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Biomedical subjects

D Owen

Publications and source records attributed to D Owen.

At least 37 records · Page 2Linked to original sources
PubMed2001

Dysregulation of integrin-linked kinase (ILK) signaling in colonic polyposis.

Mutation of the adenomatous polyposis coli (APC) gene and the subsequent dysregulation of beta-catenin are well-documented abnormalities in familial adenomatous polyposis (FAP), as well as sporadic polyposis. Intriguingly, overexpression of the integrin-linked kinase (ILK) has been shown to modulate beta-catenin subcellular localization and function. However, the significance of this finding for human carcinogenesis remains unclear. Here, we report the increased biochemical activity and expression of ILK protein in polyps from FAP patients. Furthermore, dramatic increases in ILK immunoreactivity were observed in all abnormal crypts from sporadic polyps, when compared with the normal appearing crypts within the same resected specimens. As sulindac and aspirin are the two most important therapeutic/chemopreventative agents demonstrated in colorectal carcinogenesis, in both humans and animals, further investigation revealed that these non-steroidal anti-inflammatory drugs (NSAIDs) target ILK and ILK-mediated events in vivo. These include inhibition of, both the biochemical activation of ILK, inhibition of serine 9 GSK3beta phosphorylation and the enhancement of TCF-4 transcriptional activity. In conclusion, ILK protein hyperexpression appears to be an early event in colonic polyposis. Additionally, ILK signaling is shown to undergo modulation by sulindac (and aspirin) for the first time, indicating that it is likely to be one of the targets affected by these agents in vivo.

Adenomatous Polyposis Coli↗
PubMed2001

Evidence that inhibition of cathepsin-B contributes to the neuroprotective properties of caspase inhibitor Tyr-Val-Ala-Asp-chloromethyl ketone.

During the use of tetrapeptide and other proprietary caspase inhibitors in the study of neurodegeneration, we had concluded that mechanisms other than the inhibition of caspases contributed to the protective effects of certain caspase inhibitors. Here we report our studies to identify a target for and hence a mechanism by which the tetrapeptide inhibitor tyrosine-valine-alanine-aspartate-chloromethyl ketone (Ac-YVAD-cmk) is able to rescue neuronal cell cultures from cell death. Ac-YVAD-cmk rescued neuronal cells from cell death in response to oxidative stress and oxygen/glucose deprivation. Affinity labeling with biotinylated YVAD-cmk demonstrated distinct binding proteins for the inhibitor in cells from the central nervous system versus Jurkat cells. Binding to the novel target protein was displaced by class-specific protease inhibitors and suggested that the target is a cysteine protease. Affinity purification and sequencing identified the target as cathepsin-B. Cathepsin-B inhibitors competed with biotinylated YVAD-cmk for the target protein. The availability of the target for binding was reduced in cells that had been rescued by unlabeled inhibitor. Cathepsin-B inhibitors rescue hippocampal slices from cell death induced by oxygen/glucose deprivation. These data provide evidence to support a role for cathepsin-B in neuronal cell death, particularly that following ischemia.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Complementary and alternative medicine (CAM) in the undergraduate medical curriculum: the Southampton experience.

OBJECTIVE: We describe the planning, development and evaluation of a special study module (SSM) on Complementary and Alternative Medicine (CAM) as part of the Southampton Medical School undergraduate curriculum. AIM: To address how the module was received by students, the themes that emerged, the effect on student attitudes to CAM and how the teaching aims developed into learning objectives. METHOD: Student feedback questionnaires and regular teaching staff meetings over a period of 3 years. RESULTS: The course resulted in a significant change in student attitudes to CAM and was well received by the students. It offered a reflective insight into conventional medicine. We have developed and validated core teaching objectives. CONCLUSIONS: We hope that our well validated and well received core teaching objectives will provide a broadly applicable base for those who wish to run similar courses.

Complementary Therapies↗
PubMed2001

ARP, a peptide derived from the stress-associated acetylcholinesterase variant, has hematopoietic growth promoting activities.

BACKGROUND: Psychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated "readthrough" acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses. MATERIALS AND METHODS: We studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD34 cells of human origin. We employed in situ hybridization and immunocytochemical staining to monitor gene expression, and 5-bromo-2-deoxyuridine (BrdU), primary liquid cultures, and clonogenic progenitor assays to correlate AChE-R and ARP with proliferation and differentiation of hematopoietic progenitors. RESULTS: We identified two putative glucocorticoid response elements in the human ACHE gene encoding AChE. In human CD34+ hematopoietic progenitor cells, cortisol elevated AChE-R mRNA levels and promoted hematopoietic expansion. In mice, a small peptide crossreacting with anti-ARP antiserum appeared in serum following forced swim stress. Ex vivo, ARP was more effective than cortisol and equally as effective as stem cell factor in promoting expansion and differentiation of early hematopoietic progenitor cells into myeloid and megakaryocyte lineages. CONCLUSIONS: Our findings attribute a role to AChE-R and ARP in hematopoietic homeostasis following stress, and suggest the use of ARP in clinical settings where ex vivo expansion of progenitor cells is required.

Acetylcholinesterase↗
PubMed2000

Phosphoisoprenoid binding specificity of geranylgeranyltransferase type II.

Geranylgeranyltransferase type II (GGTase-II) modifies small monomeric GTPases of the Rab family by attaching geranylgeranyl moieties onto two cysteines of their C-terminus. We investigated to what extent GGTase-II discriminates between its native substrate geranylgeranyl pyrophosphate (GGpp) and other phosphoisoprenoids, including farnesyl pyrophosphate (Fpp). On the basis of a novel fluorescent assay, we demonstrated that GGpp binds to GGTase-II with an affinity of 8 +/- 4 nM, while Fpp is bound less strongly (K(d) = 60 +/- 8 nM). Analysis of the binding kinetics of four different phosphoisoprenoids indicated that in all cases association is rapid, with rate constants in the range of 0.15 nM(-1) s(-1). In contrast, the dissociation rates differed greatly, depending on the phosphoisoprenoid used, with weak binding substrates generally displaying an increased rate of dissociation. The affinity of GGpp and Fpp for GGTase-II was also determined in the presence of the Rab7-REP-1 complex. The affinity for GGpp was essentially unaffected by the presence of the complex; Fpp on the other hand bound less strongly to the GGTase-II under these conditions, resulting in a K(d) of 260 +/- 60 nM. In vitro prenylation experiments were used to establish that Fpp not only does bind to GGTase-II but also is transferred with an observed rate constant of 0.082 s(-1) which is very similar to that of GGpp. The implications of the low level of discrimination by GGTase-II for the in vivo specificity of the enzyme and the use of farnesyltransferase inhibitors in anti-cancer therapy are discussed.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Malignant melanoma in patients with multiple endocrine neoplasia type 1 and involvement of the MEN1 gene in sporadic melanoma.

Multiple endocrine neoplasia type 1 (MEN 1) is a familial cancer syndrome associated primarily with endocrine tumors of the parathyroids, enteropancreas and anterior pituitary. However, tumors of mesenchymal origin such as angiofibroma and collagenoma of the skin have also been associated with the syndrome. This highlights the possibility of an association between MEN 1 and some other types of tumors. Here we report 7 cases of primary malignant melanoma occurring in 7 MEN 1 families, all patients exhibiting classic features of MEN 1. Based on these findings and the previous implication of multiple melanoma tumor suppressor(s) in 11q, including the MEN1 region, we have investigated the involvement of the MEN1 gene in melanoma tumorigenesis. Mutation analysis was performed on a panel of 39 sporadic metastatic melanomas, 13 melanoma cell lines and 20 melanoma families without CDKN2A or CDK4 germline mutations. In addition, 19 sporadic metastatic tumors were screened for loss of heterozygosity (LOH) in 11q13. LOH was detected in 6 tumors (32%), and in 4 of the tumors the pattern of LOH suggested that the deletion included the MEN1 gene locus. A novel somatic nonsense mutation in exon 7 (Q349X) was identified in 1 sporadic tumor which also showed loss of the wild-type allele. We conclude that the MEN1 gene plays a role in the tumorigenesis of a small subgroup of melanoma.

Adult↗
PubMed2000

Residues in Cdc42 that specify binding to individual CRIB effector proteins.

Cdc42 is a member of the Rho family of small G proteins. Signal transduction events emanating from Cdc42 lead to cytoskeletal rearrangements, cell proliferation, and cell differentiation. Many effector proteins have been identified for Cdc42; however, it is not clear how certain effectors specifically recognize and bind to Cdc42, as opposed to Rac or Rho, or in many cases, which effector controls what cellular events. Mutations were introduced into Cdc42 at residues: Met1, Val8, Phe28, Tyr32, Val33, Thr35, Val36, Phe37, Asp38, Tyr40, Val42, Met45, Ile46, Glu127, Ala130, Asn132, Gln134, Lys135, and Leu174. Measurements were made of their equilibrium binding constants to the Cdc42 binding domains of the CRIB effectors ACK, PAK, and WASP and to the GTPase-activating protein Rho GAP. Generally, mutations in the effector loop have an equally deleterious effect on binding to all CRIB proteins tested, though the F37A mutation resulted in significant selectivity. Residues outside the effector loop were found to be important for binding of Cdc42 to CRIB containing proteins and also to contribute to selectivity. Mutations such as V42A and L174A resulted in large, selective changes in binding to specific CRIB effectors. Neither mutation resulted in alteration in PAK binding, whereas both severely disrupt binding to ACK and only L174A disrupted binding to WASP. These mutations are interpreted using the structures of the Cdc42/ACK and Cdc42/WASP complexes to give insight into how effectors can specifically recognize Cdc42. Those mutations in Cdc42 that inhibit certain interactions, while retaining others, should aid investigations of the role of specific effectors in Cdc42 signaling in vivo.

Amino Acid Motifs↗
PubMed2000

Structure of Cdc42 bound to the GTPase binding domain of PAK.

The Rho family GTPases, Cdc42, Rac and Rho, regulate signal transduction pathways via interactions with downstream effector proteins. We report here the solution structure of Cdc42 bound to the GTPase binding domain of alphaPAK, an effector of both Cdc42 and Rac. The structure is compared with those of Cdc42 bound to similar fragments of ACK and WASP, two effector proteins that bind only to Cdc42. The N-termini of all three effector fragments bind in an extended conformation to strand beta2 of Cdc42, and contact helices alpha1 and alpha5. The remaining residues bind to switches I and II of Cdc42, but in a significantly different manner. The structure, together with mutagenesis data, suggests reasons for the specificity of these interactions and provides insight into the mechanism of PAK activation.

Amino Acid Motifs↗
PubMed2000

Complementary medicine: the Southampton undergraduate experience.

We describe our experiences in the development of a complementary and alternative medicine familiarization special study module for a multidisciplinary group of students, involving medical students, nurses and chiropractors. Our observations suggest that the model we have developed is an effective, interesting and exciting experience for both students and teachers. We have defined, by consensus between students and teachers, a series of clear educational objectives. The course is now established, having run for 4 academic years, but we are concerned about its sustainability, particularly in relation to ongoing funding.

Chiropractic↗
PubMed2000

Denudations as paracellular routes for alphafetoprotein and creatinine across the human syncytiotrophoblast.

We tested two hypotheses: 1) that fibrin-containing fibrinoid-filled denudations of the syncytiotrophoblast may provide a route for paracellular diffusion and 2) that placentas from women who had elevated maternal serum alphafetoprotein (MSAFP) in midgestation had raised permeability to AFP and greater denudation than in normal pregnancy. We measured AFP and creatinine clearance across term placental cotyledons from the above groups and used light microscope morphometric analysis to determine the volume density of fibrin-containing fibrinoid deposits. There was no significant difference between the two groups in terms of AFP and creatinine clearance or volume density of fibrin-containing fibrinoid deposits. The combined data showed a significant (P < 0.05) positive correlation between creatinine clearance, but not AFP clearance, and volume density of fibrin-containing fibrinoid. We conclude that syncytiotrophoblast denudations, with associated fibrinoid, do provide a route for diffusion of small hydrophilic solutes, but that other anatomic features of the placenta are rate limiting for transfer of AFP and similarly sized molecules.

Creatinine↗
PubMed2000

Granulomatous osteonecrosis in Crohn's disease.

A 25-year-old white woman was diagnosed with Crohn's disease involving the small and large intestines. She had a complex clinical course that required treatment with multiple pharmacological agents, including intravenous, oral and rectal corticosteroids. She also received parenteral nutrition with lipid emulsions. Finally, repeated intestinal resections and drainage of perianal abscesses were required. Her disease was complicated by gallstones, urolithiasis and hip pain. After osteonecrosis was diagnosed, joint replacements were performed. Review of the pathological sections from the resected hip, however, resulted in detection of granulomatous inflammation with multinucleated giant cells - the histological 'footprint' of Crohn's disease in the gastrointestinal tract. Because prior specialized perfusion fixation pathological studies of the intestine in Crohn's disease have shown that granulomas are located in the walls of blood vessels, a possible mechanism for the pathogenesis of osteonecrosis in Crohn's disease is chronic microvascular ischemia of bone.

Adult↗
PubMed1999

Norepinephrine transporter gene polymorphism is not associated with susceptibility to major depression.

The monoamine neurotransmitters serotonin, norepinephrine and dopamine have been implicated in the pathogenesis of depression, schizophrenia and mood disorders. The mechanism of action of certain antidepressant drugs, particularly the tricyclics and the newly available norepinephrine and serotonin reuptake inhibitors (NSRIs) drugs, venlafaxine and nefazodone, suggest that the norepinephrine transporter, which is a target for these antidepressant drugs, and its malfunction may be involved in major depression. In this association study, we tested the hypothesis that variants of the human norepinephrine transporter (NET) gene confer susceptibility to major depression. One hundred and five patients with major depression and 74 unrelated matched controls were analyzed for a silent 1287G/A polymorphism (NET-8) in exon 9 of the NET gene. No significant differences in genotype or allele frequencies were found between controls and patients, nor between subgroups of depressed patients classified by suicidal ideation. In addition, 60 controls and 60 patients were genotyped for a missense substitution Thr99Ile in exon 2 of the NET gene (NET-1), but only one control was heterozygous for this variant. These results suggest that the NET gene is unlikely to be involved in the susceptibility to major depression.

Adult↗
PubMed1999

Structure of the small G protein Cdc42 bound to the GTPase-binding domain of ACK.

The proteins Cdc42 and Rac are members of the Rho family of small GTPases (G proteins), which control signal-transduction pathways that lead to rearrangements of the cell cytoskeleton, cell differentiation and cell proliferation. They do so by binding to downstream effector proteins. Some of these, known as CRIB (for Cdc42/Rac interactive-binding) proteins, bind to both Cdc42 and Rac, such as the PAK1-3 serine/threonine kinases, whereas others are specific for Cdc42, such as the ACK tyrosine kinases and the Wiscott-Aldrich-syndrome proteins (WASPs). The effector loop of Cdc42 and Rac (comprising residues 30-40, also called switch I), is one of two regions which change conformation on exchange of GDP for GTP. This region is almost identical in Cdc42 and Racs, indicating that it does not determine the specificity of these G proteins. Here we report the solution structure of the complex of Cdc42 with the GTPase-binding domain ofACK. Both proteins undergo significant conformational changes on binding, to form a new type of G-protein/effector complex. The interaction extends the beta-sheet in Cdc42 by binding an extended strand from ACK, as seen in Ras/effector interactions, but it also involves other regions of the G protein that are important for determining the specificity of effector binding.

Amino Acid Sequence↗
PubMed1999

Denervation of the skin following section of the inferior alveolar nerve leads to increased NGF accumulation without change in NGF mRNA expression.

Following inferior alveolar nerve section and capping of the nerve to prevent regeneration, amounts of nerve growth factor (NGF) and NGF mRNA have been quantified in the chin skin, a discrete target of the nerve. NGF protein in the target region increases rapidly following nerve section and to levels known to induce sprouting. NGF on the ipsilateral side increased many-fold above unoperated control and the contralateral side also increased above control. Measurement of NGF mRNA levels using quantitative Northern analysis revealed, however, that there was no change in the expression levels of NGF mRNA, indicating that the large increase in protein occurs due to a cessation of transport back to the cell bodies of the innervating neurons.

Animals↗
PubMed1999

Semiquantitative polymerase chain reaction enzyme immunoassay for the diagnosis of pertussis.

The two most commonly used targets for diagnosis of pertussis by the polymerase chain reaction have been the pertussis toxin promoter and the repeated insertion sequence IS481. A comparative assessment of these primers was performed on routinely collected nasopharyngeal swabs, stored at -20 C, using novel semiquantitative enzyme immunoassays. Both sets of primers behaved similarly with bacterial suspensions, and the 17 culture-positive nasopharyngeal swabs were also positive with the pertussis toxin promoter primers, with one exception, which had been subject to prolonged storage. Significantly more of the 69 culture-negative swabs were positive with the pertussis toxin promoter primers (n = 36) than with the IS481 primers (n = 18). To determine the effect of inhibitors, a comparative assessment of three primer pairs against human DNA (beta-globin and glyceraldehyde-3-phosphate dehydrogenase) was also performed.

Child↗