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C G Cochrane

Publications and source records attributed to C G Cochrane.

At least 37 records · Page 2Linked to original sources
PubMed1993

Signal transducing properties of the N-formyl peptide receptor expressed in undifferentiated HL60 cells.

Differentiated HL60 cells respond to challenge with ligand by mobilizing intracellular second messengers, resulting in superoxide production, degranulation, and actin polymerization with subsequent chemotaxis and phagocytosis. The functional capabilities of undifferentiated HL60 cells have not been similarly characterized due to the absence of the cell surface receptors required to initiate these processes. To investigate these properties, undifferentiated HL60 cells were transfected with one of the better characterized neutrophil chemotactic receptors, the N-formyl peptide receptor (FPR). Expression of the recombinant FPR gene product in FPR-transfected HL60 cells and the absence of the endogenous FPR in vector-transfected HL60 cells was demonstrated by Northern blot and flow cytometric analyses. FPR-transfected HL60 cells retained their ability to undergo granulocytic differentiation with dibutyryl cAMP, as determined by FMLP- and PMA-stimulated superoxide production. Furthermore, incubation of FPR-transfected HL60 cells for 5 days in the presence of FMLP resulted in limited differentiation as evidenced by the expression of functional C5a receptors. Binding studies of FPR-transfected HL60 cells demonstrated the presence of two binding affinities with dissociation constants of 0.6 and 33 nM, similar to dibutyryl cAMP differentiated HL60 cells and human neutrophils but contrasting the single high affinity state of the FPR expressed in mouse L cell fibroblasts. FPR-transfected HL60 cells displayed FMLP-dependent calcium mobilization with an EC50 of 3 nM and actin polymerization with an EC50 of approximately 10 nM. Actin polymerization was not observed in FPR-transfected L cell fibroblasts or undifferentiated vector-transfected HL60 cells. Both calcium mobilization and actin polymerization were sensitive to treatment with pertussis toxin, indicating the requirement for a Gi-like protein. Stimulation of either undifferentiated or differentiated HL60 cells with ATP resulted in pertussis toxin-insensitive calcium mobilization but was ineffective in producing actin polymerization. The results described herein show for the first time that undifferentiated HL60 cells can respond to chemoattractant receptor stimulation with many of the properties of the mature neutrophil. Transfected HL60 cells will provide an excellent system to study the characteristics of chemotactic receptors as well as the functional properties of myeloid cells.

Actins↗
PubMed1993

The role of the third intracellular loop of the neutrophil N-formyl peptide receptor in G protein coupling.

The G-protein-coupled N-formyl peptide receptor (FPR) contains one of the smallest known third intracellular loops of this class of receptors, consisting of only 15 amino acids. To study the role of this region of the receptor in G protein coupling and signal transduction, we generated a deletion mutant (D3i) in which 10 amino acids of the loop were removed, as well as a series of site-directed mutants containing substitutions of the charged and polar amino acids of this loop. The D3i mutant, expressed at normal levels on the cell surface, displayed a KD for labelled N-formyl-Met-Leu-Phe ([3H]FMLP) of 165 nM. This value compares with a KD for the wild-type FPR of 1.0 nM, or 20 nM in the presence of guanosine 5'-[gamma-thio]triphosphate, which uncouples G proteins from the receptor. These results indicate that D3i contains significant structural defects, beyond the disruption of G protein coupling, that affect ligand binding properties. Ten site-directed mutants generated in the third intracellular loop (T226A, K227E, H229A, K230Q, K235Q, S236A, S236A/S237G, R238G, R241E and S244A) displayed KD values between 0.5 and 1.0 nM, with expression levels between 22% (K227E) and 111% (H229A) of that of wild type receptor. The capacity of the mutants for signal transductions was determined by measuring intracellular Ca2+ mobilization. Eight of the ten mutants displayed EC50 values for FMLP of between 0.07 and 0.9 nM, as compared with 0.12 nM for the wild-type receptor. The two mutants K227E and R238G had EC50 values of 2.7 and 2.9 nM respectively. The increase in EC50 could be accounted for partially by the low levels of receptor expression. All ten mutants gave maximum levels of Ca2+ mobilization similar to that produced by the wild-type FPR. These results contradict the conclusions reached with other G-protein-coupled receptors and indicate that the third intracellular loop of the FPR does not have a critical role in the functional coupling of G proteins.

Amino Acid Sequence↗
PubMed1993

Multiple domains of the N-formyl peptide receptor are required for high-affinity ligand binding. Construction and analysis of chimeric N-formyl peptide receptors.

Binding of the chemotactic tripeptide fMet-Leu-Phe (fMLP) to its receptor on phagocytes activates these cells through a G protein-coupled pathway. To delineate the structural requirement of the N-formyl peptide receptor (FPR) for ligand binding and signaling, we constructed chimeric receptors between FPR and a recently identified granulocyte receptor, FPR2 (Ye, R. D., Cavanagh, S. L., Quehenberger, O., Prossnitz, E. R., and Cochrane, C. G. (1992) Biochem. Biophys. Res. Commun. 184, 582-589). FPR2 shares 69% sequence homology with the FPR; yet it binds fMLP with a low affinity (Kd = 430 nM), as compared with the high affinity (Kd = 1 nM) displayed by the FPR. This property of the FPR2 was utilized for mapping the FPR ligand binding domains. Seven chimeric FPR/FPR2 receptors were generated by sequential replacement of the FPR segments with the corresponding regions from FPR2. Three reciprocal FPR2/FPR chimeric receptors were also constructed by selective substitution of the FPR segments into FPR2. These chimeric receptors were stably expressed in transfected fibroblasts and analyzed for their ligand binding and transmembrane signaling properties. Replacement of the FPR domains, including the first and the third extracellular loops, resulted in 275- and 85-fold decrease in ligand binding affinity, respectively. Introduction of both domains into the FPR2 significantly increased ligand binding affinity (Kd = 18 nM), whereas substitution of the domains containing the first or third extracellular loop alone improved ligand binding to a lesser degree (Kd = 90 and 372 nM, respectively). In contrast, substitution of either the amino or the carboxyl-terminal regions with those of the FPR2 had little effect on ligand binding affinity. An analysis of the sequences of the two receptors revealed several key residues in the first and the third extracellular loops of the FPR and their adjacent transmembrane domains that may be essential for binding of fMLP. We propose that multiple domains of the FPR are required for high-affinity ligand binding, with a major determinant located in the first extracellular loop and its adjacent transmembrane domains.

Amino Acid Sequence↗
PubMed1993

The rabbit neutrophil N-formyl peptide receptor. cDNA cloning, expression, and structure/function implications.

The rabbit neutrophil N-formyl peptide receptor (FPR) has been well studied for its ligand binding properties. Recent gene cloning experiments have established the existence of a subfamily of G protein-coupled receptors that share extensive sequence homology with the FPR, yet lack the capability of high affinity binding to FMLP. These findings prompted us to identify the structural requirement for formyl peptide ligand binding by delineation of the primary structure of the rabbit FPR. A rabbit neutrophil cDNA library was screened with a cloned human FPR cDNA probe and the insert of one positive isolate (B6) was sequenced. The 1268-bp cDNA insert encodes a peptide of 352 amino acids. Stably transfected L cell fibroblasts expressing the rabbit cDNA displayed specific binding of the ligand fMet-Leu-[3H]Phe with two affinities (Kd = 0.31 and 7.5 nM). Addition of the nonhydrolyzable guanosine triphosphate analogue, GTP gamma S, converted > or = 85% of the high affinity sites to the low affinity sites. FMLP induced mobilization of intracellular calcium in the transfected cells (EC50 = 0.5 nM), a response sensitive to pertussis toxin. FMLP stimulation desensitized the receptor such that subsequent stimulation with the same ligand produced a significantly reduced signal. These results indicate that the cloned rabbit receptor represents a high affinity FPR, and that FPR-mediated early signal transduction events can be fully reconstituted in transfected mammalian cells. The rabbit FPR sequence is 78% identical to that of the human FPR, and 68% identical to FPR2, a homologue of FPR with a low binding affinity (Kd > or = 400 nM) for FMLP. Analysis of the aligned sequences of these three proteins revealed that: 1) the amino termini and the second extracellular loops have the lowest sequence homology; 2) sequence in the intracellular domains that couple to G protein are highly conserved; and 3) the first and the third extracellular loops and their adjacent transmembrane domains of the FPR may contain residues essential for the high affinity binding of FMLP.

Amino Acid Sequence↗
PubMed1993

Reconstitution of recombinant N-formyl chemotactic peptide receptor with G protein.

A recombinant human neutrophil N-formyl peptide receptor (rFPR) expressed in transfected mouse fibroblasts (TX2 cells) was analyzed for its ability to couple physically with the heterotrimeric G protein, Gi. Immunoprecipitation of photoaffinity-labeled rFPR and endogenous neutrophil formyl peptide receptor (nFPR) with an anti-FPR peptide antibody demonstrated that the receptors were identical in both size and extent of glycosylation. Coupling of rFPR with endogenous TX2 Gi was demonstrated by coimmunoprecipitation of the two proteins with an anti-Gi antibody. Moreover, rFPR was able to form a physical complex with purified Gi in a soluble reconstitution system. We observed similar affinities of rFPR and nFPR for Gi. This report provides the first direct evidence that rFPR associates physically with Gi and provides a foundation for analysis of the G protein coupling capacity of mutant rFPRs.

Animals↗
PubMed1992

Absence of G(i) proteins in the Sf9 insect cell. Characterization of the uncoupled recombinant N-formyl peptide receptor.

We investigated the interaction of the N-formyl peptide receptor (NFPR) with G proteins in infected Sf9 insect cells expressing the recombinant NFPR. Recombinant receptor expression of up to 27 pmol/mg protein was achieved in these cells. The receptor was recognized by an antiserum raised against an NFPR carboxyl-terminal peptide, and displayed specific and saturable binding of the formyl peptide ligand fMet-Leu-[3H]Phe. Scatchard analysis of the binding data yielded a dissociation constant of approximately 62 nM, a binding affinity of 60- to 120-fold lower than that of the high affinity sites in neutrophils and in transfected mammalian cell lines expressing the NFPR. That this low binding affinity was due to a lack of receptor coupling to G protein was suggested by the failure of guanine nucleotides to regulate receptor affinity and by the lack of formyl peptide-stimulated GTPase activity in these cells. Furthermore, immunoblotting with an anti-G(i) antibody and ADP-ribosylation experiments indicated that the approximately 40-kDa G(i) alpha subunit, which couples to the NFPR in neutrophils, is not present in Sf9 cell membranes. Thus, the current study provides for the first time evidence that a major G protein is absent in the Sf9 insect cells. Potential applications of the Sf9 system for in vitro reconstitution of the NFPR-G protein interaction are discussed.

Adenosine Diphosphate Ribose↗
PubMed1992

Isolation of a cDNA that encodes a novel granulocyte N-formyl peptide receptor.

A cDNA of 1650 base pairs was isolated by screening an HL-60 granulocyte library with an N-formyl peptide receptor (NFPR) cDNA probe under low stringency conditions. The cDNA encodes a protein of 351 amino acids tentatively named FPR2, with a calculated molecular weight of 39 kDa. Sequence analysis revealed that FPR2 is 69% identical in sequence to the human NFPR and shares extensive homology to several other chemoattractant receptors. FPR2 expressed in transfected cells mediated formyl peptide-stimulated calcium mobilization at micromolar concentrations of ligand. FPR2 messenger is detected in granulocytic HL-60 cells, but not in undifferentiated HL-60 cells. These findings suggest that FPR2 is a novel receptor for formyl peptide ligand and a new member of the chemoattractant receptor gene family.

Amino Acid Sequence↗
PubMed1992

Elevation of tissue kallikrein and kinin in the airways of asthmatic subjects after endobronchial allergen challenge.

Bronchial tissue kallikrein is the major kininogenase activity in the airways of asthmatic subjects. The relationship of IgE-mediated events to its release and/or activation is unknown, however, and is the subject of this report. Seven subjects with mild atopic asthma underwent endobronchial challenge with relevant aeroallergen. Baseline pre-allergen lavage and sequential post-challenge lavages were collected over an approximate 10-minute time course. Individual aliquots were analyzed separately and compared with saline control lavages performed in a separate lobe. In five of the seven subjects, an increase in tissue kallikrein activity, measured by cleavage of the synthetic substrate Val-Leu-Arg-pNA, was identified in the post-challenge lavages. The antigenic identity of the enzymatic activity was confirmed as a tissue kallikrein in each case by immunoblotting. Tissue kallikrein activity was highly correlated with the appearance of immunoreactive histamine and kinin (p = 0.0001). High molecular weight kininogen influx and cleavage was detected in the post-challenge samples by immunoblotting and paralleled the detection of kinin in BAL fluid. Two of the subjects, despite clinical profiles similar to those of the five positive responders, failed to react to endobronchial challenge. Saline control lavages contained detectable kallikrein, kinin, and histamine in two subjects; in each case, however, this was significantly less than in the post-allergen samples. The results demonstrate a close association between immediate type hypersensitivity events in the lower airway and the appearance of active kallikrein, kininogen substrate, and the liberation of kinin.

Adult↗
PubMed1991

Ascorbic acid enhances the effects of 6-hydroxydopamine and H2O2 on iron-dependent DNA strand breaks and related processes in the neuroblastoma cell line SK-N-SH.

Neuroblastoma cells accumulate ascorbic acid and iron. It was hypothesized that these features could be exploited for sensitizing neuroblastoma cells for therapy in combination with reactive oxygen intermediates. In the present study the effects of 6-hydroxydopamine (6-OHDA) and H2O2 on metabolic parameters critical for cell survival were investigated in cells with low and high ferritin content in the presence and absence of ascorbate. Human neuroblastoma SK-N-SH cells were pretreated with 100 microM FeSO4 and 10 microM desferrioxamine, respectively, for 24 h yielding cells with different ferritin contents. The effects of 6-OHDA and H2O2 (25 microM-250 microM) in the absence and presence of 1 mM ascorbic acid on DNA strand break formation, activation of poly(ADP-ribose) polymerase, and finally decrease in NAD+ and ATP concentration were investigated. All these parameters were influenced by 6-OHDA and H2O2 in a concentration-dependent manner in a similar way. The effects were most pronounced in ferritin-rich cells and in the presence of ascorbic acid. Using isolated CCC PM2 DNA, 6-OHDA and ascorbic acid caused strand breaks that were prevented in the presence of mannitol or desferrithiocine. H2O2-mediated strand breaks were observed only in the presence of ascorbic acid. Based on these data and data published by others a model explaining the deleterious effects of ascorbic acid on neuroblastoma cells is presented. It is suggested that continuous application of a high dosage of ascorbic acid might be a useful approach in neuroblastoma therapy.

Adenosine Triphosphate↗
PubMed1991

Pulmonary surfactant protein B (SP-B): structure-function relationships.

SP-B is a protein in pulmonary surfactant that is, in greatest part, responsible for resistance to surface tension and prevention of collapse of pulmonary alveoli. Peptides of 21 residues, synthesized following the sequence of SP-B or resembling the hydrophobic and hydrophilic domains of SP-B (such as RLLLLRLLLLRLLLLRLLLLR, R, Arg, and L, Leu), enhanced the abilities of phospholipids to reduce surface tension both in vitro and in vivo. Intermittent positively charged residues were essential for this activity. The SP-B-like peptides were found by tryptophan fluorescence to partition within the phospholipid layer in contact with both polar head groups and acyl side chains. These data, together with findings that the SP-B-related peptides increase inter- and intramolecular order of the phospholipid layer, suggest that SP-B resists surface tension by increasing lateral stability of the phospholipid layer.

Amino Acid Sequence↗
PubMed1991

Characterization of a human cDNA that encodes a functional receptor for platelet activating factor.

We have cloned a cDNA for the platelet activating factor (PAF) receptor by screening an HL-60 granulocyte cDNA library with degenerate oligonucleotide probes based on conserved sequences of rhodopsin-type receptors. The 342-amino acid receptor contains 7 putative transmembrane domains, but lacks sites for N-linked glycosylation at the N-terminus. Stably transfected fibroblasts expressing the cloned cDNA responded to sub-nanomolar PAF stimulation with calcium mobilization, which could be inhibited by the PAF antagonist L-659,989. The PAF receptor message was detected as a single species of approximately 4 kb in human placenta, lung, and differentiated HL-60 granulocytes. Expression of the cloned cDNA in undifferentiated HL-60 cells devoid of endogenous PAF receptor resulted in specific and saturable binding of the PAF antagonist WEB 2086 with a dissociation constant of 30.7 nM.

Amino Acid Sequence↗
PubMed1991

Cellular injury by oxidants.

Oxidants, generated by stimulated leukocytes, induce a variety of distinct biochemical changes in target cells. Hypochlorous acid (HOCl), produced by the action of peroxidase on hydrogen peroxide (H2O2) in the presence of chloride ions, acts at low molar concentrations (10-20 microM) to damage proteins on cell membranes and destroy their function. H2O2 rapidly permeates cells and causes inhibition of adenosine triphosphate (ATP) synthesis via both glycolytic and oxidative phosphorylation (mitochondrial) pathways. In the glycolytic pathway, damage is limited to the step involving glyceraldehyde-3-PO4 dehydrogenase (GAPDH). This results from both an attack of H2O2 on GAPDH and, indirectly, by a reduction in concentration of the GAPDH cofactor, nicotinamide adenine dinucleotide (NAD). This latter effect was found to result from activation of the enzyme, poly(adenosine diphosphate) (ADP)-ribose polymerase, an enzyme involved in deoxyribonucleic acid (DNA) repair. DNA damage in target cells was found at low concentrations of H2O2 (20-80 microM) in many cell types. Strand breaks and base hydroxylation were observed, resulting in the generation of hydroxyl radicals (.OH) from H2O2, in the presence of a transition metal. DNA damage resulted in either cell injury and death or mutations of the base sequence and amino acid residues. These latter effects led to malignant transformations in cultured cells in both tissue cultures of the cells, and in vivo in athymic mice. Exposure of a proto-oncogene, K-ras 4B, also led to the development of a malignant transformation by virtue of mutations in codon positions 12 and 61. Thus, oxidant effects on target cells can damage multiple functional pathways inside the cells, as well as give rise to malignant transformation via DNA damage.

Adenosine Triphosphate↗
PubMed1991

Transmembrane signalling by the N-formyl peptide receptor in stably transfected fibroblasts.

We investigated the requirement for N-formyl peptide receptor-mediated transmembrane signalling in transfected mouse fibroblasts that express the receptor. Stably transfected cells displayed specific binding for N-formyl-Met-Leu-[3H]Phe with a dissociation constant of 3 nM. The cells responded to ligand stimulation with mobilization of calcium from intracellular stores. Calcium mobilization was ligand dose-dependent (EC50 = 3 nM fMet-Leu-Phe) and could be inhibited by pertussis toxin treatment. These results provide the first demonstration that expression of the single-chain N-formyl peptide receptor in mouse fibroblasts is sufficient for mediating ligand-induced early transmembrane signalling events, which do not appear to require other neutrophil-specific cellular components.

Animals↗
PubMed1991

Raman spectroscopic studies of model human pulmonary surfactant systems: phospholipid interactions with peptide paradigms for the surfactant protein SP-B.

The temperature dependence of dipalmitoylphosphatidylcholine (DPPC)/phosphatidylglycerol (PG) multilayers, reconstituted with various synthetic peptides for modeling human lung surfactant, was monitored by vibrational Raman spectroscopy. The synthetic peptides consisted, respectively, of residues 59-81 of the human surfactant protein SP-B and 21 amino acid residue peptides containing repeating units of arginine separated by either four or eight leucines (RL4 or RL8). Each peptide demonstrated the ability to reduce significantly the surface tension of analogues of the phospholipid mixture used in the Raman studies. Raman spectroscopic integrated band intensities and relative peak height intensity ratios, two spectral parameters used to determine bilayer disorder, provided sensitive probes for characterizing multilayer perturbations in the reconstituted liposomes. Temperature profiles derived from the various Raman intensity parameters for the 3100-2800-cm-1 carbon-hydrogen (C-H) stretching mode region, a spectral interval representative of acyl chain vibrations, reflected lipid reorganizations due to the bilayer interactions of these peptides. For the three reconstituted multilamellar surfactant systems, the gel-to-liquid-crystalline phase-transition temperatures Tm, defined by acyl chain C-H stretching mode order/disorder parameters, increased from 35 degrees C in the peptide free system to 37-38 degrees C, indicating increased lipid headgroup constraints for the model liposomes. Although the values of Tm were similar for the three recombinant lipid/peptide assemblies, individual phase-transition cooperativities varied significantly between systems and between spectroscopically derived order/disorder parameters.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

The use of synthetic peptides in the formation of biophysically and biologically active pulmonary surfactants.

Synthetic pulmonary surfactants consisting of mixtures of phospholipids with synthetic peptides based on the amino acid sequence of human surfactant apoprotein SP-B were prepared. These surfactants were analyzed for their ability to lower surface tension on a pulsating bubble surfactometer and for their capacity to improve lung compliance and increase alveolar expansion in a fetal rabbit model of surfactant deficiency. The data demonstrate that several peptides, ranging from 17 to 45 residues in length, matching the carboxy-terminal sequence of the SP-B protein, when appropriately recombined with the phospholipid dipalmitoylphosphatidylcholine and phosphatidylglycerol (3:1), are capable of producing a synthetic surfactant with biophysical and biologic activity approaching that of human surfactant derived from amniotic fluid.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1990

Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation.

Oncogenic forms of ras proteins are synthesized in the cytosol and must become membrane associated to cause malignant transformation. Palmitic acid and an isoprenoid (farnesol) intermediate in cholesterol biosynthesis are attached to separate cysteine residues near the C termini of H-ras, N-ras, and Kirsten-ras (K-ras) exon 4A-encoded proteins. These lipid modifications have been suggested to promote or stabilize the association of ras proteins with membranes. Because preventing isoprenylation also prevents palmitoylation, examining the importance of isoprenylation alone has not been possible. However, the oncogenic human [Val12]K-ras 4B protein is not palmitoylated but is isoprenylated, membrane associated, and fully transforming. We therefore constructed mutant [Val12]K-ras 4B proteins that were not isoprenylated to examine the effects of isoprenylation in the absence of palmitoylation. The nonisoprenylated mutant proteins both failed to associate with membranes and did not transform NIH 3T3 cells. In addition, inhibition of isoprenoid and cholesterol synthesis with the drug compactin also decreased [Val12]K-ras 4B protein isoprenylation and membrane association. These results unequivocally demonstrate that isoprenylation, rather than palmitoylation, is essential for ras membrane binding and ras transforming activity. These findings clearly indicate the biological significance of ras protein modification by farnesol and suggest that this modification may be important for facilitating the processing, trafficking, and biological activity of other isoprenylated proteins. Because K-ras is the most frequently activated oncogene in a wide spectrum of human malignancies, study of this pathway could lead to important therapeutic treatments.

Animals↗
PubMed1990

Preparation of physical therapists to work with handicapped infants and their families: current status and training needs.

The purposes of this study were to determine the current status of physical therapists' preparation to work with handicapped and at-risk infants and their families and to identify needs for infant- and family-focused training materials and curricula. Results of a telephone survey of 73 physical therapy programs and a follow-up mail survey of 14 physical therapy programs with infancy specialization options are presented. Students in entry-level programs and postprofessional master's degree programs with infancy specializations commonly received instruction in infancy-related topics. Many students received minimal or no exposure to family-related content. Family assessment and intervention were identified as the areas of highest priority for development of training materials and curricula. The results of this study provide direction for the design of infant- and family-focused training materials and curricula in physical therapy.

Curriculum↗