C J Bagley

Hanson Institute, Tarndarnya, South Australia, Australia

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Publications (59)258.29 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Postnatal mesenchymal stem/stromal-like cells (MSCs) including periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and bone marrow stromal cells (BMSCs) are capable of self-renewal and differentiation into multiple mesenchymal cell lineages. Despite their similar expression of MSC-associated and osteoblastic markers, MSCs retain the capacity to generate structures resembling the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical setting. With this in mind, systematic approaches are required to identify the differential protein expression patterns responsible for lineage commitment and mediating the formation of these complex structures. This is the first study to compare the differential proteomic expression profiles of ex vivo-expanded ovine PDLSCs, DPSCs, and BMSCs derived from an individual donor. The two-dimensional electrophoresis was performed and regulated proteins were identified by liquid chromatography--electrospray-ionization tandem mass spectrometry (MS and MS/MS), database searching, and de novo sequencing. In total, 58 proteins were differentially expressed between at least 2 MSC populations in both sheep, 12 of which were up-regulated in one MSC population relative to the other two. In addition, the regulation of selected proteins was also conserved between equivalent human MSC populations. We anticipate that differential protein expression profiling will provide a basis for elucidating the protein expression patterns and molecular cues that are crucial in specifying the characteristic growth and developmental capacity of dental and non-dental tissue-derived MSC populations. These expression patterns can serve as important tools for the regeneration of particular tissues in future stem cell-based tissue engineering studies using animal models.
    Stem cells and development 10/2010; 19(10):1485-99. · 4.15 Impact Factor
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    ABSTRACT: The importance of Mn(2+) for pneumococcal physiology and virulence has been studied extensively. However, the specific cellular role(s) for which Mn(2+) is required are yet to be fully elucidated. Here, we analyzed the effect of Mn(2+) limitation on the transcriptome and proteome of Streptococcus pneumoniae D39. This was carried out by comparing a deletion mutant lacking the solute binding protein of the high-affinity Mn(2+) transporter, pneumococcal surface antigen A (PsaA), with its isogenic wild-type counterpart. We provide clear evidence for the Mn(2+)-dependent regulation of the expression of oxidative-stress-response enzymes SpxB and Mn(2+)-SodA and virulence-associated genes pcpA and prtA. We also demonstrate the upregulation of at least one oxidative- and nitrosative-stress-response gene cluster, comprising adhC, nmlR, and czcD, in response to Mn(2+) stress. A significant increase in 6-phosphogluconate dehydrogenase activity in the psaA mutant grown under Mn(2+)-replete conditions and upregulation of an oligopeptide ABC permease (AppDCBA) were also observed. Together, the results of transcriptomic and proteomic analyses provided evidence for Mn(2+) having a central role in activating or stimulating enzymes involved in central carbon and general metabolism. Our results also highlight the importance of high-affinity Mn(2+) transport by PsaA in pneumococcal competence, physiology, and metabolism and elucidate mechanisms underlying the response to Mn(2+) stress.
    Journal of bacteriology 09/2010; 192(17):4489-97. · 3.94 Impact Factor
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    ABSTRACT: Neutrophil extravasation, a critical component of innate immunity must be tightly regulated to prevent inadvertent or prolonged inflammation and subsequent tissue damage. We have shown previously that endothelial ERK1/2 signaling essential for neutrophil transendothelial migration is induced by a soluble factor produced by activated neutrophils. In this study, we demonstrate that the soluble neutrophil factor is a truncated form of annexin A1 (AnxA1) that can be generated by calpain 1 cleavage of the N terminus, thus identifying a novel proinflammatory function to AnxA1. In contrast, neither the full-length protein nor the N-terminal 26 aa peptide, previously shown to be antiinflammatory, were able to activate Erk. Our data suggest that two different fragments of AnxA1 have opposing functions in inflammation. We also provide evidence that C-terminal AnxA1 functions by increasing ICAM1 clustering around adherent neutrophils to anchor them to the endothelium and promote transmigration through the transcellular route.
    The Journal of Immunology 09/2010; 185(5):3057-63. · 5.52 Impact Factor
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    ABSTRACT: The quality of MALDI-TOF mass spectrometric analysis is highly dependent on the matrix and its deposition strategy. Although different matrix-deposition methods have specific advantages, one major problem in the field of proteomics, particularly with respect to quantitation, is reproducibility between users or laboratories. Compounding this is the varying crystal homogeneity of matrices depending on the deposition strategy used. Here, we describe a novel optimised matrix-deposition strategy for LC-MALDI-TOF/TOF MS using an automated instrument that produces a nebulised matrix "mist" under controlled atmospheric conditions. Comparisons of this with previously reported strategies showed the method to be advantageous for the atypical matrix, 2,5-DHB, and improved phosphopeptide ionisation when compared with deposition strategies for CHCA. This optimised DHB matrix-deposition strategy with LC-MALDI-TOF/TOF MS, termed EZYprep LC, was subsequently optimised for phosphoproteome analysis and compared to LC-ESI-IT-MS and a previously reported approach for phosphotyrosine identification and characterisation. These methods were used to map phosphorylation on epidermal growth factor-stimulated epidermal growth factor receptor to gauge the sensitivity of the proposed method. EZYprep DHB LC-MALDI-TOF/TOF MS was able to identify more phosphopeptides and characterise more phosphorylation sites than the other two proteomic strategies, thus proving to be a sensitive approach for phosphoproteome analysis.
    Proteomics 07/2010; 10(13):2516-30. · 4.43 Impact Factor
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    ABSTRACT: Proteins of the CLC family are comprised of two subunits, each with its own fast-gated protopore, both of these being regulated simultaneously by a slower common gate. Based on the X-ray crystal structure of a bacterial CLC, the carboxyl side chain of glutamate residue E232 has been proposed as the fast gate of hClC-1, swinging into each pore to close it and competing with chloride. We now show, using hClC-1 mutants expressed in whole-cell patch-clamped HEK293 cells, that elimination of this side chain in the E232Q mutation prevents fast gate closure at all voltages but common gating is also eliminated suggesting that E232 could be the final effector of both fast and common gating. We hypothesise that the conformational information essential for common gating flows between the two E232 protopore residues across the intra-membrane interface, rather than via any cytoplasmic carboxyl-tail interface, to drive common gating. Informed by in silico modelling, we have produced five site-directed mutants that increase the volumes of residues which might be involved in allosteric transfer (A272V, A272L, S289L, V292L and T293L) and assessed them for effects on gating. These mutations could be expected to increase molecular forces between, or torques around, the intimate L287-L287 and I290-I290 contacts that form the pseudo-asymmetric axis of the hClC-1 dimer. Common gating is practically eliminated in V292L and open probability is shifted to more depolarised potentials in A272V, S289L and T293L mainly by altering the voltage dependence of common gating.
    The international journal of biochemistry & cell biology 07/2010; 42(7):1182-8. · 4.89 Impact Factor
  • Peter S Zilm, Alex Mira, Christopher J Bagley, Anthony H Rogers
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    ABSTRACT: Fusobacterium nucleatum is a Gram-negative anaerobic organism that plays a central role in the development of periodontal diseases. The progression of periodontitis is associated with a rise in pH of the gingival sulcus which promotes the growth and expression of virulence factors by periodontopathic bacteria. We have previously reported that the expression of specific cytoplasmic proteins is altered by a shift in growth pH. In the present study we have compared cell envelope protein expression of F. nucleatum during chemostat growth at pH 7.2 and 7.8. From a total of 176 proteins resolved from the cell envelope, 15 were found to have altered expression in response to an increase in growth pH and were identified by MS. Upregulated proteins included an outer membrane porin which has been identified as playing a role in virulence, a periplasmic chaperone which assists in the folding of outer membrane proteins, and a transporter thought to be involved with iron uptake. Proteins downregulated at pH 7.8 were consistent with our previous findings that the bacterium reduces its catabolism of energy-yielding substrates in favour of energy-storage pathways. Among the downregulated proteins, two transporters which are involved in the uptake of C4 dicarboxylates and phosphate were identified. A putative protease and an enzyme associated with the metabolism of glutamate were also identified. A high proportion of the cell envelope proteins suggested by these data to play a role in the organism's response to alkaline growth pH may have arisen by lateral gene transfer. This would support the hypothesis that genes that provide an ability to adapt to the changing conditions of the oral environment may be readily shared between oral bacteria.
    Microbiology 03/2010; 156(Pt 6):1783-94. · 3.06 Impact Factor
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    ABSTRACT: Abstract The high affinity receptors for GM-CSF, IL-3 and IL-5 are heterodimers consisting of a ligand-specific a chain and a common β chain. These proteins are members of a family of proteins known as the “cytokine receptor family” which is characterized by the presence of a 200-residue ligand-binding module. The GMCSF, IL-3 and IL-5 receptor α chains constitute a distinct subgroup and share features not found in other members of the cytokine receptor family, features which we propose to be important for their interaction with the common β chain and for their binding of the structurally-related ligands. The growth hormone receptor is a well-characterized member of the cytokine receptor family. Based on the structure of the complex between growth hormone and its receptor, we have proposed sites of contact between the GM-CSF, IL-3 and IL-5 receptors and their cognate ligands.
    Growth Factors 07/2009; 8(2):87-97. · 2.20 Impact Factor
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    ABSTRACT: The following peptides have been examined in this study: GLDFG(OH), caeridin 1.1 [GLLDGLLGLGGL(NH(2))], 11 Ala citropin 1.1 [GLFDVIKKVAAVIGGL(NH(2))], Crinia angiotensin [APGDRIYVHPF(OH)] and their isoAsp isomers. It is not possible to differentiate between Asp- and isoAsp-containing peptides (used in this study) using negative ion electrospray mass spectrometry. This is because the isoAsp residue cleaves to give the same fragment anions as those formed by delta and gamma backbone cleavage of Asp. The isoAsp fragmentations are as follows: RNHCH(CO(2)H)(-)CHCONHR' --> [RNH(-)(HO(2)CCH=CHCONHR')] --> RNH(-)+HO(2)CCH=CHCONHR' and RNHCH(CO(2)H)(-)CHCONHR' --> [RNH(-)(HO(2)CCH=CHCONHR'] --> (-)O(2)CCH=CHCONHR'+RNH(2). Calculations at the HF/6-31+G(d)//AM1 level of theory indicate that the first of these isoAsp cleavage processes is endothermic (by +115 kJ mol(-1)), while the second is exothermic (-85 kJ mol(-1)). The barrier to the highest transition state is 42 kJ mol(-1). No diagnostic cleavage cations were observed in the electrospray mass spectra of the MH(+) ion of the Asp- and isoAsp-containing peptides (used in this study) to allow differentiation between these two amino acid residues.
    Rapid Communications in Mass Spectrometry 07/2009; 23(13):1993-2002. · 2.51 Impact Factor
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    ABSTRACT: HIV-1 reverse transcriptase (RT) is a heterodimer composed of p66 and p51 subunits and is responsible for reverse transcription of the viral RNA genome into DNA. RT can be post-translationally modified in vitro which may be an important mechanism for regulating RT activity. Here we report detection of different p66 and p51 RT isoforms by 2D gel electrophoresis in virions and infected cells. Major isoforms of the p66 and p51 RT subunits were observed, with pI's of 8.44 and 8.31 respectively (p66(8.44) and p51(8.31)). The same major isoforms were present in virions, virus-infected cell lysates and intracellular reverse transcription complexes (RTCs), and their presence in RTCs suggested that these are likely to be the forms that function in reverse transcription. Several minor RT isoforms were also observed. The observed pIs of the RT isoforms differed from the pI of theoretical unmodified RT (p66(8.53) and p51(8.60)), suggesting that most of the RT protein in virions and cells is post-translationally modified. The modifications of p66(8.44) and p51(8.31) differed from each other indicating selective modification of the different RT subunits. The susceptibility of RT isoforms to phosphatase treatment suggested that some of these modifications were due to phosphorylation. Dephosphorylation, however, had no effect on in vitro RT activity associated with virions, infected cells or RTCs suggesting that the phospho-isoforms do not make a major contribution to RT activity in an in vitro assay. The same major isoform of p66 and p51 RT is found in virions, infected cells and RTC's and both of these subunits are post-translationally modified. This post-translational modification of RT may be important for the function of RT inside the cell.
    Retrovirology 01/2009; 5:115. · 5.66 Impact Factor
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    ABSTRACT: Alzheimer's disease (AD) is the most common form of dementia. Amyloid-beta (A beta) peptide, generated by proteolytic cleavage of the amyloid precursor protein, is central to AD pathogenesis. Most pharmaceutical activity in AD research has focused on A beta, its generation and clearance from the brain. In particular, there is much interest in immunotherapy approaches with a number of anti-A beta antibodies in clinical trials. We have developed a monoclonal antibody, called WO2, which recognises the A beta peptide. To this end, we have determined the three-dimensional structure, to near atomic resolution, of both the antibody and the complex with its antigen, the A beta peptide. The structures reveal the molecular basis for WO2 recognition and binding of A beta. The A beta peptide adopts an extended, coil-like conformation across its major immunodominant B-cell epitope between residues 2 and 8. We have also studied the antibody-bound A beta peptide in the presence of metals known to affect its aggregation state and show that WO2 inhibits these interactions. Thus, antibodies that target the N-terminal region of A beta, such as WO2, hold promise for therapeutic development.
    Journal of Molecular Biology 04/2008; 377(1):181-92. · 3.91 Impact Factor
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    ABSTRACT: To measure and contrast maternal cortisol and corticosteroid-binding globulin (CBG) levels in pregnancies with normal outcomes, pre-eclampsia, intrauterine growth restriction (IUGR) and in gamete recipients. Prospective study of 93 women at high risk of pre-eclampsia, including gamete recipients (n = 22) and 33 controls. Plasma total and free cortisol and CBG were measured every 2 weeks from 16 weeks' gestation until delivery. Forty-two per cent of the high-risk group had complications, including pre-eclampsia (n = 11), gestational hypertension (n = 16) and small-for-gestational-age (SGA) neonates (n = 12). There were no complications in the controls. In all groups, plasma CBG concentrations increased progressively across gestation (P < 0.05), in parallel to total cortisol, but fell significantly from 36 weeks' gestation onwards, with a corresponding rise in free cortisol concentrations. In pre-eclampsia and gestational hypertension, plasma CBG, and total and free cortisol concentrations were lower from 36 weeks onwards (P < 0.05). In IUGR, plasma CBG concentrations were suppressed from 28 weeks' gestation until delivery (P < 0.05), but with no significant difference in plasma total and free cortisol. Gamete recipients had significantly lower plasma CBG from 20 weeks' gestation onwards, and plasma total and free cortisol were reduced at 24 and 32 weeks onwards, respectively. Maternal plasma CBG, total and free cortisol concentrations are reduced in pre-eclampsia/gestational hypertension, and markedly reduced in gamete recipients. Low CBG may be due to reduced synthesis or enhanced inflammation-driven degradation. Low maternal cortisol may be due to a lack of placental corticotropin-releasing hormone or reduced maternal ACTH, driving cortisol production. Low maternal cortisol may influence the foetal hypothalamic-pituitary-adrenal axis and disease patterns later in life following complicated pregnancy.
    Clinical Endocrinology 06/2007; 66(6):869-77. · 3.40 Impact Factor
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    ABSTRACT: Fusobacterium nucleatum is a saccharolytic Gram-negative anaerobic organism believed to play an important role in the microbial succession associated with the development of periodontal disease. Its genome contains niche-specific genes shared with the other inhabitants of dental plaque, which may help to explain its ability to survive and grow in the changing environmental conditions experienced in the gingival sulcus during the transition from health to disease. The pH of the gingival sulcus increases during the development of periodontitis and this is thought to occur by the metabolism of nutrients supplied by gingival crevicular fluid. In comparison with other plaque inhabitants, F. nucleatum has the greatest ability to neutralize acidic environments. The differential expression of soluble cytoplasmic proteins induced by acidic (pH 6.4) or basic (pH 7.4 and 7.8) conditions, during long-term anaerobic growth in a chemostat, was identified by two-dimensional gel electrophoresis and image analysis software. Twenty-two proteins, found to have altered expression in response to external pH, were identified by tryptic digestion and mass spectrometry. Eight differentially expressed proteins associated with increased energy (ATP) production via the 2-oxoglutarate and Embden-Meyerhof pathways appeared to be directed towards either cellular biosynthesis or the maintenance of internal homeostasis. Overall, these results represent the first proteomic investigation of F. nucleatum and the identification of gene products which may be important in the organism's persistence during the transition from health to disease in vivo.
    Microbiology 02/2007; 153(Pt 1):148-59. · 2.85 Impact Factor
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    ABSTRACT: Sphingosine kinases catalyze the formation of sphingosine 1-phosphate, a bioactive lipid involved in many aspects of cellular regulation, including the fundamental biological processes of cell growth and survival. A diverse range of cell agonists induce activation of human sphingosine kinase 1 (hSK1) and, commonly, its translocation to the plasma membrane. Although the activation of hSK1 in response to at least some agonists occurs directly via its phosphorylation at Ser225 by ERK1/2, many aspects governing the regulation of this phosphorylation and subsequent translocation remain unknown. Here, in an attempt to understand some of these processes, we have examined the known interaction of hSK1 with calmodulin (CaM). By using a combination of limited proteolysis, peptide interaction analysis, and site-directed mutagenesis, we have identified that the CaM-binding site of hSK1 resides in the region spanned by residues 191-206. Specifically, Phe197 and Leu198 are critically involved in the interaction because a version of hSK1 incorporating mutations of both Phe197 --> Ala and Leu198 --> Gln failed to bind CaM. We have also shown for the first time that human sphingosine kinase 2 (hSK2) binds CaM, and does so via a CaM binding region that is conserved with hSK1 because comparable mutations in hSK2 also ablate CaM binding to this protein. By using the CaM-binding-deficient version of hSK1, we have begun to elucidate the role of CaM in hSK1 regulation by demonstrating that disruption of the CaM-binding site ablates agonist-induced translocation of hSK1 from the cytoplasm to the plasma membrane, while having no effect on hSK1 phosphorylation and catalytic activation.
    Journal of Biological Chemistry 04/2006; 281(17):11693-701. · 4.65 Impact Factor
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    ABSTRACT: Severe systemic infection leads to hypercortisolism. Reduced cortisol binding proteins may accentuate the free cortisol elevations seen in systemic infection. Recently, low total cortisol increments after tetracosactrin have been associated with increased mortality and hemodynamic responsiveness to exogenous hydrocortisone in septic shock (SS), a phenomenon termed by some investigators as relative adrenal insufficiency (RAI). Free plasma cortisol may correspond more closely to illness severity than total cortisol, comparing SS and sepsis (S). This was a prospective study. This study took place in a tertiary teaching hospital. Patients had SS (n = 45) or S (n = 19) or were healthy controls (HCs; n = 10). The aim of the study was to compare total with free cortisol, measured directly and estimated by Coolens' method, corticosteroid-binding globulin (CBG), and albumin in patients with SS (with and without RAI) and S during acute illness, recovery, and convalescence. Comparing SS, S, and HC subjects, free cortisol levels reflected illness severity more closely than total cortisol (basal free cortisol, SS, 186 vs. S, 29 vs. HC, 13 nmol/liter, P < 0.001 compared with basal total cortisol, SS, 880 vs. S, 417 vs. HC, 352 nmol/liter, P < 0.001). Stimulated free cortisol increments varied greatly with illness category (SS, 192 vs. S, 115 vs. HC, 59 nmol/liter, P = 0.004), whereas total cortisol increments did not (SS, 474 vs. S, 576 vs. HC, 524 nmol/liter, P = 0.013). The lack of increase in total cortisol with illness severity is due to lower CBG and albumin. One third of patients with SS (15 of 45) but no S patients met a recently described criterion for RAI (total cortisol increment after tetracosactrin < or = 248 nmol/liter). RAI patients had higher basal total cortisol (1157 vs. 756 nmol/liter; P = 0.028) and basal free cortisol (287 vs. 140 nmol/liter; P = 0.017) than non-RAI patients. Mean cortisol increments in RAI were lower (total, 99 vs. 648 nmol/liter, P < 0.001; free, 59 vs. 252 nmol/liter, P < 0.001). These differences were not due to altered CBG or albumin levels. Free cortisol levels normalized more promptly than total cortisol in convalescence. Calculated free cortisol by Coolens' method compared closely with measured free cortisol. Free cortisol is likely to be a better guide to cortisolemia in systemic infection because it corresponds more closely to illness severity. The attenuated cortisol increment after tetracosactrin in RAI is not due to low cortisol-binding proteins. Free cortisol levels can be determined reliably using total cortisol and CBG levels.
    Journal of Clinical Endocrinology &amp Metabolism 02/2006; 91(1):105-14. · 6.43 Impact Factor
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    ABSTRACT: In normal plasma free cortisol accounts for less than 6% of the total with 80-90% bound to corticosteroid-binding globulin (CBG) and the remainder associated albumin. However little is known about the distribution of free cortisol in plasma where CBG is inactivated or in congenital CBG deficiency. Here we describe ligand binding experiments revealing that while free cortisol in unstressed individuals is less than 6% of total cortisol this rises markedly to 25% when CBG is totally inactivated by heat. Similar elevations of the free cortisol fraction were noted in a patient with a rare genetically determined complete lack of CBG (mean 32% on frequent circadian sampling). Following heat inactivation of CBG or in the congenital absence of CBG, there is a shift in cortisol binding from CBG to albumin. That this shift occurs is further supported by experiments adding [3H]-cortisol to physiological human serum albumin solutions, where 25% of cortisol remained in the free fraction. Taken together the data provide strong evidence that when CBG is inactivated or congenitally absent then more than 25% of the total cortisol appears in the free fraction with the remainder associated with albumin. The proportion of free cortisol measured in plasma thus reflects a simple measure of functional corticosteroid-binding globulin.
    Clinica Chimica Acta 10/2005; 359(1-2):189-94. · 2.85 Impact Factor
  • Clinica Chimica Acta 01/2005; 359(1-2):189-194. · 2.85 Impact Factor
  • E Mitchell, D J Torpy, C J Bagley
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    ABSTRACT: Corticosteroid-binding globulin (CBG) is a glycoprotein that functions as a specific carrier of cortisol in the circulation. CBG contains six sites for N-glycosylation with, on average, five sites occupied by a mixture of biantennary and triantennary oligosaccharides with variable additional terminal sialic acid residues leading to glycoforms with significant heterogeneity in mass and isoelectric points. During pregnancy, a form of CBG possessing only triantennary oligosaccharides comprising approximately 10 % of total CBG appears specifically. We describe the first application of two-dimensional gel electrophoresis to the separation of human CBG glycoforms. This technique resolved a greater degree of charge heterogeneity than previous studies, and allowed simultaneous visualization of changes to the size and isoelectric points of CBG during pregnancy. Profiles of CBG glycoforms during pregnancy showed a general increase in size followed by a shift to lower pI in a large proportion of the glycoprotein. This may result from the enhancement of triantennary glycosylation, with the extent of incorporation of sialic acid increasing with the number of available sites for its addition. The pregnancy-specific CBG previously defined probably represents a subset of the acidic and high molecular weight glycoforms we have resolved by two-dimensional electrophoresis and now describe as pregnancy-associated CBG.
    Hormone and Metabolic Research 07/2004; 36(6):357-9. · 2.15 Impact Factor
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    ABSTRACT: BACKGROUND: C-reactive protein (CRP), a well-recognized marker of atherosclerosis, has recently been suggested to have a direct proinflammatory effect. The constitutive expression of low levels of CRP in normal plasma suggests the likelihood that a natural factor exists to neutralize the effect of CRP. This factor(s) has not yet been identified. Method and Results- The proinflammatory effect of CRP was measured by the induction of inflammatory adhesion molecules in human umbilical vein endothelial cells (HUVECs). We show that CRP significantly induced upregulation of adhesion molecules in both protein and mRNA levels. The CRP-induced expression of these inflammatory adhesion molecules was completely suppressed when the cells were preincubated with a physiological concentration (1 mg/mL apolipoprotein A-I) of HDLs derived from human plasma (native HDL) or reconstituted HDL (rHDL) at a very low concentration (0.01 mg/mL apolipoprotein A-I). A novel mechanism of HDL inhibition is likely to operate, because (1) rHDL was 100 times more potent than native HDL, (2) preincubation with HDL and its sustained presence were obligatory, and (3) oxidized 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine was the fundamental active component. CONCLUSIONS: The CRP-induced upregulation of inflammatory adhesion molecules in HUVECs was completely prevented by HDL via their oxidized phospholipid components.
    Circulation 06/2004; 109(17):2116-22. · 15.20 Impact Factor
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    ABSTRACT: Poly(A) binding protein (PABP) binds mRNA poly(A) tails and affects mRNA stability and translation. We show here that there is little free PABP in NIH3T3 cells, with the vast majority complexed with RNA. We found that PABP in NIH3T3 cytoplasmic lysates and recombinant human PABP can bind to AU-rich RNA with high affinity. Human PABP bound an AU-rich RNA with Kd in the nm range, which was only sixfold weaker than the affinity for oligo(A) RNA. Truncated PABP containing RNA recognition motif domains 3 and 4 retained binding to both AU-rich and oligo(A) RNA, whereas a truncated PABP containing RNA recognition motif domains 1 and 2 was highly selective for oligo(A) RNA. The inducible PABP, iPABP, was found to be even less discriminating than PABP in RNA binding, with affinities for AU-rich and oligo(A) RNAs differing by only twofold. These data suggest that iPABP and PABP may in some situations interact with other RNA regions in addition to the poly(A) tail.
    European Journal of Biochemistry 02/2004; 271(2):450-7. · 3.58 Impact Factor
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    ABSTRACT: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine that stimulates the production and functional activity of granulocytes and macrophages, properties that have encouraged its clinical use in bone marrow transplantation and in certain infectious diseases. Despite the importance of GM-CSF in regulating myeloid cell numbers and function, little is known about the exact composition and mechanism of assembly of the GM-CSF receptor complex. We have now produced soluble forms of the GM-CSF receptor alpha chain (sGMRalpha) and beta chain (sbetac) and utilized GM-CSF, the GM-CSF antagonist E21R (Glu21Arg), and the betac-blocking monoclonal antibody BION-1 to define the molecular assembly of the GM-CSF receptor complex. We found that GM-CSF and E21R were able to form low-affinity, binary complexes with sGMRalpha, each having a stoichiometry of 1:1. Importantly, GM-CSF but not E21R formed a ternary complex with sGMRalpha and sbetac, and this complex could be disrupted by E21R. Significantly, size-exclusion chromatography, analytical ultracentrifugation, and radioactive tracer experiments indicated that the ternary complex is composed of one sbetac dimer with a single molecule each of sGMRalpha and of GM-CSF. In addition, a hitherto unrecognized direct interaction between betac and GM-CSF was detected that was absent with E21R and was abolished by BION-1. These results demonstrate a novel mechanism of cytokine receptor assembly likely to apply also to interleukin-3 (IL-3) and IL-5 and have implications for our molecular understanding and potential manipulation of GM-CSF activation of its receptor.
    Blood 03/2003; 101(4):1308-15. · 9.06 Impact Factor

Publication Stats

1k Citations
258.29 Total Impact Points

Institutions

  • 2004–2010
    • Hanson Institute
      Tarndarnya, South Australia, Australia
  • 2005
    • Royal Adelaide Hospital
      • Endocrine and Metabolic Unit
      Tarndarnya, South Australia, Australia
  • 1997–2001
    • Adelaide Cancer Centre
      Tarndarnya, South Australia, Australia
  • 2000
    • Saint Vincent's Institute
      Fitzroy, Victoria, Australia
    • University of Adelaide
      Tarndarnya, South Australia, Australia