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Biomedical subjects

S F Carroll

Publications and source records attributed to S F Carroll.

At least 55 records · Page 3Linked to original sources
PubMed1991

Improved pharmacokinetics and tumor localization of immunotoxins constructed with the Mr 30,000 form of ricin A chain.

The two naturally occurring forms of ricin A chain, Mr 33,000 and Mr 30,000 (RTA33 and RTA30) have been purified, and their chemical compositions, toxicities, and tissue distributions have been determined. As reported previously, the in vitro and in vivo toxicities of RTA30 and RTA33 are similar. However, RTA30, which contains less carbohydrate with a lower mannose content than RTA33, accumulated less in the liver than did RTA33. Monoconjugate immunotoxins (i.e., containing one RTA per monoclonal antibody molecule) were constructed between RTA30 or RTA33 and the antitumor monoclonal antibody 791T/36, which recognizes a Mr 72,000 antigen on osteosarcoma and colon carcinoma cells. The two immunotoxins had similar cytoxicities in vitro but differed substantially in their pharmacokinetics and tissue distributions in vivo in nude mice bearing C170 human colorectal carcinoma xenografts. The immunotoxin derived from RTA30 (IT30) accumulated less in the liver than the immunotoxin derived from RTA33 (IT33) and cleared more slowly from the blood; the alpha and beta half-lives for IT30 and IT33 were 0.50 and 20.5 versus 0.17 and 14.6 h, respectively. As a probable consequence, IT30 accumulated to approximately 3-fold higher levels in the C170 xenografts than IT33. The reduced clearance of IT30 by the reticuloendothelial system thus resulted in prolonged survival in the blood and enhanced tumor localization relative to IT33.

Animals↗
PubMed1991

Toxicity and efficacy of anti-T-cell ricin toxin A chain immunotoxins in a murine model of established graft-versus-host disease induced across the major histocompatibility barrier.

Graft-versus-host disease (GVHD) was induced across the murine major histocompatibility complex by injecting C57BL/6 (H-2b) bone marrow and splenocytes into lethally irradiated B10.BR (H-2k) murine recipients. An immunotoxin (IT) composed of a pan T-cell monoclonal antibody called anti-Ly1 (the murine homologue to human anti-CD5) was conjugated to ricin toxin A chain (anti-Ly1-RTA) and used to treat recipient mice. In vitro, IT was as active as free RTA, bound selectively, and inhibited T-cell proliferation even in the absence of potentiators. Mice administered anti-Ly1-RTA in vivo during ongoing GVHD, at a dose of 10 micrograms/d for 5 days, showed lower numbers of splenic Thy1.2+ T cells and significantly improved survival as compared with mice given phosphate-buffered saline (PBS) or irrelevant control RTA IT. Protection was transient because GVHD and weight loss occurred when injections ceased. Survival could not be enhanced by crosslinking RTA30, a low oligosaccharide-containing fraction of purified RTA. Treatment with anti-Ly1-RTA caused a significant elevation in neutrophils, and higher doses were associated with mild hepatotoxicity. In contrast, infusion of identical doses and schedules of another pan T-cell immunotoxin, anti-Thy1.2-RTA, caused a significant decrease in lymphocytes, but not neutrophils; a precipitous increase in weight; a decrease in total plasma protein (TPP); and an increase in pleural and peritoneal effusions reminiscent of vascular leak syndrome (VLS). Although the toxic effects of anti-Thy1.2-RTA were too severe to show a survival advantage in a GVHD model, histopathologic studies showed a definite anti-GVHD effect. The most significant decline in GVHD as compared with the PBS-treated controls was observed in skin, and to a lesser extent, in liver and lung. To investigate the cause of IT toxicity, anti-Thy1.2-RTA was administered intraperitoneally to lethally irradiated B10.BR (H-2k) recipients of syngeneic bone marrow. These recipients showed the same weight gain, hypoproteinuria, and VLS observed in the GVHD model. Death occurred at higher anti-Thy1.2-RTA doses (30 or 50 micrograms/daily injections administered days 8 through 12 posttransplant). Anti-Thy1.2-RTA had a negligible effect on renal function, but histologic studies showed patchy dropout of the renal tubules. Treatment resulted in pulmonary vascular congestion, but there was no pathologic evidence of liver, brain, or colon toxicity. Weight gain was enhanced by irradiation because nonirradiated normal mice did not undergo such a precipitous weight increase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Effect of cocarboxylase in dogs subjected to experimental septic shock.

We examined the effects of cocarboxylase treatment on both the hemodynamic variables and metabolic function during endotoxic shock in dogs. Cocarboxylase inhibited deterioration in metabolic function as reflected by improved pH and base excess as well as maintenance of normal oxygen consumption. Significant improvements in mean arterial pressure and cardiac index were also seen. Cocarboxylase is a major coenzyme of mitochondrial pyruvate dehydrogenase and may exert its beneficial effects via this complex.

Animals↗
PubMed1989

Patterns of alcohol and drug abuse in an urban trauma center: the increasing role of cocaine abuse.

Alcohol continues to play a major etiologic role in blunt trauma. It is involved in 65-70% of fatal highway crashes. The role of illicit drugs, although accepted as an etiologic factor in trauma, is less well described. This is especially true of blunt and penetrating trauma related to violent crime. During a 9-month period, in a randomly selected group, blood and urine samples were taken from 169 traumatized patients for alcohol and general toxicology screens. There were 81 cases (47.9%) related to violent crime. One hundred twenty-six patients (74.5%) tested positive for illicit or prescription drugs in their blood. These included cocaine (54.4%), cannabinoids (37.2%), barbiturates (7.1%), amphetamines (4.7%), benzodiazepines (10.1%), opiates (9%), and codeine (1%). Sixty-one patients (35.5%) tested positive for alcohol. Alcohol was found in 6.2% of violent crime-related cases. Illicit drugs were found in 80.3% of violent crime-related cases. Alcohol and drugs, especially illicit drugs, are major etiologic factors in both accidental and crime-related trauma in the urban population. All patients admitted to trauma centers, especially urban trauma centers, should be screened for alcohol and drugs.

Accidents, Traffic↗
PubMed1988

Value of creatine phosphokinase isoenzyme determinations in the diagnosis of myocardial contusion.

Myocardial contusion is an injury often missed in the patient with severe multisystem trauma. Fifty patients with physical findings or mechanisms of injury that were suggestive of possible nonpenetrating injury to the heart were studied with serial creatine phosphokinase isoenzyme (CPK/MB), continuous arrhythmia monitoring, and ECGs for at least 72 hours. Two-dimensional echocardiograms were obtained on all patients within 48 hours of admission. Seventy-two percent (36 of 50) had detectable CPK/MB. Ten patients had echocardiograms suggestive of cardiac contusion. Of these ten, three had no detectable CPK/MB and six had CPK/MB of less than 5%. CPK/MB had a sensitivity of 70% and a specificity of 27.5%. We conclude that in the patient with multisystem injury, CPK/MB determinations are not reliable. The decision to evaluate the patient with echocardiography should be made on the basis of physical findings and, more importantly, on mechanism of injury.

Adolescent↗
PubMed1988

Amino acid sequence homology between the enzymic domains of diphtheria toxin and Pseudomonas aeruginosa exotoxin A.

Despite similarities in their enzymic properties, diphtheria toxin (DT) and exotoxin A (ETA) of Pseudomonas aeruginosa have major differences in structure and action: consequently, the question of possible evolutionary relatedness of these two proteins remains unanswered. Here we report the existence of significant amino acid sequence homology between the enzymic domain of DT and that of ETA. Major segments of sequence may be aligned with high percentages of identity and of conservative substitutions. The homologous stretches in ETA form much of the active-site cleft in the X-ray crystallographic structure. This evidence implies that these domains, at least, have diverged from a common ancestral protein and that active-site residues have been strongly conserved.

ADP Ribose Transferases↗
PubMed1988

The lateral approach to the above-knee femoropopliteal bypass.

Functional salvage of the limb was successful in a patient using a lateral approach to the above-knee femoropopliteal bypass. An expanded polytetrafluoroethylene prosthesis was placed in a lateral subcutaneous tunnel. This new technique offers a direct and practical alternative to the classic approaches to the proximal part of the popliteal artery and may be useful when previous scarring, infection or unusual circumstances precludes their use.

Anastomosis, Surgical↗
PubMed1987

Active site of Pseudomonas aeruginosa exotoxin A. Glutamic acid 553 is photolabeled by NAD and shows functional homology with glutamic acid 148 of diphtheria toxin.

Photoaffinity labeling with native NAD, a method employed earlier with diphtheria toxin (DT), was used to identify an active site residue of Pseudomonas aeruginosa exotoxin A (ETA). An enzymically active fragment (Mr 27,000), derived by partial digestion of ETA with thermolysin, was irradiated with ultraviolet light (254 nm) in the presence of various radiolabeled preparations of NAD. Label from the nicotinamide moiety was efficiently transferred to the protein (maximally 0.79 mol/mol), and the label was exclusively located at position 553. This position, like that photolabeled in DT (position 148), corresponds to glutamic acid in the native protein. Chromatographically identical photo-products were generated at these positions in the two toxins. Glu-553 lies in a cleft in domain III that is believed to represent the active site of ETA, and other evidence supports the notion that Glu-553 of ETA and Glu-148 of DT are directly involved in catalysis. When Glu-553 of ETA was aligned with Glu-148 of DT, we found similarities of local primary structure not detected earlier. These results suggest that the catalytically active domains of ETA and DT may be evolutionarily related, and they provide information that should prove useful for preparing vaccines against ETA by recombinant DNA methods.

ADP Ribose Transferases↗
PubMed1986

Dimeric form of diphtheria toxin: purification and characterization.

Many preparations of diphtheria toxin were found to contain dimeric and multimeric toxin forms. The monomeric and dimeric forms were fractionated to greater than 98% purity, and their properties were compared. Dimeric toxin slowly dissociated to native monomers in solution at neutral pH and could be rapidly dissociated with dimethyl sulfoxide. In cell culture assays and rabbit skin tests, the dimer exhibited no significant toxic activity, except for that attributable to trace contamination by monomer, or partial dissociation to monomer during the incubation period. In guinea pig lethality tests, however, toxic activity varied depending upon the dose. At least 7-fold greater amounts of dimer than monomer (161 ng vs. 22 ng, respectively) were required to cause death at 18 h, whereas similar weights of the two toxin forms (22 ng) caused death at 120 h. This variability probably reflected slow dissociation of dimer to monomer in the animal. The dimer was unable to bind toxin receptors on the surface of susceptible cells, whereas it retained full activity in the ADP-ribosyltransferase, NAD-glycohydrolase, or ligand-binding assays. Thus, the lack of toxicity of the dimeric toxin may have resulted from distortion or occlusion of the receptor binding site on the B moiety. We propose that the dimer contains two monomeric units bound by hydrophobic interactions and that the points of contact involve regions of the B moieties that are normally buried in the native monomer.

Animals↗
PubMed1986

Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution.

Exotoxin A of Pseudomonas aeruginosa is a secreted bacterial toxin capable of translocating a catalytic domain into mammalian cells and inhibiting protein synthesis by the ADP-ribosylation of cellular elongation factor 2. The protein is a single polypeptide chain of 613 amino acids. The x-ray crystallographic structure of exotoxin A, determined to 3.0-A resolution, shows the following: an amino-terminal domain, composed primarily of antiparallel beta-structure and comprising approximately half of the molecule; a middle domain composed of alpha-helices; and a carboxyl-terminal domain comprising approximately one-third of the molecule. The carboxyl-terminal domain is the ADP-ribosyltransferase of the toxin. The other two domains are presumably involved in cell receptor binding and membrane translocation.

ADP Ribose Transferases↗
PubMed1985

Photoaffinity labeling of diphtheria toxin fragment A with NAD: structure of the photoproduct at position 148.

Irradiation of mixtures of diphtheria toxin fragment A and [carbonyl-14C]NAD with UV light (253.7 nm) is known to induce efficient transfer of the radiolabel to position 148, corresponding to glutamic acid in the unmodified protein. Here we report the structure of the photoproduct at position 148, as determined by chemical and photochemical methods, fast-atom-bombardment mass spectrometry, and nuclear magnetic resonance. The photoproduct [an alpha-amino-gamma-(6-nicotin-amidyl)butyric acid residue] contains the entire nicotinamide moiety of NAD linked via its number 6 carbon to the decarboxylated gamma-methylene carbon of Glu-148. No portion of the ADP-ribosyl group of NAD is present. These findings are consistent with the idea that Glu-148 lies at or near the catalytic center of diphtheria toxin.

ADP Ribose Transferases↗
PubMed1985

Antithrombin III deficiency: an etiology of Budd-Chiari syndrome.

This report documents a unique case of Budd-Chiari syndrome associated with antithrombin III deficiency and massive thrombus in the superior vena cava and right atrium. This aberration of the coagulation mechanism is proposed as an etiologic factor in the pathogenesis of hepatic venous obstruction whenever the cause is obscure. Antithrombin III was restored to a level adequate to permit thrombus extraction with cardiopulmonary bypass and relief of portal hypertension with a mesoatrial shunt. The protocol for reversing this hypercoagulable state with fresh frozen plasma and warfarin (Coumadin) is discussed, and technical innovations employed in the management of this complex problem are described.

Antithrombin III Deficiency↗
PubMed1984

NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD.

We showed earlier that exposing mixtures of NAD and diphtheria toxin fragment A to ultraviolet radiation (253.7 nm) induced the formation of covalently linked protein-ligand photoproducts. Here we report that when [carbonyl-14C]NAD was employed in such procedures, the efficiency of labeling of the protein approached 1 mol/mol, and at least 94% of the incorporated label was associated with a single residue, glutamic acid at position 148. Fragment A photolabeled in this manner was enzymically inactive. The efficiency of photolabeling was much lower (less than 0.2 mol/mol) when NAD radiolabeled in either the adenine moiety or the adenylate phosphate was used, and the label was attached to different site(s) within fragment A. Efficient photochemical transfer of label from [carbonyl-14C]NAD occurred under identical conditions with the nucleotide-free form of whole diphtheria toxin, CRM-45, or activated exotoxin A from Pseudomonas aeruginosa, but not with nucleotide-bound diphtheria toxin, CRM-197, native exotoxin A, or any of several NAD-linked dehydrogenases. On the basis of these and other results we suggest that part or all of the nicotinamide moiety of NAD is efficiently transferred to glutamate-148 of fragment A under the influence of ultraviolet irradiation and that this residue is located within the nicotinamide subsite. This location implies that glutamate-148 is at or near the catalytic center of the toxin. Our data provide direct evidence for the location of the NAD site in an ADP-ribosylating toxin and demonstrate highly efficient and specific photolabeling by [carbonyl-14C]NAD.

Binding Sites↗
PubMed1981

An endogenous dinucleotide bound to diphtheria toxin. Adenylyl-(3',5')-uridine 3'-monophosphate.

Diphtheria toxin has recently been fractionated into two forms, one of which contains tightly but noncovalently bound nucleotide-like material. Here we report identification of the major nucleotide constituent (comprising 80% of the total extractable ultraviolet-absorbing material) as adenylyl-(3',5')-uridine 3'-monophosphate (ApUp). Upon incubation with ApUp, the nucleotide-free form of toxin bound approximately one molar equivalent of the dinucleotide and was converted to a form similar or identical with the native nucleotide-bound form.

Adenosine Monophosphate↗
PubMed1981

Antibacterial peptide from normal rat serum. 1. Isolation from whole serum, activity, and microbicidal spectrum.

A procedure is described for purification of the primary bactericidal component of normal rabbit serum active in vitro against Bacillus subtilis. A 65 000-fold increase in specific bactericidal activity per milligram of serum protein was obtained, yielding a low molecular weight, heat-stable polypeptide fraction (PC-III) exhibiting biological activity at protein concentrations below 10 ng/mL. This preparation appeared homogeneous as judged by column chromatography and analytical NaDodSO4-polyacrylamide gel eletrophoresis; recovery of serum bactericidal activity was routinely greater than 80%. Analysis of dansylated or 125I-labeled samples in peptide-resolving polyacrylamide gels revealed a single band with an Mr of 1800. Optimal antibacterial activity of PC-III against B. subtilis occurred at an ionic strength of 0.24 and was absolutely dependent upon divalent cations; calcium was the most effective. Under optimum conditions, 4 ng/mL of PC-III reduced the viability of B. subtilis test innocula by 90% within 10 min at 37 degrees C. Listeria monocytogenes, Escherichia coli, and Salmonella typhimurium were all sensitive to the action of PC-III, but higher bactericide concentrations were required to produce similar reductions in viability as observed with B. subtilis. All strains were killed by PC-III concentrations well below 1 microgram/mL, roughly that found in normal serum. The activity of PC-III preparations was significantly reduced by pretreatment with trypsin or proteinase K but not by neuraminidase or periodate.

Animals↗
PubMed1981

Antibacterial peptide from normal rabbit serum. 2. Compositional microanalysis.

The composition and homogeneity of the primary bactericide (PC-III) isolated from normal rabbit serum were examined by microanalytical techniques. Dansylated acid hydrolysates of PC-III were analyzed by two-dimensional thin-layer chromatography on polyamide layers. Quantitation of the separated dansyl amino acids was achieved by elution into methanol, followed by determination of fluorescence in a filter fluorometer. The method proved inexpensive and reliable, allowing accurate analysis of peptide samples containing greater than 20 pmol of each amino acid. By use of this technique, an amino acid composition for PC-III is presented which suggests that the rabbit serum bactericide contains a single, 2000-dalton peptide composed of 17 amino acid residues, 24% basic and 35% nonpolar. One of the basic residues appears to be a modified amino acid, tentatively identified as N epsilon-methyllysine. Although small amounts of carbohydrate and lipid were also detected in PC-III by fluorescent and isotopic techniques, gel filtration or extraction with organic solvents removed much of this material without significantly affecting biological activity.

Amino Acids↗