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Biomedical subjects

J C Gutierrez

Publications and source records attributed to J C Gutierrez.

10 recordsLinked to original sources

lpr T cells vaccinate against lupus in MRL/lpr mice.

MRL/MP-lpr/lpr mice are homozygous for the lpr mutation that results in the accumulation of phenotypically abnormal cells (CD3+CD4+CD8-) in all lymphoid issues. Although no major abnormalities in the T cell receptor repertoire expressed by such lpr cells have been reported, the lpr mutation is a major disease-accelerating factor. Finally, intravenous administration of irradiated lpr cells recovered from the hyperplastic lymph nodes of adult diseased animals to young MRL/Mp-lpr/lpr mice resulted in a highly significant amelioration of disease parameters. This "T cell vaccination" approach resulted in a selective depletion of cells expressing products of the V beta 8.2 subfamily among lymph node T cells, in addition to eliciting a surge in peripheral T cells capable of conferring disease protection in adoptive transfer experiments. Thus, a strategy aimed at specifically reducing the frequency of lpr cells proved successful in mitigating the autoimmune process. These findings add to the involvement of lpr cells in the autoimmune process and constitute the first report that T cell vaccination may be beneficial to a spontaneously occurring autoimmune disease.

Animals

Heterogeneity of thymic epithelial cells in promoting T-lymphocyte differentiation in vivo.

To study in vivo the contribution of different thymic epithelial cells to T-lymphocyte differentiation, we have established several nontransformed thymic epithelial cell lines and developed an in vivo assay, not involving exposure to drugs or radiation, that permitted us to study the capacity of these epithelial lines to support T-cell differentiation. We found that cell lines EA2 and ET, which express markers of cortical epithelial cells, produce interleukin 7 mRNA and after being injected into the spleens of young athymic nude mice support in vivo generation of CD4+CD8- T-cell receptor alpha beta+ T lymphocytes (ET line) or both CD4+CD8- and CD4-CD8+ T-cell receptor alpha beta+ T cells (EA2 line). Both cell lines also supported generation of T-cell receptor gamma delta+ T cells but appear not to support development of double-positive (CD4+CD8+) cells. One cell line, EB3, which expresses markers of medullary epithelial cells, produces interleukin 1 alpha RNA transcripts but does not support T-lymphocyte differentiation. The results provide direct evidence for functional heterogeneity of thymic epithelial cells in vivo and show the involvement of different cortical epithelial cells in the differentiation of T-cell progenitors into distinct thymocyte subsets.

Animals

Development of lymphocytes in interleukin 7-transgenic mice.

We have developed and established mouse transgenic lines in which the mouse interleukin 7 gene was targeted for expression in the lymphoid cell compartment. Northern blot analysis indicate that the transgene is expressed in bone marrow (BM), spleen and thymus, but not in kidney, liver, brain or heart. Both the frequency and absolute numbers of B cell precursors and mature B lymphocytes are increased in the BM and spleen of the transgenic mice. Although there is no expansion of the pro-T lymphocyte population in the BM, the number of all major subsets of thymocytes and peripheral T lymphocytes is increased in the majority of the transgenic mice analyzed. The B and T cell lymphocytes in the transgenic mice are functionally competent. In contrast, the number of granulocytes and macrophages in the BM of transgenic mice is similar to that in control non-transgenic littermates. Our results indicate that interleukin 7 plays an important role in vivo in the development of B and T lymphocytes.

Animals

Detection of antigenic cyst wall elements in Colpoda inflata: an immunoelectron microscopic study and immunoblotting identification of cyst wall polypeptides.

By using an antiserum against isolated cyst walls from resting cysts of the ciliate Colpoda inflata, cyst wall polypeptides have been identified by immunoblotting test. Likewise, an immunoelectron microscopical study on both complete resting cysts and isolated cyst walls to localize the cyst wall proteins recognized by the antiserum, has been carried out. The immunoblotting test showed that three main polypeptide bands were recognized by the antiserum, with tentative molecular weights of 61, 66 and 70 kDa respectively. This methodology provides a better identification of cyst wall proteins after electrophoretic separation of cyst wall samples from ciliate resting cysts.

Animals

Isolation and expression of rat liver sepiapterin reductase cDNA.

Sepiapterin reductase (7,8-dihydrobiopterin: NADP+ oxidoreductase, EC 1.1.1.153) catalyzes the terminal step in the biosynthetic pathway for tetrahydrobiopterin, the cofactor necessary for aromatic amino acid hydroxylation. We report here the isolation of a cDNA clone for rat liver sepiapterin reductase. The cDNA has been excised from a lambda vector and the DNA sequence was determined. The insert contains the coding sequence for at least 95% of the rat enzyme and is fused to the Escherichia coli beta-galactosidase N-terminal segment and the lac promoter. The N-terminal region of the clone contains an extraordinarily high G + C content. The amino acid sequence deduced from the clone is in agreement with the size and composition of the enzyme and was matched to several tryptic peptide sequences. The enzyme encoded by the cDNA insert was shown to have sepiapterin reductase activity after expression in E. coli. Structural similarities were identified between this protein and several enzymes that should contain similar nucleotide and pteridine binding sites.

Alcohol Oxidoreductases

Developmental and genetic effects of alcian blue in conjugating Tetrahymena thermophila: doublet formation and macronuclear retention.

Genetic, kinetical and cortical effects of treatment with the inducer of mucocyst release, alcian blue (AB), on conjugating pairs of Tetrahymena thermophila are reported. AB induces the formation of doublet cells from pairs, and the majority of them are homopolar doublets. We present a model in order to explain the origin of these cells. Macronuclear retention (MR) is the most important genetic effect observed. Two kinds of MR can be obtained: prezygotic-MR (uniparental micronucleus) and postzygotic-MR (cross-fertilized micronucleus). Within the first group, both homokaryon and heterokaryon cells are obtained. From some abnormal conjugational configurations and the results of conjugational kinetic analysis we propose an explanation for the origin of MR cells induced by AB. Genetic effects obtained after AB treatment at different conjugational times are independent of the cortical ones. The utility of these different effects in genetical and physiological studies is discussed.

Alcian Blue

Microspectrophotometric study and kinetics of autogamy during encystment of Tetrahymena rostrata.

The process of autogamy that accompanies encystment in Tetrahymena rostrata was divided into five stages; A: first meiotic division, B: second meiotic division. C: post-meiotic division and fusion of the pronuclei, D: first and second synkaryon's mitosis and E: macronuclear-polyploidization. In this communication the kinetics of these phases is reported. The degeneration of the old-macronucleus begins about the second meiotic division (stage-B), and the development of the new-macronucleus progressively rises during about 15h.

Animals

An improved fluorogenic substrate for the alternative complement pathway C3/5 converting enzyme CVFBb.

By lengthening the sequence of a previously available tripeptide to a pentapeptide, we were able to increase the specificity of the substrate for the complement enzyme CVFBb (EC 3.4.21.47) over Factor Xa (EC 3.4.21.6). This increase in specificity was achieved by both an increase in the kcat/Km for CVFBb for the longer substrate compared to the original substrate, and a decrease in the kcat/Km for Factor Xa. The new substrate, Boc-Nle-Gln-Leu-Gly-Arg-amino methyl coumarin (AMC) was synthesized by coupling Boc-Nle-Gln-Leu-Gly to Arg-AMC in solution. p-Toluenesulfonic acid was added to the coupling mixture to improve the solubility of the arginine derivative and avoid the need for covalent protection.

Chromatography, High Pressure Liquid