Biomedical subjects

Andrew Macdonald

Publications and source records attributed to Andrew Macdonald.

13 recordsLinked to original sources

PubMed2006

Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-XL.

BACKGROUND: Pim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases. Over-expression of Pim-1 and Pim-2 in mice promotes the development of lymphomas, and up-regulation of Pim expression has been observed in several human cancers. RESULTS: Here we show that the pim kinases are constitutively active when expressed in HEK-293 cells and are able to phosphorylate the Bcl-2 family member Bad on three residues, Ser112, Ser136 and Ser155 in vitro and in cells. In vitro mapping showed that Pim-2 predominantly phosphorylated Ser112, while Pim-1 phosphorylated Ser112, but also Ser136 and Ser155 at a reduced rate compared to Ser112. Pim-3 was found to be the least specific for Ser112, and the most effective at phosphorylating Ser136 and Ser155. Pim-3 was also able to phosphorylate other sites in Bad in vitro, including Ser170, another potential in vivo site. Mutation of Ser136 to alanine prevented the phosphorylation of Ser112 and Ser155 by Pim kinases in HEK-293 cells, suggesting that this site must be phosphorylated first in order to make the other sites accessible. Pim phosphorylation of Bad was also found to promote the 14-3-3 binding of Bad and block its association with Bcl-XL. CONCLUSION: All three Pim kinase family members predominantly phosphorylate Bad on Ser112 and in addition are capable of phosphorylating Bad on multiple sites associated with the inhibition of the pro-apoptotic function of Bad in HEK-293 cells. This would be consistent with the proposed function of Pim kinases in promoting cell proliferation and preventing cell death.

14-3-3 Proteins↗

PubMed2006

Experimentally-derived functional form for a population-averaged high-temporal-resolution arterial input function for dynamic contrast-enhanced MRI.

Rapid T(1)-weighted 3D spoiled gradient-echo (GRE) data sets were acquired in the abdomen of 23 cancer patients during a total of 113 separate visits to allow dynamic contrast-enhanced MRI (DCE-MRI) analysis of tumor microvasculature. The arterial input function (AIF) was measured in each patient at each visit using an automated AIF extraction method following a standardized bolus administration of gadodiamide. The AIFs for each patient were combined to obtain a mean AIF that is representative for any individual. The functional form of this general AIF may be useful for studies in which AIF measurements are not possible. Improvements in the reproducibility of DCE-MRI model parameters (K(trans), v(e), and v(p)) were observed when this new, high-temporal-resolution population AIF was used, indicating the potential for increased sensitivity to therapy-induced change.

Abdominal Neoplasms↗

PubMed2005

Dendritic cell-intrinsic expression of NF-kappa B1 is required to promote optimal Th2 cell differentiation.

A number of receptors and signaling pathways can influence the ability of dendritic cells (DC) to promote CD4(+) Th type 1 (Th1) responses. In contrast, the regulatory pathways and signaling events that govern the ability of DC to instruct Th2 cell differentiation remain poorly defined. In this report, we demonstrate that NF-kappaB1 expression within DC is required to promote optimal Th2 responses following exposure to Schistosoma mansoni eggs, a potent and natural Th2-inducing stimulus. Although injection of S. mansoni eggs induced production of IL-4, IL-5, and IL-13 in the draining lymph node of wild-type (WT) mice, NF-kappaB1(-/-) hosts failed to express Th2 cytokines and developed a polarized Ag-specific IFN-gamma response. In an in vivo adoptive transfer model in which NF-kappaB-sufficient OVA-specific DO11.10 TCR transgenic T cells were injected into OVA-immunized WT or NF-kappaB1(-/-) hosts, NF-kappaB1(-/-) APCs efficiently promoted CD4(+) T cell proliferation and IFN-gamma responses, but failed to promote Ag-specific IL-4 production. Further, bone marrow-derived DC from NF-kappaB1(-/-) mice failed to promote OVA-specific Th2 cell differentiation in in vitro coculture studies. Last, S. mansoni egg Ag-pulsed NF-kappaB1(-/-) DC failed to prime for Th2 cytokine responses following injection into syngeneic WT hosts. Impaired Th2 priming by NF-kappaB1(-/-) DC was accompanied by a reduction in MAPK phosphorylation in Ag-pulsed DC. Taken together, these studies identify a novel requirement for DC-intrinsic expression of NF-kappaB1 in regulating the MAPK pathway and governing the competence of DC to instruct Th2 cell differentiation.

Animals↗

PubMed2005

Children's injuries in a Scottish district general hospital.

BACKGROUND: Injury is a common cause of emergency department (ED) attendance but there are few data published on the spectrum of paediatric injury in a typical district general hospital (DGH). This study aimed to provide a complete picture of injury presentations to such a centre. METHODS: Prospective questionnaire study of consecutive paediatric attendances at a DGH ED in Scotland (annual attendance 53,500 patients) due to injury or poisoning. Paediatric in this context was defined as less than 14 years on the day of presentation. Admission rates were identified from the hospital information system and information on deaths was sought from the local Procurator Fiscal (the Scottish equivalent of the Coroner). RESULTS: One thousand three hundred and seventy-eight questionnaires were completed from a potential 10,697 eligible patients. Safety devices (helmets, belts, etc.) were in use in only 99 cases. Cycle helmets were used in 26% of cycle incidents and seat belts were used in 71% of car incidents. Cycling and pedestrian incidents were more common during the summer months and outside school hours. Adult supervision was present in 49% of incidents. Seventy-three percent of incidents at school were unsupervised. There were 5.6 admissions to hospital per day in the 0-13 years age group for all causes, with little seasonal variation in admission rates. There were three deaths during the year, two from SIDS and one due to choking, all in infants. CONCLUSION: Trauma is a common cause of ED attendance in children. Preventative measures are still underutilised and could make a significant impact on the incidence of children's injuries and possibly ED attendances. Cycle helmets could play a major role in injury prevention in school age children in this area.

Accidental Falls↗

PubMed2005

Perturbation of epidermal growth factor receptor complex formation and Ras signalling in cells harbouring the hepatitis C virus subgenomic replicon.

Hepatitis C virus non-structural NS5A protein inhibits epidermal growth factor (EGF)-stimulated activation of the Ras-ERK mitogen-activated protein kinase pathway at a point upstream of Ras activation. To determine the mechanism of this inhibition, the events occurring between the EGF receptor and Ras in Huh-7 cells harbouring the HCV subgenomic replicon were investigated. It was shown that, following EGF stimulation, these cells exhibited decreased EGF receptor tyrosine phosphorylation, aberrant recruitment of the adaptor proteins ShcA and Grb2 to the EGF receptor, reduced phosphorylation of ShcA and reduced Ras activation in comparison with control cells. These data are consistent with effects of NS5A and/or other components of the replicon on multiple events occurring upstream of Ras.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Further studies on hepatitis C virus NS5A-SH3 domain interactions: identification of residues critical for binding and implications for viral RNA replication and modulation of cell signalling.

The NS5A protein of hepatitis C virus has been shown to interact with a subset of Src homology 3 (SH3) domain-containing proteins. The molecular mechanisms underlying these observations have not been fully characterized, therefore a previous analysis of NS5A-SH3 domain interactions was extended. By using a semi-quantitative ELISA assay, a hierarchy of binding between various SH3 domains for NS5A was demonstrated. Molecular modelling of a polyproline motif within NS5A (termed PP2.2) bound to the FynSH3 domain predicted that the specificity-determining RT-loop region within the SH3 domain did not interact directly with the PP2.2 motif. However, it was demonstrated that the RT loop did contribute to the specificity of binding, implicating the involvement of other intermolecular contacts between NS5A and SH3 domains. The modelling analysis also predicted a critical role for a conserved arginine located at the C terminus of the PP2.2 motif; this was confirmed experimentally. Finally, it was demonstrated that, in comparison with wild-type replicon cells, inhibition of the transcription factor AP-1, a function previously assigned to NS5A, was not observed in cells harbouring a subgenomic replicon containing a mutation within the PP2.2 motif. However, the ability of the mutated replicon to establish itself within Huh-7 cells was unaffected. The highly conserved nature of the PP2.2 motif within NS5A suggests that functions involving this motif are of importance, but are unlikely to play a role in replication of the viral RNA genome. It is more likely that they play a role in altering the cellular environment to favour viral persistence.

Amino Acid Sequence↗

PubMed2005

Hepatitis C virus NS5A-mediated activation of phosphoinositide 3-kinase results in stabilization of cellular beta-catenin and stimulation of beta-catenin-responsive transcription.

The hepatitis C virus (HCV) nonstructural NS5A protein has been shown to bind to and activate phosphoinositide 3-kinase (PI3K), resulting in activation of the downstream effector serine/threonine kinase Akt/protein kinase B. Here we present data pertaining to the effects of NS5A-mediated Akt activation on its downstream targets. Using a recombinant baculovirus to deliver the complete HCV polyprotein to human hepatoma cells in a tetracycline-regulable fashion, we confirm that expression of the complete HCV polyprotein also activates PI3K and Akt. We further show that this results in the inhibition of the Akt substrate Forkhead transcription factor and the stimulation of phosphorylation of a second key Akt substrate, glycogen synthase kinase-3beta (GSK-3beta). Phosphorylation of GSK-3beta results in its inactivation; consistent with this, we show that expression of the HCV polyprotein results in the accumulation of beta-catenin. Finally, we show that levels of beta-catenin-dependent transcription are also elevated in the presence of the HCV polyprotein. Given the prevalence of beta-catenin mutations in many human tumors, especially colon and hepatocellular carcinomas, these data implicate NS5A-mediated PI3K activation as a contributory factor in the increasingly common association between HCV infection and the development of hepatocellular carcinoma.

Baculoviridae↗

PubMed2004

The Hepatitis C virus NS5A protein activates a phosphoinositide 3-kinase-dependent survival signaling cascade.

Hepatitis C virus (HCV) establishes a persistent infection, with up to 80% of infected individuals proceeding to chronic hepatitis, which in many cases may result in liver cirrhosis and hepatocellular carcinoma (HCC); indeed HCV infection is increasingly associated with the development of HCC. The long time period (up to 30 years) between primary infection and the onset of HCC implies that HCV is not directly oncogenic but in some way predisposes patients to develop tumors, though the mechanism for this is unclear as yet. We report here that NS5A binds directly to the Src homology 3 domain of the p85 regulatory subunit of phosphoinositide 3-kinase (PI3K), and this interaction is mediated by a novel (non-proline-rich) motif within NS5A. Coimmunoprecipitation analysis revealed that NS5A bound native heterodimeric PI3K and enhanced the phosphotransferase activity of the catalytic (p110) subunit both in vitro and in human cell lines harboring a subgenomic HCV replicon or expressing NS5A alone. NS5A-mediated activation of PI3K resulted in increased phosphorylation and activity of Akt/protein kinase B and concomitantly provided protection against the induction of apoptosis in both replicon-harboring cells and cells stably expressing NS5A alone. These data suggest that stimulation of PI3K by NS5A may represent an indirect mechanism for development of HCC in HCV-infected patients and further suggests potential therapeutic strategies to counteract the occurrence of HCV-related HCC.

Amino Acid Motifs↗

PubMed2004

Introduction of replication-competent hepatitis C virus transcripts using a tetracycline-regulable baculovirus delivery system.

We have developed a baculovirus delivery system that enables tetracycline-regulated expression of polII-derived hepatitis C virus (HCV) transcripts in hepatocyte-derived cell lines (McCormick et al., 2002). As part of a study to determine whether such transcripts are replication competent, the transcription start site of the tetracycline-regulable promoter was mapped and three baculovirus transfer vectors containing a neo(R)-expressing culture adapted replicon cDNA were generated. These vectors either had the first nucleotide of the 5'UTR positioned -2 (mkI) and +1 (mkII) with respect to the transcription start site, or included a hammerhead ribozyme at the 5' end of the transcript (5'HH) that cleaves between the ribozyme-5'UTR boundary. Transfection of all of the culture-adapted replicon constructs into Huh7 cells resulted in the formation of more neomycin-resistant colonies than seen with a polymerase knock-out replicon construct, although this was less pronounced in the mkI group. Furthermore, both the positive- and negative-strands of the replicon could be detected in all neomycin-resistant polyclonal cell lines except for those derived from transfection of the polymerase knock-out construct. Transduction of Huh7 cells with recombinant baculoviruses carrying the same expression cassettes improved replicon delivery, but the relative efficiency of the constructs remained the same. The baculovirus vectors were also used to introduce the replicon transcript into HepG2 cells. Expression of the culture-adapted but not the polymerase knock-out construct induced transcription of the beta-interferon gene, a response that may contribute to this cell line being unable to maintain the replicon over long-term culture.

5' Untranslated Regions↗

PubMed2004

The hepatitis C virus NS5A protein binds to members of the Src family of tyrosine kinases and regulates kinase activity.

The hepatitis C virus (HCV) non-structural NS5A protein has been shown to associate with a variety of cellular signalling proteins. Of particular interest is the observation that a highly conserved C-terminal polyproline motif in NS5A was able to interact with the Src-homology 3 (SH3) domains of the adaptor protein Grb2. As it has previously been shown that specific polyproline motifs can interact with a range of SH3 domains, we investigated whether NS5A was capable of interacting with other SH3 domain-containing proteins. We show here that NS5A interacts with the SH3 domains of members of the Src family of tyrosine kinases: a combination of in vitro binding assays and co-immunoprecipitation experiments revealed an interaction between NS5A and Hck, Lck, Lyn and Fyn, but interestingly not Src itself. Mutational analysis confirmed that the polyproline motif responsible for binding to Grb2 also bound to the SH3 domains of Hck, Lck, Lyn and Fyn. Furthermore, a previously unidentified polyproline motif, adjacent to the first motif, was also able to mediate binding to the SH3 domain of Lyn. Using transient transfections and Huh-7 cells harbouring a persistently replicating subgenomic HCV replicon we demonstrate that NS5A bound to native Src-family kinases in vivo and differentially modulated kinase activity, inhibiting Hck, Lck and Lyn but activating Fyn. Lastly, we show that signalling pathways controlled by Src-family kinases are modulated in replicon cells. We conclude that the interactions between NS5A and Src-family kinases are physiologically relevant and may play a role in either virus replication or pathogenesis.

Animals↗

PubMed2004

Hepatitis C virus NS5A: tales of a promiscuous protein.

The non-structural 5A (NS5A) protein of hepatitis C virus (HCV) has been the subject of intensive research over the last decade. It is generally accepted that NS5A is a pleiotropic protein with key roles in both viral RNA replication and modulation of the physiology of the host cell. Our understanding of the role of NS5A in the virus life cycle has been hampered by the lack of a robust in vitro system for the study of HCV replication, although the recent development of the subgenomic replicon has at least allowed us to begin to dissect the involvement of NS5A in the process of viral RNA replication. Early studies into the effects of NS5A on cell physiology relied on expression of NS5A either alone or in the context of other non-structural proteins; the advent of the replicon system has allowed the extrapolation of these studies to a more physiologically relevant cellular context. Despite recent progress, this field is controversial, and there is much work to be accomplished before we fully understand the many functions of this protein. In this article, the current state of our knowledge of NS5A, discussing in detail its direct involvement in virus replication, together with its role in modulating the cellular environment to favour virus replication and persistence, are reviewed. The effects of NS5A on interferon signalling, and the regulation of cell growth and apoptosis are highlighted, demonstrating that this protein is indeed of critical importance for HCV and is worthy of further investigation.

Amino Acid Sequence↗

PubMed2003

The hepatitis C virus non-structural NS5A protein inhibits activating protein-1 function by perturbing ras-ERK pathway signaling.

The hepatitis C virus nonstructural 5A (NS5A) protein is a pleiotropic phosphoprotein that has been shown to associate with a wide variety of cellular signaling proteins. Of particular interest is the observation that a highly conserved C-terminal Class II polyproline motif within NS5A mediated association with the Src homology 3 domains of members of the Src family of tyrosine kinases and the mitogenic adaptor protein Grb2 (A. Macdonald, K. Crowder, A. Street, C. McCormick, and M. Harris, submitted for publication). In this study, we analyzed the consequences of NS5A expression on mitogenic signaling pathways within a variety of cell lines. Utilizing a transient luciferase reporter system, we observed that NS5A inhibited the activity of the mitogenic and stress-activated transcription factor activating protein-1 (AP1). This inhibition was dependent upon a Class II polyproline motif within NS5A. Using a combination of dominant active and negative mutants of components of the MAPK signaling pathways, selective inhibitors, together with immunoblotting with phospho-specific and phosphorylation-independent antibodies, we determined the signaling pathways targeted by NS5A to inhibit AP1. These studies demonstrated that in both stable NS5A-expressing cells and Huh-7-derived cells harboring subgenomic hepatitis C virus (HCV) replicons, this inhibition was mediated through the ERK signaling pathway. Importantly, a comparable inhibition of AP1 reporter activity was observed in hepatocyte-derived cell lines transduced with a baculovirus vector driving expression of full-length HCV polyprotein. In conclusion, these data strongly suggest a role for the NS5A protein in the perturbation of mitogenic signaling pathways in HCV-infected hepatocytes.

Amino Acid Motifs↗

PubMed2002

Antipsychotics in clinical practice: guidelines for safe and effective use.

Antipsychotic drugs have made a significant contribution to the treatment of schizophrenia but the older drugs in particular have significant side-effects. The newer atypical drugs are effective for the treatment of both positive and negative symptoms and they also have significantly fewer serious side-effects. However, these drugs are considerably more expensive that the older drugs and this has generated intense debate about their cost effectiveness. There is now good evidence that when all factors are taken into consideration the atypical drugs are both cost effective and improve the quality of life of patients with schizophrenia. Despite the importance of these drugs there is widespread variation in their use and a national consensus on this important issue is long overdue.

Adolescent↗