, are those of the phthiocerol dimycocerosate (DIM) locus. Genes involved in biosynthesis of these compounds are grouped on a 50-kilobase fragment of the chromosome containing 13 genes. Analysis of mRNA produced from this 50-kilobase fragment in the wild type strain showed that this region is subdivided into three transcriptional units. Biochemical characterization of five mutants with transposon insertions in this region demonstrated that (i) the complete DIM molecules are synthesized in the cytoplasm of M. tuberculosis before being translocated into the cell wall; (ii) the genes fadD26 and fadD28 are directly involved in their biosynthesis; and (iii) both the drrC and mmpL7 genes are necessary for the proper localization of DIMs. Insertional mutants unable to synthesize or translocate DIMs exhibit higher cell wall permeability and are more sensitive to detergent than the wild type strain, indicating for the first time that, in addition to being important virulence factors, extractable lipids of M. tuberculosis play a role in the cell envelope architecture and permeability. This function may represent one of the molecular mechanisms by which DIMs are involved in the virulence of M. tuberculosis.

Base Sequence↗
PubMed1998

Phagosomal pH determination by dual fluorescence flow cytometry.

Several methods have been developed to measure the pH of phagosomes, using fluorescein derivatives as reporter of pH, and spectrofluorimetry, fluorescence microscopy, or flow cytometry as quantification technique. All have major disadvantages, including either a slow or inaccurate response. In the present study, pH determination was achieved on J774-cell phagosomes containing dual-labeled zymozan particles using dual fluorescence flow cytometry with an argonion laser excitation wavelength at 488 nm. This allowed zymozan-containing macrophages to be distinguished from other cells and their fluorescence to be measured rapidly. The use of a new probe, namely Oregon Green 488 which has a pKa lower than carboxyfluorescein with the same maximum excitation and emission wavelengths, allowed investigation of pH value below 5. The dual labeling with Oregon Green 488 and carboxytetramethylrhodamine as pH-sensitive and pH-insensitive probes, respectively, overcame the absence of an isobestic point in the Oregon Green 488 spectrum. The phagosomal pH was determined using a calibration curve of phagosomal pH established by adding ionophores in phagocyte suspension and measuring the fluorescence intensity ratio (535 nm/585 nm) for different pHs. A phagosomal pH of 4.5 +/- 0.1 can be accurately determined. This method permits pH measurements down to 4, even in the presence of nonengulfed reporter particles.

Animals↗
PubMed1998

PCR-ribotyping of Xenorhabdus and Photorhabdus isolates from the Caribbean region in relation to the taxonomy and geographic distribution of their nematode hosts.

The genetic diversity of symbiotic Xenorhabdus and Photorhabdus bacteria associated with entomopathogenic nematodes was examined by a restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes (rDNAs). A total of 117 strains were studied, most of which were isolated from the Caribbean basin after an exhaustive soil sampling. The collection consisted of 77 isolates recovered from entomopathogenic nematodes in 14 Caribbean islands and of 40 reference strains belonging to Xenorhabdus and Photorhabdus spp. collected at various localities worldwide. Thirty distinctive 16S rDNA genotypes were identified, and cluster analysis was used to distinguish the genus Xenorhabdus from the genus Photorhabdus. The genus Xenorhabdus appears more diverse than the genus Photorhabdus, and for both genera the bacterial genotype diversity is in congruence with the host-nematode taxonomy. The occurrence of symbiotic bacterial genotypes was related to the ecological distribution of host nematodes.

Animals↗
PubMed1997

Mycobacterium smegmatis phosphoinositols-glyceroarabinomannans. Structure and localization of alkali-labile and alkali-stable phosphoinositides.

Lipoarabinomannans from fast growing Mycobacterium sp., namely AraLAMs, stimulate the early events of macrophage activation. The immunological activities of all of these AraLAMs drastically decrease with the loss of the mild alkali groups, which were believed to be restricted to the fatty acid residues from the phosphatidyl-myo-inositol anchor. This report reveals the presence and the structure of mild alkali-labile phosphoinositide units linked via the phosphate to the C-5 of the beta-D-Araf in the AraLAMs of Mycobacterium smegmatis, a fast growing mycobacterial species. Their structure was unambiguously established with a strategy based on both one-dimensional 31P and two-dimensional 1H-31P heteronuclear multiple quantum correlation spectroscopy (HMQC) and HMQC-homonuclear Hartmann-Hahn spectroscopy NMR experiments applied to native AraLAMs and to AraLAMs treated in mild alkali conditions. Next to these alkali-labile phosphoinositides estimated at three per molecule, two other mild alkali-stable phosphoinositide units were identified: the expected (myo-inositol-1)-phosphate-(3-glycerol) unit typifying the well known glycosylphosphatidylinositol anchor of the mannan core and, more surprisingly, one (myo-inositol-1)-phosphate-(5-beta-D-Araf) unit having the same structure as the alkali-labile ones. Moreover, these four phosphoinositide units were found capping the arabinan side chains. Thus, their different behavior toward mild alkaline hydrolysis was explained according to their accessibility to the alkali reagent. This novel class of LAMs, namely phosphoinositols-glyceroarabinomannans (PI-GAMs), are characterized by their phosphoinositide units but also by the absence of fatty acid residues. These PI-GAMs were found to elicit the secretion of tumor necrosis factor-alpha, suggesting that phosphoinositides are the major PI-GAM epitope involved in this process.

Antigens, Bacterial↗
PubMed1996

High-pH anion-exchange chromatographic analysis of phosphorylated compounds: application to isolation and characterization of nonpeptide mycobacterial antigens.

A rapid and sensitive high-pH anion-exchange chromatography (HPAEC) method for the separation and quantification of phosphorylated antigens in mycobacterial extracts has been developed. This method provides the separation of mono-, di-, or triphosphonucleotides and of various other phosphorylated molecules. Dual detection by conductimetry and UV absorption downstream of a chemical suppressor constitute nondegradative and highly sensitive tools for the physical detection and the quantification of phosphorylated compounds in biological samples. The lower limit of accurate quantification is around 1 nmol per sample. This method was used for the separation of several phosphorylated antigens activating human gamma delta T lymphocytes from semipurified mycobacterial fractions. Their quantification revealed that the minimal concentration activating a gamma delta T cell clone is between 1 and 5 nM. This approach can be used for more general preparative purposes with samples where minute amounts of biologically active phosphoanions are analyzed.

Antigens, Bacterial↗
PubMed1996

A novel nucleotide-containing antigen for human blood gamma delta T lymphocytes.

The stimulation of human gamma delta T cells by mycobacteria occurs through recognition of four distinct nonpeptide phosphorylated antigens termed TUBag1-4. Among these latter, TUBag4 has already been biochemically characterized as a gamma-X derivative of 5'-deoxythymidine triphosphate (Constant, P., Davodeau, F., Peyrat, M. A., Poquet, Y., Puzo, G., Bonneville, M. and Fournié, J.-J., Science 1994. 264: 267). However, despite chemical synthesis of weakly stimulatory nucleotide-containing analogs, these mycobacterial compounds remained the sole nucleotide-containing antigens actually isolated from natural sources. Here, we present the complete isolation of the TUBag3 antigen from Mycobacterium fortuitum and demonstrate that this nonpeptide molecule contains a 5'-UTP nucleotide moiety. On selected V gamma 9/V delta 2 clones, T cell responses can be triggered with nanomolar concentrations of TUBag3. Like crude mycobacterial extracts, this purified nucleotide conjugate elicits a strong polyclonal response of gamma delta PBL from healthy donors. Furthermore, we present evidence that this compound is distinct from the recently synthesized gamma-isopentenyl 5'-UTP, a nucleotide conjugate of isopentenyl pyrophosphate that was found to be stimulatory for human gamma delta T cells (Tanaka, Y., Morita, C.T., Tanaka, Y., Nieves, E., Brenner, M. B. and Bloom, B. R., Nature 1995. 375: 155). Since it appears that both mycobacterial nucleotide antigens are molecules structurally related to peculiar precursors of nucleic acid synthesis, we propose that TUBag-reactive T cells might be specifically devoted to surveillance of proliferating cells.

Antigens, Bacterial↗
PubMed1996

Plasmodium falciparum stimuli for human gammadelta T cells are related to phosphorylated antigens of mycobacteria.

The presence in Plasmodium falciparum of a mitogenic factor for the major human blood gammadelta T-cell subset has been known for years. These gammadelta T cells bearing T-cell receptor Vgamma9 and Vdelta2 variable regions also respond to Mycobacterium tuberculosis, through recognition of several phosphorylated nonpeptidic antigens. In this study, we undertook a better characterization of the malarial stimulus and show that the polygonal activation of Vgamma9/Vdelta2 gammadelta T cells by P. falciparum schizonts is also and exclusively attributable to two phosphorylated malarial compounds. The finding of such stimuli in eukaryotic cells evidence an antigenic link between intracellular parasites as different as Plasmodium and Mycobacterium species. Hence, phosphorylated antigens could be involved in a common pattern of transdisease T-cell responses against various human pathogens.

Animals↗
PubMed1995

Early activation of human V gamma 9V delta 2 T cell broad cytotoxicity and TNF production by nonpeptidic mycobacterial ligands.

Human V gamma 9V delta 2 T cells were shown recently to respond to nonpeptidic phosphorylated molecules of mycobacterial origin (previously referred to as TUBag). To investigate the early events of V gamma 9V delta 2 T cell activation, we have analyzed induction of cytotoxicity and TNF production of T cell clones by these molecules. We showed that within minutes after exposure, TUBag induced cytotoxicity of V gamma 9V delta 2 CTL (but not of CTL expressing other TCR V gamma/V delta or V alpha/V beta regions) against a broad set of target cells, including effector cells themselves. Induction of V gamma 9V delta 2 cytotoxicity by TUBag was blocked by anti-TCR mAbs and was abrogated after dephosphorylation of TUBag. Similarly, TUBag, but not dephosphorylated TUBag, induced massive TNF production by V gamma 9V delta 2 T cell clones only, which already was significant 20 min after exposure. Of note, only basal amounts of TNF were produced when cells were maintained in suspension in the presence of TUBag, indicating that efficient activation of TNF production induced by these compounds required a cell-to-cell contact. Finally, preincubation experiments allowed us to demonstrate that activation of V gamma 9V delta 2 T cells was strictly dependent on the presence of TUBag because preincubation of the targets with TUBag followed by a single wash abrogated the activation. Taken together, these results strongly suggest that activation of V gamma 9V delta 2 cells by TUBag occurs after binding of these compounds to (a) yet unidentified, highly conserved, and broadly distributed molecule(s). The results also suggest either that TUBag induces a very rapid and transient expression of a V gamma 9V delta 2 TCR ligand or, more likely, that TUBag is a low affinity component of a complex recognized by the V gamma 9V delta 2 TCR.

Antibodies, Monoclonal↗
PubMed1995

The antituberculous Mycobacterium bovis BCG vaccine is an attenuated mycobacterial producer of phosphorylated nonpeptidic antigens for human gamma delta T cells.

The mycobacterial antigens stimulating human gamma delta T lymphocytes (R. L. Modlin, C. Permitz, F. M. Hofman, V. Torigian, K. Uemura, T. H. Rea, B. R. Bloom, and M. B. Brenner, Nature (London) 339:544-548, 1989; D. H. Raulet, Annu. Rev. Immunol. 7:175-207, 1989) have been characterized recently in Mycobacterium tuberculosis H37Rv as a group of four structurally related nucleotidic or phosphorylated molecules, termed TUBag1 to -4 (tuberculous antigens 1 to 4) (P. Constant, F. Davodeau, M. A. Peyrat, Y. Poquet, G. Puzo, M. Bonneville, and J. J. Fournie, Science 264:267-270, 1994). Here, we analyzed their distribution in different mycobacterial species of the M. tuberculosis group, with special emphasis on the human vaccine Mycobacterium bovis BCG. We show that the same four TUBag1 to -4 molecules are shared by these mycobacteria. Quantitative comparison reveals, however, that while the pathogen M. bovis and M. tuberculosis species produce rather high amounts of TUBag, all of the BCG strains have a surprisingly reduced production of TUBag. These observations suggest that among tuberculous mycobacteria, the bacterial TUBag load could, to some extent, constitute an immunological determinant of mycobacterial virulence for humans.

Antigens, Bacterial↗
PubMed1994

Stimulation of human gamma delta T cells by nonpeptidic mycobacterial ligands.

Most human peripheral blood gamma delta T lymphocytes respond to hitherto unidentified mycobacterial antigens. Four ligands from Mycobacterium tuberculosis strain H37Rv that stimulated proliferation of a major human gamma delta T cell subset were isolated and partially characterized. One of these ligands, TUBag4, is a 5' triphosphorylated thymidine-containing compound, to which the three other stimulatory molecules are structurally related. These findings support the hypothesis that some gamma delta T cells recognize nonpeptidic ligands.

Antigens, Bacterial↗
PubMed1978

Comparative results of 99mTc-pertechnetate scintigraphy and computerized tomography after contrast injection in cerebral pathology.

Fifty-six patients presenting acute cerebral ischemia were investigated by means of 160 CT and 90 RN scans. CT was positive before contrast injection in 90% of cases. A total of 120 CTs were done before and after contrast injection. Only 31% of the ischemic areas were enhanced. Out of 90 RN scans, 50 showed localized uptake. The peak of scintigraphic detection was found on the 15th day of evolution, while enhancement at CT was more frequent between the 15th and 21st days. For the hematomas (13 cases) detection by RN scan depends on the size of the lesion. Among the 47 patients presenting cerebral tumor, the RN uptake depends above all on the intensity of the BBB lesion. RN uptake and CT enhancement both show the same blood-brain barrier disturbance.

Brain↗
PubMed1978

Prognostic value of rCBF measurements and CT in focal cerebral ischemia.

CT is the most effective examination technique for studying the evolution of ischemic attacks, but if performed within the first 3 weeks it does not allow prognosis of possible evolution towards necrosis. CBF measurement and vasoreactivity tests under Althesin do allow this prognosis. Thirty patients whose evolution was checked clinically and by repeated CT examinations (89 in all) underwent CBF measurements (intra-arterial xenon 133) and vasoreactivity tests. In all cases (17 patients) where vasoreactivity had completely disappeared (inverse steal), the infarct evolved towards necrosis.

Brain↗
PubMed1978

Vascular thrombosis induced by direct electric current. A new technique for therapeutic embolisation; animal experimentation; first clinical applications.

A first series of experiments showed that the passage of a direct current along a positive electrode immersed in heparinised blood caused the formation of an adhering clot whose diameter increased with time and current intensity. A second series of experiments was done on 10 rabbits as follows: under general anaesthesia, a catheter used as a guide to the positive electrode was surgically inserted in the abdominal aorta. The negative electrode was placed on the thigh. A direct current (10 mA, 10V) was applied for 10-20 min. The first two clinical applications in man of this new method of treatment by electric current thrombosis were carried out using catheterisation of branches of the external carotid and cervical arteries.

Adolescent↗
PubMed1978

Cerebral blood volume and water extraction from cerebral parenchyma by hyperosmolar contrast media.

The attenuation coefficients of cerebral parenchyma and systemic blood were simultaneously measured at varying times after injection of two contrast agents with differing osmolarities. Cerebral parenchyma was in fact denser than the theory would predict. The osmolarity of the iodinated contrast agents seems to play only a very minor part. However, liposolubility could perhaps explain the leakage of iodine across the BBB and thus the increase in the attenuation coefficient of cerebral parenchyma.

Blood Volume↗
PubMed1978

Variations in the measurement of regional cerebral blood volume by CAT.

The cerebral blood volume (CBV) has been measured by transverse axial tomography. The attenuation coefficient of 100 "pixels" located in a well determined region was measured before and after injection of a contrast material (Telebrix 38). The attenuation coefficient of blood samples was measured by CT in a phantom. Blood samples were drawn before and after injection at the same time as the tomographic section (time: half rotation of the scanner). The volume was calculated by the (formula: see text). The values obtained with very early measurements (1--5 min after injection), were very close to those measured by other methods (3--5 ml/100 ml). For 5 patients, the CBV was measured by the same method at different times: 20 measures between 2 and 50 min after the injection. The CBV seemed to vary in function with time according to the equation: CBV in ML%: ate-bt + Vo (t = time in minutes: a = integration constant, a = 1.94; b = time constant, b = 0.089; Vo = real CBV). The apparent variation of CBV was the result of two factors related to the blood-brain barrier, (1) water movement from the extravascular space towards the intravascular space, related to the hyperosmolarity of the contrast medium. The variations of osmolarity and haematocrit values measured after contrast injection did confirm this hypothesis; and (2) iodine movement from the intravascular space towards the extravascular space, due to the toxicity of the material (hyperosmolarity, Na ions, etc). The measurement of CBV by CT is only possible if the contrast materials do not modify the blood-brain barrier. The use of actual materials allows the study of the blood-brain barrier behaviour and enables to make an estimate of the cerebral extra-cellular space. But CT is not a right method of CBV measurement with the contrast medium in use nowadays.

Blood Volume↗
PubMed1978

Cerebral blood flow and metabolism during etomidate anaesthesia in man.

The effects of etomidate on regional cerebral blood flow (rc.b.f.) and cerebral metabolic rate for oxygen (CMRo2) were studied in seven patients undergoing diagnostic carotid angiography. Following determination of baseline rc.b.f. while awake, the patients were anaesthetized with a single dose of etomidate 15 mg. Thereafter, an infusion of etomidate (2 or 3 mg min-1) was administered. Etomidate decreased both rc.b.f.10 (mean decrease 34%) and CMRo2 (mean decrease 45%). It was concluded that etomidate is a potent cerebral metabolic depressant. Furthermore, the cerebrovascular reactivity to carbon dioxide was maintained under etomidate anaesthesia.

Adult↗
PubMed1978

Effects of a diazepam-fentanyl mixture on cerebral blood flow and oxygen consumption in man.

The effects of a mixture of diazepam and fentanyl on cerebral blood flow (c.b.f.) and cerebral metabolism of oxygen (CMRo2) were studied in eight normal subjects and 13 patients with organic brain disease. The coupling of flow and metabolism and the carbon dioxide responsiveness of the c.b.f. were studied also. In the normal subjects the injection of the mixture resulted in a significant decrease in c.b.f. (34%), and a similar decrease in CMRo2 (34.5%). The vasoreactivity of the brain to carbon dioxide was maintained. C.b.f. decreased in all patients with intracranial pathology.

Brain↗