The future growth of global air passenger traffic will undoubtedly be subject to unanticipated sh... more The future growth of global air passenger traffic will undoubtedly be subject to unanticipated shocks. While the nature and timing of these shocks cannot (by definition) be predicted, looking at how the industry has come through previous shock events can help to assess how it is likely to fare in the face of future shocks. As shown in Figure 1, in the past global air passenger traffic has always seemed to bounce back strongly from short-term upheavals.1 Using a similar methodology as Njegovan (2006)2 we have examined the statistical characteristics of global air passenger traffic since 1950 and find evidence that global air passenger traffic has indeed reverted to an underlying growth path over the long run (See Annex A.) Part of the reason why global air traffic has proven
Journal of molecular and cellular cardiology, 2018
The shortening of sarcomeres that co-ordinates the pump function of the heart is stimulated by el... more The shortening of sarcomeres that co-ordinates the pump function of the heart is stimulated by electrically-mediated increases in [Ca]. This process of excitation-contraction coupling (ECC) is subject to modulation by neurohormonal mediators that tune the output of the heart to meet the needs of the organism. Endothelin-1 (ET-1) is a potent modulator of cardiac function with effects on contraction amplitude, chronotropy and automaticity. The actions of ET-1 are evident during normal adaptive physiological responses and increased under pathophysiological conditions, such as following myocardial infarction and during heart failure, where ET-1 levels are elevated. In myocytes, ET-1 acts through ET- or ET-G protein-coupled receptors (GPCRs). Although well studied in atrial myocytes, the influence and mechanisms of action of ET-1 upon ECC in ventricular myocytes are not fully resolved. We show in rat ventricular myocytes that ET-1 elicits a biphasic effect on fractional shortening (initi...
Autotaxin (ATX) the lysophospholipase responsible for generating the lipid receptor agonist lysop... more Autotaxin (ATX) the lysophospholipase responsible for generating the lipid receptor agonist lysophosphatidic acid (LPA) is a secreted enzyme. Here we show that once secreted it can bind to the surface of cell secreted exosomes. Exosome-bound ATX is catalytically active and carries generated LPA. Once bound to a cell, through specific integrin interaction, ATX releases the LPA to activate cell surface G-protein coupled LPA receptors; inhibition of signaling by the receptor antagonist Ki1642 suggests these are either LPAR1 or LPAR3. The binding stimulates downstream signaling including AKT and MAPK phosphorylation, the release of intracellular stored calcium and cell migration. We propose that exosomal binding of LPA-loaded ATX provides a means of efficiently delivering the lipid agonist to cell surface receptors to promote signalling. We further propose that this is a means whereby autotaxin-LPA signaling operates physiologically.
Abstract Global insolation has been measured at many sites on a horizontal surface, but is needed... more Abstract Global insolation has been measured at many sites on a horizontal surface, but is needed on a tilted surface. A study has been made of patterns of global insolation for 12 months at Fort Hood, Texas, where measurements are made on both horizontal and tilted ...
The primary bleeding time is prolonged when tested during the infusion of both plasminogen activa... more The primary bleeding time is prolonged when tested during the infusion of both plasminogen activators and anticoagulants, and such sites frequently exhibit rebleeding after initial hemostatic control. This study describes an animal (rabbit) model which distinguishes fibrinolytic from anticoagulant hemorrhage and further applies the model to the study of hemostatic plugs of increasing age. In this model, rebleeding occurred from hemostatically-stable ear puncture sites induced prior to infusion of streptokinase (SK) or recombinant tissue-plasminogen activator (rt-PA), but not of heparin or hirudin. This distinction was apparent even for lesions induced only 15 minutes prior to the infusion and fibrinolytic bleeding was observed in such lesions induced up to 24 hours earlier. Post-infusion sites bled more quickly than did pre-infusion sites, and there was a gradual decrease in susceptibility of such prior trauma sites for rebleeding, evidenced not only by a lower proportion of sites that rebled, but also by a longer lag time after starting SK or rt-PA before such rebleeding occurred. At the dosages tested, SK showed a trend (not statistically significant) toward more sites that rebled, while rt-PA showed a trend towards a longer duration of rebleeding. Thus, this animal model of rebleeding appears to be unique for fibrinolytic agents and allows for more detailed study of the physiological mechanisms of such bleeding and for a multifaceted comparison of the bleeding potential of plasminogen activators.
A major polysaccharide containing D-galactose, D-glucose and 2-acetamido-2-deoxy-D-galactose was ... more A major polysaccharide containing D-galactose, D-glucose and 2-acetamido-2-deoxy-D-galactose was obtained after mild acid hydrolysis of the water-soluble material released by treatment of cell walls from Acinetobacter baumannii strain O11 with hot, aqueous phenol. By means of NMR studies, Smith degradation and N-deacetylation/deamination, the repeating unit of the polymer was identified as a branched pentasaccharide of the structure shown. Also present was a minor polymer containing glucose, 2-acetamido-2-deoxyglucose-and 2-acetamido-2-deoxygalactose, the structure of which was not elucidated. On serological testing, the polymeric material was shown to correspond to the O-antigenic moiety of the parent extract (assumed to be lipopolysaccharide) and circumstantial evidence indicated that O11 specificity was conferred by the major polymer. [formula: see text]
The β isoform of phosphoinositide 3-kinase may be an effective therapeutic target in inflammatory... more The β isoform of phosphoinositide 3-kinase may be an effective therapeutic target in inflammatory diseases.
Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namel... more Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namely carbamylation and deamidation, have been recently dispelled (Herbert et al., J. Proteome Res. 2002, in press). We report here, for the first time, a noxious and unexpected artefact in proteome analysis: β‐elimination (or desulfuration), which results on the loss of an H2S group (34 Da) from cysteine (Cys) residues for protein focusing in the alkaline pH region. With such an elimination event, a dehydro alanine residue is generated at the Cys site. In turn, the presence of a double bond in this position elicits lysis of the peptide bond, generating a number of peptides of fairly large size from an intact protein. The first process seems to be favored by the electric field, probably due to the continuous harvesting of the SH‐ anion produced. The only remedy found to this noxious degradation pathway is the reduction and alkylation of all Cys residues prior to their exposure to the electri...
Background Psychosis is a severe mental condition that is characterized by a loss of contact with... more Background Psychosis is a severe mental condition that is characterized by a loss of contact with reality and is typically associated with hallucinations and delusional beliefs. There are numerous psychiatric conditions that present with psychotic symptoms, most importantly schizophrenia, bipolar affective disorder, and some forms of severe depression referred to as psychotic depression. The pathological mechanisms resulting in psychotic symptoms are not understood, nor is it understood whether the various psychotic illnesses are the result of similar biochemical disturbances. The identification of biological markers (so-called biomarkers) of psychosis is a fundamental step towards a better understanding of the pathogenesis of psychosis and holds the potential for more objective testing methods. Methods and Findings Surface-enhanced laser desorption ionization mass spectrometry was employed to profile proteins and peptides in a total of 179 cerebrospinal fluid samples (58 schizophrenia patients, 16 patients with depression, five patients with obsessive-compulsive disorder, ten patients with Alzheimer disease, and 90 controls). Our results show a highly significant differential distribution of samples from healthy volunteers away from drug-naïve patients with firstonset paranoid schizophrenia. The key alterations were the up-regulation of a 40-amino acid VGF-derived peptide, the down-regulation of transthyretin at ;4 kDa, and a peptide cluster at ;6,800-7,300 Da (which is likely to be influenced by the doubly charged ions of the transthyretin protein cluster). These schizophrenia-specific protein/peptide changes were replicated in an independent sample set. Both experiments achieved a specificity of 95% and a sensitivity of 80% or 88% in the initial study and in a subsequent validation study, respectively. Conclusions Our results suggest that the application of modern proteomics techniques, particularly mass spectrometric approaches, holds the potential to advance the understanding of the biochemical basis of psychiatric disorders and may in turn allow for the development of diagnostics and improved therapeutics. Further studies are required to validate the clinical effectiveness and disease specificity of the identified biomarkers.
The pro-survival protein Bcl-x L is critical for the resistance of tumour cells to DNA damage. We... more The pro-survival protein Bcl-x L is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage-induced Bcl-x L deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-x L deamidation remains unknown and its functional consequences unclear. We show here that rBcl-x L deamidation generates an iso-Asp 52 /iso-Asp 66 species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-x L deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage-induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-x L deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-x L deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy.
Although some insights into the etiology of schizophrenia have been gained, an understanding of t... more Although some insights into the etiology of schizophrenia have been gained, an understanding of the illness at the molecular level remains elusive. Recent advances in proteomic profiling offer great promise for the discovery of markers underlying pathophysiology of diseases. In the present study, we employed two high-throughput proteomic techniques together with traditional methods to investigate cerebrospinal fluid (CSF), brain and peripheral tissues (liver, red blood cells and serum) of schizophrenia patients in an attempt to identify peripheral/surrogate disease markers. The cohorts used to investigate each tissue were largely independent, although some CSF and serum samples were collected from the same patient. To address the major confounding factor of antipsychotic drug treatment, we also included a large cohort of first-onset drug-naive patients. Apolipoprotein A1 (apoA1) showed a significant decrease in expression in schizophrenia patients compared to controls in all five tissues examined. Specifically, using SELDI-TOF mass spectrometry, apoA1 was found decreased in CSF from schizophrenia patients (À35%, P = 0.00001) and, using 2D-DIGE, apoA1 was also found downregulated in liver (À30%, P = 0.02) and RBCs (À60%, P = 0.003). Furthermore, we found a significant reduction of apoA1 in sera of first-onset drug-naive schizophrenia patients using enzyme-linked immunosorbent assay (À18%, P = 0.00008) and in two investigations of post-mortem brain tissue using western blot analysis (À35%, P = 0.05; À51%, P = 0.05). These results show that apoA1 is consistently downregulated in the central nervous system as well as peripheral tissues of schizophrenia patients and may be linked to the underlying disease mechanism.
Alterations in arterial blood flow are thought to predispose to thrombus formation, but the exact... more Alterations in arterial blood flow are thought to predispose to thrombus formation, but the exact relationships have not been fially elucidated. The effect of varying blood flows on the accumulation of thrombotic material within arteries was investigated, with use of shear rate as an index of flow across the luminal surface. Partially denuded rabbit aortas were perfused with fresh nonanticoagulated human blood for 3 minutes, with an in vitro recirculating apparatus, Indium i l l-l a b e l e d platelets, and fibrinogen I 125. Shear rates ranged from zero to 1500 sec-~, correlating with the hemodynamics of various segments of the human arterial tree. A significant correlation was observed between shear rate and platelet deposition, ranging from 5.2 + 2.8 x 106 platelets/cm 2 of vessel surface area at zero shear to a maximum of 64.7-+ 8.3 x 106 platelets/cm 2 at a shear rate of 1500 sec-(F = 5.01, p < 0.05). Fibrin deposition paralleled that of platelets, ranging from 28.2-7.6 ~g/cm 2 at zero shear to 354.11-+ 62.7 wg/cm 2 at a shear rate of 1500 sec-~ (F = 5.91, p < 0.05). These results suggest that shear rate is a most important determinant of platelet and fibrin deposition on altered arterial surfaces. (J VASC SURG
Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample p... more Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea breakdown product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.
The G protein-coupled pheromone receptor neurons (V1R and V2R) of the vomeronasal organ (VNO) are... more The G protein-coupled pheromone receptor neurons (V1R and V2R) of the vomeronasal organ (VNO) are continually replaced throughout the lifetime of the mouse. Moreover, active signalling of V2Rs via the transient receptor potential 2(TRPC2) channel is necessary for regeneration of receptors, as the TRPC2 null mutant mouse showed a 75% reduction of V2Rs by the age of two months. Here we describe V2R mediated signalling in a neuronal line established from vomeronasal stem cells taken from postnatal female mice. Cells were immunoreactive for Ga o and V2R, whereas V1R and Ga i immunoreactivity could not be detected. Biological ligands (dilute urine and its protein fractions) were found to increase proliferation and survival of these neurons. Dilute mouse urine but not artificial urine also induced ERK, Akt and CREB signalling in a dose dependent way. The volatile fraction of male mouse urine alone was without effect while the fraction containing peptides (> 5 kDa) also stimulated ERK and Akt phosphorylation. The ERK, Akt and CREB phosphorylation response was sensitive to pertussis toxin, confirming the involvement of V2R linked Ga o. Dilute mouse urine or its high molecular weight protein fraction increased survival and proliferation of these neurons. Hence, urinary pheromones, which signal important social information via mature neurons, also promote survival and proliferation of their regenerating precursors. These data show that regenerating V2Rs respond to urine and the urinary peptides by activation of the Ras-ERK and PI3-Akt pathways, which appear to be important for vomeronasal neural survival and proliferation.
The major fraction of an acidic galactoglucomannan present in lipopolysaccharide extracts from ce... more The major fraction of an acidic galactoglucomannan present in lipopolysaccharide extracts from cell walls of the O23 reference strain of Serratia marcescens has the tetrasaccharide repeating-unit shown. In a minor fraction, L-glutamic acid was amide-linked to about half of the D-glucuronic acid residues. The possible contributions of the acidic polymers and a neutral polymer produced by the organism to cross-reactions with other serogroups are discussed.
In addition to a neutral glycan, lipopolysaccharide extracts from the reference strain for Serrat... more In addition to a neutral glycan, lipopolysaccharide extracts from the reference strain for Serratia marcescens serogroup O22 contain an acidic polymer which probably defines the serogroup and is of microcapsular origin. The polymer is doubly branched with a heptasaccharide repeating unit and a galactan backbone. By means of spectroscopic and degradative studies, the structure of the repeating unit was established as that shown. [formula: see text]
Structural studies have been carried out on the putative O-specific polysaccharide of the referen... more Structural studies have been carried out on the putative O-specific polysaccharide of the reference strain (C.D.C. 3607-60) for Serratia marcescens O13. Circumstantial evidence that the O13 antigen is a microcapsular, acidic polymer, rather than an integral part of the lipopolysaccharide, has been obtained. Degradative and spectroscopic studies established that the polymer is based on the repeating unit shown, in which the glucuronic acid residue of the linear pentasaccharide carries the lateral 2-acetamido-2-deoxy-beta-D-glucopyranosyl substituent in only about half of the units. The same polymer, again with non-stoichiometric substitution, is also produced by strain IP 421 (O13:H7). The latter strain also produces a neutral polymer which appears to constitute the side chain of the lipopolysaccharide. This polymer, which has a disaccharide repeating-unit of 2-substituted beta-D-ribofuranosyl and 4-substituted 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residues, has been isolated previously from the lipopolysaccharides of the reference strains for S. marcescens serogroups O12 and O14, and appears to be the antigen known to be shared by these strains. (Formula: see text).
Both a neutral and an acidic polymer have been isolated from a lipopolysaccharide extract of Serr... more Both a neutral and an acidic polymer have been isolated from a lipopolysaccharide extract of Serratia marcescens strain S3255. The neutral polymer is a linear mannan with the repeating unit shown. The same repeating unit has been described for the O-specific polymers from Escherichia coli O8 and Klebsiella O5.
The future growth of global air passenger traffic will undoubtedly be subject to unanticipated sh... more The future growth of global air passenger traffic will undoubtedly be subject to unanticipated shocks. While the nature and timing of these shocks cannot (by definition) be predicted, looking at how the industry has come through previous shock events can help to assess how it is likely to fare in the face of future shocks. As shown in Figure 1, in the past global air passenger traffic has always seemed to bounce back strongly from short-term upheavals.1 Using a similar methodology as Njegovan (2006)2 we have examined the statistical characteristics of global air passenger traffic since 1950 and find evidence that global air passenger traffic has indeed reverted to an underlying growth path over the long run (See Annex A.) Part of the reason why global air traffic has proven
Journal of molecular and cellular cardiology, 2018
The shortening of sarcomeres that co-ordinates the pump function of the heart is stimulated by el... more The shortening of sarcomeres that co-ordinates the pump function of the heart is stimulated by electrically-mediated increases in [Ca]. This process of excitation-contraction coupling (ECC) is subject to modulation by neurohormonal mediators that tune the output of the heart to meet the needs of the organism. Endothelin-1 (ET-1) is a potent modulator of cardiac function with effects on contraction amplitude, chronotropy and automaticity. The actions of ET-1 are evident during normal adaptive physiological responses and increased under pathophysiological conditions, such as following myocardial infarction and during heart failure, where ET-1 levels are elevated. In myocytes, ET-1 acts through ET- or ET-G protein-coupled receptors (GPCRs). Although well studied in atrial myocytes, the influence and mechanisms of action of ET-1 upon ECC in ventricular myocytes are not fully resolved. We show in rat ventricular myocytes that ET-1 elicits a biphasic effect on fractional shortening (initi...
Autotaxin (ATX) the lysophospholipase responsible for generating the lipid receptor agonist lysop... more Autotaxin (ATX) the lysophospholipase responsible for generating the lipid receptor agonist lysophosphatidic acid (LPA) is a secreted enzyme. Here we show that once secreted it can bind to the surface of cell secreted exosomes. Exosome-bound ATX is catalytically active and carries generated LPA. Once bound to a cell, through specific integrin interaction, ATX releases the LPA to activate cell surface G-protein coupled LPA receptors; inhibition of signaling by the receptor antagonist Ki1642 suggests these are either LPAR1 or LPAR3. The binding stimulates downstream signaling including AKT and MAPK phosphorylation, the release of intracellular stored calcium and cell migration. We propose that exosomal binding of LPA-loaded ATX provides a means of efficiently delivering the lipid agonist to cell surface receptors to promote signalling. We further propose that this is a means whereby autotaxin-LPA signaling operates physiologically.
Abstract Global insolation has been measured at many sites on a horizontal surface, but is needed... more Abstract Global insolation has been measured at many sites on a horizontal surface, but is needed on a tilted surface. A study has been made of patterns of global insolation for 12 months at Fort Hood, Texas, where measurements are made on both horizontal and tilted ...
The primary bleeding time is prolonged when tested during the infusion of both plasminogen activa... more The primary bleeding time is prolonged when tested during the infusion of both plasminogen activators and anticoagulants, and such sites frequently exhibit rebleeding after initial hemostatic control. This study describes an animal (rabbit) model which distinguishes fibrinolytic from anticoagulant hemorrhage and further applies the model to the study of hemostatic plugs of increasing age. In this model, rebleeding occurred from hemostatically-stable ear puncture sites induced prior to infusion of streptokinase (SK) or recombinant tissue-plasminogen activator (rt-PA), but not of heparin or hirudin. This distinction was apparent even for lesions induced only 15 minutes prior to the infusion and fibrinolytic bleeding was observed in such lesions induced up to 24 hours earlier. Post-infusion sites bled more quickly than did pre-infusion sites, and there was a gradual decrease in susceptibility of such prior trauma sites for rebleeding, evidenced not only by a lower proportion of sites that rebled, but also by a longer lag time after starting SK or rt-PA before such rebleeding occurred. At the dosages tested, SK showed a trend (not statistically significant) toward more sites that rebled, while rt-PA showed a trend towards a longer duration of rebleeding. Thus, this animal model of rebleeding appears to be unique for fibrinolytic agents and allows for more detailed study of the physiological mechanisms of such bleeding and for a multifaceted comparison of the bleeding potential of plasminogen activators.
A major polysaccharide containing D-galactose, D-glucose and 2-acetamido-2-deoxy-D-galactose was ... more A major polysaccharide containing D-galactose, D-glucose and 2-acetamido-2-deoxy-D-galactose was obtained after mild acid hydrolysis of the water-soluble material released by treatment of cell walls from Acinetobacter baumannii strain O11 with hot, aqueous phenol. By means of NMR studies, Smith degradation and N-deacetylation/deamination, the repeating unit of the polymer was identified as a branched pentasaccharide of the structure shown. Also present was a minor polymer containing glucose, 2-acetamido-2-deoxyglucose-and 2-acetamido-2-deoxygalactose, the structure of which was not elucidated. On serological testing, the polymeric material was shown to correspond to the O-antigenic moiety of the parent extract (assumed to be lipopolysaccharide) and circumstantial evidence indicated that O11 specificity was conferred by the major polymer. [formula: see text]
The β isoform of phosphoinositide 3-kinase may be an effective therapeutic target in inflammatory... more The β isoform of phosphoinositide 3-kinase may be an effective therapeutic target in inflammatory diseases.
Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namel... more Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namely carbamylation and deamidation, have been recently dispelled (Herbert et al., J. Proteome Res. 2002, in press). We report here, for the first time, a noxious and unexpected artefact in proteome analysis: β‐elimination (or desulfuration), which results on the loss of an H2S group (34 Da) from cysteine (Cys) residues for protein focusing in the alkaline pH region. With such an elimination event, a dehydro alanine residue is generated at the Cys site. In turn, the presence of a double bond in this position elicits lysis of the peptide bond, generating a number of peptides of fairly large size from an intact protein. The first process seems to be favored by the electric field, probably due to the continuous harvesting of the SH‐ anion produced. The only remedy found to this noxious degradation pathway is the reduction and alkylation of all Cys residues prior to their exposure to the electri...
Background Psychosis is a severe mental condition that is characterized by a loss of contact with... more Background Psychosis is a severe mental condition that is characterized by a loss of contact with reality and is typically associated with hallucinations and delusional beliefs. There are numerous psychiatric conditions that present with psychotic symptoms, most importantly schizophrenia, bipolar affective disorder, and some forms of severe depression referred to as psychotic depression. The pathological mechanisms resulting in psychotic symptoms are not understood, nor is it understood whether the various psychotic illnesses are the result of similar biochemical disturbances. The identification of biological markers (so-called biomarkers) of psychosis is a fundamental step towards a better understanding of the pathogenesis of psychosis and holds the potential for more objective testing methods. Methods and Findings Surface-enhanced laser desorption ionization mass spectrometry was employed to profile proteins and peptides in a total of 179 cerebrospinal fluid samples (58 schizophrenia patients, 16 patients with depression, five patients with obsessive-compulsive disorder, ten patients with Alzheimer disease, and 90 controls). Our results show a highly significant differential distribution of samples from healthy volunteers away from drug-naïve patients with firstonset paranoid schizophrenia. The key alterations were the up-regulation of a 40-amino acid VGF-derived peptide, the down-regulation of transthyretin at ;4 kDa, and a peptide cluster at ;6,800-7,300 Da (which is likely to be influenced by the doubly charged ions of the transthyretin protein cluster). These schizophrenia-specific protein/peptide changes were replicated in an independent sample set. Both experiments achieved a specificity of 95% and a sensitivity of 80% or 88% in the initial study and in a subsequent validation study, respectively. Conclusions Our results suggest that the application of modern proteomics techniques, particularly mass spectrometric approaches, holds the potential to advance the understanding of the biochemical basis of psychiatric disorders and may in turn allow for the development of diagnostics and improved therapeutics. Further studies are required to validate the clinical effectiveness and disease specificity of the identified biomarkers.
The pro-survival protein Bcl-x L is critical for the resistance of tumour cells to DNA damage. We... more The pro-survival protein Bcl-x L is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage-induced Bcl-x L deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-x L deamidation remains unknown and its functional consequences unclear. We show here that rBcl-x L deamidation generates an iso-Asp 52 /iso-Asp 66 species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-x L deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage-induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-x L deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-x L deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy.
Although some insights into the etiology of schizophrenia have been gained, an understanding of t... more Although some insights into the etiology of schizophrenia have been gained, an understanding of the illness at the molecular level remains elusive. Recent advances in proteomic profiling offer great promise for the discovery of markers underlying pathophysiology of diseases. In the present study, we employed two high-throughput proteomic techniques together with traditional methods to investigate cerebrospinal fluid (CSF), brain and peripheral tissues (liver, red blood cells and serum) of schizophrenia patients in an attempt to identify peripheral/surrogate disease markers. The cohorts used to investigate each tissue were largely independent, although some CSF and serum samples were collected from the same patient. To address the major confounding factor of antipsychotic drug treatment, we also included a large cohort of first-onset drug-naive patients. Apolipoprotein A1 (apoA1) showed a significant decrease in expression in schizophrenia patients compared to controls in all five tissues examined. Specifically, using SELDI-TOF mass spectrometry, apoA1 was found decreased in CSF from schizophrenia patients (À35%, P = 0.00001) and, using 2D-DIGE, apoA1 was also found downregulated in liver (À30%, P = 0.02) and RBCs (À60%, P = 0.003). Furthermore, we found a significant reduction of apoA1 in sera of first-onset drug-naive schizophrenia patients using enzyme-linked immunosorbent assay (À18%, P = 0.00008) and in two investigations of post-mortem brain tissue using western blot analysis (À35%, P = 0.05; À51%, P = 0.05). These results show that apoA1 is consistently downregulated in the central nervous system as well as peripheral tissues of schizophrenia patients and may be linked to the underlying disease mechanism.
Alterations in arterial blood flow are thought to predispose to thrombus formation, but the exact... more Alterations in arterial blood flow are thought to predispose to thrombus formation, but the exact relationships have not been fially elucidated. The effect of varying blood flows on the accumulation of thrombotic material within arteries was investigated, with use of shear rate as an index of flow across the luminal surface. Partially denuded rabbit aortas were perfused with fresh nonanticoagulated human blood for 3 minutes, with an in vitro recirculating apparatus, Indium i l l-l a b e l e d platelets, and fibrinogen I 125. Shear rates ranged from zero to 1500 sec-~, correlating with the hemodynamics of various segments of the human arterial tree. A significant correlation was observed between shear rate and platelet deposition, ranging from 5.2 + 2.8 x 106 platelets/cm 2 of vessel surface area at zero shear to a maximum of 64.7-+ 8.3 x 106 platelets/cm 2 at a shear rate of 1500 sec-(F = 5.01, p < 0.05). Fibrin deposition paralleled that of platelets, ranging from 28.2-7.6 ~g/cm 2 at zero shear to 354.11-+ 62.7 wg/cm 2 at a shear rate of 1500 sec-~ (F = 5.91, p < 0.05). These results suggest that shear rate is a most important determinant of platelet and fibrin deposition on altered arterial surfaces. (J VASC SURG
Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample p... more Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea breakdown product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.
The G protein-coupled pheromone receptor neurons (V1R and V2R) of the vomeronasal organ (VNO) are... more The G protein-coupled pheromone receptor neurons (V1R and V2R) of the vomeronasal organ (VNO) are continually replaced throughout the lifetime of the mouse. Moreover, active signalling of V2Rs via the transient receptor potential 2(TRPC2) channel is necessary for regeneration of receptors, as the TRPC2 null mutant mouse showed a 75% reduction of V2Rs by the age of two months. Here we describe V2R mediated signalling in a neuronal line established from vomeronasal stem cells taken from postnatal female mice. Cells were immunoreactive for Ga o and V2R, whereas V1R and Ga i immunoreactivity could not be detected. Biological ligands (dilute urine and its protein fractions) were found to increase proliferation and survival of these neurons. Dilute mouse urine but not artificial urine also induced ERK, Akt and CREB signalling in a dose dependent way. The volatile fraction of male mouse urine alone was without effect while the fraction containing peptides (> 5 kDa) also stimulated ERK and Akt phosphorylation. The ERK, Akt and CREB phosphorylation response was sensitive to pertussis toxin, confirming the involvement of V2R linked Ga o. Dilute mouse urine or its high molecular weight protein fraction increased survival and proliferation of these neurons. Hence, urinary pheromones, which signal important social information via mature neurons, also promote survival and proliferation of their regenerating precursors. These data show that regenerating V2Rs respond to urine and the urinary peptides by activation of the Ras-ERK and PI3-Akt pathways, which appear to be important for vomeronasal neural survival and proliferation.
The major fraction of an acidic galactoglucomannan present in lipopolysaccharide extracts from ce... more The major fraction of an acidic galactoglucomannan present in lipopolysaccharide extracts from cell walls of the O23 reference strain of Serratia marcescens has the tetrasaccharide repeating-unit shown. In a minor fraction, L-glutamic acid was amide-linked to about half of the D-glucuronic acid residues. The possible contributions of the acidic polymers and a neutral polymer produced by the organism to cross-reactions with other serogroups are discussed.
In addition to a neutral glycan, lipopolysaccharide extracts from the reference strain for Serrat... more In addition to a neutral glycan, lipopolysaccharide extracts from the reference strain for Serratia marcescens serogroup O22 contain an acidic polymer which probably defines the serogroup and is of microcapsular origin. The polymer is doubly branched with a heptasaccharide repeating unit and a galactan backbone. By means of spectroscopic and degradative studies, the structure of the repeating unit was established as that shown. [formula: see text]
Structural studies have been carried out on the putative O-specific polysaccharide of the referen... more Structural studies have been carried out on the putative O-specific polysaccharide of the reference strain (C.D.C. 3607-60) for Serratia marcescens O13. Circumstantial evidence that the O13 antigen is a microcapsular, acidic polymer, rather than an integral part of the lipopolysaccharide, has been obtained. Degradative and spectroscopic studies established that the polymer is based on the repeating unit shown, in which the glucuronic acid residue of the linear pentasaccharide carries the lateral 2-acetamido-2-deoxy-beta-D-glucopyranosyl substituent in only about half of the units. The same polymer, again with non-stoichiometric substitution, is also produced by strain IP 421 (O13:H7). The latter strain also produces a neutral polymer which appears to constitute the side chain of the lipopolysaccharide. This polymer, which has a disaccharide repeating-unit of 2-substituted beta-D-ribofuranosyl and 4-substituted 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residues, has been isolated previously from the lipopolysaccharides of the reference strains for S. marcescens serogroups O12 and O14, and appears to be the antigen known to be shared by these strains. (Formula: see text).
Both a neutral and an acidic polymer have been isolated from a lipopolysaccharide extract of Serr... more Both a neutral and an acidic polymer have been isolated from a lipopolysaccharide extract of Serratia marcescens strain S3255. The neutral polymer is a linear mannan with the repeating unit shown. The same repeating unit has been described for the O-specific polymers from Escherichia coli O8 and Klebsiella O5.
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Papers by David Oxley