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Biomedical subjects

M Egerton

Publications and source records attributed to M Egerton.

At least 19 recordsLinked to original sources
PubMed2006

Blood 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and melatonin levels in patients with either Huntington's disease or chronic brain injury.

Following a study of oxidative tryptophan metabolism to kynurenines, we have now analysed the blood of patients with either Huntington's disease or traumatic brain injury for levels of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and melatonin. There were no differences in the baseline levels of these compounds between patients and healthy controls. Tryptophan depletion did not reduce 5-HT levels in either the controls or in the patients with Huntington's disease, but it increased 5-HT levels in patients with brain injury and lowered 5-HIAA in the control and Huntington's disease groups. An oral tryptophan load did not modify 5-HT levels in the patients but increased 5-HT in control subjects. The tryptophan load restored 5-HIAA to baseline levels in controls and patients with brain injury, but not in those with Huntington's disease, in whom 5-HIAA remained significantly depressed. Melatonin levels increased on tryptophan loading in all subjects, with levels in patients with brain injury increasing significantly more than in controls. Baseline levels of neopterin and lipid peroxidation products were higher in patients than in controls. It is concluded that both groups of patients exhibit abnormalities in tryptophan metabolism, which may be related to increased inflammatory status and oxidative stress. Interactions between the kynurenine, 5-HT and melatonin pathways should be considered when interpreting changes of tryptophan metabolism.

Administration, Oral↗
PubMed2006

Tryptophan metabolism and oxidative stress in patients with chronic brain injury.

The kynurenine pathway generates the excitotoxic N-methyl-d-aspartate receptor agonist, quinolinic acid and the glutamate antagonist, kynurenic acid, as well as free-radical generators. We investigated the status of the pathway following severe brain injury sustained at least 1 year previously in 15 patients compared with controls. At baseline, patients with brain injury showed increased levels of neopterin, erythrocyte sedimentation rate, C-reactive protein and peroxidation products in the blood compared with controls, indicating persistent inflammation and oxidative stress. At baseline and following tryptophan depletion, more tryptophan was converted to kynurenine in patients than controls, but less kynurenine was converted into the neuroprotectant, kynurenic acid. This suggests that neuroprotection by kynurenic acid may be inadequate in brain-damaged patients even many years after injury. On tryptophan loading, patients metabolized more kynurenine into kynurenic acid than controls, a process which may be neuroprotective. In addition, lower levels of 3-hydroxykynurenine and 3-hydroxyanthranilic acid in patients after tryptophan loading should be protective since these compounds generate free radicals. The results suggest that for brain-damaged patients, increased activation of the kynurenine pathway, oxidative stress and raised levels of inflammation continue many years after the original insult, possibly contributing to the continuing cerebral dysfunction in these patients.

Adult↗
PubMed2005

Tryptophan metabolism and oxidative stress in patients with Huntington's disease.

Abnormalities in the kynurenine pathway may play a role in Huntington's disease (HD). In this study, tryptophan depletion and loading were used to investigate changes in blood kynurenine pathway metabolites, as well as markers of inflammation and oxidative stress in HD patients and healthy controls. Results showed that the kynurenine : tryptophan ratio was greater in HD than controls in the baseline state and after tryptophan depletion, indicating increased indoleamine dioxygenase activity in HD. Evidence for persistent inflammation in HD was provided by elevated baseline levels of C-reactive protein, neopterin and lipid peroxidation products compared with controls. The kynurenate : kynurenine ratio suggested lower kynurenine aminotransferase activity in patients and the higher levels of kynurenine in patients at baseline, after depletion and loading, do not result in any differences in kynurenic acid levels, providing no supportive evidence for a compensatory neuroprotective role for kynurenic acid. Quinolinic acid showed wide variations in blood levels. The lipid peroxidation data indicate a high level of oxidative stress in HD patients many years after disease onset. Levels of the free radical generators 3-hydroxykynurenine and 3-hydroxyanthranilic acid were decreased in HD patients, and hence did not appear to contribute to the oxidative stress. It is concluded that patients with HD exhibit abnormal handling of tryptophan metabolism and increased oxidative stress, and that these factors could contribute to ongoing brain dysfunction.

Adolescent↗
PubMed2004

Tryptophan loading induces oxidative stress.

In previous studies tryptophan loads have been administered to human subjects in order to raise central levels of 5-hydroxytryptamine (5HT) and assess the effects of 5HT on behaviour and mood. However, tryptophan is metabolised primarily along the oxidative kynurenine pathway. In this study a 6 g oral tryptophan load was administered to 15 healthy volunteers and the levels of kynurenines and lipid peroxidation products (indicative of oxidative stress) were measured. The results demonstrate that tryptophan loading produces a highly significant increase in lipid peroxidation products in parallel with increased kynurenines. The oxidative stress may result from the generation of quinolinic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid, all of which are known to have the ability to generate free radicals. The results may have implications for the use of tryptophan loading in psychiatric practice, and for the chronic use of diets high in tryptophan.

Adult↗
PubMed2000

Expression databases--resources for pharmacogenomic R&D.

This review aims to outline the primary biological databases that are being generated to understand fundamental biology, identify new drug targets, and to look at compound profiling in a new light. We will give a brief overview of four of the main areas being studied in molecular biology: genomics, pharmacogenomics, pharmacogenetics and proteomics. Looking initially at each data set and some of its potential applications, we will go on to describe some of the potentially enormous advantages gained by fully integrating these data sets.

Animals↗
PubMed1998

Characterization of human and rat brain myristoyl-CoA:protein N-myristoyltransferase: evidence for an alternative splice variant of the enzyme.

Using 5'-rapid amplification of cDNA ends, we have identified an extended 5'-end of mRNA coding for human myristoyl-CoA:protein N-myristoyltransferase (NMT). PCR using primers based on this new 5'-sequence and reverse primers within the currently accepted coding sequence of the enzyme resulted in the identification of a novel splice variant of NMT. In vitro translation of these cDNAs resulted in the production of proteins with apparent molecular masses of 63 kDa and 48 kDa. Immunoprecipitation of NMT from human cell lines and immunoblotting of a range of rat tissues has identified proteins with molecular masses corresponding to those derived from these cDNAs, and provided evidence that their relative abundance differs among tissues. Our results provide evidence that this enzyme exists in different forms resulting from alternative splicing of the mRNA.

Acyltransferases↗
PubMed1998

Crystal structure of the anti-fungal target N-myristoyl transferase.

N-myristoyl transferase (NMT) catalyzes the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine of substrate proteins, and is found only in eukaryotic cells. The enzyme in this study is the 451 amino acid protein produced by Candida albicans, a yeast responsible for the majority of systemic infections in immuno-compromised humans. NMT activity is essential for vegetative growth, and the structure was determined in order to assist in the discovery of a selective inhibitor of NMT which could be developed as an anti-fungal drug. NMT has no sequence homology with other protein sequences and has a novel alpha/beta fold which shows internal two-fold symmetry, which may be a result of gene duplication. On one face of the protein there is a long, curved, relatively uncharged groove, at the center of which is a deep pocket. The pocket floor is negatively charged due to the vicinity of the C-terminal carboxylate and a nearby conserved glutamic acid residue, which separates the pocket from a cavity. These observations, considered alongside the positions of residues whose mutation affects substrate binding and activity, suggest that the groove and pocket are the sites of substrate binding and the floor of the pocket is the catalytic center.

Acyl Coenzyme A↗
PubMed1998

Activation of the extracellular signal-regulated kinase pathway is differentially required for TCR-stimulated production of six cytokines in primary T lymphocytes.

The extracellular signal-regulated kinase (ERK) signaling pathway is strongly activated in response to TCR stimulation in normal T cells. However, the extent to which activation of the ERK pathway is necessary for TCR-stimulated cytokine production is not clear. We have addressed this question by use of two separate methods to interfere with TCR activation of the ERK cascade. The first approach utilized transient expression of a catalytically inactive form of mitogen-activated/ERK 1 (CI-MEK1), while the second involved using the MEK1- and MEK2-specific inhibitor PD98059 to block ERK activation by the TCR. In order to assess the requirement for ERK activation in T cell cytokine production, we have measured the effect of ERK inhibition upon the production of six cytokines, IL-3, IL-4, IL-5, IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and IFN-gamma, by newly activated normal mouse T cells in response to TCR stimulation. The results of experiments using both methods to block ERK activation have revealed a requirement for intact ERK signaling for the full elicitation of TCR-stimulated cytokine production. Dose-response analyses using the MEK inhibitor PD98059 showed that the TCR-stimulated production of all cytokines measured was affected by this treatment. However, the production of IL-3 and IL-4 was only partially dependent upon ERK activation, whereas IL-5, IL-10, IFN-gamma and GM-CSF production was severely affected by diminished ERK activation. We conclude that the ERK pathway is differentially involved in the activation of different cytokine genes in normal T cells.

Animals↗
PubMed1997

Activated T cells from draining lymph nodes and an effector site differ in their responses to TCR stimulation.

We previously showed that T cells from the mediastinal lymph nodes (MLN) and lung parenchyma of influenza virus-infected mice were functionally remarkably different. Here we demonstrate that the differences in cytokine production are due to differences in the frequencies of T cells within the activated pool able to produce cytokines after TCR stimulation. FACS analysis of T cells from MLN and lung tissue demonstrated that T cells expressing any of the activation markers tested (LFA-1, CD25, CD44, CD45RB, CD49d, CD62L) always expressed high levels of CD44 and LFA-1. These double-high T cells produced >99% of all anti-CD3 mAb-induced IL-4 and IFN-gamma. Separation of T cells employing mAb against the other activation markers in combination with anti-CD44 mAb did not enable further fractionation into cytokine producers and nonproducers. Despite their similar phenotype, purified double-high lung parenchyma T cells produced markedly higher levels of IL-2, IL-4, and IFN-gamma, and contained a higher frequency of cytokine producers than their MLN counterparts. Activation of the extracellular signal-regulated kinase (ERK)-2 in response to TCR cross-linking was detected in double-high T cells from lung tissue but not MLN. The requirement for ERK signaling for maximal IFN-gamma synthesis could nevertheless be demonstrated in both populations by blockade with the inhibitor PD98509. Collectively, the data suggest that inductive and effector sites differ in the frequency of activated T cells able to induce ERK-2-regulated cytokine production after TCR ligation.

Animals↗
PubMed1997

Interaction between ATM protein and c-Abl in response to DNA damage.

The gene mutated in the autosomal recessive disorder ataxia telangiectasia (AT), designated ATM (for 'AT mutated'), is a member of a family of phosphatidylinositol-3-kinase-like enzymes that are involved in cell-cycle control, meiotic recombination, telomere length monitoring and DNA-damage response. Previous results have demonstrated that AT cells are hypersensitive to ionizing radiation and are defective at the G1/S checkpoint after radiation damage. Because cells lacking the protein tyrosine kinase c-Abl are also defective in radiation-induced G1 arrest, we investigated the possibility that ATM might interact with c-Abl in response to radiation damage. Here we show that ATM binds c-Abl constitutively in control cells but not in AT cells. Our results demonstrate that the SH3 domain of c-Abl interacts with a DPAPNPPHFP motif (residues 1,373-1,382) of ATM. The results also reveal that radiation-induction of c-Abl tyrosine kinase activity is diminished in AT cells. These findings indicate that ATM is involved in the activation of c-Abl by DNA damage and this interaction may in part mediate radiation-induced G1 arrest.

Ataxia Telangiectasia↗
PubMed1997

Analysis of the promoter region of the human VEGF-related factor gene.

We have characterised the promoters of the human and murine VRF (vascular endothelial growth factor (VEGF) related factor) gene. A series of deletions were made of a 553-bp region 5' of the VRF initiation codon and were used in a luciferase reporter gene assay to determine the minimal promoter of the VRF gene. The region between base pairs -443 and -195 was sufficient to mediate transcription in lymphocytes and the region between -550 and -443 enhanced this promoter activity. Primer extension studies identified two regions of transcription initiation, both of which are preceded by Sp1, AP-2 and Egr-1 transcription factor binding sites. The VRF promoter is similar to VEGF in that it is associated with a CpG island, contains sites for Sp1 and AP-2, and lacks a TATA box. However, it has marked differences in that the promoter contains Egr-1 sites and lacks both hypoxia-inducible factor-1 and AP-1 sites. These data may indicate that expression of these two growth factors is regulated by different physiological stimuli.

Animals↗
PubMed1997

Sequence and expression of Drosophila myristoyl-CoA: protein N-myristoyl transferase: evidence for proteolytic processing and membrane localisation.

The enzyme N-myristoyl transferase transfers the 14 carbon fatty acid myristate to an N-terminal glycine residue in a small subset of cytoplasmic proteins. Many myristoyl proteins are components of cellular signalling pathways, some of which play important roles during embryonic development, for example protein kinase A. Thus, the function of N-myristoyl transferase is probably essential for embryogenesis and it is of some interest to study the enzyme in an organism with well understood developmental biology. In this paper we report the purification of a processed form of the Drosophila enzyme from peripheral membrane fractions of embryos by affinity chromatography to a protein containing leucine rich repeats. We have also isolated the Drosophila N-myristoyl transferase gene and determined its nucleotide sequence. The predicted amino acid sequence of the Drosophila enzyme is closely related to that of mammalian and fungal N-myristoyl transferases and residues essential for enzyme function are conserved. Our findings indicate that a fraction of Drosophila NMT is bound to the membrane and they are consistent with recent results for the human enzyme. We suggest that N-myristoyl transferase may be recruited to the membrane as part of a translational complex, perhaps by binding to p34 ribosome binding protein, a leucine rich repeat receptor of the microsomal membranes. We have also studied the expression pattern of the gene in the embryo by northern blot analysis and in situ hybridization. The transcripts appear to be uniformly distributed in the pre-cellular embryo but at later stages the RNA is barely detectable with these methods. However, from about stage 14, high levels of transcript are detected in a small number of randomly distributed cells of the central and peripheral nervous system.

Acyltransferases↗
PubMed1996

Identification of the 70kD heat shock cognate protein (Hsc70) and alpha-actinin-1 as novel phosphotyrosine-containing proteins in T lymphocytes.

T cell antigen receptor (TCR) ligation results in the tyrosine phosphorylation of numerous intracellular protein substrates, and the identification of these substrates has been a major undertaking by several groups. We have used pervanadate treatment to artificially increase cellular phosphotyrosine levels and immobilized anti-phosphotyrosine monoclonal antibodies to partially purify tyrosine phosphorylated proteins in quantities suitable for amino acid sequencing. This strategy was used to identify three phosphotyrosine containing proteins, with relative molecular masses of 105, 81, and 76 kD by amino acid sequencing. Here we report the identification of pp105 as alpha-actinin-1, pp81 as the murine equivalent of the HS1 gene product, and pp76 as Hsc70. This is the first report that alpha-actinin-1 and Hsc70 are targets of activated tyrosine kinases. Furthermore, we show that Hsc70 is tyrosine phosphorylated in response to TCR ligation, which constitutes the first evidence that Hsc70 might be subject to regulation by tyrosine kinase signaling pathways.

Actinin↗
PubMed1996

A novel Hsp70 of the yeast ER lumen is required for the efficient translocation of a number of protein precursors.

The yeast genome sequencing project predicts an open reading frame (YKL073) that would encode a novel member of the Hsp70 family of molecular chaperones. We report that this 881 codon reading frame represents a functional gene expressing a 113-119 kDa glycoprotein localized within the lumen of the endoplasmic reticulum (ER). We therefore propose to designate this gene LHS1 (Lumenal Hsp Seventy). Our studies indicate that LHS1 is regulated by the unfolded protein response pathway, as evidenced by its transcriptional induction in cells treated with tunicamycin, and in various mutants defective in precursor processing (sec11-7, sec53-6 and sec59-1). LHS1 is not essential for viability, but an Lhs1 null mutant strain exhibits a coordinated induction of genes regulated by the unfolded protein response indicating a role for Lhs1p in protein folding in the ER. Furthermore, the null mutation is synthetically lethal in combination with (delta)ire1, thus activation of the unfolded protein response pathway is essential for cells to tolerate loss of Lhs1p. Synthetically lethality is also seen with mutations in KAR2, strongly suggesting that Kar2p and Lhs1p have overlapping functions. The Lhs1 null mutant exhibits a severe constitutive defect in the translocation of several secretory preproteins. We therefore propose that Lhs1p is a molecular chaperone of the ER lumen involved in both polypeptide translocation and subsequent protein folding.

Base Sequence↗
PubMed1996

Differential activation of T cell cytokine production by the extracellular signal-regulated kinase (ERK) signaling pathway.

Stimulation of T cells via the T cell receptor (TCR) activates a number of signaling pathways that are potentially involved in the elicitation of physiological responses, such as the production of cytokines. The extracellular signal-regulated kinases (ERK) are a group of molecules activated in response to TCR ligation, whose role in T cell cytokine production is controversial. In this study, we have asked whether ERK activation is coupled to the production of a number of T cell-derived cytokines, and whether particular cytokines are differentially affected by ERK activation. To address these questions, we have utilized a constitutively active version of the immediate upstream activator of both ERK1 and ERK2, mitogen-activated/extracellular signal-regulated kinase 1 (MEK1), to activate ERK signaling selectively in the absence of other TCR-activated signaling pathways. The effect of constitutive MEK/ERK activation on T cell cytokine production was measured by transiently co-transfecting newly activated mouse T cells with DNA encoding constitutively active MEK1 (CA-MEK1) and the human interleukin-2 (IL-2) receptor alpha chain (hCD25), purifying hCD25+ transfectants by flow-cytometric cell sorting, and measuring the production of IL-3, IL-4, interferon (IFN)-gamma and granulocyte/macrophage-colony-stimulating factor (GM-CSF) either in the presence or absence of ionomycin stimulation. Newly activated T cells were used in these experiments as they more closely resemble T cells activated in vivo than do transformed T cells or long-term established T cell clones. CA-MEK1 expression led to constitutive ERK activation, which acted synergystically with ionomycin treatment to stimulate cytokine production. Furthermore, these experiments revealed a hierarchy of cytokine responsiveness to MEK/ERK activation, such that the production of IL-3 was most affected, followed by GM-CSF, IFN-gamma, and IL-4.

Animals↗
PubMed1995

Further development of the locus control region/murine erythroleukemia expression system: high level expression and characterization of recombinant human calcitonin receptor.

In order to demonstrate the potential of the LCR/MEL expression system for the expression of seven-transmembrane helix receptors the human calcitonin receptor (hCTR) has been cloned and expressed at high levels in this system. Using the newly developed single-step expression vectors pEV and pNV, stable recombinant MEL cells which express the hCTR at approximately 6 pmol of receptor/mg of total cell protein (60 pmol/mg of membrane protein), or 1.9 x 10(6) receptors per cell, have been generated. Ligand binding studies have shown that the cloned receptor expressed in MEL cells has properties which are indistinguishable from those of the native receptor in T47D cells. It is suggested that MEL cells expressing seven-transmembrane helix receptors provide an efficient and readily attainable source of material for ligand binding experiments using these receptors.

Animals↗