Biomedical subjects

D S Latchman

Publications and source records attributed to D S Latchman.

At least 19 recordsLinked to original sources

PubMed2002

A protein encoded by the herpes simplex virus (HSV) type 1 2-kilobase latency-associated transcript is phosphorylated, localized to the nucleus, and overcomes the repression of expression from exogenous promoters when inserted into the quiescent HSV genome.

Herpes simplex virus (HSV) is characterized by its ability to establish a latent infection in sensory neurons, from which it can periodically reactivate. The mechanisms of latency, however, remain unclear. The HSV genome is quiescent during latency except for the expression of the latency-associated transcripts (LATs). Although the exact function of the LATs remains obscure, current evidence suggests they are multifunctional and are involved in both establishment of latency and reactivation from latency. The LATs contain several open reading frames (ORFs). One or more of the functions of the LATs could therefore be protein mediated. We have previously reported that deregulated expression of the largest of the HSV type 1 (HSV-1) LAT ORFs ( approximately 274 amino acids) greatly enhances virus growth in cell types that are normally relatively nonpermissive for HSV replication and also that it complements mutations to the immediate-early (IE) gene ICP0 (S. K. Thomas, G. Gough, D. S. Latchman, and R. S. Coffin, J. Virol. 73:6618-6625, 1999). Here we show that LAT ORF expression overcomes the repression of expression from exogenous promoters introduced into the HSV-1 genome which normally occurs in the absence of IE gene expression. To further explore LAT ORF function, we have generated an epitope-tagged LAT ORF, LATmycHis, which forms punctate structures in the infected-cell nucleus reminiscent of the structures formed by ICP0. These are associated with the appearance of a phosphorylated form of the protein and are formed adjacent to, or around the edges of, viral replication compartments. These results provide further evidence that the HSV-1 LAT ORF protein is biologically functional and that the tightly regulated expression of this protein may be important in the wild-type latency phenotype in vivo.

Animals↗

PubMed2002

Urocortin increases the expression of heat shock protein 90 in rat cardiac myocytes in a MEK1/2-dependent manner.

We have previously demonstrated that urocortin protects cultured cardiac myocytes from ischaemic and reoxygenation injury and decreases the infarct size in the rat heart exposed to regional ischaemia and reperfusion. Urocortin-mediated cardioprotection is via activation of the mitogen-activated protein kinase (MAP kinase, MEK1/2) pathway. In addition, it is well documented that heat shock protein (hsp) 70 and hsp90 are cardioprotective against lethal stress. In this study we show, for the first time, that urocortin induces the expression of hsp90 but not hsp70 in primary cultures of rat neonatal cardiac myocytes. Levels of hsp90 protein increase by 1.5-fold over untreated cells within 10 min of urocortin treatment and are sustained for 24 h with a maximal increase of 2.5-fold at 60 min (P<0.05 at all time points). The increase in hsp90 expression by urocortin was not inhibited by actinomycin D, and urocortin failed to increase hsp90 promoter activity. Urocortin induction of hsp90 was inhibited by the MEK1/2 inhibitor PD98059 (P<0.001) and by cycloheximide, and both inhibitors abrogate urocortin-mediated cardioprotection (P<0.05 for cycloheximide, P<0.001 for PD98059). Hence, MEK1/2 and protein synthesis are involved in the cardioprotective effect of urocortin against hypoxic-mediated cell death, possibly due to an increase in expression of hsp90 protein. This is the first report of heat shock protein induction by urocortin or any other member of the corticotrophin-releasing hormone family.

Animals↗

PubMed2001

A minimal Bcl-x promoter is activated by Brn-3a and repressed by p53.

The Brn-3a transcription factor stimulates the expression of the anti-apoptotic Bcl-2 and Bcl-x proteins and protects neuronal cells from apoptosis. Here we show that a minimal Bcl-x promoter is activated by Brn-3a and that this stimulation is prevented by the pro-apoptotic p53 protein. Both these effects are mediated via Bcl-x promoter sequences, which are indistinguishable from those required for minimal basal promoter activity. A newly described upstream Bcl-x promoter is also activated by Brn-3a with this activation being prevented by p53. Hence, Brn-3a-mediated activation of two distinct Bcl-x promoters and of the Bcl-2 promoter is blocked by p53 whereas this is not observed for Brn-3a activated promoters derived from genes not involved in inhibiting apoptosis. p53 therefore appears to specifically target the activation by Brn-3a of promoters derived from genes with an anti-apoptotic effect and this may be involved in the pro-apoptotic activity of p53.

Animals↗

PubMed2001

CT-1 mediated cardioprotection against ischaemic re-oxygenation injury is mediated by PI3 kinase, Akt and MEK1/2 pathways.

Cardiotrophin-1 protects cardiac myocytes from ischaemic re-oxygenation (IR) injury. CT-1 activates MEK1/2,p42/44MAPK as well as the phosphatidylinositol (PI) 3-OH kinase (PI3) protein kinase B (PKB/Akt) pathway. In this study we investigate the signalling pathways that mediate the anti-apoptotic cell survival effect of CT-1 in IR. Dominant negative gene based inhibitors of MEK1/2, PI3-kinase and Akt inhibited CT-1 mediated cardioprotection in re-oxygenation as did chemical inhibitors of the PI3-kinase pathway. Hence the PI3-kinase/Akt pathway is required in addition to MEK1/2 to mediate CT-1 cardioprotection in IR.

Animals↗

PubMed2001

The POU domain transcription factor Brn-3a protects cortical neurons from apoptosis.

We have demonstrated previously that exogenously expressed Brn-3a is capable of protecting neurons of the peripheral nervous system against apoptosis. In these previous studies Brn-3a showed a degree of neuronal sub-type specificity, in that while it could promote survival in NGF-dependent sensory neurons, no effect was observed in NGF-dependent neurons of the sympathetic nervous system. In this report, we show that Brn-3a delivered using a herpes simplex virus is capable of protecting cultures of rat cerebrocortical neurons of the central nervous system against two types of cell death stimuli, including glutamate neurotoxicity. Hence the protective effect of Brn-3a is not confined to neurons of the peripheral nervous system but can also occur in neurons of the CNS.

Animals↗

PubMed2001

Heat shock protein 27 shows a distinctive widespread spatial and temporal pattern of induction in CNS glial and neuronal cells compared to heat shock protein 70 and caspase 3 following kainate administration.

Kainate-induced status epilepticus is associated with both apoptotic and necrotic cell death and induction of heat shock proteins (HSPs) in hippocampal and cortical regions of the rodent brain. In the present study we have examined the temporal, spatial and cellular expression patterns of mRNAs for the highly inducible HSPs, HSP70 and HSP27, together with the apoptotic marker, caspase 3 (CPP32) in rat brain after systemic administration of kainate. HSP70 mRNA was transiently induced in the forebrain by kainate, principally in the CA1, CA3 and hilar cells of the hippocampal formation, in piriform cortex and discrete thalamic nuclei. Maximal expression was seen at 8 h after kainate which then declined to background levels by 7 days. Labelling was predominantly neuronal. In contrast, HSP27 mRNA expression was more widespread. Intense labelling was observed in CA1, CA3 and the hilar region at 8 h after kainate but the expression profile for HSP27 mRNA expanded considerably with intense signals seen in corpus callosum, cortex and thalamus at 24 h post kainate. Emulsion autoradiographs indicated a predominantly glial localisation for HSP27 mRNA. In the hilus, a distinct subpopulation of interneurones were found to express HSP27 mRNA. CPP32 mRNA was upregulated in CA1, CA3 and hilus of the hippocampal formation and in piriform cortex. CPP32 mRNA expression was more restricted and similar in distribution to HSP70 mRNA being localised to neurones. The present study demonstrates the unique early expression of HSP27 mRNA by glial cells and distinct populations of neurones which extends beyond those in which HSP70 and CPP32 induction occurs with subsequent cell loss.

Animals↗

PubMed2001

The Brn-3b POU family transcription factor regulates the cellular growth, proliferation, and anchorage dependence of MCF7 human breast cancer cells.

The Brn-3b POU domain containing transcription factor is expressed in the developing sensory nervous system as well as in epithelial cells of the breast, cervix, and testes. Brn-3b functionally interacts with the estrogen receptor (ER) and in association with the ER, regulates transcription from estrogen responsive genes. In addition, Brn-3b expression is elevated in breast tumours compared to levels in normal mammary cells. To explore the role of Brn-3b in breast cancer, we established stable cell lines derived from the MCF7 human breast cancer cell line which had been transfected with Brn-3b sense or anti-sense constructs. The Brn-3b over-expressing cell lines exhibited increased growth rate, reached confluence at a higher saturation density, had higher proliferative activity, and an enhanced ability to form colonies in soft agar when compared to the control empty vector transfected cells. Likewise, the Brn-3b anti-sense cell lines showed reduced cellular growth and proliferation, reached confluence at a lower density, and exhibited a decreased ability to form colonies in soft agar when compared to the vector controls. Five to ten per cent of the Brn-3b over-expressing cells exhibited a severely altered morphology characterized by reduced adherence to tissue culture plastic, increased cell size, and a vacuolar cell shape. These results thus further indicate a role for the Brn-3b transcription factor in regulating mammary cell growth and suggest that its elevation in breast cancer is of functional significance.

Adenocarcinoma↗

PubMed2001

The cyclic AMP response element in the Bcl-2 promoter confers inducibility by hypoxia in neuronal cells.

In neuronal cells, expression of the anti-apoptotic Bcl-2 gene is induced by hypoxia and produces a protective effect. We show here that this effect is dependent upon the cyclic AMP response element (CRE) in the Bcl-2 promoter since mutation of this element abolishes the response and the isolated CRE can confer the response on a heterologous promoter. Interestingly however, the CRE in the Bcl-2 promoter does not render the promoter responsive to cyclic AMP and is not essential for its response to nerve growth factor. Despite the lack of cyclic AMP responsiveness, activation of the Bcl-2 promoter via the CRE in response to hypoxia requires the CREB transcription factor and is associated with the enhanced phosphorylation of CREB on serine 133 and enhanced transcriptional activation by the CREB-binding protein, CBP, in response to hypoxia. This finding establishes the importance of the CRE in the induction of Bcl-2 gene expression by hypoxia, allowing the Bcl-2 protein to protect neuronal cells against this damaging stimulus.

Animals↗

PubMed2001

The Brn-3a transcription factor plays a key role in regulating the growth of cervical cancer cells in vivo.

The cellular Brn-3a transcription factor is known to activate transcription of the genes encoding the human papilloma virus E6 and E7 proteins and is over-expressed in women with cervical neoplasia. We show that cervical cell lines with reduced Brn-3a expression show a greatly reduced ability to form tumours in nude mice compared to control cells and also show reduced expression of the HPV E6 and cellular Bcl-2 oncogenes. These effects are also observed in cervical cells over-expressing the related Brn-3b factor, which is known to antagonize activation of HPV gene expression by Brn-3a. These results demonstrate, for the first time, that inhibition of Brn-3a expression or enhanced Brn-3b expression can inhibit cervical cell-derived tumour growth in vivo as well as in vitro. Hence they establish Brn-3a as a key factor in cervical tumorigenesis and as a potential therapeutic target in human cervical neoplasia.

Cell Division↗

PubMed2001

Cardiotrophin-1 can protect cardiac myocytes from injury when added both prior to simulated ischaemia and at reoxygenation.

OBJECTIVE: The cytokine cardiotrophin-1 (CT-1) has previously been shown to protect cultured cardiocytes from cell death induced by serum removal or hypoxia when administered prior to the damaging stimulus. We wished to test whether a similar protective effect could be observed if CT-1 was added after the ischaemic period and to investigate the signalling pathways involved in the protective effect when CT-1 is given prior to or after ischaemia. METHODS: We therefore examined the protective effect of CT-1 in cultured rat cardiocytes exposed to simulated ischaemia followed by reoxygenation when CT-1 was administered either prior to simulated ischaemia or at reoxygenation. RESULTS: We show that CT-1 can exert a protective effect against the damaging effects of simulated ischaemia/reoxygenation both when added after the simulated ischaemia at reoxygenation (P<0.05 in trypan blue, TUNEL and annexin V assays) or when added prior to the simulated ischaemia (P<0.05). In both cases, these protective effects are blocked by an inhibitor of the p42/p44 MAPK pathway (P<0.05 in all assays). CONCLUSION: CT-1 can protect cardiac cells when added either prior to simulated ischaemia or at the time of reoxygenation following simulated ischaemia and these effects are dependent upon its ability to activate the p42/p44 MAPK pathway. Hence CT-1 may have therapeutic potential when added at the time of reperfusion following ischaemic damage.

Animals↗

PubMed2001

Expression of the Fabs of human auto-antibodies in Escherichia coli: optimization and determination of their fine binding characteristics and cross-reactivity.

The Fabs of three human auto-antibodies (B3/33H11, anti-DNA; UK4, anti-phospholipid) and six related hybrids have been cloned and expressed in Escherichia coli, and their relative binding to single-stranded or double-stranded DNA or to cardiolipin has been assessed in the presence of modulators (salts and serum). We describe optimized conditions that have led to significant improvement in the quality and quantity of the purified auto-antibodies. Protein expression of the assembled and functionally active Fabs was achievable with a yield of up to 5 to 9 mg/l of culture. The comparative DNA/cardiolipin-binding analyses of the nine Fabs in the presence of modulators demonstrated that B3 and 33H11 L chains possess both anti-DNA and anti-cardiolipin activities. This is the first report of the demonstration that both anti-DNA and anti-cardiolipin activities may lie on the same light chain of a human auto-antibody. We provide evidence that the auto-antibodies that appeared to be similar, in that they bound DNA or cardiolipin in conventional ELISA immunoassays, exhibited significant difference in their cross-reactivity and binding to the antigen in the presence of modulators. Such auto- antigen specificity and/or cross-reactivity may dictate the potential of an auto-antibody to cause pathogenicity and may provide an explanation as to why apparently similar auto-antibodies behave differently in vivo.

Animals↗

PubMed2001

Induction of apoptosis and Fas receptor/Fas ligand expression by ischemia/reperfusion in cardiac myocytes requires serine 727 of the STAT-1 transcription factor but not tyrosine 701.

Previously we reported that ischemia results in apoptosis and is accompanied by phosphorylation on Tyr-701 and increased expression and transcriptional activity of the signal transducer and activator of transcription-1 (STAT-1). In the present study, we show that exposure of cardiomyocytes to ischemia induced the phosphorylation of STAT-1 at another site, Ser-727. Moreover, STAT-1 is critical for the induction of Fas receptor and Fas ligand expression by ischemia/reperfusion (I/R). Transcriptional activation of Fas and FasL was dependent on Ser-727 of STAT-1 but was independent of Tyr-701. Similarly, Ser-727 but not Tyr-701 was required for enhancement of cardiomyocyte cell death by STAT-1 during I/R. In addition, inhibition of the p38 pathway prevented the induction and transcriptional activation of Fas and FasL in cardiac cells exposed to I/R, whereas inhibition of p42/p44 MAPK had no effect. Finally, I/R also induced phosphorylation of STAT-1 on Ser-727 and expression of Fas/FasL in ventricular myocytes in the intact heart ex vivo. These results indicate that Fas/FasL genes and apoptosis are activated by STAT-1 in cardiac myocytes exposed to I/R and these effects are dependent on the Ser-727 but not the Tyr-701 phosphorylation sites of STAT-1.

Animals↗

PubMed2001

The importance of somatic mutations in the V(lambda) gene 2a2 in human monoclonal anti-DNA antibodies.

2a2 is the most commonly rearranged gene in the human V(lambda )locus. It has been postulated that certain immunoglobulin genes (including 2a2) are rearranged preferentially because their germline sequences encode structures capable of binding to a range of antigens. Somatic mutation could then increase the specificity and affinity of binding to a particular antigen. We studied the properties of five IgG molecules in which the same heavy chain was paired with different light chains derived from 2a2. The pattern of somatic mutations in 2a2 was shown to be crucial in conferring the ability to bind DNA, but two different patterns of mutation each conferred this ability.Computer-generated models of the three-dimensional structures of these antibodies illustrate the ability of 2a2 to form a DNA binding site in different ways. Somatic mutations at the periphery of the DNA binding site were particularly important. In two different light chains, mutations to arginine at different sites in the complementarity determining regions (CDRs) enhanced binding to DNA. In a third light chain, however, mutation to arginine at a different site blocked binding to DNA.

Amino Acid Sequence↗

PubMed2001

Gene delivery and gene therapy with herpes simplex virus-based vectors.

The development of efficient means of delivery genes in vivo is essential both for testing gene function in the intact animal and for human gene therapy procedures. A number of viral and non-viral gene delivery methods have been developed for this purpose. Of those herpes simplex virus (HSV)-based vectors have particular advantages for gene delivery to the nervous system including their ability to infect non-dividing neurones and establish asymptomatic latent infections. Moreover, considerable progress has been made, firstly, in disabling HSV vectors so as to prevent the damaging effects of wild type virus and secondly, to ensure long-term expression of the inserted transgene(s). These vectors thus offer a valuable tool for testing gene function in neuronal cells in vivo and may ultimately be safe enough for use in human gene therapy procedures.

Animals↗

PubMed2001

Elevated levels of the 90 kDa heat shock protein (hsp90) in SLE correlate with levels of IL-6 and autoantibodies to hsp90.

Levels of the 90 kDa heat shock protein (hsp90) are elevated in a subset of patients with systemic lupus erythematosus (SLE) due to enhanced transcription of the hsp90beta gene. In cultured cells, transcription of the hsp90beta gene is induced following exposure to IL-6 or IL-10 which are known to be elevated in SLE patients. Here we have measured the levels of hsp90 protein and of IL-6, IL-10 in SLE patients and normal controls. We demonstrate that the levels of hsp90 protein in individual patients correlate with the IL-6 level but not with the level of IL-10. Moreover, hsp90 protein levels in patients correlate with the presence of IgG autoantibodies to hsp90. These results support a model in which elevated levels of IL-6 in SLE patients induce elevated levels of hsp90 protein which in turn results in the production of autoantibodies to this protein.

Adolescent↗

PubMed2001

Heat shock protein-56 is induced by cardiotrophin-1 and mediates its hypertrophic effect.

Cardiotrophin-1 (CT-1) is an interleukin-6 family cytokine with known protective and hypertrophic effects in the heart. Previous studies have shown that CT-1 treatment increases heat shock protein 70 (hsp70) and heat shock protein 90 (hsp90) levels in cardiac cells. Due to the known protective effects of hsp90 and hsp70, induction of these proteins may be involved in the protective effects of CT-1. We show here that heat shock protein 56 (hsp56), also known as FK506 binding protein 59 (FKBP59), is induced by CT-1 treatment at both the mRNA and protein levels. It has been demonstrated previously that, unlike hsp70 and hsp90, hsp56 overexpression does not protect cardiac myocytes against stressful stimuli. The other known effect of CT-1 is hypertrophy, an increase in cell size without cell division, which occurs in many cardiac pathologies. We investigated the role of hsp56 in the hypertrophic response of primary neonatal rat cardiac myocytes, using overexpression with transiently transfected plasmid vectors and Herpes viral vectors. Overexpression of hsp56 caused a significant increase in cardiac cell size and protein:DNA ratio. Hsp27, hsp70 and hsp90 overexpression had no effect on cell size. An antisense construct to hsp56 reduced hsp56 levels when transiently transfected and blocked the hypertrophic effect of CT-1. This is the first time that a hypertrophic effect has been demonstrated for a heat shock protein and demonstrates that CT-1-induced hypertrophy involves a specific hsp, which is not involved in its protective effect.

Animals↗

PubMed2001

Brn-3a activates the expression of Bcl-x(L) and promotes neuronal survival in vivo as well as in vitro.

The determination of cell fate plays a critical role during the later stages of embryogenesis and the early postnatal period-a time during which approximately half of neurons born during neurogenesis undergo programmed cell death. It has previously been reported that the type IV POU domain transcription factor Brn-3a plays a role in the maturation and survival of sensory neuronal populations. Indeed we have shown that the long form of Brn-3a is capable of activating expression of the antiapoptotic Bcl-2 gene and enhancing neuronal survival in cultures of sensory neurons. In this study, we report the identification of another antiapoptotic family member, Bcl-x(L), as a target gene of Brn-3a in sensory neurons, providing a further mechanism by which Brn-3a determines sensory neuronal fate during development. Bcl-x(L) gene expression is activated by Brn-3a in sensory but not in sympathetic neurons and its expression is reduced by antisense inhibition of Brn-3a expression in sensory neurons. Most importantly, both Bcl-x(L) expression and neuronal survival are enhanced by the overexpression of Brn-3a in dorsal root ganglion in vivo in a model of sciatic nerve injury in the intact animal.

Animals↗

PubMed2001

Heat shock proteins and cardiac protection.

The heat shock proteins (hsps) are expressed in normal cells but their expression is enhanced by a number of different stresses including heat and ischaemia. They play important roles in chaperoning the folding of other proteins and in protein degradation. In the heart a number of studies have shown that prior induction of the hsps by a mild stress has a protective effect against a more severe stress. Moreover, over-expression of an individual hsp in cardiac cells in culture or in the intact heart of either transgenic animals or using virus vectors, also produces a protective effect, directly demonstrating the ability of the hsps to produce protection. These findings indicate the potential importance of developing procedures for elevating hsp expression in a safe and efficient manner in human individuals using either pharmacological or gene therapy procedures.

Animals↗