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Biomedical subjects

G Murphy

Publications and source records attributed to G Murphy.

At least 73 records · Page 4Linked to original sources
PubMed2000

The pattern of serum markers in patients with androgen-independent adenocarcinoma of the prostate.

In this exploratory study, our objectives were to correlate the serum and bone marrow concentrations of putative markers of prostate cancer progression in patients with advanced androgen-independent prostate cancer (AIPC), to assess the frequency and quantity of relative expression of these markers, and to correlate the expression of the markers with extent of disease (EOD) and overall survival. In a cohort of 50 patients with AIPC with bone metastases, we obtained serum and bone marrow samples and measured prostate specific antigen (PSA), serum interleukin-6 (sIL-6), bone marrow interleukin-6, serum chromogranin A (sCgA), bone marrow chromogranin A, and prostate specific membrane antigen (PSMA) by immunoassays. EOD was determined by quantifying identifiable bone lesions on radionuclide bone scans. Each variable was categorized into two groups (low and high) based on the median found in this cohort or on the cutoff based on normal limits when available. Analyses were performed in two subsets of patients with EOD either <20 or >/=20. Results showed that: (1) PSA is associated with EOD but not with outcome; (2) sIL-6 and sCgA may be intermediate markers of early progression in AIPC, because they are predictive of outcome only in patients with EOD <20; (3) elevated PSMA is associated with elevated sIL-6 but not with PSA, suggesting that PSMA may be a useful marker in AIPC; and (4) the ratio of PSA to putative markers of progression may reflect the complex clonal progression of prostate cancer. We conclude that patients with advanced AIPC exhibit one of two patterns of serologic marker expression: in some patients the disease status is reflected by PSA, and in others it is reflected by other markers. If these data are prospectively confirmed, this would help group patients with advanced AIPC into clinically relevant categories.

Journal Article↗
PubMed2000

Progress in Arabidopsis genome sequencing and functional genomics.

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.

Agriculture↗
PubMed2000

Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase.

The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein-coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713-14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691-697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563-11569). However, little is known about how syndecan ectodomain shedding is regulated. To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.

Animals↗
PubMed2000

Problems in the interpretation of HIV-1 viral load assays using commercial reagents.

During routine monitoring of human immunodeficiency virus (HIV) viral load, two problems arose. First, a number of patients, the majority being African, were found to have low viral loads by the Chiron branched-chain DNA assay in conjunction with low CD4(+) cell numbers. In order to determine whether this was due to failure of the branched-chain DNA assay to detect non-B subtypes of HIV, selected samples were subtyped and HIV RNA quantified by branched-chain DNA, NASBA, and the Roche Monitor RT-PCR assay. Twenty-eight (97%) of 29 Africans were infected with a non-B subtype of HIV and 15 (93.7%) of 16 non-Africans with subtype B. Twenty-three samples had a low viral load by branched-chain DNA, which was confirmed by the NASBA and RT-PCR assays. All three assays detected B and non-B subtypes with similar efficiency; NASBA failed to detect HIV RNA in a small number of non-B samples. Discrepancies between viral load and CD4(+) cell numbers did not appear therefore to be related to subtype. Second, while quantification of HIV RNA was being conducted using version 2 of the branched-chain DNA assay (lower detection limit 500 HIV RNA copies/ml) the manufacturers had developed a more sensitive assay and a comparative evaluation was therefore conducted. In approximately 30% of samples the viral load was up to 10 times higher with the more sensitive assay. These experiences emphasise the importance of close collaboration between the clinic and the laboratory.

CD4 Lymphocyte Count↗
PubMed2000

Localisation of matrix metalloproteinases and TIMP-2 in resorbing mouse bone.

There is strong evidence that matrix metalloproteinases (MMPs) play a crucial role during osteogenesis and bone remodelling. Their synthesis by osteoblasts has been demonstrated during osteoid degradation prior to resorption of mineralised matrix by osteoclasts and their activities are regulated by tissue inhibitors of metalloproteinases (TIMPs). For this study we developed and utilised specific polyclonal antibodies to assess the presence of collagenase (MMP13), stromelysin 1 (MMP3), gelatinase A (MMP2), gelatinase B (MMP9) and TIMP-2 in both freshly isolated neonatal mouse calvariae and tissues cultured with and without bone-resorbing agents. Monensin was added towards the end of the culture period in order to promote intracellular accumulation of proteins and facilitate antigen detection. In addition, bone sections were stained for the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). In uncultured tissues the bone surfaces had isolated foci of collagenase staining, and cartilage matrix stained for gelatinase B (MMP9) and TIMP-2. Calvariae cultured for as little as 3 h with monensin revealed intracellular staining for MMPs and TIMP-2 in mesenchymal tissues, as well as in cells lining the bone plates. The addition of cytokines to stimulate bone resorption resulted in pronounced TRAP activity along bone surfaces, indicating active resorption. There was a marked upregulation of enzyme synthesis, with matrix staining for collagenase and gelatinase B observed in regions of eroded bone. Increased staining for TIMP-2 was also observed in association with increased synthesis of MMPs. The new antibodies to murine MMPs should prove valuable in future studies of matrix degradation.

Acid Phosphatase↗
PubMed2000

Recognition and catabolism of synthetic heterotrimeric collagen peptides by matrix metalloproteinases.

BACKGROUND: The general consensus is that interstitial collagens are digested by collagenases and denatured collagen by gelatinases, although processing of fibrillar and acetic-acid-soluble collagen by gelatinase A has also been reported. One of the main difficulties in studying the mechanism of action of these matrix metalloproteinases (MMPs) derives from the physicochemical properties of the natural triple-helical collagen, which makes it difficult to handle. RESULTS: Synthetic heterotrimeric collagenous peptides that contain the collagenase cleavage site of human collagen type I and differ in the thermal stability of the triple-helical fold were used to mimic natural collagen and gelatin, respectively. Results from digestion of these substrates by fibroblast and neutrophil collagenases (MMP-1 and MMP-8), as well as by gelatinase A (MMP-2), confirmed that the two classes of enzymes operate within the context of strong conformational dependency of the substrates. It was also found that gelatinases and collagenases exhibit two distinct proteolytic mechanisms: gelatinase digests the gelatin-like heterotrimer rapidly in individual steps with intermediate releases of partially processed substrate into the medium, whereas collagenases degrade the triple-helical heterotrimer by trapping it until scission through all three alpha chains is achieved. CONCLUSIONS: The results confirm the usefulness of synthetic heterotrimeric collagenous peptides in the folded and unfolded state as mimics of the natural substrates collagen and gelatin, respectively, to gain a better a insight into the proteolytic mechanisms of matrix metalloproteinases.

Binding Sites↗
PubMed2000

Interleukin-8 overexpression is present in pyoderma gangrenosum ulcers and leads to ulcer formation in human skin xenografts.

Interleukin-8 (IL-8) is a potent chemotactic polypeptide for neutrophils. However, the role of this cytokine during inflammation remains unclear. Skin specimens from patients with pyoderma gangrenosum demonstrated IL-8 overexpression in skin ulcers, which suggests a role for IL-8 in the development of the disease. We therefore constructed a recombinant adenovirus expressing the complementary deoxyribonucleic acid encoding human IL-8 (IL-8/Ad5) that induces a 2000-fold increase in IL-8 expression of infected human fibroblasts in vitro. Human skin engrafted to severe combined immunodeficiency mice and then injected with the recombinant virus demonstrated erythema, an intense perivascular infiltration of neutrophils, and extravasation of erythrocytes after 8 hours. By 12 hours after injection, neutrophils had accumulated beneath the epidermis, which then necrotized, and one or more ulcers that remained for approximately 2 weeks were observed. Clinically and histologically, the ulcers resembled pyoderma gangrenosum. These clinical and experimental findings suggest an etiologic role of IL-8 in the pathogenesis of pyoderma gangrenosum.

Animals↗
PubMed2000

Partial lipodystrophy and renal disease.

Partial lipodystrophy is a rare disorder with both autosomal recessive and familial forms. The cutaneous findings, which are often subtle, consist of a gradual loss of subcutaneous fat from the face and upper body. Low levels of C3, occasionally low C5 and the presence of the nephritic factor help to identify these patients. Associated systemic abnormalities include the development of mesangiocapillary glomerulonephritis and an increased incidence of autoimmune diseases. Recognition of this unusual disorder is essential for diagnosis and treatment of underlying potentially life- threatening disease.

Adult↗
PubMed2000

Changes in skills for people with intellectual disability: a follow-up of the Camberwell Cohort.

The skills of a total population of children with severe intellectual disability and/or autism from Camberwell, South London, UK, and the initial follow-up data, taken when the subjects were adolescents and young adults (Shah 1986), are described in the present study. Changes in skills over time are presented within the categories of communication, self-care, and educational and cognitive skills, as assessed by the Handicaps, Behaviours and Skills schedule. The results indicated that skills had improved in many areas between times 1 and 2, but that this improvement was more noticeable for the children who had been youngest at time 1. The implications of these results and predictions for a further follow-up study are discussed.

Adolescent↗
PubMed2000

Functional significance of MMP-9 in tumor necrosis factor-induced proliferation and branching morphogenesis of mammary epithelial cells.

Tissue remodeling is a key process involved in normal mammary gland development, with matrix metalloproteinases (MMPs) playing an important role in this process. Our laboratory has demonstrated that tumor necrosis factor (TNF) stimulates branching morphogenesis of mammary epithelial cells (MEC) within a reconstituted basement membrane. Studies were therefore undertaken to determine whether MMPs might mediate the effects of TNF. Using a primary culture model in which rat MEC grow three-dimensionally within a reconstituted basement membrane, we found that TNF stimulated secretion of MMP-9 but not MMP-2. To determine whether MMP-9 was involved in TNF-induced proliferation and branching morphogenesis, we used a peptide containing the prodomain sequence of MMPs and two MMP inhibitors. Both the prodomain peptide (5 x 10(-4)-10(-3) M), as well as BB-94 (10(-8)-10(-5) M) and CGS 27023A (10(-6)-10(-5) M), inhibited TNF-induced proliferation and branching morphogenesis in a concentration-dependent manner. Finally, to verify the specific requirement for MMP-9, we demonstrated that an MMP-9 neutralizing antibody blocked TNF-induced proliferation and branching morphogenesis. Together, these data suggest that TNF-regulated MMP-9 may play a role in the controlled invasion of the fad pad that occurs during normal mammary gland development and that misregulation of MMP-9 may contribute to the invasiveness of breast cancer.

Animals↗
PubMed2000

Collagen degradation and platelet-derived growth factor stimulate the migration of vascular smooth muscle cells.

Cell migration is a key event in many biological processes and depends on signals from both extracellular matrix and soluble motogenic factors. During atherosclerotic plaque development, vascular smooth muscle cells migrate from the tunica media to the intima through a basement membrane and interstitial collagenous matrix and proliferate to form a neointima. Matrix metalloproteinases have previously been implicated in neointimal formation and in this study smooth muscle cell adhesion and migration on degraded collagen have been evaluated. Vascular smooth muscle cells adhered to native intact collagen type I and to its first degradation by-product, 3/4 fragment (generated by collagenase-3 cleavage), unwound at 35 degrees C to mimic physiological conditions. PDGF-BB pre-treatment induced a fourfold stimulation of smooth muscle cell motility on the collagen 3/4 fragment whereas no increase in smooth muscle cell motility on collagen type I was observed. Cell migration on collagen type I was mediated by alpha2 integrin, whereas PDGF-BB-stimulated migration on the 3/4 collagen fragment was dependent on alphavbeta3 integrin. alphavbeta3 integrin was organised in clusters concentrated at the leading and trailing edges of the cells and was only expressed when cells were exposed to the 3/4 collagen fragment. Tyrphostin A9, an inhibitor of PDGF receptor-beta tyrosine kinase activity, resulted in complete abolition of migration of PDGF-BB treated cells on collagen type I and 3/4 fragment. These results strongly support the hypothesis that the cellular migratory response to soluble motogens can be regulated by proteolytic modification of the extracellular matrix.

Anticoagulants↗
PubMed2000

Meltrin gamma(ADAM-9) mediates cellular adhesion through alpha(6)beta(1 )integrin, leading to a marked induction of fibroblast cell motility.

The ADAMs (A Disintegrin and Metalloprotease Domains) are a family of membrane-anchored proteins that play a role in fertilisation, myoblast fusion and ectodomain shedding of cell surface proteins. Meltrin gamma (ADAM-9) is a widely expressed member of this family and is involved in the shedding of heparin binding epidermal growth factor. Here we report that meltrin gamma can function as a cell adhesion molecule via its disintegrin domain. Using solid-phase binding assays and antibody inhibition experiments, we demonstrate that a murine meltrin gamma-Fc (Mel gamma -Fc) fusion protein binds to the integrin alpha(6)beta(1) on the surface of fibroblast cell lines, HT1080 and Wehi 164 in a specific manner. Since alpha(6)beta(1) is important for the motility of several cell types on laminin, cell migration studies using time-lapse video microscopy were performed. Cells adhering to Mel gamma-Fc displayed a rounded morphology and a marked increase (eight- to tenfold) in their motility compared to that on laminin. Furthermore, the p160 ROCK kinase inhibitor Y-27632 specifically reduced the migration of cells on meltrin gamma but had no effect on migration of cells on laminin, whilst the general tyrosine phoshorylation inhibitor, genistein, inhibited cell migration on both substrates. These results together suggest that meltrin gamma may play a role in regulating the motility of cells by binding to alpha(6)beta(1) integrin and this may be important during a variety of biological and pathological processes.

ADAM Proteins↗
PubMed2000

High preoperative plasma tissue inhibitor of metalloproteinase-1 levels are associated with short survival of patients with colorectal cancer.

The objective of the present study was to measure preoperative plasma tissue inhibitor of metalloproteinase (TIMP)-1 levels in colorectal cancer patients and relate these values to clinical and biochemical patient characteristics. TIMP-1 levels were determined by ELISA in EDTA plasma samples collected preoperatively from 588 colorectal cancer patients. Plasma TIMP-1 levels were distributed with a median value of 141.1 microg/liter (range, 53.7-788.7 microg/liter). Whereas no significant differences were found in the median plasma TIMP-1 levels among patients with Dukes' stage A, B, and C disease, patients with Dukes' stage D disease had significantly higher plasma TIMP-1 levels (P < 0.0001); however, high plasma TIMP-1 levels were not restricted to advanced disease. A relatively weak correlation between plasma TIMP-1 level and age was found (r = 0.35; P < 0.0001). There was no significant difference in TIMP-1 levels between males and females (P = 0.97). Univariate analysis demonstrated an increasing risk of mortality with increasing TIMP-1 levels [scored as the log(e)(TIMP-1); hazard ratio = 3.3; 95% confidence interval, 2.6-4.2; P < 0.0001]. Including covariates (Dukes' stage, primary tumor location, gender, age, plasminogen activator inhibitor type 1, and soluble urokinase plasminogen activator receptor) in a multivariate analysis, TIMP-1 was retained in the final model (hazard ratio = 2.5; 95% confidence interval, 1.7-3.7; P < 0.0001). This study showed a highly significant association between preoperative plasma TIMP-1 levels and survival in colorectal cancer patients, with higher plasma TIMP-1 levels being associated with poor outcome. Independent of clinical parameters including Dukes' stage, plasma TIMP-1 levels were found to strongly predict prognosis of colorectal cancer patients. Additional studies are needed to validate the clinical usefulness of plasma TIMP-1 measurements.

Adult↗
PubMed1999

Primary megakaryocytes reveal a role for transcription factor NF-E2 in integrin alpha IIb beta 3 signaling.

Platelet integrin alphaIIbbeta3 responds to intracellular signals by binding fibrinogen and triggering cytoskeletal reorganization, but the mechanisms of alphaIIbbeta3 signaling remain poorly understood. To better understand this process, we established conditions to study alphaIIbbeta3 signaling in primary murine megakaryocytes. Unlike platelets, these platelet precursors are amenable to genetic manipulation. Cytokine-stimulated bone marrow cultures produced three arbitrary populations of alphaIIbbeta3-expressing cells with increasing size and DNA ploidy: small progenitors, intermediate-size young megakaryocytes, and large mature megakaryocytes. A majority of the large megakaryocytes bound fibrinogen in response to agonists, while almost none of the smaller cells did. Fibrinogen binding to large megakaryocytes was inhibited by Sindbis virus-mediated expression of isolated beta3 integrin cytoplasmic tails. Strikingly, large megakaryocytes from mice deficient in the transcription factor NF-E2 failed to bind fibrinogen in response to agonists, despite normal surface expression of alphaIIbbeta3. Furthermore, while megakaryocytes from wild-type mice spread on immobilized fibrinogen and exhibited filopodia, lamellipodia and Rho-dependent focal adhesions and stress fibers, NF-E2-deficient megakaryocytes adhered poorly. These studies establish that agonist-induced activation of alphaIIbbeta3 is controlled by NF-E2-regulated signaling pathways that mature late in megakaryocyte development and converge at the beta3 cytoplasmic tail. Megakaryocytes provide a physiologically relevant and tractable system for analysis of bidirectional alphaIIbbeta3 signaling.

Animals↗
PubMed1999

Genetic definition and sequence analysis of Arabidopsis centromeres.

High-precision genetic mapping was used to define the regions that contain centromere functions on each natural chromosome in Arabidopsis thaliana. These regions exhibited dramatic recombinational repression and contained complex DNA surrounding large arrays of 180-base pair repeats. Unexpectedly, the DNA within the centromeres was not merely structural but also encoded several expressed genes. The regions flanking the centromeres were densely populated by repetitive elements yet experienced normal levels of recombination. The genetically defined centromeres were well conserved among Arabidopsis ecotypes but displayed limited sequence homology between different chromosomes, excluding repetitive DNA. This investigation provides a platform for dissecting the role of individual sequences in centromeres in higher eukaryotes.

Arabidopsis↗