The human apolipoprotein B-100 gene: a highly polymorphic gene that maps to the short arm of chromosome 2

Using a cloned cDNA of apolipoprotein B-100 as hydridization probe, we have found high frequence polymorphisms in the apoB-100 gene involving sites for the restriction enzymes EcoRI, BamHI, and HindIII. The major EcoRI polymorphisms involved a 17 kb vs 15 kb variant. The incidence of the various phenotypes was estimated. In addition, other complex polymorphisms involving MspI and TaqI sites were also noted. [32P]-labeled apoB-100 cDNA was used as a probe in chromosome mapping studies to detect the human apoB-100 structural gene sequence in human-Chinese hamster and human-mouse cell hybrids. Southern blot analysis of 14 hybrids localized the gene to the short arm of human chromosome 2.     

Lysis of cells infected with HIV-1 by human lymphocytes targeted with monoclonal antibody heteroconjugates

The present study was undertaken to determine whether human PBL can be specifically focused to lyse cells infected with HIV-1 by mAb heteroconjugates that can bridge target and effector cells. A mAb directed against the central portion of HIV-1 glycoprotein gp110 was chemically cross-linked to a mAb directed against the CD3/TCR complex or to a mAb directed against the CD16 Fc gamma-R expressed on large granular lymphocytes (LGL). HIV-1-infected cells, but not uninfected cells, were found to be lysed to a greater extent by PBL in the presence of the gp110 X CD3 or the gp110 X CD16 antibody heteroconjugate than in the presence of the single antibodies or a mixture of the mAb comprising the heteroconjugates. Pretreatment of PBL with anti-CD3 or IL-2 augments their ability to lyse HIV-1-infected cells in the presence of the heteroconjugates. Lysis by anti-CD3-activated PBL in the presence of the gp110 X CD3 heteroconjugate was found to be mediated by CD8+-enriched T cells, whereas lysis by IL-2-treated PBL in the presence of the gp110 X CD16 heteroconjugate is mediated by PBL enriched for CD16+ cells, which are primarily LGL. Furthermore, PBL from asymptomatic, HIV-1-infected seropositive donors were found to be functional in lysing HIV-1-infected cells in the presence of the antibody heteroconjugates. Such antibody heteroconjugates, which can target T cells or LGL to lyse HIV-1-infected cells, may be of prophylactic or therapeutic value in HIV-1-infected individuals.     

CD3-zeta surface expression is required for CD4-p56lck-mediated upregulation of T cell antigen receptor-CD3 signaling in T cells.

It has been proposed that during T cell receptor antigen recognition, CD4- or CD8-p56lck molecules interact with the T cell antigen receptor-CD3 complex (TCR-CD3) to phosphorylate various undefined substrates, which then initiate signal transduction through the TCR-CD3 complex. The ability of CD4 to modulate the TCR-CD3-induced increase in intracellular Ca2+, [Ca2+]i, and substrate tyrosine phosphorylation was studied in mutants of the human leukemic T cell line HPB-ALL characterized by their low expression of the TCR-CD3 complex on the cell surface. In TCR-CD3low cells, in which CD3-zeta was found to be associated with the TCR-CD3 complex, cross-linking CD3 with CD4 resulted in a profile of calcium mobilization, CD3-zeta, and phospholipase C-gamma 1 tyrosine phosphorylation similar to that observed in HPB-ALL cells, although the magnitude of generalized substrate tyrosine phosphorylation appeared to be smaller, as compared with wild-type cells. Responses were weak or absent when CD3 was cross-linked alone. In contrast, in a mutant in which association of CD3-zeta 2 with the TCR-CD3 was defective, cross-linking of CD3 with CD4 had a weaker effect on any of the activation parameters tested. These experiments showed that the presence of CD3-zeta 2 in the TCR-CD3 complex is of critical importance for the ability of CD4 to enhance early transducing signals inside the cell. The data also suggest that CD4-associated protein tyrosine kinase p56lck could up-regulate defective CD3-mediated induction of phospholipase C activity by increasing tyrosine phosphorylation of phospholipase C-gamma 1.     

Lymphocyte lineage-restricted tyrosine-phosphorylated proteins that bind PLC gamma 1 SH2 domains.

Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibroblasts induces tyrosine phosphorylation of PLC gamma 1 and stable association of PLC gamma 1 with the receptor protein tyrosine kinase. Similarly in lymphocytes, cross-linking of antigen receptors induces the formation of molecular complexes incorporating PLC gamma 1; however, associated kinase activity is thought to be mediated through cytoplasmic protein tyrosine kinase(s). In this report, we generated a fusion protein containing the SH2 domains of human PLC gamma 1 and human IgG1 heavy chain constant region to identify lymphocyte phosphoprotein-binding PLC gamma 1 SH2 domains following cellular activation. As in EGF- or platelet-derived growth factor-stimulated fibroblasts, PLC gamma 1 is coprecipitated in activated lymphocytes, complexed with associated tyrosine-phosphorylated proteins. One of these, a 35/36-kDa protein found prominently in T cells and at lower levels in B cells, bound to the fusion protein in immunoprecipitation experiments. The fusion protein showed lineage restricted association with a 74-kDa phosphoprotein in T cells and a 93-kDa phosphoprotein in B cells. It bound to activated EGF receptor in fibroblasts as expected, and protein tyrosine kinase activity was precipitated from EGF-stimulated cells. However, PLC gamma 1-associated protein tyrosine kinase activity was not detected in activated lymphocytes. These data suggest that lymphocyte PLC gamma 1 SH2-binding proteins are cell lineage specific and may be transiently associated with activated PLC gamma 1.     

B7 costimulates proliferation of CD4-8+ T lymphocytes but is not required for the deletion of immature CD4+8+ thymocytes.

In addition to TCR-derived signals, costimulatory signals derived from stimulation of the CD28 molecule by its natural ligand, B7, have been shown to be required for CD4+8- T cell activation. We investigate the ability of B7 to provide costimulatory signals necessary to drive proliferation and differentiation of virgin CD4-8+ T-cells that express a transgenic TCR specific for the male (H-Y) Ag presented by H-2Db class I MHC molecules. Virgin male-specific CD4-8+ T cells can be activated either with B7 transfected chinese hamster ovary (CHO) cells and T3.70, a mAb specific for the transgenic TCR-alpha chain that is associated with male-reactivity, or by male dendritic cells (DC). Activated CD4-8+ T cells proliferated in the absence of exogenously added IL-2. IL-2 activity was detected in supernatants of CD4-8+T3.70+ cells that were stimulated with T3.70 and B7+CHO cells. The response of CD4-8+T3.70+ cells to T3.70/B7+CHO or to male DC stimulation were inhibited by CTLA4Ig, a fusion protein comprising the extracellular portion of CTLA4 and human IgG C gamma 1. It has been previously shown that CTLA4Ig binds B7 with high affinity. Staining with CTLA4Ig revealed that DC express about 50 times more B7 than CD4-8+ T cells. CTLA4Ig also specifically blocked the proliferation of male-reactive cells in vivo. We have also used an in vitro deletion assay whereby immature CD4+8+ thymocytes expressing the transgenic male-specific TCR are deleted by overnight incubation with either immobilized T3.70 or male DC to investigate the participation of the CD28/B7 pathway in the negative selection of immature thymocytes. Staining with B7Ig established that both immature murine CD4+8+ and mature CD4-8+ thymocytes express a high level of CD28. However, despite the high expression of CD28 on CD4+8+ thymocytes, it was found that deletion of CD4+8+ thymocytes expressing the male-specific TCR by the T3.70 mAb was not inhibited by B7+CHO cells. Furthermore, the deletion of these thymocytes by DC also was not inhibited by CTLA4Ig. These findings provide evidence that although signaling through CD28 can costimulate a primary anti-male response in mature CD4-8+ T cells, the CD28/B7 pathway does not appear to participate in the negative selection of immature CD4+8+ thymocytes.     

Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4.

Interaction of the B7 molecule on antigen-presenting cells with its receptors CD28 and CTLA-4 on T cells provides costimulatory signals for T cell activation. We have studied the effects of B7 on antitumor immunity to a murine melanoma that expresses a rejection antigen associated with the E7 gene product of human papillomavirus 16. While this E7+ tumor grows progressively in immunocompetent hosts, cotransfection of its cells with B7 led to tumor regression by a B7-dependent immune response mediated by CD8+ cytolytic T lymphocytes. The immune response induced by E7+B7+ tumor cells also caused regression of E7+B7- tumors at distant sites and was curative for established E7+B7- micrometastases. Our findings suggest that increasing T cell costimulation through the CD28 and CTLA-4 receptors may have therapeutic usefulness for generating immunity against tumors expressing viral antigens.     

Detection of deletions and cryptic translocations in Miller-Dieker syndrome by in situ hybridization.

Fluorescence in situ hybridization (FISH) using two cosmid probes (41A and P13) from the Miller-Dieker syndrome (MDS) critical region in 17p13.3 was performed in a blinded comparison of three MDS patients with submicroscopic deletions and in four normal relatives used as controls. The controls showed both chromosome 17 homologues labeled in 85%-95% of cells, while each patient showed only one homologue labeled in 75%-80% of cells. Two MDS patients with cryptic translocations were also studied. In one case, a patient and her mother had the same der(17) (p+), but the reciprocal product of the translocation could not be identified in the mother by G-banding (i.e., it was a "half-cryptic" translocation). FISH revealed a 3q;17p translocation. The other case involved a patient with apparently normal karyotype. Because a large molecular deletion was found, a translocation involving two G-negative telomeres (i.e., a "full-cryptic" translocation) was postulated. FISH studies on her father and normal brother showed an 8q;17p translocation. These studies demonstrate that in situ hybridization is an efficient method for deletion detection in Miller-Dieker syndrome. More important, parental studies by FISH on patients demonstrating molecular deletions and a normal karyotype may identify cryptic translocation events, which cannot be detected by other molecular genetic strategies. Similar in situ strategies for deletion detection can be developed for other microdeletion syndromes, such as Prader-Willi/Angelman syndrome or DiGeorge syndrome.     

Tumor suppression by chromosome 11 is not due to cellular senescence.

Previous hybrid studies involving fusion of normal with immortal human cells indicated that the phenotype of cellular senescence is dominant and that immortality results from recessive changes in normal growth regulatory genes. We have further assigned 28 different immortal human cell lines to at least four complementation groups for indefinite division. In order to identify the chromosomes involved in regulating cell proliferation, we have introduced single human chromosomes by microcell fusion into immortal human cells representative of the different complementation groups. Our results demonstrate that the introduction of chromosome 11, implicated in tumor suppression, does not cause cellular senescence in three different immortal human cell lines tested.     

In vitro screening procedure for osmotic tolerance in Prunus

Significant growth differences (p<0.01) were observed for two micropropagated Prunus accessions after 14 days in culture when 685 mM mannitol was included in Quoirin and Lepoivre nutrient medium. While there was an 11% growth increase in fresh weight during the 28-day culture period for accession K537-067, explants from New Jersey Plumcot No. 3 increased fresh weight an average of 123%. Similar tests were conducted to determine the repeatability of this short term in vitro screening procedure. Explants of Mananna 2624 were subjected to two levels of mannitol, 275 mM and 550 mM, included in the Quoirin and Lepoivre nutrient medium. Three successive 28-day tests were conducted. Explants were examined at both 14 and 28 days after the onset of the experiment for net growth changes. Addition of mannitol to the nutrient medium at concentrations of 275 mM and 550 mM decreased explant fresh weights of Marianna 2624 to 36% and 28% of controls, respectively, at 28 days past initial culture. Initial fresh weight and fresh weight changes at day 14 were significantly different (p 0.05) between tests. No significant differences existed between tests with regard to weight changes at 28 days past initial culture. This information may aid Prunus breeders in the choice of procedures for inducing drought stress and screening large numbers of plant accessions.Abbreviations K5 K537-067 - M2624 Marianna 2624 - NJPC3 New Jersey Plumcot No. 3 - Q & L Quoirin and Lepoivre     

Molecular characterization of a 130-kb terminal microdeletion at 22q in a child with mild mental retardation.

We have analyzed a recently described 22q13.3 microdeletion in a child with some overlapping features of the cytologically visible 22q13.3 deletion syndrome. Patient NT, who shows mild mental retardation and delay of expressive speech, was previously found to have a paternal microdeletion in the subtelomeric region of 22q. In order to characterize this abnormality further, we have constructed a cosmid/P1 contig covering the terminal 150 kb of 22q, which encompasses the 130-kb microdeletion. The microdeletion breakpoint is within the VNTR locus D22S163. The cloning of the breakpoint sequence revealed that the broken chromosome end was healed by the addition of telomeric repeats, indicating that the microdeletion is terminal. This is the first cloned terminal deletion breakpoint on a human chromosome other than 16p. The cosmid/P1 contig was mapped by pulsed-field gel electrophoresis analysis to within 120 kb of the arylsulfatase A gene, which places the contig in relation to genetic and physical maps of the chromosome. The acrosin gene maps within the microdeletion, approximately 70 kb from the telomere. With the distal end of chromosome 22q cloned, it is now possible to isolate genes that may be involved in the overlapping phenotype of this microdeletion and 22q13.3 deletion syndrome.