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Biomedical subjects

A Clerc

Publications and source records attributed to A Clerc.

15 recordsLinked to original sources
PubMed2004

Human mesenchymal stem cells suppress induction of cytotoxic response to alloantigens.

Mesenchymal stem cells (MSC) fail to induce allogeneic responses in mixed lymphocyte reaction assays. Because MSC express HLA class I molecules, here we investigated whether they could be recognized as allogeneic targets by cytolytic T lymphocytes (CTL). With this aim, CTL precursor (CTLp) frequencies were measured following stimulation of T cells with either allogeneic mononuclear cells (MNC) or MSC originated from the same human bone marrow donor. Lysis of MSC was measured at day 10 of culture in standard chromium release assays. In addition, allogeneic PHA blast T cells or B-EBV lymphoblastoid cell lines (LCLs) generated from the same donor were used as positive controls of lysis. Our results showed that when allogeneic MNC were used to stimulate T cells, a high CTLp frequency was detected towards MSC targets. However, when MSC were used as stimulators, CTLp frequencies were markedly altered whatever the targets used, i.e.: MSC, PHA blast T cells or EBV-B LCLs. Moreover, when graded concentrations of MSC were added together with MNC upon stimulation of alloreactive T cells, we observed a dose-dependent decrease in CTLp frequencies towards MSC targets. This inhibition of MSC lysis was partially overcome by adding exogenous rh-IL-2 from the beginning of cultures. In addition, this suppressive effect was totally reproduced when, instead of MSC, supernatant harvested from MSC cultures was added to allogeneic MNC, upon stimulation of alloreactive T cells. In conclusion, our results demonstrate that MSC which can be recognized as targets by pre-activated alloreactive CTLs, may be able to suppress differentiation of CTL precursors into CTL effectors through secretion of suppressive factors.

Cell Differentiation↗
PubMed1998

Comparison of Randomly Amplified Polymorphic DNA with Amplified Fragment Length Polymorphism To Assess Genetic Diversity and Genetic Relatedness within Genospecies III of Pseudomonas syringae.

Recently, DNA pairing analyses showed that Pseudomonas syringae pv. tomato and related pathovars, including P. syringae pv. maculicola, form a genomic species (Pseudomonas tomato) (L. Gardan, H. L. Shafik, and P. A. D. Grimont, p. 445-448, in K. Rudolph, T. J. Burr, J. W. Mansfield, D. Stead, A. Vivian, and J. von Kietzell, ed., Pseudomonas syringae Pathovars and Related Pathogens, 1997). The genetic diversity of 23 strains belonging to this genomic species and 4 outgroup strains was analyzed with randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphic (AFLP) techniques. Simple boiling of P. syringae cells was suitable for subsequent DNA amplification to obtain reliable patterns in RAPD and AFLP analyses. In general, the grouping of P. syringae strains by both analysis techniques corresponded well with the classification obtained from an RFLP analysis of ribosomal DNA operons, DNA pairing studies, and an analysis of pathogenicity data. However, two strains of P. syringae pv. maculicola produced distinct DNA patterns compared to the DNA patterns of other P. syringae pv. maculicola strains; these patterns led us to assume that horizontal transfer of DNA could occur between bacterial populations. Both techniques used in this study have high discriminating power because strains of P. syringae pv. tomato and P. syringae pv. maculicola which were indistinguishable by other techniques, including pathogenicity tests on tomato, were separated into two groups by both RAPD and AFLP analyses. In addition, data analysis showed that the AFLP method was more efficient for assessing intrapathovar diversity than RAPD analysis and allowed clear delineation between intraspecific and interspecific genetic distances, suggesting that it could be an alternative to DNA pairing studies. However, it was not possible to distinguish the two races of P. syringae pv. tomato on the basis of an analysis of the data provided by either the AFLP or RAPD technique.

Journal Article↗
PubMed1996

Growth response of adult germ cells to rat androgen-binding protein and human sex hormone-binding globulin.

Androgen-binding protein (ABP) is produced by Sertoli cells depending on the development and the stage of the spermatogenic cycle. Germ cell proliferation is at its peak when ABP is at its peak and secreted towards the testicular basal compartment containing spermatogonia and premeiotic spermatocytes. Rat isolated adult germ cell DNA synthesis was studied in vitro in the presence of ABP with and without steroids and in the presence of pure or recombinant sex steroid hormone-binding globulin (SHBG) using thymidine incorporation. Results are: SHBG is able to promote DNA synthesis in the absence of cofactors. Testosterone reacted negatively to the stimulatory effect of SHBG. We conclude that ABP, the physiological steroid-binding protein, should be considered as a paracrine regulator of spermatogenic DNA synthesis in the adult rat.

Androgen-Binding Protein↗
PubMed1992

Interaction between cGMP-phosphodiesterase and transducin alpha-subunit in retinal rods. A cross-linking study.

Cross-linking of the different subunits of the retinal cGMP-phosphodiesterase (PDE) with its activator G alpha GTP gamma S (alpha subunit of the retinal G-protein transducin with GTP gamma S (guanosine 5'-O-(3-thiotriphosphate) bound) has been investigated using purified proteins, with a N-hydroxysuccinimide homobifunctional cross-linker, bis(sulfosuccinimidyl)suberate (BS3) and its cleavable analog 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP). Interaction of purified G-protein and PDE is achieved in the presence of lecithin vesicles, at protein concentrations sufficient for full PDE activation. Protein subunits linked with DTSSP are separated by cleavage of the disulfide bridge and identified by electrophoresis. Complexes of PDE alpha (PDE beta) with 1 and 2 molecules of activator G alpha GTP gamma S are observed, providing direct evidence for an interaction or at least a close proximity between 2 molecules of activator G alpha and each of the catalytic PDE subunits in the activated state of PDE. The results also reveal symmetrical roles of PDE alpha and PDE beta, with the existence of one site for PDE gamma and one site for G alpha on each catalytic subunit.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Activated cGMP phosphodiesterase of retinal rods. A complex with transducin alpha subunit.

Purified G-protein (transducin) activated with the nonhydrolyzable analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and cGMP phosphodiesterase (PDE) from retinal rods are added to protein-stripped disc membranes. Specific binding of the mainly soluble alpha subunit of G-protein with GTP gamma S bound (G alpha GTP gamma S, activator of the PDE) to the disc membrane in the presence of PDE is measured from gel scans or experiments with labeled G-protein alpha subunit (G alpha). Its variation as a function of G concentration matches the theoretical variation of G alpha involved in the activation of PDE calculated with previously estimated dissociation constants (Bennett, N., and Clerc, A. (1989) Biochemistry 28, 7418-7424), and the G alpha bound/PDE ratio at saturation is close to 2. No increase of G alpha binding to the membrane is observed when purified inhibitory subunit of PDE (PDE gamma) is added together with or instead of total PDE, and excess PDE gamma remains soluble. These results suggest that activated PDE is a complex with the activator G alpha GTP rather than PDE from which the inhibitory subunits have been removed. A method for purifying PDE gamma with a high yield of recovery and activity is described.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

cGMP phosphodiesterase dependent light-induced scattering changes in suspensions of retinal disc membranes.

Light-induced GTP-dependent scattering changes are studied in suspensions of retinal disc membranes to which one or both of the purified proteins involved in the phototransduction mechanism (G-protein and cGMP phosphodiesterase) are reassociated; a scattering change which depends on the presence of both G-protein (G) and inhibited cGMP phosphodiesterase (PDE) and on an ATPase-dependent process, previously described in Bennett [(1986) Eur. J. Biochem. 157, 487-495] is compared to the signal observed in the absence of PDE or of ATP and to PDE activity. The same signal can also be induced either in the dark or in the light by addition of preactivated G in the presence of inhibited PDE. This PDE-dependent scattering change is composed of two components (fast and slow); the variation of the amplitude and kinetics of both components with PDE or G concentration is similar to the variation of the active PDE state with two activator GGTP molecules (G with GTP bound), calculated with dissociation constants previously reported for the interaction between GGTP and PDE [Bennett, N., & Clerc, A. (1989) Biochemistry 28, 7418-7424]. The two components are therefore proposed to be associated with processes which depend on the formation of the active PDE state with two activators.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1990

Molecular characterization of human complement factor B subtypes.

While several laboratories have agreed that there are two subtypes of the BF*F alleles, no information has been available until now at the molecular level. The region of the BF gene corresponding to the Ba fragment [1.7 kilobases (kb)] of the BF*S, BF*FA, and BF*FB alleles has been sequenced after specific amplification using the polymerase chain reaction (PCR). A point mutation at codon 7 has been revealed converting a cytosine in the BF*S allele to a thymidine in BF*FB. At the translational level an arginine residue in BF*S is substituted for a tryptophan residue in BF*FB. The amino-terminal sequencing of factor B immunoprecipitated from serum has been carried out from microquantities of protein blotted onto polyvinylidene fluoride (PVDF) membranes. We have shown that the difference between the BF*FA and the BF*FB subtypes in characterized by a glutamine at position 7 in BF*FA and a tryptophan in BF*FB.

Base Sequence↗
PubMed1989

Activation of cGMP phosphodiesterase in retinal rods: mechanism of interaction with the GTP-binding protein (transducin).

The mechanism of activation of cGMP phosphodiesterase by the GTP-binding protein in the disc membrane of retinal rods has been investigated by measuring the light-induced phosphodiesterase activity in reconstituted systems where the concentration of either the GTP-binding protein or the phosphodiesterase is varied. The results are consistent with the existence of two activator sites per phosphodiesterase functional unit: binding of one G alpha GTP (alpha subunit of the G-protein with GTP bound) with high affinity (100 +/- 50 nM) partially activates the enzyme (Vmax1 approxmately 0.05 Vmax to 0.10V max to trypsin-activated phosphodiesterase); binding of a second G alpha GTP with lower affinity (600 +/- 100 nM) induces maximal activation (Vmax2 approximately Vmax of trypsin-activated phosphodiesterase). The two different states of activated phosphodiesterase have the same Km for cGMP and the same pH dependence; they differ in their sensitivity to GMP. Micromolar concentration of protamines increases the affinity of the two activator sites and slightly increases Vmax1. When G-protein is activated with GTP-gamma S instead of GTP, the affinities of the two activator sites are not significantly modified, while Vmax1 appears to be increased.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1989

Direct activation of cGMP-dependent channels of retinal rods by the cGMP phosphodiesterase.

The cationic conductances of purified bovine retinal rod membranes were studied by incorporation of vesicles into planar lipid bilayers. When the membranes were stripped of all peripheral proteins [guanine nucleotide-binding protein (G protein) and cGMP phosphodiesterase (3',5'-cyclic-GMP 5'-nucleotidohydrolase), EC 3.1.4.35], sodium and calcium fluxes were almost only observed in the presence of cGMP. Reconstitution experiments in which purified cGMP phosphodiesterase alone or with G protein were reassociated to the vesicles in proportions similar to those found in the native rod provide evidence for a direct interaction between the cGMP-dependent channel protein and the phosphodiesterase. (i) In its inhibited state, phosphodiesterase markedly stimulates the activity of the channels in the presence of cGMP (situation in the dark-adapted rod) but is not capable of activating the channels in the absence of cGMP. (ii) In the absence of cGMP, activation of the phosphodiesterase by G protein with GTP bound (equivalent to photoexcitation) induces the opening of cation channels that have the same conductance for sodium ions as cGMP-activated channels (20-22 pS, with two sublevels of about 7 pS and 13 pS).

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1987

Biochemical and biological properties of an alpha 1-antitrypsin concentrate.

alpha 1-Antitrypsin (AAT) has been purified from human plasma supernatant A (equivalent to COHN fraction II + III) by a large-scale chromatographic procedure involving anion-exchange adsorption on DEAE Sepharose CL-6B fast flow and size-exclusion chromatography on Sephacryl S-200. Before freeze-drying, the liquid concentrate was heat-treated at 60 degrees C for 10 h to reduce the risk of transmission of blood-born viral diseases. Using this procedure, AAT is recovered with 80-90% purity in 65-75% yield from supernatant A. The heterogeneity of AAT is preserved across the purification steps. In addition, purified AAT exhibits inhibitory activities against trypsin and elastase equivalent to that of the serum protein. The mean association rate constant for elastase was found as high as 2.15 X 10(5) M-1 s-1. Thus, purifying active AAT from supernatant A contributes to improving the availability of this protein which may be potentially useful in the treatment of hereditary emphysema.

Chromatography, DEAE-Cellulose↗
PubMed1987

Large scale chromatographic purification of alpha 1-antitrypsin from human plasma supernatant II + III.

We describe a large-scale chromatographic method to purify alpha 1-antitrypsin (AAT) from human plasma supernatant II + III. Supernatant II + III was injected onto DEAE sepharose CL 6B to get an albumin-rich fraction as well as a fraction that contained about 45% AAT. This intermediate purity AAT fraction was further purified by size-exclusion chromatography on Sephacryl S-200. The product was heated in solution at 60 degrees C for 10 hours in the presence of a stabilizer to lower the risk of transmission of viral diseases. Purity of the AAT concentrate is over 85%; clinical testing of its safety and efficacy in the treatment of PiZ patients is underway.

Blood Proteins↗
PubMed1981

Haptoglobin polymorphism among Saharian and West African groups. Haptoglobin phenotype determination by radioimmunoelectrophoresis on Hp O samples.

The haptoglobin (Hp) polymorphism is investigated in 11 African groups living in an area from the Algerian Sahara to Central Africa. More than 4,000 samples were examined. In the Saharian samples, the Hp1 gene frequency is higher than in any other African group. From north to south, a decrease in the Hp1 gene frequency is observed; in the Pygmy sample only, this frequency is lower than the frequency of the Hp2 gene. By means of a sensitive radioimmunoelectrophoresis, the presence of a residual Hp in Hp O sera in which the Hp polymorphism can also be determined can be revealed. Absence of Hp 1-1 and significant excess of Hp 2-2 individuals were observed. More Hp 2-1M phenotypes were detected in the Hp O population than in the non-Hp O population examined. In the Hp O samples, the influence of the phenotype distribution on the Hp gene frequencies is discussed. The heavy polymers of the Hp related to the presence of the alpha 2 chain (Hp2 gene product) are involved only in the biological mechanisms responsible for the presence of Hp O and Hp 2-1 M phenotypes among African groups.

Africa, Central↗
PubMed1976

[Chromatographic analysis of urinary amino acids in Paget's disease. I : Biochemical study. Isolation and amino acid composition of a urinary peptide specific to this bone disease].

The authors demonstrated in the urine of patients presenting Paget's bone disease a peptide rich in hydroxyproline. The level of this compound expressed as "norleucine equivalent" was determined by chromatographic analysis of the urinary amino acids. There was a very good correlation between the total hydroxyproline level and the quantity of this peptide in the urine. The authors isolated this peptide and determined that it contained 3 hydroxyprolines for every 2 glutamines. This peptide thus seems to indicate an anomaly in the chain of synthesis; a disorder of the collagen metabolism would produce in these patients large quantities of these molecules that are excreted via the kidney. At the present stage of the author's studies this urinary peptide appears to be "specific" to Pagets disease.

Amino Acids↗