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Biomedical subjects

S Corey

Publications and source records attributed to S Corey.

At least 19 recordsLinked to original sources
PubMed2003

Productive infection of cerebellar granule cell neurons by JC virus in an HIV+ individual.

BACKGROUND: In the setting of severe immunosuppression, the polyomavirus JC (JCV) can cause a lytic infection of oligodendrocytes. This demyelinating disease of the CNS white matter (WM) is called progressive multifocal leukoencephalopathy (PML). JCV has a very narrow host-cell range and productive infection of neurons has never been demonstrated. Patient, methods, and results: An HIV-1-infected patient presented with signs of pyramidal tract and cerebellar dysfunction. Brain MRI revealed T2 hyperintensities in the WM of both frontal lobes and cerebellar atrophy. His disease progressed despite therapy and he died 6 months later. In addition to classic PML findings in the frontal lobe WM, autopsy revealed scattered foci of tissue destruction in the internal granule cell layer (IGCL) of the cerebellum. In these foci, enlarged granule cell neurons identified by the neuronal markers MAP-2 and NeuN reacted with antibodies specific for the polyomavirus VP1 capsid protein. Electron microscopy showed 40 nm viral particles, consistent with polyomaviruses, in these granule cell neurons. In addition, JCV DNA was detected by PCR after laser capture microdissection of cells from the areas of focal cell loss. Finally, in situ hybridization studies demonstrated that many granule cell neurons were infected with JCV but did not contain viral proteins. Sequence analysis of the JCV regulatory region from cerebellar virions showed a tandem repeat pattern also found in PML lesions of the frontal lobe WM. CONCLUSION: JCV can productively infect granule cell neurons of the IGCL of the cerebellum. This suggests a role for JCV infection of neurons in cerebellar atrophy occurring in HIV-infected individuals.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Perivascular macrophages are the primary cell type productively infected by simian immunodeficiency virus in the brains of macaques: implications for the neuropathogenesis of AIDS.

The macrophage is well established as a target of HIV and simian immunodeficiency virus (SIV) infection and a major contributor to the neuropathogenesis of AIDS. However, the identification of distinct subpopulations of monocyte/macrophages that carry virus to the brain and that sustain infection within the central nervous system (CNS) has not been examined. We demonstrate that the perivascular macrophage and not the parenchymal microglia is the primary cell productively infected by SIV. We further demonstrate that although productive viral infection of the CNS occurs early, thereafter it is not easily detectable until terminal AIDS. The biology of perivascular macrophages, including their rate of turnover and replacement by peripheral blood monocytes, may explain the timing of neuroinvasion, disappearance, and reappearance of virus in the CNS, and questions the ability of the brain to function as a reservoir for productive infection by HIV/SIV.

AIDS Dementia Complex↗
PubMed2001

The Stoichiometry of Gbeta gamma binding to G-protein-regulated inwardly rectifying K+ channels (GIRKs).

G-protein-coupled inwardly rectifying K(+) (GIRK; Kir3.x) channels are the primary effectors of numerous G-protein-coupled receptors. GIRK channels decrease cellular excitability by hyperpolarizing the membrane potential in cardiac cells, neurons, and secretory cells. Although direct regulation of GIRKs by the heterotrimeric G-protein subunit Gbetagamma has been extensively studied, little is known about the number of Gbetagamma binding sites per channel. Here we demonstrate that purified GIRK (Kir 3.x) tetramers can be chemically cross-linked to exogenously purified Gbetagamma subunits. The observed laddering pattern of Gbetagamma attachment to GIRK4 homotetramers was consistent with the binding of one, two, three, or four Gbetagamma molecules per channel tetramer. The fraction of channels chemically cross-linked to four Gbetagamma molecules increased with increasing Gbetagamma concentrations and approached saturation. These results suggest that GIRK tetrameric channels have four Gbetagamma binding sites. Thus, GIRK (Kir 3.x) channels, like the distantly related cyclic nucleotide-gated channels, are tetramers and exhibit a 1:1 subunit/ligand binding stoichiometry.

Animals↗
PubMed1999

Structure, G protein activation, and functional relevance of the cardiac G protein-gated K+ channel, IKACh.

The muscarinic-gated atrial potassium channel IKACh has been well characterized functionally, and has been an excellent model system for studying G protein/effector interactions. Complementary DNAs encoding the composite subunits of IKACh have been identified, allowing direct probing of structural and functional features of the channel. Here, we highlight recent approaches taken in our laboratory to determine the oligomeric structure of native cardiac IKACh, the mechanism of activation of IKACh by G proteins, and the relevance of IKACh to cardiac physiology.

Amino Acid Sequence↗
PubMed1999

GIRK4 confers appropriate processing and cell surface localization to G-protein-gated potassium channels.

GIRK1 and GIRK4 subunits combine to form the heterotetrameric acetylcholine-activated potassium current (IKACh) channel in pacemaker cells of the heart. The channel is activated by direct binding of G-protein Gbetagamma subunits. The GIRK1 subunit is atypical in the GIRK family in having a unique ( approximately 125-amino acid) domain in its distal C terminus. GIRK1 cannot form functional channels by itself but must combine with another GIRK family member (GIRK2, GIRK3, or GIRK4), which are themselves capable of forming functional homotetramers. Here we show, using an extracellularly Flag-tagged GIRK1 subunit, that GIRK1 requires association with GIRK4 for cell surface localization. Furthermore, GIRK1 homomultimers reside in core-glycosylated and nonglycosylated states. Coexpression of GIRK4 caused the appearance of the mature glycosylated form of GIRK1. [35S]Methionine pulse-labeling experiments demonstrated that GIRK4 associates with GIRK1 either during or shortly after subunit synthesis. Mutant and chimeric channel subunits were utilized to identify domains responsible for GIRK1 localization. Truncation of the unique C-terminal domain of Delta374-501 resulted in an intracellular GIRK1 subunit that produced normal IKACh-like channels when coexpressed with GIRK4. Chimeras containing the C-terminal domain of GIRK1 from amino acid 194 to 501 were intracellularly localized, whereas chimeras containing the C terminus of GIRK4 localized to the cell surface. Deletion analysis of the GIRK4 C terminus identified a 25-amino acid region required for cell surface targeting of GIRK1/GIRK4 heterotetramers and a 25-amino acid region required for cell surface localization of GIRK4 homotetramers. GIRK1 appeared intracellular in atrial myocytes isolated from GIRK4 knockout mice and was not maturely glycosylated, supporting an essential role for GIRK4 in the processing and cell surface localization of IKACh in vivo.

Animals↗
PubMed1998

Identification of native atrial G-protein-regulated inwardly rectifying K+ (GIRK4) channel homomultimers.

G-protein-regulated inwardly rectifying K+ (GIRK) channels play critical inhibitory roles throughout the nervous system, heart, and pancreas. They are believed to be heterotetramers consisting of GIRK1 (Kir3.1) and either GIRK2 (Kir3.2), GIRK3 (Kir3.3), or GIRK4 (Kir3.4) subunits. The GIRK1 subunit is hypothesized to be critical to form GIRK channels with normal channel kinetics based on heterologous expression studies. However, GIRK2 and GIRK3 proteins are present in areas of the brain where no GIRK1 has been detected. Here we demonstrate that GIRK tetramers lacking GIRK1 can be purified from bovine heart atria. We have found that only half of GIRK4 is purified as the GIRK1-GIRK4 heterotetramer, whereas the remaining GIRK4 forms a high molecular weight, SDS-resistant complex that does not contain GIRK1. These GIRK4 complexes, most likely GIRK4 homotetramers, were previously not seen because of their aberrant migration on SDS-polyacrylamide gels. We propose that all of GIRK1 and half of GIRK4 proteins in atria combine to form the heterotetramer IKACh, whereas the remaining GIRK4 forms a novel tetrameric complex. GIRK4 homotetramers form channels with unusual single channel behavior, and their contribution to native currents requires further investigation.

Animals↗
PubMed1998

Number and stoichiometry of subunits in the native atrial G-protein-gated K+ channel, IKACh.

The G-protein-regulated, inwardly rectifying K+ (GIRK) channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK channels are distributed predominantly throughout the heart, brain, and pancreas. In recent years, GIRK channels have received a great deal of attention for their direct G-protein betagamma (Gbetagamma) regulation. Native cardiac IKACh is composed of GIRK1 and GIRK4 subunits (Krapivinsky, G., Gordon, E. A., Wickman, K. A., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). Here, we examine the quaternary structure of IKACh using a variety of complementary approaches. Complete cross-linking of purified atrial IKACh protein formed a single adduct with a total molecular weight that was most consistent with a tetramer. In addition, partial cross-linking of purified IKACh produced subsets of molecular weights consistent with monomers, dimers, trimers, and tetramers. Within the presumed protein dimers, GIRK1-GIRK1 and GIRK4-GIRK4 adducts were formed, indicating that the tetramer was composed of two GIRK1 and two GIRK4 subunits. This 1:1 GIRK1 to GIRK4 stoichiometry was confirmed by two independent means, including densitometry of both silver-stained and Western-blotted native atrial IKACh. Similar experimental results could potentially be obtained if GIRK1 and GIRK4 subunits assembled randomly as 2:2 and equally sized populations of 3:1 and 1:3 tetramers. We also show that GIRK subunits may form homotetramers in expression systems, although the evidence to date suggests that GIRK1 homotetramers are not functional. We conclude that the inwardly rectifying atrial K+ channel, IKACh, a prototypical GIRK channel, is a heterotetramer and is most likely composed of two GIRK1 subunits and two GIRK4 subunits.

Animals↗
PubMed1997

The use of counterflow centrifugal elutriation for the depletion of T cells from unrelated donor bone marrow.

Transplantation of marrow from unrelated donors is associated with an increased incidence and severity of graft-versus-host disease (GVHD). In an attempt to minimize GVHD without compromising engraftment, unrelated donor marrow was depleted of lymphocytes by counterflow centrifugal elutriation (CCE), and a fixed dose of 0.5 x 10(6) CD3+ T cells/kg, as measured in real time by flow cytometry, was added back to the graft. Patients received cyclosporine (CYA) and corticosteroids for GVHD prophylaxis and to facilitate engraftment. In the first cohort (study I), 7 patients received busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) (CY) and one patient received CY (200 mg/kg) + 1260 cGy fractionated TBI. Of 6 who were evaluable for both engraftment and rejection, 4 rejected their graft. The study was terminated, and the protocol was modified (study II) by the addition of antithymocyte globulin (ATG) to the pre-BMT and post-BMT therapy. Twelve patients received CY + TBI as above plus ATG given pre-BMT and post-BMT. Ten of twelve who received ATG engrafted. Twelve patients from studies I and II were evaluable for acute GVHD. Two developed grade I acute GVHD. Two patients developed grade II acute GVHD, 2 patients developed grade III GVHD, and 1 patient developed grade IV acute GVHD. Two of three cases of acute GVHD (> grade II) occurred later than day 100 after BMT concomitant with reduction of immunosuppressive therapy. The rate of engraftment was significantly higher in study II (p = .054). In numbers of CD34+ cells infused, numbers of CFU-GM infused, and numbers of nucleated cells did not correlate with engraftment. We conclude that (a) in contrast to the results seen in recipients of marrow from HLA-matched sibling donors, the depletion of unrelated donor marrow of all but 0.5 x 10(6) CD34+ cells/kg together with CYA + corticosteroids was not sufficient to facilitate engraftment. The use of a more immunosuppressive regimen containing TBI and ATG appeared to improve engraftment. (b) The reduction of the graft T cell dose to 0.5 x 10(6) CD34+ cells/kg resulted in a higher incidence of acute GVHD than that seen in recipients of marrow from genotypically identical donors whose marrow was similarly processed.

Adolescent↗
PubMed1995

FK506 (tacrolimus) in the treatment of steroid-resistant acute graft-versus-host disease in children undergoing bone marrow transplantation.

Steroid-resistant graft-versus-host disease (GVHD) is an often lethal complication of bone marrow transplantation (BMT). FK506 (tacrolimus) is a new potent immunosuppressant which has been shown to be superior to conventional immunosuppression in the prevention and treatment of graft rejection in recipients of solid organ transplants. To determine whether FK506 is effective in the treatment of steroid-resistant acute GVHD, 6 children with biopsy-proven severe GVHD were studied. FK506 was administered as intravenous or oral therapy and the dose was adjusted to achieve serum levels between 0.5 and 1.0 microgram/ml by ELISA. Steroid doses were tapered based on clinical grading in each organ. Within 1-2 days, improvement occurred in skin and gut in all patients, and in liver in 3 patients. Toxicity attributable to FK506 was similar to that described in solid organ transplant patients and included neurotoxicity, nephrotoxicity and gastrointestinal effects. While FK506 is effective in the treatment of steroid-resistant acute GVHD, toxicity may limit its use. Further studies evaluating FK506 as GVHD prophylaxis and treatment of less advanced GVHD are needed.

Adolescent↗
PubMed1995

Divergent signal transduction pathways and effects on natural killer cell functions induced by interaction of Fc receptors with physiologic ligands or antireceptor antibodies.

Natural killer (NK) cells express receptors that bind to the Fc portion of immunoglobulin molecules (FcR), and their function can potentially be modulated positively or negatively by such receptor:ligand interactions. This review discusses recent developments in the understanding of expression of various forms and isoforms of FcRs by NK cells, and the sequelae of binding of physiologic ligands. Particular attention is paid to the significance of FcR:Ig interactions in both activating and inactivating NK cells under various conditions.

Animals↗
PubMed1993

Receptor stimulated accumulation of phosphatidylinositol (3,4,5)-trisphosphate by G-protein mediated pathways in human myeloid derived cells.

Phosphoinositide 30H-kinase (PI3K) activities are thought to be critical regulatory enzymes in a new intracellular signalling pathway, the activation of which results in the rapid accumulation of a putative signalling molecule, phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5) P3]. To date, activation of PI3K has always correlated with its recruitment into complexes containing protein tyrosine kinases (PTK). Here we report that agonists which utilize G-protein mediated transduction pathways can stimulate very rapid and large accumulations of PtdIns(3,4,5)P3 via a novel mechanism, possibly involving direct coupling between the G-protein and a PI3K activity. In addition, some of these agonists also stimulate small increases in PI3K activity in anti-phosphotyrosine and anti-src-type PTK antibody directed immunoprecipitates, indicating activation of PI3K via a 'conventional' PTK mediated mechanism; these pathways however, play only a minor role in the initial, agonist sensitive production of PtdIns(3,4,5)P3 in myeloid derived cells.

Adenosine Triphosphate↗
PubMed1993

Granulocyte macrophage-colony stimulating factor stimulates both association and activation of phosphoinositide 3OH-kinase and src-related tyrosine kinase(s) in human myeloid derived cells.

The signalling pathways used by the GM-CSF receptor are currently unknown. Here we show that in human myeloid derived cells GM-CSF can stimulate; (i) the accumulation of PtdIns(3,4,5)P3; (ii) increases in p53/p56lyn and p62c-yes directed protein tyrosine kinase activities in anti-lyn and anti-c-yes antibody directed immunoprecipitates, respectively and; (iii) increases in phosphoinositide 3OH-kinase activity in antiphosphotyrosine, anti-p53/p56lyn and anti-p62c-yes antibody directed immunoprecipitates. These results suggest that GM-CSF can stimulate formation of protein tyrosine kinase co-ordinated signalling complexes, that contain p53/p56lyn, p62c-yes and an activated PtdInsP2 directed phosphoinositide 3OH-kinase, which can drive the accumulation of the putative second-messenger PtdIns(3,4,5)P3.

Cells, Cultured↗