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R J Collier

Publications and source records attributed to R J Collier.

At least 91 records · Page 5Linked to original sources
PubMed1995

Structure of the isolated catalytic domain of diphtheria toxin.

The structure of the isolated catalytic domain of diphtheria toxin at pH 5.0 was determined by X-ray crystallography at 2.5 A resolution and refined to an R-factor of 19.7%. The domain is bound to its endogenous inhibitor adenylyl(3'-->5')uridine 3'-monophosphate (ApUp). The structure of this 190-residue domain, which was expressed in and isolated from Escherichia coli, is essentially identical to the structure of the catalytic domain within whole diphtheria toxin determined at pH 7.5. However, there are two adjacent surface loops (loop 66-78 and loop 169-176) that exhibit clear differences when compared to the structure of the catalytic domain in whole diphtheria toxin. Although both loops are at the surface of the protein and are relatively flexible, the chain trace is well-defined in the electron density. The main structural difference is the closer approach of loops 66-78 and 169-176. We ascribe this structural change mainly to the absence of the neighboring transmembrane domain in the isolated catalytic domain as compared to whole diphtheria toxin. We suggest that this change represents the first step of the structural transition from the catalytic domain in whole diphtheria toxin to the translocated form of the domain. The changes are described in detail, and their implications for membrane translocation are discussed.

Binding Sites↗
PubMed1995

Effects of somatotropin on the conceptus, uterus, and ovary during maternal recognition of pregnancy in cattle.

Effects of recombinant bovine somatotropin (rbST) on ovarian and uterine function and the production of components of the insulin-like growth factor (IGF) system were examined during the period of maternal recognition of pregnancy in cattle. Lactating dairy cows were treated with 25 mg/d rbST (n = 8) or saline (n = 8) for 16 d after estrus. Ovaries, uteri, and conceptuses were collected on Day 17 after estrus. The length (millimeters) of the conceptus was recorded. The concentration of IGF-I and the content of IGF-binding proteins (BP) in uterine flushings were determined. Corpora lutea (CL) were weighed, and the number of follicles (> or = 2 mm in diameter) were counted. Follicular fluid from the largest and second-largest follicles was assayed for the concentration of IGF-I, IGFBP, progesterone, and estradiol. The length of the conceptus and the total amount of IGF-I in uterine fluid were similar for rbST and control. Recombinant bST increased 1) the weight of the CL, 2) the number of largest follicles (10 to 15 mm in diameter), 3) the concentration of IGF-I in the follicular fluid, 4) the follicular fluid content of IGFBP of the largest estrogenic follicle, and 5) the quantity of IGFBP in uterine flushings. The concentration of progesterone in the follicular fluid tended to be increased in rbST-treated cows, whereas the concentration of estradiol was similar to that of control cows. The concentration of progesterone in plasma was similar for rbST compared with control. In conclusion, the administration of rbST in lactating dairy cows for 16 d after estrus did not alter the growth of the conceptus collected on Day 17. The greatest responses to rbST were found within the ovary, where rbST increased the weight of the CL and altered the amount of IGF-I and IGFBP in the follicular fluid.

Animals↗
PubMed1995

Pituitary response to thyrotropin, corticotropin, and gonadotropin-releasing hormones in lactating cows treated with sometribove for a fourth consecutive lactation.

The effect of chronic treatment with recombinant methionyl bovine somatotropin (USAN, sometribove) on anterior pituitary secretions and its target organs was investigated in six control and six sometribove-treated British Friesian cows. Cows averaged 112 and 119 d postpartum in their fourth lactation of treatment and, except for one control, had active corpora lutea. During each lactation, treated cows received sometribove injections (500 mg) every 2 wk (injection cycle) starting 60 +/- 3 d postpartum. On Day 9 of one injection cycle, blood was sampled for 390 min, starting 30 min before an intravenous injection of thyrotropin (TRH, 0.33 microgram/kg), corticotropin (100 microgram), and gonadotropin (GnRH, 200 micrograms)-releasing hormones. Baseline somatotropin (bST) and adrenocorticotropin (ACTH) were higher in sometribove-treated cows vs. controls (3.27 vs. 1.03 ng/ml and 35.24 vs. 19.28 pg/ml, respectively). Baseline total thyroxine, free thyroxine, triiodothyronine, prolactin, follicle stimulating and luteinizing hormones, estradiol, and progesterone (P4) were similar across treatments. Circulating cortisol levels did not differ between control and sometribove cows, indicating a reduced adrenal ACTH responsiveness in the latter. Releasing factors induced similar changes across treatments in hormones studied with the following exceptions: a bST spike was seen in control cows only, cortisol response to ACTH was reduced in treated cows, and a significantly higher P4 concentration was detected in the plasma of sometribove-treated cows, suggesting increased ovarian responsiveness to GnRH-stimulated P4 output. The study demonstrated reduced bST response to TRH, consistent with physiologic feedback mechanisms, whereas the release profiles of the other pituitary hormones were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1995

Protective antigen-binding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino- or carboxy-terminus.

The edema factor (EF) and lethal factor (LF) components of anthrax toxin require anthrax protective antigen (PA) for binding and entry into mammalian cells. After internalization by receptor-mediated endocytosis, PA facilitates the translocation of EF and LF across the membrane of an acidic intracellular compartment. To characterize the translocation process, we generated chimeric proteins composed of the PA recognition domain of LF (LFN; residues 1-255) fused to either the amino-terminus or the carboxy-terminus of the catalytic chain of diphtheria toxin (DTA). The purified fusion proteins retained ADP-ribosyltransferase activity and reacted with antisera against LF and diphtheria toxin. Both fusion proteins strongly inhibited protein synthesis in CHO-K1 cells in the presence of PA, but not in its absence, and they showed similar levels of activity. This activity could be inhibited by adding LF or the LFN fragment (which blocked the interaction of the fusion proteins with PA), by adding inhibitors of endosome acidification known to block entry of EF and LF into cells, or by introducing mutations that attenuated the ADP-ribosylation activity of the DTA moiety. The results demonstrate that LFN fused to either the amino-terminus or the carboxy-terminus of a heterologous protein retains its ability to complement PA in mediating translocation of the protein to the cytoplasm. Besides its importance in understanding translocation, this finding provides the basis for constructing a translocation vector that mediates entry of a variety of heterologous proteins, which may require a free amino- or carboxy-terminus for biological activity, into the cytoplasm of mammalian cells.

Amino Acid Sequence↗
PubMed1995

Effect of supplemental dietary fat or protein on the short-term milk production response to bovine somatotropin.

Effects of supplemental energy or protein on the milk production response to bST administration were examined in two separate trials. In trial 1, 40 cows were used in a 2 x 2 factorial, completely randomized design to determine the effects of bST and fat supplementation. The study consisted of a 7-d pretreatment period and a 42-d treatment period. Fat was top-dressed at 3.0 Mcal/d of NEL, and bST was administered. Supplemental fat had no effect on milk production, and NEL intakes were unaffected. Administration of bST increased milk production by 7.1 kg/d, and the milk production response was unaffected by supplemental fat. In trial 2, 4 cows were used in four periods with a 2 x 2 factorial arrangement in which water or casein was infused into the abomasum of cows fed for ad libitum intake or at 80% of their requirements. Diets and infusions were initiated simultaneously and continued for 11 d. All cows were given bST during the last 5 d. Infusion of water or casein did not alter the milk production response to bST, but restricted feeding reduced the bST response (3.2 vs. 7.2 kg/d). Concentrations of IGF-I in plasma were increased by bST administration, and the increase was greatest for cows fed for ad libitum intake. The milk production response to bST was not increased by additional energy or protein offered to cows fed well-balanced diets.

Animals↗
PubMed1995

Specific endometrial binding sites for bovine placental lactogen are antigenically similar to the growth hormone receptor.

Bovine liver growth hormone receptors bind both bovine growth hormone (bGH) and bovine placental lactogen (bPL) with high affinity (Kd = 1.4 x 10(-11) M and 3.0 x 10(-11) M, respectively). By contrast, the uterine endometrium of pregnant cattle has high-affinity (Kd = 8.0 x 10(-11) M) binding sites for bPL but, displays negligible binding of bGH. A polyclonal antiserum raised against the extracellular domain of the bGH receptor, was used to determine if there was antigenic similarity between the liver bGH receptor and the endometrial bPL binding site(s). On Western blots, this antiserum displayed cross-reactivity with a 180,000-mol wt protein (nonreducing conditions) in detergent-solubilized extracts of microsomal membranes from both tissues. However, different detergents (Triton X-100 for endometrium and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate [CHAPS] for liver) were required to solubilize the cross-reacting protein in the two tissues. The purified immunoglobulin fraction from this same antiserum also blocked binding of [125I]bPL to microsomal membrane preparations from both liver and endometrium. These results indicate that the endometrial binding site for bPL is antigenically similar to the bGH receptor and raise the possibility that it may be a modified GH receptor.

Animals↗
PubMed1995

Effect of long-term administration of a prolonged release formulation of bovine somatotropin (sometribove) on clinical lameness in dairy cows.

A matched case-control study design was used to assess the effects of long-term administration of a prolonged release formulation of bovine somatotropin (sometribove) on clinical lameness and limb lesions in dairy cows. Cows treated with sometribove for at least 2 lactations (cases) and nontreated dairy cows matched by herd, parity, age, and stage of lactation (controls) in 8 herds were evaluated for clinical lameness (as assessed by gait abnormality) and limb lesions by 2 observers, using a standardized scoring procedure at a single herd visit. Although a high proportion of the study cows were clinically lame (43%), an association was not detected between chronic administration of sometribove and prevalent lameness. Of 21 types of limb lesions identified, 2 were positively associated and 2 were negatively associated with long-term sometribove use. Superficial laceration of the tarsus (odds ratio [OR] = 2.1) and superficial swelling of the metatarsophalangeal joint (OR = 4.5) were positively associated with sometribove treatment, whereas femoral lesions (OR = 0.2) and superficial lacerations of the femur (OR = 0.14) were negatively associated with sometribove treatment.

Age Factors↗
PubMed1994

Active-site mutations of diphtheria toxin: role of tyrosine-65 in NAD binding and ADP-ribosylation.

Previous studies have suggested that tyrosine-65 (Tyr-65) of diphtheria toxin (DT) is located at the active site. To investigate the role of Tyr-65 in NAD binding and the ADP-ribosylation of elongation factor-2 (EF-2), we changed this residue to alanine and phenylalanine by site-directed mutagenesis of a synthetic gene encoding the catalytic fragment of DT (DTA). The alanine mutant was greatly diminished in ADP-ribosylation activity (350-fold) and NAD-glycohydrolase activity (88-fold), whereas the phenylalanine mutant was reduced in these activities only slightly. Dissociation constants (Kd) for NAD binding were 15 microM for wild-type DTA, 26 microM for the phenylalanine mutant, and greater than 800 microM NAD for the alanine mutant. However, both mutant enzymes were found to bind adenosine with nearly equal affinity as wild-type DTA. These results support a model of ADP-ribosylation in which the phenolic ring of Tyr-65 interacts with the nicotinamide ring of NAD, orienting the N-glycosidic bond of NAD for attack by the incoming nucleophile in a direct displacement mechanism.

Adenosine↗
PubMed1994

Participation of lysine 516 and phenylalanine 530 of diphtheria toxin in receptor recognition.

To identify amino acid residues within diphtheria toxin that participate in receptor recognition, we made alanine replacements for each of 12 solvent-accessible residues within a loop (residues 516-530) of the toxin's R domain, which prominently extends from the main surface of the domain. Amino acid replacements were generated in an enzymatically attenuated form of the toxin (in compliance with regulations for cloning in Escherichia coli), and the mutant toxins were purified and assayed for toxicity on Vero cells. The largest effects were seen with K516A and F530A, which caused approximately 22- and approximately 10-fold increases, respectively, in the toxin concentration required for half-maximal inhibition of protein synthesis (IC50). Smaller effects were seen at certain other sites and no effect at still others. K516A caused approximately 500-fold reduction in ability to compete with radiolabeled wild-type toxin for receptors, and F530A gave approximately 100-fold reduction. The small differences in IC50 with the mutants, compared with differences in receptor binding, are attributable to nonlinearity in the cytotoxicity assay with the enzymatically attenuated toxin. K516A and F530A also inhibited the receptor-blocking activity of the isolated R domain. Neither mutation caused a change in the circular dichroism spectrum of the R domain. These results indicate important roles for Lys-516 and Phe-530 in receptor recognition. In addition, on the basis of cytotoxicity data, four other residues (Tyr-514, Val-523, Asn-524, and Lys-526) are proposed play roles in receptor binding. These findings support the notion that the toxin's receptor binds to the solvent-exposed face of the R domain opposite the catalytic domain.

Amino Acid Sequence↗
PubMed1994

Interaction of the protein kinase Raf-1 with 14-3-3 proteins.

Members of a family of highly conserved proteins, termed 14-3-3 proteins, were found by several experimental approaches to associate with Raf-1, a central component of a key signal transduction pathway. Optimal complex formation required the amino-terminal regulatory domain of Raf-1. The association of 14-3-3 proteins and Raf-1 was not substantially affected by the activation state of Raf.

14-3-3 Proteins↗
PubMed1994

Association of the protein kinases c-Bcr and Bcr-Abl with proteins of the 14-3-3 family.

In this study, a protein that interacts with sequences encoded by the first exon of the protein kinase Bcr was cloned. The Bcr-associated protein 1 (Bap-1) is a member of the 14-3-3 family of proteins. Bap-1 interacts with full-length c-Bcr and with the chimeric Bcr-Abl tyrosine kinase of Philadelphia chromosome (Ph1)-positive human leukemias. Bap-1 is a substrate for the Bcr serine-threonine kinase and is also phosphorylated on tyrosine by Bcr-Abl but not by c-Abl. Bap-1 may function in the regulation of c-Bcr and may contribute to the transforming activity of Bcr-Abl in vivo. 14-3-3 proteins are essential for cell proliferation and have a role in determining the timing of mitosis in yeast. Through direct binding to sequences present in Bcr and in other proteins implicated in signaling, the mammalian 14-3-3 proteins may link specific signaling protein components to mitogenic and cell-cycle control pathways.

14-3-3 Proteins↗
PubMed1994

Dynamic transitions of the transmembrane domain of diphtheria toxin: disulfide trapping and fluorescence proximity studies.

Translocation of the catalytic domain of diphtheria toxin across the endosomal membrane to the cytosolic compartment depends on low-pH-triggered insertion of the toxin's T (transmembrane) domain into the membrane. The T domain, consisting of nine alpha-helices arranged in three layers, was cloned and expressed as a discrete protein in Escherichia coli, and mutant forms were prepared and characterized. To investigate the relative movements of the three layers under various conditions, we generated two mutant forms of the domain, each containing an artificial intramolecular disulfide bridge linking the buried apolar hairpin (TH8-TH9) to one of the other two layers. Both disulfides inhibited exposure of the domain's apolar regions in solution at low pH, as determined by 2-p-toluidinylnaphthalene-6-sulfonate binding, and blocked its ability to form channels in artificial bilayers. Reduction of the bridges abolished these effects. Reduced forms of the mutant proteins were reacted with pyrenylmaleimide, a fluorescent probe, to monitor separation of the layers. Strong excimer bands seen in both mutants at neutral pH were undiminished at pH 5, indicating the retention of gross conformation in solution under acidic conditions. The addition of phospholipid vesicles at pH 5, but not at pH 7.5, quenched excimer fluorescence, reflecting the physical separation of the TH8-TH9 hairpin from the other layers upon the T domain's interaction with the bilayer. The results indicate that (i) the conformation of the isolated T domain closely resembles that seen in the whole toxin, (ii) the TH8-TH9 hairpin separates from both of the other layers of the domain as an essential step of membrane insertion, and (iii) this separation is triggered by contact of the domain with the membrane under acidic conditions.

Amino Acid Sequence↗
PubMed1994

Active-site mutations of diphtheria toxin. Tryptophan 50 is a major determinant of NAD affinity.

The two active-site tryptophans of diphtheria toxin, Trp-50 and Trp-153, were individually or jointly replaced with phenylalanine or alanine by directed mutagenesis of a synthetic gene for the toxin's catalytic A fragment. Substitution of Trp-50 with alanine (W50A) decreased the ADP-ribosyltransferase activity by nearly 10(5)-fold and reduced NAD-glycohydrolase activity beyond the limits of our detection. Effects of the W153A mutation on these activities were less dramatic, < 40-fold decrease in ADP-ribosylation and < 10-fold decrease in NAD glycohydrolysis. The W50F and W153F substitutions caused only minimal reductions (< 2-fold) in enzyme activities and NAD affinity. Decreases in affinity for NAD in the initial, ground state complex, as measured by intrinsic protein fluorescence, correlated well with the reductions in enzyme activity. None of the mutations caused greater than a 10-fold decrease in NAD affinity for the ternary Michaelis complex in the ADP-ribosylation reaction; and none caused significant increase in susceptibility to proteolytic digestion by trypsin. The results indicate that Trp-50 is a major determinant of NAD affinity. Also, they identify this residue as a candidate for modification in the development of inactive forms of the toxin for use in vaccine development.

Binding Sites↗
PubMed1994

Mutational analysis of the helical hairpin region of diphtheria toxin transmembrane domain.

Entry of the catalytic domain of diphtheria toxin into the cytoplasma of eukaryotic cells depends on insertion of the T (transmembrane) domain into the endosomal membrane, a process triggered by low pH. To probe the mechanism of insertion, we mutated ionizable residues within the helical hairpin region of the T domain. Only three mutations caused significant effects on cytotoxicity, D295K, E349K, and D352K. Each of these represents a substitution of a basic for an acidic residue at the tip of a helical hairpin. Substitution of Lys for Glu349 or Asp352, in the TH8/9 hairpin, reduced toxicity for Vero cells > 100-fold, whereas a Lys substitution for Asp295, one of 3 acidic residues in the TH5/6/7 hairpin, caused a less marked reduction. All three mutations also altered the pH-dependent formation, and/or ion conductance, of channels formed by the toxin in artificial bilayers or the plasma membrane. E349K or D352K did not alter the pH dependence of conformational changes in the toxin occurring near pH 5. Our findings support the hypothesis that the TH8/9 hairpin inserts into the endosomal membrane after low pH-mediated partial unfolding of the T domain. A positive residue at the tip of this hairpin apparently inhibits insertion and blocks toxin action. The ion-conducting properties of channels formed by selected mutants, described elsewhere, are consistent with this model. The status of the TH5/6/7 hairpin in the integral membrane form of the T domain remains uncertain.

Amino Acid Sequence↗
PubMed1994

Anthrax protective antigen forms oligomers during intoxication of mammalian cells.

The protective antigen component (PA) of anthrax toxin binds to receptors on target cells and conveys the toxin's edema factor (EF) and lethal factor (LF) components into the cytoplasm. PA (83 kDa) is processed by a cellular protease, yielding a 63-kDa fragment (PA63), which binds EF and/or LF. When exposed to acidic pH, PA63 inserts into membranes and forms ion-conductive channels. By electron microscopy, a significant fraction of purified PA63 was found to be in the form of a multi-subunit ring-shaped oligomer (outer diameter, 10.4 nm). The rings are heptameric, as judged by inspection and by rotational power spectra. Purified PA63 showed a high M(r) band, apparently corresponding to the oligomer, on SDS-polyacrylamide gels, and oligomer of similar size was formed in cells in a time-dependent manner after addition of full-length PA. Inhibitors of internalization and endosome acidification blocked conversion of cell-associated PA to a high molecular weight species, and medium at pH 5.0 induced oligomer formation in the presence or absence of the inhibitors. These results correlate PA63 oligomerization with conditions required for translocation of EF and LF across lipid bilayers, implying that the PA63 oligomer may function in translocation.

Animals↗