Search PubMed⌕ Search

Biomedical subjects

J M Lord

Publications and source records attributed to J M Lord.

At least 109 records · Page 6Linked to original sources
PubMed1993

Expression and activity of pre-dianthin 30 and dianthin 30.

Dianthin 30 is a ribosome inactivating protein (RIP 1) found in different tissues of the carnation (Dianthus caryophyllus). Recently we have isolated and sequenced a cDNA clone from a lambda gt11 expression library [Legname et al. (1991) Biochim. Biophys. Acta 1090, 119-122]. Here we describe specific PCR amplifications of either the full length pre-dianthin 30 or dianthin 30, the mature polypeptide lacking the 23 amino acid signal peptide. In vitro expression of both proteins in reticulocyte lysate generated products of the expected molecular weight. Moreover, the activity of both proteins has been evaluated confirming the characteristics of the natural product. A first attempt to produce recombinant dianthin 30 in Escherichia coli is described.

Animals↗
PubMed1992

Cell surface and intracellular functions for ricin galactose binding.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.

Animals↗
PubMed1992

Biological activity of recombinant Ricinus communis agglutinin A chain produced in Escherichia coli.

DNA encoding Ricinus communis agglutinin A chain was ligated into the E. coli expression vector pDS 5/3. Induced E. coli 71.18 cells which had been transformed with this plasmid express Ricinus communis agglutinin A chain in a soluble and biologically active form. Recombinant Ricinus communis agglutinin A chain had ribosomal RNA N-glycosidase activity and was approximately 10-fold less active than ricin A chain in a cell-free protein synthesis inhibition assay.

Cell-Free System↗
PubMed1992

Recombinant ricin B chain fragments containing a single galactose binding site retain lectin activity.

Ricin B chain is an N-glycosylated galactose-specific lectin. Examination of the amino acid sequence of the protein has shown it to be the product of a series of gene duplication events based on an original galactose binding peptide. The X-ray crystallographic structure of the protein reveals that it consists of two globular domains, each composed of three smaller subdomains. In each globular domain only one of the three subdomains has retained its ability to bind galactose. Through DNA manipulation we have created a series of fusions of portions of ricin B chain, each carrying only one galactose binding site, to the ricin signal sequence. Transcripts synthesized in vitro using SP6 RNA polymerase were injected into Xenopus oocytes where the recombinant proteins were produced in a mature form. The products were shown to be N-glycosylated and produced in a soluble stable form. Also, they retained the ability to bind galactose. Preliminary experiments on the reassociation of these ricin B chain fragments with ricin A chain to create a modified holotoxin were also carried out.

Amino Acid Sequence↗
PubMed1992

Addition of an ER retention signal to the ricin A chain increases the cytotoxicity of the holotoxin.

With the exception of diphtheria toxin, which translocates from acidified endosomes, the intracellular organelle from which the catalytic moieties of several plant and bacterial toxins enter the target cell during endocytic uptake has not been identified. We have recently proposed that some toxins may travel the entire secretory pathway in reverse, moving from the cell surface to the lumen of the ER, before entering the cytosol. Several bacterial toxins have the ER retention sequence KDEL or a related analogue at their carboxyl termini, suggesting that the KDEL receptor may play a role in delivering these toxins to the ER. Here we provide further support for this possibility since the cytotoxicity of ricin, which lacks a KDEL sequence, can be significantly increased by adding KDEL to the C-terminus of its A chain.

Amino Acid Sequence↗
PubMed1992

Changes in the phosphorylation status of a 19 kD cytosolic protein are linked to the growth arrest of HL-60 cells.

A major 19 kD cytosolic protein (p19) has been described in a number of cell systems with respect to its rapid phosphorylation when protein kinase C is activated and has been proposed as a key substrate of this enzyme. Phosphorylation of p19 occurs when the growth of cells is affected by 12-O-tetradecanoylphorbol-13-acetate (TPA) and it has been proposed that increased phosphorylation of p19 relates to the cessation of cell growth. This study delineates precisely the relationship between p19 phosphorylation changes in the growth and differentiation status of cells. Changes in the levels of two phosphorylated forms of p19 were assessed in HL-60 promyelocytic cells and a variant HL-60 cell line which stopped growing and differentiated in response to TPA and were compared to changes seen in HL-60 variant lines which merely growth arrested when treated with TPA. In lines which either did or did not differentiate, in response to TPA, the p19 protein was rapidly and transiently phosphorylated. Thus, this alteration in the phosphorylation status of p19 is associated with the process of growth arrest and not related to the onset of cell differentiation. The p19 protein and the enzymes which effect its phosphorylation status modulate the growth of cells and possible disregulation of p19 and/or its kinases and phosphatases is of interest as regards the leukaemic transformation of cells.

Cell Differentiation↗
PubMed1992

Cytotoxic proteins.

Cytotoxic proteins, which enter eukaryotic cells and catalytically inactivate protein synthesis, are being increasingly studied using a combination of molecular biology, cell biology and structural approaches. The creation of genetically engineered fusions with alternative cell-binding ligands paves the way for tailor-made, cell-type-specific killing agents.

Bacterial Proteins↗
PubMed1992

Toxin entry: how reversible is the secretory pathway?

A number of proteins produced by plants and bacteria are extremely toxic to eukaryotic cells. Their potency arises from their ability to catalyse the modification of crucial cellular components. Only a few toxin molecules are required to kill a cell, but to do so they must first reach the cytosol. How such proteins are translocated across the target cell membrane is poorly understood, but we argue here that some toxins may travel the secretory pathway in reverse, passing all the way from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol.

Journal Article↗
PubMed1992

Molecular cloning of ricin.

A variety of strategies have been used to obtain cDNA and genomic clones encoding ricin. Since their isolation these sequences have been manipulated to allow expression of A chain (19) and A chain mutants (15,20,34), B chain (14,21-23) and proricin (24). Utilizing structural information (35), precise changes have been introduced into both A and B chains with the aim of probing catalytic and sugar-binding residues, respectively. In the longer term, such manipulations, coupled with successful expression and purification schemes, will allow the delineation of functional residues and domains, ensuring that ricin remains the prototype plant toxin with which to study cellular intoxication and ribosome inactivation and to utilize in pharmaceutical product development.

Amino Acid Sequence↗
PubMed1991

A method for detecting the expression of a toxic gene in cultured cells.

We have devised a rapid method for examining the expression of a toxin gene following in vitro transfection using a bacterial beta-galactosidase (lacZ) gene as a reporter gene. Ricin A chain DNA and the lacZ gene, both under the control of the immunoglobulin gene promoter and enhancer, were transfected into mouse fibroblast cells (L cells). Transient expression of the lacZ gene was detected 2 days after transfection by histochemical staining of the transfectants with 5-bromo-3-indolyl-beta-D-galactoside. Cotransfection of the ricin A chain gene resulted in a progressive reduction in the number of lacZ transfectants as the expressed toxin killed the cells. A ricin construct with the intervening sequence from the human beta-actin gene required 4 days instead of 2 days to produce the toxic effect. This is a useful method for examining the expression of toxin gene in a cell.

Animals↗
PubMed1991

Mutational analysis of the galactose binding ability of recombinant ricin B chain.

Ricin B chain (RTB) is an N-glycosylated galactose-specific lectin which folds into two globular domains. Each domain binds one galactoside. The x-ray crystallographic structure has shown that the two binding sites are structurally similar and contain key binding residues which hydrogen bond to the sugar, and a conserved tripeptide, Asp-Val-Arg. We have used oligonucleotide site-directed mutagenesis to change either the binding residues or the homologous tripeptide in one or other or in both of the sites. The 5' signal sequence and RTB coding region were excised from preproricin cDNA and fused in frame to generate preRTB cDNA. Transcripts synthesized in vitro from wild-type or mutant preRTB cloned into the Xenopus transcription vector pSP64T using SP6 RNA polymerase, were microinjected into Xenopus oocytes. The recombinant products were segregated into the oocyte rough endoplasmic reticulum and core-glycosylated, and the N-terminal signal peptide was removed. Mutating sugar binding sites individually did not abrogate the lectin activity of RTB. When both sites were changed simultaneously, RTB was produced which was soluble and stable but no longer able to bind galactose. Changing the Asn residues of the two RTB N-glycosylation sites to Gln showed that oligosaccharide side chains were essential for both the stability and biological activity of recombinant RTB.

Animals↗
PubMed1991

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3' end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A2660, which lies in a sequence that is highly conserved in all species.

Abrin↗
PubMed1991

Changes in the levels of inositol lipids and phosphates during the differentiation of HL60 promyelocytic cells towards neutrophils or monocytes.

HL60 cells were adapted to grow in a serum-free medium containing 1 mg l-1 inositol, in which they differentiated normally towards neutrophils (in 0.9% by volume dimethylsulphoxide) and towards monocytes (in 10 nM phorbol myristate acetate). Cells that had been equilibrium-labelled with [2-3H]myo-inositol contained a complex pattern of inositol metabolites, several of which were at relatively high concentrations. These included InsP5 and InsP6, which were present at concentrations of about 25 microM and 60 microM, respectively. Striking and different changes occurred in the levels of some of the inositol polyphosphates as the cells differentiated towards either neutrophils or monocytes. Most notable were a large but gradual accumulation of Ins(1,3,4,5,6)P5 as HL60 cells decreased in size and acquired neutrophil characteristics, and much more rapid and sequential declines in InsP4, InsP5 and InsP6 as the cells started to take on monocyte character. There was a marked accumulation of free inositol and of phosphatidylinositol in the cells during neutrophil differentiation, probably caused at least in part by an increased rate of inositol uptake providing an increased intracellular inositol supply. The same accumulation of Ins(1,3,4,5,6)P5 occurred during neutrophil differentiation, whether it was induced by dimethylsulphoxide or by a combination of retinoic acid and a T-lymphocyte cell line-derived differentiation factor. Ins(1,4,5)P3, a physiological intracellular mediator of Ca2+ release from membrane stores, did not change in concentration during these differentiation processes. These observations suggest that some of the more abundant cellular inositol polyphosphates play some important, but not yet understood, role either in the processes of haemopoietic differentiation or in the expression of differentiated cell character in myeloid cells.

Cell Differentiation↗
PubMed1991

Nucleotide sequence of cDNA coding for dianthin 30, a ribosome inactivating protein from Dianthus caryophyllus.

Rabbit antibodies raised against dianthin 30, a ribosome inactivating protein from carnation (Dianthus caryophyllus) leaves, were used to identify a full length dianthin precursor cDNA clone from a lambda gt11 expression library. N-terminal amino acid sequencing of purified dianthin 30 and dianthin 32 confirmed that the clone encoded dianthin 30. The cDNA was 1153 basepairs in length and encoded a precursor protein of 293 amino acid residues. The first 23 N-terminal amino acids of the precursor represented the signal sequence. The protein contained a carboxy-terminal region which, by analogy with barley lectin, may contain a vacuolar targeting signal.

Amino Acid Sequence↗
PubMed1991

The effects of N-glycosylation on the lectin activity of recombinant ricin B chain.

Soluble, biologically-active recombinant ricin B chain has been produced by expressing B chain-encoding DNA in heterologous eukaryotic or prokaryotic hosts. N-Glycosylated recombinant ricin B chain expressed in Xenopus oocytes bound to both immobilized asialofetuin and immobilized lactose. Non-glycosylated ricin B chain expressed in either E. coli or in tunicamycin-treated oocytes did not bind to immobilized lactose. However, it did bind to asialofetuin, and increasing concentrations of free lactose did not reduce this asialofetuin binding dramatically, in contrast to the effect of free lactose on the binding of either glycosylated recombinant B chain or native ricin B chain.

Amino Acid Sequence↗
PubMed1991

Preproabrin: genomic cloning, characterisation and the expression of the A-chain in Escherichia coli.

Synthetic oligonucleotides representing all possible sequences of an N-terminal and an internal region of the A-chain of abrin C were used to generate a probe specific for abrin-related sequences using the polymerase chain reaction on Abrus precatorius genomic DNA. A lambda phage library constructed from genomic DNA isolated from leaf tissue of A. precatorius was screened and positive hybridising clones were characterised by restriction enzyme analysis. The coding regions of unique clones were characterised by DNA sequencing. One clone encodes a preproprotein closely related to abrin C with 83% similarity between the A-chain sequences. Based on similarity with the ricin toxins and Ricinus communis agglutinin, the preproabrin consists of an A-chain of 251 amino acids preceded by 34 amino acids containing an N-terminal signal peptide, followed by a 14-amino-acid linker and a B-chain of 263 amino acids. The mature A-chain of the preproabrin has been expressed cytoplasmically in Escherichia coli and the soluble recombinant protein was produced at levels exceeding 6% of total cell protein. The recombinant A-chain has been purified to homogeneity and its ability to depurinate 28S rRNA in rat liver ribosomes has been demonstrated in vitro.

Abrin↗