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R H Spencer

Publications and source records attributed to R H Spencer.

9 recordsLinked to original sources
PubMed1998

Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel.

Mechanosensitive ion channels play a critical role in transducing physical stresses at the cell membrane into an electrochemical response. The MscL family of large-conductance mechanosensitive channels is widely distributed among prokaryotes and may participate in the regulation of osmotic pressure changes within the cell. In an effort to better understand the structural basis for the function of these channels, the structure of the MscL homolog from Mycobacterium tuberculosis was determined by x-ray crystallography to 3.5 angstroms resolution. This channel is organized as a homopentamer, with each subunit containing two transmembrane alpha helices and a third cytoplasmic alpha helix. From the extracellular side, a water-filled opening approximately 18 angstroms in diameter leads into a pore lined with hydrophilic residues which narrows at the cytoplasmic side to an occluded hydrophobic apex that may act as the channel gate. This structure may serve as a model for other mechanosensitive channels, as well as the broader class of pentameric ligand-gated ion channels exemplified by the nicotinic acetylcholine receptor.

Amino Acid Sequence↗
PubMed1997

Purification, visualization, and biophysical characterization of Kv1.3 tetramers.

The voltage-gated K+ channel of T-lymphocytes, Kv1.3, was heterologously expressed in African Green Monkey kidney cells (CV-1) using a vaccinia virus/T7 hybrid expression system; each infected cell exhibited 10(4) to 5 x 10(5) functional channels on the cell surface. The protein, solubilized with detergent (3-[cholamidopropyl)dimethylammonio]-1-propanesulfonic acid or cholate), was purified to near-homogeneity by a single nickel-chelate chromatography step. The Kv1.3 protein expressed in vaccinia virus-infected cells and its purified counterpart are both modified by a approximately 2-kDa core-sugar moiety, most likely at a conserved N-glycosylation site in the external S1-S2 loop; absence of the sugar does not alter the biophysical properties of the channel nor does it affect expression levels. Purified Kv1.3 has an estimated size of approximately 64 kDa in denaturing SDS-polyacrylamide electrophoresis gels, consistent with its predicted size based on the amino acid sequence. By sucrose gradient sedimentation, purified Kv1.3 is seen primarily as a single peak with an approximate mass of 270 kDa, compatible with its being a homotetrameric complex of the approximately 64-kDa subunits. When reconstituted in the presence of lipid and visualized by negative-staining electron microscopy, the purified Kv1.3 protein forms small crystalline domains consisting of tetramers with dimensions of approximately 65 x 65 A. The center of each tetramer contains a stained depression which may represent the ion conduction pathway. Functional reconstitution of the Kv1.3 protein into lipid bilayers produces voltage-dependent K+-selective currents that can be blocked by two high affinity peptide antagonists of Kv1.3, margatoxin and stichodactylatoxin.

Animals↗
PubMed1993

Immunological Identification of the Shaker-Related Kv1.3 Potassium Channel Protein in T and B Lymphocytes, and Detection of Related Proteins in Flies and Yeast

Shaker-related potassium (K+) channel proteins contain sequences which exhibit remarkable conservation across species. We have generated polyclonal anti-peptide antibodies (Abs) which cross-react with peptide epitopes of several Shaker-related channels (Kv1.1, 1.2 and 1.3), in addition to a Kv1.3-specific Ab. The Kv1.3-specific Abs react with a protein expressed in human T-cells (Jurkat and PBLs), as well as in mouse T-cells (EL-4) and pre-B cells (230.37). The cross-reactive Abs detect the Shaker protein in Drosophila melanogaster, in addition to an immunologically related protein in the yeast Saccharomyces cerevisiae. Abs which recognize these shared epitopes could serve for the identification and biochemical characterization of Shaker-related proteins in diverse organisms.

Amino Acid Sequence↗
PubMed1992

Induction of thymus-reestablishing lymphoid tumors by a murine leukemia virus carrying the avian v-myc oncogene.

Primary lymphoid tumor cells induced by wild type or recombinant Moloney murine leukemia viruses were inoculated intravenously into sublethally irradiated, syngeneic mice and compared for their ability to reestablish in the recipient thymus and spleen. Lymphoid tumors induced by one recombinant, M-MuLV(myc), containing the v-myc oncogene from the avian MC29 retrovirus consistently reestablished in the thymus and spleen of recipient mice. Since tumors in the recipient thymus and spleen could have arisen by infection of host cells, or could reflect an expansion of a minor subpopulation from the donor tumor, we verified the donor and clonal origin of these tumors by Southern blot analysis using a virus-specific probe. As few as 500 of these tumor cells could migrate to the thymus via the blood and flourish in that microenvironment. In contrast, the majority of tumors induced by wild type M-MuLV or two other M-MuLV recombinants not containing v-myc sequences, inefficiently reestablished in the thymus of recipient mice following i.v. inoculation, although splenic tumors developed. These tumors could, however, multiply within the recipient thymus when inoculated intrathymically. Thus, the inability of tumors induced by wild type M-MuLV or M-MuLV recombinants lacking v-myc to reestablish in the thymus following i.v. inoculation appears to reflect inefficient 'thymic homing' rather than the ability to grow in the thymic microenvironment. Collectively, our data suggest that the thymic homing ability of M-MuLV(myc)-induced tumor cells is related to the presence of v-myc sequences or to the cell type transformed by M-MuLV(myc). Thus, we suggest that these cells may provide a useful model for studying how blood-borne metastatic cells, and possibly normal lymphocytes, enter the thymus by way of the microvasculature.

Animals↗
PubMed1990

A family of three mouse potassium channel genes with intronless coding regions.

To understand the molecular mechanisms responsible for generating physiologically diverse potassium channels in mammalian cells, mouse genomic clones have been isolated with a potassium channel complementary DNA, MBK1, that is homologous to the Drosophila potassium channel gene, Shaker. A family of three closely related potassium channel genes (MK1, MK2, and MK3) that are encoded at distinct genomic loci has been isolated. Sequence analysis reveals that the coding region of each of these three genes exists as a single uninterrupted exon in the mouse genome. This organization precludes the generation of multiple forms of the protein by alternative RNA splicing, a mechanism known to characterize the Drosophila potassium channel genes Shaker and Shab. Thus, mammals may use a different strategy for generating diverse K+ channels by encoding related genes at multiple distinct genomic loci, each of which produces only a single protein.

Amino Acid Sequence↗
PubMed1989

A T lymphoid cell line responds to a thymic stromal cell line by expression of Thy-1 and CD4.

We have cloned both T lymphoid and stromal lines from a single murine thymic tumor that was induced by a retrovirus carrying the v-myc oncogene (M-MuLV(myc]. The T lymphoid line, L4, was cloned by growth in agar. L4 cells were initially negative for Thy-1.2 and CD4 (although they contained rearranged TCR-beta genes), and they remained so if passaged in medium alone. However, cocultivation of these Thy-1.2- CD4- cells with the cloned stromal cell line, St3, resulted in sequential expression of Thy-1.2 and CD4 in subpopulations of cells. Thy-1.2+ CD4- and Thy-1.2+ CD4+ L4 subclones were obtained from the cocultures by subsequent cloning in agar. Derivation of these subclones from the starting Thy-1.2- CD4- clone was verified by Southern blot analyses specific for TCR-beta gene rearrangements and for M-MuLV(myc) proviral integration sites. Continuous cocultivation of Thy-1.2+ CD4+ L4 subclones with the St3 stromal cells was necessary for maintenance of CD4 on the cell surface. Furthermore, CD4 expression which was lost when CD4+ L4 cells were removed from the stroma could be reinduced if they were again cultured on St3 stroma. These cells may provide a model system for studying thymocyte-stromal cell interactions in induction and maintenance of expression of Thy-1 and CD4 molecules.

Animals↗
PubMed1989

The Initial Blood Storage Experiment--the spaceflight hardware program.

The Initial Blood Storage Experiment (IBSE) was conceived to investigate the effects of microgravity on the formed elements of human blood. The experiment flew on the January 1986, 61-C mission of the Space Shuttle Columbia. The experiment hardware was designed to provide a closely controlled temperature and air flow environment for all blood samples. During the mission, two IBSE modules were on board the orbiter and an identical set of hardware and blood samples were maintained on Earth as a control. This paper describes the development and performance of the IBSE hardware which was converted from a conceptual design to an on-orbit, man-rated, mid-deck locker experiment in 17 months.

Blood Platelets↗
PubMed1988

Characterization of lymphoid tumors induced by a recombinant murine retrovirus carrying the avian v-myc oncogene. Identification of novel (B-lymphoid) tumors in the thymus.

Lymphoid tumors induced by a recombinant murine retrovirus carrying the v-myc oncogene of avian MC29 virus were characterized. The Moloney murine leukemia virus myc oncogene (M-MuLV (myc], carried by an amphotropic MuLV helper, induced tumors in NIH Swiss and NFS/N mice after a relatively long latency (8 to 24 wk). Tumor masses appeared in the thymus, spleen, and lymph nodes. Flow cytometry of the tumor cells indicated that approximately 50% were positive for Thy 1.2. Most of these tumors also expressed one or more other cell surface markers of thymocytes and mature T cells (CD4, CD8). Southern blot hybridization revealed genomic rearrangements for the TCR beta genes. The TCR beta analysis suggested that the M-MuLV(myc)-induced Thy 1.2+ tumors were derived from somewhat less mature cells than tumors induced by M-MuLV, which is a classical non-acute retrovirus lacking an oncogene. The remainder of the M-MuLV(myc)-induced tumors were Thy 1.2-, but they were positive for Ly-5 (B220) and also for MAC-2. The Thy 1.2- tumors were characteristically located in the thymus. However, they were negative for TCR beta gene rearrangements. Some, but not all, of the Thy 1.2- tumors contained rearrangements for Ig genes. Additionally, they typically expressed mRNA specific for B but not for T cells. Thus, these thymic tumors had characteristics of the B cell lineage. Tumor transplantation experiments demonstrated that the Thy 1.2- tumor cells could reestablish in the thymus and spleen of irradiated hosts, and low level expression of the Thy 1 molecule was observed in the thymus but not the spleen on the first passage. After serial passage, one Thy 1- tumor altered its cell surface phenotype to Thy 1low B220-.

Animals↗