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Biomedical subjects

R J Collier

Publications and source records attributed to R J Collier.

At least 163 records · Page 9Linked to original sources
PubMed1990

The effect of bovine lactoferrin on muscle growth in vivo and in vitro.

Lactoferrin was found to be a potent stimulator of proliferation for L6 myoblasts. Both apo and holo-forms of lactoferrin were equipotent. By contrast, only the holo-form of transferrin (a structurally related iron binding protein) stimulated proliferation, apo-transferrin was without activity. Holo-transferrin was also less stimulatory than lactoferrin. Purified lactoferrin was administered to mature female rats and to neonatal rats by daily subcutaneous injection to determine if there was a measurable effect on muscle cell growth in vivo. Results from the in vivo studies suggest that lactoferrin has little or no effect on muscle cell growth in the whole animal.

Animals↗
PubMed1990

Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine.

Glutamic acid-148, an active-site residue of diphtheria toxin identified by photoaffinity labeling with NAD, was replaced with aspartic acid, glutamine, or serine by directed mutagenesis of the F2 fragment of the toxin gene. Wild-type and mutant F2 proteins were synthesized in Escherichia coli, and the corresponding enzymic fragment A moieties (DTA) were derived, purified, and characterized. The Glu----Asp (E148D), Glu----Gln (E148Q), and Glu----Ser (E148S) mutations caused reductions in NAD:EF-2 ADP-ribosyltransferase activity of ca. 100-, 250-, and 300-fold, respectively, while causing only minimal changes in substrate affinity. The effects of the mutations on NAD-glycohydrolase activity were considerably different; only a 10-fold reduction in activity was observed for E148S, and the E148D and E148Q mutants actually exhibited a small but reproducible increase in NAD-glycohydrolytic activity. Photolabeling by nicotinamide-radiolabeled NAD was diminished ca. 8-fold in the E148D mutant and was undetectable in the other mutants. The results confirm that Glu-148 plays a crucial role in the ADP-ribosylation of EF-2 and imply an important function for the side-chain carboxyl group in catalysis. The carboxyl group is also important for photochemical labeling by NAD but not for NAD-glycohydrolase activity. The pH dependence of the catalytic parameters for the ADP-ribosyltransferase reaction revealed a group in DTA-wt that titrates with an apparent pKa of 6.2-6.3 and is in the protonated state in the rate-determining step.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1990

Cloning and in vivo expression of bovine growth hormone receptor mRNA.

A cDNA for the bovine growth hormone (bGH) receptor has been cloned out of a cDNA library prepared from liver of a pregnant Holstein heifer. The cDNA clone hybridizes to a single 4.5 kb mRNA species and shares a high degree of sequence homology with growth hormone receptors cloned from other species. Utilizing the bGH receptor cDNA as a probe, a relatively high level of bGH-receptor mRNA was detected in bovine liver. In comparison to liver values, lower concentrations of bGH-receptor mRNA were detected in bovine kidney, anterior pituitary, and mammary gland. Because specific binding sites for bGH have not been convincingly demonstrated in isolated cell membranes from whole bovine mammary tissue, mammary tissue from two pregnant heifers (separate experiments) was separated into fractions enriched for epithelium, stroma, and blood components. These fractions were then probed for growth hormone receptor mRNA using solution hybridization-nuclease protection assays performed on isolated RNA. The assay results indicated that a low level of bGH-receptor mRNA is relatively evenly distributed throughout the mammary tissues of the two cows studied. In contrast, experiments using a probe to bovine insulin-like growth factor-I (IGF-I) indicate that the IGF-I mRNA is localized in the stromal/blood component of the mammary gland. These data suggest a possible paracrine mechanism for bGH action in the mammary gland.

Amino Acid Sequence↗
PubMed1990

Pseudomonas aeruginosa exotoxin A: alterations of biological and biochemical properties resulting from mutation of glutamic acid 553 to aspartic acid.

Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) was identified earlier as a putative active-site residue by photoaffinity labeling with NAD. Here ETA-E553D, a cloned form of the toxin in which Glu-553 has been replaced by aspartic acid, was purified from Escherichia coli extracts and characterized. Cytotoxicity of the mutant toxin for mouse L-M cells was less than 1/400,000 that of the wild type. The mutation caused a 3200-fold reduction in NAD:elongation factor 2 ADP-ribosyltransferase activity, as estimated by assays with an active fragment derived from the toxin by digestion with thermolysin. NAD glycohydrolase activity was reduced somewhat less, by a factor of 50, and photoaffinity labeling with NAD by a factor of 2. We detected less than 2-fold change in the values of KM for NAD or elongation factor 2 and no change in KD for NAD, as determined by quenching of protein fluorescence. The drastic reduction of ADP-ribosyltransferase activity therefore results primarily from an effect of the mutation on kcat, implying that Glu-553 plays an important and possibly direct role in catalyzing this reaction. The effects of the E553D mutation are similar to those of the E148D mutation in diphtheria toxin, supporting the notion that these two Glu residues perform the same function in their respective toxins.

ADP Ribose Transferases↗
PubMed1990

Effect of farm and simulated laboratory cold environmental conditions on the performance and physiological responses of lactating dairy cows supplemented with bovine somatotropin (BST).

A study was conducted to evaluate the effect of bovine somatotropin (BST) supplementation in twelve lactating dairy cows maintained in cold environmental conditions. Six cows were injected daily with 25 mg of BST; the other six were injected with a control vehicle. Cows were maintained under standard dairy management during mid-winter for 30 days. Milk production was recorded twice daily, and blood samples were taken weekly. Animals were then transferred to environmentally controlled chambers and exposed to cycling thermoneutral (15 degrees to 20 degrees C) and cycling cold (-5 degrees to +5 degrees C) temperatures for 10 days in a split-reversal design. Milk production, feed and water intake, body weights and rectal temperatures were monitored. Blood samples were taken on days 1, 3, 5, 8 and 10 of each period and analyzed for plasma triiodothyronine (T3), thyroxine (T4), cortisol, insulin and prolactin. Under farm conditions, BST-treated cows produced 11% more milk than control-treated cows and in environmentally controlled chambers produced 17.4% more milk. No differences due to BST in feed or water intake, body weights or rectal temperatures were found under laboratory conditions. Plasma T3 and insulin increased due to BST treatment while no effect was found on cortisol, prolactin or T4. The results showed that the benefits of BST supplementation in lactating dairy cows were achieved under cold environmental conditions.

Animals↗
PubMed1990

Characterization of glycosylated bovine placental lactogen and the effect of enzymatic deglycosylation on receptor binding and biological activity.

Bovine placental lactogen (bPL) is a glycoprotein hormone that has both somatogenic and lactogenic properties. Purified preparations of the hormone contain many isoforms that are separated by isoelectric focusing. The sequence for bPL contains one consensus site for an N-linked oligosaccharide and many potential sites for O-linked sugars. To determine whether the isoforms are the result of differences in glycosylation, the oligosaccharide portion of bPL was partially characterized. In addition, a number of the isoforms were isolated and enzymatically deglycosylated to determine the effect of O- and N-linked glycosylation on biological activity. Biological activity was assessed in a somatotropin radioreceptor assay and also in the Nb2 lymphoma lactogenic bioassay. The structure of N-linked oligosaccharide was found to be sialylated and triantennary and appeared to be the same for all of the different charge isomers. Compositional analysis suggested that O-linked oligosaccharides were also present. Treatment of the intact hormone with neuraminidase resulted in the loss of some, but not all, of the isoforms, suggesting that a large degree of the charge heterogeneity is due to posttranslational modifications unrelated to glycosylation. Enzymatic removal of N-linked oligosaccharides from native bPL resulted in a 1.2-2.3-fold increase in binding to the somatotropin receptor, whereas receptor binding was unaffected by enzymatic removal of O-linked oligosaccharide. Lactogenic activity was affected very little by the removal of either type of oligosaccharide. The data suggests that glycosylation of bPL may have a small effect on receptor specificity, but that overall its presence does not dramatically affect receptor binding or biological activity.

Animals↗
PubMed1990

Effect of acute challenge with an extreme dose of somatotropin in a prolonged-release formulation on milk production and health of dairy cattle.

Eight pregnant Holstein cows were given weekly injections of 15 g of recombinant bST over a 2-wk period for a total dose of 30 g to determine signs of acute toxicity. Cows were monitored intensively throughout the study, and samples were taken for analyses of hormones, metabolites, chemistries, hematology, and urine analytes. Animal health throughout the study was generally excellent. Mean rectal temperatures were significantly higher in treated cows (38.7 vs. 39.2 degrees C). Least squares means for 3.5% FCM production were 15.9 and 23.0 kg/d, and net energy intakes were 29.4 and 26.9 Mcal/d for control and treated cows, respectively. Somatotropin concentrations reached more than 250 ng/ml on d 10 and remained above 200 ng/ml. Insulin and glucose concentrations were also increased but returned toward baseline values. Free fatty acid concentrations were higher in treated cows, but beta-hydroxybutyrate was not affected. Most hematological measurements were unaffected except for a reduction in erythrocyte number in treated cows and decreases in hematocrit and hemoglobin, but values were within clinically normal ranges. Although cows received in 2 wk a dose that was equivalent to the amount administered during more than 2 yr of continuous use, no signs of acute toxicity to bST were observed.

Animals↗
PubMed1990

Structure and interaction with model membranes of a CNBR peptide of diphtheria toxin B fragment.

The mechanism by which diphtheria toxin (DT) crosses the endosomal membrane to exert its biological activity in the cell cytoplasm is still poorly understood. By measuring the change in conductance of planar lipid bilayers induced by cyanogen bromide peptides of fragment B of DT, we have identified a domain that could be involved in the pH-dependent membrane interaction of DT. Moreover, infrared spectroscopy has allowed us to demonstrate that, at low pH, in the presence of a lipid bilayer, this domain is mainly helical with the axis of the helices oriented parallel to the lipid acyl chains. On the basis of these results, we have designed mutants of DT which should provide information about the molecular mechanism of the DT membrane translocation process.

Diphtheria Toxin↗
PubMed1990

Food safety and pharmacokinetic studies which support a zero (0) meat and milk withdrawal time for use of sometribove in dairy cows.

Sometribove (SB) is a synthetic form of bovine somatotropin (BST) whose amino acid sequence is the same for 190 of the 191 amino acids in BST. Administration of 500 mg of SB to dairy cows every 14 d increases the efficiency of milk production. Regulatory agencies have authorized a zero (0) milk and meat withdrawal time for investigational use of SB. The scientific basis for this authorization is as follows: 1) BST and other non-primate somatotropins are not active in humans, due to differences in the amino acid sequence from human somatotropin, which limits the ability of BST to bind to receptors on human tissues. 2) SB is not orally active, as it is degraded like other proteins when eaten. Administration of 50,000 microgram/kg/d SB to rats for 90 d produced no growth response. 3) Residual levels of SB in meat/milk are very low (ppb) and comparable to endogenous BST levels. 4) Residual levels (ppb) of insulin-like growth factor I (IGF-I) in meat and milk are only marginally increased by SB treatment (somatotropin stimulates local production of IGF-I in tissues to mediate some of its biological effects. 5) IGF-I was not orally active when fed to rats at doses ranging from 200 to 2,000 microgram/kg for 14 d.

Animals↗
PubMed1990

Purification and partial characterization of a bovine epidermal growth factor-like polypeptide.

A heterologous radioreceptor assay was developed to follow the purification of an EGF-like polypeptide from bovine kidney. Purification of the growth factor was facilitated by the use of a novel affinity column using fixed A431 cells attached to sephadex beads. The mol. wt. of the purified EGF-LP was estimated to be 5480 from the amino acid composition. The purified EGF-like polypeptide stimulated the proliferation of bovine mammary epithelial cells and appeared to be equipotent to mouse EGF. Available evidence suggests that the purified molecule is distinct from bovine TGF-alpha.

Amino Acids↗
PubMed1989

Formation of active diphtheria toxin in vitro based on ligated fragments of cloned mutant genes.

An intact gene coding for wild type diphtheria toxin was reconstituted in vitro from fragments of cloned inactive diphtheria toxin mutants with defects in different parts of the gene. The reconstituted DNA template was amplified using the Taq DNA polymerase chain reaction, providing a virtually unlimited supply. The toxin was expressed in vitro by transcription from a T3 RNA polymerase promoter, followed by translation of the mRNA in a rabbit reticulocyte lysate system. Diphtheria toxin synthesised in this manner was shown to be as toxic as natural toxin and behaved like natural toxin in every way tested. This can serve as a general approach for study of toxic proteins, opening up the possibility of in vitro gene manipulation. Reconstitution of the wild type gene from fragments followed by polymerase chain reaction amplification bypasses the hazards of cloning protein toxins.

Animals↗
PubMed1989

X-ray grade crystals of the enzymatic fragment of diphtheria toxin.

The enzymatic fragment of diphtheria toxin, fragment A (Mr = 21,167), complexed to the dinucleotide adenosine 3',5'-uridine (ApU), has been crystallized at two different values of pH by hanging drop vapor diffusion. Crystals grown at a pH value of 5.0 (from I) belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 71.2 A, b = 73.0 A, c = 139.8 A and four protomers in the asymmetric unit. Crystals grown at a pH value of 8.1 (form II) belong to the monoclinic space group C2, with unit cell parameters a = 65.2 A, b = 85.6 A, c = 34.6 A, beta = 103.0 degrees and one protomer in the asymmetric unit. Both crystal forms diffract to 2.5 A resolution. The molecular structures of fragment A obtained from these two crystal forms may illuminate the pH-dependent transition of diphtheria toxin during membrane translocation.

Crystallization↗
PubMed1989

Cloned diphtheria toxin within the periplasm of Escherichia coli causes lethal membrane damage at low pH.

Acidic pH within endosomal vesicles of sensitive animal cells triggers a conformational change in diphtheria toxin (DT) that is believed to cause the B chain to insert into the vesicular membrane and the enzymic A chain to be released into the cytosol. In artificial lipid bilayers, DT forms ion-conductive channels under mildly acidic conditions (pH approximately 5). Here we report a related phenomenon in Escherichia coli strains that secrete certain cloned DT-related proteins into their periplasm: the cells are rapidly killed at pH 5 but remain unharmed at pH 7. Expression of full-length DT (an active-site mutant, to comply with the National Institutes of Health recombinant DNA guidelines) causes acid-sensitivity, whereas expression of the A chain alone does not. The killed cells are not lysed, but inner-membrane functions are impaired (membrane potential, active transport, and ion impermeability). We propose that acidification of DT within the periplasm induces its insertion into the inner membrane, lethally damaging the permeability barrier. This discovery provides a potentially important selection procedure for mutations affecting the membrane insertion function of DT. Similar approaches may be useful in studying other proteins that undergo condition-dependent interaction with membranes.

Biological Transport↗
PubMed1989

Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers.

The three separate proteins that make up anthrax toxin--protective antigen (PA), edema factor (EF), and lethal factor (LF)--act in binary combinations to produce two distinct reactions in experimental animals: edema (PA + EF) and death (PA + LF). PA is believed to interact with a membrane receptor, and after proteolytic processing, to mediate endocytosis and subsequent translocation of EF or LF into the cytosol. PA can be separated, after mild trypsinolysis, into two fragments, PA65 (65 kDa) and PA20 (20 kDa). We demonstrate that trypsin-cleaved PA is capable of forming cation-selective channels in planar phospholipid bilayer membranes and that this activity is confined to the PA65 fragment; PA20, LF, and EF are devoid of channel-forming activity. These PA65 channels exhibit pH-dependent and voltage-dependent activity--a property reminiscent of the channels formed by the two-chain proteins diphtheria, tetanus, and botulinum toxins.

Antigens, Bacterial↗
PubMed1989

Photolabeling of Glu-129 of the S-1 subunit of pertussis toxin with NAD.

UV irradiation was shown to induce efficient transfer of radiolabel from nicotinamide-labeled NAD to a recombinant protein (C180 peptide) containing the catalytic region of the S-1 subunit of pertussis toxin. Incorporation of label from [3H-nicotinamide]NAD was efficient (0.5 to 0.6 mol/mol of protein) relative to incorporation from [32P-adenylate]NAD (0.2 mol/mol of protein). Label from [3H-nicotinamide]NAD was specifically associated with Glu-129. Replacement of Glu-129 with glycine or aspartic acid made the protein refractory to photolabeling with [3H-nicotinamide]NAD, whereas replacement of a nearby glutamic acid, Glu-139, with serine did not. Photolabeling of the C180 peptide with NAD is similar to that observed with diphtheria toxin and exotoxin A of Pseudomonas aeruginosa, in which the nicotinamide portion of NAD is transferred to Glu-148 and Glu-553, respectively, in the two toxins. These results implicate Glu-129 of the S-1 subunit as an active-site residue and a potentially important site for genetic modification of pertussis toxin for development of an acellular vaccine against Bordetella pertussis.

Cyanogen Bromide↗
PubMed1989

Effects of eliminating a disulfide bridge within domain II of Pseudomonas aeruginosa exotoxin A.

Cysteines 265 and 287 of Pseudomonas aeruginosa exotoxin A (ETA) were substituted by serine, thereby eliminating a disulfide bridge within domain II, the putative membrane insertion-translocation domain. Purified mutant toxin was 80-fold less toxic for mouse L cells than was wild-type ETA while retaining the same specific activity in the ADP-ribosyltransferase reaction as did wild-type toxin. Binding of the nonionic detergent Triton X-114 by mutant ETA occurred at a slightly higher pH than did binding by wild-type ETA, suggesting that the mutant protein more readily undergoes a conformational change exposing hydrophobic regions. Data are presented supporting the notion that the mutant and wild-type toxins enter from the same intracellular compartment. The lower cytotoxicity of the mutant protein could be due to accelerated intracellular degradation or abortive, premature membrane insertion.

ADP Ribose Transferases↗
PubMed1989

Effects of thermal stress and level of feed intake on portal plasma flow and net fluxes of metabolites in lactating Holstein cows.

Six Holstein cows (averaging 475 kg body weight, 2.3 parities and 96 d in lactation) fitted with catheters in the hepatic portal vein, mesenteric vein and intercostalis posterior artery were exposed to treatments of thermal comfort environments with libitum or restricted (75% of ad libitum) DM intake and a thermal stress environment with ad libitum intake in two balanced 3 x 3 latin squares to evaluate effects of thermal stress on portal plasma flow and net fluxes of metabolites. Portal plasma flow was measured by administering a primed, continuous infusion of para-aminohippurate into a mesenteric vein and determining its concentration and dilution in portal vein plasma. Thermal stress treatment increased rectal temperatures and respiration rates. Dry matter intake decreased from thermal comfort ad libitum level (15.1 kg/d) to lower levels in thermal comfort restricted (11.5 kg/d) and thermal stress (11.1 kg/d) treatments. Portal plasma flow was related directly to level of DM intake, declining about 14% with thermal comfort restricted intake and thermal stress environment treatments compared with the thermal comfort restricted intake and thermal stress environment treatments compared with the thermal comfort ad libitum intake treatment. Net flux of alpha-amino N was reduced 20 and 35% by thermal comfort restricted intake and thermal stress treatments compared with the thermal comfort ad libitum intake treatment. Net fluxes of urea N, ammonia N and glucose were not affected by experimental treatments. A portion of the negative effects of thermal stress on milk production can be explained by decreased nutrient intake and decreased nutrient uptake by the portal-drained viscera of the cow.

Amino Acids↗