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Biomedical subjects

W A Carter

Publications and source records attributed to W A Carter.

At least 91 records · Page 5Linked to original sources
PubMed1976

Interaction of human interferons with immobilized hydrophobic amino acids and dipeptides.

Human fibroblast interferon binds to L-tryptophan, D-tryptophan, L-phenylalanine, and L-tyrosine, all immobilized directly to cyanogen bromide-activated agarose, as well as to L-tryptophan and D-tryptophan methyl ester, both immobilized via molecular arms. The retention of fibroblast interferon is selective and results in a 2300-fold purification. Human leukocyte interferon binds neither to L-tryptophan attached directly to an agarose matrix nor to L-tryptophan immobilized via a molecular arm; it binds, however, to immobilized L-tryptophyl-L-tryptophan and L-tryptophyl-L-tryrosine. When retained, both interferons cannot be displaced unless ethylene glycol is included in the eluant, indicating a hydrophobic interaction. The interaction takes place under physiologic solvent conditions, thus revealing the high intrinsic hydrophobicity of both interferons.

Amino Acids↗
PubMed1976

Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction.

Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside, human fibroblast interferon could not be recovered from concanavalin A-agarose coupled at pH 9.0. When concanavalin A was immobilized via molecular arms, in tetrameric as well as dimeric forms, the binding of interferon again occurred exclusively through carbohydrate recognition. Thus, the hydrophobic interaction can be eliminated by appropriate immobilization of the lectin, and then adsorbed glycoproteins, as exemplified here by interferon, can be recovered readily with methyl mannoside alone.

Binding Sites↗
PubMed1976

RNA tumor virus-like activities in human solid tissues: endogenous RNA:DNA polymerase activities in the prostate.

Several human prostatic tissues have been examined for possible particles and associated DNA polymerizing activity generally associated with the C-type RNA tumor virus family. Partially purified tissue extracts, when centrifuged to equilibrium in sucrose gradients, yield fractions which contain actinomycin D resistant, endogenous DNA polymerase activity; this activity bands at a density of 1.15-1.18 gm/cm3. Further analysis of the endogenous products by sucrose gradient sedimentation suggested the presence of high molecular weight RNA:DNA hybrids generally felt to be indicative of a faithful copy of a lengthy stretch of viral specific RNA. However, most of the DNA products synthesized in these endogenous reactions sedimented in much lower molecular weight regions of these sucrose gradients. Clearly, the relative distributions of "high" and "low" molecular weight products could critically depend on the nuclease content of the subcellular fraction under study, and the prostate may be relatively enriched in nucleases. Further, oligo (dT) stimulated the endogenous DNA polymerase activity contained in these extracts, and omission of one of the DNA precursor nucleotides depressed it. Thus, it seems unlikely that terminal transferase activity, rather than genuine DNA polymerization, was being measured primarily. Because of the spectrum of molecular weight classes formed by these DNA:RNA hybrids, as well as their apparent presence in normal prostatic tissue, we find it difficult to ascribe their presence with certainty either to the presence of typical C-type RNA viruses or to the exclusive behavior of the neoplastic prostatic tissue. Thus, our studies lend support to the growing evidence for functions similar to those of C-type RNA viruses being relatively widespread in human tissues without the apparent necessity for a possible etiologic role in neoplastic production (Strand and August, 1974; Sherr et al., 1974). At the same time, our current studies emphasize the need for caution in drawing conclusions from results utilizing probes generally felt quite useful in scoring for presence of virus in lower animals at least in the human prostate.

Centrifugation, Density Gradient↗
PubMed1976

Purification and characterization of mouse interferon with novel affinity sorbents.

Several novel selective sorbents for mouse interferon are described that exploit the hydrophobic property and glycoprotein nature of this molecule. Low-molecular-weight ligands (hydrocarbons) and high-molecular-weight ligands (bovine serum albumin) immobilized on agarose bind selectively mouse L-cell interferon. The high selectivity of binding is due primarily to a hydrophobic effect, although electrostatic forces are also apparently involved. Mouse L-cell interferon binds to immobilized serum albumin and can be completely recovered by raising the ionic strength of the eluant. The specific activity of interferon preparations can be increased 2,000-fold to a value of 3 x 10(8) reference units per mg of protein in a single step with full recovery of the antiviral activity. A selective adsorption, although to a lesser degree, can be also obtained on hydrocarbon-coated agarose (Affi-Gel 202), resulting in 300-fold purification on desorption. The existence of two major components of mouse interferon was revealed upon its chromatography on the following sorbents: (i) bovine serum albumin-agarose, (ii) omega-carboxypentyl-agarose; and (iii) Bandeiraea simplicifolia lectin-agarose. This report thus provides for the first time a means for efficient and clear-cut separation of interferon components, thus enabling their further characterization.

Adsorption↗
PubMed1976

Nature of the molecular heterogeneity of human leukocyte interferon.

The existence of two components of human leukocyte interferon has been recently reported. In the present study, the nature of this molecular heterogeneity was explored by affinity chromatography on immobilized micro- and macroligands, ion-exchange chromatography, and molecular sieving. Chromatography on a series of alkyl-agarose adsorbents shows, for the first time, the intrinsic hydrophobicity of human leukocyte interferon. Additionally, the separation of two interferon components is achieved by use of the alkyl-agarose as well as by the omega-aminoalkyl-agarose adsorbents. Clear-cut separation of the two components was also achieved by chromatography on BSA-CH-Sepharose and on DEAE-Bio Gel A. An important feature of these separations is that they do not require the use of denaturing conditions. The molecular weights of the leukocyte interferon components, as determined on Sephadex G-75, are quite similar or identical, approximately 26,000. Thus, the molecular heterogeneity of human leukocyte interferon can be attributed, at least in part, to differences in the hydrophobicity and ionic properties of its two components.

Chromatography, Affinity↗
PubMed1975

Hydrophobic binding sites on human interferon.

Human interferon binds to a omega-carboxpentyl-agarose column at low ionic strength (0.15 M NaCl) and is still retained when the ionic strength is raised (to 1.0 M NaCl). The binding can be reversed, however, by ethylene glycol, indicating a hydrophobic interaction. The binding of human interferon to omega-aminohexyl-agarose is weak, even at a low ionic strength, and is probably exclusively electrostatic. This disparate binding behavior may be caused by the presence of a positive charge, adjacent to the hydrophobic binding site, on human interferon. The interaction of human interferon with omega-carboxypentyl-agarose is quite selective, inasmuch as the majority of proteins present in interferon preparations pass through the column unretained. Hydrophobic chromatography of human interferon may thus be useful in its purification.

Binding Sites↗
PubMed1975

Friend leukemia: rapid development of erythropoietin-independent hematopoietic precursors.

Infection of mice with the polycythemia-inducing strain of Friend leukemia virus caused a rapid emergence of new erythroid precursor cells. These cells which, in the absence of erythropoietin, proliferated in vitro to form colonies and even differnetiated, quickly out-numbered the usual erythropoietin-dependent hematopoietic elements in bone marrow and spleen. Ultimately, the marrow and spleen were probably totally repopulated with this erythropoietin-independent cell.

Animals↗
PubMed1975

Activity of pure streptovaricins and fractionated streptovaricin complex against Friend virus.

Chromatographic fractionation of streptovaricin complex yields two stable components enriched (4- to 16-fold) in activity directed against the polycythemic strain of Friend virus; both components apparently contain no streptovaricins. When compared with their unfractionated parent streptovaricin complex, eight individual intact streptovaricins (A through G and J) show at least a 30-fold reduction in antiviral activity. These results further support the conclusion that the diversified biological properties of streptovaricin complex probably reside in different molecular structures.

Animals↗
PubMed1975

Molecular structure of human fibroblast and leukocyte interferons: probe by lectin and hydrophobic chromatography.

Structural differences between human leukocyte virus-induced interferon and human fibroblast polyinosinic-polycytidylic acid (rIn-rCn)-induced interferon have been noted in previous studies. This study reports the behavior of human leukocyte and fibroblast interferon, induced by virus and by rIn-rCn, in several lectin and hydrophobic chromatographic systems. Differences in both glycosylation and in hydrophobicity of human leukocyte and fibroblast interferons are documented. Human fibroblast interferon is a glycoprotein, whereas our evidence suggests that human leukocyte interferon probably is not. Also, fibroblast interferon is more hydrophobic than leukocyte interferon, as probed on several hydrophobic adsorbents. The possible relationships of these differences to each other and to antigenic variations are discussed. Generally, the differences appear to be attributable to the cell type in which the interferon was induced. However, our results suggest that at least subtle differences in the processing of the induction signal (virus or rIn-rCn) within the same cell type may occur, slightly altering some structural features.

Chromatography↗
PubMed1975

The interferon refractory state. II. Biological characterization of a refractoriness-inducing protein.

When L929 cells were treated with samples collected from cells or mice producing interferon, almost complete refractoriness to subsequent interferon induction was observed. The relationship of this refractoriness-inducing product (RIP) to the antiviral activity of the samples was determined. Using interferon in the pretreatment sample as a measure of RIP concentration, a semilog plot of the pretreatment interferon titer and interferon subsequently produced, resulted in an approximately linear relationship between 10 and 100 units of interferon in the pretreatment sample. This relationship was largely independent of multiplicity of Newcastle disease virus (NDV) used to produce interferon in the treated monolayers. A 2- to 6-fold temporal differentiation in the refractoriness-inducing activity and the antiviral activities was obtained both in vitro and in vivo. Supernatants collected 48 hr after NDV infection of L929 cells had 2- to 4-fold more refractoriness-inducing activity than those collected earlier. In serum of ICR mice given poly rIn:rCn-poly-D-lysine, a quantitatively similar temporal increase in refractoriness-inducing activity was observed. Treatment of L929 cells with cycloheximide resulted in a 4-fold increase in interferon production. At equivalent interferon concentrations, supernatants from the cycloheximide-treated monolayers inhibited interferon production 4-fold less than did comparable supernatants from non-cycloheximide-treated monolayers. Soluble cell extracts inhibited interferon production to a greater degree than did extracellular preparations. The protein nature of the refractoriness-inducing activity was suggested by its trypsin sensitivity. These findings suggest that the refractoriness-inducing and antiviral activities of interferon preparations result from different proteins.

Antiviral Agents↗
PubMed1975

Inhibition of RNA-dependent DNA polymerase reaction by 6-(p-hydroxyphenylazo)-uracil: A result of drug induced dithiothreitol oxidation.

6-(P-hydroxyphenylazo)-uracil (HPUra) reduced by dithiothreitol inhibited AMV or RLV virion associated exogenous RNA-dependent DNA polymerase reactions. However, the inhibition was variable from experiment to experiment and was not consistent with the base specificity of HPUra seen for inhibition of gram positive DNA-dependent DNA polymerases. Increasing the concentration of dithiothreitol reversed the inhibition. Furthermore, at non-toxic concentrations, HPUra did not influence the plating efficiency of RLV in tissue culture, as measured by the ability to induce foci on sarcoma virus positive-leukemia virus negative cells. Oxidation of dithiothreitol in the presence or absence of HPUra was followed spectrophotometrically under enzyme conditions. HPUra catalyzed the oxidation of dithiothreitol under these conditions. Since dithiothreitol is required for optimum reaction rates, as well as complete disruption of virus in some polymerase assay systems, the oxidation of dithiothreitol in the presence of HPUra is sufficient to explain the inhibition seen.

Animals↗