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Biomedical subjects

A Fallon

Publications and source records attributed to A Fallon.

At least 19 recordsLinked to original sources
PubMed1999

Activation of the erythropoietin receptor by the gp55-P viral envelope protein is determined by a single amino acid in its transmembrane domain.

The spleen focus forming virus (SFFV) gp55-P envelope glycoprotein specifically binds to and activates murine erythropoietin receptors (EpoRs) coexpressed in the same cell, triggering proliferation of erythroid progenitors and inducing erythroleukemia. Here we demonstrate specific interactions between the single transmembrane domains of the two proteins that are essential for receptor activation. The human EpoR is not activated by gp55-P but by mutation of a single amino acid, L238, in its transmembrane sequence to its murine counterpart serine, resulting in its ability to be activated. The converse mutation in the murine EpoR (S238L) abolishes activation by gp55-P. Computational searches of interactions between the membrane-spanning segments of murine EpoR and gp55-P provide a possible explanation: the face of the EpoR transmembrane domain containing S238 is predicted to interact specifically with gp55-P but not gp55-A, a variant which is much less effective in activating the murine EpoR. Mutational studies on gp55-P M390, which is predicted to interact with S238, provide additional support for this model. Mutation of M390 to isoleucine, the corresponding residue in gp55-A, abolishes activation, but the gp55-P M390L mutation is fully functional. gp55-P is thought to activate signaling by the EpoR by inducing receptor oligomerization through interactions involving specific transmembrane residues.

Amino Acid Sequence↗
PubMed1998

The anemic Friend virus gp55 envelope protein induces erythroid differentiation in fetal liver colony-forming units-erythroid.

The gp55 envelope proteins of the spleen focus-forming virus initiate erythroleukemia in adult mice. Because the gp55 from the polycythemic strain (gp55-P), but not from the anemic strain (gp55-A), activates the erythropoietin receptor (EpoR) for proliferation of hematopoietic cell lines, the mechanism by which gp55-A initiates erythroleukemia has remained a mystery. We show here that gp55-A activates the EpoR in fetal liver cells. In contrast to previous studies using bone marrow cells from phenylhydrazine-treated, anemic mice, we find that both gp55-A and gp55-P induce erythroid differentiation from colony-forming unit-erythroid (CFU-E) progenitors in fetal liver cells. The effects on CFU-Es of both gp55-A and -P are mediated by the EpoR, because no colonies are seen upon expression of either gp55 in EpoR-/- fetal liver cells. However, only gp55-P induces erythroid bursts from burst-forming unit-erythroid progenitors and only gp55-P induces Epo independence in Epo-dependent cell lines. Using chimeric gp55 P/A proteins, we extend earlier work showing that the transmembrane sequence determines the capacity of gp55 proteins to differentially activate EpoR signaling. We discuss the possibilities for different signaling capacities of gp55-A and -P in fetal liver and bone marrow-derived erythroid progenitor cells.

Animals↗
PubMed1996

Termination in inpatient groups.

This article provides the therapist with a three-factor framework for understanding and constructively responding to members' termination in the inpatient group. The first factor is the context in which the inpatient group takes place. It will be argued that how the broader system defines and treats termination affects the group process at the time of termination. The second factor is the therapist's countertransference. Three especially common reactions (disappointment, abandonment anxiety, and powerlessness) will be described. The third factor is the characteristic need of inpatients at termination time. Each factor is described in terms of its potential impact on the termination process. Techniques and suggestions are offered that aim to mitigate the destructive forces of each factor and to enhance the therapeutic value of the termination event for the patient.

Countertransference↗
PubMed1993

Geographic variation in human immunodeficiency virus seroconversion rates in the U.S. Navy.

The Navy population is centered around 19 U.S. home ports and several inland duty stations. This is a study of variation in human immunodeficiency virus (HIV) seroconversions in Navy enlisted men by location in the United States, based on 949,570 enzyme-linked immunosorbent assays and 812 seroconversions during 1987-90. Seroconversion rates were higher in personnel in San Francisco (p = 0.0004), Washington, D.C. (p = 0.001), and Orlando, FL (p = 0.04) than in other areas. They were lower in Charleston, SC, New London, CT, Seattle; and Brunswick, ME (p < 0.05). Black men had triple the seroconversion rate of all other men (p < 0.0001). After adjustment for race, age, marital status, and occupation, risk of seroconversion remained high in San Francisco (p = 0.02) and Orlando (p = 0.03). The risk of seroconversion in San Francisco was twice that of other areas in a cohort that did not change location (p = 0.01). In contrast to declining trends overall in the Navy, rates did not decrease in Washington, D.C., a result consistent with previous calculations indicating a delayed second wave of HIV infection in the region.

Adolescent↗
PubMed1992

Purification and analysis of proteinase-resistant mutants of recombinant platelet-derived growth factor-BB exhibiting improved biological activity.

Recombinant platelet-derived growth factor (PDGF)-BB was expressed and secreted from yeast in order to study the structure-function relationships of this mitogen. A simple purification scheme has been developed which yields greater than 95% pure PDGF-BB. Analysis of this recombinant PDGF-BB shows partial proteolysis after arginine-32. Substitution of this arginine residue, or arginine-28 [a potential KEX2 (lysine-arginine endopeptidase) cleavage site], prevents or reduces cleavage of PDGF-BB respectively. These mutations result in a 5-fold increase in expression levels of PDGF-BB, and the resulting mutant proteins show higher activity in a number of biological assays than the cleaved wildtype PDGF-BB. These data are in accord with previous work by Giese, LaRochelle, May-Siroff, Robbins & Aaronson [(1990) Mol. Cell Biol. 10, 5496-5501] suggesting that the region isoleucine-25-phenylalanine-37 is involved in PDGF-receptor binding.

3T3 Cells↗
PubMed1992

Characterization of the structure and conformation of platelet-derived growth factor-BB (PDGF-BB) and proteinase-resistant mutants of PDGF-BB expressed in Saccharomyces cerevisiae.

A detailed biophysical study of the secondary and tertiary structures of recombinant platelet-derived growth factor (PDGF)-BB produced in yeast has been carried out. The secondary structure of the molecule is composed of 54% beta-sheet with less than 5% ordered helix. The single tryptophan residue has been shown to be solvent-accessible; however, the ability of the side chain to rotate is severely restricted. The fluorescence emission is quenched at pH 7.0 and in the presence of high salt, but dequenched by titration to lower pH with a pK of 5.8. Two proteinase-resistant mutants of PDGF [( Ser28]- and [Pro32]-PDGF-BB) have also been characterized and shown to have secondary and tertiary structures indistinguishable from wild-type PDGF-BB. These are, therefore, suitable stable background molecules in which to carry out structure-activity-relationship studies on PDGF-BB.

Mutagenesis, Site-Directed↗
PubMed1991

Structure-function relationships in human epidermal growth factor studied by site-directed mutagenesis and 1H NMR.

In order to elucidate the mechanism of interaction between human epidermal growth factor (EGF) and its receptor, selected variants of EGF, differing by single amino acid substitutions, have been made by site-directed mutagenesis. The receptor affinity of these mutants was determined by a receptor binding competition assay, and the effects of the substitution on the structure of the protein were assessed by 1H nuclear magnetic resonance techniques. Various substitutions of Arg-41 resulted in substantial reduction in receptor affinity of EGF whereas change of Tyr-13 did not affect binding to the receptor. The 1H resonances of all nonexchangeable protons of the Tyr-13----Leu, Arg-41----His, and Leu-47----Glu variants were assigned and compared in order to assess the structural integrity of these mutants, which possess very different spectral and biological properties. In the case of the Leu-47----Glu mutant, only minor localized spectral changes were observed, confirming that the tertiary structure of the protein is preserved upon mutation. In contrast, for both the Arg-41----His and Tyr-13----Leu variants, significant and strikingly similar spectra changes were observed for many residues located far away from the mutated residues. This implies that similar structural alterations have taken place in both proteins, an idea further supported by hydrogen-exchange experiments where the exchange rates of hydrogen-bonded amide protons for both the Tyr-13----Leu and the Arg-41----His mutants were found to be about 4 times faster than in the wild-type protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Two PDGF-B chain residues, arginine 27 and isoleucine 30, mediate receptor binding and activation.

PDGF may be involved in the pathogenesis of a variety of disorders including atherosclerosis and certain types of cancer. There is currently little understanding of the molecular structure of PDGF and of the critical amino acid residues involved in receptor binding and cell activation. Two such PDGF-B chain residues, arginine 27 and isoleucine 30, have been identified by a site-directed mutagenesis programme. Substitutions in these positions can lead to PDGF mutants defective in both receptor affinity and cell activation as judged by displacement of [125I]PDGF-BB, mitogenic assay and inositol lipid turnover. Circular dichroism and fluorescence spectroscopy show that such mutations do not disrupt the structure of PDGF.

3T3 Cells↗
PubMed1990

Structure-function relationships in epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha).

The solution structures of the homologous growth factors human epidermal growth factor (hEGF) and human transforming growth factor-alpha (hTGF-alpha), as determined by high resolution NMR and various computational methods, are described. Knowledge of these structures and the sequences of other homologous proteins leads to predictions about growth factor residues which may be involved in the receptor/ligand interface. Recent experiments designed to check these predictions are described briefly. These involve site-specific mutagenesis, receptor binding assays and high resolution NMR studies.

Amino Acid Sequence↗
PubMed1990

Structure-function analysis of epidermal growth factor: site directed mutagenesis and nuclear magnetic resonance.

The role of leucine-47 in determining the structure and activity of human epidermal growth factor was examined using site-directed mutagenesis. Wild type protein and four variants in which Leu47 was replaced by valine, glutamate, aspartate and alanine were produced from yeast. 1H NMR experiments demonstrated that substitution of Leu47 had little effect on the protein structure. The observed reduction in receptor binding affinity caused by the substitutions could thus be attributed to perturbation of a residue directly involved in receptor interactions.

Binding, Competitive↗
PubMed1984

Investigation of the parameters for reversed-phase high-performance liquid chromatography of collagen types I and III.

A detailed study was carried out on the behaviour of collagen and its subcomponents on large pore size reversed-phase high-performance liquid chromatographic columns. Investigation of laboratory-prepared C18 and CN 50-nm pore size supports using a simple solvent system showed that heat-denatured collagen chains could be retained and resolved and that the C18 packing had a greater selective capacity. However, because of inconsistencies in the chromatography obtained using laboratory-prepared supports commercial 33-nm pore size packings were subsequently studied with both pyridine-based and trifluoroacetic acid-based solvent systems. An optimised solvent was defined and used to check the resolving capacity of both C18 and CN commercial columns. These studies led to the description and interpretation of the effects of pH, counter-ion concentration, solvent strength, type of support and molecular weight of protein solutes on the chromatographic behaviour of this large protein and its CNBr peptides. An explanation of the major forces acting to bring about retention and resolution is presented and suggestions are made for the application of this methodology to other proteins.

Animals↗
PubMed1984

Collagen stimulating factors in hepatic fibrogenesis.

Four factors which stimulate collagen synthesis and prolyl hydroxylase activity in cultures of human and mouse fibroblasts have been isolated by molecular sieve chromatography from animal and human fibrotic and cirrhotic livers. These factors do not stimulate protein or DNA synthesis or total DNA in these cultures. It has also been shown that these factors, designated collagen stimulating factors F1-F4, do not owe their activity to ascorbate or glutamine. Collagen stimulating factors are heat stable, and F1 and F2 have apparent molecular weights of about 4000 and 1000 respectively. Since these factors are not present in normal animal or human liver it is suggested that they may be responsible for increased collagen production in vivo in hepatic fibrosis and cirrhosis.

Animals↗
PubMed1983

Separation of native types I and III collagens and denatured chains by reverse-phase high-performance liquid chromatography.

A simple and effective high-performance-liquid-chromatography system has been developed for the separation of the native forms of Types I and III collagens. This separation is achieved on a commercially available 330-A-pore-size C18 reverse-phase support using a solvent system consisting of ammonium bicarbonate (0.05 M) and trifluoroacetic acid (0.4%) with tetrahydrofuran as the eluting solvent. This simple system can also be applied to the separation of mixtures of the denatured chains of both collagen types.

Animals↗
PubMed1983

Isolation of collagen stimulating factors from healing wounds.

Five factors (collagen stimulating factors) have been isolated from healing murine skin wounds which stimulate prolyl hydroxylase activity and collagen synthesis in mouse fibroblasts in vitro. These factors stimulate general protein synthesis to a much smaller extent. Collagen stimulating factors are detectable in wounds three days after healing begins and disappear after six days when healing is complete. These data indicate that these factors may modulate collagen production during wound healing.

Animals↗