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T N Wells

Publications and source records attributed to T N Wells.

At least 109 records · Page 6Linked to original sources
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

Inhibition of phosphomannose isomerase by mercury ions.

Mercury ions can inhibit Candida albicans phosphomannose isomerase (PMI) by two different processes at sub-micromolar concentrations. Kinetic studies show that mercury ions are in rapid equilibrium with the enzyme and cause a clear partial noncompetitive inhibition when mannose 6-phosphate is used as the substrate. The inhibition constants at 37 degrees C in 50 mM Hepes buffer, pH 8.0, are 35 and 57 nM for Kii and Kis, respectively. In addition to this inhibition at rapid equilibrium, mercury ions also inactivate C. albicans PMI by a much slower process, involving an irreversible mechanism. This is shown to be a two-step process, proceeding via an intermediate complex with a dissociation constant of 5.6 microM, with a maximum rate of inactivation of 0.15 min-1. The rate of irreversible inactivation can be slowed by the addition of the substrate, mannose 6-phosphate. Incubation of the enzyme with [203Hg]Cl2 causes the formation of a stable adduct with one atom of mercury incorporated into each enzyme molecule during the inactivation. Since cysteine-150 is the only iodoacetate-modifiable cysteine in the protein, we propose that this is where the mercury ion reacts during the irreversible inactivation process. In the Escherichia coli enzyme this cysteine is replaced by an asparagine, and the enzyme cannot be irreversibly inactivated by mercury ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Isolation of a lipid-soluble histamine release factor from human platelets.

We have isolated a histamine release factor from human platelet supernatants by heparin column chromatography, gel filtration and reversed phase HPLC. Fractions from each chromatographic step were assayed for histamine release factor activity (HRF). A peak of HRF activity was detected at a molecular mass of 60 kDa. Subsequent HPLC purification showed that the factor co-eluted with human serum albumin, but was totally extractable into the lipid phase. Comparison of biological activity with known basophil and eosinophil-activating hydroxyeicosanoids demonstrated similar activity with 5(S)-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5(S)-HPETE). The identification of this lipid soluble HRF demonstrates the novel potential role of an as yet unidentified lipid as an HRF, in the absence of priming stimuli.

Amino Acid Sequence↗
PubMed1994

Arginine 304 is an active site residue in phosphomannose isomerase from Candida albicans.

The reaction catalyzed by Candida albicans phosphomannose isomerase (PMI) (EC 5.3.1.8) has a bell-shaped pH dependence, with pKa's at 5.6 and 8.7. The enzyme can be inhibited in a time-dependent manner using the arginine-specific modification reagent phenylglyoxal. This modification takes place with a rate constant of 0.022 +/- 0.002 min-1 mM-1 at 37 degrees C in 50 mM Hepes buffer, pH 8.5. The enzyme can be protected from this inactivation by the addition of the substrate mannose 6-phosphate at concentrations close to its Km value. The pH dependence of the inactivation reaction shows a single pKa at 9.1 +/- 0.1, which is close to one of the values for the pH dependence of the enzyme-catalyzed reaction. Using [7-14C]phenylglyoxal, it is shown that a single molecule is incorporated into the enzyme in the absence of substrate and that this inactivates the enzyme. This incorporation of radioactivity is prevented by the coincubation with substrate. The modified protein has then been reduced with sodium borohydride to fix the modification and then cleaved with Asp-N protease. The resultant peptides were separated by HPLC, and the radioactivity was counted. Sequencing of the peptide with the highest incorporation level identified it as DNVVRAGFTPKFK, which corresponds to amino acids 300-312 of phosphomannose isomerase. Radioactive counting of the phenylthiohydantoin amino acid derivatives confirmed that the modified amino acid was arginine 304. The role of this residue in the catalytic reaction of phosphomannose isomerase is discussed.

Amino Acid Sequence↗
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

Crystallization and preliminary X-ray analysis of Candida albicans phosphomannose isomerase.

Crystals of recombinant phosphomannose isomerase from Candida albicans have been obtained in a form suitable for X-ray diffraction analysis. The enzyme plays a key role in the biosynthesis of the mannan component of the fungal cell wall. It crystallizes in monoclinic space group C2, with cell dimensions a = 124.9 A, b = 52.9 A, c = 85.9 A and beta = 127.4 degrees. The crystals diffract to Bragg spacings beyond 1.7 A, native data have been collected to 2.4 A and a search for heavy-metal derivatives is in progress. The asymmetric unit contains one molecule of the enzyme (M(r) approximately 49,000) with a Vm of 2.3 A3/Da.

Candida albicans↗
PubMed1994

Preliminary X-ray diffraction studies of recombinant 19 kDa human fibroblast collagenase.

Crystals of the catalytic domain of human fibroblast collagenase have been grown in the presence and absence of an inhibitor. Crystals of the inhibitor complex grew from 0.2 M ammonium sulfate and 15 to 30% PEG 8000 at 22 degrees C as bipyramids in the space group P6(2) or P6(4). Crystals of the unligated enzyme grew as rods in the space group P4(1)2(1)2 or P4(3)2(1)2 from 1.0 to 2.0 M sodium formate at 4 degrees C. Both crystal forms grew quite slowly over a period of months, but ultimately yielded crystals that diffracted beyond 2.5 A. The collagenase samples used in these studies were heterogeneous at the amino terminus. Three major species (full length, N-1 and N-2) were identified by mass spectrometry and Edman sequencing. Analysis of dissolved crystals revealed the native crystal form selectively crystallized as the N-2 species; however, no selectivity of N-terminal forms was observed for crystals of the inhibitor complex.

Collagenases↗
PubMed1994

Purification, cDNA cloning and heterologous expression of human phosphomannose isomerase.

Phosphomannose isomerase catalyses the interconversion of fructose-6-P and mannose-6-P and has a critical role in the supply of D-mannose derivatives required for many eukaryotic glycosylation reactions. Three classes of enzymes possessing phosphomannose-isomerase activity have been identified in bacteria and lower eukaryotes. We have purified human phosphomannose isomerase to homogeneity from placental tissue. Protein sequence information obtained from internal fragments of the protein was used to design degenerate oligonucleotides which were used to amplify a fragment of a human phosphomannose-isomerase cDNA. A full-length cDNA was isolated from a human testes lambda gt11 library using this fragment as a probe. The cDNA encoded a protein with significant sequence identity to fungal and some bacterial phosphomannose isomerases but was unrelated to those from other bacteria. Based on amino acid sequence identity we propose a classification system for enzymes with phosphomannose-isomerase activity. The cDNA, under the control of the GAL1 promoter, was expressed in a Saccharomyces cerevisiae strain from which the native gene encoding phosphomannose isomerase had been deleted. The human enzyme was found to be able to functionally substitute for the yeast enzyme. Phosphomannose-isomerase mRNA was found in all human tissues tested but was more highly expressed in heart, brain and skeletal muscle. The cDNA was expressed in Escherichia coli permitting the isolation of pure recombinant protein which will be used for kinetic and structural studies.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of fungal and mammalian phosphomannose isomerases.

Phosphomannose isomerase (PMI) is essential for the production of yeast cell walls. An inhibitor which inhibits the fungal enzyme without altering the activity of the mammalian enzyme would be a potential fungicidal agent, increasingly important in view of the increasing mortality from visceral mycoses in immunosuppressed patients. We have purified human, porcine, and Candida albicans enzymes 29,000-fold to homogeneity, and characterized their physical properties, as well as their kinetic parameters, inhibition constants, and pH dependences. Surprisingly, in view of the large differences between Pseudomonas aerugenosa and Saccharomyces cerevisiae PMI, the human and C. albicans enzymes are almost identical. We suggest therefore that species-selective inhibition of the fungal rather than mammalian enzyme may require molecules which bind away from the substrate binding pocket of the enzyme.

Amino Acid Sequence↗
PubMed1994

Influence of surface and protein modification on immunoglobulin G adsorption observed by scanning force microscopy.

Scanning force microscopy has been used successfully to produce images of individual protein molecules. However, one of the problems with this approach has been the high mobility of the proteins caused by the interaction between the sample and the scanning tip. To stabilize the proteins we have modified the adsorption properties of immunoglobulin G on graphite and mica surfaces. We have used two approaches: first, we applied glow discharge treatment to the surface to increase the hydrophilicity, favoring adhesion of hydrophilic protein molecules; second, we used the arginine modifying reagent phenylglyoxal to increase the protein hydrophobicity and thus enhance its adherence to hydrophobic surfaces. We used scanning force microscopy to show that the glow discharge treatment favors a more homogeneous distribution and stronger adherence of the protein molecules to the graphite surface. Chemical modification of the immunoglobulin caused increased aggregation of the proteins on the surface but did not improve the adherence to graphite. On mica, clusters of modified immunoglobulins were also observed and their adsorption was reduced. These results underline the importance of the surface hydrophobicity and charge in controlling the distribution of proteins on the surface.

Adsorption↗
PubMed1994

Interleukin-8 and RANTES induce the adhesion of the human basophilic cell line KU-812 to human endothelial cell monolayers.

Basophils are implicated in the pathogenesis of the late-phase allergic reaction, but the mechanisms by which circulating basophils adhere to vascular endothelium and migrate to lesional sites remain unclear. In order to assess the biological similarity of the basophilic cell line KU-812 to normal human basophils, we have compared the adhesion response of this cell line and normal basophils, following challenge with interleukin-8 (IL-8) and RANTES. We demonstrate here that IL-8 and RANTES are able to stimulate the adherence of the basophilic cell line, KU-812, to cytokine-activated human umbilical vein endothelium (HUVEC). The chemokine-induced increase in adhesion was dose-related and was maximal after prior priming with IL-5. The stimulation of adhesion was partially inhibited by co-incubation with anti-CD18 and anti-CD11c antibodies and antibodies to the beta 1-integrins. In comparison, the chemokine-induced adhesion of normal human basophils was only inhibited by the beta 2-integrins. These chemokines were also able to induce the migration of KU-812 in a dose-dependent manner, but only after prior treatment with phorbol myristate acetate (PMA) or IL-5. In all cases tested, IL-8 was more potent and efficacious than RANTES. We conclude from these studies that these members of the chemokine superfamily may play an important role in the recruitment of reactive leukocytes in allergic inflammation, by stimulating their adhesion and subsequent migration from the vasculature into the inflammatory sites. However, it is apparent that KU-812 is not an adequate substitute for normal human basophils in order to investigate chemokine biology.

Base Sequence↗
PubMed1993

Identification of Cys-150 in the active site of phosphomannose isomerase from Candida albicans.

Candida albicans phosphomannose isomerase (PMI) (EC 5.3.1.8) has been recently cloned and overexpressed in Escherichia coli. The enzyme can be irreversibly inactivated by iodoacetate in 50 mM borate buffer, pH 9.0, in a time-dependent manner at a rate of 4.2 +/- 0.03 min-1 M-1. This inhibition can be prevented by the substrate mannose 6-phosphate with a Ks of 0.22 +/- 0.05 mM, slightly lower than its Km value. However, metals such as zinc and cadmium, which are reversible, competitive inhibitors for PMI, do not protect the enzyme against modification. The protein has been labeled by using [2-14C]iodoacetate, in the presence or absence of substrate, and the protein is fully inactivated when 1.0 thiol group is modified per molecule of enzyme. Tryptic maps of the modified protein have been produced. The protected peptide has been identified and sequenced, and the phenylthiohydantoin amino acids have been collected. The modified amino acid is Cys-150. This cysteine residue is conserved in mammalian and yeast phosphomannose isomerases, but not in bacterial species where it is replaced with asparagine. We therefore purified PMI from E. coli and showed that this enzyme is not sensitive to inactivation by iodoacetate. The iodoacetate is presumably inhibiting PMI by sterically blocking the mannose 6-phosphate binding site. Multiple sequence alignment procedures were used to try to identify potential ligands of the zinc atom that is essential for enzyme activity and thus to delineate the active site region.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗