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J M Lord

Publications and source records attributed to J M Lord.

At least 163 records · Page 9Linked to original sources
PubMed1986

Expression of a 215,000-dalton nuclear envelope protein decreases during cell maturation.

The monoclonal antibody AGF2.3 identifies a nuclear envelope protein expressed at high levels in most transformed cell lines and weakly expressed in or absent from normal tissues and primary cell cultures. Here we report that expression of AGF2.3 antigen relates to the maturation status of cells. When HL60 cells were induced to mature loss of AGF2.3 antigen correlated with the appearance of neutrophils in culture. Acute myeloid leukaemia cells were intensely labelled by AGF2.3 antibody whereas chronic myeloid leukaemia cells were weakly reactive. Staining of tonsil epithelium showed a progressive loss of antigen during epithelial cell maturation. High levels of AGF2.3 antigen in most permanent cell lines suggest that such lines represent transformation of immature cells.

Antibodies, Monoclonal↗
PubMed1985

The primary sequence of Ricinus communis agglutinin. Comparison with ricin.

A mixture of synthetic oligonucleotides representing all possible sequences of a peptide present in the ricin B chain has been used to screen a cDNA library constructed using ripening castor bean seed poly(A+) RNA. The eight largest recombinant plasmids selected, by hybridization, a single mRNA species whose translational product was identified as preprolectin by immunoprecipitation. Restriction enzyme analysis of these clones demonstrated that two classes were present representing sequences complementary to two distinct but closely related preprolectin mRNA species. The nucleotide sequence of the cloned cDNA from one of these classes encodes preproricin and has been presented elsewhere (Lamb, F. I., Roberts, L. M., and Lord, J. M., (1985) Eur. J. Biochem. 148, 265-270). The nucleotide sequence of the second class is presented here and shown to represent prepro-Ricinus communis agglutinin. The entire coding sequence was deduced from two overlapping cDNA clones having inserts of 1668 and 1151 base pairs. The coding region defines a preproprotein with a 24-amino acid N-terminal signal sequence preceding the A chain (266 amino acids) which is joined to the B chain (262 amino acids) by a 12-amino acid linking peptide. The protein was confirmed as R. communis agglutinin since the deduced B chain N-terminal sequence corresponds exactly with that determined for purified R. communis agglutinin B chain over a region where several residue differences occur in the ricin B chain. The nucleotide and deduced amino acid sequences of the R. communis agglutinin precursor are compared with those of the ricin precursor.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence of cloned cDNA coding for preproricin.

The primary structure of a precursor protein that contains the toxic (A) and galactose-binding (B) chains of the castor bean lectin, ricin, has been deduced from the nucleotide sequence of cloned DNA complementary to preproricin mRNA. A cDNA library was constructed using maturing castor bean endosperm poly(A)-rich RNA enriched for lectin precursor mRNA by size fractionation. Clones containing lectin mRNA sequences were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing all possible sequences for a peptide of the ricin B chain. The entire coding sequence of preproricin was deduced from two overlapping cDNA clones having inserts of 1614 and 1049 base pairs. The coding region (1695 base pairs) consists of a 24-amino-acid N-terminal signal sequence (molecular mass 2836 Da) preceding the A chain 267 amino acids, molecular mass 29 399 Da), which is joined to the B chain (262 amino acids, molecular mass 28 517) by a 12-amino-acid linking region (molecular mass 1385 Da).

Amino Acids↗
PubMed1985

Synthesis and intracellular transport of lectin and storage protein precursors in endosperm from castor bean.

The biosynthesis of the lectins and the other major storage proteins, the 11S globulins and the 2S albumins, which are found in protein bodies has been studied in developing castor bean endosperm cells. Newly synthesized proteins were radiolabelled by incubating intact endosperm tissue with [35S]methionine. The intracellular distribution of radiolabelled proteins was determined after fractionating endosperm homogenates by sucrose density gradient centrifugation. Pulse-chase experiments revealed that all the major protein body components are initially segregated in precursor form into the lumen of the endoplasmic reticulum. The lectin precursors appeared as a group of 64 000-68 000-Mr glycosylated polypeptides, the 11S globulins as a group of 46 000-55 000-Mr polypeptides and the 2S albumins as a single 32 500-Mr polypeptide. These precursors were transferred from the endoplasmic reticulum to a population of transporting vesicles. The subsequent disappearance of the precursors from this vesicle fraction was accompanied by the accumulation of mature polypeptides in the protein body matrix (lectins and 2S albumins) or in the insoluble protein body crystalloid complexes (11S globulins). The castor bean proteins studied all exist as heterodimers in the protein bodies. After intracellular transport an endoproteolytic step is required to release each subunit of the heterodimer from the appropriate single polypeptide precursor.

Albumins↗
PubMed1985

Precursors of ricin and Ricinus communis agglutinin. Glycosylation and processing during synthesis and intracellular transport.

During synthesis in vivo the castor bean lectin precursors initially appear in the endoplasmic reticulum as a group of core glycosylated polypeptides of relative molecular mass 64 000-68 000. Pretreatment of intact castor bean endosperm tissue with tunicamycin partially inhibits the cotranslational core glycosylation step and results in the accumulation of a single sized unglycosylated precursor polypeptide of relative molecular mass 59 000. The glycosylated precursors in the endoplasmic reticulum were enzymically converted to the 59 000-Mr form by incubation with endoglucosaminidase H. Intracellular transport of the glycosylated lectin precursors from the endoplasmic reticulum to a denser vesicle fraction was accompanied by modifications to the oligosaccharide moieties which conferred resistance to the action of endoglucosaminidase H. The post-translational addition of fucose to the carbohydrate chain was identified as one of the oligosaccharide modification steps. Fucose addition was catalysed by a glycosyltransferase associated with a smooth-surfaced membrane fraction which was distinct from the endoplasmic reticulum and which was tentatively identified as the Golgi apparatus. Glycosylation was not essential for intracellular transport of the lectin precursors: unglycosylated precursor synthesized in the presence of tunicamycin gave rise to unglycosylated lectin subunits in the protein bodies.

Agglutination Tests↗
PubMed1985

Postreceptor effect of metformin on insulin action in mice.

To investigate the possibility that metformin (dimethylbiguanide) modifies insulin-mediated glucose metabolism by an effect that is independent of insulin receptor binding, glycogenesis and insulin binding were measured in soleus muscles isolated from streptozocin diabetic mice after treatment with 60 mg kg-1 metformin daily for 10 weeks. This dose of metformin increased insulin-stimulated glycogenesis but did not affect insulin binding in the soleus muscles of streptozocin diabetic mice. The results suggest that metformin can influence postreceptor sites of insulin action independently of insulin receptor binding.

Animals↗
PubMed1985

Effect of age on hepatocyte and soleus muscle insulin receptor binding in lean and genetically obese diabetic (ob/ob) mice.

Hepatocyte and soleus muscle insulin binding and the rate of insulin stimulated glycogen synthesis were examined in normal lean and genetically obese diabetic (ob/ob) mice with age. In ob/ob mice a significant decrease in the concentration of high affinity hepatocyte insulin receptors and insulin binding to soleus muscle at 10-20 and 10-40 weeks of age respectively was associated with an age related development of insulin resistance characterized by increased body weight, plasma insulin and plasma glucose. In lean mice a significant reduction in the concentration of low affinity hepatocyte receptors and soleus muscle insulin binding was observed at 60 weeks and associated with a marginally increased plasma insulin concentration and unchanged level of glycaemia. Insulin stimulated glycogen synthesis was unchanged with age in lean mouse soleus muscle but in ob/ob was significantly reduced by 5 weeks and preceded the reduction in insulin binding in this tissue. In 40 week ob/ob mice insulin sensitivity was improved and characterized by a reduction in body weight, plasma insulin and glucose, increased hepatocyte high affinity insulin receptor concentration and decreased affinity, Ke. Although insulin binding to soleus muscle was not increased and there was no improvement in insulin stimulated glycogen synthesis at this age, the increased hepatocyte insulin binding was probably not the primary cause of the improved insulin sensitivity which was most likely mediated at post-receptor loci.

Aging↗
PubMed1983

Ricin and Ricinus communis agglutinin subunits are all derived from a single-size polypeptide precursor.

Antibodies have been raised in rabbits against the individually purified A and B subunits of the toxic castor bean lectin, ricin, and against the A' and B' subunits of Ricinus communis agglutinin type I. Each of the antisera recognised a single polypeptide species of Mr 60 500 when maturing castor bean endosperm mRNA was translated in vitro in a rabbit-reticulocyte-derived system. When dog pancreatic microsomal vesicles were included in the translational system, each subunit antiserum precipitated a group of 66 000-68 000-Mr core-glycosylated polypeptides which had been translocated into the lumen of the vesicles. The 60 500-Mr polypeptide appeared to be a common precursor to all four individual lectin subunits since (a) its glycosylated (66 000-68 000-Mr) forms were readily detected in the endoplasmic reticulum fraction isolated from maturing castor bean endosperm and (b) pulse-chase studies showed that the glycosylated precursors disappeared from the endoplasmic reticulum fraction with the concomittant appearance of authentic lectin subunits in a soluble protein fraction which included protein body matrix components. Antiserum prepared against whole R. communis agglutinin, type I, also precipitated the 65 000-Mr precursor in vitro and in vivo, but in addition precipitated a non-glycosylated 34 000-Mr polypeptide. This smaller protein is not a lectin subunit precursor, contradicting an earlier suggestion. It is most probably a precursor to the 2-S albumin storage proteins found in castor bean endosperm protein bodies.

Acetylglucosaminidase↗
PubMed1983

Effect of metformin on insulin receptor binding and glycaemic control in type II diabetes.

To investigate the effect of metformin on insulin receptor binding and diabetic control, eight obese type II diabetic patients were studied before treatment, after one and four weeks of taking metformin (500 mg thrice daily), and four weeks after withdrawal of the drug. After one and four weeks of treatment the number of erythrocyte insulin receptors had increased by 116% and 184% respectively. This was due almost entirely to an increase in the number of low affinity binding sites. The number of receptors was still raised four weeks after metformin had been withdrawn. Diabetic control as assessed by urinary glucose, glycosylated haemoglobin (HbA1), and glucose tolerance values was significantly improved during metformin treatment, while plasma insulin concentrations were not altered. These results indicate that metformin produces a rapid and protracted increase in low affinity insulin receptors in type II diabetes, associated with greater insulin sensitivity and improved diabetic control.

Aged↗
PubMed1983

Genetic factors in the population of Plati, Greece.

One-thousand, thirty-eight individuals from Plati, Greece were examined for the following red cell antigens, serum proteins, and red cell enzymes A A1 Ai B H; MNSs Mg Henshaw Nya Mur Vw; CCwcDEeCe; K k Kpa Kpb Jsa Jsb; P1; Lua; Fy1 Fy2; Jka Jkb; Wra; Zt; Vel; Swa; Jensen, Radin, Gerbich, Diego, Gregory, Haptoglobin, Transferrin, Acid phosphatase, Adenylate kinase, Adenosine deaminase, Esterase-D, Glucose-6-phosphate dehydrogenase, Phosphoglucomutase, 6-Phosphogluconate dehydrogenase, Phosphohexose isomerase, Lactate dehydrogenase, Malate dehydrogenase, and Superoxide dismutase. The results are discussed in detail and compared with other Greek and neighbouring populations. Because of the Plati population's long history of residence in the Cappadocian area of Turkey the data have been compared, whenever possible, with results for that region.

Adolescent↗
PubMed1983

Effect of metformin on hepatocyte insulin receptor binding in normal, streptozotocin diabetic and genetically obese diabetic (ob/ob) mice.

The effect of metformin on hepatocyte insulin receptor binding was examined in normal, streptozotocin diabetic and genetically obese diabetic (ob/ob) mice. In normal mice, chronic administration of metformin (60 mg X kg-1 X day-1 for 50 weeks) increased the number of low affinity receptors by 148%. During acute studies, metformin increased (30%) the number of low affinity receptors after 24 h. When metformin was withdrawn after treatment for 96 h, the number of low affinity receptors decreased, approaching control values by 48 h. In severely insulin resistant ob/ob mice, the concentrations of high and low affinity receptors were reduced by 60% and 27%, respectively. A high dose of metformin (240 mg X kg-1 X day-1 for 4 weeks) increased the concentration of high and low affinity receptors in ob/ob mice by 63% and 86%, respectively. However, the hypoglycaemic response to exogenous insulin was not altered. In streptozotocin-diabetic mice, the number of low affinity receptors was increased by 68% compared with normal mice. Metformin (60 mg X kg-1 X day-1 for 10 weeks) did not significantly alter the number of insulin receptors in streptozotocin-diabetic mice, but the hypoglycaemic response to exogenous insulin was improved by 94%. The results raise the possibility that metformin might affect post-receptor sites of insulin action independently of effects at the receptor level.

Animals↗
PubMed1983

Heterogeneous distribution of glycosyltransferases in the endoplasmic reticulum of castor bean endosperm.

Endoplasmic reticulum membranes stripped of attached ribosomes were isolated from homogenates of germinating castor bean (Ricinus communis L.) endosperm by sucrose density gradient centrifugation. The isolated endoplasmic reticulum fraction was further separated into two major membrane subfractions by centrifugation on a flotation gradient. Both subfractions appeared to be derived from the endoplasmic reticulum inasmuch as they share several enzymic markers including cholinephosphotransferase, NADH-cytochrome c reductase, and glycoprotein fucosyl-transferase and phase separation of membrane polypeptides using Triton X-114 revealed a striking similarity in both their hydrophilic and hydrophobic protein components. The endoplasmic reticulum membrane subfractions contain glycoproteins which were readily labeled by incubating intact endosperm tissue with radioactive sugars prior to fractionation.Castor bean endosperm endoplasmic reticulum apparently exhibits a degree of enzymic heterogeneity, however, since the enzymes responsible for the synthesis of dolicholpyrophosphate N-acetylglucosamine and dolicholmonophosphate mannose together with their incorporation into the oligosaccharide-lipid precursor of protein N-glycosylation were largely recovered in a single endoplasmic reticulum subfraction.

Journal Article↗
PubMed1981

The synthesis of Ricinus communis agglutinin, cotranslational and posttranslational modification of agglutinin polypeptides.

Polyadenylated RNA isolated from the endosperm tissue of maturing castor bean seeds was translated in a cell-free rabbit reticulocyte lysate system. Rabbit antibodies raised against Ricinus communis agglutinin were used to identify nascent agglutinin chains. In contrast to the authentic agglutinin polypeptides with molecular weights of 31000 (A chains) and 37000 (glycosylated B chains), immunoreactive translational products of Mr 33500 and 59000 were observed. The inclusion of canine pancreatic microsomes in the translational system resulted in the cotranslational segregation of these immunoreactive products into the lumen of the vesicles and their modification, to molecular weights of 32000 and 66000--69000 respectively. These cotranslational size modifications resulted from the cleavage of leader sequences and, in the case of the larger product, concomittant core glycosylation, 32000-Mr and 66000--69000-Mr proteins were also observed amongst the immunoreactive products initially formed during the labelling of intact endosperm tissue in vivo, together with 37000-Mr and 39000-Mr proteins. Pulse-chase experiments showed that 66000--69000-Mr proteins slowly disappeared while the smaller proteins were further cleaved to chains of Mr 31000 (authentic A chain), 34000 and 37000 (authentic glycosylated B chains). It was concluded that R. communis agglutinin polypeptides were synthesized in precursor form, possibly as a 'giant' precursor in the case of the B chain, on membrane-bound polysomes. Cotranslational translocation across the endoplasmic reticulum membrane was accompanied by proteolysis to remove leader sequences and, where appropriate, core glycosylation. Even after cotranslational processing agglutinin polypeptides were still in precursor form. Processing to authentic size appeared to occur posttranslationally.

Animals↗
PubMed1981

Synthesis and posttranslational segregation of glyoxysomal isocitrate lyase from castor bean endosperm.

Total polyadenylated RNA isolated from 3-day-old germinating castor bean endosperm was translated in the rabbit reticulocyte lyase cell-free protein-synthesizing system in either the presence or absence of canine pancreatic microsomal vesicles. Isocitrate lyase was immunoprecipitated from the translational products using rabbit antibodies raised against the purified glyoxysomal enzyme. Isocitrate lyase synthesized in vitro had the same apparent molecular weight as the authentic glyoxysomal enzyme irrespective of whether synthesis occurred in the presence or absence of microsomes. Furthermore, in contrast to several proteins encoded by mRNA isolated from maturing seed endosperm tissue, isocitrate lyase was not cotranslationally segregated into the lumen of the microsomal vesicles. Intact 3-day-old endosperm tissue was labelled in vivo by adding [35S]methionine and at various times the tissue was homogenized and the cellular organelles fractionated. Separated fractions were assayed for newly synthesized isocitrate lyase. Immunoreactive product was initially detected in the cytosolic fraction and subsequently began to accumulate in glyoxysomes. The results indicate that glyoxysomal isocitrate lyase is synthesized on free polysomes and released into the cytosol. Transfer to the matrix of the glyoxysome involves a posttranslational membrane translocation step which is not accompanied by proteolytic cleavage of the polypeptide chain.

Animals↗