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Biomedical subjects

R M Gibson

Publications and source records attributed to R M Gibson.

At least 19 recordsLinked to original sources
PubMed2001

RhoGDI-binding-defective mutant of Cdc42Hs targets to membranes and activates filopodia formation but does not cycle with the cytosol of mammalian cells.

We have identified a mutant of the human G-protein Cdc42Hs, R66E, that fails to form a detectable complex with the GDP-dissociation inhibitor RhoGDI in cell-free systems or in intact cells. This point mutant is prenylated, binds guanine nucleotide and interacts with GTPase-activating protein in a manner indistinguishable from wild-type Cdc42Hs. Immunofluorescence localization studies revealed that this RhoGDI-binding-defective mutant is found predominantly in the Golgi apparatus, with a staining pattern similar to that of the wild-type protein. However, unlike wild-type Cdc42Hs, which is distributed in both the microsomal membrane and cytosolic fractions, studies using differential centrifugation show that prenylated R66E Cdc42Hs is found exclusively in association with lipid bilayers. Additionally, whereas the overexpression of RhoGDI results in an apparent translocation of wild-type Cdc42Hs from the Golgi apparatus into the cytosol, identical RhoGDI-overexpression conditions do not alter the Golgi localization of the R66E mutant. Furthermore, overexpression of this RhoGDI-binding-defective mutant of Cdc42Hs seems to activate redistribution of the actin cytoskeleton and filopodia formation in fibroblasts in a manner indistinguishable from the wild-type protein. Taken together, these results suggest that the interaction of Cdc42Hs with RhoGDI is not essential for proper membrane targeting of nascent prenylated Cdc42Hs in mammalian cells; neither is this interaction an essential part of the mechanism by which Cdc42Hs activates filopodia formation. However, it does seem that redistribution of Cdc42Hs to the cytosolic compartment is absolutely dependent on RhoGDI interaction.

Animals↗
PubMed2001

Disruption of focal adhesions mediates detachment during neuronal apoptosis.

Apoptosis participates in the development of the nervous system and in neurodegeneration. The aim of this study was to investigate the mechanisms of detachment of neuronal cells from the extracellular matrix (ECM) during apoptosis. Detachment of Ntera2 neuronal cells was accompanied by decreased surface expression of the beta1 integrin and redistribution of proteins from focal adhesions (FA). FA proteins were cleaved in a discrete sequence: p130cas, then paxillin, then talin. Caspase inhibition prevented detachment and cleavage of paxillin and p130cas, whilst calpain inhibition blocked talin cleavage. Neuronal cells therefore detach as a result of redistribution and caspase-dependent cleavage of focal adhesion proteins. Cleavage occurs sequentially such that critical ECM-integrin survival signalling cascades are severed before disruption of focal adhesion-cytoskeletal links.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Microsatellite analysis of female mating behaviour in lek-breeding sage grouse.

We used microsatellite DNA markers to genotype chicks in 10 broods of lek-breeding sage grouse, Centrocercus urophasianus, whose mothers' behaviour was studied by radio-tracking and observing leks. Previous behavioural studies suggested that almost all matings are performed by territorial males on leks and that multiple mating is rare. Two broods (20%) were sired by more than one male. Genetic analyses of the broods of eight females that visited an intensively studied lek were consistent with behavioural observations. Four females observed mating produced singly sired broods and males other than the individual observed copulating were excluded as sires for most or all of their chicks. Territorial males at the study lek were excluded as sires of broods of four other females that visited the lek but were not observed mating there. Radio-tracking suggested that two of these females mated at other leks. Our results confirm the reliability of mating observations at leks, but do not rule out a possible unseen component of the mating system.

Animals↗
PubMed2000

Phosphorylation of human gp130 at Ser-782 adjacent to the Di-leucine internalization motif. Effects on expression and signaling.

The receptor for leukemia inhibitory factor (LIF) consists of two polypeptides, the LIF receptor and gp130. Agonist stimulation has been shown previously to cause phosphorylation of gp130 on serine, threonine, and tyrosine residues. We found that gp130 fusion proteins were phosphorylated exclusively on Ser-782 by LIF- and growth factor-stimulated 3T3-L1 cell extracts. Ser-780 was required for phosphorylation of Ser-782 but was not itself phosphorylated. Ser-782 is located immediately N-terminal to the di-leucine motif of gp130, which regulates internalization of the receptor. Transient expression of chimeric granulocyte colony-stimulating factor receptor (G-CSFR)-gp130(S782A) receptors resulted in increased cell surface expression in COS-7 cells and increased ability to induce vasoactive intestinal peptide gene expression in IMR-32 neuroblastoma cells when compared with expression of chimeric receptors containing wild-type gp130 cytoplasmic domains. These results identify Ser-782 as the major phosphorylated serine residue in human gp130 and indicate that this site regulates cell surface expression of the receptor polypeptide.

3T3 Cells↗
PubMed1999

Caspase activation is downstream of commitment to apoptosis of Ntera-2 neuronal cells.

Death by apoptosis is widespread among different cell types, including neurones. Apoptosis consists of a phase during which cells commit to die, followed by an execution phase, characterized by conserved morphological changes. To prevent neuronal loss during disease, it is important to identify the events which define irreversible commitment to death. The present study has investigated the events accompanying the commitment and execution phases of the neuronal cell line Ntera-2. In response to serum starvation, Ntera-2 cells enter the execution phase and detach into the culture supernatant with an apoptotic morphology. This phase is associated with activation of caspases. The remaining adherent cells have a normal morphology and can adhere to extracellular matrix substrates. However, after 96 h of serum deprivation, 95% of these adherent cells fail to form colonies in a single cell cloning assay. When refed with serum, 70% of these cells become apoptotic within 24 h, suggesting that they had previously committed to die. A further 20% of the cells escape from commitment to apoptosis by beginning to differentiate. Inhibition of caspases fails to delay commitment. In response to serum deprivation, therefore, neuronal cells either differentiate or commit to cell death, and events upstream of caspase activation regulate this irreversible commitment. These results have significant therapeutic implications since they suggest that caspase inhibitors may not promote long-term survival of every neuronal cell type in every situation.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Recent population trends in nonmetropolitan cities and villages: from the turnaround, through reversal, to the rebound.

"The purpose of this study is to track and contrast the patterns of local concentration and deconcentration in nonmetropolitan America between 1950 and 1996. We consider the growth of places by initial size as well as the growth of population living in the countryside or in unincorporated hamlets.... To determine how widespread and consistent the trends are, we compare patterns of growth by nearness to metropolitan areas, and by region of the country. We also examine differences among a subset of nonmetropolitan places distinguished by the primary socioeconomic character of their county. Using a detailed data file from the 1990 census, we are able to give some consideration to commuting."

Americas↗
PubMed1997

Dissecting the cooperative reassociation of the regulatory and catalytic subunits of cAMP-dependent protein kinase. Role of Trp-196 in the catalytic subunit.

The catalytic (C) subunit of cAMP-dependent protein kinase requires two distinct surfaces to form a stable complex with its physiological inhibitors, the regulatory (R) subunits and the heat-stable protein kinase inhibitors. In addition to a substrate-like segment that is common to both inhibitors, R requires a peripheral recognition site, PRS2. This surface is comprised of the essential phosphorylation site, Thr-197, His-87, Trp-196, and several surrounding basic residues. To probe the role of Trp-196 in the recognition of R, Trp-196 was replaced with Arg and Ala. Although both rC(W196A) and rC(W196R) were inhibited readily with cAMP-free R, they failed to form an inhibited holoenzyme complex with native R under conditions in which wild-type holoenzyme formed readily. Pairing rC(W196R) with mutant forms of R lacking domain B or having defects in cAMP binding sites A or B highlighted the importance of the conformation of R, and, in particular, the accessibility of site A. One of these mutants, rR(R333K), having a defect in cAMP binding site B formed a stable complex with rC(W196R) in the absence of cAMP. However, unlike wild-type holoenzyme, this complex was active.

Catalysis↗
PubMed1997

Interaction of the regulatory and catalytic subunits of cAMP-dependent protein kinase. Electrostatic sites on the type Ialpha regulatory subunit.

Since a basic surface on the catalytic (C) subunit of cAMP-dependent protein kinase is important for binding to the regulatory (R) subunit, acidic residues in R were sought that might contribute to R-C interaction. Using differential labeling by a water-soluble carbodiimide (Buechler, T. A., and Taylor, S. S. (1990) Biochemistry 29, 1937-1943), seven specific carboxylates in RIalpha were identified that were protected from chemical modification in the holoenzyme; each was then replaced with Ala. Of these, rRI(E15A/E106A/D107A)), rRI(E105A), rRI(D140A), rRI(E143A), and rRI(D258A) all were defective in holoenzyme formation and define negative electrostatic surfaces on RIalpha. An additional conserved carboxylate, Glu101 in RIalpha and the equivalent, Glu99 in RIIalpha were mutated to Ala. Replacement of Glu101 had no effect while rRII(E99A) was very defective. RIalpha and RIIalpha thus differ in the molecular details of how they recognize C. Unlike wild-type RI, two additional mutants, rRI(D170A) and rRI(K242A), inhibited C-subunit stoichiometrically in the presence of cAMP and show increases in both on- and off-rates. Asp170, which contributes directly to the hydrogen bonding network in cAMP-binding site A, thus contributes also to holoenzyme stability.

Amino Acid Sequence↗
PubMed1997

Identification of electrostatic interaction sites between the regulatory and catalytic subunits of cyclic AMP-dependent protein kinase.

Two classes of molecules inhibit the catalytic subunit (C) of the cyclic AMP-dependent protein kinase (cAPK), the heat-stable protein kinase inhibitors (PKIs) and the regulatory (R) subunits. Basic sites on C, previously identified as important for R/C interaction in yeast TPK1 and corresponding to Lys213, Lys217, and Lys189 in murine C alpha, were replaced with either Ala or Thr and characterized for their kinetic properties and ability to interact with RI and PKI. rC(K213A) and rC(K217A) were both defective in forming holoenzyme with RI but were inhibited readily with PKI. This contrasts with rC(R133A), which is defective in binding PKI but not RI (Wen & Taylor, 1994). Thus, the C-subunit employs two distinct electrostatic surfaces to achieve high-affinity binding with these two types of inhibitory molecules even though all inhibitors share a common consensus site that occupies the active site cleft. Unlike TPK1, mutation of Lys189 had no effect. The mutant C subunits that were defective in binding RI, rC(K213A) and rC(K217A), were then paired with three RI mutants, rRI(D140A), rRI(E143A), and rRI(D258A), shown previously to be defective in recognition of C. Although the mutations at Asp140 and Asp258 in RI were additive with respect to the C mutations. rC(K213A) and rRI(E143A) were compensatory, thus identifying a specific electrostatic interaction site between RI and C. The results are discussed in terms of the RI and C crystal structures and the sequence homology between the yeast and mammalian enzymes.

Alanine↗
PubMed1995

Lectin and epidermal growth factor domains of P-selectin at physiologic density are the recognition unit for leukocyte binding.

P-selectin is an integral membrane glycoprotein on stimulated platelets and endothelial cells that serves as a receptor for leukocytes. To estimate the density of P-selectin in membranes necessary to support adhesion, we incorporated purified P-selectin at varying concentrations into phospholipid bilayers that encapsulated glass microspheres. Maximal binding of these lipospheres to HL60 cells, a P-selectin ligand-expressing cell line, was approached at a P-selectin density of about 100 molecules per microns 2; half-maximal binding was observed at about 50 to 60 molecules per microns 2. Compatible results were obtained with P-selectin expressed on Chinese hamster ovary cells. The P-selectin density on stimulated platelets was estimated to be 150 to 200 molecules/microns 2. To identify the domains of P-selectin required for HL60 cell binding, chimeras of P-selectin and L-selectin were stably expressed in Chinese hamster ovary cells and clones that expressed the chimeras at the estimated physiologic density were selected. Chimeras containing the P-selectin lectin and epidermal growth factor (EGF) domains or the lectin, EGF, and short consensus repeats bound HL60 cells equivalently, but a chimera containing the P-selectin lectin domain alone bound HL60 cells much less well. These results indicate that at a physiologically relevant P-selectin density on membrane surfaces, the lectin, and EGF domains of P-selectin are together required for optimal leukocyte binding.

Animals↗
PubMed1994

A role for the epidermal growth factor-like domain of P-selectin in ligand recognition and cell adhesion.

The selectin family of adhesion molecules mediates the initial interactions of leukocytes with endothelium. The extracellular region of each selectin contains an amino-terminal C-type lectin domain, followed by an EGF-like domain and multiple short consensus repeat units (SCR). Previous studies have indirectly suggested a role for each of the extracellular domains of the selectins in cell adhesion. In this study, a panel of chimeric selectins created by exchange of domains between L- and P-selectin was used to directly examine the role of the extracellular domains in cell adhesion. Exchange of only the lectin domains between L- and P-selectin conferred the adhesive and ligand recognition functions of the lectin domain of the parent molecule. However, chimeric selectins which contained both the lectin domain of L-selectin and the EGF-like domain of P-selectin exhibited dual ligand-binding specificity. These chimeric proteins supported adhesion both to myeloid cells and to high endothelial venules (HEV) of lymph nodes and mesenteric venules in vivo. Exchange of the SCR domains had no detectable effect on receptor function or specificity. Thus, the EGF-like domain of P-selectin may play a direct role in ligand recognition and leukocyte adhesion mediated by P-selectin, with the lectin plus EGF-like domains collectively forming a functional ligand recognition unit.

Animals↗
PubMed1992

A novel Bacillus subtilis expression vector based on bacteriophage phi 105.

We have developed a novel expression vector based on the bacteriophage phi 105, and employed it for the production of mutant beta-lactamases in Bacillus subtilis. Expression of the beta-lactamase-encoding gene was low when cloned into the prophage under the control of its own promoter. However, expression was considerably elevated when the gene was inserted into the phage genome in the same orientation as phage transcription. A defective phi 105 vector was constructed with a deletion removing a region needed for cell lysis, and with a mutation in the immunity repressor, rendering it temperature sensitive. Production of beta-lactamase could then be induced by a shift in temperature and without concomitant cell lysis, facilitating purification of the protein from the culture supernatant. This phage has considerable potential for development as a vector for controllable production of heterologous proteins in B. subtilis.

Bacillus subtilis↗
PubMed1992

P-selectin and E-selectin. Distinct but overlapping leukocyte ligand specificities.

P-selectin on platelets and endothelial cells and E-selectin on endothelial cells are leukocyte receptors that recognize lineage-specific carbohydrates on neutrophils and monocytes. The proposed ligands for these receptors contain the Le(x) core and sialic acid. Since other investigators have shown that both E-selectin and P-selectin bind to sialylated Le(x), we evaluated whether E-selectin and P-selectin recognize the same counter-receptor on leukocytes. The interaction of HL60 cells with Chinese hamster ovary (CHO) cells expressing P-selectin or E-selectin was studied. To determine whether a protein component is required in addition to sialyl Le(x) for either P-selectin or E-selectin recognition, HL60 cells or neutrophils were digested with proteases, including chymotrypsin, elastase, proteinase Glu-C, ficin, papain, or thermolysin. Cells treated with these proteases bound E-selectin but not P-selectin. Fucosidase or neuraminidase treatment of HL60 cells markedly decreased binding to both E-selectin- and P-selectin-expressing CHO cells. Growth of HL60 cells in tunicamycin inhibited the ability of these cells to support P-selectin-mediated binding and, to a lesser extent, E-selectin-mediated binding. Purified P-selectin inhibited CHO:P-selectin binding to HL60 cells, but incompletely inhibited CHO:E-selectin binding to HL60 cells. However, purified soluble E-selectin inhibited CHO:P-selectin and CHO:E-selectin binding to HL60 cells equivalently and completely. COS cells, unable to bind to E-selectin or P-selectin, bound E-selectin but not P-selectin upon transfection with alpha-1,3-fucosyltransferase or alpha-1,3/1,4-fucosyltransferase. Similarly, LEC 11 cells expressing sialyl Le(x) bound E-selectin- but not P-selectin-expressing CHO cells. Sambucus nigra lectin, specific for the sialyl-2,6 beta Gal/GalNAc linkage, inhibited P-selectin but not E-selectin binding to HL60 cells. Although sialic acid and Le(x) are components of the P-selectin ligand and the E-selectin ligand, these results indicate that the ligands are related, having overlapping specificities, but are structurally distinct. A protein component containing sialyl Le(x) in proximity to sialyl-2,6 beta Gal structures on the P-selectin ligand may contribute to its specificity for P-selectin.

Animals↗