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Biomedical subjects

J M Lord

Publications and source records attributed to J M Lord.

At least 127 records · Page 7Linked to original sources
PubMed1991

Changes in inositol transport during DMSO-induced differentiation of HL60 cells towards neutrophils.

[3H]Inositol uptake by HL60 cells was measured during DMSO-induced differentiation towards neutrophils. The values for Km (53.2 microM) and Vmax (5.3 pmol/min per 10(6) cells) obtained for control HL60 cells are in good agreement with previously published figures for this cell line. Inositol transport into HL60 cells was an active, saturable and specific process which was unaffected by extracellular glucose concentrations. Inositol transport rates changed during DMSO-induced differentiation of HL60 cells towards neutrophils. An increase in inositol transport rates occurred during the first 4 days of exposure to 0.9% DMSO and was concommitant with the period leading to growth arrest and prior to the acquisition of the differentiated phenotype. These changes preceded the rise in intracellular inositol concentration from 10.9 to 132.7 microM seen between day 1 and day 5. After 4 days exposure to DMSO the rate of inositol transport fell to a value of 3.2 +/- 0.3 pmol/min per 10(6) cells at day 7, this was accompanied by a small reduction in intracellular inositol from a peak value of 132.7 to 112 microM. The inositol transport rate, thus, appears to closely accompany changes in the intracellular concentration of inositol. Inositol transport in human peripheral blood neutrophils was an order of magnitude slower than the value for uninduced HL60 cells, but the Km for inositol transport was similar in both cell types and was unchanged during HL60 differentiation. This suggests that changes in inositol transport rate are achieved by the modulation of a commonly expressed inositol transporter, one consequence of which is the alteration of intracellular inositol concentrations.

Biological Transport, Active↗
PubMed1991

Alteration of an amino acid residue outside the active site of the ricin A chain reduces its toxicity towards yeast ribosomes.

Yeast transformants containing integrated copies of a galactose-regulated, ricin toxin A chain (RTA) expression plasmid were constructed and used in an attempt to isolate RTA-resistant yeast mutants. Analysis of RNA from mutant strains demonstrated that approximately half contained ribosomes that had been partially modified by RTA, although all the strains analysed transcribed full-length RTA RNA. The mutant strains could have mutations in yeast genes giving rise to RTA-resistant ribosomes or they could contain alterations within the RTA-encoding DNA causing production of mutant toxin. Ribosomes isolated from mutant strains were shown to be susceptible to RTA modification in vitro suggesting that the strains contain alterations in RTA. This paper describes the detailed analysis of one mutant strain which has a point mutation that changes serine 203 to asparagine in RTA protein. Although serine 203 lies outside the proposed active site of RTA its alteration leads to the production of RTA protein with a greatly reduced level of ribosome modifying activity. This decrease in activity apparently allows yeast cells to survive expression of RTA as only a proportion of the ribosomes become modified. We demonstrate that the mutant RTA preferentially modifies 26S rRNA in free 60S subunits and has lower catalytic activity compared with native RTA when produced in Escherichia coli. Such mutations provide a valuable means of identifying residues important in RTA catalysis and of further understanding the precise mechanism of action of RTA.

Amino Acids↗
PubMed1991

Ribosome inactivating proteins of plants.

Many plant tissues produce single chain proteins which can enzymatically remove a specific adenine residue from ribosomal RNA. Although these proteins are potently toxic to isolated ribosomes, they are non-toxic to intact cells, being unable to gain access to their ribosomal substrate. In certain plants however, the gene for the ribosome inactivating protein has fused with a gene encoding a galactose-specific lectin. This generates heterodimeric proteins which can bind to and enter target cells, and which are among the most potent cytotoxins known.

Amino Acid Sequence↗
PubMed1990

Cytotoxicity of a recombinant ricin-A-chain fusion protein containing a proteolytically-cleavable spacer sequence.

Chimeric proteins composed of ricin toxin A chain (RTA) and staphylococcal protein A (PA) have been produced in E. coli. Constructs consisting of N-terminal RTA and C-terminal PA (RTA-PA) or N-terminal PA and C-terminal (PA-RTA) were capable of binding to immunoglobulin G (via PA) and of specifically depurinating 28 S ribosomal RNA (via RTA). However, neither fusion protein was cytotoxic to antigen-bearing target cells in the presence of an appropriate monoclonal antibody presumably because the RTA could not be released from the PA within the cytosol where the ribosomal substrate of RTA is located. The overcome this, a short amino acid sequence from diphtheria toxin was engineered between the RTA and PA to produce a disulfide-linked loop containing a trypsin sensitive cleavage site. Cleavage of this fusion protein with trypsin converted the RTA-DT-PA to the two chain form consisting of RTA linked by a disulfide bond to PA. The cleaved fusion protein was highly toxic to Daudi cells coated with anti-immunoglobulin antibody suggesting that the RTA could be released from the PA by reduction within the cytosol.

Amino Acid Sequence↗
PubMed1990

Inositol lipids and phosphates in the regulation of the growth and differentiation of haemopoietic and other cells.

Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis is an important signalling reaction involved in the responses of cells to some, but not all, stimuli that promote cell proliferation. Active agents in this regard include antigens activating T and B lymphocytes, angiotensin (employing a receptor encoded by the mas oncogene), bombesin and platelet-derived growth factor PDGF). However, accumulating evidence suggests that inositol lipids and phosphates also have other roles in the regulation of cell growth and differentiation. Growth factor receptors that encode tyrosine kinases (such as that for PDGF) activate a kinase that synthesises phosphatidylinositol 3-phosphate, a novel lipid, and loss of this kinase-activating function abolishes growth-promoting activity. Human interleukin-4, a lymphokine that activates B lymphocytes, appears to employ phosphatidylinositol 4,5-bisphosphate hydrolysis as a brief initial signal that is followed by a sustained rise in cyclic adenosine monophosphate (cAMP): both signals are needed for the successful induction of the surface antigen CD23. Moreover, the same inositol lipid signalling pathway as is employed by antigen-stimulated mature T lymphocytes to provoke proliferation may be redeployed in immature T cells to trigger their elimination when they encounter self-antigens. Finally, studies of HL60 promyelocytic cells have shown that these cells contain high concentrations of inositol 3,4,5,6-tetrakisphosphate, 1,3,4,5,6-pentakisphosphate and hexakisphosphate, three inositol polyphosphates that are probably formed independently of inositol lipid metabolism. When these cells are induced to differentiate either towards neutrophils (in the presence of dimethylsulphoxide) or macrophages (in phorbol myristate acetate), cessation of growth and acquisition of differentiated characteristics are accompanied by large and different changes in the concentrations of these inositol phosphates that may be characteristic of these two pathways of differentiation.

Animals↗
PubMed1990

The Ricinus communis 2S albumin precursor: a single preproprotein may be processed into two different heterodimeric storage proteins.

The Ricinus communis (castor bean) 2S albumin is a heterodimer of glutamine-rich, disulphide-linked 4 and 7 kDa polypeptide. A cDNA library was constructed using mRNA from maturing castor bean endosperm as template. Clones containing sequences complementary to albumin mRNA were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing sequences predicted for a peptide present in the 2S albumin large subunit. The nucleotide sequence contained an open reading frame encoding a preproprotein of 258 amino acid residues. The preproprotein included both polypeptides of the previously sequenced 2S albumin. In addition, this precursor included two further glutamine-rich sequences which, in term of their size and conserved cystein residues typically found in seed proteins of the 2S albumin superfamily, possible represent the small and large polypeptide subunits of a second heterodimeric storage protein. A post-translational processing scheme is proposed which would result in a single preproprotein generating two distinct heterodimeric 2S albumins. The generation of a second heterodimer seems likely since polypeptide candidates for its small and large subunits were found in the Ricinus 2S albumin fraction, and N-terminal protein sequencing confirmed the existence of the putative small subunit.

2S Albumins, Plant↗
PubMed1990

The influence of anti-(ricin toxin A chain) monoclonal antibodies on the pharmacokinetics of ricin toxin A chain and recombinant ricin A chain in mice.

Two monoclonal antibodies against ricin toxin A chain (RTA) have been examined for their effects on the blood survival and biodistribution of RTA and recombinant ricin A chain in mice. When admixed with the toxins at 1:1 molar ratios prior to intravenous injection, the antibodies prolonged blood survival and whole-body retention of both species of RTA, and this was due essentially to reduced renal clearance of the toxins. Immune complexes were identified by gel filtration chromatography and immune precipitation with anti-IgG antiserum in mixtures prior to injection and in the serum of mice injected with the mixtures. An irrelevant monoclonal antibody showed no complex formation, and no effect on biodistribution. These studies have shown that immune complexes formed between monoclonal antibodies and protein antigens of molecular mass up to at least 30 kDa survive in the circulation, rather than being cleared by the reticuloendothelial system. Such antibodies could be used to modulate the biodistribution of toxic molecules such as ribosome-inhibiting proteins like RTA. This might be exploited therapeutically, for example in the construction of bispecific antibodies against ribosomal inhibiting proteins and tumour-associated antigens.

Animals↗
PubMed1990

Models of haemopoiesis.

To date, various models have been proposed to explain the diversification of haemopoietic stem cells along one of at least six pathways of differentiation. Consideration of evidence for and against particular models leads to the conclusion that a precise lineage map for the haemopoietic system is, as yet, unavailable. However, recently available cell and molecular biology techniques provide the means to resolve this problem.

Hematopoiesis↗
PubMed1990

Phorbol myristate acetate treatment of normal human myeloid blast cells promotes monopoiesis and inhibits granulopoiesis.

Fractionation of mononuclear cells from human fetal liver provides a cell population at early stages of myeloid differentiation which, when cultured, generates neutrophils and macrophages for up to a month. These studies describe the further purification of an undifferentiated myeloid blast cell population by rosette sedimentation of unwanted cells, after coating these cells with monoclonal antibodies which identify macrophages and erythroblasts. In culture, the purified blast cells generated only neutrophils and macrophages. When treated with 10 nM PMA, 62% of the purified cells were induced to differentiate towards macrophages within 48 h. PMA-induced cells acquired morphological features of macrophages and synthesized alpha-naphthyl acetate esterase. The differentiation of the remaining blast cells towards neutrophils, seen in untreated cultures, was completely inhibited by PMA, as revealed by the absence of increases in the numbers of cells expressing lactoferrin and an antigen which appears at the promyelocyte stage of differentiation. Thus, PMA effects intracellular changes which both promote monopoiesis and inhibit granulopoiesis, suggesting a reciprocal interaction between intracellular processes which regulate the capacity for the two pathways of maturation. The purified blast cell population provides a good model system for studies of molecular events which regulate the expression of macrophage characteristics.

Cell Differentiation↗
PubMed1990

Immunotoxins: status and prospects.

Immunotoxins, which are conjugates of cell-binding antibodies and toxins, show considerable promise in the treatment of certain cancers. Genetic engineering is increasingly being used to refine and modify these conjugates, and it is now possible to design, express and purify completely recombinant therapeutic molecules.

Animals↗
PubMed1989

Recombinant proricin binds galactose but does not depurinate 28 S ribosomal RNA.

Preproricin transcripts microinjected into Xenopus oocytes were expressed and the product was segregated by the oocyte endoplasmic reticulum and core glycosylated. Recombinant proricin was soluble, stabilised by intramolecular disulfide bonds and biologically active in that it could bind to immobilized lactose (selectin 2) or immobilized asialofetuin. Affinity-purified proricin did not catalyse the depurination of 28 S ribosomal RNA unless it was reduced, when slight but significant activity was observed. Gel filtration of the reduced proricin fraction showed that this depurination activity was not associated with proricin. The activity was apparently due to ricin A chain released by reduction from mature ricin which was, in turn, generated from proricin, presumably via endogenous oocyte endoprotease activity.

Animals↗
PubMed1989

Expression of ricin B chain in Escherichia coli.

DNA encoding ricin B chain was fused to that encoding the E. coli OmpA signal peptide using the expression secretion vector pIN-111-ompA. When induced, E. coli cells transformed with the recombinant plasmid express ricin B chain. The recombinant product accumulates in the periplasmic space in a soluble, biologically active form.

Escherichia coli↗
PubMed1989

Ribosome inactivation by ricin A chain: a sensitive method to assess the activity of wild-type and mutant polypeptides.

When recombinant ricin A chain transcripts are translated in a rabbit reticulocyte lysate the ribosomes are rapidly inactivated as shown by their inability to support translation of yeast preproalpha factor or chicken lysozyme transcripts added subsequently. In contrast, ribosomes which have translated transcripts encoding non-toxic polypeptides such as ricin B chain, readily translate the second transcript under identical conditions. Ribosome inactivation is accompanied by a highly specific modification of 28S rRNA which occurs at the same position as the N-glycosidic cleavage of an adenine residue and which is thought to cause inactivation of the ribosomes. Protein synthesis by wheat germ ribosomes was not inhibited under the conditions which inhibit reticulocyte ribosomes confirming earlier observations that plant cytoplasmic ribosomes are much less sensitive to inhibition by ricin A chain than are mammalian ribosomes. Using the same assay we have shown that deleting an internal hexapeptide, which shares homology with hamster elongation factor-2, completely abolishes catalytic activity. Deleting a second pentapeptide conserved between ricin A chain and the ribosome-inactivating plant toxin trichosanthin, had no effect. Deleting the first nine residues from the N-terminus of A chain did not affect toxicity whereas deleting a further three residues inactivated the polypeptide. Point mutations which individually converted arginine 48 and arginine 56 of ricin A chain to alanine residues or which deleted arginine 56 were also without effect on the catalytic activity of the toxin.

Amino Acid Sequence↗