Search PubMed⌕ Search

Biomedical subjects

R J Collier

Publications and source records attributed to R J Collier.

At least 127 records · Page 7Linked to original sources
PubMed1993

Variants of somatotropin in cattle: gene frequencies in major dairy breeds and associated milk production.

The amino acid sequence of bovine somatotropin (bST) varies at position 127 where either valine or leucine is found. The frequencies of leucine127 and valine127 bST gene alleles in cows (n = 302) and sires (n = 70) from major dairy breeds (Holstein, Brown Swiss, Guernsey, Jersey, and Ayrshire) were determined using DNA extracted from whole blood or spermatozoa. A 428 base pair fragment of the bST gene was amplified using polymerase chain reaction (PCR) and variants of the bST gene were detected as polymorphisms by Alu I restriction endonuclease digestion of PCR products. Restriction enzyme DNA fragments for the leucine127 variant were 265, 96, 51, and 16 base pair and for the valine127 variant were 265, 147, and 16 base pair as a polymorphism of bST was present in the 147 base pair DNA fragment. Frequencies of leucine127 and valine127 alleles for cows (n = 302) were 1.0 and 0 for Brown Swiss, .93 and .07 for Holstein, .92 and .08 for Guernsey, .79 and .21 for Ayrshire, and .56 and .44 for Jersey, respectively. In Holstein sires used for artificial insemination (n = 70), the frequency of leucine127 and valine127 alleles was .96 and .04. Estimates of transmitting ability for milk production tended to be greater for Holstein cows that were homozygous for leucine127 bST and Jersey cows that were homozygous for valine127 bST whereas Holstein sires with different bST genotypes were similar. In summary, frequencies of alleles for the bST gene were not similar in different dairy breeds and estimates of milk production were correlated with bST gene variant in cows but not sires.

Alleles↗
PubMed1993

Immunohistochemical and nucleic acid analysis of somatotropin receptor populations in the bovine ovary.

Ovaries were analyzed for somatotropin receptor protein and mRNA through use of immunohistochemistry, solution hybridization/nuclease protection, Northern blotting, and reverse transcriptase polymerase chain reaction (RT-PCR). As indicated by immunoperoxidase staining, CL expressed immunoreactive somatotropin receptor (positive stain). Ovarian stroma, connective tissue, endothelium, and erythrocytes did not express somatotropin receptor (negative stain). Within the CL, somatotropin receptor protein was expressed primarily in large luteal cells whereas small luteal cells were negative. Most follicles (1-5 mm, after fixation) were negative for somatotropin receptor. On the basis of solution hybridization/nuclease protection, the mRNA for somatotropin receptor was found in greatest abundance in CL and large luteal cells and was nearly undetectable in small luteal cells or follicles (class 1, 3-5 mm; class 2, 6-9 mm; and class 3, > or = 10 mm). Northern blotting of mRNA for somatotropin receptor showed expression of somatotropin receptor mRNA transcripts in whole ovary (4.7 and 4.4 kb), CL (4.7 and 4.4 kb), and liver (4.4 kb); and RT-PCR amplified a single amino acid coding region for somatotropin receptor in CL and liver. In summary, somatotropin receptor (both immunoreactive protein and mRNA) is found primarily in the large luteal cell, and lesser amounts of the expressed receptor or its message are found in the follicle. Alternative sizes of mRNA for somatotropin receptor suggest novel mRNA processing in the bovine ovary.

Animals↗
PubMed1993

pH-dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen.

Protective antigen (PA) of anthrax toxin forms ion-conductive channels in planar lipid bilayers and liposomes under acidic pH conditions. We show here that PA has a similar permeabilizing action on the plasma membranes of CHO-K1 and three other mammalian cell lines (J774A.1, RAW264.7 and Vero). Changes in membrane permeability were evaluated by measuring the efflux of the K+ analogue, 86Rb+, from prelabelled cells, and the influx of 22Na+. The permeabilizing activity of PA was limited to a proteolytically activated form (PAN) and was dependent on acidic pH for membrane insertion (optimal at pH 5.0), but not for sustained ion flux. The flux was reduced in the presence of several known channel blockers: tetrabutyl-, tetrapentyl-, and tetrahexylammonium bromides. PAN facilitated the membrane translocation of anthrax edema factor under the same conditions that induced changes in membrane permeability to ions. These results indicate that PAN permeabilizes cellular membranes under conditions that are believed to prevail in the endosomal compartment of toxin-sensitive cells; and they provide a basis for more detailed studies of the relationship between channel formation and translocation of toxin effector moieties in vivo.

Animals↗
PubMed1993

Membrane translocation and channel-forming activities of diphtheria toxin are blocked by replacing isoleucine 364 with lysine.

A mutant of diphtheria toxin in which Ile-364 was replaced by Lys was at least 500-fold less toxic to Vero cells than the parental toxin. Its ability to undergo low-pH-triggered translocation across the plasma membrane was greatly diminished, as was its ability to form ion-conductive channels. In addition, the mutant toxin was inactive in the pH-dependent killing of Escherichia coli.

Animals↗
PubMed1993

Stimulation of food intake and weight gain in mature female rats by bovine prolactin and bovine growth hormone.

Recombinant bovine prolactin (rbPRL) or bovine growth hormone (rbGH) was administered to mature female rats (10/treatment group) by daily subcutaneous injection for 10 days. Doses ranged from 7 to 5,000 micrograms/day (0.03-24 mg/kg body wt). Both rbPRL and rbGH increased body weight gain and food intake, but these parameters were increased at lower doses of rbPRL (7-63 micrograms/day) than rbGH (> 190 micrograms/day). Weight gain and food intake were maximally stimulated by 190 micrograms/day rbPRL, whereas maximal increased weight gain was obtained with the highest dose of rbGH (5,000 micrograms/day). Total carcass protein was increased by both hormones; however, protein as a percentage of body weight was unchanged. Similarly, neither rbPRL nor rbGH changed the percentage of carcass moisture. Percentage of body fat was increased by rbPRL but was decreased by rbGH. Weight of the gastrointestinal tract and kidneys was increased by both hormones, but increases were in proportion to body weight gain. These data confirm that ungulate prolactin is a hyperphagic agent in the female rat. In addition, they suggest that, while prolactin stimulates growth in mature female rats, this growth is probably not via a somatogenic mechanism.

Aging↗
PubMed1993

Performance, clinical chemistry, and carcass responses of finishing lambs to recombinant bovine somatotropin and bovine placental lactogen.

Bovine placental lactogen (PL) is a partial somatotropin agonist in the cow and decreases urea nitrogen, indicating increased nitrogen retention. In the present study, the somatogenic effects of bovine PL (bPL; 4 and 8 mg/d) were compared with those of bovine somatotropin (bST; 4 and 8 mg/d) in finishing lambs. Measures of comparison included growth performance, carcass composition, and growth-related clinical chemistry traits. Although feed efficiency during the first 3 wk of treatment with bPL was improved by 14% (P < .05), feed efficiency for the full 6-wk treatment period did not differ from that of control lambs. Responsiveness to bPL may have been attenuated by high titer antibodies present after 2 wk of treatment. However, bPL also did not influence growth-related clinical chemistry traits during short-term (7 d) treatment, strongly suggesting that bPL was ineffective in finishing lambs at the doses tested. In contrast, bST improved 6-wk feed efficiency by an average of 17% (P < .05) and decreased feed intake by an average of 12% (P < .05). In addition, measures of carcass composition including longissimus muscle area, specific gravity of the rack, kidney and pelvic fat, and fat thickness demonstrated that bST, but not bPL, treatment decreased carcass fatness and increased carcass leanness. Treatment with bST, but not with bPL, affected IGF-I, insulin, glucose, and urea nitrogen in a dose-related manner. Thus, daily injections of bPL did not affect either performance or carcass quality, whereas performance and carcass responses of finishing lambs to bST were consistent with those reported by others.

Adipose Tissue↗
PubMed1993

Plasma growth hormone, insulin-like growth factor, insulin, and thyroid hormone association with body protein and fat accretion in steers undergoing compensatory gain after dietary energy restriction.

Eighteen Chianina crossbred steers were used to examine the interrelationship between hormonal status and empty body protein (EBPRT) and fat (EBFAT) accretion during an 88-d controlled realimentation (REAL; DMI was adjusted weekly on an individual BW basis) period. Body composition was determined by monitoring endogenous whole-body 40[K]. At the end of restriction (REST) and on d 31 and 59 of REAL blood samples were taken and analyzed for concentrations of growth hormone (GH), IGF-I, IGF-II, insulin (INS), thyroxine (T4), triiodothyronine (T3), reverse triiodothyronine (rT3), nonesterified fatty acids (NEFA), glucose, and urea nitrogen. Depositions of EBPRT and EBFAT were decreased (P < .001) in REST (92 d) animals compared with nonrestricted (NR) controls. During REST, plasma levels of glucose, IGF-I, INS, T4, T3, and rT3 were decreased (P < .05); plasma urea nitrogen (PUN), NEFA, and GH levels were increased (P < .05), and IGF-II concentrations were similar between REST and NR steers. A transient compensatory response in BW gain and protein deposition occurred in repleted steers between d 35 and 62 of REAL. During this period, PUN levels markedly declined (P < .01) in REAL steers and glucose concentration increased (P < .01) to levels similar to those in NR controls. This response prevailed even though apparent digestible nutrients were decreased in repleted animals. During energy repletion, plasma levels of T4, T3, rT3, IGF-I, and INS were positively, and of GH was negatively, correlated with EBPRT and EBFAT gain in repleted animals. Unlike IGF-I, IGF-II was not correlated with compensatory body growth. These results suggest that compensating steers are more metabolically efficient; rapid body tissue gain occurs during a period of reduced nutrient digestibility. Additionally, plasma levels of IGF-I, GH, INS, and thyroid hormones, but not of IGF-II, are markedly affected by alteration of energy intake and are highly correlated with empty body gain and protein deposition in compensating late-maturing steers.

Adipose Tissue↗
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↗