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Biomedical subjects

M I Greene

Publications and source records attributed to M I Greene.

At least 91 records · Page 5Linked to original sources
PubMed1994

Considerations in the design and production of small anti-receptor antibody forms: optimizing gains while reducing size.

Recent advances which have lead to the cloning of numerous immunoglobulin genes has resulted in the production of a variety of recombinant small antibody-like molecules. These smaller immunoglobulin derived molecules have potential for the development of diagnostic and pharmaceutical compounds. The considerations in the design and production of smaller antibody-like molecules and their clinical applications will be summarized in this review.

Antibodies, Monoclonal↗
PubMed1994

Kinase-deficient neu proteins suppress epidermal growth factor receptor function and abolish cell transformation.

p185c-neu and epidermal growth factor receptor (EGFR) associate into an active heterodimer, and overexpression of these two receptors leads to a transformed phenotype. However, the association of EGFR and kinase-deficient Neu proteins (point mutant N757 or cytoplasmic domain deletion mutant N691stop) results in a defective or inactive heterodimeric complex. In this report we explore the biological consequences of heterodimerization between EGFR and wild-type (WT) or kinase-deficient mutant Neu proteins in living cells. We show that co-expression of EGFR and kinase-deficient Neu proteins abolished the synergistic transformation and tumorigenicity. Moreover, the normal responses of EGFR to ligand were significantly suppressed, e.g., loss of EGF-dependent transformation, reduced rate of receptor endocytosis and turnover, diminished DNA synthesis, and decreased EGF binding affinity. These results provide the first evidence that kinase-deficient Neu proteins suppress normal EGFR function and display a dominant negative mutant phenotype. Together with the stimulatory effects observed in cells forming active heterodimers, these studies provide a role for heterodimerization of EGFR and Neu/c-erbB2 in interreceptor activation and synergistic signaling which may be responsible for the transition from normal receptor function into oncogenesis.

Animals↗
PubMed1994

The neu-oncogene: signal transduction pathways, transformation mechanisms and evolving therapies.

A variety of pathways exist to transmit biological signals. One mechanism used for the regulated control of cell growth and differentiation is through the transduction of signals resulting from the binding of soluble polypeptide growth factors to their cognate receptors. The specificity of growth factor action is mediated by the interaction of ligand with cognate receptors which can lead to exquisite control in a tissue- and developmental-specific manner. In addition, individual receptors on the cell surface can form complex assemblies with other receptor/signal transduction molecules that potentially lead to additional levels of signal transmissions. Biological signaling by peptide ligands can be mediated through the enzymatic activation of the receptor resulting in the triggering of a defined biochemical pathway. Ultimately, a mitogenic or differentiation signal is delivered to the nucleus, completing the biological action of the growth factor. The biochemical mechanisms of signal transduction by the p185 neu/c-erbB-2 growth factor receptor and the subsequent physiological responses are the topics of this review. Study of the p185 growth factor receptor has helped to illustrate the functional role of receptor homo- (and hetero-) dimerization in enzyme activation and, in malignant cells, the detrimental results of structural mutations or aberrant gene expression which may effect this dimerization. The ability of one type of growth factor receptor to affect the activity of another (as illustrated by the p185/epidermal growth factor receptor heterodimeric complex) is likely to be a common regulatory feature of growth factor receptor action. The nomenclature to be used in this review will refer to the oncogenic mutated form of the rat protein as 'p185neu', the proto-oncogenic rat protein as 'p185c-neu' and the human form as 'p185c-erbB-2'. The term 'p185' will be used to refer to any type of protein, regardless of the source.

Animals↗
PubMed1993

Reduced CD3-mediated protein tyrosine phosphorylation in anergic CD4+ and CD8+ T cells.

Mice inoculated i.v. with superantigens exhibit long lived Ag-specific T cell tolerance. An in vitro model for this phenomenon is the ensuing unresponsiveness of Th1 T cell clones activated via the TCR/CD3 complex in the absence of co-stimulation. We have previously demonstrated alterations in TCR-mediated early protein tyrosine phosphorylation events in Th1 clones anergic for IL-2 production. In this study, we demonstrate unresponsiveness in CD4+ and CD8+ T cells from V beta 8.1 transgenic mice inoculated i.v. with the superantigen Mls-1a. The unresponsiveness of both CD4+ and CD8+ T cells involves defective IL-2 production upon restimulation, with CD4+ T cells exhibiting an additional defect in IL-2 utilization. The transgenic model allowed study of T cell signaling in a relatively homogeneous population of unresponsive cells without elaborate purification of Ag-reactive populations. Both CD4+ and CD8+ T cells exhibit altered tyrosine phosphorylation of two protein substrates upon CD3-mediated restimulation. The substrates involved, p38 and p75, are of identical size to substrates similarly affected in anergic Th1 clones. Altered tyrosine phosphorylation is therefore closely associated with defective IL-2 production in these three anergic T cell types, and may play a role in the maintenance of anergy.

Animals↗
PubMed1993

Interaction of the neu/p185 and EGF receptor tyrosine kinases: implications for cellular transformation and tumor therapy.

Growth factor receptors such as the epidermal growth factor receptor (EGFR) and the p185c-neu protein serve vital roles in the transduction of differentiation, developmental, or mitogenic signaling within normal cells. Two methods of analysis suggest that the inappropriately high expression of either protein tyrosine kinase promotes malignant transformation. First, data from in vitro experiments indicate that overexpression of either EGFR or p185c-neu (or the human homolog c-erbB-2) transforms cell-lines. Second, analysis of primary tumors and tumor cell-lines derived from many epithelial tissues (breast, stomach, ovary, and pancreas) show growth factor receptor gene amplification and elevated protein levels. The physical and functional interaction of p185c-neu and EGFR leads to the formation of a highly active, heterodimeric tyrosine kinase complex which synergistically activates cellular transformation. Anti-receptor antibodies have shown potential utility for the down modulation of these cell-surface proteins and suppression of the malignant phenotype. Design of organic antibody "mimetics" based on the structure of antireceptor antibodies may provide useful therapies and biological reagents to affect growth factor receptor function.

Antibodies, Monoclonal↗
PubMed1993

Reovirus receptors.

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Amino Acid Sequence↗
PubMed1993

Frequent deletion of the transgene in T cell receptor beta chain transgenic mice.

TCR V beta 8.1 transgenic mice were generated using a genomic TCR V beta gene construct under the control of its promoter and enhancer. Among three lines of transgenic mice, one line expressed the transgenic TCR on only approximately 70% of peripheral T cells, while the other two lines expressed it on almost all mature T cells. T cells which lacked expression of the transgenic TCR beta chain expressed endogenous TCR beta chains. The molecular basis underlying the lack of transgene expression in T cells of this line of transgenic mice was investigated. The transgenic TCR- cells were isolated by two methods. First, Thy-1+ V beta 8.1/8.2- cells were purified from peripheral T cells using cell sorting. Second, transgenic TCR- T cell clones were established. In both cases, Southern blotting indicated that V beta 8.1- T cells had deleted the transgenic TCR gene. Thus, deletion of the transgenic TCR can occur in a high proportion of T cells, which allows rearrangement and expression of endogenous TCR beta chains.

Animals↗
PubMed1993

A structural and kinetic comparison of proto-oncogenic and oncogenic neu holo-receptors expressed in insect cells.

The proto-oncogenic and oncogenic forms of the rat neu receptors were expressed in the baculovirus system to characterize their structural and enzymatic differences. The epitopes of their extracellular domains, their molecular weights, and kinase activities were similar to rat neu receptors expressed in fibroblasts. The receptors were partially purified using a phospho-agarose column and were analyzed to compare kinetic parameters using ATP as a substrate. The oncogenic form of the receptor showed a significant increase in Vmax (56%) over the proto-oncogenic form. Structural analysis of these proteins using sucrose gradients showed the oncogenic receptors to have a 62.6% increase in aggregated receptors when compared to the proto-oncogenic receptors. These studies are the first to link enzymatic activation and the physical form of the receptor using isolated receptor species.

Animals↗
PubMed1992

Differential effects of Bcl-2 on T and B cells in transgenic mice.

We have produced bcl-2 transgenic mice by using a construct which mimics the t(14;18) translocation in human follicular lymphomas. Although lymphoid tissues from all transgenic mice contained high levels of human Bcl-2 protein, transgene expression was differentially regulated within the B- and T-cell compartments of lines derived from various founder mice. We have characterized the phenotypes of two lines of bcl-2 transgenic mice (line 2 and line 6) in which bcl-2 transgene expression was restricted primarily to the T- or B-cell lineages, respectively. Analysis of line 6 lymphocytes revealed a polyclonal expansion of B cells, and these B cells exhibited prolonged survival in vitro. In line 2 mice, numbers of T cells in the peripheral lymphoid tissues were more moderately elevated despite enhanced T-cell survival in vitro. Line 2 transgenic mice also showed significantly increased proportions of thymocytes with a mature phenotype. Taken together, these findings suggest different roles for bcl-2 in the in vivo regulation of B- and T-cell development and homeostasis.

Animals↗
PubMed1992

Carboxyl-terminal deletion and point mutations decrease the transforming potential of the activated rat neu oncogene product.

The rat neu oncogene encodes a constitutively activated growth factor receptor/transmembrane tyrosine kinase, p185Tneu, that is structurally similar to yet distinct from the epidermal growth factor receptor. To explore the role of the carboxyl-terminal region and of putative autophosphorylation sites in regulating the activity of the rat p185Tneu (T, transforming) protein, we used site-directed mutagenesis to generate a p185Tneu mutant in which a putative tyrosine autophosphorylation site (residue 1253) at the extreme carboxyl terminus was replaced by a phenylalanine residue and a mutant in which the carboxyl-terminal 122 amino acids were deleted. These proteins were expressed in NIH 3T3 cells at comparable levels and exhibited similar autophosphorylation activity, exogenous substrate phosphorylation ability, oligomerization levels, and responsiveness to a partially purified neu-activating factor. However, the mutant p185Tneu proteins displayed a decreased transforming capacity both in vitro and in vivo. This analysis demonstrated that the carboxyl-terminal domain and at least one putative tyrosine autophosphorylation site of p185Tneu play a role in positively regulating the cell growth-regulating properties of the neu protein.

3T3 Cells↗
PubMed1992

Inhibition of thymocyte apoptosis and negative antigenic selection in bcl-2 transgenic mice.

The bcl-2 gene, which is overexpressed in human follicular B-cell lymphomas, has been found to extend cellular lifespan through inhibition of apoptosis, or programmed cell death. However, the physiological role of the Bcl-2 protein in lymphocyte development is unclear. We have established a transgenic mouse line that expresses high levels of the Bcl-2 protein in both cortical and medullary thymocytes, disrupting the normal pattern of expression of this gene. We found that in these mice, immature thymocytes became resistant to apoptosis mediated by corticosteroids and calcium ionophores. Untreated thymocytes also exhibited a survival advantage in suspension cultures compared with controls. In addition, overexpression of bcl-2 enabled a proportion of thymocytes and peripheral T cells to escape the process of clonal deletion, which normally eliminates self-reactive T cells during thymocyte maturation. These findings implicate the Bcl-2 protein in regulating the lifespan of maturing thymocytes and in the antigenic-selection process.

Animals↗
PubMed1992

Design and synthesis of a CD4 beta-turn mimetic that inhibits human immunodeficiency virus envelope glycoprotein gp120 binding and infection of human lymphocytes.

Poor bioavailability, rapid degradation, antigenicity, and high cost often limit the use of proteinaceous pharmaceuticals. One goal of structural biochemistry is the reduction of complex molecules to small functional units that are amenable to high-resolution structural analysis and rapid modification. The dissection of complex proteins into small synthetic conformationally restricted components is an important step in the design of low molecular weight nonpeptides that mimic the activity of the native protein. We have developed a reverse-turn mimetic system to explore peptide and protein structure-function relationships. We now report the design and synthesis of a small molecule (M(r) 810, as its trifluoroacetate salt), water soluble, proteolytically stable mimetic of residues Gln40-Thr45 of the complementarity-determining 2-like region of CD4. This mimetic has a low micromolar Kd for human T-lymphotropic virus type IIIB gp120 and reduces syncytium formation.

Binding, Competitive↗
PubMed1992

p185c-neu and epidermal growth factor receptor associate into a structure composed of activated kinases.

The protein product of the neu protooncogene, p185c-neu, is structurally similar to the epidermal growth factor receptor (EGFR). Overexpression of these two receptor tyrosine kinases, but not either separately, leads to transformation and tumorigenicity. Heterodimerization of p185c-neu and EGFR occurs in M1 cells, which express both receptors. We have individually identified the two components of the heterodimer as EGFR and p185c-neu. Analysis of this association with relatively nondenaturing detergents and in the absence of cross-linkers indicates that noncovalent interactions are primarily responsible for heterodimer formation. The rapid reversible heterodimerization was promoted by EGF binding to its receptor. Functionally, the heterodimer is a highly active protein kinase for receptor autophosphorylation and exogenous substrate phosphorylation in vitro. The isolated heterodimer was highly phosphorylated on tyrosine residues in vivo. These results indicate that the physical association between EGFR and p185c-neu is of functional significance and define enzymatic features of complex receptor formation.

Cross-Linking Reagents↗
PubMed1992

Inhibition of abnormal T cell development and autoimmunity in gld mice by transgenic T cell receptor beta chain.

Mice homozygous for the gld (generalized lymphoproliferative disease) mutation developed systemic autoimmune disease and severe lymphadenopathy due to an age-related accumulation in the peripheral lymphoid organs of polyclonal T cells bearing a unique phenotype (CD4-CD8-TCR alpha beta+B220+). These T cells overexpress T cell receptor (TcR) alpha beta chain RNA, proto-oncogenes c-myb and fyn, and proliferate poorly in response to TcR-mediated stimulation. The origin of these T cells is poorly understood. To study the influence of a functionally rearranged TcR beta chain on the T cell developmental abnormality of the gld mutation and autoimmunity, we have backcrossed TcR V beta 8.1-transgenic mice to C3H-gld/gld to homozygosity (transgenic gld mice). In transgenic gld mice, lymphadenopathy was markedly inhibited and the accumulation of CD4-CD8- T cells did not occur, although the remaining T cells overexpressed c-myb and proliferated poorly in response to TcR occupancy. These features indicate that the pattern of proto-oncogene expression and abnormal function persist in phenotypically normal T cells in transgenic gld mice, and that these characteristics can be dissociated from the accumulation of CD4-CD8- T cells. The hypergammaglobulinemia and anti-double-stranded DNA (anti-dsDNA) antibody production was partially improved in transgenic gld mice, supporting the critical role of T cells in abnormal B cell activation described in autoimmunity-prone mice. To investigate further the mechanisms underlying the inhibition of CD4-CD8- T cell accumulation in transgenic gld mice, the fetal ontogeny of T cells in transgenic mice was compared with that of non-transgenic mice. In transgenic thymus, development of TcR alpha beta+ cells was accelerated as detected by earlier expression of CD4, CD8 and TcR in fetal thymus. In contrast, the number of TcR gamma delta+ cells was reduced. We suggest that altered T cell development in transgenic mice directly or indirectly inhibits the accumulation of abnormal T cells in gld mice.

Animals↗
PubMed1992

Modulation of T cell responses with MHC-derived peptides.

T cells are activated by an interaction of their TCRs with a complex made up of antigenic peptide bound to the interhelical groove of MHC molecules. The helices lining the antigen binding groove of MHC molecules are felt to contribute several contact residues for TCR binding. Peptides derived from the amino acid sequences of these helices may be capable of modulating immune responses and aiding in the dissection of immune recognition. These studies address the effects of a peptide derived from the sequence of amino acids 68-83 of the IAk beta 1 domain (IAk 68-83) predicted to represent a portion of an antigen-binding helix on the IAk molecule. The IAk 68-83 peptide is bound by a monoclonal anti-IAk antibody and inhibits its binding to IAk-bearing cells. The IAk 68-83 peptide inhibits antigen-dependent activation of the IAk+con-albumin restricted T cell clone D10.G4, and this effect is more pronounced at lower doses of antigen-presenting cells. The free peptide has a small effect in limiting binding of anticlonotypic antibodies to D10.G4, and a multivalent form bound to BSA has a more pronounced effect in this regard. The BSA-peptide conjugate, when fluoresceinated, specifically stained D10.G4 cells, and this was specifically competed by unfluoresceinated IAk 68-83 peptide-BSA conjugate, as well as by anticlonotype. These results suggest that peptides derived from the predicted helical region of MHC class II molecules may have a direct interaction with T cell receptors. Such peptides may be capable of modulating immune responses in a physiologically significant manner.

Amino Acid Sequence↗
PubMed1992

Ocular tissue involvement in HIV infection: immunological and pathological aspects.

The CNS afflictions in AIDS are myriad and suggest a tropism of HIV to neural tissue. Ocular involvement is a frequent manifestation of the HIV infection, resulting in a high incidence of blindness within this patient population. Ocular lesions include cotton wool spots, presumably from HIV-induced microvasculopathy, retinal hemorrhage in cytomegalovirus retinitis and conjunctival Kaposi's sarcoma. These manifestations have been noted in up to 71% of AIDS patients. In fact, ocular disease is often the presenting symptom in an HIV-infected individual. Despite the high incidence of ocular involvement in AIDS patients, the etiology and pathogenesis of these manifestations are not well understood. The immunosuppressive action of HIV is the most likely primary cause for the development of ocular complications in AIDS. Here we review some of the important immunological and pathological features of AIDS affliction in the eye.

AIDS-Related Opportunistic Infections↗