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Biomedical subjects

B Burke

Publications and source records attributed to B Burke.

At least 19 recordsLinked to original sources
PubMed2006

Current research priorities in chronic fatigue syndrome/myalgic encephalomyelitis: disease mechanisms, a diagnostic test and specific treatments.

Chronic fatigue syndrome (CFS) is an illness characterised by disabling fatigue of at least 6 months duration, which is accompanied by various rheumatological, infectious and neuropsychiatric symptoms. A collaborative study group has been formed to deal with the current areas for development in CFS research--namely, to develop an understanding of the molecular pathogenesis of CFS, to develop a diagnostic test and to develop specific and curative treatments. Various groups have studied the gene expression in peripheral blood of patients with CFS, and from those studies that have been confirmed using polymerase chain reaction (PCR), clearly, the most predominant functional theme is that of immunity and defence. However, we do not yet know the precise gene signature and metabolic pathways involved. Currently, this is being dealt with using a microarray representing 47,000 human genes and variants, massive parallel signature sequencing and real-time PCR. It will be important to ensure that once a gene signature has been identified, it is specific to CFS and does not occur in other diseases and infections. A diagnostic test is being developed using surface-enhanced, laser-desorption and ionisation-time-of-flight mass spectrometry based on a pilot study in which putative biomarkers were identified. Finally, clinical trials are being planned; novel treatments that we believe are important to trial in patients with CFS are interferon-beta and one of the anti-tumour necrosis factor-alpha drugs.

Fatigue Syndrome, Chronic↗
PubMed2004

Transgenic mice showing inflammation-inducible overexpression of granulocyte macrophage colony-stimulating factor.

We used the promoter of the human C-reactive protein (CRP) gene to drive inflammation-inducible overexpression of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) in transgenic mice. Transgenic mice carrying a CRP/GM-CSF fusion gene show a >150-fold increases in circulating levels of GM-CSF within 6 h of intraperitoneal inoculation with 25 microg of lipopolysaccharide. However, some of the transgenic mice also display relatively high basal levels of GM-CSF in the absence of any obvious inflammatory stimulus. Raised basal levels of GM-CSF are associated with a number of pathological changes, including enlarged and histologically abnormal livers and spleens and with increases in the number and activation state of macrophages and granulocytes in the peripheral blood. Despite problems associated with the expression of such a potent pleiotropic cytokine as GM-CSF, the principle of inflammation-inducible expression of chimeric constructs has been shown to be feasible. Inducible expression systems such as that described here could be of potential use in the study of the role of cytokines in health and disease and in the development of disease-resistant strains of livestock.

Amino Acid Sequence↗
PubMed2003

Cytokine gene polymorphisms associated with symptomatic parvovirus B19 infection.

BACKGROUND: The immune system has been implicated in the pathogenesis of certain clinical manifestations of parvovirus B19 infection, including rash and arthralgia. Cytokines feature in the pathogenesis of parvovirus B19 infection, so inherited variability in cytokine responses to B19 infection might have a bearing on the symptomatology of parvovirus B19 infection. AIMS: To investigate the possible role of cytokine gene polymorphisms in the clinical manifestations of parvovirus B19 infection. METHODS: Thirty six patients with a variety of symptoms at acute infection and follow up (mean, 22.0 months) and controls (99-330, depending on the locus) were examined for the following cytokine polymorphisms: tumour necrosis factor alpha (TNF alpha), -308; interferon gamma (IFN-gamma), +874; interleukin 6 (IL-6), -174; IL-10, -592, -819, and -1082; and transforming growth factor beta1 (TGF beta 1), +869 (codon 10) and +915 (codon 25). RESULTS: The TNF alpha -308*A allele occurred in 13.9% of the parvovirus group compared with 27.0% of the control group (odds ratio (OR), 0.44; p = 0.02). The TGF beta 1 CG/CG haplotype was more frequent in the parvovirus group than in the controls (16.7% v 5%; OR, 4.8; p = 0.02). Within the B19 infected group, the TGF beta 1 +869 T allele was associated with skin rash at acute infection (p = 0.005), whereas at follow up the IFN-gamma +874 T allele was associated with the development of anti-B19 non-structural protein 1 antibodies (p = 0.04). CONCLUSIONS: The results of the present study suggest that inherited variability in cytokine responses may affect the likelihood of developing symptoms during parvovirus infection.

Adolescent↗
PubMed2002

Regulation of a rat VL30 element in human breast cancer cells in hypoxia and anoxia: role of HIF-1.

Novel approaches to cancer gene therapy currently exploit tumour hypoxia to achieve transcriptional targeting using oxygen-regulated enhancer elements called hypoxia response elements. The activity of such elements in hypoxic cells is directly dependent on upregulation of the hypoxia-inducible transcription factor-1 However tumours also contain areas of anoxia, which may be considered a more tumour-selective transcriptional stimulus than hypoxia for targeting gene therapy to tumours. Another element, from the rat virus-like retrotransposon, VL30 (termed the "secondary anoxia response element") has been reported to be more highly inducible in rat fibroblasts under anoxia than hypoxia. To investigate anoxia as a potential transcriptional target in human tumours, we have examined secondary anoxia response element inducibility in two human breast cancer cell lines, MCF-7 and T47D, under anoxia, hypoxia and normoxia. In both cell types, the trimerised secondary anoxia response element showed greater inducibility in anoxia than hypoxia (1% and 0.5% O(2)). The anoxic response of the secondary anoxia response element was shown to be dependent on hypoxia-inducible transcription factor-1 and the presence of a hypoxia-inducible transcription binding site consensus (5'-ACGTG-3'). Mutational analysis demonstrated that the base immediately 5' to this modulates the anoxic/hypoxic induction of the secondary anoxia response element, such that TACGTG>GACGTG>>CACGTG. A similar correlation was found for erythropoietin, phosphoglycerate kinase 1, and aldolase hypoxia response elements, which contain these respective 5' flanking bases.

Animals↗
PubMed2002

Macrophages in gene therapy: cellular delivery vehicles and in vivo targets.

The appearance and activation of macrophages are thought to be rapid events in the development of many pathological lesions, including malignant tumors, atherosclerotic plaques, and arthritic joints. This has prompted recent attempts to use macrophages as novel cellular vehicles for gene therapy, in which macrophages are genetically modified ex vivo and then reintroduced into the body with the hope that a proportion will then home to the diseased site. Here, we critically review the efficacy of various gene transfer methods (viral, bacterial, protozoan, and various chemical and physical methods) in transfecting macrophages in vitro, and the results obtained when transfected macrophages are used as gene delivery vehicles. Finally, we discuss the use of various viral and nonviral methods to transfer genes to macrophages in vivo. As will be seen, definitive evidence for the use of macrophages as gene transfer vehicles has yet to be provided and awaits detailed trafficking studies in vivo. Moreover, although methods for transfecting macrophages have improved considerably in efficiency in recent years, targeting of gene transfer specifically to macrophages in vivo remains a problem. However, possible solutions to this include placing transgenes under the control of macrophage-specific promoters to limit expression to macrophages or stably transfecting CD34(+) precursors of monocytes/macrophages and then differentiating these cells into monocytes/macrophages ex vivo. The latter approach could conceivably lead to the bone marrow precursor cells of patients with inherited genetic disorders being permanently fortified or even replaced with genetically modified cells.

Adenoviridae↗
PubMed2001

Divergent adaptation of tRNA recognition by Methanococcus jannaschii prolyl-tRNA synthetase.

Analysis of prolyl-tRNA synthetase (ProRS) across all three taxonomic domains (Eubacteria, Eucarya, and Archaea) reveals that the sequences are divided into two distinct groups. Recent studies show that Escherichia coli ProRS, a member of the "prokaryotic-like" group, recognizes specific tRNA bases at both the acceptor and anticodon ends, whereas human ProRS, a member of the "eukaryotic-like" group, recognizes nucleotide bases primarily in the anticodon. The archaeal Methanococcus jannaschii ProRS is a member of the eukaryotic-like group, although its tRNA(Pro) possesses prokaryotic features in the acceptor stem. We show here that, in some respects, recognition of tRNA(Pro) by M. jannaschii ProRS parallels that of human, with a strong emphasis on the anticodon and only weak recognition of the acceptor stem. However, our data also indicate differences in the details of the anticodon recognition between these two eukaryotic-like synthetases. Although the human enzyme places a stronger emphasis on G35, the M. jannaschii enzyme places a stronger emphasis on G36, a feature that is shared by E. coli ProRS. These results, interpreted in the context of an extensive sequence alignment, provide evidence of divergent adaptation by M. jannaschii ProRS; recognition of the tRNA acceptor end is eukaryotic-like, whereas the details of the anticodon recognition are prokaryotic-like. This divergence may be a reflection of the unusual dual function of this enzyme, which catalyzes specific aminoacylation with proline as well as with cysteine.

Amino Acid Sequence↗
PubMed2001

The A-type lamins: nuclear structural proteins as a focus for muscular dystrophy and cardiovascular diseases.

Mutations in the lamin A (LMNA) gene are associated with the tissue-specific diseases Emery-Dreifuss muscular dystrophy (EDMD), limb girdle muscular dystrophy (LGMD-1B), dilated cardiomyopathy with conduction system disease (DCM-CD), and Dunnigan's familial partial lipodystrophy (FPLD). Lamins A and C, the products of the LMNA gene, are nuclear intermediate filament proteins and are the major structural components of the lamina network that underlies and supports the nuclear envelope. Nuclear fragility and mislocalization of the nuclear envelope protein emerin are two defects induced by a lack of the A-type lamins. These observations reveal that organization and structural integrity of the nucleus are critical factors in the origins of certain dystrophic and cardiovascular diseases.

Animals↗
PubMed2001

The nuclear envelope in muscular dystrophy and cardiovascular diseases.

Considerable interest has been focused on the nuclear envelope in recent years following the realization that several human diseases are linked to defects in genes encoding nuclear envelope specific proteins, most notably A-type lamins and emerin. These disorders, described as laminopathies or nuclear envelopathies, include both X-linked and autosomal dominant forms of Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy with conduction system defects, limb girdle muscular dystrophy 1B with atrioventricular conduction disturbances, and Dunnigan-type familial partial lipodystrophy. Certain of these diseases are associated with nuclear structural abnormalities that can be seen in a variety of cells and tissues. These observations clearly demonstrate that A-type lamins in particular play a central role, not only in the maintenance of nuclear envelope integrity but also in the large-scale organization of nuclear architecture. What is not obvious, however, is why defects in nuclear envelope proteins that are found in most adult cell types should give rise to pathologies associated predominantly with skeletal and cardiac muscle and adipocytes. The recognition of these various disorders now raises the novel possibility that the nuclear envelope may have functions that go beyond housekeeping and which impact upon cell-type specific nuclear processes.

Animals↗
PubMed2001

Nuclear envelope defects associated with LMNA mutations cause dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy.

Nuclear lamin A and C alleles that are linked to three distinct human diseases have been expressed both in HeLa cells and in fibroblasts derived from Lmna null mice. Point mutations that cause dilated cardiomyopathy (L85R and N195K) and autosomal dominant Emery-Dreifuss muscular dystrophy (L530P) modify the assembly properties of lamins A and C and cause partial mislocalization of emerin, an inner nuclear membrane protein, in HeLa cells. At the same time, these mutant lamins interfere with the targeting and assembly of endogenous lamins and in this way may cause significant changes in the molecular organization of the nuclear periphery. By contrast, lamin A and C molecules harboring a point mutation (R482W), which gives rise to a dominant form of familial partial lipodystrophy, behave in a manner that is indistinguishable from wild-type lamins A and C, at least with respect to targeting and assembly within the nuclear lamina. Taken together, these results suggest that nuclear structural defects could contribute to the etiology of both dilated cardiomyopathy and autosomal dominant Emery-Dreifuss muscular dystrophy.

Animals↗
PubMed2001

Nuclear envelope dynamics.

The nuclear envelope (NE) provides a semi permeable barrier between the nucleus and cytoplasm and plays a central role in the regulation of macromolecular trafficking between these two compartments. In addition to this transport function, the NE is a key determinant of interphase nuclear architecture. Defects in NE proteins such as A-type lamins and the inner nuclear membrane protein, emerin, result in several human diseases that include cardiac and skeletal myopathies as well as lipodystrophy. Certain disease-linked A-type lamin defects cause profound changes in nuclear organization such as loss of peripheral heterochromatin and redistribution of other nuclear envelope components. While clearly essential in maintenance of nuclear integrity, the NE is a highly dynamic organelle. In interphase it is constantly remodeled to accommodate nuclear growth. During mitosis it must be completely dispersed so that the condensed chromosomes may gain access to the mitotic spindle. Upon completion of mitosis, dispersed NE components are reutilized in the assembly of nuclei within each daughter cell. These complex NE rearrangements are under precise temporal and spatial control and involve interactions with microtubules, chromatin, and a variety of cell-cycle regulatory molecules.

Animals↗
PubMed2000

Evolutionary coadaptation of the motif 2--acceptor stem interaction in the class II prolyl-tRNA synthetase system.

Known crystal structures of class II aminoacyl-tRNA synthetases complexed to their cognate tRNAs reveal that critical acceptor stem contacts are made by the variable loop connecting the beta-strands of motif 2 located within the catalytic core of class II synthetases. To identify potential acceptor stem contacts made by Escherichia coli prolyl-tRNA synthetase (ProRS), an enzyme of unknown structure, we performed cysteine-scanning mutagenesis in the motif 2 loop. We identified an arginine residue (R144) that was essential for tRNA aminoacylation but played no role in amino acid activation. Cross-linking experiments confirmed that the end of the tRNA(Pro) acceptor stem is proximal to this motif 2 loop residue. Previous work had shown that the tRNA(Pro) acceptor stem elements A73 and G72 (both strictly conserved among bacteria) are important recognition elements for E. coli ProRS. We carried out atomic group "mutagenesis" studies at these two positions of E. coli tRNA(Pro) and determined that major groove functional groups at A73 and G72 are critical for recognition by ProRS. Human tRNA(Pro), which lacks these elements, is not aminoacylated by the bacterial enzyme. An analysis of chimeric tRNA(Pro) constructs showed that, in addition to A73 and G72, transplantation of the E. coli tRNA(Pro) D-domain was necessary and sufficient to convert the human tRNA into a substrate for the bacterial synthetase. In contrast to the bacterial system, base-specific acceptor stem recognition does not appear to be used by human ProRS. Alanine-scanning mutagenesis revealed that motif 2 loop residues are not critical for tRNA aminoacylation activity of the human enzyme. Taken together, our results illustrate how synthetases and tRNAs have coadapted to changes in protein-acceptor stem recognition through evolution.

Adaptation, Biological↗
PubMed2000

Recombinant Nup153 incorporates in vivo into Xenopus oocyte nuclear pore complexes.

Nup153 is a molecular constituent of the nuclear basket of the nuclear pore complex (NPC) that plays a critical role in nuclear export of RNAs and proteins. In an effort to map this nucleoporin more precisely within the nuclear basket we have developed an experimental approach for localizing Nup153 expressed and incorporated in vivo into Xenopus oocyte NPCs. This approach involves the microinjection into the cytoplasm of Xenopus oocytes of in vitro synthesized mRNA from a vector encoding an epitope-tagged cDNA. Here we present results obtained by Western blots, fluorescence microscopy, and immuno-electron microscopy, which clearly document that the heterologous protein is properly expressed, targeted, and incorporated into preexisting Xenopus NPCs. This new approach for localizing nucleoporins within the structure of the NPC overcomes limitations of previous techniques and allows for greater specificity and resolution than have been possible with previous methods.

Animals↗
PubMed2000

Prospects for treating acquired pendular nystagmus with servo-controlled optics.

PURPOSE: To determine whether a device featuring electronically controlled motor-driven prisms can reduce oscillopsia and improve acuity in patients with acquired pendular nystagmus (APN). METHODS: A device was developed that senses eye movements and, by the use of motor-driven prisms, oscillates the image of the world in lockstep with the pathologic nystagmus, to negate its deleterious visual effects. Unlike existing optical and surgical treatments for nystagmus, the device negates only the pathologic movements. Voluntary and normal reflex eye movements required for normal vision are unaffected. The benefits of the device were assessed by its impact on acuity in five patients with medication-refractory APN. RESULTS: All patients reported decreases in oscillopsia when the device was in operation. Averaged across patients, the device increased the percentage of time in which retinal image velocity was within +/-4 degrees/sec from 12.8% to 33.3%. Acuities improved in four of five patients, by an average of 0.21 logMAR units. CONCLUSIONS: The symptoms of pendular nystagmus can be treated with a servomechanical device. Further refinements in the device should result in greater improvements in acuity, and a portable, wearable version is feasible using existing technologies.

Adult↗