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Biomedical subjects

S F Carroll

Publications and source records attributed to S F Carroll.

66 records · Page 4Linked to original sources
PubMed1981

Antibacterial peptide from normal rabbit serum. 3. Inhibition of microbial electron transport.

The influence of the primary rabbit serum bactericide, PC-III, on the respiratory activity of Bacillus subtilis has been examined. Glucose- or lactate-dependent respiration by whole cells was rapidly and completely inhibited by concentrations of the bactericide producing significant cell death. Similar results were observed with membrane vesicles oxidizing NADH. In both cases, bactericide-induced inhibition of respiration was calcium dependent and blocked electron transport between cytochromes b and a. PC-III competed with oxidized Saccharomyces cytochrome c when the latter was used as an electron acceptor in cytochrome c reductase reactions catalyzed by B. subtilis membrane vesicles. Competitive inhibition by PC-III was also observed when reduced Saccharomyces cytochrome c was used as electron donor in the cytochrome c oxidase reaction. At an ionic strength of 0.13, PC-III exhibits a Ki of 25.9 and 102 nM for the reductase and oxidase complexes, respectively. Increasing the ionic strength to that producing optimal antibacterial action against whole cells (0.24) increased the Ki of PC-III for the reductase (75.4 nM), while the oxidase decreased (92.3 nM).

Animals↗
PubMed1980

Ligand interactions of diphtheria toxin. I. Binding and hydrolysis of NAD.

Prior studies showed that diphtheria toxin could be separated into ATP-binding and nonbinding fractions (Fractions II and I, respectively) by affinity chromatography on ATP-Sepharose (Lory, S., and Collier, R. J. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 267-271). Here we show that the two fractions also differ in their interactions with NAD. Fraction II bound a single molecule of NAD (Kd about 9 microM) as assayed by flow dialysis or fluorescence quenching and catalyzed both NAD-glycohydrolase and auto-ADP-ribosylation reactions. Fraction I was deficient in NAD-binding and NAD-related reactions. The ratio of the two fractions vried widely among toxin preparations and was independent of the proportion of toxin in the nicked state. Properties of th NAD site on Fraction II were similar to, but not identical with, those of the corresponding site on free Fragment A.

Binding Sites↗
PubMed1980

Ligand interactions of diphtheria toxin. II. Relationships between the NAD site and the P site.

Prior studies have described two functionally distinct ligand-binding sites on whole diphtheria toxin, the NAD site, which catalyzes the intracellular ADP-ribosylation reaction, and the P site, which affects toxin binding to sensitive cells. Occupancy of the P site by ATP or other phosphorylated compounds inhibits toxin attachment to cells. Here we show that binding of NAD site and P site ligands is competitive; and we characterize ligand-binding properties of two mutant forms of the toxin, CRM 45 and CRM 197. The data suggest that the NAD site, on the A moiety, lies immediately adjacent to the P site, formed by a strongly cationic region on the COOH-terminal half of B. The cationic character of the P site slightly alters the substrate specificity of the NAD site, and occupancy of either of the sites blocks ligand binding to the other. Possible roles of the P site in toxin attachment are discussed.

Adenosine Diphosphate Ribose↗
PubMed1980

Ligand interactions of diphtheria toxin. III. Direct photochemical cross-linking of ATP and NAD to toxin.

The locations of ATP- and NAD-binding sites on diphtheria toxin were investigated by ultraviolet irradiation of ligand . toxin complexes. Illumination of ATP with ultraviolet light (253.7 nm) in the presence of various proteins resulted in photoinduced cross-linking only with Fraction II of diphtheria toxin. Under the same conditions, NAD was cross-linked most effectively to Fragment A, followed by Fraction II and CRM 45. For both ATP and NAD, the degree of protein labelling correlated well with binding data, suggesting that photoinduced cross-linking ocurred only at the high affinity binding sites for these ligands. Nonspecific labeling of unrelated proteins was consistently less than 5% of that observed for Fraction II. Analysis of nicked and reduced Fraction II . ligand complexes on SDS polyacrylamide gels demonstrated that essentially all of the cross-linked label migrated with the A fragment, whether photolysis was performed with ATP or NAD.

Adenosine Triphosphate↗
PubMed1980

Sequential metabolic expressions of the lethal process in human serum-treated Escherichia coli: role of lysozyme.

Several metabolic parameters indicative of Escherichia coli function and integrity were kinetically examined in response to treatment with normal human serum in the presence and absence of functional human lysozyme. Specific inhibition of this enzyme in bacteriolytic and bactericidal reactions was accomplished by using purified rabbit anti-human lysozyme immunoglobulin G. Initiation of the complement-mediated alterations of cytoplasmic membrane integrity, as judged by the leakage of 86Rb from prelabeled cells or the hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by a cryptic strain, was found to be independent of lysozyme action. Furthermore, inhibition of macromolecular synthesis by E. coli in response to serum treatment occurred at the same time regardless of the functional state of lysozyme. Although the rate and extent of bacteriolysis were reduced in the absence of lysozyme, the bactericidal kinetics was unaffected. These results demonstrate that the lethal events associated with the action of antibody and complement on gram-negative bacteria are independent of lysozyme, suggesting an accessory role for this enzyme in immune reactions. A possible temporal sequence of complement-induced effects occurring at the cell surface is presented.

Bacterial Proteins↗
PubMed1979

Purification and properties of rabbit alveolar macrophage lysozyme.

Lysozyme was isolated from Bacillus Calmette-Guerin-elicited rabbit alveolar macrophages by acid extraction and purified to homogeneity by a single-column procedure. Yields of the purified enzyme averaged between 20 and 30 mg per rabbit, values far in excess of those obtained with previously published methods. Rabbit lysozyme has a molecular weight of 14,300 and exhibits optimal lytic activity against Micrococcus lysodeikticus at an ionic strength of 0.04, pH 6.5. Our results indicate that lysozyme and other granule components can be fractionated from elicited alveolar macrophages by using simple techniques, suggesting methods for the bulk purification of lysosomal constituents.

Amino Acids↗
PubMed1979

Role of rabbit lysozyme in in vitro serum and plasma serum bactericidal reactions against Bacillus subtilis.

The antibacterial activity of purified rabbit lysozyme was kinetically investigated at concentrations comparable to those in normal rabbit serum and plasma serum. The bactericidal capability, lysozyme content, and electrophoretic composition of "purified beta-lysin," fractionated from normal rabbit serum, were also examined. In contrast to the extensive antibacterial activity of dilute normal rabbit serum observed in vitro, rabbit lysozyme was only weakly bactericidal for Bacillus subtilis. Inhibition of lysozyme enzymatic and bactericidal activities in normal rabbit serum by antilysozyme immunoglobulin G slightly reduced the initial rate of killing. The addition of neutralizing antibody or histamine (another lysozyme inhibitor) to partially purified bactericidal serum fractions had no effect on killing kinetics. Increasing the ionic strength of reaction mixtures containing normal serum or partially purified bactericidal fractions to levels which completely inhibited lysozyme activity resulted in stimulation of their respective killing kinetics. The addition of inhibitors to normal rabbit plasma serum completely eliminated its bactericidal activity. With regard to the killing of B. subtilis by rabbit and human blood fractions, these analyses clearly demonstrated that (i) although lysozyme is not a significant antibacterial component of normal rabbit serum, it represents the principal factor in normal rabbit plasma serum; (ii) different primary bactericidal mechanisms which are not detectable by singlepoint analyses operate in the sera of different species; and (iii) purified beta-lysin isolated from normal rabbit serum by the classical procedure is a heterogenous mixture of components.

Animals↗
PubMedDate not supplied

Substituted 2-iminothiolanes: reagents for the preparation of disulfide cross-linked conjugates with increased stability.

Much attention has been focused recently on the stability of immunotoxin (antibody-toxin) conjugates linked by a disulfide bridge. Conflicting reports have appeared regarding the in vivo stability of such conjugates prepared with the two most commonly used cross-linking reagents, SPDP and 2-iminothiolane. We have developed (i) a series of reagents based on 2-iminothiolane substituted at the 4- and/or 5-positions (X2ITs) which, based on model studies with simple amines, should show enhanced disulfide stability when conjugated with antibodies or other proteins and (ii) a real-time method for monitoring the rate and extent of conjugation of these reagents with amino groups. Depending upon the substituent, the stability of model-activated disulfides relative to unsubstituted 2-iminothiolane was increased from 5- to 4000-fold as measured by glutathione-induced release of thionitrobenzoic acid. This family of cross-linking reagents should allow the construction of disulfide cross-linked toxin, drug, or enzyme conjugates with enhanced stability in vivo.

Chemical Phenomena↗
PubMedDate not supplied

Cysteine analogs of recombinant barley ribosome inactivating protein form antibody conjugates with enhanced stability and potency in vitro.

Antibody immunoconjugates were made with native and recombinant forms of the type-I ribosome inactivating protein from barley (BRIP) and with three recombinant BRIP (rBRIP) analogs engineered to contain a unique cysteine residue near the C terminus (at amino acid 256, 270, or 277). rBRIP and all three cysteine analogs (rBRIPc256, rBRIPc270, and rBRIPc277) were produced in E. coli, with yields of soluble protein as high as 1 g/L, and were as active as native BRIP in inhibiting protein synthesis in vitro. Interestingly, the position of the engineered cysteine influenced not only the efficiency of conjugation to antibody but also the efficacy and disulfide bond stability of the immunoconjugates. Anti-CD5 antibody conjugates prepared with native and rBRIP were relatively inactive against antigen-positive target cells, while the conjugate made with rBRIPc277 was 5-fold more cytotoxic. Anti-CD7 antibody conjugates made with rBRIPc277 or rBRIPc270 also exhibited improved potency and stability compared to the conjugate with native BRIP. These results indicate that engineering a cysteine residue into selected positions near the C-terminus of a type-IRIP such as BRIP can improve immunoconjugate yield, disulfide bond stability, and potency.

Animals↗
PubMedDate not supplied

Enhanced stability in vitro and in vivo of immunoconjugates prepared with 5-methyl-2-iminothiolane.

Substituted 2-iminothiolanes (X2ITs) are new heterobifunctional crosslinking agents designed for the preparation of disulfide-linked conjugates with enhanced resistance to reduction. Based upon 2-IT substituted at the 4 and/or 5 position, these reagents appear to function by sterically protecting the conjugate disulfide bond from attack by thiolate nucleophiles. Here, we have used the X2ITs to prepare and evaluate a series of immunoconjugates (antibody-cytotoxin conjugates) between the murine monoclonal antibody 791/T36, which recognizes a 72-kDa surface antigen present on many human tumor cells, and RTA30, the naturally occurring 30-kDa glycoform of ricin A chain. The X2IT-linked conjugates were also compared to immunoconjugates prepared with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) and 4-[(succinimidyloxy)carbonyl]-alpha-methyl-alpha-(2- pyridyldithio)toluene (SMPT), as well as with methyl- and dimethyl-substituted structural analogs of SPDP. In vitro, 791-(X2IT)-TNB model compounds exhibited a 6000-fold range of stabilities. In contrast, the corresponding 791-(X2IT)-RTA30 immunoconjugates were up to 20-fold more stable than conjugates made with unhindered linkages. These improvements resulted in immunoconjugates with prolonged serum half-lives in animals. Our data indicate that one of the crosslinking agents, 5-methyl-2-iminothiolane (M2IT), has optimal properties for the preparation of disulfide crosslinked immunoconjugates intended for therapeutic use in that (i) it is highly water soluble and reacts rapidly with protein amino groups at neutral pH, preserving the positive charge, (ii) it forms conjugates with RTA30 efficiently, and (iii) its conjugates exhibited enhanced disulfide bond stability in vitro and in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗