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Biomedical subjects

C Baron

Publications and source records attributed to C Baron.

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

Graft-host tolerance in bone marrow transplant chimeras. Absence of graft-versus-host disease is associated with unresponsiveness to minor histocompatibility antigens expressed by all tissues.

Because bone marrow (BM) transplantation is used with increasing frequency, it is important to elucidate the mechanisms involved in the establishment of tolerance to host minor histocompatibility antigens (MiHA) in recipients transplanted with T-cell-undepleted marrow grafts. We have previously shown that BM chimeras transplanted across MiHA barriers showed specific unresponsiveness to MiHA expressed on recipient-type concanavalin A blasts. Because expression of many MiHA is tissue-specific, we wanted to determine if chimera T lymphocytes would be tolerant to MiHA expressed by all host tissues and organs. To investigate this issue, we measured in vivo proliferation of lymphoid cells from normal C57BL/10 (B10) mice and (B10-->LP) chimeras in tissues and organs of lethally irradiated syngeneic and allogeneic recipients. Donor B10 cells were either untreated, or depleted with anti-Thy-1.2, anti-CD4, or anti-CD8 antibodies. Transplantation of B10 cells in LP recipients triggered an important T-cell-dependent 125I-dUrd uptake in several organs that involved both CD4+ and CD8+ cells. Using Thy-1-congeneic mice we showed that in long-term chimeras practically all CD4+ and CD8+ T lymphocytes were derived from hematopoietic progenitors and not from mature T cells present in the BM graft. When (B10-->LP) BM chimera cells were injected to secondary recipients, no proliferation was observed in any organ of LP hosts whereas normal proliferation was seen in H-2k allogeneic hosts. Thus, in these BM chimeras, tolerance encompasses MiHA expressed by all organs.

Animals↗
PubMed1994

Eukaryotic selenocysteine inserting tRNA species support selenoprotein synthesis in Escherichia coli.

Although the tRNA species directing selenocysteine insertion in prokaryotes differ greatly in their primary structure from that of their eukaryotic homologues they share very similar three-dimensional structures. To analyse whether this conservation of the overall shape of the molecules reflects a conservation of their functional interactions it was tested whether the selenocysteine inserting tRNA species from Homo sapiens supports selenoprotein synthesis in E. coli. It was found that the expression of the human tRNA(Sec) gene in E.coli can complement a lesion in the tRNA(Sec) gene of this organism. Transcripts of the Homo sapiens and Xenopus laevis tRNA(Sec) genes synthesised in vitro were amino-acylated by the E.coli seryl-tRNA ligase although at a very low rate and the resulting seryl-tRNA(Sec) was bound to and converted into selenocysteyl-tRNA(Sec) by the selenocysteine synthase of this organism. Selenocysteyl-tRNA(Sec) from both eukaryotes was able to form a complex with translation factor SELB from E.coli. Although the mechanism of selenocysteine incorporation into seleno-proteins appears to be rather different in E.coli and in vertebrates, we observe here a surprising conservation of functions over an enormous evolutionary distance.

Animals↗
PubMed1994

Acquired C1 inhibitor deficiency as a result of an autoantibody to the reactive center region of C1 inhibitor.

An autoantibody that we hypothesize to react with the reactive center of the plasma serine proteinase inhibitor, C1 inhibitor (C1INH), has been found in a patient with acquired C1INH deficiency. The Ab blocks the ability of C1INH to inhibit the hydrolysis of N-carbobenzyloxy-L-lysine thiobenzylester by purified C1s. A cryoprecipitate from the patient's plasma as well as the Ig fraction were able to block C1INH inhibition of C1s. The immunoaffinity purified Ab to C1INH from the patient's plasma Ig fraction neutralizes the inhibitory activity of C1INH in a dose-dependent manner and blocks the ability of normal C1INH to form a complex with C1s. The neutralizing activity of the purified Ab is reversed by a synthetic peptide that corresponds to the amino acid sequence in the P1 to P15 positions of the reactive center of C1INH but not by a 34-amino-acid trypsin peptide or 37-amino-acid elastase peptide derived from the C-terminus of C1INH. Western blot analysis indicated that the Ab is an oligoclonal Ig with kappa light chains.

Adult↗
PubMed1994

The formation of diselenide bridges in proteins by incorporation of selenocysteine residues: biosynthesis and characterization of (Se)2-thioredoxin.

A system was devised which allows the efficient substitution of cysteine residues in a protein by selenocysteine. It involves overexpression of the respective gene with the aid of the T7 promotor/polymerase system in a cysteine auxotrophic strain. The induction of the T7 polymerase formation was performed in cysteine-supplemented medium followed by wash-out of the cysteine and production of the desired gene product in the presence of selenocysteine. The system was applied to substitute the two cysteine residues in Escherichia coli thioredoxin. Analysis of the purified gene product by electrospray mass spectrometry and HPLC revealed that both cysteine residues were replaced in approximately 75-80% of the protein, only one cysteine residue was substituted in about 5-10%, and no substitution had taken place in 12-17% of the protein. The occurrence of diselenide, seleno-sulfur, and disulfide bridges in the purified gene product was revealed by ES/MS and chemical modification studies. The diselenide bridge represents an entity in protein structures which has hitherto not been described. The redox property of the selenocysteine variant of thioredoxin [(Se)2-thioredoxin] was found to be substantially different from that of thioredoxin. Only the latter could be reduced under native conditions in the presence of an excess of beta-mercaptoethanol. The oxidized (Se)2-thioredoxin was then separated from the selectively reduced and carboxymethylated protein by anion-exchange chromatography. The purity of the isolated (Se)2-thioredoxin was at least 92%.

Chromatography, High Pressure Liquid↗
PubMed1994

[Cancer after transplantation].

Transplantation is associated with an increased risk of cancer. The major factor favouring carcinogenesis is the degree of immunosuppression rather than the nature of the drug used. A large number of these tumours are virus-induced, reflecting an immune system deficiency in combating viral infection. The transplanted patient should be closely followed, both early after transplantation and consistently thereafter, for early detection of such complications.

Humans↗
PubMed1994

Recognition of the mRNA selenocysteine insertion sequence by the specialized translational elongation factor SELB.

In Escherichia coli the unusual amino acid selenocysteine is incorporated cotranslationally at an in-frame UGA codon. Incorporation of selenocysteine relies, in part, on the interaction between a specialized elongation factor, the SELB protein, and a cis-acting element within the mRNA. Boundary and toeprint experiments illustrate that the SELB-GTP-Sec-tRNA(Sec) ternary complex binds to the selenoprotein encoding mRNAs fdhF and fdnG, serving to increase the concentration of SELB and Sec-tRNA(Sec) on these mRNAs in vivo. Moreover, toeprint experiments indicate that SELB recognizes the ribosome-bound message and that, upon binding, SELB may protrude out of the ribosomal-mRNA track so as to approach the large ribosomal subunit. The results place the mRNA-bound SELB-GTP-Sec-tRNA(Sec) ternary complex at the selenocysteine codon (as expected) and suggest a mechanism to explain the specificity of selenocysteine insertion. Cis-acting mRNA regulatory elements can tether protein factors to the translation complex during protein synthesis.

Bacterial Proteins↗
PubMed1994

Evaluation of two new chromogenic media for detection of Salmonella in stools.

A clinical collaborative study was conducted to compare two new chromogenic agar media, Rambach agar and the Salmonella Detection and Identification Medium (SMID) (bioMérieux, France), with two conventional media, Salmonella-Shigella agar and Hektoen agar. Thirty-nine Salmonella strains involving 14 serotypes were isolated from 1,454 stool specimens. After enrichment in a selective broth, 100% sensitivity was obtained with each medium. The SMID and Rambach agars are considerably more specific than the conventional media. Although SMID agar detects all Salmonella serotypes, it is not as specific as Rambach agar, which requires a complementary test (C8 esterase test) to detect all serotypes.

Agar↗
PubMed1994

Effects of lymphocyte subpopulations on the clonal assay of HPRT mutants: occupational exposure to cytostatic drugs.

The mutagenic effect of occupational exposure to antineoplastic agents was studied in chemotherapy nurses and pharmacists using the T-lymphocyte clonal assay. A significant increase in mutant frequency was observed compared to controls. However, in the present study, cloning efficiency without selection (CEU) was significantly reduced in exposed personnel raising the possibility of an overestimation of the calculated MF. Changes in lymphocyte populations and clonal potential of T-cells were also observed following exposure. CEU was related to % CD4 cells but CE with selection (CETG) was not. Differences in clonal ability of T-cells under selective and unselective conditions coupled with differential lethal effect of antineoplastic agents on lymphocyte subsets may result in inaccurate estimation of MF.

Adult↗
PubMed1994

Flow cytometric evaluation of the acrosome reaction of human spermatozoa: a new method using a photoactivated supravital stain.

A flow cytometric assay using a double-stain method for the measurement of the acrosome reaction of human spermatozoa is described. The use of a stable photoactivated stain, ethidium monoazide, allowed evaluation of the viability of spermatozoa. This stain was more stable in fixed samples than propidium iodide, which is not bound covalently to DNA and is therefore removed readily during the washing procedure. The permeabilized acrosome was labelled with Pisum sativum agglutinin conjugated with fluoroisothiocyanate. Since this lectin binds to the acrosome and acrosomal contents, a decrease in the fluorescence intensity allows the cytometric evaluation of the acrosome reaction. Microscopic analysis and flow cytometric analysis were well correlated and cell sorting was performed to ensure the homogeneity of each different subpopulation encountered.

Acrosome↗
PubMed1994

Prospective study of pulmonary function for cyclomegalovirus infection after renal transplantation.

Pulmonary involvement remains the main complication of cytomegalovirus (CMV) infection in renal transplantation (RT). Early diagnosis is required for the best management of patients, and preemptive therapy could be a successful approach. In order to define the predictive value of lung alveolocapillary abnormalities. We prospectively studied 26 renal transplant recipients for their diffusing capacity of carbon monoxide (DLCO) and their pulmonary clearance of a 99mTc-DTPA aerosol. Patients were studied before transplantation and then every 2 weeks up to the end of the 3rd month following RT. Viral blood cultures and serological determinations were performed every week during these first 3 months. Bronchoalveolar lavage (BAL) was done in case of CMV disease. Statistic analysis was done using Student's t-test and Pearson's chi-square test. During the 3-month follow-up, 13 patients remained free of CMV infection. Three non-infected and 8 infected patients showed DTPA clearance abnormalities (P < 0.05). Six non-infected and 7 infected patients showed DLCO disturbance (P > 0.50). DTPA clearance were significantly modified on days 45 and 60 in the infected group. Serial DTPA scanning, in association with viral blood cultures, could be a useful test to avoid unnecessary BAL in a preemptive therapy strategy.

Bronchoalveolar Lavage↗
PubMed1994

Split tolerance in chimeric GVH mice.

The i.v. inoculation of parental spleen cells into unirradiated adult F1 hybrid mice results in a graft-versus-host reaction (GVHR). In the strain combination B10D2 --> (B10.BRx B10.D2) F1, this reaction is associated with thymic injury and transient but profound cellular immune deficiency. We further analysed the immune status of these mice 60 days after GVHR induction. Phenotypic studies of spleen cells showed that these mice were repopulated with parental lymphocytes resulting in a high degree of chimerism (85%). At this time, the mice looked healthy and recovered a normal cytotoxic T cell response (CTL) against allogeneic cells. GVH chimeric splenocytes were unresponsive against F1 hybrid cells in mixed lymphocyte culture (MLC), but exhibited anti-F1 CTL reactivity. We also analysed the anti-F1 reactivity of these mice in vivo. GVH chimeric splenocytes were unable to induce GVHR after injection into a new F1 hybrid and F1 GVH mice specifically rejected F1 bone marrow (BM) cells after lethal irradiation. Grafting a neonatal parental thymus prevented the rejection of F1 BM cells and restored CTL alloreactivity. It is concluded that the chimeric state induced by GVHR is associated with a split tolerance and that a radiosensitive thymic-dependent mechanism is involved in maintaining self-tolerance in these mice.

Animals↗
PubMed1993

Solution structure of selenocysteine-inserting tRNA(Sec) from Escherichia coli. Comparison with canonical tRNA(Ser).

Selenocysteine-inserting tRNAs (or tRNA(Sec)) are structurally untypical tRNAs that are charged by seryl-tRNA synthetase before being recognized by the selenocysteine synthase that converts serine into selenocysteine. tRNA(Sec) from Escherichia coli contains 95 nucleotides and is the longest tRNA known to date, in contrast to canonical tRNA(Ser), 88 nucleotides-long. We have studied its solution conformation by chemical and enzymatic probing. Global structural features were obtained by cobra venom and S1 nuclease mapping, as well as by probing with Pb2+. Accessibilities of phosphate groups were measured by ethylnitrosourea probing. Information about positions in bases involved in Watson-Crick pairing, in stacking or in tertiary interactions were obtained by chemical probing with dimethylsulfate, diethylpyrocarbonate, kethoxal and carbodiimide. On the basis of these chemical data, a three-dimensional model was constructed by computer modeling and compared to that of canonical tRNA(Ser). tRNA(Sec) resembles tRNA(Ser) at the level of its T-arm and anticodon-arm conformations, as well as at the joining of the D- and T-loops by a tertiary Watson-Crick G19-C56 interaction. Its extra-long variable arm is a double-stranded structure closed by a four nucleotide loop that is linked to the body of the tRNA in a way different from that found in tRNA(Ser). As anticipated from the peculiar features of the sequence in the D-loop and at the junction of amino acid and D-arms, tRNA(Sec) possesses a novel but restricted set of tertiary interactions in the core of its three-dimensional structure: a G8-A21-U14 triple pair and a novel interaction between C16 of the D-loop and C59 of the T-loop. A third triple interaction involving C15-G20a-G48 is suggested but some experimental evidence for it is still lacking. It is furthermore concluded that the D-arm has six base-pairs instead of three, as in canonical class II tRNA(Ser), with the D-loop containing only four nucleotides. Finally, the amino acid accepting arm forms a stack of eight Watson-Crick base-pairs (instead of 7 in other tRNAs). The biological relevance of this model with regard to interaction with seryl-tRNA synthetase and enzymes from the selenocysteine metabolism is discussed.

Adenine↗
PubMed1993

Interaction of translation factor SELB with the formate dehydrogenase H selenopolypeptide mRNA.

The SELB protein from Escherichia coli is a specialized elongation factor required for the UGA-directed insertion of the amino acid selenocysteine into selenopolypeptides. Discrimination of the UGA codon requires the presence of a recognition element within the mRNA, which is located at the 3' side of the UGA codon; a hairpin structure can be formed within this mRNA region. By gel shift assays, a specific interaction between SELB and the mRNA recognition element could be demonstrated. Footprinting experiments, using nucleases or iodine as cleaving agents, showed that SELB binds to the loop region of the hairpin structure. In the presence of selenocysteinyl-tRNA, SELB formed a complex with the charged tRNA and the mRNA. The results indicate that targeted insertion of selenocysteine is accomplished by the binding of the SELB protein to this mRNA recognition element, resulting in the formation of a selenocysteinyl-tRNA.SELB complex at the mRNA in the immediate neighborhood of the UGA codon.

Bacterial Proteins↗
PubMed1993

Germline deletion in a neurofibromatosis type 2 kindred inactivates the NF2 gene and a candidate meningioma locus.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease which predisposes to the development of schwannomas, meningiomas, ependymomas, and juvenile cataracts. The NF2 gene (NF2) has recently been isolated and maps to chromosome 22q12 between the loci D22S212 and D22S32. Deletion studies in sporadic and NF2 associated schwannomas and meningiomas, and the presence of inactivating mutations in NF2 in patients suggest that it acts as a tumor suppressor gene. A candidate meningioma gene (MEN) has also been isolated from the same interval. A new highly polymorphic (CA)n marker, D22S268, which maps very near to NF2, has allowed us to identify a kindred with three living affected individuals, where the disease is presumably caused by a large germline deletion. Fluorescence in situ hybridization and pulsed field gel electrophoresis confirm the presence of a 700kb deletion which includes the neurofilament heavy chain subunit gene locus (NEFH), D22S268, NF2 and the putative MEN gene. The absence of meningiomas in this pedigree raises doubts as to the existence of a separate MEN locus in this region. These results support the hypothesis that NF2 results from the inactivation of a tumor suppressor gene on chromosome 22q.

Cells, Cultured↗
PubMed1992

Coding from a distance: dissection of the mRNA determinants required for the incorporation of selenocysteine into protein.

Incorporation of selenocysteine into proteins is directed by specifically 'programmed' UGA codons. The determinants for recognition of the selenocysteine codon have been investigated by analysing the effect of mutations in fdhF, the gene for formate dehydrogenase H of Escherichia coli, on selenocysteine incorporation. It was found that selenocysteine was also encoded when the UGA codon was replaced by UAA and UAG, provided a proper codon-anticodon interaction was possible with tRNA(Sec). This indicates that none of the three termination codons can function as efficient translational stop signals in that particular mRNA position. The discrimination of the selenocysteine 'sense' codon from a regular stop codon has previously been shown to be dependent on an RNA secondary structure immediately 3' of the UGA codon in the fdhF mRNA. It is demonstrated here that the correct folding of this structure as well as the existence of primary sequence elements located within the loop portion at an appropriate distance to the UGA codon are absolutely required. A recognition sequence can be defined which mediates specific translation of a particular codon inside an mRNA with selenocysteine and a model is proposed in which translation factor SELB interacts with this recognition sequence, thus forming a quaternary complex at the mRNA together with GTP and selenocysteyl-tRNA(Sec).

Anticodon↗