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Biomedical subjects

Mark Harris

Publications and source records attributed to Mark Harris.

At least 19 recordsLinked to original sources
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

A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria.

We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this in vitro activity is inhibited by amantadine. Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted trans-membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy.

Amantadine↗
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↗
PubMed2003

SNP cherry picker: maximizing the chance of finding an association with a disease SNP.

UNLABELLED: The high cost of genotyping single nucleotide polymorphisms (SNPs) generally prohibits the systematic mapping of entire genetic linkage regions in order to find the polymorphisms associated with increased risk of disease. In practice, SNPs are selected at approximately equal spacing across the linkage region to try to locate a SNP lying in the haplotype block of the disease SNP. The size of the haplotype block may not be known, however, and SNPs taken from public domain sources may not in fact be polymorphic. Our program will choose a subset of the SNPs in a linkage region so as to maximize the expected proportion of the sequence that lies within a given distance of a real SNP. AVAILABILITY: The software is available, free of charge, for academic use on request from the authors. SUPPLEMENTARY INFORMATION: www.oxagen.co.uk

Algorithms↗
PubMed2003

Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D.

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations. The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.

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↗
PubMed2003

Iodine deficiency and goitre in schoolchildren in Melbourne, 2001.

OBJECTIVE: To assess iodine status and goitre prevalence in a sample of schoolchildren in Melbourne. DESIGN: Cross-sectional study of urinary iodine excretion and presence of goitre in a sample of schoolchildren from Years 5-12 attending two urban schools. PARTICIPANTS: 607 children aged 11-18 years consented to thyroid gland palpation and 577 provided a urine sample on the day of examination in August 2001. OUTCOME MEASURE: Iodine status of the study population, based on median urinary iodine values categorised as normal (> or = 100 microg/L), mild (50-99 microg/L) or moderate-severe (< 50 microg/L), and classified according to sex, school year and presence of goitre. RESULTS: 76% (439/577) of students had abnormal urinary iodine values, with 27% (156/577) having values consistent with moderate-severe deficiency. The median urinary iodine excretion for the total group was 70 microg/L, with values for school years 5-12 ranging from 62 microg/L (Year 12) to 76 microg/L (Year 9). The median urinary iodine value in girls was lower than that in boys (64 microg/L v 82 microg/L), and girls had significantly lower urinary iodine values overall (P < 0.002). There was no association between goitre grade and moderate-severe (< 50 microg/L; P = 0.39) or mild (50-99 microg/L; P = 0.07) urinary iodine deficiency. CONCLUSIONS: We found mild iodine deficiency in a cohort of schoolchildren in Melbourne. Our results support other data showing mild iodine deficiency in Sydney and Tasmania and the argument for a national study of iodine nutrition.

Adolescent↗
PubMed2003

HIV-1 Nef induces the release of inflammatory factors from human monocyte/macrophages: involvement of Nef endocytotic signals and NF-kappa B activation.

It has been recently reported that the endogenous expression of HIV-1 Nef in human monocyte/macrophages induces the release of chemokines and other as yet unidentified soluble factors leading to multiple effects of pathogenic significance, such as the recruitment and activation of quiescent lymphocytes. However, the description of underlying molecular mechanisms remained elusive. We recently demonstrated that human monocyte-derived macrophages (MDM) efficiently internalize soluble rNef, thereby inducing effects largely resembling those observed in cells endogenously expressing Nef. By exploiting the rNef/MDM model, we sought to gain more insights on the molecular mechanisms underlying the response of MDM to Nef. Array analysis for the detection of transcripts from a large number of monokines, chemokines, cytokines, and receptors thereof showed that MDM promptly responded to rNef treatment by increasing the transcription of genes for several inflammatory factors. Analysis of supernatants revealed that rNef treatment induced the release of macrophage inflammatory proteins 1alpha and 1beta, IL-1beta, IL-6, and TNF-alpha. Conversely, rNefs mutated in domains critical for the interaction with the endocytotic machinery (i.e., EE155-156QQ, and DD174-175AA) were ineffective. Interestingly, we found that the Nef-dependent release of inflammatory factors correlated with the activation of the NF-kappaB transcription factor, mainly in its p50/p50 homodimeric form, and in a de novo protein synthesis-independent manner. Our data add new hints supporting the idea that the presence of Nef is per se heavily detrimental for monocyte/macrophages and relative cross-talking cell types.

Adult↗
PubMed2003

The di-leucine motif in the cytoplasmic tail of CD4 is not required for binding to human immunodeficiency virus type 1 Nef, but is critical for CD4 down-modulation.

The human immunodeficiency virus type 1 (HIV-1) nef gene encodes a 205 residue, myristoylated phosphoprotein that has been shown to play a critical role in the replication and pathogenesis of the virus. One of the most studied functions of the Nef protein is the down-modulation of cell surface CD4. Nef has been reported to interact with both the cytoplasmic tail of CD4 and proteins that are components of the endocytic machinery, thereby enhancing the endocytosis of CD4 through clathrin-coated pits. A di-leucine motif in the cytoplasmic tail of CD4 (residues 413/414) was reported to be essential both for Nef mediated down-modulation and for Nef binding. In order to further characterize the involvement of this di-leucine motif in CD4 down-modulation we generated a CD4 mutant in which the leucines were substituted by alanines, termed CD4(LL-AA). We demonstrate here that, contrary to previous data obtained with the cytoplasmic tail of CD4 alone, full-length CD4(LL-AA) bound to Nef both in vivo, in recombinant baculovirus-infected Sf9 cells, and in vitro. In contrast the di-leucine motif was required for both Nef-mediated and phorbol ester-induced CD4 down-modulation, suggesting that the essential requirement for the di-leucine motif in CD4 down-modulation reflects the fact that this motif is needed for the interactions of CD4 with the endocytic machinery, not for the interaction with Nef. We have also exploited the observation that CD4(LL-AA) is refractory to Nef-mediated down-modulation to provide the first experimental evidence for a physical interaction between Nef and CD4 in intact mammalian cells.

Amino Acid Motifs↗
PubMed2003

Reversible metaphyseal dysplasia, a novel bone phenotype, in two unrelated children with autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy: clinical and molecular studies.

We report the association of an undescribed, reversible metaphyseal dysplasia (RMD) with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) in two patients, one homozygous and one heterozygous for a 13-bp deletion in exon 8 of the autoimmune regulator (AIRE) gene. One patient also had a novel deletion in exon 6, resulting in a frameshift mutation and introduction of a STOP codon in exon 10. Their APECED phenotypes differed, but both patients developed progressive skeletal deformities and growth failure from early childhood. Radiological examination suggested a generalized abnormality of endochondral ossification, with irregular, flared, radioopaque regions in the metaphyses, subjacent to the growth plates. Histopathology in patient 1 showed islands of calcified cartilage within bone, consistent with impaired coupling of cartilage resorption with vascular invasion and ossification. Despite discordance for puberty, both patients experienced radiological resolution of their bone disease in their mid-teens, with improvement in histopathology in patient 1. RMD may constitute a rare phenotypic variation of APECED, possibly resulting from autoimmunity directed against skeletal proteins. We also demonstrated AIRE expression in chondrocytes derived from human fetal growth plates, primary culture of human chondrocytes, and two chondrosarcoma cell lines, suggesting a potential role for abnormal AIRE expression in the development of RMD.

Adult↗
PubMed2003

Prostate cancer.

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Aged↗
PubMed2002

Randomised controlled trial of home based care of patients with chronic obstructive pulmonary disease.

OBJECTIVES: To evaluate usefulness of limited community based care for patients with chronic obstructive pulmonary disease after discharge from hospital. DESIGN: Randomised controlled trial. SETTING: Liverpool Health Service and Macarthur Health Service in outer metropolitan Sydney between September 1999 and July 2000. PARTICIPANTS: 177 patients randomised into an intervention group (84 patients) and a control group (93 patients) which received current usual care. INTERVENTIONS: Home visits by community nurse at one and four weeks after discharge and preventive general practitioner care. MAIN OUTCOME MEASURES: Frequency of patients' presentation and admission to hospital; changes in patients' disease-specific quality of life, measured with St George's respiratory questionnaire, over three months after discharge; patients' knowledge of illness, self management, and satisfaction with care at discharge and three months later; frequency of general practitioner and nurse visits and their satisfaction with care. RESULTS: Intervention and control groups showed no differences in presentation or admission to hospital or in overall functional status. However, the intervention group improved their activity scores and the control group worsened their symptom scores. While intervention group patients received more visits from community nurses and were more satisfied with their care, involvement of general practitioners was much less (with only 31% (22) remembering receiving a care plan). Patients in the intervention group had higher knowledge scores and were more satisfied. There were no differences in general practitioner visits or management. CONCLUSIONS: This brief intervention after acute care improved patients' knowledge and some aspects of quality of life. However, it failed to prevent presentation and readmission to hospital.

Adult↗
PubMed2002

Xtrack - a web-based crystallographic notebook.

Xtrack is a web-based database-backed crystallographic notebook system that allows groups of workers to keep track of crystallographic project data from the crystallization stage through to data bank deposition. A working demonstration version is available from http://xray.bmc.uu.se/xtrack and the code is available for those who wish to set up their own databases.

Crystallography, X-Ray↗
PubMed2002

Age-related differences in vision and proprioception in a lower limb interceptive task: the effects of skill level and practice.

This study examined age-related differences in the role of visual proprioception during a lower limb interceptive action and how this varies as a function of skill and practice. In Experiment 1, skilled and less-skilled 8-, 10-, and 12-year-old boys controlled a soccer ball using their preferred foot under full vision and when sight of the effector was occluded. With the exception of the high-skilled 12-year-olds and low-skilled 8-year-olds, participants showed a decrement in peformance when denied access to visual proprioception. In Experiment 2, the effect of practicing under different informational constraints was examined for 12 year-old boys. Children performed varying amounts of practice under full vision, or in a condition where sight of the foot was occluded, before being transferred to the alternative viewing condition. Participants who practiced under occluded viewing conditions showed greater relative improvement in performance over practice and transfer sessions compared with a full vision control group. Some support is provided for the manipulation of visual informational constraints as an effective pedagogical approach to motor learning.

Age Factors↗
PubMed2002

Australian GPs are satisfied with their job: even more so in rural areas.

BACKGROUND: Australia has seen a constriction of the scope of practice and of professional prerogatives for GPs in the 1990s, which is said to have led to lower job satisfaction, particularly for rural GPs. OBJECTIVE: The aim of this study was to determine levels of job satisfaction and mental health in a rural and an urban sample of GPs, and to correlate population and practice characteristics with lower job satisfaction and morale. METHODS: A postal survey of GP members of two urban and four rural Divisions of General Practice in New South Wales was carried out using the Wall-Cook-Warr scale and the 12-item General Health Questionnaire (GHQ-12). The overall response rate was 74%. RESULTS: Both urban and rural Australian GPs were generally satisfied with their jobs; however, rural GPs had significantly higher job satisfaction scores. The highest levels of satisfaction were recorded for factors denoting autonomy. The lowest ranked factors were hours of work, income and government policy in general practice. GPs without psychological disturbance were significantly more satisfied with their work than those who were disturbed. Lower job satisfaction was significantly associated with urban GPs, GPs who worked full-time and those who used a language other than English during consultations. Poor mental health and working in an urban area were predictive of lower job satisfaction in men, and bulk-billing all patients (i.e. not charging patients a co-payment, but making a consolidated claim to the government funder Medicare) and working full-time were predictive of lower job satisfaction in women. CONCLUSIONS: Both urban and rural GPs derive a large measure of satisfaction from their job. In particular, rural GPs are satisfied, especially so with their autonomy. Poor mental health was the strongest predictor of lower job satisfaction. The high percentage of GPs with psychological disturbances raises the problem of their access to appropriate mental health services, in particular in rural areas.

Female↗
PubMed2002

Efficient delivery and regulable expression of hepatitis C virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors.

Baculovirus vectors have been used as efficient delivery vehicles for constitutive gene expression in a variety of mammalian cells. We have further developed the system to allow for regulable expression by placing the gene of interest under the control of an inducible promoter, and complementing it with a second baculovirus vector providing the control elements necessary for promoter activity. We have used this system to express (a) the lacZ gene, (b) a 'minigenome' derived from hepatitis C virus (HCV) and carrying lacZ or (c) the full-length HCV viral genome, in human hepatocyte cell lines in an inducible fashion. Control systems that rely on either the absence of tetracycline or presence of ponasterone to induce gene expression were tested. Expression of lacZ was controlled by ponasterone, but beta-galactosidase activity was limited to 10-20% of cells. In contrast, the tetracycline-controlled expression system gave a low basal activity and was highly inducible in almost 100% of cells. Inducible expression was also obtained in almost 100% of cells infected with baculoviruses in which an HCV minigenome was placed downstream of the tetracycline-inducible promoter and upstream of either a hammerhead or hepatitis delta virus ribozyme. Northern blot analysis was consistent with accurate cleavage of the minigenome transcript by the hepatitis delta virus ribozyme. Finally, regulable transcript production and viral polypeptide processing could be demonstrated in HepG2 cells infected with baculoviruses bearing the full-length HCV genome. This system thus provides a novel tool for the analysis of HCV replication and host-cell interactions.

Animals↗