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Biomedical subjects

G Brady

Publications and source records attributed to G Brady.

At least 19 recordsLinked to original sources
PubMed2006

Serum levels of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs), -1, -2, -3, in oral cancer.

INTRODUCTION: Insulin-like growth factor (IGF) possesses mitogenic properties promoting cellular proliferation and inhibiting cellular apoptosis. Reported evidence suggests that cancer (non-oral) is associated with high circulating levels of IGF-1 and low levels of IGFBP-3. AIMS: This study measured circulating levels of IFG-1 and IGFBPs -1, -2 and -3 in oral cancer patients. METHODOLOGY: Blood was collected from surgical patients and controls. Samples were assayed for IGF-1 and IGFBPs -1, -2, -3, using commercially available enzyme-linked immunosorbant assays (ELISA; R&D Systems Europe, Oxon, UK). RESULTS: Twenty-seven oral cancer and 31 age- and sex-matched patients were recruited. Mean IGF-1 and IGFBP-3 levels were significantly lower in cancer patients than controls (85.3 ng/ml and 2008 ng/ml versus 191 ng/ml and 2935 ng/ml, P<0.001). In contrast, levels of IGFBPs 1 and 2 were significantly higher in cancer patients than in controls. No significant association was demonstrated with tumour size or nodal metastases. DISCUSSION: This study has shown that in contrast to other cancers, circulating levels of IGF-1 and IGFBP-3 may both be lowered in patients with head and neck cancer.

Biomarkers, Tumor↗
PubMed2006

Gene expression analysis of myeloid and lymphoid lineage markers during mouse haematopoiesis.

Expression profiling of haematopoietic cells is hampered by the heterogeneous nature of haematopoietic tissues and the absolute rarity of early unrestricted progenitors. To overcome this, the expression profile of lymphoid and myeloid-associated genes (LEF1, EBF, CD19, Sox-4, B29, CD45, C-fms, lysozyme, PU.1 and CD5) were investigated in 40 mouse myeloid haematopoietic precursors covering the entire haematopoietic hierarchy from multipotential to committed single lineages. The lineage-specific expression seen in single-cell studies was confirmed by examining fractionated bone marrow, whole tissues and differentiation of the multipotent cell line FDCP (Factor Dependent Cell Paterson) mix. Analysis of the 40 single myeloid precursors failed to detect expression of lymphoid-associated genes, LEF1, EBF, CD19 and CD5, despite detection in lymphoid cell controls. Surprisingly, the lymphoid-associated genes, Sox-4 and B29 were detected in the single myeloid precursors, which was confirmed in bone marrow and a multipotential myeloid cell line. The pattern of Sox-4 and B29, is consistent with a potential role in the commitment of bipotential granulocytic/macrophage precursors towards the granulocyte or macrophage lineage. In addition to providing baseline values for myeloid and lymphoid lineage markers during mouse haematopoiesis, these results highlight the importance of single-cell analysis in the study of complex tissues.

Animals↗
PubMed2003

The parathyroid hormone-related protein receptor is expressed in breast cancer bone metastases and promotes autocrine proliferation in breast carcinoma cells.

Overproduction of parathyroid hormone-related protein (PTHRP) occurs in a high proportion of primary breast cancers (PBC) and is strongly implicated in their metastatic spread to bone. Although the PTHRP-receptor (PTHRP-R) is often coexpressed with PTHRP in PBC, its role in regulating breast cancer cell proliferation and metastases to bone remains unclear. The aims of this study were to determine the expression of the PTHRP-R in breast cancer bone metastases (BM) and to investigate the effects of PTHRP-R overexpression on breast cancer cell proliferation. PTHRP-R expression occurred in 85% (11 out of 13) of BM compared with 58% (39 out of 67) of PBC. Median expression was higher (P<0.05) in BM compared with PBC. PTHRP increased cAMP accumulation and DNA synthesis in MCF-7 cells stably overexpressing the PTHRP-R (MCF-7(WTR)) but not in MCF-7(VEC) control cells. The increase in DNA synthesis was mimicked by the cAMP pathway activator forskolin. The receptor antagonist PTHRP(7-34) reduced DNA synthesis in MCF-7(WTR) cells, but not MCF-7(VEC) cells, indicating that receptor overexpression promotes autocrine PTHRP activity. MCF-7(WTR) cells showed increased mitogenic responsiveness to fetal calf serum and reduced doubling times. PTHRP induced weak activation of ERK1 and ERK2 and potentiated their activation by serum growth factors. Collectively these results show that the PTHRP-R is frequently expressed in breast cancer BM and indicate that receptor overexpression drives proliferation via autocrine signals that are mediated via cAMP and ERK pathways.

Autocrine Communication↗
PubMed2001

Quantitative amplification of single-stranded DNA (QAOS) demonstrates that cdc13-1 mutants generate ssDNA in a telomere to centromere direction.

We have developed a method that allows quantitative amplification of single-stranded DNA (QAOS) in a sample that is primarily double-stranded DNA (dsDNA). Single-stranded DNA (ssDNA) is first captured by annealing a tagging primer at low temperature. Primer extension follows to create a novel, ssDNA-dependent, tagged molecule that can be detected by PCR. Using QAOS levels of between 0.2 and 100% ssDNA can be accurately quantified. We have used QAOS to characterise ssDNA levels at three loci near the right telomere of chromosome V in budding yeast cdc13-1 mutants. Our results confirm and extend previous studies which demonstrate that when Cdc13p, a telomere-binding protein, is disabled, loci close to the telomere become single stranded whereas centromere proximal sequences do not. In contrast to an earlier model, our new results are consistent with a model in which a RAD24-dependent, 5' to 3' exonuclease moves from the telomere toward the centromere in cdc13-1 mutants. QAOS has been adapted, using degenerate tagging primers, to preferentially amplify all ssDNA sequences within samples that are primarily dsDNA. This approach may be useful for identifying ssDNA sequences associated with physiological or pathological states in other organisms.

Cell Cycle Proteins↗
PubMed2001

Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction.

The recognition of microbial pathogens by the innate immune system involves Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns. Different TLRs recognize different pathogen-associated molecular patterns, with TLR-4 mediating the response to lipopolysaccharide from Gram-negative bacteria. All TLRs have a Toll/IL-1 receptor (TIR) domain, which is responsible for signal transduction. MyD88 is one such protein that contains a TIR domain. It acts as an adapter, being involved in TLR-2, TLR-4 and TLR-9 signalling; however, our understanding of how TLR-4 signals is incomplete. Here we describe a protein, Mal (MyD88-adapter-like), which joins MyD88 as a cytoplasmic TIR-domain-containing protein in the human genome. Mal activates NF-kappaB, Jun amino-terminal kinase and extracellular signal-regulated kinase-1 and -2. Mal can form homodimers and can also form heterodimers with MyD88. Activation of NF-kappaB by Mal requires IRAK-2, but not IRAK, whereas MyD88 requires both IRAKs. Mal associates with IRAK-2 by means of its TIR domain. A dominant negative form of Mal inhibits NF-kappaB, which is activated by TLR-4 or lipopolysaccharide, but it does not inhibit NF-kappaB activation by IL-1RI or IL-18R. Mal associates with TLR-4. Mal is therefore an adapter in TLR-4 signal transduction.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Facial features of widespread neuroblastoma: a case report.

A case report of stage IV neuroblastoma which presented with periorbital swelling and ecchymosis originally misdiagnosed as facial trauma. The child soon developed a sinister pancytopenia, which following extensive investigations was revealed to be due to an underlying neuroblastoma. Periorbital ecchymosis associated with neuroblastoma is termed 'raccoon eyes' and is a diagnostic trap for the unwary.

Aortic Diseases↗
PubMed2001

Transactivation by the p65 subunit of NF-kappaB in response to interleukin-1 (IL-1) involves MyD88, IL-1 receptor-associated kinase 1, TRAF-6, and Rac1.

We have examined the involvement of components of the interleukin-1 (IL-1) signaling pathway in the transactivation of gene expression by the p65 subunit of NF-kappaB. Transient transfection of cells with plasmids encoding wild-type MyD88, IL-1 receptor-associated kinase 1 (IRAK-1), and TRAF-6 drove p65-mediated transactivation. In addition, dominant negative forms of MyD88, IRAK-1, and TRAF-6 inhibited the IL-1-induced response. In cells lacking MyD88 or IRAK-1, no effect of IL-1 was observed. Together, these results indicate that MyD88, IRAK-1, and TRAF-6 are important downstream regulators of IL-1-mediated p65 transactivation. We have previously shown that the low-molecular-weight G protein Rac1 is involved in this response. Constitutively active RacV12-mediated transactivation was not inhibited by dominant negative MyD88, while dominant negative RacN17 inhibited the MyD88-driven response, placing Rac1 downstream of MyD88 on this pathway. Dominant negative RacN17 inhibited wild-type IRAK-1- and TRAF-6-induced transactivation, and in turn, dominant negative IRAK-1 and TRAF-6 inhibited the RacV12-driven response, suggesting a mutual codependence of Rac1, IRAK-1, and TRAF-6 in regulating this pathway. Finally, Rac1 was found to associate with the receptor complex via interactions with both MyD88 and the IL-1 receptor accessory protein. A pathway emanating from MyD88 and involving IRAK-1, TRAF-6, and Rac1 is therefore involved in transactivation of gene expression by the p65 subunit of NF-kappaB in response to IL-1.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Global cDNA amplification combined with real-time RT-PCR: accurate quantification of multiple human potassium channel genes at the single cell level.

We have developed a sensitive quantitative RT-PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel mRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitative RT-PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of samples, we have adapted the approach to allow use of TaqMan real-time quantitative PCR. We demonstrate that the approach represents a major improvement over existing conventional and real-time quantitative PCR approaches, since it can be applied to samples equivalent to a single cell, is able to accurately measure expression levels equivalent to less than 1/100th copy/cell (one specific cDNA molecule present amongst 10(8) total cDNA molecules). Furthermore, since the initial step involves a global amplification of all expressed genes, a permanent cDNA archive is generated from each sample, which can be regenerated indefinitely for further expression analysis.

Breast↗
PubMed2000

Expression profiling of single mammalian cells--small is beautiful.

Increasingly mRNA expression patterns established using a variety of molecular technologies such as cDNA microarrays, SAGE and cDNA display are being used to identify potential regulatory genes and as a means of providing valuable insights into the biological status of the starting sample. Until recently, the application of these techniques has been limited to mRNA isolated from millions or, at very best, several thousand cells thereby restricting the study of small samples and complex tissues. To overcome this limitation a variety of amplification approaches have been developed which are capable of broadly evaluating mRNA expression patterns in single cells. This review will describe approaches that have been employed to examine global gene expression patterns either in small numbers of cells or, wherever possible, in actual isolated single cells. The first half of the review will summarize the technical aspects of methods developed for single-cell analysis and the latter half of the review will describe the areas of biological research that have benefited from single-cell expression analysis.

Animals↗
PubMed2000

Expression of the imprinted tumour-suppressor gene H19 is tightly regulated during normal haematopoiesis and is reduced in haematopoietic precursors of patients with the myeloproliferative disease polycythaemia vera.

cDNA subtraction was employed to uncover differences in gene expression between myeloproliferative polycythaemia vera (PV) and normal haematopoietic precursors. Following cDNA subtraction using mRNAs isolated from PV and normal CD34+/CD33- bone-marrow cells, expression of the tumour suppressor H19 was found to be low or absent in the PV sample. Low levels of H19 expression in PV patients were confirmed by in situ hybridization. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to examine expression in the pluripotent haematopoietic cell line FDCP-mix and single bone-marrow precursors, unambiguous IGF2 and H19 expression was demonstrated in normal haematopoietic precursors. Examination of individual bone-marrow precursors revealed that all IGF2-expressing haematopoietic precursors also co-expressed H19, indicating that H19 and IGF2 may be co-ordinately regulated during haematopoiesis. Analysis of FDCP-mix undergoing differentiation and single pluripotent and committed bone-marrow precursors revealed that the pattern of H19 expression coincided with the commitment to a single lineage. Taken together, these observations demonstrate that H19 and IGF2 are specifically expressed during haematopoiesis and that low levels of H19 expression are associated with PV and may contribute to the pathology of the disease.

Animals↗
PubMed1999

Further investigation of endothelium-derived hyperpolarizing factor (EDHF) in rat hepatic artery: studies using 1-EBIO and ouabain.

1. The characteristics of endothelium-dependent hyperpolarization in rat hepatic artery have been further investigated in the presence of inhibitors of cyclo-oxygenase and nitric oxide synthase. 2. Using sharp micro-electrodes, the smooth muscle hyperpolarization induced by acetylcholine, KCl or 1-ethyl-2-benzimidazolinone (1-EBIO) in intact hepatic arteries was abolished by 30 micronM barium plus 500 nM ouabain. 3. In vessels without endothelium, the smooth muscle hyperpolarization induced by KCl was not reduced by 30 micronM barium alone. However, in the presence of barium the effects of KCl were partially inhibited by 100 nM ouabain and essentially abolished by 500 nM ouabain. 4. Using sharp micro-electrodes, the hyperpolarization of both the smooth muscle and the endothelium induced by 1-EBIO or by acetylcholine was unaffected by 100 nM iberiotoxin. However, in the presence of 100 nM charybdotoxin, the effects of 1-EBIO were abolished whereas those of acetylcholine were only partially reduced. The hyperpolarization induced by levcromakalim was unaffected by either charybdotoxin or iberiotoxin. 5 Under whole-cell patch-clamp recording conditions, 1-EBIO induced a voltage-insensitive, charybdotoxin-sensitive K+ current in cultured endothelial cells but was without effect on K+ currents in smooth muscle cells isolated from hepatic arteries. 6 It is concluded that the endothelium-dependent hyperpolarization of smooth muscle induced by either acetylcholine or by 1-EBIO in rat hepatic artery is initially associated with the opening of endothelial calcium-sensitive K+-channels insensitive to iberiotoxin. The resulting accumulation of K+ in the myoendothelial space activates an isoform of Na+/K+-ATPase which is sensitive to low concentrations of ouabain.

Acetylcholine↗
PubMed1998

Blood cells with reduced mitochondrial membrane potential and cytosolic cytochrome C can survive and maintain clonogenicity given appropriate signals to suppress apoptosis.

Reduction of mitochondrial membrane potential (Psim) and release of cytochrome c from mitochondria appear to be key events during apoptosis. Apoptosis was induced in IC.DP premast cells by the withdrawal of interleukin-3 (IL-3). Psim decreased by 12 hours and cytochrome c was detected in the cytosol at 18 hours. Despite these changes in the mitochondria after 18 hours of IL-3 deprivation, clonogenicity was unaffected when IL-3 was replenished at 18 hours. Activation of v-Abl tyrosine kinase (v-Abl TK) in IC.DP cells before IL-3 depletion led to increased levels of Bcl-XL, prevented reduction of Psim and the release of mitochondrial cytochrome c, and suppressed apoptosis. Activation of v-Abl TK 18 hours after withdrawal of IL-3 when </=10% of the cells had died restored Psim in the remaining cells. More than 40% of cells thus rescued by v-Abl TK between 18 and 42 hours could subsequently form colonies in the presence of IL-3. These data suggest that reduction in Psim precedes loss of mitochondrial cytochrome c in IC.DP cells; that v-Abl TK activation, probably via upregulation of Bcl-XL, prevents loss of Psim and blocks the release of cytochrome c from mitochondria; and that neither of these mitochondrial events is sufficient for commitment to apoptosis.

Animals↗
PubMed1998

Human granulocytic ehrlichiosis in New York.

BACKGROUND: Human granulocytic ehrlichiosis (HGE), a potentially fatal tick-borne disease, was first described in the upper Midwest in 1994. Following reports of suspected cases of ehrlichiosis from New York physicians, descriptive and case-control studies were conducted to characterize the epidemiology and risk factors for HGE in New York residents. METHODS: Descriptive data were gathered from surveillance and laboratory reports and hospital records. A confirmed case was defined as either (1) a 4-fold change in total antibody titer to Ehrlichia equi by indirect immunofluorescence or (2) a polymerase chain reaction assay positive for Ehrlichia phagocytophila/E equi group DNA. A probable case was defined as an acute febrile illness and either (1) a single E equi titer greater than or equal to 80 or (2) morulae on a peripheral blood smear. The case-control study included patients with confirmed HGE 18 years of age or older with the onset of disease in 1995 and 2 to 3 neighborhood-matched controls. RESULTS: During 1994 and 1995, the New York State Department of Health, Albany, received reports of 241 residents who were tested for HGE; 30 met the confirmed case definition and 34 met the probable case definition. The median age of patients was 46 years (age range, 9-90 years), 35 (55%) were male, and 25 (45%) were hospitalized. Fever, headache, malaise, and myalgia were the most frequently reported symptoms. Fifty-six (88%) of the 64 patients resided in areas in which Lyme disease is hyperendemic. In the case-control analysis, cases were more likely than controls to have sustained a tick bite during 1995 (matched odds ratio, 5.0; 95% confidence interval, 0.9-49.8). Cases and controls did not differ by occupational exposure to ticks, underlying chronic diseases, or measures taken to prevent tick bites. CONCLUSIONS: This study, which, to our knowledge, is the first population-based study of HGE, demonstrates the recent recognition of HGE in the state of New York. Control measures should be integrated with those for Lyme disease and should focus on minimizing contact with ticks and obtaining early treatment for infection.

Adolescent↗
PubMed1998

Homology between a human apoptosis specific protein and the product of APG5, a gene involved in autophagy in yeast.

Apoptosis specific proteins (ASP) are expressed in the cytoplasm of cultured mammalian cells of various lineages following induction of apoptosis. The cDNA encoding ASP has been cloned from a human expression library and has significant homology to the Saccharomyces cerevisiae APG5 gene which is essential for yeast autophagy. The ASP gene, known as hAPG5, can be transcribed to give mRNAs of 3.3 kbp, 2.5 kbp and 1.8 kbp which are present at comparable levels in viable and apoptotic cells, demonstrating that protein expression must be regulated at the translational level. These data indicate a possible relationship between apoptosis and autophagy and suggest evolutionary conservation in mammalian apoptosis of a degradative process present in yeast.

Animals↗
PubMed1998

Commitment to cell death measured by loss of clonogenicity is separable from the appearance of apoptotic markers.

Kinetic analysis of dexamethasone-induced apoptosis in the human lymphoblastoid cell line CCRF CEM C7A has revealed a point when cells, morphologically indistinguishable from untreated cells, have irreversibly engaged a program leading to death, measured by a loss of clonogenicity. Since all cells that fail to clone eventually died through apoptosis, measurements of clonogenicity in this system provide an accurate measure of commitment to apoptotic death. Inhibition of caspases by peptide inhibitors blocked proteolysis of endogenous substrates and reduced nuclear condensation yet did not alter either dexamethasone-induced changes in clonogenicity or mitochondrial membrane potential. In contrast to the results with caspase inhibitors, expression of BCL-2 in CCRF CEM C7A cells proved sufficient to block all changes associated with apoptosis, including loss of both clonogenicity and changes in mitochondrial membrane potential. These results demonstrate that commitment to cell death can precede the key biochemical or morphological features of apoptosis by several hours and indicate that separate regulators govern cellular commitment to clonogenic death and the subsequent execution phase characterised as apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Expression of the Id family helix-loop-helix regulators during growth and development in the hematopoietic system.

To better understand the molecular mechanism(s) by which growth and differentiation of the primitive hematopoietic stem cell is initiated, as well as the means by which the maturing cell can commit to development along a specific cell lineage, we elected to study the Id family of helix-loop-helix (HLH) transcriptional regulators. Some members of the HLH family are expressed in a stage-specific manner during hematopoietic development and can regulate the ability of immature hematopoietic cells to terminally differentiate. None of the four Id family genes were detected in the most primitive progenitors. Id-1 was widely expressed in proliferating bi- and unipotential progenitors, but its expression was downregulated in cells of increasing maturity; conversely, Id-2 and, to a limited extent, Id-3 gene expression increased as cells matured and lost proliferative capacity. Id-2 expression ran counter to that of Id-1 not only during maturation, but during periods of cell growth and arrest as well. This is quite distinct from the nonhematopoietic tissues, in which these two factors are coordinately expressed and suggests that Id-1 and Id-2 might be regulating very different events during hematopoiesis than they regulate in other cell types.

Animals↗