Biomedical subjects

R J Collier

Publications and source records attributed to R J Collier.

At least 199 records · Page 11Linked to original sources

PubMed1987

The concentration of bovine placental lactogen and the incidence of different forms in fetal cotyledons and in fetal serum.

The concentration of bovine placental lactogen (bPL) was determined in fetal placentomes, allantoic fluid, amniotic fluid, maternal and fetal plasma throughout pregnancy. In addition, chromatofocusing chromatography was used to separate the different forms of bPL found both in fetal serum and in placental homogenates in order to determine whether the different forms that have been reported to exist in the cotyledon are also found in the fetal circulation. Reproductive tracts were collected from cows between 109 and 247 days of pregnancy. The concentration of bPL in the fetal cotyledonary tissue was measured by both radioreceptor assay and radioimmunoassay, both assays showed that the concentration of bPL in the fetal portion of the placentomes remained constant throughout the period of pregnancy tested. The mass of the placenta increased approximately 10-fold during the period of study but the concentration of bPL in the maternal plasma was low (0.9 +/- 0.1 ng/ml) at all stages of pregnancy tested. The mean concentration of bPL (Mean +/- S.E.M.) in amniotic and allantoic fluid was 0.4 +/- 0.1 and 1.2 +/- 0.2 ng/ml respectively. Fetal blood contained the highest concentrations of bPL, from 11.6 to 18.4 ng/ml, and the concentration tended to decrease with advancing gestation (slope = 0.07, P = 0.001). Several forms of bPL were found in the fetal circulation; however, a higher percentage of forms with more acidic isoelectric points were found in the fetal serum than in placental homogenates. These results suggest that either some forms of bPL are more stable or that the hormone isolated from placental tissue is not representative of the final secreted product.

Allantois↗

PubMed1987

Exotoxin A of Pseudomonas aeruginosa: evidence that domain I functions in receptor binding.

We have constructed defined deletions in the structural gene of Pseudomonas aeruginosa exotoxin A (ETA) in order to probe the function of Domain I of this protein. Three forms of the gene containing specific deletions were expressed in a strain of Escherichia coli K12 with lesions in the htpR and Ion genes; extracts containing the gene products were tested for ADP-ribosylation activity, cytotoxicity, and ability to protect sensitive cells from the cytotoxic action of authentic ETA. Two of the mutant ETAs gave concentration-dependent protection against authentic ETA, and protection correlated with the presence of the bulk of Domain I. The results support the notion that Domain I functions in binding the toxin to specific cell-surface receptors.

ADP Ribose Transferases↗

PubMed1987

Expression of the S-1 catalytic subunit of pertussis toxin in Escherichia coli.

The S-1 subunit of pertussis toxin was expressed as a fusion protein in a strain of Escherichia coli deficient in protein degradation. The fusion protein reacted with anti-pertussis toxin antibody, and, like authentic pertussis toxin, it ADP-ribosylated a 41,000-molecular-weight membrane protein from human erythrocytes.

Bordetella pertussis↗

PubMed1987

Expression of a mutant, full-length form of diphtheria toxin in Escherichia coli.

A mutant, full-length form of diphtheria toxin was cloned into Escherichia coli K-12 and expressed under BL-1 + EK-1 containment. A gene fragment encoding the catalytic domain of the toxin was subjected to oligonucleotide-directed mutagenesis to produce a three-base mutation of an active site residue; Glu-148 was thereby replaced by Ser. Ser-148 fragment A had less than 1% of the ADP-ribosyltransferase activity of wild-type fragment A. Next, the complementary portion of the toxin structural gene was spliced with the mutated DNA fragment downstream of codon 148 to produce a construct that encoded mutant whole toxin with Ser at position 148. The mutant toxin was indistinguishable from authentic diphtheria toxin by Western blot analysis, but was about 800-fold less cytotoxic than wild-type toxin for BS-C-1 cells. Evidence from subunit exchange experiments indicated that a substantial fraction of the mutant toxin contained a fully functional B moiety, capable of mediating the entry of wild-type fragment A into sensitive mammalian cells. This combination of approaches provides a means of applying recombinant DNA methods in E. coli to study structure-function relationships in whole diphtheria toxin.

ADP Ribose Transferases↗

PubMed1987

Exotoxin A of Pseudomonas aeruginosa: active, cloned toxin is secreted into the periplasmic space of Escherichia coli.

We subcloned the structural gene for exotoxin A (ETA) of Pseudomonas aeruginosa in front of the tac promoter in an Escherichia coli expression vector and studied the intracellular location and properties of the protein product. The E. coli K-12 strain that carried this recombinant plasmid produced an immunoreactive protein that was identical to authentic ETA in size and in cytotoxic and ADP-ribosyl transferase activities per unit of immunoreactive material. The protein was predominantly in the periplasmic fraction; and a mutation in the secA gene blocked secretion, processing, and conversion of the protein to a fully toxic conformation. The results indicate that expression of the ETA gene in E. coli yields native ETA, which is localized within the periplasmic space. This organism may therefore serve as a useful host for studying structure and function in ETA.

ADP Ribose Transferases↗

PubMed1987

Exotoxin A of Pseudomonas aeruginosa: substitution of glutamic acid 553 with aspartic acid drastically reduces toxicity and enzymatic activity.

Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) has been identified by photoaffinity labeling as a residue within the NAD binding site (S.F. Carroll and R.J. Collier, J. Biol. Chem. 262:8707-8711, 1987). To explore the function of Glu-553 we used oligonucleotide-directed mutagenesis to replace this residue with Asp in cloned ETA and expressed the mutant gene in Escherichia coli K-12. ADP-ribosylation activity of Asp-553 ETA in cell extracts was about 1,800-fold lower and toxicity for mouse L-M929 fibroblasts was at least 10,000-fold lower than that of the wild-type toxin. Extracts containing Asp-553 ETA inhibited the cytotoxicity of authentic ETA on L-M929 fibroblasts, suggesting that the mutant toxin competes for ETA receptors. The results indicate that Glu-553 is crucial for ADP-ribosylation activity and, consequently, cytotoxicity of ETA. Substitution or deletion of this residue may be a route to new ETA vaccines.

ADP Ribose Transferases↗

PubMed1987

Secretion and mammary gland uptake of prolactin in dairy cows during lactogenesis.

Mammary arteriovenous differences of prolactin concentration and net mammary uptake of prolactin from blood were quantified near parturition in 9 dairy cows. Six cows were milked once daily for at least 6 d before parturition, and prepartum lactogenesis occurred in 3 of 6 cows. Prepartum milking 2 or more d before parturition abruptly increased secretion of prolactin into blood, but milkings within 1 d before or after parturition did not increase prolactin secretion. Concentrations of prolactin in whole milk sampled over 8 d before parturition (64.5 ng/ml) were substantially greater than those occurring several days after parturition (19 ng/ml). Milk prolactin concentrations were unaffected by the successful induction of prepartum lactogenesis, which greatly increased prepartum yields of milk (2 to 8 kg/milking). Therefore, the alveolar lumenal content of prolactin was greatest in pregnant cows with prepartum lactogenesis. This enhanced content of intraalveolar prolactin before parturition was associated with an absence of mammary uptake of prolactin immediately prior to ejection of the prolactin-containing milk from the alveoli. However, prolactin uptake was quickly restored to about 2 micrograms/min per half udder shortly after milk ejection. During the prepartum period, an enhanced intraalveolar reservoir of 200 to 400 micrograms prolactin, due to induction of prepartum lactogenesis, appears to saturate temporarily all putative sites for uptake of prolactin from blood.

Animals↗

PubMed1987

Periparturient changes in secretion and mammary uptake of insulin and in concentrations of insulin and insulin-like growth factors in milk of dairy cows.

Mammary uptake of insulin from blood was quantified near parturition in nine dairy cows, some of which were milked prepartum to induce prepartum lactogenesis. Milk samples were immunoassayed for concentrations of insulin, insulin-like growth factor I, and insulin-like growth factor II. Cows milked prepartum, but in which prepartum lactogenesis did not occur (denoted as unsuccessful group), had higher arterial concentrations and greater mammary uptake of insulin during several days immediately preceding parturition than did those with prepartum lactogenesis. Although mammary uptake of insulin was measurable before parturition, estimated uptake on postpartum d 4 through 6 was negligible. Colostrum and milk contained very substantial quantities of insulin, insulin-like growth factor I, and insulin-like growth factor II. Cows in which prepartum lactogenesis was induced (denoted as successful group) had lower concentrations of both forms of insulin-like growth factor in prepartum samples of milk. Cows of the successful group also had a decreasing prepartum profile of milk insulin, but the unsuccessful group had an increasing trend until parturition. Milk concentrations of insulin and both forms of insulin-like growth factor declined rapidly after parturition and completion of lactogenesis.

Animals↗

PubMed1986

Diphtheria toxin can simultaneously bind to its receptor and adenylyl-(3',5')-uridine 3'-monophosphate.

Diphtheria toxin that was bound to receptors on BS-C-1 cells was able to bind approximately 1 molar equiv of adenylyl-(3',5')-uridine 3'-monophosphate (ApUp). In contrast, receptor-bound CRM197, a mutant form of toxin with greatly diminished affinity for dinucleotides, did not bind ApUp. Affinity of the dinucleotide for receptor-bound toxin differed from that for free toxin by less than an order of magnitude. These results indicate that the receptor site and the ApUp site on the toxin do not significantly overlap.

Animals↗

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↗

PubMed1986

Effect of number of conceptuses on maternal hormone concentrations in the pig.

This study examined the effect of number of conceptuses on maternal concentrations and profiles of estrogen sulfate, estrone, estradiol-17 beta, progesterone and prolactin in gilts. Estradiol-valerate injections were used to induce pseudopregnancy (O conceptuses; n = 5) and oviduct ligation or no treatment were utilized to obtain pregnant gilts with 4 to 7 (n = 4), or 8 to 11 (n = 4) conceptuses, respectively. Blood samples were collected every 10 d from d 10 through 110 of pregnancy or pseudopregnancy. At 110 d after onset of estrus, all gilts were slaughtered and numbers and(or) weights of fetuses, corpora lutea, placentae and the empty uterus were determined. Concentrations of estrogen sulfate and estrone, but not progesterone or prolactin, were associated with fetal number, total fetal weight, total placental weight or empty uterine weight. In contrast, only progesterone was highly correlated with number of corpora lutea. Results suggest that most conjugated estrogen, estrone and estradiol were of fetal-placental origin, whereas little, if any, placental production of progesterone or prolactin occurred. Increases in estrogen sulfate and estrone concentrations were observed at gestation d 30 and from d 70 to 100. The latter increase coincides with previously established increases in the rate of maternal mammary development and fetal growth.

Animals↗

PubMed1986

Factors influencing passive transfer in dairy calves.

Records from 2105 calves born over 2 yr on a large dairy in a subtropical climate were examined to delineate factors that influence passive transfer of colostral immunoglobulins and the effect of passive transfer of immunoglobulin on calf mortality. Seasonal effects on immunoglobulin absorption were detected with highest serum total protein occurring in February and March. Lower total protein concentrations were associated with elevated environmental temperatures in the summer months. Maternal antibody was highest in calves from second parity cows; and dystocia appeared to decrease the amount of immunoglobulin absorbed by the neonatal calf. Calves that died of infectious disease during the first 14 wk of life had significantly lower serum total protein concentrations than those that lived. Heritability of serum protein concentration in calves was .02 +/- .03.

Animals↗

PubMed1986

Effect of dry period versus continuous milking on periparturient concentrations of bovine prolactin in milk from half udders.

Four multiparous cows were used to determine whether continuous twice daily milking of half the udder throughout the dry period would alter the prepartum accumulation of prolactin in secreted milk. The control half of each udder was allowed a dry period of 43 d, and twice daily milking was reinitiated in this half udder 11 d before parturition, whereas the treated half was milked continuously throughout. Milk from the treated half decreased to very low yields despite continuous milking, whereas concentrations of prolactin in milk remained low and without much day-to-day variation within each cow. Concentrations of prolactin in milk and yields of milk increased during the week before parturition in both halves of the udder, but there were no significant differences due to previous continuous milking. Therefore, prepartum accumulation of high concentrations of prolactin in secreted milk did not appear to depend on prior occurrence of a dry period. Elevated concentrations of prolactin in milk declined rapidly and equally in each half udder after parturition.

Animals↗

PubMed1986

Ultrastructural changes in porcine mammary tissue during lactogenesis.

Ultrastructural changes occurring in porcine mammary tissue were characterised between Day 90 of pregnancy and Day 4 of lactation. Porcine mammary tissue on Day 90 of pregnancy was composed of alveoli which contained negligible to moderate amounts of secretion. Epithelial cells of these alveoli were relatively undifferentiated. The appearance and distribution of cellular organelles suggested that mammary epithelial differentiation had been initiated by Day 105 of pregnancy in the pig. A further increase in intracellular lipid droplets and granular endoplasmic reticulum suggested that differentiation had progressed by Day 112. On the day of parturition, secretions within the alveolar lumina assumed the appearance of normal milk (as opposed to colostrum) and the epithelia displayed a distinct cellular polarity characteristic of lactating mammary tissue. By Day 4 of lactation, differentiation of epithelial cells appeared to be complete, with dilated cisternae of the granular endoplasmic reticulum and with numerous secretory vesicles. Elongated microvilli were present and numerous cells contained lipid droplets which were being extruded into the lumina. Data from this and previous studies indicate that lactogenesis in the pig occurs in two stages. Stage 1 occurs between Days 90 and 105 of pregnancy, and Stage 2 between Days 112 of pregnancy and early lactation when the predominant feature is active milk secretion.

Animals↗

PubMed1985

Diphtheria toxin. Effect of substituting aspartic acid for glutamic acid 148 on ADP-ribosyltransferase activity.

Photoaffinity labeling experiments with diphtheria toxin fragment A have implicated glutamic acid 148 as a constituent of the NAD binding site. To evaluate the role of this residue in ADP-ribosylation of elongation factor 2, we replaced it with aspartic acid by in vitro mutagenesis of a toxin gene fragment cloned in Escherichia coli. Fragment A containing aspartic acid at position 148 had less than 0.6% the ADP-ribosylation activity of wild-type fragment A. The mutation produced no change in sensitivity of fragment A to trypsin and little, if any, reduction in affinity of fragment A for NAD. These results indicate that glutamic acid 148 is essential for the ADP-ribosylation of elongation factor 2 and are consistent with other data suggesting that this residue may be at or near the catalytic center of the toxin.

Aspartic Acid↗

PubMed1985

Circular dichroism of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and various derivatives.

We have recorded the near- and far-ultraviolet circular dichroism spectra of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and derivatives of these toxins. The far-ultraviolet spectra of various forms of diphtheria toxin were virtually identical, implying that no major changes in secondary structure accompany proteolytic nicking or dimerization of toxin, or binding of the endogenous dinucleotide, adenylyl-(3'-5')-uridine 3'-monophosphate (AdoPUrdP). Alpha-helix content was estimated to be 29%, as compared with 8% for fragment A. Near-ultraviolet spectra were identical between nicked and intact diphtheria toxin. A broad negative transition with a minimum at 304 nm was assigned to the intrachain disulfide bridge within the B moiety. Dimeric diphtheria toxin showed perturbations of aromatic residues. Binding of AdoPUrdP to monomeric diphtheria toxin or of adenylyl-(3',5')-uridine (AdoPUrd) to fragment A perturbed one or more tryptophans. The latter results correlate with evidence for involvement of a tryptophan in NAD binding. Native exotoxin A was estimated to have 16% alpha-helix, and the activated form of exotoxin A, 11%. An enzymically active, 31 kDa proteolytic fragment of exotoxin A showed similar alpha-helix content (7%) to that of diphtheria toxin fragment A.

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↗