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R J Collier

Publications and source records attributed to R J Collier.

At least 109 records · Page 6Linked to original sources
PubMed1994

Association of polyomavirus middle tumor antigen with 14-3-3 proteins.

To carry out its transformation function, the middle tumor antigen (MT) of murine polyomavirus associates with a number of cellular proteins involved in regulation of cell proliferation, including pp60c-Src, phosphatidylinositol 3-kinase, protein phosphatase 2A, Src homologous and collagen protein and growth factor receptor-binding protein 2. Here, two additional MT-associated proteins were identified as members of the 14-3-3 family of proteins. Yeast homologs of 14-3-3 proteins have recently been shown to play a role in the timing of mitosis. Thus, regulation of 14-3-3 protein function by MT may contribute to the development of neoplasia.

14-3-3 Proteins↗
PubMed1994

Reaction of diphtheria toxin channels with sulfhydryl-specific reagents: observation of chemical reactions at the single molecule level.

The diphtheria toxin channel is believed to be a homooligomer of its T domain in which each subunit consists of two alpha-helices, lying within the membrane, connected by a short interhelical loop of four amino acids (residues 349-352). To investigate the validity and implications of this model, we singly mutated each of these amino acids to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and added to the trans compartment sulfhydryl-specific reagents [methanethiosulfonate derivatives (MTS-ER)] that introduce a positive or negative charge to reacted cysteines. The introduction of a positive charge at residue 351 or 352 (through the MTS-ER reactions) resulted in a step decrease in single-channel conductance, whereas the introduction of a negative charge resulted in a step increase. The opposite sign of these effects indicates the predominantly electrostatic nature of the phenomenon and implies that residues 351 and 352 lie close to the channel entrance. The same reactions at residue 350 resulted in very little change in channel conductance but instead changed the character of the natural rapid flickering of the channel between open and closed states to one in which the channel spent more time in the closed state; this may have resulted from the group introduced at position 350 acting as a tethered channel blocker. The MTS derivatives had no effect on channels containing a cysteine at position 349, suggesting that this residue faces away from the channel entrance. We propose that the step changes in conductance or flickering pattern result from the chemical reaction of one MTS-ER molecule with one cysteine, and thus a bimolecular chemical reaction is being witnessed at the single molecule level. From the distribution of waiting times between the appearance (i.e., the opening) of a channel and the step change in its conductance or flickering pattern, we can calculate a pseudo-first-order rate constant, which can then be converted to a second-order rate constant, for the chemical reaction.

Amino Acid Sequence↗
PubMed1994

Active-site mutations of the diphtheria toxin catalytic domain: role of histidine-21 in nicotinamide adenine dinucleotide binding and ADP-ribosylation of elongation factor 2.

Diphtheria toxin (DT) has been studied as a model for understanding active-site structure and function in the ADP-ribosyltransferases. Earlier evidence suggested that histidine-21 of DT is important for the ADP-ribosylation of eukaryotic elongation factor 2 (EF-2). We have generated substitutions of this residue by cassette mutagenesis of a synthetic gene encoding the catalytic A fragment (DTA) of DT, and have characterized purified mutant forms of this domain. Changing histidine-21 to alanine, aspartic acid, leucine, glutamine, or arginine diminished ADP-ribosylation activity by 70-fold or greater. In contrast, asparagine proved to be a functionally conservative substitution, which reduced ADP-ribosylation activity by < 3-fold. The asparagine mutant was approximately 50-fold-attenuated in NAD glycohydrolase activity, however. Dissociation constants (Kd) for NAD binding, determined by quenching of the intrinsic protein fluorescence, were 15 microM for wild-type DTA, 160 microM for the asparagine mutant, and greater than 500 microM NAD for the alanine, leucine, glutamine, and arginine mutants. These and previous results support a model of the ADP-ribosylation of EF-2 in which histidine-21 serves primarily a hydrogen-bonding function. We propose that the pi-imidazole nitrogen of His-21 hydrogen-bonds to the nicotinamide carboxamide, orienting the N-glycosidic bond of NAD for attack by the incoming nucleophile in a direct displacement mechanism, and then stabilizing the transition-state intermediate of this reaction.

Adenosine Diphosphate Ribose↗
PubMed1994

Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain.

Diphtheria Toxin (DT) is a 535 amino acid exotoxin, whose active form consists of two polypeptide chains linked by an interchain disulphide bond. DT's N-terminal A fragment kills cells by enzymatically inactivating their protein synthetic machinery; its C-terminal B chain is required for the binding of toxin to sensitive cells and for the translocation of the A fragment into the cytosol. This B fragment, consisting of its N-terminal T domain (amino acids 191-386) and its C-terminal R domain (amino acids 387-535) is responsible for the ion-conducting channels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channels formed by deletion mutants of DT in lipid bilayer membranes under several pH conditions. Channels formed by mutants containing only the T domain (i.e., lacking the A fragment and/or the R domain), as well as those formed by mutants replacing the R domain with Interleukin-2 (IL-2), have single channel conductances and selectivities essentially identical to those of channels formed by wild-type DT. Furthermore, deleting the N-terminal 118 amino acids of the T domain also has minimal effect on the single channel conductance and selectivity of the mutant channels. Together, these data identify a 61 amino acid stretch of the T domain, corresponding to the region which includes alpha-helices TH8 and TH9 in the crystal structure of DT, as the channel-forming region of the toxin.

Amino Acid Sequence↗
PubMed1994

Structure function relationships in diphtheria toxin channels: II. A residue responsible for the channel's dependence on trans pH.

Ion-conducting channels formed in lipid bilayers by diphtheria toxin are highly pH dependent. Among other properties, the channel's single channel conductance and selectivity depend on proton concentrations on either side of the membrane. We have previously shown that a 61 amino acid fragment of DT is sufficient to form a channel having the same pH-dependent single channel properties as that of the intact toxin. This region corresponds to an alpha-helical hairpin in the recently published crystal structure of DT in solution; the hairpin contains two alpha-helices, each long enough to span a membrane, connected by a loop of about nine residues. This paper reports on the single channel effects of mutations which alter the two negatively charged residues in this loop. Changing Glutamate 349 to neutral glutamine or to positive lysine has no effect on the DT channel's single channel conductance or selectivity. In contrast, mutations of Aspartate 352 to neutral asparagine (DT-D352N) or positive lysine (DT-D352K) cause progressive reductions in single channel conductance at pH 5.3 cis/7.2 trans (in 1 M KCl), consistent with this group interacting electrostatically with ions in the channel. The cation selectivity of these mutant channels is also reduced from that of wild-type channels, a direction consistent with residue 352 influencing permeant ions via electrostatic forces. When both sides of the membrane are at pH 4, the conductance difference between wild-type and DT-D352N channels is minimal, suggesting that Asp 352 (in the wild type) is neutral at this pH. Differences observed between wild-type and DT-D352N channels at pH 4.0 cis/7.2 trans (with a high concentration of permeant buffer in the cis compartment) imply that residue 352 is on or near the trans side of the membrane. Comparing the conductances of wild-type and DT-D352K channels at large (cis) positive voltages supports this conclusion. The trans location of position 352 severely constrains the number of possible membrane topologies for this region.

Diphtheria Toxin↗
PubMed1994

Structure-function relationships in diphtheria toxin channels: III. Residues which affect the cis pH dependence of channel conductance.

The conductance of channels formed by diphtheria toxin (DT) in lipid bilayer membrane depends strongly on pH. We have previously shown that a 61 amino acid region of the protein, denoted TH8-9, is sufficient to form channels having the same pH-dependent conductance properties as those of whole toxin channels. One residue in this region, Aspartate 352, is responsible for all the dependence of single channel conductance on trans pH, whereas another, Glutamate 349, has no effect. Here, we report that of the seven remaining charged residues in the TH8-9 region, mutations altering the charge on H322, H323, H372, and R377 have minimal effects on single channel conductance; mutations of Glutamates 326, 327, or 362, however, significantly affect single channel conductance as well as its dependence on cis pH. Moreover, Glutamate 362 is titratable from both the cis and trans sides of the membrane, suggesting that this residue lies within the channel; it is more accessible, however, to cis than to trans protons. These results are consistent with the membrane-spanning topology previously proposed for the TH8-9 region, and suggest a geometric model for the DT channel.

Aspartic Acid↗
PubMed1994

Extended function of the corpus luteum and earlier development of the second follicular wave in heifers treated with bovine somatotropin.

Effects of recombinant bovine somatotropin (bST) on growth of the corpus luteum (CL) and development of ovarian follicles were tested. Starting at estrus (Day=0), the following treatments were administered: control (saline injected Days 0 to 19, n=5); bST[0-9] (25 mg bST injected Days 0 to 9, saline injected Days 10 to 19, n=5); bST[10-19] (saline injected Days 0 to 9, 25 mg bST injected Days 10 to 19, n=5); and bST[0-19] (25 mg bST injected Days 0 to 19, n=6). Blood was collected daily for progesterone analysis, and ultrasound examinations were performed daily for measurement of follicles and CL. Compared with the heifers treated with saline, those treated with bST had larger CL and more progesterone during the early (</= Day 10) and late (Days 15 to 21) phases of the estrous cycle. At mid-cycle (Days 10 to 15), luteal tissue area and progesterone levels were similar for bST- and saline-treated heifers. Ovarian follicular growth was similar for bST- and saline-treated heifers during the first follicular wave. However, during recruitment for the second follicular wave (Days 10 to 14), second largest follicles emerged sooner (P<0.001), the number of Class-3 (>/=10 mm) follicles was greater (P<0.01) and largest follicles were smaller (P<0.001) in bST than in saline-treated heifers. Estrous cycle length and ovulation rate were similar for each group. In conclusion, bST increased initial development of the CL and extended its function. Furthermore, the second follicular wave was earlier with bST.

Journal Article↗
PubMed1994

In vivo measurements of the phase constants of transverse mechanical waves in a human tibia from 100 to 1000 Hz.

This paper contains the details of a new technique for measuring the mechanical phase constants of transverse waves in human tibia, in vivo. The measurements are made in the frequency range from 100 to 1000 Hz. The importance of these measurements is that healthy tibia has quite small phase delays along the bone at these frequencies. On the other hand, a fractured tibia has a large phase delay at the fracture site. This paper establishes a set of typical phase delays for a range of tibia so that subsequent measurements on fractured patients can be interpreted. It contains a description of the method of measuring the phase delay and a detailed analysis of errors. In particular, it includes an account of the phase delay through the soft tissues as well as the bone and how the former can be eliminated. It is not an exhaustive account of this topic but contains sufficient results to show that the measurements can be made relatively easily. Finally, there is a theoretical model which can be used to predict the phase shift. However, in order to use the model various other measurements are necessary and a direct measurement of phase is easier than using a model.

Electric Impedance↗
PubMed1994

Placental lactogen and somatotropin: hormone binding to the corpus luteum and effects on the growth and functions of the ovary in heifers.

The effects of recombinant bovine placental lactogen (rbPL) and recombinant bovine somatotropin (rbST) on development of ovarian follicles and CL were tested in heifers. Estrus (day = 0) was synchronized and heifers were treated (Days 0-21) with either saline (control; n = 7), rbST (25 mg/day; n = 6), or rbPL. (50 mg/day; n = 8). Blood was collected daily for analyses of progesterone, estradiol, ST, PL, and insulin-like growth factor (IGF)-I; ultrasound was performed daily for measurement of follicles and CL. PL in plasma (mean +/- SE; ng/ml) averaged 4.1 +/- 0.2 for rbPL-treated heifers, and ST in plasma (ng/ml) averaged 2.7 +/- 0.3 for rbST-treated heifers. IGF-I in plasma (ng/ml) was increased for rbST-treated (198 +/- 10; p < 0.001) and rbPL-treated (143 +/- 9; p < 0.06) heifers compared to controls (117 +/- 9). After Day 9 of the estrous cycle, heifers treated with rbPL had larger CL (p < 0.001) and more progesterone in plasma (p < 0.001) than controls, whereas rbST-treated heifers were intermediate for these measures. Largest follicles were decreased in size (mm) throughout the estrous cycle for rbPL-treated heifers (12.9 +/- 0.4) compared to controls (14.2 +/- 0.5; p < 0.06) or heifers given rbST (14.0 +/- 0.5; p < 0.11). After Day 17 (preovulatory period), concentrations of estradiol in serum (pg/ml) were decreased for rbST-treated (2.7 +/- 0.3; p < 0.01) and rbPL-treated (2.9 +/- 0.2; p < 0.02) heifers compared to controls (3.8 +/- 0.3).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Unilateral control of ovarian oxytocin release and the facilitatory effects of insulin-like growth factor-I in sheep.

Prostaglandin F2 alpha (PGF2 alpha)-induced release of ovarian oxytocin was investigated to determine whether the effect in vivo was local. [3H]PGF2 alpha infused downstream into a single ovarian lymphatic was transferred into the adjacent ovarian vasculature (estimated transfer 1.1 and 1.7%, two experiments). When unlabelled PGF2 alpha was infused in a similar manner (76 pmol min-1), there was a prompt eightfold increase in ovarian oxytocin release from the adjacent ovary containing a corpus luteum, but no effect on the opposite corpus luteum, showing that the effect was local. Instillation of 2% lignocaine into the ovarian vascular pedicle did not affect PGF2 alpha-induced oxytocin release, supporting the idea that neural mechanisms are not involved. Repeated doses of PGF2 alpha given close-arterially produced a successive reduction in oxytocin release. This effect was prevented by a prior infusion of insulin-like growth factor-I (IGF-I), which itself gave a small, but significant, increase in oxytocin release. The results show that PGF2 alpha in ovarian lymphatics acts locally and directly to stimulate ovarian oxytocin secretion, that repeated exposure of the corpus luteum to pulses of PGF2 alpha can result in tachyphylaxis, and that this latter effect can be ameliorated by IGF-I infused in vivo.

Animals↗
PubMed1994

Stimulation of mammogenesis and lactogenesis by recombinant bovine placental lactogen in steroid-primed dairy heifers.

A model of induced lactation was modified to examine the effects of bovine prolactin (bPRL) and bovine placental lactogen (bPL) on mammary growth and differentiation. Thirty-two peripubertal, non-pregnant Holstein heifers were given daily s.c. injections of oestradiol (0.05 mg/kg) and progesterone (0.25 mg/kg) for 7 days to initiate mammary growth. Treatment with bromocriptine (40 mg/3 days) reduced serum PRL concentrations to approximately 25% of pretreatment levels, for the duration of the study. On the day following the last steroid injection, groups of eight heifers were given twice daily s.c. injections of either saline (negative control), recombinant bPRL (rbPRL; 80 mg/day) or recombinant bPL (rbPL; 80 and 160 mg/day) for 7 days. At the end of this period (day 15), growth and differentiation of the mammary glands were assessed. Treatment with rbPL increased total mammary DNA above control value by 50 and 60% for the 80 and 160 mg/day doses respectively. However, total DNA was not different for the control and rbPRL-treated groups. The blood serum concentration of alpha-lactalbumin was measured daily throughout the study and used as an index of mammary differentiation. Both rbPRL and rbPL stimulated mammary differentiation (i.e. induction of milk synthesis), although rbPRL appeared to be more potent than rbPL. These results indicate that rbPL is lactogenic in vivo and strongly suggest that bPL is a mammary mitogen.

Animals↗
PubMed1994

Clinical mastitis in cows treated with sometribove (recombinant bovine somatotropin) and its relationship to milk yield.

Effect of sometribove (methionyl bovine somatotropin) on mastitis in 15 full lactation trials (914 cows) in Europe and the US and 70 short-term studies (2697 cows) in eight countries was investigated. In full lactation studies, sometribove (500 mg/2 wk) was given for 252 d, commencing 60 d postpartum. Although herds varied considerably, incidence of clinical mastitis within a herd was similar for cows receiving control and sometribove treatments. Relative risk analyses indicated no treatment effect, and percentage of mastitis during treatment was similar for control and sometribove groups. A positive linear relationship existed between peak milk yield and mastitis incidence (percentage of cows contracting mastitis or cases per 100 cow days); sometribove treatment did not alter this relationship. Increases in mastitis related to milk yield increase from sometribove or related to genetic selection were similar. When expressed per unit of milk, mastitis incidence declined slightly as milk yield increased; this relationship was not altered by sometribove. No effect on clinical mastitis was observed in 70 commercial herds utilizing sometribove for 84 d. However, effects were significant for stage of lactation and milk yield. Overall, studies represented a wide range of research and commercial situations demonstrating that sometribove had no effect on incidence of clinical mastitis during the lactation of treatment. Furthermore, sometribove did not alter typical relationships between milk yield or herd factors and incidence of clinical mastitis.

Animals↗
PubMed1994

Role of macrophage oxidative burst in the action of anthrax lethal toxin.

BACKGROUND: Major symptoms and death from systemic Bacillus anthracis infections are mediated by the action of the pathogen's lethal toxin on host macrophages. High levels of the toxin are cytolytic to macrophages, whereas low levels stimulate these cells to produce cytokines (interleukin-1 beta and tumor necrosis factor-alpha), which induce systemic shock and death. MATERIALS AND METHODS: Experiments were performed to assess the possibility that the oxidative burst may be involved in one or both of lethal toxin's effects on macrophages. Toximediated cell lysis, superoxide anion and cytokine production were measured. Effects of antioxidants and macrophage mutations were examined. RESULTS: RAW264.7 murine macrophages treated with high levels of toxin released large amounts of superoxide anion, beginning at about 1 hr, which correlates with the onset of cytolysis. Cytolysis could be blocked with various exogenous antioxidants or with N-acetyl-L-cysteine and methionine, which promote production of the endogenous antioxidant, glutathione. Mutant murine macrophage lines deficient in production of reactive oxygen intermediates (ROIs) were relatively insensitive to the lytic effects of the toxin, whereas a line with increased oxidative burst potential showed elevated sensitivity. Also, cultured blood monocyte-derived macrophages from a patient with Chronic Granulomatous Disease, a disorder in which the phagocyte's oxidative burst is disabled, were totally resistant to toxin, in contrast to control monocytes. CONCLUSIONS: These results imply that the cytolytic effect of the toxin is mediated by ROIs. Additionally, cytokine production and consequent pathologies showed partial dependence on macrophage ROIs. Antioxidants moderately inhibited toxin-induced cytokine production in vitro, and BALB/c mice pretreated with N-acetyl-L-cysteine or mepacrine showed partial protection against lethal toxin. Thus ROIs are involved in both the cytolytic action of anthrax lethal toxin and the overall pathologic process in vivo.

Animals↗
PubMed1993

On the role of macrophages in anthrax.

Bacillus anthracis, the causative agent of anthrax, produces systemic shock and death in susceptible animals, primarily through the action of its lethal toxin. This toxin, at high concentrations, induces lysis of macrophages in vitro but shows little or no effect on other cells. We found that when mice were specifically depleted of macrophages by silica injections, they became resistant to the toxin. Sensitivity could be restored by coinjection of toxin-sensitive cultured macrophages (RAW 264.7 cells) but not by coinjection of other cell lines tested. These results implied that macrophages mediate the action of lethal toxin in vivo and led us to investigate their role in death of the mammalian host. Sublytic concentrations of lethal toxin, orders of magnitude lower than those required to induce lysis of RAW 264.7 cells, were found to induce these cells to express interleukin 1 (IL-1) and tumor necrosis factor in vitro. Passive immunization against IL-1 or injection of an IL-1 receptor antagonist protected mice from toxin challenge, whereas anti-tumor necrosis factor provided little, if any, protection. These results imply that systemic shock and death from anthrax result primarily from the effects of high levels of cytokines, principally IL-1, produced by macrophages that have been stimulated by the anthrax lethal toxin.

Animals↗
PubMed1993

Diphthamide synthesis in Saccharomyces cerevisiae: structure of the DPH2 gene.

A gene involved in diphthamide biosynthesis, DPH2, was cloned from Saccharomyces cerevisiae by complementation of a diphthamide mutant. DPH2 exists as a single-copy gene in the yeast genome and is located on the left arm of chromosome XI. Sequence analysis of the DPH2 locus predicts that the DPH2 gene product is a 534-amino acid (aa) protein, with a calculated M(r) of 59,772. This conclusion was supported by Northern blot analysis of the DPH2 transcript and gel analysis of the DPH2 protein overproduced in Escherichia coli. Gene disruption studies indicate that the DPH2 gene is not essential for viability of yeast. The role of DPH2 in diphthamide biosynthesis is discussed.

Amino Acid Sequence↗
PubMed1993

The eukaryotic host factor that activates exoenzyme S of Pseudomonas aeruginosa is a member of the 14-3-3 protein family.

Exoenzyme S (ExoS), which has been implicated as a virulence factor of Pseudomonas aeruginosa, catalyzes transfer of the ADP-ribose moiety of NAD+ to many eukaryotic cellular proteins. Its preferred substrates include Ras and several other 21- to 25-kDa GTP-binding proteins. ExoS absolutely requires a ubiquitous eukaryotic protein factor, termed FAS (factor activating ExoS), for enzymatic activity. Here we describe the cloning and expression of a gene encoding FAS from a bovine brain cDNA library and demonstrate that purified recombinant FAS produced in Escherichia coli activates ExoS in a defined cell-free system. The deduced amino acid sequence of FAS shows that the protein (245 residues, calculated molecular mass 27,743 Da) belongs to a highly conserved, widely distributed eukaryotic protein family, collectively designated as 14-3-3 proteins. Various functions have been reported for members of the 14-3-3 family, including phospholipase A2 activity and regulation of tyrosine hydroxylase, tryptophan hydroxylase, and, possibly, protein kinase C activities. Identification of FAS as a 14-3-3 protein establishes an additional function for this family of proteins--the activation of an exogenous ADP-ribosyltransferase. Elucidation of the precise role of FAS in activating ExoS will contribute to understanding the molecular mechanisms by which P. aeruginosa causes disease.

14-3-3 Proteins↗
PubMed1993

DNA fragmentation and cytolysis in U937 cells treated with diphtheria toxin or other inhibitors of protein synthesis.

We treated the human monoblastoid cell line, U937, with various cytotoxic proteins or drugs that specifically inhibit protein synthesis and monitored the cells for degradation of chromosomal DNA and other changes. In cells treated with diphtheria toxin (DT), Pseudomonas aeruginosa exotoxin A, ricin toxin, and abrin toxin the chromosomal DNA was degraded into oligonucleosome-sized fragments, the chromatin became condensed, and the cell nuclei fragmented. All of these changes are characteristic of cells undergoing apoptosis, or programmed cell death. Various drugs, including puromycin, cycloheximide, emetine, and anisomycin produced similar changes. An enzymically attenuated mutant of DT, DT-E148S, produced effects identical to those produced by the native toxin, except that a higher concentration of toxin was required, corresponding to the reduction in ADP-ribosylation activity. In all cases, DNA degradation and other changes were observed only after the rate of protein synthesis was reduced to low levels, approximately 10% or less of normal levels. These results imply that inhibition of protein synthesis in U937 cells induces apoptosis, regardless of the mechanism of action of the inhibitor. Differences in the kinetics of induction of apoptosis by the various inhibitors may reflect secondary effects on other aspects of cellular physiology.

Apoptosis↗