PubMed Health⌕ Search

Biomedical subjects

D S Latchman

Publications and source records attributed to D S Latchman.

At least 181 records · Page 10Linked to original sources
PubMed1996

Nerve growth factor treatment of sensory neuron primary cultures causes elevated levels of the mRNA encoding the ATP synthase beta-subunit as detected by a novel PCR-based differential cloning method.

The mRNA encoding the rat ATP synthase beta-subunit was rapidly induced by nerve growth factor, within 60 min, in cultured adult rat dorsal root ganglion neurons. ATP synthase beta-subunit cDNA clones were isolated from a lambda library. The library was constructed using rat dorsal root ganglion mRNA that was differentially screened with cDNA-derived probes from untreated and nerve-growth-factor-treated primary cultures of adult rat dorsal root ganglion sensory neurons. Radiolabelled probes were made from submicrogram quantities of RNA, by a novel PCR-based technique, which allows small amounts of primary tissue to be used for library screening. The use of this technique in isolating novel differentially expressed mRNAs is discussed.

Animals↗
PubMed1996

Induction of the Oct-2 transcription factor in primary sensory neurons during inflammation is nerve growth factor-dependent.

The mRNA encoding the POU family transcription factor Oct-2 is induced in cultured adult sensory neurons following treatment with nerve growth factor (NGF) but not with a variety of other growth factors. We show here that the Oct-2 mRNA is also upregulated in vivo in sensory neurons innervating inflamed tissue following intraplantar injection of complete Freund's adjuvant. This rise is abolished by systemic administration of anti-NGF neutralizing antibodies indicating that it is an NGF-dependent effect. Hence a very specific aspect of the NGF response occurs in sensory neurons innervating inflamed tissue in vivo. In turn, the induction of Oct-2 may play a role in the other changes observed in such neurons both in gene expression and in their ability to respond to painful stimuli.

Animals↗
PubMed1996

Specific induction of the 70-kD heat stress proteins by the tyrosine kinase inhibitor herbimycin-A protects rat neonatal cardiomyocytes. A new pharmacological route to stress protein expression?

Heat shock protein (hsp) induction by stressful stimuli such as heat and ischemia is known to protect cardiac cells from severe stress. The ability to induce hsp's in the heart directly by "nonstressful" means would potentially have important clinical implications. In noncardiac cells, the tyrosine kinase inhibitor herbimycin-A has been shown to induce the 72-kD hsp. We therefore examined whether herbimycin-A and another tyrosine kinase inhibitor, genistein, could induce 70-kD hsp's in primary cultures of rat neonatal cardiomyocytes, and whether these treatments protect against severe stress. Primary cardiomyocytes were incubated with herbimycin-A or genistein. hsp induction was measured 16-20 h later by Western blotting. Cell survival after subsequent lethal heat stress or simulated ischemia was assessed using trypan blue exclusion and released lactate dehydrogenase activity. Our results indicate that, in cardiac cells, herbimycin-A induces 70-kD hsp's but not hsp90, -60, -25, or glucose-regulated protein 78, whereas genistein has no effect on hsp's. Moreover, hsp induction correlated with the ability of herbimycin-A to protect cells against severe stress, whereas genistein has no protective effects. This suggests that herbimycin-A may induce 70-kD hsp's via a tyrosine kinase-independent mechanism. These results indicate the possibility of a pharmacological approach to HSP70 induction and cardiac protection, which may ultimately be of clinical relevance.

Animals↗
PubMed1996

Differential protection of primary rat cardiocytes by transfection of specific heat stress proteins.

The aim of this study was to examine the individual contribution of specific heat stress proteins in primary rat cardiocytes to any protection observed following lethal heat stress or simulated lethal ischaemia. cDNAs for the inducible heat stress protein 70, for heat stress proteins 90 or 60, or a control plasmid were contransfected with a detectable marker, alkaline phosphatase, into cardiocytes. Survival assays were performed after the lethal stress. Transfection of the inducible heat stress protein 70 was found to increase survival following a lethal heat stress by 2.45-fold (P < 0.01) and against lethal ischaemia by 2.71-fold (P < 0.01). Transfection of heat stress protein 90 improved survival against heat by 2.55-fold (P < 0.05) but failed to have any effect on survival against lethal ischaemia. Transfection of heat stress protein 60 offered no protection against either stress.

Alkaline Phosphatase↗
PubMed1996

Alternative splicing of the Brn-3a and Brn-3b transcription factor RNAs is regulated in neuronal cells.

The closely related and functionally antagonistic POU family transcription factors Brn-3a and Brn-3b are encoded by two distinct genes that are expressed primarily in neuronal cells. In addition, however, the primary transcript of each of these genes is alternatively spliced to produce two distinct mRNAs encoding long and short isoforms that differ at the N-terminus of the protein. We show that this process is regulated so that different proportions of the mRNAs encoding the long and short forms of either Brn-3a or Brn-3b are produced in different rat tissues. Similarly, the ratio of each of these forms can be modulated by specific stimuli in both a neuronal cell line and primary neurons. The significance of these effects is discussed in relation to the functional differences between the two forms of each factor.

Alternative Splicing↗
PubMed1996

Trigeminal ganglion neurons are protected by the heat shock proteins hsp70 and hsp90 from thermal stress but not from programmed cell death following nerve growth factor withdrawal.

A prior mild thermal stress (heat shock) can protect neuronal cells against a subsequent exposure to either severe thermal stress or the induction of programmed cell death (apoptosis). By micro-injecting trigeminal ganglion neurons with expression constructs we show that over-expression of the individual heat shock proteins hsp70 and hsp90 can protect these cells against severe thermal stress but not against apoptosis. However, the protective effect of prior heat shock against subsequent apoptosis is dependent upon its ability to induce heat shock protein (hsp) synthesis rather than, for example, the inhibition of other protein synthesis associated with heat shock. The significance of these effects is discussed in terms of the role of different hsps in protecting neuronal cells from distinct stresses.

Animals↗
PubMed1996

Inhibitory transcription factors.

Although the majority of the transcription factors that were initially characterized had a stimulatory effect on gene expression, it is now clear that transcription factors that inhibit gene transcription are at least as important in regulating a wide variety of processes, including development. These factors can act either by interfering with the action of a positively acting factor (indirect repression) or by directly interfering with transcription by interacting with the basal transcriptional complex of RNA polymerase and associated factors (direct repression). Indirect repression often operates by the negative factor preventing the positively acting factor binding to DNA. This can involve reorganization of chromatin structure, blockage of the binding site in the DNA by binding of the inhibitory factor or formation of a non-DNA binding protein-protein complex. Indirect repression can also occur via quenching of the activity of a positive factor that remains bound to DNA. Direct repression can be produced by factors that interact with the basal transcriptional complex to reduce its activity or stability. This can be achieved either by factors that interact with the complex following binding to DNA or by those which bind directly to it. Factors that act by each of these means, and their mechanisms of action, are discussed.

Animals↗
PubMed1996

The Oct-2 transcription factor.

The Oct-2 transcription factor is a member of the POU (Pit-Oct-Unc) family of transcription factors and is expressed only in B lymphocytes and in neuronal cells but not in other cell types. The primary RNA transcript of the gene is subject to alternative splicing to yield different variants which can either activate or repress gene expression. The forms produced in B lymphocytes have a predominantly activating effect on gene expression whereas those produced in neuronal cells have a predominantly inhibitory effect and can repress the expression of both the herpes simplex virus immediate-early genes and the cellular tyrosine hydroxylase gene. Thus Oct-2 plays an important role in the regulation of cellular gene expression in both B cells and neuronal cells as well as in the control of viral latency.

Amino Acid Sequence↗
PubMed1996

Sequences of monoclonal antiphospholipid antibodies: variations on an anti-DNA antibody theme.

Antiphospholipid antibodies (aPL) are recognized increasingly as a probable cause of clinical features such as thrombosis and recurrent miscarriages, particularly in a subset of patients with systemic lupus erythematosus (SLE) and those with the antiphospholipid antibody syndrome (APS). A powerful method of studying the origin of these antibodies and delineating their binding sites is to sequence monoclonal aPL. The few reports of mouse aPL sequences suggest that gene families J558 and Vk23 may be used preferentially but without extensive mutation of complementarity determining regions (CDR). Polyreactive human aPL, which bind DNA as well as phospholipids, generally use germline genes with few mutations. Specific immunoglobulin (Ig) M aPL also tend to use relatively unmutated genes but often have high concentrations of positive residues in CDR, which may enhance binding to anionic phospholipids. IgG aPL show many more antigen-selected mutations, particularly in heavy chain CDR. This difference between isotypes is similar to that seen in anti-DNA antibodies, but the role of positively charged residues in aPL is less evident, and additional motifs are likely to be important in antigen binding.

Animals↗
PubMed1996

Focal cerebral ischaemia increases the levels of several classes of heat shock proteins and their corresponding mRNAs.

The induction of focal cerebral ischaemia in rats by middle cerebral artery occlusion has previously been shown to increase, over time, the mRNA levels of the heat shock proteins (HSPs) 27 and 70. However, the levels of HSP90 mRNA remain constant. In contrast, during global ischaemia, HSP70 and HSP90 mRNA levels are both raised, particularly in the CA1 neurons in the hippocampus, an area that is resistant to the insult in comparison to the surrounding regions. HSP27 mRNA is raised in the neuroglia in the subregions of the hippocampus. However, the protein levels of HSP27, 70 and 90 have not been characterised in focal ischaemia. With this data in mind, we have carried out a comparative study of HSP27, 56, 60, 70 and 90 mRNA and protein levels during focal cerebral ischaemia in rats, up to 24 h post-occlusion. We have shown that HSP70 and HSP27 mRNA levels are increased and also that HSP60 mRNA levels (which had also not previously been characterised in this model of focal ischaemia) are significantly raised. HSP90 and HSP56 mRNAs were not significantly elevated. On Western blot analysis, the inducible HSP72 protein was first detected at 8 h post-occlusion, HSP27 protein was detected only at 24 h post-occlusion and HSP60 protein, although constitutive, appeared to increase at 24 h post-occlusion. HSP56 protein levels appeared to rise on the occluded side, but HSP90 protein levels remained constant.

Animals↗
PubMed1996

Do heat shock proteins have a role in breast cancer?

It is clear therefore that hsps are overexpressed in patients with malignant tumours compared with healthy controls and this overexpression does show some correlation with disease features. Furthermore, expression of hsps has been reported on the cell surface of tumour cell lines. This could be associated with the immune response which has been reported with hsp90 and which also correlates with some disease features. It now appears that hsps may be involved in the presentation of tumour antigens leading to the possibility of hsps being used as a means of therapy. Hsp65 expression has not been investigated in patients with breast cancer. However, transfection of bacterial hsp65 into a tumour cell line resulted in the hsp65-expressing tumour cells losing their tumorigenicity in mice (Lukacs et al., 1993). Thus, hsps and the immune response to them are of interest as diagnostic and prognostic tools as well as a novel form of immunotherapy.

Animals↗
PubMed1996

Functional interaction between the HIV-1 Tat transactivator and the inhibitory domain of the Oct-2 cellular transcription factor.

OBJECTIVES: To examine the interaction of the HIV Tat protein and the inhibitory domain of the cellular transcription factor Oct-2. DESIGN: HIV reporter plasmids were co-transfected with expression vectors encoding wild-type and mutant Tat proteins and vectors encoding the inhibitory domain either alone or linked to a DNA-binding domain. METHODS: The activity of the different reporters was compared in the presence or absence of Tat or the inhibitory domain, or both. RESULTS: In the presence of the Tat protein, the Oct-2 inhibitory domain enhances the activation of Tat-responsive promoters by Tat. This effect is not observed when either the inhibitory domain or Tat cannot bind to the target gene, but is observed when one or both of the factors can bind. No activation is observed when the Oct-2 domain is co-transfected with Tat mutants that lack the Tat activation domain, indicating that the Oct-2 domain cannot substitute for the Tat activation domain. CONCLUSION: These data indicate that the Oct-2 inhibitory domain can functionally interact with Tat to form a complex with enhanced transactivating ability.

DNA-Binding Proteins↗
PubMed1996

Gene delivery to the heart in vivo and to cardiac myocytes and vascular smooth muscle cells in vitro using herpes virus vectors.

Herpes simplex virus 1 (HSV1), while usually thought of as neurotrophic, can also efficiently infect a wide variety of non-neuronal cell types and so might be developed as a vector for gene delivery to non-neuronal as well as neuronal cells. Here we have tested three different disabled HSV vectors for their ability to deliver a lacZ gene to primary cardiac myocytes and vascular smooth muscle cells in vitro, and used the most efficient virus to transfect the rat heart in vivo. We also assessed the degree of cytopathic effect of the various viruses on the cardiac myocytes in vitro by testing the effects on the frequency of beating in synchronously beating myocyte cultures. While an HSV mutant in which the essential immediate-early gene IE2 had been deleted gave high efficiency gene transfer to the cardiac myocytes in vitro and the rat heart in vivo, viruses in which ICP34.5 or ICP34.5 and VMW65 were inactive (and which were also unable to replicate in these cells) gave a much lower efficiency of gene transfer, mirroring the degree of cytopathic effect observed in the beating myocyte cultures. Gene transfer to the vascular smooth muscle cells was considerably less efficient than to the myocytes in all cases. These results indicate that while HSV may be inappropriate for highly efficient gene transfer to the arterial wall, efficient gene transfer can be achieved in the myocardium, and thus that HSV vectors may be suitable for the alteration of cardiac cell physiology in vivo.

Animals↗
PubMed1996

Gene delivery to the central and peripheral nervous systems of mice using HSV1 ICP34.5 deletion mutant vectors.

Herpes simplex virus 1 (HSV1) ICP34.5 deletion mutants are avirulent upon inoculation of both the central and peripheral nervous systems of mice, but they replicate to near wild-type titres in a number of non-neuronally derived cell lines in culture. Thus these mutants might be suitable for development as safe vectors for gene transfer to the nervous system. However, the mechanism of this avirulent phenotype in neuronal cells is at present poorly understood, although it has been suggested that nonpermissive cells infected with these mutants may undergo apoptosis, the function of ICP34.5 being to prevent this response and to allow continued virus replication. If this were the case ICP34.5 null mutants might be unsuitable for gene transfer as infected cells would quickly die, limiting the expression of a transgene. Here we have inserted a beta-galactosidase marker gene into a nonessential gene of an HSV1 strain 17+ mutant in which ICP34.5 has been deleted and also into a second mutant in which the virion transactivator protein VMW65 is also inactive. While all the mutants grew to high titre in tissue culture, mice inoculated by the foot-pad or intracranial route at high titre remained healthy until the end of the experiment. Moreover, beta-galactosidase was expressed either in the brain or in the dorsal root ganglia, depending on the site of inoculation. This suggests that in vivo the absence of ICP34.5 does not prevent the expression of a transgene in neuronal tissue and indicates that non-neurovirulent mutants lacking this gene may be suitable for further development as safe vectors for gene therapy in vivo.

Animals↗
PubMed1995

The degree of protection provided to neuronal cells by a pre-conditioning stress correlates with the amount of heat shock protein 70 it induces and not with the similarity of the subsequent stress.

A mild thermal stress protects primary cultures of dorsal root ganglion (DRG) neurons against a subsequent lethal heat stress as well as to a lesser extent against a subsequent lethal ischaemia. In contrast, a mild ischaemic stress protects DRG neurons only against a subsequent severe thermal stress and not against severe ischaemia. A greater induction of heat shock protein (hsp) synthesis was observed in these cells following mild temperature stress compared to mild ischaemia. This suggests that the protective effect observed is dependent on hsp synthesis resulting in the observed cross-protective effect and does not involve a particular pre-stress specifically protecting against a subsequent, more severe application of the same stress. Moreover, a particular level of hsp induction produces a better protective effect against lethal heat stress than against lethal ischaemia.

Animals↗