The y1 gene of maize codes for phytoene synthase.

The cloned y1 locus of maize was sequenced and found to encode phytoene synthase. Different "wild-type" alleles of the locus were found to differ by the insertion of transposable elements in their promoter and polyA addition regions, and by the length of a CCA tandem repeat series, without any obvious effect on function of the gene. A dominant Y1 ("wild-type") allele was observed to be expressed at highest levels in the seedling but also in the embryo and endosperm. The Mu3 transposable element insertion responsible for a pastel allele of y1, which gives lowered levels of carotenoids in the endosperm of kernels and seedlings grown at high temperatures, was located in the 5' end of the gene. Although the size of the transcript from this y1 mutation suggests that the Mu3 element provides the promoter for this allele, leaf tissue in this mutant line contained approximately normal amounts of y1 mRNA. A recessive allele of y1, which conditions normal levels of carotenoids in the embryo and seedling, but almost no carotenoids in the endosperm, was found to accumulate normal amounts of y1 mRNA in the seedling and embryo, while y1 transcripts were not detected in the endosperm.

Lymphocyte aging in bone marrow chimeras.

Chimeric mice provide a unique approach to the analysis of genetic factors associated with aging since cells with two genetically distinct backgrounds can be analyzed in the same animal. In this study, bone marrow chimeras were produced by reconstituting lethally irradiated female B6AF1 [(C57BL/6 female x A male)F1] mice with varying mixtures of T cell-depleted bone marrow cells from A (short-lived) and C57BL/6 (long-lived) mice. The phenotypic composition of the peripheral blood lymphocytes was analyzed using either a cytotoxicity assay or flow cytometry with indirect immunofluorescence. The percentage of A-derived lymphocytes in the peripheral blood following reconstitution was generally higher than the percentage of A bone marrow cells with which the irradiated mice were inoculated, suggesting that the cells from the A donor bone marrow were more efficient at marrow reconstitution than the cells from the C57BL/6 donor bone marrow. In order to determine whether the percentage of A- versus C57BL/6-derived cells changed with age in each animal, the chimeric mice were bled for phenotype analysis of peripheral blood lymphocytes between 2-6 months following reconstitution and at 2-3 month intervals until death. For most animals [93/127 (73%)], there was no consistent pattern of increase or decrease (> 20%) with regard to the percentage of A lymphocytes in the peripheral blood over time. However, in 34/127 (27%) of the chimeras, a change greater than 20% in the phenotypic composition of the peripheral blood lymphocytes was observed and these animals were considered unstable. Among these 34 unstable animals, 6 (18%) showed an overall increase in A-derived lymphocytes, 24 (71%) showed an overall decrease in A-derived lymphocytes, and 4 (12%) showed fluctuating increases and decreases over their lifespan. While the lifespans of the chimeric animals in these studies were considerably shorter than those reported for untreated mice of the same strain and gender, in these animals increased proportions of A cells were associated with significantly longer lifespans. In addition, the lifespan of the B6AF1 chimeric mice was a function of the proportion of A lymphocytes present in the peripheral blood over the course of the animal's life.

Induction of HL-60 cell differentiation by water-soluble and nitrogen-containing conjugates of retinoic acid and retinol.

Retinoids induce the promyelocytic cell line, HL-60, to differentiate along the granulocytic pathway in vitro. A number of water-soluble and nitrogen-containing retinoids were synthesized in our laboratory [retinoyl-glucose (RAGL), retinyl-glucose (ROGL), retinoyl-adenosine (RADS), retinoyl-adenine (RAD), retinoyl-beta-glucuronide (beta RAG), and retinoyl-alpha-glucuronide (alpha RAG)]. These retinoids (10(-5) to 10(-8) M), as well as retinoic acid (RA) and retinol (ROL), were tested for their ability to induce the differentiation of HL-60 cells in vitro and to affect cell growth and viability during a 24- to 72-h incubation period. Differentiation was assessed by measuring the percentage of cells expressing the Mac-1 antigen on their cell surfaces. RA and the conjugates of RA were all quite active in inducing HL-60 cell differentiation, whereas ROL and ROGL had much less activity at equimolar concentrations. beta RAG, alpha RAG, RADS, and RAD were less toxic, whereas the glucose conjugates of retinol and retinoic acid (ROGL and RAGL) were both considerably more toxic than either RA or ROL at equimolar concentrations. All retinoids affected cell growth in a dose-dependent fashion. At 24 h, free RA or ROL was not detected in the cells after incubation with any of the retinoid conjugates.

Major histocompatibility complex antigen expression on lymphocytes from aging strain A mice.

Molecules encoded by the major histocompatibility complex (MHC) are crucial for the proper functioning of the immune response. In this study, the levels of class I and class II major histocompatibility antigens on lymphocytes from strain A mice were measured as a function of age. Class I protein levels increased significantly on both peripheral blood and spleen (T cells and B cells) lymphocytes with age. This increase in MHC class I protein levels was accompanied by an increase in class I mRNA levels. On the other hand, class II protein levels did not show a significant change with age. Moreover, while the percentage of class I-expressing spleen lymphocytes stayed at a steady-state level of 100% with age, the percentage of class II-expressing spleen lymphocytes decreased from 85% in young animals to 70% in old animals. This decrease was due to a decrease in the relative proportion of B cells compared to T cells in the spleen lymphocyte population of old mice. When class II mRNA levels were measured, it was found that these levels decreased markedly with age. Overall, it is clear that the regulation of MHC class I and class II expression changes with age in A strain mice. Since optimal levels of MHC expression are crucial for the proper functioning of cellular and humoral immune responses, it will be most interesting to understand how the control of MHC gene expression changes with age and whether MHC gene expression can be modulated in old individuals to restore better immune function.

Alteration of bronchoalveolar lavage cell populations following bleomycin treatment in mice.

Clinical use of the antitumor antibiotic bleomycin (BLM) is associated with the development of interstitial pulmonary fibrosis. Shortly after acute lung damage caused by intratracheal (it) administration of BLM to experimental animals there is an influx of inflammatory cells which are believed to modulate the process of fibrosis. This study was undertaken to determine what subpopulations of lymphocytes were in the bronchoalveolar lavage (BAL) cell population of C57BL/6J mice at various times after a single it dose of BLM and to determine whether BAL T-lymphocytes were activated after BLM treatment. The BAL lymphocyte population was analyzed by differential cell analysis and flow cytometry utilizing monoclonal antibodies specific for lymphocyte subpopulations. The majority of lymphocytes in the BAL of control and BLM-treated mice were T-lymphocytes, with less than 10% being B-cells. During the first 7 days after BLM the number of Lyt-2+ T-cells exceeded L3T4+ T-cells while in control mice the reverse was observed. The percentage of BAL lymphocytes expressing the IL-2 receptor did not change significantly at 3 and 7 days after BLM treatment, but was significantly decreased at Day 14. In contrast, the total number of lymphocytes expressing the IL-2 receptor was increased at all time points investigated. These results demonstrated that the majority of lymphocytes in the BAL were T-cells and that while the percentage of activated lymphocytes did not increase following BLM treatment, the absolute number did and this increased number of activated lymphocytes may be important in the disease process.

Effect of cytotoxic monoclonal antibody depletion of T-lymphocyte subpopulations on bleomycin-induced lung damage in C57BL/6J mice.

Treatment of cancer patients with the antitumor antibiotic bleomycin (BLM) is associated with lung damage which can progress to pulmonary fibrosis. Shortly after intratracheal (it) administration of BLM to experimental animals there is an influx of inflammatory cells into the lung. These inflammatory cells, consisting primarily of polymorphonuclear cells, monocytes and lymphocytes, are believed to modulate the pathogenesis of pulmonary fibrosis. The objective of the present study was to determine the role of specific T-lymphocyte subpopulations in this disease process following a single it administration of BLM to C57BL/6J mice. Specific in vivo T-lymphocyte subpopulation depletion was accomplished by multiple intraperitoneal administrations of cytotoxic monoclonal antibodies to mice prior to and following BLM administration. Acute lung damage was assessed by measuring levels of angiotensin-converting enzyme and total protein in the bronchoalveolar lavage fluid 7 days after BLM treatment while chronic fibrosis was assessed by total lung hydroxyproline 28 days after BLM. Although we were able to deplete lymph nodes and BAL of specific T-lymphocyte subpopulations we were unable to detect a difference in the extent or severity of either the acute or chronic stage of BLM-induced lung damage. These results suggest that BLM lung disease progresses unabated in C57BL/6J mice despite virtually complete depletion of either L3T4+ or Lyt-2+ T-lymphocytes. Although a greater than 80% decrease in Thy-1.2+ T-lymphocytes was accomplished, there was a residual population of Thy-1.2+ lymphocytes resistant to the cytotoxic antibody. It is possible, therefore, that this population of cells does play a role in the development of BLM lung disease.