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D M Neville

Publications and source records attributed to D M Neville.

At least 73 records · Page 4Linked to original sources
PubMed1981

Kinetics of protein synthesis inactivation by diphtheria toxin in toxin-resistant L cells. Evidence for a low efficiency receptor-mediated transport system.

In an attempt to gain an understanding of the mechanism by which toxins enter cells, we have analyzed the intoxication of diphtheria toxin in the toxin-resistant L cell and compared these results with those previously reported for toxin-sensitive HeLa and KB cells (Uchida, T., Pappenheimer, A. M., Jr., and Harper, A. A. (1973) J. Biol. Chem. 248, 3845-3850; Moehring, J. M., and Moehring, T. J. (1976) Infect. Immun. 13, 221-228). The inactivation of protein synthesis in diphtheria toxin-treated L cells is characterized by first order inactivation kinetics and a concentration-dependent lag phase. The log rate of inactivation of protein synthesis when plotted versus toxin concentration yields a complex hyperbola with a saturable component detected in the range of 5 to 10 X 10(-5) M. We conclude that the intoxication of L cells by diphtheria toxin in the range of 10(-8) to 5 X 10(-6) M occurs via a saturable process and that the saturable component probably represents a plasma membrane toxin receptor with an affinity 200-fold lower than that found in sensitive HeLa cells. Since L cells require a 10(4)- to 10(5)-fold higher toxin dose than HeLa cells, a 200-fold affinity difference cannot fully explain the resistance of L cells to diphtheria toxin. In addition, L cells must have either fewer toxin receptors or a deficiency in the transport step(s). Because toxin-exposed L cells in contrast to sensitive cells exhibit different behavior with respect to pH and pH reversal of NH4Cl protection, a deficiency in the transport step is considered the more likely case.

Ammonium Chloride↗
PubMed1981

Studies on the galactose-binding site of ricin and the hybrid toxin Man6P-ricin.

N-acetylimidazole (NAI) was used to O-acetylate the plant seed toxin ricin. O-acetylation of one to two tyrosine residues per molecule of ricin inhibited ricin binding to Sepharose 4B and decreased toxicity by 90% in a protein synthesis inhibition assay in HeLa cells. Lactose, known to block the binding site on the ricin B subunit, protected ricin from NAI modification of binding or toxicity. Thus NAI, under these conditions, can be a lactose site-specific inhibitor. The lactose site-specific modification of the hybrid toxin, Man6P-ricin, performed under the same conditions, exhibited the same 90% inhibition of Man6P receptor-mediated toxicity as the galactose-containing receptor-mediated toxicity of either Man6P-ricin or ricin. Thus the ricin B chain lactose-binding site appears to be essential for the high potency of Man6P-ricin via the new cell type-specific Man6P receptor. Treatment of fibroblasts with neuraminidase exposes galactose residues, thus increasing the sensitivity to ricin eight fold. The Man6P receptor-mediated toxicity of Man6P-ricin is not affected by this treatment, although the galactose-inhibited route is potentiated eight fold. The Man6P-ricin hybrid appears to require the ricin B chain galactose-binding site to enter the cytosol after initially binding to the Man6P receptor. These data provide some insights into the proper design of hybrid toxins. We discuss a number of possible models for hybrid toxin entry.

Acetylation↗
PubMed1980

Mannose 6-phosphate receptor-mediated uptake of modified low density lipoprotein results in down regulation of hydroxymethylglutaryl-CoA reductase in normal and familial hypercholesterolemic fibroblasts.

Monophosphotetramannosyl-1-deoxymannitol-1-yl-low density lipoprotein (Man-6-P-LDL) was prepared by covalent attachment of the pentasaccharide omega-(6-phospho-tetra(alpha 1-3)mannosyl(alpha 1-2)mannose to amino groups on low density lipoprotein. Normal human fibroblasts were shown to specifically bind, internalize, and degrade 125I-labeled Man-6-P-LDL. Specificity for the mannose 6-phosphate (Man-6-P) receptor was demonstrated by competitive displacement with cold Man-6-P-LDL, Man-6-P, or mannose. No displacement was seen with cold LDL. Kd is estimated to be less than or equal to 2 X 10(-9) M. Degradation of 125I-labeled Man-6-P-LDL in familial hypercholesterolemic fibroblasts showed the same time course and specificity as observed in normal fibroblasts. Man-y-P-LDL was also able to deliver cholesterol to the cytosol where down regulation of the enzyme 3-hydroxy-3-methylglutaryl CoA reductase was observed in both normal and familial hypercholesterolemic fibroblasts. Down regulation could be blocked by Man-6-P in both cell lines. The possible uses of agents such as Man-6-P-LDL as research probes and therapeutic tools directed to specific cell types are discussed.

Binding, Competitive↗
PubMed1980

Anti-Thy 1.2 monoclonal antibody linked to ricin is a potent cell-type-specific toxin.

The cell-type specificity of the toxin ricin, which ordinarily binds, enters, and kills cells through receptors containing galactose, has been altered by covalently binding it to a monoclonal antibody and by reversibly binding it to lactose. The antibody, a monoclonal rat IgG2b directed against the Thy 1.2 antigen, provides ricin with a new binding site for the murine thymus cell surface. Addition of lactose saturates the galactose-binding site on ricin and inhibits ricin from binding and killing cells via the galactose-containing receptors. The antibody-ricin hybrid protein, anti-Thy 1.2-ricin, formed with a thioether linkage, has been purified by size exclusion and affinity chromatography. When assayed by inhibition of protein synthesis of EL-4 cells, which express the Thy 1.2 antigen anti-Thy 1.2-ricin is equally as toxic as ricin on a molar basis. The hybrid protein toxicity is unchanged in the presence of 100 mM lactose, whereas unmodified ricin toxicity is reduced to 1% of its toxicity in the absence of lactose. This demonstrates the altered receptor specificity of the ricin hybrid. The cell-type specificity of the anti-Thy 1.2-ricin inhibition of protein synthesis correlates with the presence of the Thy 1.2 antigen. Anti-Thy 1.2-ricin at 4 microgram/ml in the presence of lactose inhibits protein synthesis within 3.5 hr by 60-80% in EL-4 cells but does not affect Thy 1.1 alloantigen and HeLa cells that lack the Thy 1 antigen. Anti-Thy 1.2-ricin in the presence of lactose selectively kills EL-4 cells at concentrations that do not kill AKR-K36 cells. This selectivity, expressed as the ratio of anti-Thy 1.2-ricin concentrations required to kill 40% of both cell types, is 700. Ricin-monoclonal antibody hybrids of this type combine a high degree of cell-type selectivity and toxicity and may have pharmacologic utility as antitumor reagents.

Animals↗
PubMed1979

Receptor-mediated transport of the hybrid protein ricin-diphtheria toxin fragment A with subsequent ADP-ribosylation of intracellular elongation factor II.

A hybrid protein of ricin and the enzymatically active fragment A of diphtheria toxin (toxin A) has been synthesized and purified. The diphtheria toxin A fragment of the hybrid protein is shown to enter the cytosol compartment of HeLa cells, its presence assayed by the fall of intracellular elongation factor II (EF-2) and the rise of ADP-ribosylated EF-2. Hybrid entrance to HeLa cells is blocked by lactose which blocks receptor-mediated entry of ricin but not by NH4Cl which blocks the transport of diphtheria toxin. It is concluded that the diphtheria toxin fragment A moiety of the hybrid enters the cell cytosol via the ricin receptor-mediated transport system. The kinetics of intracellular ADP-ribosylation of EF-2 by diphtheria toxin have also been studied. Ribosylation is preceded by a toxin dose-dependent lag period. The data suggest that the time constant responsible for the lag period is in the transport step. Models consistent with these data are discussed.

Adenosine Diphosphate Ribose↗
PubMed1979

Ricin linked to monophosphopentamannose binds to fibroblast lysosomal hydrolase receptors, resulting in a cell-type-specific toxin.

The receptor specificity of the plant seed toxin ricin, which ordinarily binds to galactose-containing receptors, has been altered by coupling monophosphopentamannose residues to ricin by reductive amination and by reversibly binding lactose to the modified ricin. The added monophosphopentamannose residues provide ricin with the recognition factor common to fibroblast lysosomal hydrolases and enable the modified ricin (Man6P-ricin) to bind to the fibroblast Man6P receptor and inhibit protein synthesis in the cells via this receptor. The addition of lactose to Man6P-ricin saturates the galactose site on Man6P-ricin and prevents the binding of Man6P-ricin to cells via galactose-containing ricin receptors. The Man6P receptor-mediated toxicity of Man6P-ricin, identified in human fibroblasts by competition by Man6P and blockade by alkaline phosphatase treatment, was not detected in HeLa cells or human amnion cells. Consequently, in the presence of lactose, the fibroblasts were 8 and 13 times more sensitive than amnion and HeLa cells, respectively. These results show that highly toxic cell-type-specific reagents can be made by the proper alteration of toxin receptor specificities. An attempt to construct a highly toxic altered toxin by adding Man6P residues to diphtheria toxin fragment A was unsuccessful. A possible explanation is that in Man6P-ricin the ricin B chain performs some entry function, even though the initial binding step occurs at the Man6P receptor.

Biological Transport↗
PubMed1978

Demonstration of diphtheria toxin receptors on surface membranes from both toxin-sensitive and toxin-resistant species.

A detailed binding study of 125I-labeled diphtheria toxin to isolated cell surface membrane-enriched fractions is reported. The study was undertaken to determine if toxin-resistant species exhibit a defet in either the binding step or the transport step of the intoxication process. Surface membrane fractions were obtained from liver and mammary glands of toxin-sensitive species, rabbit and giunea pig, and toxin-resistant species, rat mouse. All membrane fractions exhibited reversible binding of 125I-toxin which was competitively inhibited by unlabeled toxin. Toxin receptors from liver co-purified with plasma membranes and the plasma membrane marker 5'-nucleotidase. One-half saturation of all receptors occurred between 5 x 10(-8) and 1.8 x 10(-7) M. Scatchard plots were nonlinear and concave upwards. Total receptor sites ranged from 3.4 to 16 pmol/mg of membrane protein, tissue differences being more pronounced than difference between sensitive and nonsensitive species. Over 95% of the toxin specific binding was inhibited by removal of divalent cation from the medium or by the inclusion of 1 mM ATP, procedures which have been shown to protect sensitive cells from intoxication by diphtheria toxin. We conclude that the rat and mouse have surface membrane receptors for diphtheria toxin and that the toxin insensitivity of these species results from a defect in or a lack of the transport process.

Animals↗
PubMed1978

Alterations in insulin binding induced by changes in vivo in the levels of glucocorticoids and growth hormone.

Previous studies have shown that the sensitivity of tissues to insulin is diminished in states of glucocorticoid and GH excess and is increased when these hormones are deficient. To evaluate the role of the insulin receptor in these states, we have studied [125I]insulin binding to purified liver plasma membranes obtained from rats with a variety of perturbations of both glucocorticoids and GH. Glucocorticoid excess was produced in rats by administration of ACTH (40 U/day for 4 days) or dexamethasone (1 mg/day for 4 days). This resulted in an insulin-resistant state. Associated with this insulin resistance, there was a 50-60% decrease in insulin binding to its specific receptors in liver. Conversely, adrenalectomy, which produces an increase in insulin sensitivity, was associated with an increase in insulin binding to liver. Computer-assisted Scatchard analysis using a negative cooperative model for the inulin-receptor interaction indicated that, in contrast to our findings with obesity, the changes in insulin binding in these states were most likely due entirely to changes in receptor affinity, with no change in receptor concentration. GH administration also produced mild insulin resistance and a decrease in receptor concentration. This was associated with a reciprocal increase in receptor affinity and thus, no major alteration in insulin binding occurred at low physiological insulin concentrations. Hypophysectomized rats, on the other hand, showed an increase in receptor concentration and a decrease in affinity, and GH treatment only partially corrected these changes. Rats implanted with the MtT tumor (which secretes ACTH, GH, and PRL) have the combined effects of excess glucocorticoids and GH and are very insulin resistant. Liver membranes prepared from these rats showed a decrease in insulin binding and receptor affinity similar to that observed in other states of glucocorticoid excess. Further, adrenalectomy of the tumor-bearing rats resulted in an increase in insulin binding despite the persistence of the elevated levels of GH, ACTH, and PRL. These findings suggest that alterations in insulin receptor affinity and number may play a major role in the states of altered insulin sensitivity which accompany glucocorticoid excess and deficiency, and follow hypophysectomy. In contrast, the insulin resistance associated with GH excess is mediated at either a site on the receptor distal to the insulin-binding site (i.e. transduction) or at one or more of the intracellular reactions important in insulin action.

Adrenalectomy↗
PubMed1977

Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. I. Synthesis of diphtheria toxin fragment A-S-S-human placental lactogen with methyl-5-bromovalerimidate.

In order to study the mechanism of entry of plant seed and bacterial toxins into mammalian cells, methods have been developed to synthesize artificial protein hybrid conjugates containing a moiety which binds to a cell membrane receptor and an active fragment of a toxin protein. Utilizing methyl-5-bromovalerimidate, a disulfide cross-linked conjugate of human placental lactogen (hPL) and diphtheria toxin fragment A (toxin A) was synthesized. The reagent was prepared from 5-bromovaleryl nitrile by Pinner synthesis and then used to amidinate hPL. The bromo group thus introduced was converted to S-sulfonate by nucleophilic displacement with 1 M aqueous sodium thiosulfate at room temperature overnight. The S-sulfonated hPL reacted readily with the-SH gorup of reduced toxin A to form a 1 mol/mol of disulfide conjugate in high yield. Thus when reduced toxin A was incubated with a 4-fold excess of the hPL S-sulfonate at 4 degrees and pH 6.5 for 120 h, a conjugate yield of 50% relative to the toxin A input was obtained. Homopolymer formation was negligible and the product was purified by gel filtration on Sephadex G-150. Purity of the conjugate estimated by quantitative analysis of sodium dodecyl sulfate gels was 90%. The toxin A-hPL conjugate retained the activities of both toxin A and hPL, as reported in the accompanying paper. This method of preparing protein hybrid conjugates appeared to have advantages over previous methods utilizing bifunctional reagents with respect to both yield and freedom from homopolymer formation.

Animals↗
PubMed1977

Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. II. Biochemical and biologic properties of diphtheria toxin fragment A-S-S-human placental lactogen.

The biochemical and biologic properties of a purified disulfide conjugate of diphtheria toxin fragment A and human placental lactogen (toxin A-hPL) have been studied by (a) assaying the ADP-ribosyltransferase activity of the intact conjugate, (b) assaying the binding of the intact conjugate to mammary gland plasma membrane lactogenic receptors, and (c) assaying the effect of the conjugate on the rate of protein synthesis in rabbit mammary gland explants maintained in organ culture. The toxin A-hPL conjugate retains one-third of the NAD+:EF-2 ADP-ribosyltransferase activity of toxin A, and 26% of the hPL-binding activity to lactogenic receptors. Binding activity was demonstrated by radioreceptor assay and by assaying toxin A activity bound to membranes which was competitively displaced by excess hPL. Since the toxin A-hPL conjugate retained activities of its separate subunits, it could be regarded as a structural analogue of nicked diphtheria toxin with replacement of the original membrane-binding chain by another binding chain that is specific for lactogenic receptor. However, the conjugate failed to inhibit protein synthesis in organ-cultured mammary gland explants, although these were sensitive to native diphtheria toxin and could bind hPL. It is concluded from these results that the toxin A-hPL conjugate does not act as a functional analogue of diphtheria toxin with altered receptor specificity, and that the hPL receptor cannot mediate the entry of toxin A or toxin A-hPL from membrane-bound conjugate into the cytosol site of action of toxin A.

Adenosine Diphosphate Sugars↗
PubMed1975

The NSILA-s receptor in liver plasma membranes. Characterization and comparison with the insulin receptor.

NSILA-s (nonsuppressible insulin-like activity, soluble in acid ethanol) is a serum peptide that has insulin-like and growth-promoting activities. We have demonstrated previously that liver plasma membranes possess separate receptors for NSILA-s and insulin and have characterized the insulin receptor in detail. In the present study we have characterized the properties and specificity of the NSILA-s receptor and compared them to those of the insulin receptor in the same tissue. Both 125I-NSILA-s and 125I-insulin bind rapidly and reversibly to their receptors in liver membranes; maximal NSILA-s binding occurs at 20 degrees while maximal insulin binding is seen at 1-4 degrees. The pH optimum for NSILA-s binding is broad (6.0 to 8.0), in contrast to the very sharp pH optimum (7.5 to 8.0) for insulin binding. Both receptors exhibit a high degree of specificity. With the insulin receptor, NSILA-s and insulin analogues compete for binding in proportion to their insulin-like potency: insulin greater than proinsulin greater than NSILA-s. With the NSILA-s receptor, NSILA-s is most potent and the order is reversed: NSILA-s greater than proinsulin greater than insulin. Furthermore, six preparations of NSILA-s which varied 70-fold in biological activity competed for 125I-NSILA-s binding in order of their potencies. NSILA-s which had been inactivated biologically by reduction and aminoethylation and growth hormone were less than 1/100,000 as potent as the most purified NSILA-s preparation. Purified preparations of fibroblast growth factor, epidermal growth factor, nerve growth factor, and somatomedins B and C were less than 1% as effective as NSILA-s in competing for the 125I-NSILA-s suggesting that these factors act through other receptors. In contrast, somatomedin A was 10% as active as NSILA-s and multiplication-stimulating activity was fully as active as NSILA-s in competing for the NSILA-s receptor. Analysis of the data suggests that there are approximately 50 times more insulin receptors than NSILA-s receptors per liver cell, while the apparent affinity of NSILA-s receptors is somewhat higher than that of the insulin receptor.

Animals↗
PubMed1975

Insulin binding to liver plasm membranes in the obese hyperglycemic (ob/ob) mouse. Demonstration of a decreased number of functionally normal receptors.

In previous studies, the insulin resistance of the obese hyperglycemic mouse (ob/ob) was found to be associated with decreased insulin binding to liver, fat, and lymphocytes. The present study demonstrates that the insulin receptors in the liver membranes of the ob/ob mouse are decreased in number, but are indistinguishable from normal by other criteria including affinity, kinetics of association and dissociation, temperature dependence of binding, and biological specificity of the binding reaction. The receptor in liver membranes of the ob/ob mouse is also indistinguishable with respect to insulin receptor site-site interactions. Degradation of both insulin and of receptor sites was studied and did not account for differences observed in binding. We conclude that the insulin receptor from the ob/ob mouse is a functionally normal receptor and that its presence in diminished number accounts for the observed decrease in insulin binding to liver plasma membranes.

Animals↗