Search PubMed⌕ Search

Biomedical subjects

J Guardiola

Publications and source records attributed to J Guardiola.

At least 37 records · Page 2Linked to original sources
PubMed1988

Molecular genotyping of the HLA-DQ alpha gene region.

Restriction fragment analysis has been applied to genomic DNA extracted from human tumor cell lines. Polymorphic restriction fragments encompassing the HLA-DQ alpha gene were observed upon digestion with Bgl II, Eco RI, and Hind III. Analysis of these polymorphic fragments (or allogenotopes) showed that for each restriction enzyme a series of three differently sized allogenotopes existed. Clusters of cosegregating allogenotopes belonging to the different allelic series defined three different allogenotypes. Each allogenotype exhibited a distinctive restriction map generated by digestion with five restriction enzymes. Comparison of these restriction maps showed that generation of the polymorphisms observed at the HLA-DQ alpha region in these sets of cell lines is not caused by a single event. In some B- and T-lymphoma cell lines a fourth allogenotype was found. The restriction site map of genomic DNA from these cell lines suggested that the latter distribution of restriction enzyme sites was most probably generated by recombination between two of the previously observed allogenotypes at a crossover site(s) adjacent to the HLA-DQ alpha gene.

Alleles↗
PubMed1987

Distinct mechanisms regulate MHC class II gene expression in B cells and macrophages.

In a previous series of studies, we had shown that the constitutive Ia expression in an immunoselected Ia-human B cell variant, RJ 2.2.5, could be restored by somatic cell hybridization with mouse B cells. These experiments allowed us to show the existence of a transacting activator factor(s) operating across species barriers and encoded by the aIr-1 locus located on mouse chromosome 16. The aim of the present study was to investigate whether the B cell constitutive Ia expression and the inducible Ia expression, as seen in macrophages treated with IFN-gamma, are controlled by similar intracellular factors. To this purpose, we constructed an interspecies somatic cell hybrid between the human Ia-RJ 2.2.5 B cells and the mouse Ia-P388 D1 macrophage cells. These murine cells transiently express Ia antigens when incubated with IFN-gamma. Our results show that RJ 2.2.5 X P388 D1 cell hybrids do not express either human or mouse class II gene products. Treatment with human recombinant IFN-gamma did not modify the MHC phenotype of either the hybrid cells or the human parental cells. On the other hand, treatment of the hybrid cells with murine recombinant IFN-gamma resulted in de novo expression of mouse Ia mRNA and corresponding cell surface antigens without, however, reinduction of the human class II-positive phenotype. Furthermore, treatment with the mouse lymphokine significantly increased the levels of human HLA class I mRNA and corresponding cell surface antigens in the hybrid cells, further reinforcing the notion of the existence of non-species-specific secondary mediators generated after receptor-ligand interaction in the IFN-gamma system. Together, these results indicate that in macrophages, the intracellular events taking place after binding of IFN-gamma with its own receptor and leading to the expression of a class II-positive phenotype do not operate via an activation of the aIr-1 locus and/or its products. Thus, at least in our experimental system, we can firmly establish a first, relevant distinction between constitutive and inducible class II gene expression. This difference, dictated by the specific differentiation program of each cell type, may be relevant for the understanding of the function of class II gene products.

Animals↗
PubMed1987

T cell receptor beta chain gene rearrangements and V beta gene usage in horse cytochrome c-specific T cell hybridomas.

Comparison of T cell receptor alpha and beta-chain genes in murine major histocompatibility complex (MHC) class I and class II-restricted T cell clones and hybridomas recognizing different antigens indicates that no simple correlation exists between the observed antigen/MHC specificity and the expression of certain alpha and beta-chain heterodimers. We have attempted to establish a possible correlation by analyzing T cell receptor beta chain gene rearrangements and V beta gene usage in five T cell hybridomas with identical antigen/MHC specificity and another hybridoma recognizing a different antigenic determinant in association with the same restriction molecule. We report here that in each of the five clones a uniquely rearranged beta chain gene is expressed in combination with at least two different V beta gene segments. The presence of the differently rearranged T cell receptor beta chain genes correlated with the finding of distinct fine specificity pattern of antigen recognition in each of the hybridomas. Interestingly, two hybridomas specific for different epitopes showed identical beta chain D-J rearrangements indicating that the differences might be encoded by the alpha chain gene or/and the V beta gene element.

Animals↗
PubMed1987

GC/MS, HPLC and FAB mass spectrometric analysis of organic micropollutants in Barcelona's water supply.

Water samples from Llobregat river entering two water work plants, Barcelona tap water and waste dumping samples taken along the river course were analyzed for trace organic contaminants by different procedures, liquid-liquid extraction, adsorption on granular activated carbon followed by GC/MS/DS. Ether insoluble organic fractions were analyzed and fractionated by HPLC with diode-array detection, followed by FAB and FAB-CID-MIKE characterisation. Results, after two years monitoring, proved that surfactants, plasticizers, ethyleneglycol derivatives, phosphates, hydrocarbons and other miscellaneous compounds can be considered as chronic pollutants of Llobregat river. Some of the identified compounds by GC/MS and FAB mass spectrometry have not been previously reported to occur in water.

Chromatography, High Pressure Liquid↗
PubMed1986

aIr-1, a newly found locus on mouse chromosome 16 encoding a trans-acting activator factor for MHC class II gene expression.

RJ 2.2.5 is a human B cell line that has lost the capacity to express MHC class II genes. The human class II-positive phenotype is restored in somatic cell hybrids between RJ 2.2.5 and mouse spleen cells. By karyotype and molecular studies of an informative family of hybrids we have now shown that the reexpression of human class II gene products, as well as the maintenance of the mouse class II-positive phenotype, correlates with the presence of mouse chromosome 16. Thus, the existence on this mouse chromosome of a newly found locus, designated by us aIr-1, that determines a trans-acting activator function for class II gene expression, is established. Possible implications of this finding are discussed.

Animals↗
PubMed1986

Stable integration of mouse DNA into Ia-negative human B-lymphoma cells causes reexpression of the human Ia-positive phenotype.

RJ 2.2.5, a variant of the human B-lymphoma cell line Raji, does not express the HLA-DR, -DQ, and -DP class II (or Ia) histocompatibility antigens, as a result of a defect in the transcription of the corresponding genes. This defect is corrected after fusion of RJ 2.2.5 cells with mouse Ia-positive cells. Previous work showed that the trans-acting transcriptional activator supplied by the mouse cells is encoded by a locus on mouse chromosome 16. We show here that reexpression of human major histocompatibility complex class II genes by RJ 2.2.5 cells can also be achieved by stable integration of mouse genomic sequences into the RJ 2.2.5 genome after DNA-mediated gene transfer.

Animals↗
PubMed1985

Trans-acting element(s) operating across species barriers positively regulate expression of major histocompatibility complex class II genes.

Raji, a human B lymphoma line, expresses high levels of major histocompatibility complex (MHC) class II antigens. Conversely, none of the detectable human Ia antigens is present in RJ 2.2.5, an immunoselected Raji variant. Clonal analysis, biochemical characterization, and nucleic acid hybridization studies of hybrids between mouse spleen cells and RJ 2.2.5 show that MHC class II gene expression is regulated in trans by a factor which, as judged by dominance studies, has the characteristics of an activator. Such a positive trans acting factor is expressed in mouse spleen cells, and is able to implement MHC class II gene expression across species boundaries. Expression of this factor in spleen cells strongly suggests that it plays a role in in vivo regulation of Ia expression. Additional data suggest that different subsets of class II genes such as DR and DQ may, in part, be regulated by different mechanisms. It has also been possible to show that the amount of In chain-specific mRNA, present at reduced levels in RJ 2.2.5 cells compared to the parental Raji cells, drastically increased in human X mouse cells hybrids reexpressing human Ia antigens, suggesting that the In chain gene and the class II genes, although located on different chromosomes, are regulated in a concerted fashion, either directly through the same implementing factor, or indirectly through a cascade mechanism.

Animals↗
PubMed1985

Reactivation by a trans-acting factor of human major histocompatibility complex Ia gene expression in interspecies hybrids between an Ia-negative human B-cell variant and an Ia-positive mouse B-cell lymphoma.

Raji, a human B-lymphoma cell line, expresses high levels of class II (Ia) antigens. The expression of Ia antigens is totally abolished at the level of specific mRNA accumulation in RJ 2.2.5, a variant cell line derived from Raji after mutagenesis and immunoselection. We report here that the human Ia antigen expression can be restored in interspecies somatic cell hybrids between RJ 2.2.5 cells and BALB/c-derived M12.4.1 (I-A+, I-E+) B-lymphoma cells. Two hybrid clones were studied in detail. In both clones Ia molecules of the DR and NG2 type were easily detected by cell surface immunofluorescence and specific immunoprecipitation. In contrast, the DQ1 molecules were not detected with the above technique. DNA hybridization experiments using specific probes indicated that alpha-chain DR and beta-chain DQ genes were present in the hybrids. However, RNA hybridization experiments revealed that beta-chain DQ mRNA was present in the hybrids at very low amount compared to alpha-chain DR-specific mRNA. These results indicate that at least several genes of the class II gene cluster are coordinately regulated by trans-acting factor(s) that operate across species barriers. The basis of the mechanisms controlling the expression of class II antigens in these human-mouse hybrids might be related to the extinction (lack of expression) or activation of tissue-specific traits that take place when genomes of cells of dissimilar developmental potentials are brought together.

Animals↗
PubMed1985

Identification and preliminary characterization of a sperm-binding protein in normal human semen.

In the seminal plasma of normal men a protein, immunologically related to a major protein (RSV-IV) secreted from the rat seminal vesicle epithelium, was detected by competition with RSV-IV in a specific radioimmunoassay using a rabbit anti RSV-IV antiserum. The protein was partly (80%) purified by column chromatography; characterization by gel electrophoresis indicated that the protein is slightly basic and has a molecular weight of 140 000. The protein was present on the surface of human spermatozoa as well as in the ejaculates of azoospermic men and it is believed to be a sperm-binding protein.

Antigens, Surface↗
PubMed1984

The 11S rat seminal vesicle mRNA directs the in vitro synthesis of two precursors of the major secretory protein IV.

The 11s mRNA extracted from the rat seminal vesicles directs the synthesis of two different precursors of the major secretory protein RSV-IV. These two precursors are not interconvertible and seemingly originate from different translational events. Sucrose gradients, polyacrylamide gel electrophoresis and positive hybridization translation experiments do not allow the separation of the two putatively different mRNAs. It is concluded that the two RSV-IV precursors either derive from two extremely similar, but physically not separable mRNA species, or from two different modes of translation of the same mRNA molecule.

Animals↗
PubMed1984

Synthesis of a testosterone-dependent secretory protein by rat seminal vesicle-derived cell lines.

Primary cell cultures were established from explants of rat seminal vesicle. The establishment of primary cell cultures required, among other factors, the presence of testosterone. Two cell populations were detected in such primary cultures: fibroblast-like cells and epithelial-like cells; the latter encompassed a subtype of small cells and a subtype of large squamous cells (most likely the result of a degenerative process acting upon the former). Histochemical, as well as electron-microscopical observations, indicated the presence of a persistent secretory activity in the small epithelial cells; fibroblast and large squamous epithelial cells were inactive in this respect. Staining of the cells with a peroxidase-conjugated antibody and analysis of the proteins produced in the presence of labelled methionine, showed that one of the major rat seminal vesicle secretory proteins, namely RSV-IV, was also produced. Conditions which favoured the growth of epithelial cells, rather than of fibroblasts, were determined. The use of nearly homogeneous cell populations and the use of collagen-coated Petri dishes, allowed the cloning of two independently obtained permanent cell lines, namely SVC-1 and SVC-2. The in vitro growth rate of both cell lines was modulated by the amount of testosterone in the medium. Both cell lines were able to synthesize a significant amount of RSV-IV protein under testosterone control.

Animals↗
PubMed1984

Polymorphism of rat seminal vesicle secretory proteins: characterization of svp-1 and svp-2 and their identification with the major secretory proteins IV and V.

The proteins secreted by the rat seminal vesicle can be separated into five denaturing conditions. Two polymorphic proteins, svp-1 and svp-2, also present in the mouse, are produced by the seminal vesicle as well, but the procedure used for their identification makes it impossible to ascertain whether they correspond to any of the major fractions mentioned above. We show here that, on the basis of molecular weight measurements and of amino acid composition determinations, svp-1 and RSV-V are indeed the same protein. We also show that svp-2 is strictly related to another major secretory protein, RSV-IV, whose amino acid composition is almost identical, but for a few amino acid residues, to that of svp-2. We thus conclude that the latter protein is a variant of RSV-IV that can be expressed only in rats homozygous for a given allele at the svp-2 locus. This paper thus brings together published information on the genetics of the loci coding for svp-1 and for svp-2 and on the molecular biology of RSV-IV and RSV-V and of their corresponding gene.

Amino Acids↗
PubMed1982

Expression in male and genomic organization of the gene(s) coding for a major protein secreted by the rat seminal vesicle epithelium.

Double strand cDNA copies of lls poly(A)+mRNA purified from adult rat seminal vesicles (RSV), have been cloned in E.coli C600 using the Pst I site of pBR322. Filter hybridization, nucleotide sequence analysis and positive hybridization translation were used to demonstrate that one of the recombinant plasmids obtained (pRSV25) contained a 260 bp long insert coding for a significant part of the precursor to the protein IV present in the RSV secretion. By using labelled pRSV25 DNA we have found that high levels of RSV IV mRNA were present only in the rat seminal vesicle epithelium. The amounts of RSV IV mRNA present in other tissues of the same organism were below the levels detectable by the methods used. In addition, other data reported here indicate that the RSV IV gene(s) is present in both sexes, probably with a different organization.

Animals↗
PubMed1979

Threonine deaminase: autogenous regulator of the ilv genes in Escherichia coli K-12.

In this paper we analyze the effect of mutations in three genes, ilvO, ilvA and rho, on the expression of the ilvEJGDA gene cluster of Escherichia coli K-12. The ilvO603 mutation causes a cis-dominant derepression of the ilvEJGDA genes. In particular, the ilvG gene, not expressed in the wild type, becomes expressed in the ilvO603 strain. We have introduced ilvA mutations (ilvA454 or ilvA628) in the ilvO603 strain and we show that ilvG expression requires the presence in cis of both an ilvO603 mutation and of an ilvA+ allele. The ilvG gene is not expressed when in trans is present an ilvO+, ilvA+ genotype. However, it is expressed when the chromosome in trans is ilvO603, ilvA+ (ilvG-). We suggest that ilvO603 is part of ilvA, the structural gene for threonine deaminase, and that threonine deaminase from the ilvO603 mutant binds the ilvO603 site and not the ilvO+ site. Therefore, the ilvA gene product would be a cis-acting protein. Mutations in the rho gene cause derepression of the ilvEJGDA gene cluster without a concomitant expression of the ilvG gene. We show that introduction of either a rho-218 or a rho-115 mutation into the ilvO603, ilvA454 double mutant causes expression of ilvG. We therefore suggest that the ilvA gene product, threonine deaminase, is involved in termination of transcription as an antagonist of the rho gene product. Introduction of ilvA454 into an ilvO603 strain causes also a decrease in expression of the ilvE, ilvJ and ilvD genes. This effect is maximum in the case of the ilvD gene and we studied it in detail in isogenic strains containing also the rho-218 mutation.

Escherichia coli↗
PubMed1978

Dual autogenous regulatory role of threonine deaminase in Escherichia coli K-12.

We describe the regulatory properties of two strains carrying either the ilvA624 or the ilvA625 mutations, located in the structural gene for threonine deaminase. Crude extracts of both these strains possess a threonine deaminase activity migrating on polyacrylamide gels, differently from the wild type enzyme. Growth studies demonstrate that these mutations do not cause a limitation of isoleucine biosynthesis, suggesting normal catalytic activity of deaminase. A regulatory consequence of the ilvA624 allele is a derepression of the isoleucine-valine biosynthetic enzymes, which is recessive to an ilvA+ allele. The ilvA625 mutation causes a derepression which is dominant in an ilvA625/ILVA+ diploid. We interpret these data assuming that threonine deaminase, previously shown to be an autogenous regulator of the ilv genes, lacks a repressor function in the ilvA624 mutant, while in the ilvA625 mutant it is a better activator than wild type threonine deaminase. The data are discussed in terms of a model requiring that threonine deaminase, or a precursor of it, is in equilibrium between two forms, one being an activator of gene expression and the other being a repressor.

Escherichia coli↗