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Biomedical subjects

R A Walker

Publications and source records attributed to R A Walker.

At least 73 records · Page 4Linked to original sources
PubMed1998

The erbB/HER type 1 tyrosine kinase receptor family.

The erb type 1 tyrosine kinase receptors are important for mediating proliferation and differentiation, and aberrant activation may contribute to tumour development and progression. They comprise epidermal growth factor receptor (EGFR), c-erbB-2, c-erbB-3, and c-erbB-4. There are overlaps and differences in the cellular distribution of the four receptors, which is of significance since they can form heterodimers that give differing responses to different ligands. The different distributions could be of particular importance in cancer if therapeutic modalities are identified to inhibit or stimulate the different ligands.

Adenocarcinoma↗
PubMed1998

Ca2+/calmodulin regulation of the Arabidopsis kinesin-like calmodulin-binding protein.

The kinesin family motor protein KCBP (kinesin-like calmodulin binding protein) was identified during a screen for Arabidopsis calmodulin-binding proteins [Reddy, et al., 1996b: J. Biol Chem. 271:7052-7060]. KCBP contains a C-terminal motor domain and is unique among kinesin motors in that it has a calmodulin-binding site. We expressed the KCBP motor domain in Escherichia coli and examined its microtubule (MT) binding and ATPase activity. KCBP bound MTs in an ATP-dependent manner and exhibited MT-stimulated ATPase activity. Ca2+/ calmodulin inhibited binding of KCBP to MTs under conditions that normally favor tight motor-MT interactions, and the extent of inhibition was dependent on the concentration of calcium and calmodulin. Ca2+/calmodulin did not affect KCBP's basal ATPase activity, but reduced the motor's MT-stimulated ATPase activity. The substantial reduction in affinity of KCBP for MTs in the presence of Ca2+/calmodulin suggests that Ca2+/calmodulin may modulate the activity of KCBP in vivo by regulating the motor's association with MTs. KCBP is the first MT-dependent motor protein found to be regulated by direct binding of Ca2+/calmodulin to its motor subunit.

Adenosine Triphosphatases↗
PubMed1998

Comparison between oral and intramuscular dexamethasone in suppressing unconjugated estriol levels during the third trimester.

OBJECTIVES: Unconjugated estriol production depends on fetal adrenal androgen precursors. Fetal exposure to exogenous glucocorticoids results in adrenal suppression with a subsequent decrease in maternal serum unconjugated estriol levels. We compared the efficacy between oral and intramuscular dexamethasone in maternal serum unconjugated estriol suppression at 48 hours after the initial dose among women at risk for preterm delivery. STUDY DESIGN: Twenty-four gravidas at risk for preterm delivery were randomized to receive either 6 mg intramuscular or 8 mg oral dexamethasone every 12 hours for a total of 4 doses. Blood samples (9 mL) were obtained before the initial dexamethasone administration and again after the fourth dose. Serum was separated and frozen at -70 degreesC and subsequently underwent batch analysis. Unconjugated estriol levels were determined by radioimmunoassay with intra-assay and interassay coefficients of variation of 7.9% and 5.5%, respectively. All values are reported as mean +/- SD. The primary statistical analysis was a t test, with P <.05 considered significant. RESULTS: At the time of dexamethasone administration, gestational ages in both groups were similar. Predexamethasone and postdexamethasone unconjugated estriol levels were also similar between the intramuscular and oral groups (5.39 +/- 3.99 vs 1.80 +/- 2.49 ng/mL and 6.05 +/- 3.00 vs 1.61 +/- 1.03 ng/mL, respectively, P >.05). No difference in percent decrease in unconjugated estriol levels was found between the intramuscular (0.67 +/- 0.24) and oral (0.65 +/- 0.39) groups. CONCLUSION: Oral dexamethasone (8 mg) produces similar maternal serum unconjugated estriol suppression compared with intramuscular dexamethasone (6 mg) when evaluated 48 hours after administration.

Administration, Oral↗
PubMed1997

Modulation of myoepithelial-associated alpha6beta4 integrin in a breast cancer cell line alters invasive potential.

In normal breast, cell-stromal contact is mediated by myoepithelial cells which strongly express alpha2beta1, alpha3beta1, and alpha6beta4 integrins, while epithelial cells exhibit alpha2beta1 and alpha3beta1 integrins at cell-cell borders, but do not express alpha6beta4 integrin. Breast carcinomas consistently show down-regulation of all integrins. We have investigated the modulatory effect of stromal proteins, hormones, and transforming growth factor beta (TGF-beta) on integrin expression in breast cancer cell lines MCF-7, T47-D, and MDA-MB 231 using indirect immunofluorescence and confocal laser scanning microscopy. MCF-7 and T47-D cells displayed low levels of both alpha2beta1 and alpha3beta1 integrins, and no alpha6beta4 integrin, and this profile remained unchanged by modulatory agents. The MDA-MB 231 cells exhibited stronger staining for alpha2beta1 and alpha3beta1 integrins and focal staining for alpha6beta4 integrin under control conditions, but markedly enhanced reactivity for the alpha6beta4 complex in the presence of TGF-beta. This was associated with acquisition of a spread cellular morphology and localization of alpha6beta4 at the cell periphery in a discrete punctate distribution. There was associated enhanced expression of epiligrin, the ligand for alpha6beta4, with similar localization to the cell periphery. Cell invasion assays through a Matrigel barrier revealed significantly reduced invasive potential of TGF-beta-treated cells, an effect largely reversed following preincubation of the treated cells with anti-beta4 integrin antibody. We conclude that alpha6beta4 integrin can be up-regulated by TGF-beta and has an anti-invasive effect on MDA-MB 231 cells. In addition to alpha6beta4, MDA-MB 231 cells exhibit other myoepithelial markers including cytokeratin 14, vimentin, and weak expression of CALLA. These findings support the concept of a subgroup of breast carcinomas displaying features of myoepithelial differentiation.

Antigens, Surface↗
PubMed1997

A metastable intermediate state of microtubule dynamic instability that differs significantly between plus and minus ends.

The current two-state GTP cap model of microtubule dynamic instability proposes that a terminal crown of GTP-tubulin stabilizes the microtubule lattice and promotes elongation while loss of this GTP-tubulin cap converts the microtubule end to shortening. However, when this model was directly tested by using a UV microbeam to sever axoneme-nucleated microtubules and thereby remove the microtubule's GTP cap, severed plus ends rapidly shortened, but severed minus ends immediately resumed elongation (Walker, R.A., S. Inoué, and E.D. Salmon. 1989. J. Cell Biol. 108: 931-937). To determine if these previous results were dependent on the use of axonemes as seeds or were due to UV damage, or if they instead indicate an intermediate state in cap dynamics, we performed UV cutting of self-assembled microtubules and mechanical cutting of axoneme-nucleated microtubules. These independent methods yielded results consistent with the original work: a significant percentage of severed minus ends are stable after cutting. In additional experiments, we found that the stability of both severed plus and minus ends could be increased by increasing the free tubulin concentration, the solution GTP concentration, or by assembling microtubules with guanylyl-(alpha,beta)-methylene-diphosphonate (GMPCPP). Our results show that stability of severed ends, particularly minus ends, is not an artifact, but instead reveals the existence of a metastable kinetic intermediate state between the elongation and shortening states of dynamic instability. The kinetic properties of this intermediate state differ between plus and minus ends. We propose a three-state conformational cap model of dynamic instability, which has three structural states and four transition rate constants, and which uses the asymmetry of the tubulin heterodimer to explain many of the differences in dynamic instability at plus and minus ends.

Animals↗
PubMed1997

Control of matrix metalloproteinase activity in cancer.

Remodelling of extracellular matrix (ECM) and basement membranes is a key component of the process of tumour cell invasion and metastasis. Matrix metalloproteinases (MMPs) are one of the major classes of enzymes involved in degrading ECM, having different substrate specificities and being inhibited by naturally occurring tissue inhibitors (TIMPs). Elevated levels of MMPs have been associated with poor prognosis for a variety of malignancies. However, the expression and effective action of MMPs are influenced by multiple factors: most are synthesized by stroma rather than tumour cells, suggesting tumour cell-stromal cell co-operation; receptors (MT-MMPs) have to be present on tumour cells for binding and activation of MMP; co-ordination of tissue proteolysis and subsequent integrin binding will aid cell movement through a matrix; integrin receptors can directly moderate the production of MMP. These various components need to be considered when trying to determine the key events regulating matrix proteolysis and hence invasion.

Extracellular Matrix↗
PubMed1997

n-ethylmaleimide and ethacrynic acid inhibit kinesin binding to microtubules in a motility assay.

Treatment of proteins in vitro with sulfhydryl (SH)-reactive compounds has been used successfully to determine protein regions critical for normal function. To probe structure-function relationships in the microtubule (MT) motor kinesin, the motor was treated with two SH reactive compounds, n-ethylmaleimide and ethacrynic acid, and its function was assayed by motility and co-sedimentation techniques. In the motility assay, treatment of kinesin either before or after adsorption to the glass surfaces of a flow cell was found to inhibit the ability of coverslip-bound kinesin to bind to MTs. Inactivation of MT binding was slow, required high molar excess of the SH-reactive drug, and was very sensitive to temperature. Inhibition of MT binding occurred well after complete modification of kinesin light chain, but paralleled modification of the kinesin heavy chain. The results point to a model in which one critical cysteine per kinesin heavy chain is relatively inaccessible to solvent. Surprisingly, when the interaction between modified kinesin and MTs was examined by a co-sedimentation assay, kinesin retained the ability to bind MTs. These contrasting results may be due to conformational differences in the kinesin molecule that exist in the two assays.

Animals↗
PubMed1997

Molecular pathology of breast cancer and its application to clinical management.

Breast cancer is a major cause of morbidity and mortality in women in many parts of the world. Breast carcinomas are heterogenous in their biological and clinical behaviour and a greater understanding of how they develop and progress could lead to more directed forms of screening and therapy. It is important to determine the molecular mechanisms underlying the natural history of breast cancer. Developments in the techniques for molecular analysis have meant that they can now be applied to a large range of clinical material such as cytological preparations and fixed, embedded material, so increasing the potential for relating any molecular alterations to clinical behaviour and response to therapy. In this review we consider recent developments in three areas of importance to breast cancer; genetic analysis-oncogenes, tumour suppressor genes, loss of heterozygosity, microsatellite instability, familial breast cancer; steroid receptors, oestrogen regulated proteins, epidermal growth factor receptor, growth factors particularly transforming growth factor beta; and cell adhesion, invasion and metastasis-E-cadherin, integrins, proteases. These are discussed in relation to potential for screening, prognosis and treatment.

Breast Neoplasms↗
PubMed1997

Expression of chalcone synthase and chalcone isomerase proteins in Arabidopsis seedlings.

Antibodies have been developed against the first two enzymes of flavonoid biosynthesis in Arabidopsis thaliana. Chalcone synthase (CHS) and chalcone isomerase (CHI) were overexpressed and purified from Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. The recombinant proteins were then used to immunize chickens and the resulting IgY fraction was purified from egg yolks. Immunoblots of crude protein extracts from Arabidopsis seedlings carrying wild-type and null alleles for CHS and CHI showed that the resulting antibody preparations provide useful tools for characterizing expression of the flavonoid pathway at the protein level. An initial analysis of expression patterns in seedlings shows that CHS and CHI proteins are present at high levels during a brief period of early seedling germination that just precedes the transient accumulation of flavonoid end-products.

Acyltransferases↗
PubMed1997

Loss of heterozygosity at chromosome 6q in preinvasive and early invasive breast carcinomas.

We have used polymerase chain reaction (PCR) analysis to study the incidence of allelic imbalance at four polymorphic microsatellite markers on chromosome 6q25.1-27, three dinucleotide repeats and one trinucleotide repeat, for microdissected tumour foci from a group of 75 'early' breast carcinomas. The tumours comprised 16 preinvasive cases of ductal carcinoma in situ (DCIS) and 59 mammographically detected early invasive carcinomas. Loss of heterozygosity (LOH) was detected at all four loci and in all types and grade of disease. The frequency of LOH ranged from 23% to 50% depending on the marker studied. The highest frequency of LOH was observed at the D6S186 locus for the cases of DCIS and at the oestrogen receptor locus for the invasive carcinomas. These data suggest that the inactivation of tumour-suppressor genes within this region on chromosome 6q is important for the development of these early lesions.

Adenocarcinoma↗
PubMed1997

Lack of cyclin E immunoreactivity in non-malignant breast and association with proliferation in breast cancer.

Cyclin E is a G1 cyclin that is essential for the transition from G1 to S phase in the cell cycle. Alterations to cyclin E expression or regulation could be important in tumorigenesis. Previous immunohistochemical and immunoblotting studies have investigated the expression of cyclin E in breast carcinomas. In this study, cyclin E has been investigated in a range of non-malignant and malignant breast using immunohistochemistry. Normal and benign tissue from pre- and post-menopausal women (39 cases), non-involved tissue from cancer-containing breasts (47 cases), ductal carcinoma in situ (22 cases) and invasive breast carcinomas (109 cases) have been examined. There was no reactivity in any of the non-malignant breast. Only one ductal carcinoma in situ contained more than 5% reactive cells. A total of 28% of invasive carcinomas had > 5% of reactive cells (range 0-88% positive cells, mean 12.59%, median 1.0%). A significant association was found with poorer differentiation (P < 0.001), high MIB1 index (P < 0.001), lack of oestrogen receptor (0.05 > P > 0.025) and the presence of p53 protein (0.05 > P > 0.025). Virtually all cases with cyclin E and p53 were poorly differentiated. The presence of cyclin E is therefore only found in breast malignancies and is associated with more aggressive features, including high proliferation.

Adult↗
PubMed1997

Loss of heterozygosity at the mannose 6-phosphate insulin-like growth factor 2 receptor gene correlates with poor differentiation in early breast carcinomas.

Chromosome 6q has been shown to be one of the most frequent sites for allelic loss in human breast cancer. The mannose 6-phosphate/insulin-like growth factor 2 receptor (IGF2R) gene, which maps to chromosome 6q26-27, functions in the activation of TGF-beta1, a potent growth inhibitor for most cell types, the degradation of the mitogen IGF2 and the intracellular trafficking of lysosomal enzymes. Loss of heterozygosity (LOH) at the IGF2R locus with mutations in the remaining allele have been reported in liver cancers and recently in two high-grade cases of ductal carcinoma in situ of the breast. We have sought to confirm that allelic loss of IGF2R is an early event in the aetiology of breast cancer by screening a group of 'early' lesions for LOH at a polymorphic microsatellite marker within the IGF2R gene using polymerase chain reaction (PCR). Several microdissected tumour foci were analysed for each of 40 mammographically detected invasive carcinomas and 22 cases of pure ductal carcinoma in situ (DCIS). None of 25 (62.5%) informative early invasive carcinomas showed any evidence of LOH. This group comprised predominantly of well- to moderately differentiated cases (95%). However, 4 out of 18 informative DCIS cases (22%) showed clear evidence of LOH. Three of these were poorly differentiated (high-grade) lesions. These data suggest that loss of heterozygosity at the IGF2R gene is associated with poor differentiation at this early stage of breast cancer development and progression.

Breast Neoplasms↗
PubMed1997

A minimally obtrusive, secure mask for prevention of access to the mouth.

Several childhood neurological conditions can lead to problems with unintended access to the mouth. Earlier workers have demonstrated benefits in the use of a helmet restricting mouth access, within a behavioural modification programme. Conventionally, such headgear is very obtrusive. This paper presents a simple technique using conventional plaster-based moulding, which has been successfully used in making secure, minimally obtrusive masks. Masks produced for two children have been readily accepted and have been successful. Any such headgear should be used only within a managed behavioural modification programme.

Adolescent↗
PubMed1996

Intraoperative duplex scanning as a means of quality control during carotid endarterectomy.

OBJECTIVES: To identify correctable technical errors following carotid endarterectomy using intraoperative colour duplex sonography (ATL, UM9, HDI). Results were compared with intraoperative flow measurements using an operative flow meter and with middle cerebral artery velocity measured by trans-cranial Doppler (TCD). DESIGN: Prospective study. MATERIALS AND METHODS: 50 consecutive patients undergoing carotid endarterectomy were investigated. Follow-up was performed at 6 weeks using duplex scanning and clinical evaluation. RESULTS: Significant intraoperative technical errors were detected in three patients and were re-explored. Two scans demonstrated kinking or pinching at the distal endarterectomy site requiring patch-plasty and the third revealed a large mass of intramural thrombus. A further 18 endarterectomies yielded 21 additional minor abnormalities. CONCLUSIONS: Duplex sonography provides a sensitive intraoperative technique for detecting thrombus and technical errors. It yields both anatomic and hemodynamic details and is superior to intraoperative flow measurements and transcranial doppler.

Aged↗