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Biomedical subjects

M I Greene

Publications and source records attributed to M I Greene.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1991

Isolation and characterization of a neu protein-specific activating factor from human ATL-2 cell conditioned medium.

The rat neu gene product is a 185 kD membrane bound tyrosine kinase that is closely related to, yet distinct from the epidermal growth factor receptor. The biochemical and cellular effects of a neu protein-specific activating factor (NAF) detected in human ATL-2 cell conditioned medium were recently described (1). To further characterize NAF, some of its physicochemical properties were examined and a method for purifying this factor from ATL-2 cell conditioned medium was developed. In these studies NAF was found to be heat stable and sensitive to the protease chymotrypsin. In addition, a method for purifying this activity was developed using a quantifiable, in vitro autophosphorylation assay system to measure NAF activity in fractions following ion-exchange and then reverse-phase HPLC.

Cell Line↗
PubMed1991

Design and synthesis of a mimetic from an antibody complementarity-determining region.

A technique for producing non-peptide compounds (mimetics) of designed specificities was developed that permitted the synthesis of a conformationally restricted molecule that mimicked the binding and functional properties of monoclonal antibody (MAb) 87.92.6, which recognizes the reovirus type 3 cellular receptor. Binding of either MAb 87.92.6, peptide analogs, or 87.1-mimetic to the cellular receptor inhibited cellular proliferation. The mimetic was a synthetic beta-loop structure that mimics the second complementarity-determining region of the MAb. These studies may lead to strategies for the synthetic design of antibody complementarity regions, ligands, and other pharmacologically active agents that are water soluble, resistant to proteolysis, and nonimmunogenic.

Antibodies, Monoclonal↗
PubMed1991

Contact residues and predicted structure of the reovirus type 3-receptor interaction.

Sequence similarity between the reovirus type 3 hemagglutinin (HA3) and a anti-idiotypic monoclonal antibody (87.92.6) has been shown to define the site of interaction with a neutralizing (idiotypic) monoclonal antibody (9B.G5) and the cellular receptor for the virus. A synthetic peptide (VL peptide) derived from the anti-idiotypic sequence inhibits viral binding to the receptor. In this study, variants of the VL peptide were utilized to probe specific amino acid residues involved in binding the neutralizing antibody and the receptor. These studies indicate that the--OH groups of several residues are involved in contacting the reovirus type 3 receptor, including Tyr49, Ser50, Ser52, and Thr53 in the anti-idiotypic sequence, corresponding to Tyr326, Ser327, Ser329, and Ser325 in HA3, respectively. In contrast, only Ser50 of the anti-idiotypic sequence, corresponding to Ser327 of HA3, significantly altered neutralizing antibody binding. Additional studies implicate sialic acid as a potential reovirus type 3 receptor on some cells. This includes inhibition of binding of reovirus type 3 and 87.92.6 to L cells by heavily sialylated glycoproteins. Sialic acid was therefore utilized as a candidate receptor to analyze potential interaction schemes with HA3/87.92.6. Sequence similarity to other immunoglobulin structures with similar sequences allowed modeling of the three-dimensional structure of these epitopes. These structures, in combination with peptide studies, allow the development of a model of the interaction of these epitopes with sialic acid, which serves as a reovirus type 3 receptor. These models reveal that similar amino acid residues and side-chain geometries may be utilized by the reovirus type 3 and influenza hemagglutinins in their interactions with cell-surface receptors.

Amino Acid Sequence↗
PubMed1991

Design of bioactive peptides based on antibody hypervariable region structures. Development of conformationally constrained and dimeric peptides with enhanced affinity.

The variable regions of antibody molecules bind antigens with high affinity and specificity. This binding is imparted largely by the hypervariable portions of the variable region. Hypervariable regions typically fold into reverse turn or loop structures. Peptides derived from antibody hypervariable region sequences can bind antigens with similar specificity, albeit with markedly lower affinity. In this study, cyclic and dimeric peptide analogs of an anti-idiotypic/antireceptor antibody hypervariable region were developed. This antibody (87.92.6) binds to reovirus type 3 receptors on cells as well as to a neutralizing anti-reovirus type 3 monoclonal antibody (9B.G5). The cyclic peptides were utilized to probe the optimal conformation for binding to both the receptor and 9B.G5. By dimerizing or constraining the conformation of these peptides, higher affinity binding was produced. By utilizing several different cyclic peptides, the optimal conformation for binding was established. The conformationally optimized cyclic peptide possessed greater than 40-fold higher affinity for the receptor and the idiotype than the linear analog. This study suggests that conformationally constrained and dimeric peptides derived from antibody hypervariable loop sequences can bind antigens (including receptors) with reasonable affinity. hypervariable loop sequences can bind antigens (including

Amino Acid Sequence↗
PubMed1991

Anti-reovirus receptor antibody accelerates expression of the optic nerve oligodendrocyte developmental program.

Previous studies showed that the cell-surface receptor for reovirus serotype 3 (Reo3R) appears at an early stage of oligodendrocyte differentiation and that anti-Reo3R antibodies and Reo3R-binding peptides induce galactocerebroside expression by developing oligodendrocytes. In the present studies, anti-Reo3R antibodies are shown to stimulate additional features of the program of oligodendrocyte development, including the loss of the A2B5 marker and expression of myelin basic protein. In anti-Reo3R antibody-treated cultures, galactocerebroside was expressed by cells having the morphology of immature oligodendrocyte precursors. Reo3R binding did not appear directly to inhibit or stimulate proliferation of glial progenitor cells or to affect their lineage commitment. Cell-surface structures utilized as a receptor by reovirus type 3 appear to play a role in the regulation of the initiation or rate of execution of the oligodendrocyte developmental program.

Animals↗
PubMed1991

Expression of c-erbB-2 in human pancreatic adenocarcinomas.

The c-erbB-2 (neu) gene encodes a transmembrane phosphoglycoprotein (p185erbB-2) which resembles a growth factor receptor-like molecule closely related to the epidermal growth factor receptor. Overexpression of c-erbB-2 induces cell transformation in vitro. Poorer survival rates and elevated recurrence rates following treatment have been shown in patients whose breast adenocarcinomas demonstrate increased c-erbB-2 expression. Using immunoprecipitation and immunoperoxidase staining, we surveyed human cell lines for p185erbB-2. Cell lines from most tumor types (e.g. lymphomas, neuroblastomas, melanomas) demonstrated negligible p185erB-2; however, 3 of 6 pancreatic cell lines overexpressed c-erbB-2. Southern blot analysis revealed that c-erbB-2 was amplified in two of these cell lines and was both rearranged and amplified in one of them. Based on these findings, we examined tissue sections from archival specimens of primary human pancreatic adenocarcinomas. A substantial proportion of specimens had increased p185erbB-2, as judged by increased immunostaining of the tumor cells. In such pancreatic tumors p185erbB-2 may contribute to the malignant phenotype and could provide a target for immunodiagnostic or immunotherapeutic strategies.

Adenocarcinoma↗
PubMed1991

Human genes other than CD4 facilitate HIV-1 infection of murine cells.

The human CD4 glycoprotein is a specific receptor for the HIV family of retroviruses. When expressed on human cell lines, this molecule binds virus through direct interactions with the gp 120 viral envelope glycoprotein thus allowing virus infection to occur. Subsequent to binding, conformational changes in the viral envelope glycoproteins are thought to facilitate virus entry into the target cell through direct fusion of the virus with the cell membrane. In contrast to the infection observed in human cell lines, infection of murine cell lines even in the presence of the CD4 receptor does not readily occur. We have examined this species tropism of HIV infection. We report that the inability to infect murine cells is not a function of the receptor for HIV or a suppressive function of the murine cellular background. Human CD4 expression, configuration and down-modulation on the murine background are similar if not identical to expression on the human cell background. Utilizing a panel of interspecific cell hybridomas, we have been able to bypass the barrier to infectivity of human CD4-positive murine cells. We demonstrate that there are at least two different restrictions to infectivity on the mouse background which can be complemented by the human genome. One restriction appears to be an extremely early postbinding function likely to be molecules necessary for viral entry into cells, the second restriction is necessary for high levels of virus production. Our mapping studies suggest that fewer than five human chromosomes are necessary for reconstituting infectivity in the murine background. These results have implications for models of HIV-induced pathogenesis and infectivity.

Acquired Immunodeficiency Syndrome↗
PubMed1990

p185neu expression in human lung adenocarcinomas predicts shortened survival.

p185neu is the protein product of the HER2/neu protooncogene. This protein has characteristics of a tyrosine kinase growth factor receptor and is postulated to be important in human carcinogenesis. To define the significance of the expression of this protein in human non-small cell lung cancer, 55 tumors from patients with squamous cell carcinoma (16), adenocarcinoma (29), or large cell carcinoma (10) of the lung were examined for p185neu using immunohistological methods. Five of 16 squamous cell carcinomas and 10 of 29 adenocarcinomas were found to overexpress p185neu relative to levels of expression seen in uninvolved bronchiolar epithelium. For the adenocarcinomas, p185neu expression was associated with older age (66.6 +/- 10.1 versus 57.5 +/- 10.8 years) (P = 0.04) and shortened survival (83.7 +/- 94.1 versus 188.5 +/- 120 weeks) (P = 0.01). In this group, using Cox's multivariate survival analysis, p185neu expression was found to be a significant determinant of survival (P = 0.04) even after accounting for the effect of tumor stage. For the squamous cell carcinomas, p185neu expression was not correlated with any of our clinicopathological parameters. Our findings indicate that non-small cell lung cancers which express p185neu do so at levels higher than that found in normal bronchiolar epithelium, and expression in adenocarcinomas of the lung is independently associated with diminished survival intervals.

Adenocarcinoma↗
PubMed1990

Intermolecular association of the p185neu protein and EGF receptor modulates EGF receptor function.

We have used cross-linking reagents on cell lines expressing both p185neu and EGFR. The lysates of the cells were precipitated with anti-p185neu or anti-EGFR antibodies. These precipitates included a high molecular weight complex that was identified as an EGFR-p185neu heterodimer. Heterodimerization was found to be induced by exposure to EGR. The EGFR of these cells displayed three affinity states for EGF: low (Kd, approximately 10(-9) M), high (Kd, 10(-9) to 10(-10) M), and very high (Kd, 10(-11) M), as determined by Scatchard analyses. Relatively small levels of EGF had a dramatic biological effect on cells expressing very high affinity EGFR. The very high affinity EGFR disappeared after the cells were treated with anti-p185neu monoclonal antibodies that selectively down-regulated p185neu. EGF and TPA had differential effects on down-modulation of the EGFR in cells that express either one or both species of receptor proteins.

Animals↗