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Biomedical subjects

M I Greene

Publications and source records attributed to M I Greene.

At least 19 recordsLinked to original sources
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↗
PubMed1992

Receptor utilization by reovirus type 3: distinct binding sites on thymoma and fibroblast cell lines result in differential compartmentalization of virions.

Reovirus type 3/D infects cells following binding to specific cell-surface receptors. The characteristics of these receptors may play an important role in determining post-binding events critical to the viral life cycle. Some cell lines, i.e. L-cells, appear to bind reovirus type 3/D utilizing sialylated proteins as specific receptors for viral adsorption. Such binding results in productive infection. Other cell lines, i.e. R1.1 thymoma cells, bind reovirus type 3/D in a sialic acid independent manner which does not result in productive infection. Yet, a peptide analogue of the viral binding site is capable of inhibiting binding of reovirus type 3 to both cell types, suggesting the same viral epitope interacts with both cellular receptors. When binding of reovirus is studied by electron microscopy, the virus particles enter the L cells via coated pits, and are later seen in large accumulations in endocytic vesicles near the transGolgi network. In contrast, R1.1 cells appear to divert the reovirus particles to a cell membrane elaboration, with reovirus remaining bound to the cell membrane. At later time points with R1.1 cells, there are no apparent intracellular accumulations. These studies demonstrate that viruses can attach to different cells utilizing distinct receptors, and this may play a role in the ability of the virions to productively infect the cells. The capacity of virus to be adsorbed to cellular receptors which do not lead to internalization may be an important mechanism for the sequestration and clearance of virus. These observations have implications for the tissue tropism demonstrated by reovirus type 3/D and other viruses.

Amino Acid Sequence↗
PubMed1992

Loops and secondary structure mimetics: development and applications in basic science and rational drug design.

One goal of protein design and structural biochemistry is the reduction of complex molecules to small functional units that are amenable to high resolution analysis and rapid modification. We have developed a variety of small molecules which biochemically and biologically mimic the combining sites of proteins of the immunoglobulin superfamily. The chemical and biological properties of peptide mimetics suggest that these analogs can be used as indicators for new pharmaceutical agents. Mimetics are powerful tools for the study of molecular recognition since they are small in size, maintain solubility in physiologic fluids and are amenable to detailed structural studies. As such, they represent a step toward the rational design of low molecular weight non-peptide pharmaceutical agents devoid of some of the shortcomings of conventional peptides. Here we discuss the rationale and approaches for the development of these molecules, and their current and future applications.

Amino Acid Sequence↗
PubMed1992

Response of V beta 8.1+ T cell clones to self Mls-1a: implications for the origin of autoreactive T cells.

Clonal deletion and anergy are two major mechanisms of self-tolerance. However, the molecular mechanisms underlying clonal deletion and anergy, as well as the threshold of TCR affinity/avidity required for these processes, are not known. Expression of the V beta 8.1 TCR correlates with the reactivity of the T cells to the minor lymphocyte stimulating locus-1a (Mls-1a) and T cells expressing this TCR are deleted in the thymus of Mls-1a mice. Similarly, in TCR V beta 8.1 transgenic mice, the number of CD4+CD8-T cells is reduced in Mls-1a mice. However, small numbers of CD4+CD8-T cells remain in the periphery of adult Mls-1a transgenic mice. We have generated T cell clones from TCR V beta 8.1 transgenic mice by stimulation of lymph node T cells with C57BL/6 alloantigens. Interestingly, CD4+CD8-V beta 8.1+ clones isolated from the transgenic mice of Mls-1a background responded to the self-antigen Mls-1a, to which they did not respond in primary assay. Reactive patterns of the clones were compared with clones derived from Mls-1b mice. Proliferation and cytokine production of the clones from Mls-1a mice to the self-antigen Mls-1a were generally reduced when compared with clones from Mls-1b mice. More importantly, T cell clones from Mls-1a mice required more Mls-1a antigen for their activation, and were more susceptible to the inhibitory effects of anti-CD4 antibody on the proliferative responses to Mls-1a than those from Mls-1b mice. These results suggest that the T cell receptor on clones derived from Mls-1a mice have functional but reduced affinity/avidity for self-antigen Mls-1a.

Animals↗
PubMed1992

Expression of c-erbB-2 in in situ and in adjacent invasive ductal adenocarcinomas of the female breast.

Several lines of evidence have demonstrated that expression of the c-erbB-2 gene product contributes to the malignant phenotype. We and others have determined that c-erbB-2 is substantially expressed in most ductal in situ carcinomas of the comedo type, but not in other patterns of ductal carcinoma in situ or in atypical ductal hyperplasia of the breast. In the present investigation, by immunohistochemistry we inquired whether invasive ductal adenocarcinomas retained the c-erbB-2 expression status of the in situ carcinomas from which they derived. Of twelve specimens containing both cribriform/micropapillary in situ and derivative invasive adenocarcinomas in the same section, all tumor cells were negative for c-erbB-2 expression. In thirteen in situ carcinomas of the comedo type, with identifiable invasive components, ten had definite c-erbB-2 expression, and in every case there was comparable c-erbB-2 protein staining of in situ and invasive components; in three of these ten cases the staining in the in situ component tended to be more intense. These findings imply that a significant proportion of invasive mammary adenocarcinomas expressing c-erbB-2 protein is derived from ductal in situ carcinomas of the comedo type.

Breast Neoplasms↗
PubMed1992

In vivo modulation of oligodendrocyte function by an anti-receptor antibody.

The receptor for reovirus serotype 3 (Reo3R) is biochemically, pharmacologically, and antigenically related to the adrenergic receptors. Previous studies have demonstrated that anti-Reo3R antibodies and Reo3R-binding peptides alter oligodendrocyte differentiation in culture. In the present studies, antibodies and peptides that bind the Reo3R were found to alter myelin morphology in vivo. Microinjection of purified anti-Reo3R antibody into guinea pig optic nerves produced expansion of the adaxonal oligodendrocyte cytoplasm, separation of myelin lamellae, widening of Schmidt-Lanterman clefts, myelin vesiculation, and demyelination. A divalent Reo3R-binding peptide reproduced some of these changes. Anti-Reo3R antibodies and Reo3R-binding peptides alter oligodendrocyte function in vivo resulting in myelin changes. These effects appear to be mediated directly by Reo3R perturbation, at least in part, rather than through activation of additional effector mechanisms.

Amino Acid Sequence↗
PubMed1992

Synthetic cyclodextrin derivatives inhibit HIV infection in vitro.

The AIDS pandemic has stimulated the search for safe potent antiviral agents. To date, only AZT has been approved as a therapeutic agent for the treatment of HIV infection. It is likely that a large number of antiviral compounds would be necessary to control a life-long infection. We have utilized the rigid structure of the cyclodextrin molecule to determine the minimal components necessary for anti-HIV activity. Utilizing this targeted approach to drug design, we demonstrate the antiviral effects of candidate compounds from the family of cyclodextrins. We report that polysulfated cyclodextrins mediate significant anti-HIV effects which include blocking infectivity and syncytia formation mediated by the HIV viruses. Several other substituted forms of cyclodextrins did not mediate significant antiviral effects. Further results demonstrate that the polysulfated cyclodextrins mediated no specific antiviral effects against already infected human cells. These results demonstrate that the antiviral activities of this class of compounds are centered around early events in the viral life cycle. These in vitro results suggest that such molecules may be of importance in antiretroviral therapeutic regimes.

Antiviral Agents↗
PubMed1992

Expression, purification, and characterization of Bacneu. A soluble protein tyrosine kinase domain encoded by the neu-oncogene.

To further characterize the structure and regulation of the tyrosine kinase encoded by the rodent neu oncogene, its cytoplasmic tyrosine kinase domain has been expressed as a soluble protein, called Bacneu, in Sf9 insect cells, using the baculovirus expression system. Expression of Bacneu was detected by immunoblotting with anti p185neu antisera and in vitro autophosphorylation analysis as early as 24 h postinfection. Maximal expression was observed at 48 h postinfection. The soluble kinase was purified to near homogeneity by sequential chromatography on DEAE-Sepharose, phosphocellulose, poly-L-lysine, and Sephacryl 300, yielding 0.55 mg Bacneu per L of Sf9 cells (4% yield). The kinase is more active in the presence of Mn2+ compared to Mg2+ ions. The specific activity of the kinase using poly(Glu4Tyr1) as a substrate is 179 nmol/min/mg. Maximal incorporation of 1.4 mol of phosphate per mol of enzyme by autophosphorylation was found to increase the activity of the enzyme 1.5- to twofold. These results indicate that the Bacneu kinase is activated by phosphorylation. Therefore, it will be a useful reagent for characterizing the effects that phosphorylation by other cellular kinases and dephosphorylation by phosphatases have on its activity.

Animals↗
PubMed1991

Characterization of a neu/c-erbB-2 protein-specific activating factor.

The neu oncogene encodes a tyrosine kinase with growth factor receptor-like properties. A neu protein-specific activating factor (NAF) was partially purified from medium conditioned by the transformed human T-cell line ATL-2. NAF was able to stimulate the tyrosine-specific kinase activity of the neu protein (p185neu), induce dimerization and internalization, and increase the growth of cells bearing the neu protein. The effects of NAF were mediated by an interaction with the p185neu extracellular domain. NAF had no effect on the epidermal growth factor receptor kinase activity and no effect on cells that express that receptor. Further analysis of NAF and of other recently described neu protein-activating activities should help clarify the role of the neu protein in cell growth and transformation.

Cell Division↗