Biomedical subjects

M I Greene

Publications and source records attributed to M I Greene.

At least 55 records · Page 3Linked to original sources

PubMed1997

Transduction of activation signal that follows HIV-1 binding to CD4 and CD4 dimerization involves the immunoglobulin CDR3-like region in domain 1 of CD4.

The role of CD4 during the human immunodeficiency virus type 1 (HIV-1) life cycle in T cells is not restricted to binding functions. HIV-1 binding to CD4 also triggers signals that lead to nuclear translocation of NF-kappaB and are important to the productive infection process. In addition to its cytoplasmic tail, in the ectodomain, the immunoglobulin (Ig) CDR3-like region of CD4 domain 1 seemed to play a role in this cascade of signals. We demonstrate in this work that the structural integrity of the CDR3-like loop is required for signal transduction. Substitutions of negatively charged residues by positively charged residues within the CDR3-like loop either inhibited NF-kappaB translocation after HIV-1 and gp120-anti-gp120 immune complexes binding to E91K,E92K mutants or induced its constitutive activation for E87K,D88K mutants. Moreover, A2.01-3B cells expressing the E91K,E92K mutant exhibited a lower HIV-1Lai replication. These cells, however, expressed p56(lck), demonstrated NF-kappaB translocation upon PMA stimulation, bound HIV-1Lai envelope glycoprotein with high affinity, and contained HIV-1 DNA 24 h after exposure to virus. E91K, E92K, and E87K,D88K mutant CD4 molecules were unable to bind a CD4 synthetic aromatically modified exocyclic, CDR3.AME-(82-89), that mimics the CDR3-like loop structure and binds to native cell surface CD4. This result together with molecular modeling studies indicates that the CDR3.AME-(82-89) analog binds to the CDR3-like loop of CD4 and strongly suggests that this region represents a site for CD4 dimerization. The negative charges on the CDR3-like loop thus appear critical for CD4-mediated signal transduction most likely related to CD4 dimer formation.

Amino Acid Sequence↗

PubMed1997

Relationship of p215BRCA1 to tyrosine kinase signaling pathways and the cell cycle in normal and transformed cells.

We have analysed the relationship of the products of two genes, neu and BRCA1, known to be important in human breast cancer. Highly specific antibodies that recognized both the rodent and human form of the BRCA1 gene product (Mr 215 kDa, p215BRCA1) were developed to facilitate these efforts. p215BRCA1 was identified as a tyrosine phosphorylated protein primarily localized in the nucleus of several breast cancer cell lines. In transformed murine and human cells, levels of p215BRCA1 tyrosine phosphorylation were inversely correlated with the activity of the erbB family receptor-tyrosine-kinases and with the transformed growth features of these cells. Regulation of p215BRCA1 tyrosine phosphorylation was also related to events in the cell cycle. Increased levels of p215BRCA1 phosphotyrosine content were observed in NIH3T3 cells arrested at the G2/M transition. These findings indicate that the products of BRCA1, neu, and erbB breast cancer genes participate in a common or shared signaling pathway important in cell growth and its regulation.

3T3 Cells↗

PubMed1997

Trans receptor inhibition of human glioblastoma cells by erbB family ectodomains.

Our aim has been to understand the features of erbB receptor homo- and heterodimer assembly to develop approaches to disrupt receptor activation. We have developed a general approach to cause erbB receptor-specific trans inhibition of human neoplasia. The clonal progression of human astrocytomas to a more malignant phenotype often involves the amplification and overexpression of the epidermal growth factor receptor (EGFr) gene. We have selectively targeted the EGFr in human glioblastoma cells with kinase-deficient mutants of the erbB family derived from the ectodomain of the Neu oncogene that are able to form heterodimers with EGFr and inhibit EGFr-dependent phenotypes. In EGFr-positive U87MG human glioblastoma cells, expression of the Neu ectodomain inhibits EGF-, but not platelet-derived growth factor-, induced DNA synthesis; inhibits cell proliferation in the presence of EGF, but not platelet-derived growth factor; inhibits the ability of U87MG to form colonies in soft agar; and inhibits transforming efficiency in athymic mice. These studies establish that EGFr-mediated signal transduction is important in the maintenance of malignant glioma, and that trans receptor inhibition is a novel way to abrogate abnormal growth of these tumors. Neu ectodomains will be useful in determining the manner in which the EGFr contributes to glial tumorigenesis and in the design of pharmaceuticals that disable erbB family oncoproteins. In addition, these studies provide a rationale for the application of the Neu ectodomain in gene therapy approaches to human malignant glioma and, potentially, to other systemic epithelial malignancies expressing erbB family receptors.

Animals↗

PubMed1997

Identification of a structural constituent and one possible site of postembryonic formation of a teleost otolithic membrane.

A gelatinous otolithic membrane (OM) couples a single calcified otolith to the sensory epithelium in the bluegill sunfish (Lepomis macrochirus) saccule, one of the otolithic organs in the inner ear. Though the OM is an integral part of the anatomic network of endorgan structures that result in vestibular function in the inner ear, the identity of the proteins that make up this sensory accessory membrane in teleosts, or in any vertebrate, is not fully known. Previously, we identified a cDNA from the sunfish saccular otolithic organ that encoded a new member of the collagen family of structural proteins. In this study, we examined biochemical features and the localization of the saccular collagen (SC) protein in vivo using polyclonal antisera that recognize the noncollagenous domains of the SC protein. The SC protein, in vivo, was identified as a 95-kDa glycoprotein in sunfish whole-saccule lysate and in homogenates of microdissected saccular OMs. Immunohistochemical analyses demonstrated that the SC protein was localized within one of the two distinct layers of the sunfish saccular OM. The SC protein was also detected within the cytoplasm of supporting cells at the edges of the saccular sensory epithelium, indicating that these cells are a primary site for the synthesis of this structural protein. Further studies of the organization of this matrix molecule in the OM may help clarify the role of this sensory accessory membrane in vestibular sensory function.

Animals↗

PubMed1997

Therapeutic peptides and peptidomimetics.

Peptidomimetics are one set of probes used in the transition pathway of small molecule drug design. Cyclization of the peptide backbone and its modification with aromatic residues constitutes an effective approach to mimetic drug design and circumvents obstacles associated with delivery and formulation of peptides and peptidomimetics. In the past year, examples of mimicking beta-turn structures has led to combining design strategies with molecular libraries, demonstrating that peptidomimetics can provide valuable clues about receptor similarities not revealed by their endogenous ligands. This information can lead to the development of dual inhibitors. In addition, this work suggests that the use of libraries and rational design need not be mutually exclusive approaches to lead discovery.

Animals↗

PubMed1997

Synthetic CD4 exocyclics inhibit binding of human immunodeficiency virus type 1 envelope to CD4 and virus replication in T lymphocytes.

CD4 functions as a major T-cell surface receptor for human immunodeficiency virus by binding the human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 with relatively high affinity. We have developed constrained aromatically modified analogs of the secondary structures of the first domain of CD4 in order to analyze surfaces involved in binding of gp120. Complementarity determining-like regions (CDRs) of the D1 domain of CD4 were reproduced as synthetic aromatically modified exocyclic (AMEs) forms. The exocyclic CDR3.AME(82-89), derived from the CDR3 (residues 82-89) region of CD4 D1 domain, specifically inhibited binding of recombinant gp120 to both recombinant soluble CD4, and CD4+ Jurkat cells, and blocked syncytium formation and virus particle production caused by HIV-1 infection. We have previously shown that the CDR3.AME analog binds to the CD4 CDR3 region and creates a disabled CD4 heterodimer. We propose that the AME prevents the formation of an essential homodimeric surface needed for efficient HIV binding. Additionally the disabled CD4 receptor may be less able to signal the cell to allow HIV replication and HIV infection. Such compounds may represent a new receptor specific approach to modulate biological functions.

Amino Acid Sequence↗

PubMed1997

Structure-based design and characterization of exocyclic peptidomimetics that inhibit TNF alpha binding to its receptor.

Exocyclic small peptidomimetics corresponding to three critical binding sites of tumor necrosis factor (TNF)-receptor(I) have been designed based on atomic features deduced from the crystal structures of TNF alpha and the TNF beta/TNF-receptor(I) complex and a model of an anti-TNF alpha monoclonal antibody. TNF alpha antagonistic activities were evaluated by binding assays using soluble receptor or intact receptor on cells as well as an apoptosis/cytotoxicity assay. The most critical interaction site for rational design of peptidomimetics was localized to the loop1/domain3 of the TNF-receptor. The best antagonist showed 5 microM inhibition in the binding assay. Biologically, the mimetics inhibited TNF alpha-mediated apoptosis.

Amino Acid Sequence↗

PubMed1997

Identification of p185neu sequences required for monoclonal antibody- or ligand-mediated receptor signal attenuation.

Anti-p185neu antibodies downmodulate constitutively active p185neu receptors from the cell surface, which is associated with a reduction in the transformed phenotype. We have analyzed a group of mutant p185neu forms with carboxyl (C)-terminal truncations and/or an internal deletion of amino acids 1008-1057. Receptor endocytosis and degradation were examined by flow cytometric analysis and pulse-chase assays following anti-p185neu monoclonal antibody (MAb) treatment. Deletion of a sequence within the distal carboxyl terminus, including three known autophosphorylation sites, did not affect MAb-mediated receptor surface downmodulation and degradation of surface receptor. However, kinase-active deletion mutants with elimination of the putative internalization sequence (Tint delta), or Tint delta mutants also containing a large C-terminal truncation, displayed markedly impaired receptor endocytosis in response to MAb treatment. Cells expressing endocytosis-defective mutant proteins became insensitive to anti-p185neu MAb-mediated inhibition of anchorage-independent growth and were more oncogenic in vivo. Cells expressing endocytosis-defective mutant EGFR/neu chimeric proteins were more transforming upon EGF addition when compared to cells expressing wild-type EGFR/neu receptors. Taken together, these data suggest that, in addition to kinase activity, p185neu receptor endocytosis requires a functional modular structure, i.e., an internalization sequence, possibly to serve as target for endocytotic adapter proteins. Unattenuated signaling from oncogenic p185neu forms resulting from prolonged surface localization may result in enhanced cellular transformation and desensitization to MAb-mediated downregulation and phenotypic reversion.

Animals↗

PubMed1997

Murine thioredoxin peroxidase delays neuronal apoptosis and is expressed in areas of the brain most susceptible to hypoxic and ischemic injury.

Thioredoxin peroxidase (TPx) is an antioxidant protein that limits the activity of reactive oxygen species (ROS). We cloned the cDNA encoding the mouse homolog of TPx from an E14.5 brain cDNA library and analyzed its distribution and function in murine tissues. Comparison of the amino acid sequence of mouse TPx with those of other species revealed that TPx was highly conserved across all species. Mouse TPx had broad tissue distribution, but its expression was especially marked in cells that metabolize oxygen molecules at high levels such as erythroid cells, renal tubular cells, cardiac and skeletal muscle cells, and certain types of neurons. Levels of increased expression of TPx in the brain were coincident with regions known to be especially sensitive to hypoxic and ischemic injury in humans. Models of erythroid differentiation and neuronal survival were employed to study the function of TPx. Murine erythroleukemia cells (MEL cells) increased TPx transcription when in a chemically differentiated state. Furthermore, expression of mouse TPx in PC12 pheochromocytoma cells prolonged their survival in the absence of nerve growth factor (NGF) and serum, indicating that TPx could promote neuronal cell survival. We propose that TPx contributes to antioxidant defense in erythrocytes and neuronal cells by limiting the destructive capacity of oxygen radicals. These findings identify a novel gene that appears to be relevant to hypoxic brain injury and may be of importance in development of new approaches to abrogate the effects of ischemic- and hypoxic-related injury in the central nervous system (CNS).

Amino Acid Sequence↗

PubMed1997

Principles of receptor-based inhibition of erbB family receptor kinases: prospects for new therapies for human cancers.

The erbB family of receptor tyrosine kinases is composed of growth factor receptors that are overexpressed or mutated in a large variety of human cancers. The study of erbB-mediated signal transduction will lead to an understanding of the role played by this family of receptors in normal and transformed cells. In this article, we discuss the contemporary understanding of the structure and function of these receptors and how these features might be exploited in receptor-based strategies of growth inhibition. The first part of our study details the structure of erbB receptors as it relates to the process of transformation of cells and the malignant phenotype in humans. In the second part, we discuss two rational approaches to therapy for cancers in which erbB receptors are overexpressed or mutated: first, the potential for antireceptor immunity and the evolution of small molecules for receptor-based immunotherapy; second, a strategy of ectodomain association that leads to the formation of defective kinase assemblies. These studies provide a basis for the application of receptor-based strategies of growth inhibition in erbB-expressing human cancers.

Enzyme Inhibitors↗

PubMed1996

Inhibition of p185neu kinase activity and cellular transformation by co-expression of a truncated neu protein.

The rat neu oncogene product encodes a 185 kDa receptor tyrosine kinase which is constitutively activated as a result of a single amino acid substitution (Val664-->Glu) within the transmembrane region. In this study, we show that the transforming activity of oncogenic p185neu (also termed Tneu) can be inhibited by co-expression of a truncated neu protein with a large cytoplasmic deletion (termed T691stop) which includes the tyrosine kinase domain. In cell lines co-expressing full-length and truncated neu proteins, we observed co-dimerization between full-length p185neu and truncated T691 stop, resulting in the formation of a kinase-inactive heteromeric complex. Phenotypic analysis of several different clones showed that the degree of inhibition of transformation in vitro and tumorigenicity in vivo was related to the ratio of full-length and truncated p185 proteins co-expressed in cells. These results provide evidence that expression of kinase-deficient neu proteins leads to co-dimerization that results in suppression of kinase activation and oncogenicity of associated p185neu-activated receptors. The mutant neu protein mediates inhibition in both transfected fibroblasts expressing oncogenic p185neu and mammalian cancer cells derived from a rat primary neuroglioblastoma expressing oncogenic p185neu. This truncated peptide may be important for the design of future therapies directed against erbB family oncoproteins.

Animals↗

PubMed1996

Control of MHC restriction by TCR Valpha CDR1 and CDR2.

Individual T cell receptor (TCR) Valpha elements are expressed preferentially in CD4 or CD8 peripheral T cell subsets. The closely related Valpha3.1 and Valpha3.2 elements show reciprocal selection into CD4 and CD8 subsets, respectively. Transgenic mice expressing site-directed mutants of a Valpha3.1 gene were used to show that individual residues in either the complementarity-determining region 1 (CDR1) or CDR2 were sufficient to change selection from the CD4 subset to the CD8 subset. Thus, the germline-encoded Valpha elements are a major influence on major histocompatibility class complex (MHC) restriction, most likely by a preferential interaction with one or the other class of MHC molecule.

Animals↗

PubMed1996

Structural analysis of p185c-neu and epidermal growth factor receptor tyrosine kinases: oligomerization of kinase domains.

The epidermal growth factor receptor (EGFR) and p185c-neu proteins associate as dimers to create an efficient signaling assembly. Overexpression of these receptors together enhances their intrinsic kinase activity and concomitantly results in oncogenic cellular transformation. The ectodomain is able to stabilize the dimer, whereas the kinase domain mediates biological activity. Here we analyze potential interactions of the cytoplasmic kinase domains of the EGFR and p185c-neu tyrosine kinases by homology molecular modeling. This analysis indicates that kinase domains can associate as dimers and, based on intermolecular interaction calculations, that heterodimer formation is favored over homodimers. The study also predicts that the self-autophosphorylation sites located within the kinase domains are not likely to interfere with tyrosine kinase activity, but may regulate the selection of substrates, thereby modulating signal transduction. In addition, the models suggest that the kinase domains of EGFR and p185c-neu can undergo higher order aggregation such as the formation of tetramers. Formation of tetrameric complexes may explain some of the experimentally observed features of their ligand affinity and hetero-receptor internalization.

Amino Acid Sequence↗

PubMed1996

Inhibition of apoptosis and augmentation of lymphoproliferation in bcl-2 transgenic Fas/Fas ligand-defective mice.

Mice defective in Fas (CD95 or APO-1) or its ligand (lpr or gld mice) develop age-dependent lymphadenopathy and systemic autoimmune disease. T cells accumulating in the lymph nodes of these mice express reduced levels of Bcl-2 protein and are susceptible to spontaneous and glucocorticoid-induced apoptosis. We backcrossed bcl-2 transgenic mice to lpr and gld mice to homozygosity to determine the effects of Bcl-2 overexpression. T cells in these mice were resistant to spontaneous and glucocorticoid-induced apoptosis in vitro. Moreover, the accumulation of CD4(-)CD8(-) T cells in the lymph nodes and the spleens was augmented, suggesting that a Bcl-2-dependent mechanism regulating the number of T cells residing in the peripheral lymphoid organs in addition to the Fas-mediated pathway exists.

Animals↗

PubMed1996

Synthetic CD4 exocyclic peptides antagonize CD4 holoreceptor binding and T cell activation.

We have developed peptide analogs to analyze precise human CD4 substructures involved in MHC class II binding. Forms of the complementarity determining-like regions (CDRs) of the D1 domain of human CD4 were reproduced as synthetic aromatically modified exocyclic (AME) analogs and tested for their ability to block CD4-MHC II interactions and T cell activation. The exocyclic derived from CDR3 (residues 82-89) of human CD4, which specifically associated with CD4 on the T cell surface to create a heteromeric CD4 complex, blocked IL-2 production and antagonized the normal function of the CD4 receptor. The approach of creating novel synthetic antagonistic receptor complexes may represent a new receptor specific pharmaceutical approach to modulate biological function.

Amino Acid Sequence↗

PubMed1996

Association of signaling proteins with a nonmitogenic heterodimeric complex composed of epidermal growth factor receptor and kinase-inactive p185c-neu.

The functional consequences of heterodimer formation between the epidermal growth factor receptor (EGFr) and the p185c-neu receptor tyrosine kinase include increased mitogenic and transformation potencies. To determine the possible alteration of signal transduction pathways resulting from this heteromeric complex, the capacity of several signaling proteins to associate with the heterodimeric receptors has been assayed. The in vivo interaction with the EGFr/p185c-neu heterodimer of several signal transduction proteins, including phospholipase C-gamma 1 (PLC-gamma 1), the p85 subunit of phosphotidylinositol 3-kinase, the ras GTPase activating protein, SHC, NCK, p72RAF, and the tyrosine phosphatase SHPTP2, was measured by coimmunoprecipitation. The binding of these signaling proteins to a complex composed of EGFr and a kinase-inactive form of p185 (p185K757M) was not impaired, even though the mitogenic and transformation activity of this complex had been abrogated. In addition, the EGF-induced phosphorylation of GAP, p85, and PLC-gamma 1 did not correlate with the dominant-negative action of p185K757M on EGFr function. Thus, substrate association and phosphorylation do not correlate stringently with the mitogenic and transforming activity of this receptor complex, suggesting additional pathways or mechanisms vital to EGFr/p185c-neu heterodimeric signaling.

Animals↗

PubMed1996

Cloning of m-ehk2 from the murine inner ear, an eph family receptor tyrosine kinase expressed in the developing and adult cochlea.

To identify receptor tyrosine kinases (RTKs) present in the murine inner ear, a degenerate polymerase chain reaction (PCR) methodology was employed to clone partial cDNAs encoding RTKs from embryonic day-17.5 mouse whole inner ear RNA. At least 20 distinct TKs were identified within the first 50 subcloned PCR products obtained by this analysis (Davis/Lee et al., 1996). One of the receptor RTKs identified encoded an eph-related kinase not previously described in the mouse. Analysis of full-length cDNAs revealed that this RTK is the mouse homolog of the rat ehk-2 gene product (Maisonpierre et al., 1993). Differences in the carboxyl terminal of the mouse and rat ehk2 RTKs suggest that differential splicing of this gene may occur resulting in transcripts encoding truncated and nontruncated forms of the ehk2 RTK. Multiple transcripts corresponding to this RTK were detected by Northern blot analysis only in the mouse brain. RT-PCR analysis revealed the presence of transcripts encoding this kinase in adult mouse brain, inner ear, testes, ovary, thymus, and spleen. Transcripts encoding this kinase were localized using in situ hybridization to the postembryonic day 1 cochlear ganglion neurons in the inner ear and to neurons in discrete regions of the nervous system. This is the first report of eph-related RTK in inner ear tissue that is present in both the developing and adult inner ear tissue. Because this is a member of a family of RTKs that is implicated in establishing the specificity of neuron-target cell interactions (Garrity and Zipursky, 1995), additional studies to determine if the ehk-2 gene product is involved in such processes in the murine cochlea are warranted.

Amino Acid Sequence↗

PubMed1996

A p65/p95 neural surface receptor is expressed at the S-G2 phase of the cell cycle and defines distinct populations.

A surface receptor complex of Mr approximately 65 000 (p65) and approximately 95 000 (p95) is expressed in cells of the central nervous system of mice. This receptor is recognized by monoclonal antibody 87.92.6 or by reovirus type 3 haemagglutinin as unnatural ligands. The p65/p95 receptor is expressed mostly in neural embryonic precursors undergoing proliferation, especially those in the S-G2 phase of the cell cycle. Receptor expression decreases progressively throughout embryogenesis to low but detectable levels in the adult brain. Biochemical characterization revealed that the neural p65/p95 receptor complex is indistinguishable from the p65/p95 receptor expressed in T cells, where receptor ligation leads to a mitogenic block. In neural and lymphoid tissues the p65/p95 receptor (or an associated protein) possesses a tyrosine kinase enzymatic activity. Receptor ligation in neural cells resulted in the rapid tyrosine phosphorylation of cellular proteins which are different from substrates phosphorylated in T cells. Differential substrate coupling to the receptor may account for differences in signal transduction and biology between neural cells and T cells. Further study of this receptor complex may help define important features of neural proliferation, differentiation and survival.

Animals↗