Biomedical subjects

David S Latchman

Publications and source records attributed to David S Latchman.

At least 19 recordsLinked to original sources

PubMed2007

p300 activation by Presenilin 1 but not by its M146L mutant.

The transcriptional co-activator p300 plays an important role in regulating gene expression in a number of different cell types. We have shown that wild type (WT) Presenilin 1 (PS1) stimulates the transcriptional activity ability of CREB Binding Protein (CBP), a close homolog of p300, whereas the Alzheimer's disease (AD) associated mutant of PS1 does not have this effect. A recent report has suggested that mutant PS1 can also disrupt the TCF/beta-catenin/CBP interaction but has no effect on the TCF/beta-catenin/p300 interaction. This suggests that the malregulation of CBP, but not of p300, caused by mutation in PS1 may be involved in the disease process. Here we show that wild type PS1 stimulates the transcriptional activity ability of p300 whereas an Alzheimer's disease-associated mutant of PS1 did not produce this effect. To our knowledge, this is the first report that shows regulation of p300 activity by WT PS1 and not by mutant PS1, indicating that like CBP, p300 can be differentially regulated by WT PS1 compared to its AD-associated mutant.

Alzheimer Disease↗

PubMed2007

Binding of antiphospholipid antibodies to discontinuous epitopes on domain I of human beta(2)-glycoprotein I: mutation studies including residues R39 to R43.

OBJECTIVE: Pathogenic antiphospholipid antibodies (aPL) bind the self antigen N-terminal domain (domain I) of beta(2)-glycoprotein I (beta(2)GPI), with residues G40-R43 being important. However, peptides homologous to other regions of domain I have also been shown to bind aPL. Furthermore, there are no published reports of the effects of altering R39, which has greater surface exposure than the G40-R43 residues. METHODS: We used a novel, efficient method of production and purification of human domain I by Escherichia coli to create multiple mutants of domain I. These domain I mutants were then screened for binding to a range of polyclonal IgG purified from patients with antiphospholipid syndrome, using both solid-phase and fluid-phase assays. RESULTS: E coli-expressed purified domain I selectively bound IgG derived from patients with antiphospholipid syndrome. In region R39-R43, the R39S mutation had the greatest effect in terms of reducing binding to a panel of aPL in the fluid phase (mean +/- SD inhibition 14 +/- 18.5% versus 44.1 +/- 31.7% for G40E and 62.9 +/- 25.7% for wild-type domain I). Conversely, altering both D8 and D9 to S8 and G9, respectively, had the effect of enhancing binding to aPL in the fluid phase. Adding the remainder of the domain I-II interlinker resulted in enhanced binding over wild-type in the solid phase but not the fluid phase. CONCLUSION: The binding of aPL to beta(2)GPI domain I is complex and likely to involve discontinuous epitopes that include R39 in addition to G40-R43, the domain I-II interlinker, and possibly D8 and D9. Domain I variants with enhanced binding to aPL compared with wild-type domain I may aid in the development of novel therapies.

Antibodies, Antiphospholipid↗

PubMed2006

Role of the JAK-STAT pathway in myocardial injury.

Cardiovascular pathologies are an enormous burden in human health and despite the vast amount of research; the molecular mechanisms and pathways that control the underlying pathologies are still not fully appreciated. The Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway has recently been shown to be an integral part of the response of the myocardium to various cardiac insults, including myocardial infarction, oxidative damage, myocarditis, hypertrophy and remodeling, in addition to having a prominent role in cardioprotective therapies such as ischaemic preconditioning. Here, recent advances in the understanding of how the JAK-STAT pathway orchestrates the response to cellular damage in the myocardium are discussed, along with the potential benefits and challenges in manipulating this pathway in cardiovascular therapy.

Animals↗

PubMed2006

Brn-3b enhances the pro-apoptotic effects of p53 but not its induction of cell cycle arrest by cooperating in trans-activation of bax expression.

The Brn-3a and Brn-3b transcription factor have opposite and antagonistic effects in neuroblastoma cells since Brn-3a is associated with differentiation whilst Brn-3b enhances proliferation in these cells. In this study, we demonstrate that like Brn-3a, Brn-3b physically interacts with p53. However, whereas Brn-3a repressed p53 mediated Bax expression but cooperated with p53 to increase p21cip1/waf1, this study demonstrated that co-expression of Brn-3b with p53 increases trans-activation of Bax promoter but not p21cip1/waf1. Consequently co-expression of Brn-3b with p53 resulted in enhanced apoptosis, which is in contrast to the increased survival and differentiation, when Brn-3a is co-expressed with p53. For Brn-3b to cooperate with p53 on the Bax promoter, it requires binding sites that flank p53 sites on this promoter. Furthermore, neurons from Brn-3b knock-out (KO) mice were resistant to apoptosis and this correlated with reduced Bax expression upon induction of p53 in neurons lacking Brn-3b compared with controls. Thus, the ability of Brn-3b to interact with p53 and modulate Bax expression may demonstrate an important mechanism that helps to determine the fate of cells when p53 is induced.

Animals↗

PubMed2006

Free radical scavenging inhibits STAT phosphorylation following in vivo ischemia/reperfusion injury.

The signal transducer and activator of transcription (STAT) family are latent transcription factors involved in a variety of signal transduction pathways, including cell death cascades. STAT1 has been shown to have a crucial role in regulating cardiac cell apoptosis in the myocardium exposed to ischemia/reperfusion (I/R) injury. The free radical scavenger, tempol, is known to have cardioprotective properties, although little is known about the molecular mechanism(s) by which it acts. In the present study, we assessed the levels of phosphorylated STAT1 and STAT3 and examined whether tempol was able to affect STAT activation after in vivo cardiac I/R injury. We observed a reperfusion time-dependent increase in the tyrosine phosphorylation of STAT1 and STAT3 at residues 701 and 705, respectively. Here we show for the first time that tempol dramatically reduced STAT1 and 3 phosphorylation. The reduction in STAT1 and 3 phosphorylation was accompanied by a concomitant decrease in cellular malondialdehyde (MDA) levels. To verify the role of STAT1 in modulating the cardioprotective effect of tempol, rats were injected with the STAT1 activator, IFN-gamma, and tempol during I/R injury. We found that the presence of IFN-gamma abrogated the protective effects of tempol, suggesting that the protective effects of tempol may partly operate by decreasing the phosphorylation of STAT1. This study demonstrates that careful dissection of the molecular mechanisms that underpin I/R injury may reveal cardioprotective targets for future therapy.

Animals↗

PubMed2006

STAT signalling in the heart and cardioprotection.

During the past six years, great progress has been made in defining the key cellular and molecular signalling pathways that determine the fate of cardiac myocytes after ischaemia-reperfusion (IR) and consequently the severity of myocardial infarction. Among the complex network of kinases and transcription factors that mediate signals during IR is the STAT family of 'signal transducers and activators of transcription'. During IR they are inducibly activated by Janus kinases and have the capability of transcriptionally regulating pro-survival and apoptotic gene expression. In this review we focus on recent progress made in elucidating the regulation and role of the STAT family of proteins during cardioprotective signalling events in myocardial preconditioning and during IR injury in cardiac myocyte cells and the myocardium. Moreover, we also highlight developments in understanding how this signalling pathway might be amenable to therapeutic intervention, permitting cardiac myocyte survival following IR.

Animals↗

PubMed2006

CREB-binding protein activation by presenilin 1 but not by its M146L mutant.

The transcriptional coactivator CREB-binding protein plays a key role in regulating gene expression in a number of different cell types. Recently, a report has suggested a link between CREB-binding protein and presenilins, which are mutated in many cases of early onset Alzheimer's disease. Thus, presenilin 1 and 2 double knockout mice showed reductions in CREB-binding protein levels and in cAMP response element-dependent gene expression in the cerebral cortex, which is likely to contribute to the subsequent neuronal degeneration. This suggests that the inactivation of CREB-binding protein caused by mutation in presenilin 1 may be involved in the disease process. We have shown that wild-type presenilin 1 stimulates the transcriptional activity ability of CREB-binding protein whereas presenilin 1 M146L mutant did not produce such an effect. The activation of CREB-binding protein by wild-type presenilin 1 involves the PI 3-kinase, p38 mitogen-activated protein kinase (MAPK) and p42/p44 MAPK pathways and targets primarily the C terminus of CREB-binding protein. To our knowledge, this is the first report that shows regulation of CREB-binding protein activity by wild-type presenilin 1 and not by its M146L mutant, and suggests a mechanism in which mutation of presenilin 1 may lead to neurodegeneration.

Animals↗

PubMed2006

Urocortin inhibits Beclin1-mediated autophagic cell death in cardiac myocytes exposed to ischaemia/reperfusion injury.

Autophagy is known to be a feature of cardiomyopathies and chronic ischaemia. Here we demonstrate that autophagy is also induced by a single cycle of ischaemia/reperfusion (I/R in neonatal and adult rat cardiac myocytes). Consistent with the critical role for Beclin1 in autophagocytosis, reduction of Beclin1 expression in cardiac myocytes by RNAi reduces I/R-induced autophagy and this is associated with enhanced cell survival. Autophagy is also reduced by urocortin, an endogenous cardiac peptide which we have previously shown to reduce other forms of myocyte cell death induced by I/R. The inhibition of autophagy by urocortin is mediated in part by inhibition of Beclin1 expression, an effect which is mediated by activation of the PI3 kinase/Akt pathway but which does not involve activation of p42/p44 MAPK.

Animals↗

PubMed2006

The Brn-3b POU family transcription factor represses plakoglobin gene expression in human breast cancer cells.

The Brn-3b transcription factor has been shown to be overexpressed in human breast cancer cells and contributes toward cell growth regulation. Using micro-arrays, more than 50 cancer-related genes regulated by Brn-3b in human breast cancer cells have been identified. For example, Brn-3b activates the cell cycle regulator CDK4 that provides a mechanism by which Brn-3b controls the growth of breast cancer cells. Here, we show that Brn-3b regulates plakoglobin (gamma-catenin), a member of the catenin family involved in cell-cell adhesion and signal transduction. Brn-3b expression inversely correlates with plakoglobin expression at both mRNA and protein levels in breast cancer cell lines and human breast biopsies. In contrast, no significant correlation was observed between Brn-3b expression and beta-catenin, or between Brn-3b expression and E-cadherin expression. Brn-3b represses the plakoglobin promoter via a Brn-3 consensus binding site contained within the region -965 to -593 relative to the transcriptional start site. Both repression of the promoter and binding of Brn-3b are lost when this site is mutated. To our knowledge, this is the first time that a Brn-3b POU family transcription factor has been shown to regulate a member of the catenin family, which provides insight into the molecular mechanisms by which Brn-3b expression may favour breast cancer progression and tumor invasion.

Binding Sites↗

PubMed2006

A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli.

BACKGROUND: The antiphospholipid syndrome (APS), characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI) of human beta 2 glycoprotein I (beta2GPI) is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL), which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL. METHODS: Using a computer programme called Juniper, sequentially overlapping primers were designed to be used in a recursive polymerase chain reaction (PCR) to produce a synthetic DI gene. Specifically Juniper incorporates 'major' codons preferred by bacteria altering 41 codons out of 61. This was cloned into the expression plasmid pET(26b) and expressed in BL21(DE3) Escherichia coli (E. coli). By virtue of a pelB leader sequence, periplasmic localisation of DI aided disulphide bond formation and toxicity was addressed by tightly regulating expression through the high stringency T7lac promoter. RESULTS: Purified, soluble his-tagged DI in yields of 750 microg/L bacterial culture was obtained and confirmed on Western blot. Expression using the native human cDNA sequence of DI in the same construct under identical conditions yielded significantly less DI compared to the recombinant optimised sequence. This constitutes the first description of prokaryotic expression of soluble DI of beta2GPI. Binding to murine monoclonal antibodies that recognise conformationally restricted epitopes on the surface of DI and pathogenic human monoclonal IgG aPL was confirmed by direct and indirect immunoassay. Recombinant DI also bound a series of 21 polyclonal IgG samples derived from patients with APS. CONCLUSION: By producing a synthetic gene globally optimised for expression in E. coli, tightly regulating expression and utilising periplasmic product translocation, efficient, soluble E. coli expression of the eukaryotic protein DI of beta2GPI is possible. This novel platform of expression utilising pan-gene prokaryote codon optimisation for DI production will aid future antigenic studies. Furthermore if DI or peptide derivatives of DI are eventually used in the therapeutic setting either as toleragen or as a competitive inhibitor of pathogenic aPL, then an E. coli production system may aid cost-effective production.

Amino Acid Sequence↗

PubMed2006

A heterozygous effect for PINK1 mutations in Parkinson's disease?

OBJECTIVE: To investigate the significance of PINK1 mutations in sporadic Parkinson's disease (PD). METHODS: We determined the frequency of PINK1 mutations by direct sequencing in a large series of PD patients with apparently sporadic disease (n = 768). RESULTS: Twelve heterozygous mutations were identified, nine in PD patients and three in control subjects. INTERPRETATION: Given the difficulty in interpreting the pathogenic significance of the heterozygous mutations that have already been reported in parkin and DJ-1, we first determined the frequency of heterozygous PINK1 mutations in the general population by sequencing a large number of control subjects (n = 768), then subsequently assessed their functional significance by examining their effects on stress-induced alterations to the mitochondrial membrane potential (DeltaPsim). We demonstrate an enrichment of heterozygous mutations in sporadic PD patients compared with matched control subjects (1.2% in PD vs 0.4% in control subjects). Furthermore, we show that they adversely affect DeltaPsim in a similar way to the familial G309D mutation. Although it remains difficult to conclusively demonstrate the pathogenicity of heterozygous mutations, the results of this study and the previously reported subclinical nigrostriatal dysfunction in carriers of heterozygous PINK1 mutations suggest the possibility that these heterozygous mutations are a significant risk factor in the development of later onset PD.

Adult↗

PubMed2006

Neuroprotective effects of virally delivered HSPs in experimental stroke.

Heat shock proteins (HSPs) are molecular chaperones with essential roles in modulating the proteolytic machinery and accelerating cell repair. Heat shock protein overexpression has been observed in vivo and in vitro under stresses including heat, nutrient deprivation and ischemia. Experiments in in vivo models of stroke indicate that transgenically overexpressed or virally delivered HSPs can enhance cell survival, but cannot always reduce lesion size. This study aims to assess the effects of virally delivered HSPs in a rat middle cerebral artery occlusion model of reversible focal cerebral ischemia using noninvasive magnetic resonance imaging. Attenuated herpes simplex virus carrying HSP27, HSP70, or a LacZ control was microinjected into the striatum 3 days before ischemia. Multislice T(2)-weighted images at 24 h after ischemia indicated that lesion volume was reduced by 44% in HSP27-treated animals compared with controls (P = 0.019). No significant differences were found between HSP70-treated and control animals (P = 0.88). Immunohistochemistry and Western blots revealed that HSP27 and HSP70 expression levels were equally high in injected hemispheres, but only the former had an effect on lesion size. This is the first evidence of the efficacy of gene therapy with any viral vector expressing HSP27 in an experimental model of stroke.

Animals↗

PubMed2006

Induction of parkin expression in the presence of oxidative stress.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders. Gene mutations have been found in rare familial forms of PD, with mutations in parkin being the most common cause. Oxidative stresses have also been implicated as an important contributing factor in the pathogenesis of PD. Currently, there is accumulating evidence that parkin may play a role in maintaining mitochondrial function and preventing oxidative stress. We demonstrated here that parkin is up-regulated when SH-SY5Y dopaminergic neuroblastoma cells are exposed to the oxidant dopamine. The up-regulation of parkin appeared to be due to transcriptional activation as luciferase assays confirmed that specific parkin promoter constructs could confer enhanced transcriptional activation in response to dopamine. Moreover, this effect was also seen when SH-SY5Y cells were subjected to another oxidative stress, 1-methyl-4-phenylpyridinium. In parallel with these studies, we also observed similar transcriptional activation of the parkin coregulated gene by oxidative stress. This is the first demonstration that parkin expression is up-regulated by oxidative stresses and may suggest that this might be a general neuroprotective response of parkin to oxidative stresses.

1-Methyl-4-phenylpyridinium↗

PubMed2006

Altered cleavage and localization of PINK1 to aggresomes in the presence of proteasomal stress.

Following our identification of PTEN-induced putative kinase 1 (PINK1) gene mutations in PARK6-linked Parkinson's disease (PD), we have recently reported that PINK1 protein localizes to Lewy bodies (LBs) in PD brains. We have used a cellular model system of LBs, namely induction of aggresomes, to determine how a mitochondrial protein, such as PINK1, can localize to aggregates. Using specific polyclonal antibodies, we firstly demonstrated that human PINK1 was cleaved and localized to mitochondria. We demonstrated that, on proteasome inhibition with MG-132, PINK1 and other mitochondrial proteins localized to aggresomes. Ultrastructural studies revealed that the mechanism was linked to the recruitment of intact mitochondria to the aggresome. Fractionation studies of lysates showed that PINK1 cleavage was enhanced by proteasomal stress in vitro and correlated with increased expression of the processed PINK1 protein in PD brain. These observations provide valuable insights into the mechanisms of LB formation in PD that should lead to a better understanding of PD pathogenesis.

Animals↗

PubMed2006

Targeting Brn-3b in breast cancer therapy.

The Brn-3b POU domain transcription factor is elevated in a significant proportion of breast cancers and in neuroblastoma tumours, where it is associated with increased proliferation, anchorage-independent growth, faster and larger tumour growth in xenograft models, resistance to growth inhibitory stimuli and increased migratory potential. These effects are associated with the ability of Brn-3b to regulate specific genes associated with these processes. Reducing Brn-3b can reverse many of these effects, suggesting that it may be possible to alter the growth and behaviour of tumour cells by abrogating Brn-3b in these cancers. This review discusses the effect of altering Brn-3b in these cancer cells and possible approaches to targeting Brn-3b as a strategy for therapy in treatment of breast cancers.

Amino Acid Sequence↗

PubMed2006

Hsf1-mediated stress response can transiently enhance cellular radioresistance.

To elucidate how the heat-shock transcription factor 1 (Hsf1)-mediated stress response affects cellular radioresistance, mouse embryo fibroblasts with Hsf1-gene knockout (Hsf1(-/-) cells) or with normal wild-type Hsf1 expression (Hsf1 wild-type cells) were preconditioned by heating (43 degrees C, 30 min) without or with quercetin (an inhibitor of Hsf1) and then exposed to gamma radiation (4 or 6 Gy). Some cell samples were infected with virus-based vectors to overexpress the constitutively active (mutant) form of Hsf1 or individual heat-shock proteins (Hsps). The heat preconditioning transiently up-regulated the Hsp levels in Hsf1 wild-type cells and significantly improved their postirradiation survival; these effects could be abolished by quercetin or simulated (without preheating) by the Hsf1 overexpression. In contrast, no enhanced radioresistance was found in heat-preconditioned Hsf1(-/-) cells that were unable to trigger Hsf1-mediated Hsp induction after heating. However, when the constitutively active Hsf1 was overexpressed in Hsf1(-/-) cells, the latter accumulated stress-inducible Hsps and became more radioresistant like heat-preconditioned Hsf1 wild-type cells. The overexpression of Hsp70 or/ and Hsp27 also enhanced radioresistance of both cell cultures. Thus the preirradiation stress response resulting in the intracellular Hsp accumulation can improve survival of severely irradiated mammalian cells.

Animals↗

PubMed2005

IL-6 induces PI 3-kinase and nitric oxide-dependent protection and preserves mitochondrial function in cardiomyocytes.

OBJECTIVE: Interleukin-6 (IL-6) is a pro-inflammatory cytokine which is a prognostic marker associated with left ventricular contractile dysfunction and heart failure. On the other hand, IL-6 activates signalling pathways which mediate delayed ischemic preconditioning. We have therefore studied the cellular mechanisms of IL-6-induced cardioprotection. METHODS: Inducible nitric oxide synthase (iNOS) expression, cardiomyocyte calcium handling, mitochondrial energetics, and the activation of protective signalling pathways in response to IL-6 were studied in a model of simulated ischemia/reperfusion (sI/R) in isolated neonatal rat ventricular cardiomyocytes. RESULTS: Reperfusion after sI/R induced a rise in cytosolic [Ca2+], a loss of cell morphology and integrity, and a transient increase in mitochondrial potential (Deltapsi m), followed by mitochondrial swelling and collapse of Deltapsi m. Pre-treatment of cardiomyocytes with 10 ng/ml IL-6 for 6 h, 24 h prior to sI/R prevented the secondary rise in cytosolic [Ca2+] and induced expression of iNOS and NO-dependent protection against sI/R injury. The protection against sI/R was concomitant with a NO-dependent reduction in the amplitude of cytosolic Ca2+ transients. IL-6 induced an increase in inner mitochondrial membrane polarisation and increased mitochondrial Ca2+ loading (rhod-2 fluorescence) at baseline, but prevented the reperfusion-induced changes in mitochondrial function. IL-6 pre-treatment also resulted in activation of the phosphatidylinositol (PI) 3-kinase/Akt pathway, and both iNOS induction and IL-6-dependent protection were blocked by the PI 3-kinase inhibitor wortmannin. CONCLUSION: IL-6 induces a PI 3-kinase and NO-dependent protection of cardiomyocytes, which is associated with alterations in mitochondrial Ca2+ handling, inhibition of reperfusion-induced mitochondrial depolarisation, swelling and loss of structural integrity, and suppression of cytosolic Ca2+ transients.

Androstadienes↗

PubMed2005

Phosphorylation of the Brn-3a transcription factor is modulated during differentiation and regulates its functional activity.

Brn-3a is a transcription factor expressed in a subset of neurons of the peripheral nervous system. Its role encompasses the activation of genes involved in neuronal differentiation and survival. While a lot of data have been produced on Brn-3a target promoters, very little is known about the upstream regulatory signals that mediate its activation in response to differentiation. In this work, we describe for the first time that Brn-3a is phosphorylated in IMR-32 neuroblastoma cells in response to differentiation induced by retinoic acid treatment and that its post-translational modification is potentially mediated by the activation of the MAPK/ERK pathway. Furthermore, we show that the mutation of a putative phosphorylated amino acid strongly reduces the ability of Brn-3a to mediate the differentiation of IMR-32 cells.

Amino Acid Sequence↗