Biomedical subjects

S Ballester

Publications and source records attributed to S Ballester.

14 recordsLinked to original sources

PubMed1998

NF-kappaB in Th2 cells: delayed and long-lasting induction through the TCR complex.

Nucelar NFkappaB was analyzed in murine Th2 cells after stimulation via the TCR pathway. Signals delivered through the TCR/CD3 complex induced active NFkappaB translocation to the nucleus of Th2 cells after a late phase (24 h) of the activation process, which is in contrast to the rapid appearance of nuclear NFkappaB (3 h) in Th1 cells after the same stimulation. The slow kinetic of NFkappaB nuclear uptake in Th2 cells was not accelerated by CD28 triggering or under stimulation with antigen plus antigen-presenting cells. Th1 and Th2 cells were also different in the composition of NFkappaB complexes induced. Whereas in Th1 cells TCR triggering induced the presence of nuclear p50.p65 heterodimers, in Th2 cells the complexes induced were shown to be composed of p65 plus another NFkappaB protein distinct from p50. The delayed NFkappaB induction in Th2 cells was dependent on protein synthesis and the significance of this is discussed.

Animals↗

PubMed1997

Modulation of lymphocyte interaction with endothelium and homing by HIV-1 gp120.

We have previously shown that HIV-1 glycoprotein 120 (gp120) induces CD4 association with several molecules on the surface of CD4+ lymphocytes. Since one of these molecules was CD38, involved in lymphocyte/endothelium interaction, this article examines the possibility that gp120/CD4 binding alters CD4+ T cell interaction with vascular endothelium in vitro and in vivo. Cocapping experiments showed that gp120 induced CD4 association with CD38, CD29, CD49d, and CD11a in peripheral blood CD4+ T cells. Two in vitro binding assays were used to evaluate the effect of gp120. A static binding assay, performed at 37 degrees C, evaluated stable interactions mediated by integrins, and a dynamic binding assay, performed at 4 degrees C on a rocking shelf, evaluated weak interactions mediated by constitutively active molecules such as selectins and CD38. Gp120 increased dynamic binding and inhibited static binding to the endothelium of peripheral blood CD4+ T cells and SUPT-1 cells. Binding inhibition with mAbs suggested that the gp120 effect on dynamic binding involved CD38, CD31, and CD49d, whereas the effect on static binding involved CD11a and CD49d. In vivo experiments showed that treatment of 2D4 cells, a CD4- CD8- mouse T cell clone transfected with the human CD4, with gp120 increased their homing into the spleen, intestine, and mesenteric lymph nodes, whereas it decreased homing into peripheral lymph nodes. Alteration of lymphocyte homing may contribute to immune deficiency in HIV-1+ patients by decreasing the probability of an encounter between Ags and lymphocytes and inhibiting the spread of effector lymphocytes into tissues.

Animals↗

PubMed1997

CD4 dependence of activation threshold and TCR signalling in mouse T lymphocytes.

The effect of CD4 expression on the activation threshold of mouse T lymphocytes has been analysed. To do this, the authors studied the response to antigen and other T cell receptor (TCR) ligands in a series of CD4- mutants obtained from the SR.D10 clone. This non-tumour clone spontaneously arose from the Th2 clone D10.G4.1, and characteristically shows a low threshold for antigen activation as well as reactivity to syngeneic antigen presenting cells (APC). Although SR.D10 CD4- mutant cells can be stimulated by antigen, they need higher antigen concentration or more APC than SR.D10 or CD4 transfectants to yield optimal antigen responses. Furthermore, CD4- clones are not activated by syngeneic APC or by clonotypic antibodies. These effects do not correlate with changes in the expression of cell surface molecules implicated in antigen recognition, like TCR/CD3, CD2, LFA-1, or CD45, or with lower p56lck or p59fyn activity in the mutant cells. Since inhibition experiments using anti-CD4 antibodies have previously shown that activation of the CD4+ T cell clone D10.G4.1 by antigen or alloantigens is largely dependent on CD4, our results indicate that activation by antigen-plus self MHC may become CD4-independent if the activation threshold is lowered enough, e.g. in cells like SR.D10. Expression of CD4 further lowers the activation threshold of the cells, allowing the detection of low-affinity TCR reactivities like those directed at self MHC. Moreover, by using anti-TCR/CD3 antibodies, the authors have confirmed the importance of CD4-associated tyrosine kinase activity in early TCR/CD3 signalling in this Th2 cell line, as (1) upon TCR/CD3 ligation, tyrosine phosphorylation is detected only in those CD3 chains co-precipitating with CD4; and (2) CD4 expression is needed for efficient early tyrosine phosphorylation and detectable p56lck-TCR co-precipitation.

Animals↗

PubMed1995

A hyperreactive variant of a CD4+ T cell line is activated by syngeneic antigen presenting cells in the absence of antigen.

A variant of the murine CD4+ T helper cell clone D10.G4.1 (D10) has been isolated and cloned. This line, which we have named "syngeneic-reactive D10", or SR.D10, maintains the I-Ak-restricted specificity for Conalbumin and the allogeneic specificities characteristic of D10 cells. However, it is hyperreactive to TCR-dependent and-independent stimuli, indicating a lower activation threshold than the original D10.G4.1 clone. The hyperreactivity of SR.D10 runs in parallel with the acquisition of a reactive phenotype against syngeneic antigen presenting cells (APCs). As in antigen activation, reactivity to syngeneic APCs can be inhibited by anti-TCR, anti-CD4, or anti-class II monoclonal antibodies. The role of CD4 in this phenomenon is highlighted as "syngeneic reactivity" disappears in CD4- mutants of SR.D10 and is recovered in CD4 transfectants. The expression of several cell surface molecules involved in T cell activation show qualitative and/or quantitative differences between SR.D10 and the original D10. No significant differences in quantity and activity of p56lck and p59fyn were detected between the hyperreactive and the original clone. Our results suggest that high sensitivity to activation, concomitant with expression of CD4, might allow the acquisition of an autoreactive phenotype and confirm the important contribution of coreceptors to determine the activation threshold of the cells. The characteristics of SR.D10 and the possibility of growing them in the presence of interleukins make this cell line a experimental model of great interest for analyzing activation mechanisms in T cells.

Animals↗

PubMed1994

Genetic and immunochemical evidence for CD4-dependent association of p56lck with the alpha beta T-cell receptor (TCR): regulation of TCR-induced activation.

Recent observations suggest that the tyrosine kinase p56lck is involved in the transduction of transmembrane signals through the antigen specific T cell receptor (TCR) in CD4+ T cells. By means of in vitro kinase assays, we have found that p56lck coprecipitated with the TCR from lysates of a murine CD4+ T cell line in the absence of TCR-mediated stimuli. Analysis of CD4- mutants and CD4-transfected cells shows that p56lck-TCR association occurred only when CD4 was present. The functional importance of CD4:p56lck-TCR association was demonstrated by low activating potential of rare clonotypic antibodies which did not coprecipitate CD4:p56lck, as well as by total or partial loss of anti-TCR or antigen induced stimulation in CD4- cells, which could be recovered by CD4 transfection. Complementation assays using different anti-TCR antibodies suggest that cross linking of TCR-p56lck:CD4 plus structural changes in the complex are needed for efficient transduction of activating signals through the TCR in these cells.

Animals↗

PubMed1993

Replication of the promiscuous plasmid pLS1: a region encompassing the minus origin of replication is associated with stable plasmid inheritance.

Deletion of a region of the promiscuous plasmid pLS1 encompassing the initiation signals for the synthesis of the plasmid lagging strand led to plasmid instability in Streptococcus pneumoniae and Bacillus subtilis. This defect could not be alleviated by increasing the number of copies (measured as double-stranded plasmid DNA) to levels similar to those of the wild-type plasmid pLS1. Our results indicate that in the vicinity of, or associated with the single-stranded origin region of pLS1 there is a plasmid component involved in its stable inheritance. Homology was found between the DNA gyrase binding site within the par region of plasmid pSC101 and the pLS1 specific recombination site RSB.

Bacillus subtilis↗

PubMed1992

Genetic alterations by human papillomaviruses in oncogenesis.

The integration sites in the cellular genome of human papillomavirus are located in chromosomal regions always associated with oncogenes or other known tumor phenotypes. Two regions, 8q24 and 12q13, are common to several cases of cervical carcinoma and can have integrated more than one type of papillomavirus DNA. These two chromosomal regions contain several genes implicated in oncogenesis. These observations strongly imply that viral integration sites of DNA tumor viruses can be used as the access point to chromosomal regions where genes implicated in the tumor phenotype are located, a situation similar to that of non-transforming retroviruses.

Chromosomes, Human, Pair 12↗

PubMed1991

Influence of PCR parameters on amplifications of HIV-1 DNA: establishment of limiting sensitivity.

We have investigated the optimal reaction conditions and the limiting sensitivity for detection of HIV-1 DNA by PCR. The amplification systems studied were gag (SK38/SK39); pol (P3/P4); and two other systems described here for the first time, LTR (LTR1/LTR2) and nef (Nef1/Nef2), which amplify fragments of 115 bp, 308 bp, 632 bp and 643 bp, respectively. Two PCR profiles were assayed, and the requirements for deoxynucleoside triphosphate and MgCl2 concentrations for each amplification reaction were determined. Optimal reaction conditions were oriented toward selecting maximal amplification of the expected size fragment. Limiting sensitivity was estimated by testing the decreasing copy number of a plasmid containing HIV-1 genome and obtaining a positive amplification signal with at least 5, 5, 10 and 5 copies for LTR, gag, pol and nef, respectively. We conclude that the establishment of the detection sensitivity on a PCR is an important parameter to be considered for the interpretation of results on HIV-1 infection.

Base Sequence↗

PubMed1990

Comparative expression of the pC194 cat gene in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli.

The expression of the cat gene of the staphylococcal plasmid pC194 present in the pLS1-pC194 composite plasmid pJS37 was lower in Streptococcus pneumoniae and Escherichia coli than in Bacillus subtilis. Different transcription start points (and, by inference, different promoter utilization) of the cat mRNA synthesized in S. pneumoniae or B. subtilis were detected. Plasmid pJS37 is prone to deletion formation when host cells are grown in the presence of chloramphenicol (Cm). The analysis of the expression of the cat gene carried by the deleted derivatives of pJS37 has shown that a new promoter for the synthesis of cat mRNA is involved in the selective advantage conferred to the host by those deleted plasmids. Characterization of either in vivo or in vitro deleted plasmids has shown that the nucleotide sequence that could encode for a putative leader peptide is required for the Cm-induced pC194 cat gene expression.

Amino Acid Sequence↗

PubMed1989

Plasmid structural instability associated with pC194 replication functions.

The hybrid plasmid pJS37 is composed of the streptococcal plasmid pLS1, which confers tetracycline resistance, and the staphylococcal plasmid pC194, which confers chloramphenicol resistance. When gram-positive bacteria containing pJS37 were grown in the presence of chloramphenicol, four different deleted derivatives accumulated. The deletions in the plasmid enhanced resistance to chloramphenicol by placing the cat gene of pC194 near promoters of pLS1. All four deletions shared a common endpoint that corresponded to the putative target site for DNA strand nicking by the pC194 replication protein, RepH. At the other, variable endpoint, the DNA sequence was similar to the putative RepH target sequence. Alteration of the RepH protein, by in vitro modification of the gene encoding it, eliminated this class of deletions. By extending a previously proposed model for the generation of a different but related class of deletions (B. Michel and S.D. Ehrlich, EMBO J. 5:3691-3696, 1986), a comprehensive model that could generate both classes of deletions is suggested. It proposes that a nicking-closing activity of the plasmid replication protein at its normal target site and, aberrantly, at sites with similar sequence can generate deletions either proximal or distal to the aberrant site during rolling-circle replication of the plasmid.

Bacterial Proteins↗

PubMed1986

Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids.

A hybrid plasmid, pJS37, was made by combining pLS1, which confers tetracycline (Tc) resistance, and pC194, which confers chloramphenicol (Cm) resistance. Both pJS37 (7.3 kb) and its derivative pJS140 (6.0 kb), from which pC194 replication genes were removed, were structurally and segregationally stable when introduced into Streptococcus pneumoniae and grown either in the presence of Tc or in the absence of drug. However, both hybrid plasmids underwent systematic deletion when grown in the presence of Cm. One of the deleted forms, pJS4 (3.4 kb), could not be maintained in the absence of a helper plasmid; two others, pJS3 (4.1 kb) and pJS5 (3.8 kb), lost the tet gene but retained the replication functions of pLS1. They both expressed very high levels of Cm acetyltransferase (CAT), which, in the case of pJS5, were constitutive. Nucleotide sequence determination of the deletion junctions in pJS3 and pJS5 indicated that the deletions occurred, presumably by recombination, between short direct repeats of 6 and 9 bp, respectively. In both cases the tet promoter was juxtaposed to the cat gene. In the case of pJS5, the deletion removed a sequence that sequestered the ribosome-binding site (RBS) for cat, thereby rendering constitutive the production of CAT. The increased resistance to Cm afforded by the hyperexpression of the cat gene apparently provided a positive selective advantage for the accumulation of the deleted forms in the plasmid pool.

Acetyltransferases↗

PubMed1986

Inhibition of transformation of Bacillus subtilis by ethidium bromide.

Incubation of donor DNA with ethidium bromide before addition to competent cells, strongly inhibited chromosomal transformation, plasmid transfer and phage-SPP1 DNA mediated transfection. There was no effect of the dye on the binding and entry of DNA into the cells. Analysis of the fate of transforming DNA treated with ethidium bromide showed a reduction in the amount of donor DNA associated with the recipient chromosome. The formation of this donor-recipient complex seems to be slowed down by the dye.

Bacillus subtilis↗

PubMed1985

Binding of transforming DNA to protoplasts isolated from competent Bacillus subtilis.

Transforming 3H-labelled DNA binds specifically to protoplasts isolated from competent cultures of Bacillus subtilis. The bound DNA is fully accessible to added DNase I, indicating that protoplasts bind, but do not process donor DNA. A bi-phasic pattern of competition for binding with increasing amounts of unlabelled and labelled DNA was found. In these conditions, two levels of saturation appeared which correspond to two kinds of DNA receptor sites. Homologous DNA binds preferentially to the first kind of receptor (specific) and only at higher concentrations would bind to the second (unspecific) binding sites. Bound DNA forms a rather stable complex with some constituent(s) of the protoplasts.

Bacillus subtilis↗

PubMed1983

Transformation in Bacillus subtilis: different affinities for DNA-binding to competent cells and to membrane vesicles.

Comparison between dose-response curves to homologous and to T*4 (non-glucosylated) DNA in cells and in membrane vesicles, isolated from competent Bacillus subtilis, indicated the presence of two kinds of DNA receptors in the membrane vesicles system. This was confirmed by competition experiments. In addition, concentration dependence for binding of low-molecular weight homologous DNA also revealed the existence of more than one DNA receptor site in competent cells of B. subtilis. However, no differences in the uptake of sheared or intact DNA was observed, which indicates that only one kind of receptors is involved in the entry of donor DNA. Competition experiments in binding, uptake, and transformation, with the possible combinations of sheared or intact DNAs, suggested that the former has more affinity for the binding sites than the latter. This finding is also supported by the results obtained in membrane vesicles, either in competition or in chasing experiments.

Bacillus subtilis↗