PubMed Health⌕ Search

Biomedical subjects

C A Power

Publications and source records attributed to C A Power.

At least 55 records · Page 3Linked to original sources
PubMed1996

A molecular switch of chemokine receptor selectivity. Chemical modification of the interleukin-8 Leu25 --> Cys mutant.

Interleukin-8 (IL-8), a member of the CXC chemokine family, is a key activator of neutrophils. We have previously shown that two novel CC chemokine-like properties, namely monocyte chemoattraction and binding to CC CKR-1, are introduced into IL-8 by mutating Leu25 to the conserved tyrosine present in CC chemokines. To further investigate the role of this position in receptor selectivity, we have mutated Leu25 to cysteine. The protein folds correctly with two disulfide bonds and a free thiol group at Cys25. This mutant behaves overall like wild-type IL-8, with little change in neutrophil chemotaxis and IL-8 receptor binding, and has no effect on CC CKR-1. These data are consistent with cysteine being approximately isosteric with the natural amino acid leucine. However, modification of the cysteine by addition of a fluorescent N-methyl-N-(2-N-methyl, N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)aminoethyl)acetamido (NBD) group lowers potency in neutrophil chemotaxis and affinity in IL-8 receptor binding assays by 2 orders of magnitude. This Leu25 --> Cys-NBD mutant introduces monocyte chemoattractant activity and the ability to displace 125I-labeled macrophage inflammatory protein-1 alpha from the recombinant CC CKR-1 receptor. Additionally, we show a specific interaction between the fluorescent mutant and the N-terminal 34-amino acid peptide from CC CKR-1. This confirms the importance of this region in IL-8 in receptor binding and in conferring specificity between CXC and CC chemokines. Circular dichroism spectra of the IL-8 mutants having CC chemokine-like activity show a consistent drop in alpha-helical content compared with the spectra for wild-type IL-8. This suggests that distortion of the C-terminal helix may play a role in chemokine receptor-ligand selectivity.

4-Chloro-7-nitrobenzofurazan↗
PubMed1996

Extension of recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist.

Extension of recombinant human RANTES by a single residue at the amino terminus is sufficient to produce a potent and selective antagonist. RANTES is a proinflammatory cytokine that promotes cell accumulation and activation in chronic inflammatory diseases. When mature RANTES was expressed heterologously in Escherichia coli, the amino-terminal initiating methionine was not removed by the endogenous amino peptidases. This methionylated protein was fully folded but completely inactive in RANTES bioassays of calcium mobilization and chemotaxis of the promonocytic cell line THP-1. However, when assayed as an antagonist of both RANTES and macrophage inflammatory polypeptide-1 alpha (MIP-1 alpha) in these assays, the methionylated RANTES (Met-RANTES) inhibited the actions of both chemokines. T cell chemotaxis was similarly inhibited. The antagonistic effect was selective since Met-RANTES had no effect on interleukin-8- or monocyte chemotractant protein-1-induced responses in these cells. Met-RANTES can compete with both [125I]RANTES and [125I]IMP-1 alpha binding to THP-1 cells or to stably transfected HEK cells recombinantly expressing their common receptor, CC-CKR-1. These data show that the integrity of the amino terminus of RANTES is crucial to receptor binding and cellular activation.

Chemokine CCL5↗
PubMed1996

Molecular cloning of murine CC CKR-4 and high affinity binding of chemokines to murine and human CC CKR-4.

We have cloned the murine homologue of human CC Chemokine Receptor-4 (CC CKR-4). In equilibrium competition binding assays performed in undifferentiated HL-60 cells transfected with human and murine CC CKR-4 cDNA, the IC50 values for the binding of [125I]macrophage inflammatory protein-1 alpha to human and murine CC CKR-4 were 14.5 +/- 9.0 nM and 10.1 +/- 3.0 nM, respectively, and the IC50 values for the binding of [125I]RANTES to human and murine CC CKR-4 were 9.3 +/- 3.0 nM and 5.7 +/- 2.6 nM, respectively. The cDNA clone for murine CC CKR-4 is 1531 bp, and the largest open reading frame encodes a protein of 360 amino acids that is 85% identical to human CC CKR-4. Murine CC CKR-4 was detected in the thymus and T-cell lines by Northern blot analysis. This first report of direct binding of chemokines to CC CKR-4 demonstrates that the highly homologous human and murine receptors have similar binding characteristics and tissue distribution.

Amino Acid Sequence↗
PubMed1996

Selectivity and antagonism of chemokine receptors.

The chemokine superfamily can be subdivided into two groups based on their amino terminal cysteine spacing. The CXC chemokines are primarily involved in neutrophil-mediated inflammation and, so far, two human receptors have been cloned. The CC chemokines tend to be involved in chronic inflammation, and recently we have cloned a fourth leukocyte receptor for this group of ligands. Understanding what makes one receptor bind its range of agonists is important if we are to develop potent selective antagonist. We have started to investigate the molecular basis of this receptor selectivity by looking at why CC chemokines do not bind to the CXC receptors in several ways. First, we looked at the role of the three-dimensional structure of the ligand, and have solved the three dimensional structure of RANTES using nuclear magnetic resonance spectroscopy. The structure is similar to that already determined for the CC chemokine macrophage inflammatory protein-1 beta, and it has a completely different dimer interface to that of the CXC chemokine interleukin-8 (IL-8). However, the monomer structures of all the chemokines are very similar, and at physiological concentrations the proteins are likely to be monomeric. Second, by examining all the known CC and CXC chemokines, we have found a region that differs between the two subfamilies. Mutations of one of the residues in this region, Leu-25 in IL-8, to tyrosine (which is conserved at this position in CC chemokines) enables the mutant IL-8 to bind CC chemokine receptor-1 (CC-CKR-1) and introduces monocyte chemoattractant activity. Using other mutations in this region, we can show a direct interaction with the N-terminus of CC-CKR-1. Third, we have found that modification of the amino terminus of RANTES by addition of one amino acid makes it into an antagonist with nanomolar potency. Taken together, this data suggests a two-site model for receptor activation and for selectivity between CC and CXC chemokines, with an initial receptor contact provided by the main body of the chemokine, and activation provided by the amino terminal region.

Amino Acid Sequence↗
PubMed1996

Chemokine receptors - the new frontier for AIDS research.

CD4 is widely known as the HIV receptor, but is insufficient to allow viral infection. Recently, members of the family of chemokine receptors have been identified as the missing co-receptors, which act with CD4 to allow the virus to enter cells. These discoveries open up the possibilities of novel therapeutic strategies to combat HIV infection and AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Characterisation of the RANTES/MIP-1 alpha receptor (CC CKR-1) stably transfected in HEK 293 cells and the recombinant ligands.

The CC chemokines RANTES and MIP-1 alpha are known to activate certain leucocytes and leucocytic cell lines. We have produced and fully characterised the recombinant proteins expressed in E. coli. They induce chemotaxis of the pro-monocytic cell line, THP-1 and T cells. THP-1 cells express three of the known CC chemokine receptors. In order to study the activation of a single receptor, we have expressed the shared receptor (CC CKR-1) for RANTES and MIP-1 alpha stably in the HEK 293 cell line. We have examined the effects of RANTES and MIP-1 alpha on the CC CKR-1 transfectants by equilibrium binding studies and in a chemotaxis assay. RANTES competes for [125I]RANTES with an IC50 of 0.6 +/- 0.23 nM, whereas MIP-1 alpha competes for its radiolabelled counterpart with an IC50 of 10 +/- 1.6 nM in the transfectants. These affinities are the same as those measured on the THP-1 cell line. The stably transfected HEK 293 cells respond to both these chemokines in the chemotaxis assay with the same EC50 values as those measured for THP-1 cells. This indicates that this cellular response can be mediated through the CC CKR-1 receptor.

Base Sequence↗
PubMed1995

Molecular cloning and functional expression of a novel CC chemokine receptor cDNA from a human basophilic cell line.

We report the cloning and characterization of a novel basophil CC chemokin receptor, K5-5, from the human immature basophilic cell line KU-812. The predicted protein sequence of K5-5 shows only 49% identity to the macrophage inflammatory protein-1 alpha/RANTES receptor (CC CKR-1) and 47% identity to monocyte chemotactic protein-1 receptor (b form), suggesting that this cDNA encodes a novel member of the CC chemokine receptor family. Analysis of K5-5 mRNA expression indicates that it is restricted to leukocyte-rich tissues. In addition, we have shown significant levels of K5-5 mRNA in human basophils, which were up-regulated by treatment with interleukin-5. The CC chemokines, Macrophage inflammatory protein-1 alpha, RANTES, and monocyte chemotactic protein-1 were able to stimulate a Ca(2+)-activated chloride channel in Xenopus laevis oocytes injected with K5-5 cRNA, whereas no signal was detected in response to monocyte chemotactic protein-2, macrophage inflammatory protein-1 beta, or the CXC chemokine, interleukin-8. Taken together, these results indicate for the first time the presence of a CC chemokine receptor on basophils, which functions as a "shared" CC chemokine receptor and may therefore be implicated in the pathogenesis of basophil-mediated allergic diseases.

Amino Acid Sequence↗
PubMed1995

IL-8-induced signal transduction in T lymphocytes involves receptor-mediated activation of phospholipases C and D.

We have characterized the IL-8-induced signal transduction processes in T lymphocytes. A basal level of IL-8 receptor expression was shown on mixed PBL, as identified by using phycoerythrin (PE)-coupled IL-8, and this expression was increased following IL-2 stimulation. Scatchard analysis of T cells revealed competitive binding of IL-8 with a Kd of 0.55 nM, with approximately 1200 receptors per cell, on freshly isolated T cells. After 24 h in culture following purification, reverse transcriptase PCR (RT-PCR) analyses show the mRNA for only the type B IL-8R on these cultured T lymphocytes and the cell line MOLT-4. Stimulation of T lymphocytes or T cell clones with IL-8 led to generation of inositol trisphosphate and calcium flux. In addition, when T cells were prelabeled with [3H]oleic acid, IL-8 caused a long lasting, time- and dose-related increase in [3H]phosphatidylethanol (PtE), indicating activation of phospholipase D (PLD). By contrast, this IL-8-dependent PLD activity was undetectable in IL-8-stimulated neutrophils. PLD activation appeared to be downstream of protein kinase C, because several inhibitors abrogated the increase in [3H]PtE, whereas guanosine-5'-O-(3-thiotriphosphate (GTP(gamma)S) and inositol trisphosphorothioate (IP3S3) both increased the generation of [3H]PtE. Together, these results demonstrate that the IL-8RB receptor is sufficient to mediate phospholipase C (PLC) and PLD activation in T lymphocytes, but not in neutrophils, and indicate an important difference in receptor usage and signal transduction pathways between IL-8-stimulated lymphocytes and neutrophils.

CD4-Positive T-Lymphocytes↗
PubMed1995

Mutation of Leu25 and Val27 introduces CC chemokine activity into interleukin-8.

Interleukin-8 (IL-8) is a member of the CXC branch of the chemokine superfamily and activates neutrophils but not monocytes. The related CC chemokine branch, which includes monocyte chemoattractant protein-1 (MCP-1) and RANTES are potent chemoattractants for monocytes but not neutrophils. Examination of the sequences of the CXC chemokines reveals that the highly conserved leucine, corresponding to Leu25 in IL-8, is always replaced by tyrosine in CC chemokines. There is also a high degree of conservation among the CXC chemokines of the adjacent Val27 residue, which points out from the same side of the beta-sheet as Leu25. In RANTES, Val27 is also replaced by a tyrosine. In order to investigate the role of these residues in controlling cell specificity, we have made the single mutants Leu25-->Tyr, Val27-->Tyr and the double mutant Leu25-->Tyr, Val27--> Tyr of IL-8. These proteins have been expressed in Escherichia coli and purified to homogeneity from inclusion body material. All three mutants have lower potency and efficacy in chemotaxis and calcium mobilization assays using neutrophils. The mutants also show lowered affinity to both IL-8 receptors A and B expressed recombinantly in HL-60 cells and to neutrophils in [125I]IL-8 competition assays. Additionally, the Leu25-->Tyr mutation introduces a novel monocyte chemoattractant activity into IL-8. We therefore studied the displacement of [125I]MIP-1 alpha by IL-8 Leu25-->Tyr from the CC-CKR-1 receptor. The mutant displaces MIP-1 alpha ligand with an affinity only 12-fold less than MIP-1 alpha itself. This suggests that mutations in this region of IL-8 are involved in receptor binding and activation and in the control of specificity between CC and CXC chemokines.

Amino Acid Sequence↗
PubMed1995

A fluorescent interleukin-8 receptor probe produced by targetted labelling at the amino terminus.

Interleukin-8 is the most extensively characterised member of the structurally related chemotactic and pro-inflammatory proteins collectively called chemokines. It binds to two closely related members of the seven transmembrane chemokine receptor family found on a variety of leukocyte cell types. In order to study the interaction of interleukin-8 with its receptors, and their distribution, we have produced a fluorescently labelled protein as an alternative to the radioactive 125I-interleukin-8 ligand. Interleukin-8 is naturally produced as two forms, a 72-residue polypeptide by monocytes and a 77-residue form produced by endothelial cells which has an extension of five amino acids at the amino terminal. Both forms are active at nanomolar concentrations, implying that chemical modification to the amino terminus of the 72-residue form will not destroy activity. The 72-residue interleukin-8 sequence starts with a serine residue, which can be oxidised under mild conditions to give a reactive glyoxylyl function which is then reacted with a nucleophilic fluorescein derivative. The site-specifically labelled protein was easily isolated by reverse-phase HPLC. The dissociation constant of the fluorescently labelled interleukin-8 from its receptors on neutrophils was measured by displacement of 125I-interleukin-8 and found to be 10 nM compared to 1 nM for the unmodified protein. The modified protein is highly active in in vitro bioassays using human neutrophils, giving an EC50 of 7 nM in chemotaxis and an EC50 of 0.62 nM for shape change. The binding of the fluorescent protein to neutrophils can also be measured by fluorescent automatic cell sorter (FACS) analysis, and can be competed by unlabelled interleukin-8. The amino-terminal modification of interleukin-8 has produced a reagent which is useful for the quantification of interleukin-8 receptor expression, and will also be useful in monitoring the fate of the ligand after receptor binding.

Amino Acid Sequence↗
PubMed1995

Chemokine and chemokine receptor mRNA expression in human platelets.

In order to study the role of platelets in inflammation we constructed a cDNA library from human platelet mRNA. By polymerase chain reaction (PCR) analysis of the library we have shown that platelets express mRNAs for the following chemokines: connective tissue activating peptide-III (CTAP-III), epithelial-derived neutrophil activating factor-78 (ENA-78), RANTES and monocyte chemotactic protein-3 (MCP-3). Platelets also express mRNAs for interleukin 8 receptor A (IL-8RA) and a novel chemokine receptor K5.5. These results suggest that chemokines may not only play an important role in platelet activation but can also influence the nature of the leukocyte infiltrate to sites of inflammation and infection, by the production of multiple chemokines with overlapping specificities.

Base Sequence↗
PubMed1994

Cloning of a full-length cDNA encoding the neutrophil-activating peptide ENA-78 from human platelets.

Here we describe the cloning of a full-length cDNA encoding a neutrophil chemoattractant peptide, ENA-78, from human platelets. The cDNA encodes a predicted sequence of 114 amino acids and contains the Cys motif C-X-C found in other members of the alpha-chemokine family which also includes interleukin 8 (IL-8). ENA-78 has a high degree of sequence identity with other platelet-derived chemokines which also share overlapping chemotactic activities such as GRO alpha and the neurophil-activating peptide 2 (NAP-2; derived by proteolytic cleavage of the connective-tissue-activating peptide III (CTAP-III)).

Amino Acid Sequence↗
PubMed1994

Eotaxin: cloning of an eosinophil chemoattractant cytokine and increased mRNA expression in allergen-challenged guinea-pig lungs.

Eotaxin was recently identified as the major eosinophil chemoattractant in bronchoalveolar lavage fluid obtained 3h after allergen challenge of sensitised guinea-pigs. We now report the cDNA cloning of this C-C chemokine. The 777 base-pair clone, pEo3122, consists of a 40 base 5' untranslated region, an open reading frame of 288 bases predicting a 73 amino acid mature protein plus a 23 amino acid signal peptide, and a 3' untranslated region of 449 bases containing a poly A tail. Northern blot analysis showed eotaxin mRNA in the lungs of naive and sensitised guinea-pigs, which was considerably increased after allergen challenge. Eotaxin may be an important mediator of eosinophil accumulation and activation in allergic reactions. As eotaxin stimulates human eosinophils, this chemokine and related molecules may be involved in human diseases such as asthma where eosinophil accumulation is a prominent feature.

Allergens↗
PubMed1994

Crystallization and preliminary X-ray diffraction studies of human RANTES.

The chemotactic cytokine RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted) is a potent chemoattractant and activator of a number of leukocytes, with a molecular mass of 8 kDa. Crystals of this protein have been grown from 100 mM sodium acetate buffer (pH 4.6) containing 200 mM magnesium acetate, with 20% (w/v) PEG 4000 and 6% (v/v) glycerol. The crystals grow as thick rods, which diffract to at least 1.8 A resolution on a rotating anode X-ray source. The crystals belong to space group p2(1)2(1)2(1) with unit cell dimensions a = 95.14 A, b = 57.58 A and c = 24.01 A with alpha = beta = gamma = 90 degrees. The asymmetric unit contains two molecules of the RANTES monomer, with a VM of 2.0 A(3)/Da.

Chemokine CCL5↗
PubMed1994

Molecular cloning and characterisation of a neutrophil chemotactic protein from porcine platelets.

In our search for novel chemoattractant factors, we have purified a heparin-binding protein from porcine platelets which is a potent chemoattractant for human neutrophils. The protein has 80 amino acids and a molecular mass of 8597.5Da as measured by electrospray mass spectrometry. It has been characterised by amino acid sequencing and shown to have highest identity to members of the human platelet basic-protein-family. Its N-terminal sequence is intermediate in length between the human connective-tissue-activating polypeptide III (CTAP-III) and neutrophil-activating polypeptide-2 (NAP-2). The porcine NAP-2/CTAP-III shows the classic CXC cysteine spacing found towards the N-terminus in the chemokine alpha family and contains the ELR motif which has been shown to be essential for neutrophil chemotaxis. We have isolated mRNA from porcine platelets and constructed a cDNA library containing 1.0 x 10(6) independent clones. Using probes based on the protein sequence we have isolated a full length-clone for this gene, with an open reading frame containing 119 amino acids. Despite overall similarity between the human and porcine proteins, the N-terminal region is almost completely different between the two species, with only two identical amino acids. The proteolytic cleavage sites required for processing of human platelet basic protein are completely missing in the porcine homologue, implying a different processing pathway or mechanism. The porcine protein is capable of agonizing certain effects of both NAP-2 and CTAP-III when incubated with human cells indicating that the same porcine protein may be involved in both processes.

Amino Acid Sequence↗
PubMed1994

Identification of the horse epidermal growth factor (EGF) coding sequence and its use in monitoring EGF gene expression in the endometrium of the pregnant mare.

The PCR technique and highly degenerate oligonucleotide primers were used to amplify a 282 bp fragment of the horse (Equus caballus) epidermal growth factor (EGF) cDNA. The clone corresponded to 94 amino acids of the EGF precursor molecule. The deduced amino acid sequence of the 53 residue EGF mitogenic peptide within the precursor sequence showed 60-70% identity with five other published EGF sequences. The PCR cDNA fragment hybridized to a 4.9 kb transcript in horse kidney and endometrial RNA which was of a similar size to the mature EGF transcript found in other mammalian species. The horse cDNA clone was used in Northern blots to monitor EGF expression in the endometrium of pregnant mares up to day 83 of gestation (term = 330-340 days). The level of expression increased from day 33 and showed a further dramatic increase between days 35 and 45, which coincides with the onset of implantation and placentation in this species. Levels remained elevated up to day 83. The horse DNA sequence was used to design sense and antisense oligonucleotide probes (45-mers) for in situ hybridization studies. The antisense probe showed specific hybridization to the glandular, but not lumenal, epithelial cells of the endometrium and there was no signal in fetal membranes. The in situ hybridization signal increased between days 35 and 45 to a similar degree to that observed in the Northern blot analysis. This dramatic increase in EGF expression in the glandular epithelium of the mare's endometrium during pregnancy may provide a mitogenic stimulus to the endometrium and/or trophoblast to facilitate placental differentiation and attachment. Alternatively, the precursor could be involved in the endometrial gland secretory process which is necessary to produce uterine milk for fetal sustenance. The PCR cloning methods used in this study should be generally applicable to the cloning of EGF cDNAs from other species.

Amino Acid Sequence↗