Increased near-ultraviolet induced DNA fragmentation in xeroderma pigmentosum variants.

Immediate fragmentation of parental DNA by near-ultraviolet irradiation at 313 nm was measured in cultured skin fibroblasts from normal individuals, patients with Xeroderma pigmentosum of complementation group A (XPA) and Xeroderma pigmentosum variants (XPV) by the alkaline elution procedure. For a dose of 2.25 KJm^?2 given at O^o fragmentation was comparable in all cell strains. However, fragmentation was strongly increased relative to O^o in XPV but not in normal fibroblasts and the XPA strains when irradiation was carried out at 37^o. From our results it appears that a step in the repair of $\text{parental}$ DNA is abnormal in XPV.     

Molecular mechanisms involved in T cell activation. I. Evidence for independent signal-transducing pathways in lymphokine production vs proliferation in cloned cytotoxic T lymphocytes

It is well-established that activated T cells proliferate in response to interleukin 2 (IL 2) and produce various soluble lymphokines such as macrophage-activating factor (MAF) in response to antigen. Prior to investigating the molecular events involved in signaling the initiation of these responses in cloned murine cytotoxic T lymphocytes (CTL), we determined whether these responses could occur independently, and we established for each response the time during which signal transducing mechanisms may function. It was found that this cloned CTL population was in a resting state (G1 phase of cell cycle) 7 days after stimulation with antigen plus IL 2. At this time, the incubation of these resting CTL with IL 2 for 4 to 6 hr resulted in a maximal proliferative response that was not accompanied by the production of MAF. Conversely, the incubation of resting CTL with antigen or lectin (in the absence of IL 2) for at least 8 hr resulted in the maximal production of MAF at 24 hr without inducing a proliferative response. In addition, antigen or lectin, but not IL 2, triggered an immediate (less than 1 min) and sustained (at least 8 hr) mobilization of intracellular calcium. The kinetics of this calcium response paralleled the minimum time (8 hr) that was required for resting CTL to interact with either antigen or lectin in order to produce maximal titers of MAF. These results indicate that proliferation and lymphokine (MAF) production in cloned murine CTL are independent events. In these resting CTL, the signal mechanisms that mediate the production of lymphokines are most likely restricted to the initial 8 hr of stimulation by antigen or lectin and involve the rapid and prolonged mobilization of cytoplasmic calcium. Proliferative signals, however, are probably complete within 4 to 6 hr after stimulation by IL 2 and do not involve readily demonstrable fluxes of cytoplasmic calcium, as determined by the fluorescent calcium probe Quin 2.     

Mechanism of cyclosporin A-induced immunosuppression. Cyclosporin A inhibits receptor-mediated and non-receptor-mediated lymphokine production as well as interleukin-2-induced proliferation in cloned T lymphocytes

The effects of cyclosporin A (CyA) on the activation processes of cloned murine cytotoxic T lymphocytes (CTL) have been examined. With the use of Day 7 resting cloned CTL it was possible to separate the functions of lymphokine production (macrophage-activating factor, MAF) and interleukin 2 (IL-2)-induced proliferation of these cells. The effect of CyA on each of these activities was analyzed independently. CyA was found to inhibit both receptor-mediated MAF production in response to stimulation with antigen and lectin and MAF production in response to non-receptor-mediated stimulation (by anti-Thy-1 antibodies, ionophore, and phorbol ester). Further, CyA was observed to inhibit the re-entry of these resting CTL into the cell cycle upon stimulation with IL-2. The effect of CyA on MAF production did not appear to be due to inhibition of the signal-transducing mechanism involved in this process (i.e., inositol lipid hydrolysis, calcium mobilization, and protein phosphorylation). The action of CyA on the IL-2-induced proliferation was not due to inhibition of IL-2 receptor expression or the binding of IL-2 to its receptor. Thus, CyA appeared to mediate its suppressive effects on MAF production and IL-2-induced proliferation through an action on some later step(s) in the signal pathways of these activities.     

Glomerular podocytes in cultured rat kidney slices

Summary The ultrastructure of rat glomerular epithelial cells (podocytes) in kidney slices in vitro was examined using qualitative and quantitative electron microscopy. The kidney slices were cultured in Medium 199 with Hanks' salts in a 5% CO₂/95% O₂ environment for up to 14 days. Few changes in podocyte ultrastructure occurred in the first 12 h of culture, but by 24 h cell bodies were rounded, microvilli were present on all podocyte surfaces, and some foot processes had been replaced by flattened expanses of cytoplasm. These changes were more pronounced by 3 days, when some podocytes had developed pseudopodal extensions and appeared to be migrating from glomeruli onto the slice surface. Podocytes could still be identified after 8, 10 and 14 days of culture, although relatively few glomeruli remained at 14 days. Morphometric methods were used to analyse podocyte shape, volume and surface area during the first 4 days of culture. The most significant change involved loss of foot processes: the number of filtration slits per 100 m of basement membrane decreased from 211.8 ± 15.0 (mean ± SD) at the commencement of culture, to 55.3 ± 22.6 after 2 days (P < 0.001). These data provide baseline information for in vitro studies on the effects of nephrotoxins on podocytes.     

Segments of the POU domain influence one another''s DNA-binding specificity.

The ubiquitously expressed mammalian POU-domain protein Oct-1 specifically recognizes two classes of cis-acting regulatory elements that bear little sequence similarity, the octamer motif ATGCAAAT and the TAATGARAT motif. The related pituitary-specific POU protein Pit-1 also recognizes these two motifs but, unlike Oct-1, binds preferentially to the TAATGARAT motif. Yet in our assay, Pit-1 still binds octamer elements better than does the octamer motif-binding protein Oct-3. The POU domain is responsible for recognizing these diverse regulatory sequences through multiple DNA contacts that include the two POU subdomains, the POU-specific region, and the POU homeodomain. The DNA-binding properties of 10 chimeric POU domains, in which different POU-domain segments are derived from either Oct-1 or Pit-1, reveal a high degree of structural plasticity; these hybrid proteins all bind DNA well and frequently bind particular sites better than does either of the parental POU domains. In these chimeric POU domains, the POU-specific A and B boxes and the hypervariable POU linker can influence DNA-binding specificity. The surprising result is that the influence a particular segment has on DNA-binding specificity can be greatly affected by the origin of other segments of the POU domain and the sequence of the binding site. Thus, the broad but selective DNA-binding specificity of Oct-1 is conferred both by multiple DNA contacts and by dynamic interactions within the DNA-bound POU domain.     

Expression of the full-length rabbit prolactin receptor and its specific domains in baculovirus infected insect cells

The prolactin receptor is a membrane protein mainly involved in the development of the mammary gland and in lactation in mammals. We used specific cDNA constructs and the insect/baculovirus expression system and produced independently and in large amounts several recombinant forms of the rabbit mammary gland prolactin receptor: the full-length receptor (L1, L2), a truncated membrane form (S), a secretable form of the extracellular domain (E) and two forms of the intracellular domain (I1, I2). Of these forms, the L1 and L2 are associated with the membrane fraction, the E is predominantly secreted into the medium and the I1 and I2 are expressed as soluble proteins and surprisingly, a great portion accumulates in the culture medium. The molecular mass (94 kDa) of the expressed full-length receptor corresponds to the translation product of the entire cDNA coding region. The receptor biochemically identified in the rabbit mammary gland is however much shorter. Thus, in the mammary gland, the receptor presumably undergoes post-translational modifications. The receptor forms L1, L2 and S bind prolactin with specificity and affinity similar to those reported for the native receptor. They also interact with two monoclonal antibodies, M110 and A917, specific for the native conformation of the hormone-binding site. The I1 and I2 forms do not bind prolactin, whereas the E form does. Thus, the hormone binding site is located in the extracellular domain which can function autonomously as a PRL-binding soluble protein. However, the E form binds prolactin with a higher affinity than the native receptor and it does not bind one of the two antireceptor monoclonal antibodies, known to be hormone binding-site specific. Thus, the conformation of the native receptor and that of the E form differ.     

Poly ADP-ribosylation of histones in tumor promoter phorbol-12-myristate-13-acetate treated mouse embryo fibroblasts C3H10T1/2

The tumor promoter phorbol-12-myristate-13-acetate (PMA) increases the poly ADP-ribosylation of acid extractable (0.2N H2SO4) nuclear proteins in mouse embryo fibroblasts C3H10T1/2. Catalase suppresses the reaction by approximately 50%. Polyacrylamide gel electrophoresis reveals that the core histones H2B, A24 and H3d serve as major poly ADP-ribose acceptors. Smaller amounts of poly ADP-ribose are associated with histones H2A/H3 and H1. Poly ADP-ribosylation of histones may change the nucleosomal structure and function and play a role in PMA induced modulation of gene expression in promotion.     

Suppression of tumor promoter phorbolmyristate acetate-induced chromosome breakage by antioxidants and inhibitors of arachidonic acid metabolism

To gain insight into the mechanism of formation of chromosomal aberrations by the tumor promoter phorbolmyristate acetate (PMA) in human lymphocytes, we investigated the effect of antioxidants and inhibitors of arachidonic acid metabolism. Among the antioxidants bovine erythrocyte CuZn superoxide dismutase, glutathione peroxidase, mannitol (a scavenger of hydroxyl radicals), butylated hydroxytoluene and butylated hydroxyanisole were anticlastogenic while catalase and dimethylfuran (a scavenger of singlet oxygen) were inactive. These results show that the induction of aberrations by PMA occurs via indirect action, i.e. the intermediacy of superoxide and hydroxyl radicals. The following inhibitors of arachidonic acid metabolism were strongly anticlastogenic: the cyclo-oxygenase inhibitors indomethacin and flufenamic acid and the lipoxygenase inhibitor BN1015. Imidazole, nordihydroguaiaretic acid BN 1048 and 5,8,11,14-eicosatetraynoic acid were moderately active. The inhibitor of phospholipase A₂, fluocinolone acetonide, was also anticlastogenic.

We conclude that the oxidative metabolism of arachidonic acid is involved in the induction of chromosomal aberrations by PMA in human lymphocytes. However, because of the limited selectivity of these drugs, it is not yet possible to identify unambiguously the step(s) in the arachidonic acid cascade responsible for PMA clastogenicity.