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Biomedical subjects

M C Riedy

Publications and source records attributed to M C Riedy.

13 recordsLinked to original sources
PubMed1999

Activin A and TGF-beta stimulate phosphorylation of focal adhesion proteins and cytoskeletal reorganization in rat aortic smooth muscle cells.

Activin A and Transforming Growth Factor-beta (TGF-beta) are members of a common family of cytokines that bind to and stimulate serine/threonine kinase receptors. Activin A and TGF-beta are important during embryonic development exerting both positive and negative effects on cell growth. In the adult organism, they function in processes such as tissue repair, cellular proliferation, and differentiation. Although activin A and TGF-beta often induce opposite functional outcomes in specific cells; proliferation or differentiation, both were found to stimulate the formation of actin stress fibers and focal adhesions in serum-starved rat aortic smooth muscle (RASM) cells. These structural changes were accompanied by phosphorylation of the focal adhesion proteins, paxillin, and p130(cas). Similar cytoskeletal and biochemical changes were observed with the vasoactive agonist angiotensin II. Activation of the ERK/MAP kinase pathway has been implicated in the migration in certain cell types. However, while activin A, TGF-beta, and angiotensin II all stimulated ERK activity in RASM cells, only activin A and angiotensin II stimulated migration. TGF-beta failed to illicit a chemotactic response. Furthermore, pharmacologic inhibition of MEK activity failed to block migration in response to activin A and angiotensin II, indicating RASM migration can occur independent of ERK activity. These results suggest that TGF-beta and activin A share several signaling pathways with angiotensin II leading to cytoskeletal remodeling and ERK activation, but there are distinct differences regarding the effect of these agonists on cellular migration.

Actins↗
PubMed1999

Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.

Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.

ADP-Ribosylation Factors↗
PubMed1997

Tumor-induced suppression of T lymphocyte proliferation coincides with inhibition of Jak3 expression and IL-2 receptor signaling: role of soluble products from human renal cell carcinomas.

The proliferative capacity of T cells infiltrating human tumors is known to be impaired, possibly through their interaction with tumor. Here we demonstrate that soluble products derived from renal cell carcinoma (RCC-S) explants but not normal kidney can inhibit an IL-2-dependent signaling pathway that is critical to T cell proliferation. A major target of the immunosuppression was the IL-2R-associated protein tyrosine kinase, Janus kinase 3 (Jak3). RCC-S suppressed basal expression of Jak3 and its increase following stimulation with anti-CD3/IL-2. Jak3 was most sensitive to suppression by RCC-S; however, reduction in expression of p56(lck), p59(fyn), and ZAP-70 was observed in some experiments. Expression of other signaling elements linked to the IL-2R (Jak1) and the TCR (TCR-zeta, CD3-epsilon, and phospholipase C-gamma) were minimally affected. In naive T cells, RCC-S also partially blocked induction of IL-2R alpha-, beta- and gamma-chain expression when stimulating via the TCR/CD3 complex with anti-CD3 Ab. To determine whether RCC-S suppressed IL-2-dependent signaling, primed T cells were employed since RCC-S had no effect on IL-2R expression but did down-regulate Jak3 expression and, to a lesser degree, p56(lck) and p59(fyn). Reduction in Jak3 correlated with impaired IL-2-dependent proliferation and signal transduction. This included loss of Jak1 kinase tyrosine phosphorylation and no induction of the proto-oncogene, c-Myc. These findings suggest that soluble products from tumors may suppress T cell proliferation through a mechanism that involves down-regulation of Jak3 expression and inhibition of IL-2-dependent signaling pathways.

Carcinoma, Renal Cell↗
PubMed1996

Genomic sequence, organization, and chromosomal localization of human JAK3.

Members of the Janus (JAK) protein tyrosine kinase family including JAK3 have recently emerged as important components in cytokine signal transduction. Mutations of JAK3 have been found in a number of patients who present with severe combined immunodeficiency. To facilitate the further identification of JAK3-SCID patients and to understand the structure of JAK3 better, we undertook the determination of the genomic sequence, organization, and chromosomal localization of the JAK3 gene. The JAK3 gene was found to consist of 19 exons and 18 introns. Interestingly, the organization of the kinase-(JH1) and pseudokinase-(JH2) domains were found to be dissimilar. In addition, the JAK3 gene was localized to human chromosome 19p13.1. These data should facilitate the identification of patients with this new form of immunodeficiency and will provide insight into the structure of this kinase.

Chromosome Mapping↗
PubMed1996

Signaling by IL-2 and related cytokines: JAKs, STATs, and relationship to immunodeficiency.

Cytokines that bind to the interleukin-2 (IL-2) receptor common gamma chain (gamma c), including IL-2, IL-4, IL-7, IL-9, and IL-15, are important for the growth and differentiation of T and B lymphocytes, natural killer cells, macrophages, and monocytes. These cytokines have overlapping biological effects that in part result from the use of the shared receptor subunit gamma c. Recently it has become clear that these cytokines activate a number of important intracellular signaling molecules, including the Janus kinases JAK1 and JAK3 and members of the transcription factor family of signal transducers and activators of transcription (STATs). The discovery of these signaling pathways has led to important new insights into their role in lymphocyte maturation, as it has emerged that mutations in the genes encoding both gamma c and JAK3 result in similar forms of severe combined immunodeficiency (SCID). In this review we examine the structure and function of cytokine receptors and the signaling pathways involved in their regulation of gene expression. Furthermore, we discuss recent advances that have led to a better understanding of how cytokines elicit intracellular responses, as well as their role in normal lymphoid development.

Cell Transformation, Neoplastic↗
PubMed1995

Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development.

Males with X-linked severe combined immunodeficiency (XSCID) have defects in the common cytokine receptor gamma chain (gamma c) gene that encodes a shared, essential component of the receptors of interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. The Janus family tyrosine kinase Jak3 is the only signaling molecule known to be associated with gamma c, so it was hypothesized that defects in Jak3 might cause an XSCID-like phenotype. A girl with immunological features indistinguishable from those of XSCID was therefore selected for analysis. An Epstein-Barr virus (EBV)-transformed cell line derived from her lymphocytes had normal gamma c expression but lacked Jak3 protein and had greatly diminished Jak3 messenger RNA. Sequencing revealed a different mutation on each allele: a single nucleotide insertion resulting in a frame shift and premature termination in the Jak3 JH4 domain and a nonsense mutation in the Jak3 JH2 domain. The lack of Jak3 expression correlated with impaired B cell signaling, as demonstrated by the inability of IL-4 to activate Stat6 in the EBV-transformed cell line from the patient. These observations indicate that the functions of gamma c are dependent on Jak3 and that Jak3 is essential for lymphoid development and signaling.

Amino Acid Sequence↗
PubMed1995

Quantitative RT-PCR for measuring gene expression.

Classical Northern blot analysis for measuring mRNA requires too many cells to be practical for cell sorting. Yet, measurement of gene expression in small subsets within a heterogeneous population of cells is often desired. The PCR in combination with prior reverse transcription (RT-PCR) of the mRNA of interest provides a means for measuring gene expression using as few as one cell. When RT-PCR is performed, the reliability of the data can be highly subjective due to the efficiency of both RT and PCR steps. This subjectivity can be eliminated by a technique for quantitating specific RNA molecules using an internal RNA competitive reference standard (RNA-CRS), which is identical to the sequence of interest except for a deletion of 80 bases. Here we illustrate a strategy for quantitative PCR using a RNA-CRS, synthesized solely using nonplasmid-based PCR techniques. The competitive reaction consists of a constant quantity of wild-type mRNA (from 100-1000 cells) added individually to tubes containing a serially decreasing amount of RNA-CRS. The RT-PCR is performed on these samples, then the resulting product is analyzed by gel electrophoresis and densitometry. The procedure for preparing the RNA-CRS and subsequent RT-PCR steps are described in detail.

Animals↗
PubMed1992

Inhibitory role of interleukin-6 in macrophage proliferation.

We have shown that there are two forms of progenitor cells for macrophages. The first is characterized by a short lag period (about 1 day) before initiating the cell cycle, forms large colonies, is found in the bone marrow, and is in the nonadherent fraction. The second progenitor cell, found primarily in the adherent cell fraction of bone marrow and in peripheral tissues, forms small colonies after 14 days. We investigated the effect of combining interleukin-6 (IL-6) with colony-stimulating factor 1 (CSF-I) on macrophage proliferation. We found that IL-6 inhibited the proliferation of both types of progenitor cells, as well as more differentiated macrophages. This inhibitory effect was reversible because macrophages could initiate a proliferative response after removal of IL-6 from the culture medium. The introduction of anti-IL-6 into macrophage cultures containing IL-6 allowed proliferation, indicating that the effect was IL-6 specific. These results suggest that IL-6 may play a regulatory role in vivo by suppressing the production of bone marrow and tissue macrophages.

Bone Marrow Cells↗
PubMed1991

Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations.

Flow cytometric determination of viable versus nonviable cells in fixed samples can be accomplished by utilizing the irreversible binding of photoactivated ethidium monoazide (EMA). EMA is a positively charged molecule which is excluded by cells with intact membranes (viable cells), included by cells with damaged membranes, and can be photochemically crosslinked to nucleic acids using visible light. EMA fluorescence can be excited using a standard argon laser operating at 488 nm and is able to be distinguished from fluorescein and phycoerythrin. Fixation is important when analyzing cells from a potentially infectious origin. EMA is photochemically crosslinked and therefore unable to leak out of cells when removed from the extracellular media, unlike propidium iodide (PI) or other viability stains, which were heretofore commonly used. We demonstrate the usefulness of EMA in combination with fluoresceinated and phycoerythrin labeled monoclonal antibodies in immunophenotyping. The photoaffinity labeling technique allows for a quick and efficient means of identifying nonviable cells which cannot be distinguished on the basis of light-scattering properties.

Affinity Labels↗
PubMed1988

Lymphocyte phenotyping of infants with congenital heart disease: comparison of cell preparation techniques.

The diagnostic evaluation of infants with suspected DiGeorge syndrome includes peripheral blood lymphocyte phenotype analysis by flow cytometry. Mononuclear cells are typically concentrated from infants' blood samples by Ficoll-Hypaque (FH) density gradient centrifugation prior to monoclonal antibody (Mab) staining. Having observed that lymphocyte percentages in samples prepared by this technique were low more often than anticipated based on the prevalence of the suspected diagnosis, we undertook a prospective study of 55 infants with congenital heart disease comparing FH with a whole-blood (WB) technique. Sixty healthy adult controls were similarly studied. We report the observation that FH-prepared mononuclear cells from blood samples of infants less than 4 months of age yield substantially different phenotypes than WB-prepared samples. The differences are related to red blood cell (RBC) contamination. No such difference was seen with adult samples. Unlike FH-prepared adult samples, contaminating RBCs are indistinguishable from lymphocytes through at least 4 months of age when assessed by the standard cytometric gating parameters of forward and 90 degrees light scatter. The use of a WB ammonium chloride lysis technique is recommended as most appropriate for the preparation of infants' blood samples.

Antigens, Surface↗
PubMed1986

Griffonia simplicifolia I isolectin as a functionally monovalent probe for use in flow cytometry.

The inherent tendency of lectins to agglutinate cells has limited their use as reagents for the detection of carbohydrate groups on cell surfaces by flow cytometry. In the current study, we demonstrate a method for the use of a fluoresceinated tetrameric isolectin (Griffonia simplicifolia I-A3B, FITC-GS I-A3B) as a functionally monovalent, nonagglutinating probe in flow cytometry. This isolectin contains three A subunits and one B subunit. Both types of subunits bind alpha-D-galactopyranosyl (alpha-D-galp-) end groups with similar affinities; however, the A subunits have a 1,000-fold greater affinity for N-Acetyl-D-galactosamine (GalNAc) than does the B subunit. The addition of low (1-2 mM) concentrations of GalNAc to the FITC-GS I-A3B isolectin results in blockage of the three A subunits without significantly affecting the B subunit; this yields a functionally monovalent probe for the detection of cell surface alpha-D-Galp end groups. This approach has been used to examine two types of cells: Ehrlich ascites tumor cells and rat alveolar macrophages, both of which are known to express cell surface alpha-D-Galp end groups. Lectin binding, as determined by number of positive cells and fluorescence intensity, was dependent upon concentration of the lectin and haptenic sugar. Specificity of the staining was demonstrated by the ability of methyl alpha-D-galactopyranoside (Met alpha-D-Galp) to abolish the binding of the lectin to the cells. Elimination of both GalNAc and Met alpha-D-Galp from the staining solution resulted in agglutination of the cells, indicating that the A subunits were active in the absence of GalNAc.

Animals↗