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Biomedical subjects

D S Latchman

Publications and source records attributed to D S Latchman.

At least 109 records · Page 6Linked to original sources
PubMed1999

Herpes simplex virus latency-associated transcript encodes a protein which greatly enhances virus growth, can compensate for deficiencies in immediate-early gene expression, and is likely to function during reactivation from virus latency.

Herpes simplex virus types 1 and 2 (HSV1 and HSV2) enter and reactivate from latency in sensory neurons, although the events governing these processes are little understood. During latency, only the latency-associated transcripts (LATs) are produced. However, although the LAT RNAs were described approximately 10 years ago, their function remains ambiguous. Mutations affecting the LATs have minimal effects other than a small reduction in establishment of and reactivation from latency in some cases. Mutations in putative LAT-contained open reading frames (ORFs) have so far shown no effect. The LATs consist of a large species from which smaller (approximately 2 kb), nuclear, nonlinear LATs which are abundant during latency are spliced. Thus, translation of ORFs in these smaller LATs would not usually be expected to be possible, and if expressed at all, their expression might be tightly regulated. Here we show that deregulated expression of the largest HSV1 2-kb LAT-contained ORF in various cells of neuronal and nonneuronal origin greatly enhances virus growth in a manner specific to HSV1-the HSV1 LAT ORF has no effect on the growth of HSV2. Similar results of enhanced growth were found when the HSV1 LAT ORF was constitutively expressed from within the HSV1 genome. The mechanism of LAT ORF action was strongly suggested to be by substituting for deficiencies in immediate-early (IE) gene expression (particularly ICP0), because deregulated LAT ORF expression, as well as enhancing wild-type virus growth, was also found to allow efficient growth of viruses with mutations in ICP0 or VMW65. Such viruses otherwise exhibit considerable growth defects. IE gene expression deficiencies are often the block to productive infection in nonpermissive cells and are also evident during latency. These results, which we show to be protein- rather than RNA-mediated effects, strongly suggest a function of the tightly regulated expression of a LAT ORF-encoded protein in the reactivation from HSV latency.

Animals↗
PubMed1999

Equine herpesvirus 1 gene 12 can substitute for vmw65 in the growth of herpes simplex virus (HSV) type 1, allowing the generation of optimized cell lines for the propagation of HSV vectors with multiple immediate-early gene defects.

Herpes simplex virus (HSV) has often been suggested for development as a vector, particularly for the nervous system. Considerable evidence has shown that for use of HSV as a vector, immediate-early (IE) gene expression must be minimized or abolished, otherwise such vectors are likely to be highly cytotoxic. Mutations of vmw65 which abolish IE promoter transactivating activity may also be included to reduce IE gene expression generally. However, when vmw65 mutations are combined with an IE gene deletion, such viruses are hard to propagate, even on cells which otherwise complement the IE gene deletion effectively. We have found that vmw65 mutants can be effectively grown on cell lines expressing equine herpesvirus 1 gene 12, a non-HSV homologue of vmw65 with little sequence similarity to its HSV counterpart. This prevents repair by homologous recombination of vmw65 mutations in the virus, which would occur if mutations were complemented by vmw65 itself. The gene 12 protein is not packaged into HSV virions, which is important if viruses grown on such cells are to be used as vectors. These results not only further strengthen the evidence for direct functional homology between and similar modes of action of the two proteins but have allowed the generation of gene 12-containing cell lines in which ICP4 and ICP27 expression is induced by virus infection (probably by ICP0) and which give efficient growth of viruses deficient in ICP27, ICP4, and vmw65 (the viruses also have ICP34.5/ORFP deleted). Efficient growth of such viruses has not previously been possible. As these viruses are highly deficient in IE gene expression generally, such virus-cell line combinations may provide an alternative to HSV vectors with deletions of all four of the regulatory IE genes which, for optimal growth, require cell lines containing all four IE genes but which are hard to generate due to the intrinsic cytotoxicity of each of the proteins.

Animals↗
PubMed1999

Transcriptional regulation of the heat shock protein genes by STAT family transcription factors.

We have previously demonstrated that interleukin-6 (IL-6) increases the levels of the heat shock protein 90 (Hsp90) and activates the Hsp90beta promoter via the IL-6-activated transcription factors NF-IL6 and STAT-3. In addition, interferon-gamma (IFN-gamma) treatment increases the levels of Hsp70 and Hsp90 and also enhances the activity of the Hsp70 and Hsp90beta promoters with these effects being dependent on activation of the STAT-1 transcription factor by IFN-gamma. The effect of IL-6/STAT-3 and IFN-gamma/STAT-1 was mediated via a short region of the Hsp70/Hsp90 promoters, which also mediates the effects of NF-IL6. This region also contains a binding site for the stress-activated transcription factor HSF-1. Furthermore, STAT-1 and HSF-1 interact with one another via a protein-protein interaction and produce a strong activation of transcription. In contrast, STAT-3 and HSF-1 antagonize one another and reduce the activation of both the Hsp70 and Hsp90 promoters. Thus, STAT-1 or STAT-3 activation alone or together results in the activation of Hsp promoters. However, STAT-1 or STAT-3 interact differently with HSF-1 to regulate Hsp promoter activity. These results indicate that STATs are able to moduate the Hsp70 and Hsp90 gene promoters and that these transcription factors are likely to play a very important role in Hsp gene activation by nonstressful stimuli and the integration of these responses with the stress response of these genes.

Animals↗
PubMed1999

Gene therapy with herpes simplex virus vectors.

Gene delivery to the nervous system represents perhaps the ultimate challenge of gene therapy in view of the complexity of this system, the wide variety of intractable neurological diseases and the need to deliver the gene to non-dividing cells. Although a variety of systems for such gene delivery are under development, herpes simplex virus has unique advantages in terms of its large genome size and its ability to enter a latent state in neuronal cells. Considerable progress has been made in the effective disablement of this virus whilst retaining its ability to deliver genes and in producing long term expression of the foreign gene. Although much remains to be achieved in the further disablement of the virus and its testing in rodent and primate models of human diseases, it is likely that these viruses may ultimately be of use in human gene therapy procedures for otherwise intractable neurological diseases.

Animals↗
PubMed1998

Activation of the iNOS gene promoter by Brn-3 POU family transcription factors is dependent upon the octamer motif in the promoter.

The promoter of the gene encoding the inducible nitric oxide synthase (iNOS) contains an octamer motif which is of importance for its activation by specific stimuli. We show that in contrast to the promoter of the neuronal nitric oxide synthase gene (nNOS) which is strongly activated by the Oct-2 octamer-binding POU family transcription factor, the iNOS gene is only weakly activated by Oct-2 via its octamer motif. Unlike the nNOS promoter, however, the iNOS promoter is strongly activated by the POU family transcription factors Brn-3a and Brn-3b. This activation is dependent upon the octamer motif in the iNOS promoter and requires the activation domain located within the POU domain of Brn-3a or Brn-3b but not the N-terminal activation domain of Brn-3a. Thus different but related POU proteins play important roles in the regulation of the genes encoding different forms of nitric oxide synthase.

Animals↗
PubMed1998

Nerve growth factor up-regulates the transcriptional activity of CBP through activation of the p42/p44(MAPK) cascade.

Cyclic AMP response element-binding protein-binding protein (CBP) functions as a transcriptional coactivator through interactions with a number of cellular and viral transcription factors. It has been suggested to play a central integrative role in gene regulation. However, little is known about signal cascades that can regulate CBP activity. Here we show that either nerve growth factor (NGF) or cAMP treatment led to enhanced activity of CBP in PC12 cells. The C-terminal glutamine-rich activation domain of CBP was shown to be responsible for induction by NGF and cAMP. NGF-induced enhancement of CBP activity was also observed in protein kinase A (PKA)-deficient PC12 cells, whereas cAMP failed to increase the transcriptional activity of CBP in these cells. Moreover, the specific PKA inhibitor H-89 blocked cAMP-induced but not NGF-induced up-regulation of CBP activity. The up-regulation of CBP transcriptional activity in response to NGF was, however, prevented by the specific inhibitor of mitogen-activated protein kinase (p42/44(MAPK)) activation, PD98059, which had no effect on the up-regulation induced by cyclic AMP, indicating that activation of the mitogen-activated protein kinase signal pathway is specifically involved in the NGF-induced activation of CBP. In addition, expression of a dominant-negative interfering mutant of p42/44(MAPK) can prevent the NGF-mediated induction of the CBP activity, whereas expression of a p42/44(MAPK) constitutively active mutant can enhance the transcriptional activity of CBP. These data indicate that activation of the p42/p44(MAPK) cascade mediates the up-regulation of the transcriptional activity of CBP by NGF, whereas the similar up-regulation induced by cyclic AMP is mediated by PKA activation.

Activating Transcription Factor 2↗
PubMed1998

The N-terminal domain unique to the long form of the Brn-3a transcription factor is essential to protect neuronal cells from apoptosis and for the activation of Bbcl-2 gene expression.

The ability of the POU family transcription factor Brn-3a to stimulate neurite outgrowth and the expression of the genes encoding neuronal proteins such as the neurofilaments and SNAP-25 has previously been shown to be dependent upon the C-terminal POU domain which can mediate both DNA binding and transcriptional activation. We show here, however, that the ability of Brn-3a to activate Bcl-2 expression and protect neuronal cells from apoptosis (programmed cell death) requires a distinct N-terminal activation domain. Bcl-2 gene activation and protection from apoptosis are thus produced only by the long form of Brn-3a which contains this domain and not by a naturally occurring short form lacking this domain or by the isolated POU domain, although all these forms of Brn-3a can stimulate neurite outgrowth. Hence Brn-3a is a multi-functional transcription factor with different regions of the factor mediating its different effects and two distinct forms with different properties being generated by alternative splicing.

Animals↗
PubMed1998

The expression of constitutively active isotypes of protein kinase C to investigate preconditioning.

The role of protein kinase C (PKC) in ischemic preconditioning remains controversial because of difficulties with both its measurement and pharmacological manipulation. We investigated preconditioning in isolated neonatal rat cardiocytes by expressing constitutively active isotypes of PKC. Observations at differing durations of simulated ischemia suggested beta-galactosidase (beta-gal) activity reflected viability within transfected myocytes. Preconditioning with 90 min of ischemia significantly increased beta-gal activity and myocyte survival after 6 h of ischemia; an effect abolished by PKC inhibitors. After co-transfection with plasmids encoding beta-gal and either constitutively active mutants of PKC-delta, PKC-alpha, wild type PKC-delta, or empty vector, cardiocytes were subjected to 6 h of ischemia. Only PKC-delta, rendered constitutively active by a limited deletion within the pseudosubstrate domain, consistently increased resistance to simulated ischemia (beta-gal activity was 85.6 +/- 11.9% versus 53.7 +/- 6.5% (p </= 0.01) and dead myocytes 46.8 +/- 3.4% versus 68.7 +/- 2.8% (p </= 0.01)). Since transfection was apparent in only 5-12% of cells, the results suggested a protective bystander effect that was confirmed by co-culture of transfected myocytes with untransfected myocytes. In neonatal cardiocytes expression of active PKC-delta increases resistance to simulated ischemia. This observation may provide further insight into the mechanism and possible avenues for therapeutic exploitation of preconditioning.

Animals↗
PubMed1998

Functional role of position 22 in the homeodomain of Brn-3 transcription factors.

The Brn-3a POU family transcription factor activates a number of neuronal promoters which are repressed by the closely related Brn-3b factor. Although transcriptional activation by mutant forms of Brn-3a/Brn-3b can occur with a number of different amino acids at position 22 of the POU homeodomain, an isoleucine at this position is of critical importance for transcriptional repression. In addition, this isoleucine residue can mediate an interaction with the herpes simplex virus Vmw65 regulatory protein whereas the valine found in Brn-3a cannot do so. Thus, position 22 in the homeodomain plays a critical role in the function of neuronally expressed POU factors by controlling their interaction with viral and cellular co-activator/co-repressor proteins.

Animals↗
PubMed1998

Bcl-2 transcription from the proximal P2 promoter is activated in neuronal cells by the Brn-3a POU family transcription factor.

The BCL-2 protein is able to protect neuronal and other cell types from apoptotic programmed cell death and plays a key role in regulating the rate of apoptosis during development of the nervous system. We have previously demonstrated that the Brn-3a POU domain transcription factor protects sensory neurons from apoptotic programmed cell death induced by nerve growth factor withdrawal. We report here that Bcl-2 transcription is predominantly initiated from the Bcl-2 P2 promoter in both the ND7 neuronal cell line and primary dorsal root ganglion neurons, in contrast to the predominant use of the Bcl-2 P1 promoter in other cell types. Moreover, Bcl-2 transcription initiated from the P2 region increases in ND7 cells stably overexpressing Brn-3a, resulting in enhanced BCL-2 protein levels. Similarly, the Bcl-2 P2 promoter is directly activated by Brn-3a in co-transfection assays in both ND7 cells and dorsal root ganglion neurons. Analysis of the Bcl-2 regulatory sequence revealed a binding site for Brn-3a that is required for maximal activation by Brn-3a both in transfected cells and during differentiation of ND7 cells. Together these data identify Brn-3a as the first transcription factor regulating Bcl-2 activity specifically in neuronal cells and indicate that the anti-apoptotic effect of Brn-3a is likely to be mediated, at least in part, via the up-regulation of Bcl-2 expression.

Base Sequence↗
PubMed1998

Adjacent proline residues in the inhibitory domain of the Oct-2 transcription factor play distinct functional roles.

A 40 amino acid region of Oct-2 from amino acids 142 to 181 functions as an active repressor domain capable of inhibiting both basal activity and activation of promoters containing a TATA box, but not of those that contain an initiator element. Based on our observation that the equivalent region of the closely related Oct-1 factor does not act as an inhibitory domain, we have mutated specific residues in the Oct-2 domain in an attempt to probe their importance in repressor domain function. Although mutations of several residues have no or minimal effect, mutation of proline 175 to arginine abolishes the ability to inhibit both basal and activated transcription. In contrast, mutation of proline 174 to arginine confers upon the domain the ability to repress activation of an initiator-containing promoter by acidic activation domains, and also suppresses the effect of the proline 175 mutation. Hence, adjacent proline residues play key roles in the functioning of the inhibitory domain and in limiting its specificity to TATA-box-containing promoters.

Amino Acid Sequence↗
PubMed1998

Cell type specific repression of the varicella zoster virus immediate early gene 62 promoter by the cellular Oct-2 transcription factor.

The cellular transcription factor Oct-2.1 has previously been shown to repress the transactivation of the varicella zoster virus (VZV) immediate early gene promoter by viral transactivators but not to inhibit its basal activity. In the case of the related virus herpes simplex virus (HSV), the effect of Oct-2 on the IE promoters has been shown to be cell type specific and to differ between the different alternatively spliced forms of Oct-2. Here we show that as well as Oct-2.1, the Oct-2.4 and 2.5 isoforms which are expressed in neuronal cells can inhibit transactivation of the VZV immediate early promoter regardless of the cell type used. In contrast, all the isoforms of Oct-2 can inhibit basal activity of the VZV promoter in neuronal cells but not in other cell types indicating that this effect is cell type specific. These effects are discussed in terms of the differential regulation of latent infections with HSV or VZV in dorsal root ganglia.

Animals↗
PubMed1998

The HPV-activating cellular transcription factor Brn-3a is overexpressed in CIN3 cervical lesions.

The cervical cellular transcription factors Brn-3a and Brn-3b have antagonistic effects on transcription of the human papilloma virus types 16 and 18 E6 and E7 oncogenes, with Brn-3a activating expression and Brn-3b repressing it. We therefore measured expression of Brn-3a and Brn-3b mRNAs in biopsies from 16 women with no detectable cervical abnormality, and in 14 women with cervical intraepithelial neoplasia grade 3 (CIN3) lesions. Although the mean level of Brn-3b expression was similar in both groups, the mean level of Brn-3a expression was over 300-fold higher in the CIN3 samples when compared with normals. Elevated expression of Brn-3a was also detected in 16 histologically normal regions of the cervix adjacent to the CIN3 lesions, indicating that elevation of Brn-3a levels is not confined to the lesion in women with CIN3, and is thus not a consequence of the oncogenic process. The elevated levels of Brn-3a in the CIN3 patient samples, together with the activating effect of Brn-3a on HPV-16 and -18 oncogene expression, suggest that induction of this factor is involved in activating HPV-16 and -18 oncogene expression in the cervix, and hence in the production of cervical cancers induced by HPV.

Base Sequence↗
PubMed1998

The Brn-3c transcription factor contains a neuronal-specific activation domain.

Brn-3a, Brn-3b and Brn-3c are closely related POU family transcription factors which are expressed predominantly in neuronal cells. Previously Brn-3a has been shown to activate a number of gene promoters in co-transfections carried out in different cell types, while Brn-3b inhibits the basal activity of these promoters. We show here that Brn-3c activates the same promoters as Brn-3a but only in co-transfections into neuronal cells and not in other cell types. This effect is dependent upon an N-terminal region of Brn-3c which acts as a neuronal cell specific activation domain when linked to a heterologous DNA binding domain. To our knowledge this is the first report of an activation domain with apparently neuronal specific activity.

Animals↗
PubMed1998

The nuclear factor interleukin-6 (NF-IL6) and signal transducer and activator of transcription-3 (STAT-3) signalling pathways co-operate to mediate the activation of the hsp90beta gene by interleukin-6 but have opposite effects on its inducibility by heat shock.

The levels of the 90 kDa heat-shock protein (hsp90) and the activity of the hsp90beta gene promoter are increased in response to treatment by interleukin (IL)-6. The hsp90beta gene promoter contains binding sites for the transcription factors nuclear factor IL-6 (NF-IL6) and signal transducer and activator of transcription 3 (STAT-3), which are activated respectively by the mitogen-activated-protein-kinase and Jak-kinase pathways following IL-6 treatment. Both these factors can activate the hsp90 promoter and have a strong synergistic effect on its activity, which appears to be critical for IL-6-mediated activation of the promoter. Interestingly, the two factors interact differently with the heat-shock factor (HSF) and a heat-shock stress. Thus STAT-3 reduces the stimulatory effect of heat shock whereas NF-IL6 enhances it. When applied together, heat shock and IL-6 produce only weak activation of the hsp90 promoter compared with either stimulus alone, indicating that the inhibitory effect of STAT-3 on HSF predominates under these conditions. In contrast, IL-1, which activates only the NF-IL6 pathway, synergizes with heat shock to produce strong activation of hsp90. These effects are discussed in terms of the multiple stimuli that may regulate the hsp90 promoter in unstressed cells and their interaction with its stress-mediated activation.

CCAAT-Enhancer-Binding Proteins↗
PubMed1998

Genetic, structural and functional properties of an IgG DNA-binding monoclonal antibody from a lupus patient with nephritis.

Antibodies binding to double-stranded (ds) DNA are strongly associated with renal involvement in patients with systemic lupus erythematosus (SLE). We have generated two new IgG DNA-binding monoclonal antibodies (mAb), RH-14 and DIL-6, from the peripheral blood lymphocytes of two SLE patients with glomerulonephritis using the heteromyeloma cell line CB-F7. RH-14 is an IgG1 lambda antibody which also bound to single-stranded DNA, histones and nucleosomes. DIL-6 is an IgG3 lambda antibody with restricted antigen binding specificity. cDNA encoding the variable regions of the heavy (V(H)) and light (V(L)) chains of RH-14 was sequenced and the antigen binding site of this mAb was computer modelled. Sequence analysis of V(H) and V(L) regions of RH-14 showed that V(H) is derived from germ-line gene V3-7, a member of the V(H)3 family, and V(L) is derived from DPL 11, a member of the V(lambda)2 family. Somatic mutations and basic amino acid residues are identified in the complementarity-determining regions of both V(H) and V(L) regions. The nephritogenic properties of these mAb were analyzed by implanting and growing the hybridoma cells secreting the mAb in the peritoneum of SCID mice. The animals that received the RH-14 hybridoma produced higher levels of proteinuria (3 to > or = 4) (p < 0.001) compared to the groups that received DIL-6 (trace to > or = 1) or CB-F7 (trace). Electron microscopy of kidney sections from all the RH-14-implanted animals showed granular immunoglobulin deposition in the renal glomerular capillaries and mesangium. In this study we have shown for the first time using electron microscopy that a human IgG anti-dsDNA mAb, RH-14, is nephritogenic and that deposition of such an antibody alone is sufficient to induce renal damage.

Amino Acid Sequence↗
PubMed1998

Elevation of IL-6 in transgenic mice results in increased levels of the 90 kDa heat shock protein (hsp90) and the production of anti-hsp90 antibodies.

Treatment of human peripheral blood lymphocytes (PBL) in vitro with the cytokine interleukin-6 (IL-6) induces increased levels of the 90 kDa heat shock protein (hsp90). Hsp90 levels are also elevated in PBLs of human patients with systemic lupus erythematosus (SLE) and in MRL/lpr mice with autoimmune disease. Although IL-6 is elevated in both these situations it has not been shown that it is involved in stimulating elevation of hsp90 levels in vivo. Here we show directly that the elevation of IL-6 in vivo either in mice transgenic for the IL-6 gene or in knock-out mice lacking a functional gene for the transcription factor C/EBP beta (NF-IL-6) does indeed result in elevated hsp90 levels. This overexpression is associated with the specific production of autoantibodies to hsp90 in these mice which is also observed in SLE patients and MRL/1pr mice. Hence IL-6 is likely to play a critical role in the regulation of hsp90 levels both in autoimmune disease states and potentially in normal cells in vivo. In turn the elevated levels of hsp90 produced in autoimmune diseases are likely to be responsible for the observed production of anti-hsp90 autoantibodies.

Animals↗