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R J Collier

Publications and source records attributed to R J Collier.

At least 19 recordsLinked to original sources
PubMed1992

pH-dependent insertion of proteins into membranes: B-chain mutation of diphtheria toxin that inhibits membrane translocation, Glu-349----Lys.

To investigate how diphtheria toxin (DT) undergoes pH-dependent membrane translocation in mammalian cells, we have isolated and characterized mutants of the toxin that are defective in acidic-pH-dependent killing of Escherichia coli. Cloned DT secreted to the periplasm of E. coli kills the bacteria under acidic conditions (near pH 5.0) by inserting into and permeabilizing the inner membrane (a mechanism independent of the toxin's ADP-ribosylation activity). Mutant forms of DT with reduced lethality for E. coli were selected by plating the bacteria under acidic conditions. CRM503, one of the full-length mutants selected by this protocol, also showed diminished cytotoxicity for mammalian cells. We traced the altered cytotoxicity of CRM503 to a Glu-349----Lys mutation (E349K), one of three point mutations, within the B fragment. The E349K mutation alone inhibited cytotoxicity and membrane translocation in mammalian cells and lethality for E. coli but did not affect enzymic activity or receptor binding. The recently determined crystallographic model of DT shows that Glu-349 resides within a short loop connecting two long hydrophobic alpha-helices of the translocation domain. Protonation of Glu-349 and two other nearby acidic residues, Asp-352 and Glu-362, may enable these helices to undergo membrane insertion and the intervening loop to be transferred to the opposite face of the bilayer. The E349K mutation introduces a positive charge at this site, which would be expected to inhibit membrane insertion and the insertion-dependent activities of DT. These results suggest that protonation of Glu-349 and nearby acidic residues may be important in triggering the translocation step of toxin action.

Animals↗
PubMed1992

Reversion of recombinant toxoids: mutations in diphtheria toxin that partially compensate for active-site deletions.

Deleting an important active-site residue of diphtheria toxin, glutamic acid-148, reduces the toxin's ADP-ribosyltransferase activity by a factor of greater than 10(4). We considered using this mutation to construct a recombinant toxoid for expression by live attenuated vaccines and explored second-site mutations that might cause reversion. Activity was partially restored by substituting glutamic acid for valine-147 or by extending the deletion by five residues toward the NH2 terminus, thereby placing glutamic acid-142 immediately adjacent to tyrosine-149. In both mutants the indicated glutamic acid may occupy a spatial locus similar to that of glutamic acid-148 in the unmutated protein. Simply deleting a crucial residue does not, therefore, provide confidence that a second-site mutation could not readily restore activity to a toxoid.

Amino Acid Sequence↗
PubMed1992

The crystal structure of diphtheria toxin.

The crystal structure of the diphtheria toxin dimer at 2.5 A resolution reveals a Y-shaped molecule of three domains. The catalytic domain, called fragment A, is of the alpha + beta type. Fragment B actually consists of two domains. The transmembrane domain consists of nine alpha-helices, two pairs of which are unusually apolar and may participate in pH-triggered membrane insertion and translocation. The receptor-binding domain is a flattened beta-barrel with a jelly-roll-like topology. Three distinct functions of the toxin, each carried out by a separate structural domain, can be useful in designing chimaeric proteins, such as immunotoxins, in which the receptor-binding domain is substituted with antibodies to target other cell types.

Bacterial Toxins↗
PubMed1992

Mutations affecting the activity of the Shiga-like toxin I A-chain.

Like ricin, Escherichia coli Shiga-like toxin I (SLT-I) inactivates eukaryotic ribosomes by catalytically depurinating adenosine 4324 in 28S rRNA. Although the primary structure of the enzymatic portion of the molecule (Slt-IA) is known to contain regions of significant homology to the ricin A chain (RTA), and although certain residues have been implicated in catalysis, the crystal structure of Slt-IA has not been solved nor has the geometry of its active site been well defined. In order to derive a more complete understanding of the nature of the Slt-IA active site, we placed the slt-IA gene under control of an inducible promoter in Saccharomyces cerevisiae. Induction of the cloned element was lethal to the host. This lethality was the basis for selection of an attenuated mutant of Slt-IA changed at tyrosine 77, a locus not previously linked to the active site. As well, it permitted evaluation of the toxicity of a number of mutant Slt-IA cassettes that we constructed in vitro. Putative active-site residues implicated in this fashion and in other studies were mapped to an energy-minimized computer model of Slt-IA that had been generated on the basis of the known crystal structure of RTA. A cleft was identified on one face of the protein in which all implicated residues clustered, irrespective of their distances from one another in the primary structure of the molecule. Many of the chemical features anticipated in the active site of an RNA N-glycosidase are indeed present on the amino acid side chains occupying the cleft.

Amino Acid Sequence↗
PubMed1992

Conformational integrity of a recombinant toxoid of Pseudomonas aeruginosa exotoxin A containing a deletion of glutamic acid-553.

A mutant form of Pseudomonas aeruginosa exotoxin A (ETA) carrying a deletion of glutamic acid-553, an important active-site residue, was expressed in an ETA-negative strain of P. aeruginosa and shown to be exported from the cells as efficiently as wild-type ETA. The mutant protein, purified from the culture medium, was devoid of ADP-ribosyltransferase activity. Protein conformation was barely perturbed by the deletion, as determined by a number of measures, including affinity for substrate NAD, proteinase sensitivity, absorbance and fluorescence spectroscopy, and differential scanning calorimetry. The conformational integrity and stability of the mutant toxin are consistent with potential use of the protein in vaccines or as a carrier in preparing conjugate vaccines.

ADP Ribose Transferases↗
PubMed1992

Locating a residue in the diphtheria toxin channel.

We are studying structure-function relationships in the Diphtheria Toxin (DT) channel using a combination of site-directed mutagenesis and electrophysiology in artificial lipid bilayers. We made site-directed mutations of charged residues in the toxin's channel-forming B fragment, and after expressing the mutant proteins in Escherichia coli, we analyzed the single channels they formed in lipid bilayers. Changing aspartate 352, which is located in a short hydrophilic loop separating two hydrophobic stretches, to asparagine or lysine dramatically reduces the single-channel conductance of the pore at pH 5.3 cis, 7.2 trans (5.3/7.2). Lowering the pH on both sides of the membrane essentially eliminates the difference between wild-type and D352N; this finding is consistent with the idea that an aspartate with a (protonated) neutral side-chain and the always neutral asparagine have similar electrostatic influences on permeant ions. Using a high concentration of permeant buffer to clamp the pH of the cis compartment and the pore, and varying the pH on the trans side, we have located D352 at or near the trans compartment. We further find that D352N channels, in contrast to wild-type, display conductances independent of trans pH. This observation allows us to determine the titration curve of aspartate 352 in the wild-type toxin, establishing its pKa at approximately 5.5.

Amino Acid Sequence↗
PubMed1992

Comparative mitogenic and galactopoietic effects of IGF-I, IGF-II and Des-3-IGF-I in bovine mammary gland in vitro.

Insulin-like growth factors (IGFs) I and II (IGF-I, IGF-II) and Des-3-IGF-I at physiological concentrations are potent mitogens of bovine undifferentiated mammary epithelial cells cultured in collagen in a serum-free medium. Des-3-IGF-I was found to be as potent as IGF-I, while IGF-II was significantly less active. All three factors acted either synergistically or additively with epidermal growth factor (EGF), cholera toxin and fetal calf serum (FCS). Indirect evidence indicates that despite its lower mitogenic activity the action of IGF-II is mediated through IGF-I receptors. The galactopoietic activity of Des-3-IGF-I and IGF-II was studied in an organ culture of bovine lactating mammary glands using lactogen-responsive fat synthesis as a test. Neither Des-3-IGF-I nor IGF-II exhibited galactopoietic activity nor did they affect the galactopoietic activity of prolactin.

Animals↗
PubMed1992

Biochemical and physiological changes induced by anthrax lethal toxin in J774 macrophage-like cells.

Experiments were performed to probe the mechanism by which Bacillus anthracis Lethal Toxin (LeTx) causes lysis of J774 macrophage-like cells. After incubation of cells with saturating concentrations of the toxin, two categories of effects were found, which were distinguishable on the basis of chronology, Ca(2+)-dependence, and sensitivity to osmolarity. The earliest events (category I), beginning 45 min postchallenge, were an increase in permeability to 22Na and 86Rb and a rapid conversion of ATP to ADP and AMP. Later events (category II) included alterations in membrane permeability to 45Ca, 51Cr, 36Cl, 35SO4, 3H-amino acids, and 3H-uridine, beginning at 60 min; inhibition of macromolecular synthesis, leakage of cellular lactate dehydrogenase and onset of gross morphological changes, at approximately 75 min; and cell lysis, beginning at 90 min. Category II events exhibited an absolute requirement for extracellular Ca2+ and were blocked by addition of 0.3 M sucrose to the medium, whereas category I events were attenuated, but not blocked, by either of these conditions. On the other hand, both ATP depletion and the category II events were blocked in osmotically stabilized medium that was also isoionic for Na+ and K+. This suggests that permeabilization of the plasma membrane to monovalent cations and water may be the earliest of the physiological changes described here. The resulting influx of Na+ and efflux of K+ would be expected to cause depletion of ATP, via increased activity of the Na+/K+ pump. Subsequently the influx of Ca2+, induced by depletion of ATP, imbalances in monovalent cautions, and/or more dramatic changes in permeability due to influx of water, would be expected to trigger widespread changes leading ultimately to cytolysis.

Adenosine Diphosphate↗
PubMed1992

Impact of somatotropins on nutrient requirements in domestic animals.

Somatotropins increase nitrogen retention, decrease carcass fat and increase lean/fat ratio in growing ruminants and swine. However, responses are much more variable in growing ruminants. Maintenance requirements in both ruminants and swine are increased slightly with somatotropin treatment in conjunction with increased lean mass. This is associated with increases in partial efficiency of protein accretion, resulting in increased efficiency of growth. Additional information is required to accurately assess effects of somatotropin on nutrient requirements for growing ruminants and swine. Future studies should target degradable and nondegradable protein requirements for growing ruminants and the impact of somatotropin on these requirements; definition of dose, pattern and formulation of somatotropin for both ruminants and swine and interaction of somatotropin with other factors affecting feed intake in swine and ruminants.

Animals↗
PubMed1992

DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.

A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant. A null mutation of the chromosomal DPH5 gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fully active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.

Amino Acid Sequence↗
PubMed1992

Destripeptide insulin-like growth factor-I in milk from bovine somatotropin-treated cows.

Total somatomedins from milk of bovine somatotropin-treated cows were isolated and characterized to determine the relative amount of the three amino acid N-terminally truncated form of IGF-I (destripeptide IGF-I). The somatomedin fraction was isolated using organic solvent and solid-phase extractions followed by preparative reverse phase HPLC and affinity chromatography. The overall yield of IGF-I was 28%, and destripeptide IGF-I was recovered with similar efficiency. The isolated somatomedins were resolved by capillary zonal electrophoresis and identified using recombinant somatomedin standards. The concentration of destripeptide IGF-I relative to full length IGF-I was determined by amino terminal sequencing and by bioassay. Results from these experiments indicated that the level of destripeptide IGF-I in milk from somatotropin-treated cows was less than 3% of the IGF-I concentration. Destripeptide IGF-I is therefore a minor component of the somatomedins present in milk from treated cows and does not contribute significantly to the proliferative activity of this milk.

Animals↗
PubMed1992

Serum half-life and in-vivo actions of recombinant bovine placental lactogen in the dairy cow.

The clearance rate of recombinant bovine placental lactogen (rbPL) from the blood serum of four lactating dairy cows was measured using a specific radioimmunoassay. Two animals were non-pregnant, while the other two were at approximately 120 days of gestation. The rbPL was administered as an i.v. bolus injection (4 mg total) via an indwelling jugular catheter. Blood samples were taken periodically for 180 min and assayed for rbPL. Analysis of the clearance curves for the bolus injection suggested a single-compartment model and a serum half-life of 7.25 min. In a second experiment with the same animals, following cessation of lactation, rbPL or bovine GH (bGH) were administered by s.c. injection (50 mg/day) for 5 consecutive days. Blood samples were taken twice per day during the treatment period and a 3-day pretreatment period. Samples were analysed for glucose, blood urea nitrogen (BUN), non-esterified fatty acids (NEFA), creatinine, insulin, insulin-like growth factor-I (IGF-I) and IGF-II, tri-iodothyronine (T3), progesterone and IGF-binding protein-2 (IGFBP-2) to determine whether rbPL mediates similar metabolic effects to those of bGH. Administration of bGH stimulated an increase in NEFA, glucose, T3 and insulin, whereas none of these variables was affected by rbPL. The plasma concentrations of IGF-I and IGF-II were both increased by treatment with rbPL but, to a lesser extent than occurred with bGH. Interestingly, BUN and IGFBP-2 concentrations were reduced equally by bGH and rbPL. These results suggest that rbPL does not necessarily act as a GH agonist but, rather, may have distinct effects on intermediary metabolism that could be mediated through another specific receptor.

Animals↗
PubMed1992

Effect of growth hormone treatment on the distribution of insulin-like growth factor-I between plasma and lymph of lactating sheep.

Plasma and mammary efferent lymph concentrations of insulin-like growth factor I (IGF-I) were determined in lactating ewes before and after treatment with GH (10 mg/day) for 3 days. The lymph:plasma ratio of IGF-I increased from 0.34 to 0.47 after GH treatment when the IGF-I content of plasma increased by 19.4 nmol/l (from 32.1 nmol/l) and lymph by 13.7 nmol/l (from 10.7 nmol/l). This increase in the relative content of IGF-I in lymph was associated with increased lymph content of IGF-I in a lower molecular mass pool (nominally 50 kDa) derived by size exclusion chromatography. GH treatment increased the total binding capacity for IGF-I in both high (150 kDa) and low (50 kDa) molecular mass pools of plasma and the 150 kDa pool in lymph but there was a proportionally greater increase in 50 kDa total binding in lymph relative to plasma. Further, GH treatment increased the 'saturation' of the 50 kDa binding proteins but decreased the 'saturation' of the 150 kDa fraction, in both plasma and lymph. Ligand blot analysis of IGF-binding proteins (IGFBPs) in plasma and lymph showed that GH treatment of lactating sheep increased IGFBP-3 and decreased IGFBP-2 in plasma and lymph. Radioimmunoassay of IGFBP-2 showed that while GH treatment reduced the plasma content of IGFBP-2 by about half, the lymph:plasma ratio was increased from 0.68 to 0.87. GH treatment of lactating ewes not only increased the IGF-I content of plasma but increased the apparent efficiency of transfer of IGF-I across capillary endothelium to mammary efferent lymph.

Animals↗
PubMed1992

Ruminant placental lactogens: structure and biology.

Ruminant placental lactogens (PL) are members of the somatotropin, prolactin gene family that are synthesized by trophectodermal binucleate cells. The structure and biology of PL has been studied in the cow, sheep, and goat. Ruminant PL have greater structural identity to prolactin than somatotropin, although they bind to both lactogenic and somatogenic receptors. The molecular weights of ovine and caprine PL are approximately 23,000, whereas bovine PL is larger (31,000 to 34,000) due to glycosylation. Placental lactogen is secreted into both the fetal and maternal circulations. The concentration of PL in the fetus decreases with advancing gestation, whereas PL concentration peaks in the maternal circulation during the last third of pregnancy then reaches a plateau. Furthermore, the maternal concentration of PL is 100- to 1,000-fold higher in sheep and goats than in cows. The precise factors that modulate secretion of PL are unknown, although placental mass and nutrition seem to play a role. Ruminant PL have both lactogenic and somatogenic biological activities and may also have unique activities mediated through a specific receptor. There is circumstantial evidence to suggest that PL plays a role in stimulating mammogenesis. Placental lactogen secreted into the fetal compartment may also help regulate fetal growth. Direct experimental data indicate that PL can regulate maternal intermediary metabolism. Thus, it may act as a partitioning agent to regulate nutrient supply for fetal growth. The precise biological function of PL in ruminants, therefore, still needs to be defined.

Amino Acid Sequence↗
PubMed1992

Long-term effects of sometribove in lactating cows during a fourth consecutive lactation of treatment: insulin and somatotropin responses to glucose infusion.

The effect of sometribove (USAN, methionyl bST) on the endocrine pancreas and blood bST concentrations was investigated in 6 control and 6 treated Friesian cows, averaging 111 and 118 d postpartum in their fourth lactation of treatment. Each lactation the treated cows received sometribove injections (500 mg) every 2 wk (injection cycle) starting 60 +/- 3 d postpartum, increasing milk yield 3.3, 5.9, 1.9, and 4.2 kg/d in lactations 1, 2, 3, and 4, respectively. On d 8 of a fourth lactation injection cycle, blood was sampled for 390 min, starting 30 min before an intravenous glucose infusion (100 mg/kg) over a 20-min period. Preinfusion concentrations of glucose, insulin, and bST were elevated in sometribove-treated cows versus controls: 82.1 versus 74.4 mg/dl, 28.1 versus 19.7 microIU/ml, and 4.6 versus .9 ng/ml, respectively. Glucose infusion resulted in a rapid increase in blood glucose and insulin concentrations, followed by a sharp decline to preinfusion values across both treatments, resulting in similar net area under the curves for glucose and insulin. Blood bST concentrations remained unchanged. This study supports the concept that sometribove increases milk yield in dairy cows by chronically influencing homeorhetic mechanisms.

Animals↗
PubMed1992

Stimulation of milk yield and feed intake by bovine placental lactogen in the dairy cow.

A 6 x 6 Latin square design was used to test the effects of recombinant bovine placental lactogen on milk yield, milk composition, feed intake, and blood hormone and metabolite levels in nonpregnant lactating cows. The six treatments (5, 10, 20, and 40 mg/d of placental lactogen, water as negative control, and 20 mg/d of bST as positive control) were administered by subcutaneous injection twice daily for 9 d. Blood samples were taken during the last 5 d of the treatment period. The three highest doses of placental lactogen increased milk yield, and there was a linear dose effect, although placental lactogen was less potent than bST. Milk concentrations of lactose, protein, and fat were not altered by any of the treatments. Dry matter intake was increased by two of the doses of placental lactogen, but not by bST. Blood urea N concentration was decreased in a dose-dependent manner by placental lactogen and was also decreased by bST. Similarly, serum insulin-like growth factor-I was increased in a dose-dependent manner by placental lactogen and was also increased by bST. Plasma concentrations of NEFA and glucose were increased by bST, but placental lactogen had little or no effect on either of these parameters. Thus, placental lactogen appears to act, in part, as a weak somatotropin agonist; however, it also appears to have specific activities, e.g., stimulating feed intake.

Animals↗
PubMed1992

Effect of high doses of a sustained-release bovine somatotropin on antibody formation in dairy cows.

Eighty-two lactating Holstein cows received either one, three, or five concurrent, intramuscular injections of a unit dose (.6 g) of zinc methionyl bST (some-tribove) or five doses of the vehicle. Injections were administered at 14-d intervals from 60 d postpartum until the end of lactation or necropsy. Thirty-eight cows continued on the same treatment for a 2nd yr. Blood bST antibodies developed within the first 7 wk of treatment, and the number of cows with anti-bST binding generally declined with time. Thirteen out of 59 cows receiving bST developed binding activity > 25% (positives) during the 1st yr. At the .6-g dose level, no binding was detected after wk 15. Seven of the 13 positive cows were among the group randomly selected to continue on study during yr 2. In the 2nd yr, only 2 out of 24 bST-treated cows were positive. Binding activity was associated with the IgG fraction in serum. Binding capacities of antibodies ranged from .625 to 3.04 mg of bST/L, and affinities ranged from 1.14 x 10(8) to 3.14 x 10(8) L/mol. Cows considered to be clinically positive had performance similar to those of their herdmates having binding < 25%. No evidence of a pathologic effect of antibodies existed in treated cows, their calves, or fetuses. The presence of anti-bST antibodies did not affect milk production of the cow or growth of the calves conceived during bST treatment.

Animals↗