Molecular detection of microscopic and submicroscopic deletions associated with Miller-Dieker syndrome.

Miller-Dieker syndrome (MDS), a disorder manifesting the severe brain malformation lissencephaly ("smooth brain"), is caused, in the majority of cases, by a chromosomal microdeletion of the distal short arm of chromosome 17. Using human chromosome 17-specific DNA probes, we have begun a molecular dissection of the critical region for MDS. To localize cloned DNA sequences to the MDS critical region, a human-rodent somatic cell hybrid panel was constructed which includes hybrids containing the abnormal chromosome 17 from three MDS patients with deletions of various sizes. Three genes (myosin heavy chain 2, tumor antigen p53, and RNA polymerase II) previously mapped to 17p were excluded from the MDS deletion region and therefore are unlikely to play a role in its pathogenesis. In contrast, three highly polymorphic anonymous probes, YNZ22.1 (D17S5), YNH37.3 (D17S28), and 144-D6 (D17S34), were deleted in each of four patients with visible deletions, including one with a ring chromosome 17 that is deleted for a portion of the single telomeric prometaphase subband p13.3. In two MDS patients with normal chromosomes, a combination of somatic cell hybrid, RFLP, and densitometric studies demonstrated deletion for YNZ22.1 and YNH37.3 in the paternally derived 17's of both patients, one of whom is also deleted for 144-D6. The results indicate that MDS can be caused by submicroscopic deletion and raises the possibility that all MDS patients will prove to have deletions at a molecular level. The two probes lie within a critical region of less than 3,000 kb and constitute potential starting points in the isolation of genes implicated in the severe brain maldevelopment in MDS.     

Significance testing for correlated binary outcome data

Multiple logistic regression is a commonly used multivariate technique for analyzing data with a binary outcome. One assumption needed for this method of analysis is the independence of outcome for all sample points in a data set. In ophthalmologic data and other types of correlated binary data, this assumption is often grossly violated and the validity of the technique becomes an issue. A technique has been developed (Rosner, 1984) that utilizes a polychotomous logistic regression model to allow one to look at multiple exposure variables in the context of a correlated binary data structure. This model is an extension of the beta-binomial model, which has been widely used to model correlated binary data when no covariates are present. In this paper, a relationship is developed between the two techniques, whereby it is shown that use of ordinary logistic regression in the presence of correlated binary data can result in true significance levels that are considerably larger than nominal levels in frequently encountered situations. This relationship is explored in detail in the case of a single dichotomous exposure variable. In this case, the appropriate test statistic can be expressed as an adjusted chi-square statistic based on the 2 X 2 contingency table relating exposure to outcome. The test statistic is easily computed as a function of the ordinary chi-square statistic and the correlation between eyes (or more generally between cluster members) for outcome and exposure, respectively. This generalizes some previous results obtained by Koval and Donner (1987, in Festschrift for V. M. Joshi, I. B. MacNeill (ed.), Vol. V, 199-224.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colchicine inhibits ionophore-induced formation of leukotriene B4 by human neutrophils: the role of microtubules

Neutrophils which ingest particles (serum-treated zymosan, monosodium urate crystals) or are exposed to calcium ionophore A23187 generate leukotriene B4 (LTB4). Earlier work has shown that cells exposed to colchicine before exposure to monosodium urate crystals produce less LTB4; the formation of 5-HETE is unaffected. To determine whether inhibition by colchicine of LTB4 generation was stimulus-specific and was mediated by microtubule integrity, the effects of colchicine (10 microM, 60 min) on the release of lipoxygenase products from neutrophils exposed to ionophore A23187 (10 microM, 5 min) were examined. In the presence of exogenous arachidonic acid (100 microM, 15 min), colchicine decreased LTB4 to 48% +/- 11.7 of control and 5-HETE to 60.5% +/- 5.7 of control (mean +/- SEM); 15-HETE was also decreased to 61% +/- 10.3 of control. In the absence of exogenous arachidonate, LTB4 was decreased to 22.2% +/- 11.7 of control and 5-HETE to 13% +/- 4.8 of control. Lumicolchicine did not significantly affect formation of 5-HETE or LTB4. However, vinblastine sulfate (20 microM, 60 min), another microtubule-disruptive agent, decreased the formation of both 5-lipoxygenase products. The effects of colchicine and vinblastine were not due to impairment of cell viability because the release of cytoplasmic lactic dehydrogenase was unaffected. Ultrastructural analysis of centriolar microtubules showed that decrements in microtubule numbers of colchicine- and vinblastine-treated cells paralleled decrements in 5-lipoxygenase products. These pharmacologic manipulations suggested that functional microtubules might be required for optimal lipoxygenase activity. Consequently, we prepared neutrophil-derived cytoplasts, devoid of an intact microtubule system. No significant decreases in the 5- or 15-lipoxygenase products were found when cytoplasts were exposed to colchicine in the presence of exogenous arachidonate and A23187. The data show that colchicine inhibits the formation of lipoxygenase products from neutrophils stimulated with A23187, most likely via its effect on microtubules, the integrity of which appears necessary for full expression of 5- and 15-lipoxygenases.     

Regulation of primary cytotoxic T lymphocyte responses generated during mixed leukocyte culture with H-2d identical Qa-1-disparate cells

Cytotoxic lymphocyte (CTL) responses are not usually generated during primary mixed leukocyte culture (MLC) with H-2 identical cells. Thus NZB mice are unusual in that their spleen cells do mount CTL responses during primary MLC with H-2d identical stimulator cells; the predominant target antigen for these NZB responses is Qa-1b. Considering the numerous immunoregulatory defects in NZB mice, we postulated that these NZB anti-Qa-1 primary CTL responses were due to an abnormality in T suppressor cell activity. Cellular interactions capable of suppressing NZB anti-Qa-1 primary CTL responses were investigated by using one-way and two-way MLC with spleen cells from NZB mice and other H-2d strains. Although H-2d identical one-way MLC with the use of NZB responders resulted in substantial CTL responses, only minimal CTL responses were detected from two-way MLC with the use of NZB spleen cells plus nonirradiated spleen cells from other H-2d mice. Thus the presence of non-NZB spleen cells in the two-way H-2d identical MLC prevented the generation of NZB CTL. Noncytotoxic mechanisms were implicated in the suppression of the NZB CTL responses during two-way MLC, because only minimal CTL activity was generated when NZB spleen cells were cultured with semiallogeneic, H-2d identical (e.g., NZB X BALB) F1 spleen cells. The observed suppression could be abrogated with as little as 100 rad gamma-irradiation to the non-NZB spleen cells. The phenotype of these highly radiosensitive spleen cells was Thy-1+, Lyt-1+, Lyt-2-, L3T4+. The functional presence of these cells in the spleens of semiallogeneic, H-2d identical F1 mice indicated that their deficiency in NZB mice was a recessive trait. These data suggest that NZB mice lack an L3T4+ cell present in the spleens of normal mice that is capable of suppressing primary anti-Qa-1 CTL responses. This model system should facilitate additional investigations of the cellular interactions and immunoregulatory mechanisms responsible for controlling primary CTL responses against non-H-2K/D class I alloantigens. The model may also provide insight into the immunoregulatory defects of autoimmune NZB mice.     

Cellular requirements for the generation of primary cell-mediated lympholysis responses to Qa-1 antigens

Mixed leukocyte cultures (MLC) between NZB responder spleen cells and Qa-1-disparate stimulator spleen cells were employed to determine the cellular requirements for the generation of primary anti-Qa-1 cell-mediated lympholysis (CML) responses. Although primary anti-Qa-1 cytotoxic lymphocytes (CTL) were generated during H-2-homologous stimulation, anti-Qa-1 CTL were not detectable from MLC in which the stimulators were H-2 allogeneic. Anti-Qa-1 CTL also were not generated from MLC in which the stimulators were semiallogeneic. Thus, H-2 identity between responder and stimulator cells was not sufficient to permit the generation of primary anti-Qa-1 CTL when H-2 disparity was also present. The capacity for H-2 disparity to prevent anti-Qa-1 CML responses was further demonstrated in MLC containing both H-2-allogeneic and H-2-homologous stimulator cells. Therefore, in subsequent studies we employed NZB responders and H-2-homologous, Qa-1-disparate stimulators. When various subpopulations of stimulator cells were studied for their ability to induce anti-Qa-1 CTL, nylon wool-adherent cells were found to be potent stimulators, but nylon wool-nonadherent cells were not. Furthermore, depletion of macrophages from the stimulator population abrogated the generation of anti-Qa-1 CML responses, despite the presence of responder macrophages in the culture. In contrast, all fractionated subpopulations stimulated anti-H-2 CML responses. When macrophage-enriched cells were used as stimulators, anti-Qa-1 CTL could be generated with approximately 80-fold fewer stimulator cells than when unfractionated splenocytes were used as stimulators. These findings indicated that stimulator macrophages were essential for the generation of primary anti-Qa-1 CTL. Direct evidence for macrophage expression of Qa-1-antigens was obtained by using a Qa-1b-specific CTL clone. These studies provide i) the first evidence for Qa-1 expression on macrophages, ii) a basis for comparison of the cellular interactions necessary to generate CTL against H-2K/D-encoded vs Qa-1-encoded class 1 antigens, and iii) a model for investigating the mechanisms responsible for the immunodominance of H-2K/D alloantigens.     

Polymorphism and tissue distribution of maternally transmitted antigen defined by cytotoxic T lymphocyte lines

We have developed cytotoxic T lymphocyte (CTL) lines specific for two determinants of the maternally transmitted antigen (Mta) and have used these CTL lines to study the tissue distribution of Mta. In previous reports, we characterized CTL lines specific for the Mta.1 determinant. Here, we describe CTL lines specific for the newly defined Mta.2 determinant. Mta.2-specific CTL lines lysed target cells from F1 mice of an NZB (Mtf beta) mother but did not lyse target cells from reciprocal F1 mice of any Mtf alpha mother. Backcross mice were used as the source of target cells to demonstrate that the Mta.2-specific CTL were H-2 nonrestricted in their recognition. Mta.-1- and Mta.2-specific CTL lines were used to demonstrate Mta expression on lymphoid cells taken from spleen, thymus, lymph nodes, and bone marrow. In addition, Mta was expressed on cultured cell lines of myeloid, epithelial, and mesenchymal origin. Our results suggest that Mta is not a differentiation marker restricted to lymphocyte subpopulations.     

Meiotic Gene Conversion Mutants in SACCHAROMYCES CEREVISIAE . I. Isolation and Characterization of pms1-1 and pms1-2

The pms1 mutants, isolated on the basis of sharply elevated meiotic prototroph frequencies for two closely linked his4 alleles, display pleiotropic phenotypes in meiotic and mitotic cells. Two isolates carrying recessive mutations in PMS1 were characterized. They identify a function required to maintain low postmeiotic segregation (PMS) frequencies at many heterozygous sites. In addition, they are mitotic mutators. In mutant diploids, spore viability is reduced, and among survivors, gene conversion and postmeiotic segregation frequencies are increased, but reciprocal exchange frequencies are not affected. The conversion event pattern is also dramatically changed in multiply marked regions in pms1 homozygotes. The PMS1 locus maps near MET4 on chromosome XIV. The PMS1 gene may identify an excision-resynthesis long patch mismatch correction function or a function that facilitates correction tract elongation. The PMS1 gene product may also play an important role in spontaneous mitotic mutation avoidance and correction of mismatches in heteroduplex DNA formed during spontaneous and UV-induced mitotic recombination. Based on meiotic recombination models emphasizing mismatch correction in heteroduplex DNA intermediates, this interpretation is favored, but alternative interpretations involving longer recombination intermediates in the mutants are also considered.     

Isolation of Z-DNA binding proteins from SV40 minichromosomes: evidence for binding to the viral control region

Proteins dissociated from SV40 minichromosomes by increasing NaCl concentration were tested for their binding to Z-DNA [Br-poly(dG-dC)] and B-DNA [poly (dG-dC)]. Z-DNA binding proteins are largely released in 0.2 M NaCl whereas most B-DNA binding proteins are not released until 0.6 M NaCl. Incubation of SV40 minichromosomes with Z-DNA-Sephadex in low salt solution results in proteins with Z-DNA binding activity (PZ proteins). These proteins bind to negatively supercoiled DNAs containing left-handed Z-DNA but not to relaxed DNAs. They compete with anti-Z-DNA antibodies in binding to negatively supercoiled DNAs. The binding is tighter to negatively supercoiled SV40 DNA than to other plasmids, suggesting sequence-specific Z-DNA binding. PZ proteins binding to negatively supercoiled SV40 DNA interfere with cleavage at the Sph I sites, within the 72 bp repeat sequences of the viral control region, but not with cleavage at the Bgl I site, at the origin of replication. Removal of PZ proteins also exposes the Sph I sites in the SV40 minichromosomes while addition of PZ proteins makes the sites inaccessible.     

Immunoglobulin G-induced experimental chronic immune synovitis: cell-mediated immunity to native interstitial collagen molecules and their constituent polypeptide chains

In vitro cell-mediated immune responses to homologous rabbit immunoglobulin G (IgG), purified protein derivative (PPD), native Type I, II, and III collagen, and denatured Type I, II, and III collagen were studied in an IgG-induced animal model of immune synovitis. Immune response was measured as augmented [3H]thymidine incorporation by spleen cells on exposure to antigen. Immune responses were observed in vitro after 72 hr of culture with antigen, while a majority of responses to antigens occurred after 96 hr of incubation. Separation of spleen cell subpopulations showed that measured immune responses were of T-cell origin. In vitro cell-mediated immune responses were observed for native and denatured collagen in splenic cell cultures from six of seven synovitic rabbits (P less than 0.01) but not in control spleen cell cultures derived from normal, adjuvant-primed or IgG-immune nonsynovitic rabbits. The incidence of cellular reactivity to incubation with native interstitial collagens was as follows: Type I, 43%; Type II, 43%; Type III, 57%. The incidence of in vitro immune responses to denatured collagens in cultures derived from rabbits with synovitis was: Type I, 50%; Type II, 50%; Type III, 67%. The relatively high incidence of immune response to both native and denatured collagens suggests that immunity to structural components of the synovial membrane and the adjacent surface of articular cartilage may play a role in the inflammation observed in immune synovitis.     

Diacylglycerol modulates binding and phosphorylation of the epidermal growth factor receptor

Tumor promoters cause a variety of effects in cultured cells, at least some of which are thought to result from activation of the Ca2+-phospholipid-stimulated protein kinase C. One action of tumor promoters is the modulation of the binding and phosphorylation of the epidermal growth factor (EGF) receptor in A431 cells. To determine if these compounds act on the EGF receptor by substituting for the endogenous activator of C kinase, diacylglycerol, we compared the effects of the potent tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) with those of the synthetic diacylglycerol analog 1-oleyl 2-acetyl diglycerol (OADG). When A431 cells were treated with TPA, the subcellular distribution of C kinase activity shifted from a predominantly cytosolic location to a membrane-associated state; OADG also caused the disappearance of cytosolic C kinase activity. The shift in the subcellular distribution of C kinase, caused by TPA or OADG, correlated with changes in binding and phosphorylation of the EGF receptor. OADG, like TPA, caused loss of binding to an apparent high affinity class of receptors, blocked EGF-induced tyrosine phosphorylation of the EGF receptor, and stimulated phosphorylation of the EGF receptor at both serine and threonine residues. No difference between the phosphopeptide maps of receptors from cells treated with OADG or TPA was observed. Thus, it appears that tumor promoters can exert their effects on the EGF receptors by substituting for diacylglycerol, presumably by activating protein kinase C. Further, these results suggest that endogenously produced diacylglycerol may have a role in normal growth regulatory pathways.