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R A Walker

Publications and source records attributed to R A Walker.

At least 127 records · Page 7Linked to original sources
PubMed1991

Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate.

Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build-up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP-tubulin association.

Guanosine Triphosphate↗
PubMed1991

Loss of chromosome 17p13 sequences and mutation of p53 in human breast carcinomas.

DNAs from ninety seven primary breast carcinoma biopsies have been examined for loss of sequences on 17p13. In addition, immunohistochemical analysis has been carried out on the majority of these cases to determine whether p53 gene expression can be detected. Detection of p53 expression is taken to indicate mutation of p53 leading to stabilisation of the protein and thus detectable levels of p53 in the cell. In 86% of breast carcinoma samples where both allele loss and expression data were available, loss of sequences on 17p13 and/or expression of p53 was detected. Alterations to p53, whether loss of one allele or mutation, are therefore by far the most common changes so far detected in primary human breast tumours. In three cases where expression of p53 could be detected by immunohistochemistry, the precise mutation to p53 was identified. All three mutations fall within the regions which are highly conserved in p53, encoded by exons 5 to 8. Two are single base changes leading to misense mutations, and the third is a single base-pair deletion. The expression of the latter gene would result in production of a truncated protein which should lack normal biological activities.

Alleles↗
PubMed1990

The Drosophila claret segregation protein is a minus-end directed motor molecule.

A product encoded at the claret locus in Drosophila is needed for normal chromosome segregation in meiosis in females and in early mitotic divisions of the embryo. The predicted amino-acid sequence of the segregation protein was shown recently to be strikingly similar to Drosophila kinesin heavy chain. We have expressed the claret segregation protein in bacteria and have found that the bacterially expressed protein has motor activity in vitro with several novel features. The claret motor is slow (4 microns min-1), unlike either kinesin or dyneins. It has the directionality, the ability to generate torque and the sensitivity to inhibitors reported previously for dyneins. The finding of minus-end directed motor activity for a protein with sequence similarity to kinesin suggests that the dynein and kinesin motor domains are ancestrally related. The minus-end directed motor activity of the claret motor is consistent with a role for this protein in producing chromosome movement along spindle microtubules during prometaphase and/or anaphase.

Adenosine Triphosphatases↗
PubMed1990

Effects of magnesium on the dynamic instability of individual microtubules.

We investigated the effect of magnesium ion (Mg) on the parameters of dynamic instability of individual porcine brain microtubules. Rates of elongation and rapid shortening were measured by using video-enhanced DIC light microscopy and evaluated by using computer-generated plots of microtubule length vs time. Increasing [Mg] from 0.25 to 6 mM increased the second-order association rate constant for elongation about 25% at each end. At plus ends, this resulted in a 1.5-2-fold increase in elongation rates over the tubulin concentrations explored. Rapid shortening rates were more dramatically affected by Mg. As [Mg] was increased from 0.25 to 6 mM, the average rate of rapid shortening increased about 3-fold at plus ends and 4-5-fold at minus ends. The ends had roughly equivalent average rates at low [Mg], of 30-45 microns/min. At any Mg concentration, rates of disassembly varied from one microtubule to another, and often an individual microtubule would exhibit more than one rate during a single shortening phase. Individual rates at 6 mM Mg varied from 12 to 250 microns/min. Over the concentration range explored, Mg affected the frequencies of transition from elongation to shortening and back only at minus ends. Minus ends were relatively stable at low [Mg], having 4 times the frequency of rescue than at high [Mg], and a lower frequency of catastrophe (particularly evident at low tubulin concentrations). Plus ends, surprisingly, were highly unstable at all Mg concentrations investigated, having about the same transition frequencies as did the least stable (high Mg) minus ends. Our results have implications for models of the GTP cap, again emphasizing that GTP caps cannot build up in proportion to elongation rate, and must be constrained to the tips of growing microtubules.

Animals↗
PubMed1990

Ultrastructural localization of milk fat globule membrane antigens in human breast carcinomas.

The localization of milk fat globule membrane components has been assessed using post-fixation immunoelectron microscopy with three different antibodies for a group of breast carcinomas of different type and histological differentiation. For well differentiated carcinomas localization was in relation to the cell membrane, with polarization being evident in a proportion of cases. Moderately differentiated carcinomas showed a combined picture of cell membrane, vesicular, and intracytoplasmic luminal localization. The latter is a feature of infiltrating lobular carcinomas. Poorly differentiated carcinomas exhibited vesicular labelling throughout the cytoplasm, with no cell membrane localization. No labelling was seen over endoplasmic reticulum. It is proposed that carcinomas exhibit defects in intracellular transport of milk fat globule membrane components resulting in failure of expression at the cell surface and accumulation of vesicles within the cytoplasm, the extent of change relating to tumour differentiation.

Antigens, Neoplasm↗
PubMed1990

Post embedding immunoelectron microscopy of human breast cancer: a comparison of three acrylic resins.

The suitability of three acrylic resins for the immunoelectron microscopical localization of cell surface and cytoskeletal antigens in surgically excised, immersion fixed human breast cancer, using an immunogold system, has been assessed. Good localization of milk fat globule membrane was achieved with LR White, LR Gold and Lowicryl K11M, although the embedding schedule for LR White had to be modified. The best results were achieved with Lowicryl K11M. Only scanty labelling of actin and cytokeratin was seen in LR White embedded tissue, whereas there was clear localization in LR Gold and Lowicryl K11M embedded samples. Tubulin and alpha-actinin was detected at low level in tissues in the low temperature embedding resins, but not in LR White embedded samples. The morphology of the latter was poorer, and there was greater variability in ultrastructure and labelling. Of the two low temperature embedding resins, Lowicryl K11M gave slightly better results. However, the advantages could be outweighed by the problem incurred in achieving the low temperatures, and by poorer handling properties than LR Gold.

Acrylic Resins↗
PubMed1990

A sequence previously identified as metastasis-related encodes an acidic ribosomal phosphoprotein, P2.

We have used a metastasis-related human cDNA isolated from a liver metastasis from a colonic adenocarcinoma to screen a human breast carcinoma cDNA library for homologous sequences. Nucleotide sequence analysis of positive clones revealed that the cDNA represents a ribosomal phosphoprotein. P2. The expression of P2 mRNA was significantly higher (Student's t test, one tail; P less than or equal to 0.01) in seven fibroadenomas than in seven carcinomas, with an average five-fold difference. This enhanced expression level P2 mRNA in benign fibroadenomas compared with malignant carcinomas is contrary to that expected, based on earlier work with normal colonic mucosa, colorectal carcinoma and hepatic metastasis. The identification of gene transcripts which differ in abundance and correlate with the metastatic phenotype may be of considerable importance both as diagnostic aids and in defining the changes associated with tumour progression and metastasis at the molecular level. The possible role that ribosomal proteins may play in the progression of carcinoma of the breast is discussed.

Base Sequence↗
PubMed1990

Is apocrine differentiation in breast carcinoma of prognostic significance?

Apocrine differentiation in human breast cancers has been assessed using immunohistochemistry to detect zinc alpha 2 glycoprotein and the findings related to standard prognostic factors, disease free interval (DFI) and survival in 145 women with early breast cancer. Breast tumour samples from women with a minimum follow-up of 5 years were assessed. Routinely fixed and processed tissue was used throughout. Sixty-six (45%) tumours did not stain with the antibody. Fifty-two (36%) exhibited positive apocrine staining while for 27 (19%) only a few cells were reactive. The presence of apocrine differentiation was unrelated to lymph node status, menstrual status, tumour grade or size, oestrogen receptor (E2R) or progesterone receptor status. However, patients whose tumours exhibited apocrine staining had a shorter disease-free interval (DFI) (P = 0.03) and survival (P = 0.03). A Cox's multiple regression analysis of the data found that the presence of staining added significantly (P = 0.047) to the predictive value of node status (P = 0.0001), menstrual status (P = 0.0001), tumour size (P = 0.0026) and E2R status (P = 0.0014) for patient survival. The other seven prognostic factors tested did not reach significance and were rejected from the model. Apocrine differentiation in breast cancer appears to be an independent predictor of poor prognosis tumours.

Biomarkers, Tumor↗
PubMed1990

Assessment of milk fat globule membrane antibodies and lectins as markers of short-term prognosis in breast cancer.

The milk fat globule membrane antibodies HMFG1, HMFG2, NCRC 11 and four of the Mam 6 series, and the lectins peanut agglutinin, wheat germ agglutinin, Concanavalin A, Lotus tetragonolobus and Ulex europaeus I have been applied to 115 stage I and II breast carcinomas (median follow up = 36 months) to assess their value as prognostic markers. Of the milk fat globule membrane antibodies only NCRC 11 staining showed a relationship to development of recurrent disease and overall survival, but this did not act as an independent indicator over and above that provided by histological grade. None of the lectins gave prognostic information, including those whose binding related to node status or grade. It is concluded that for short-term prognosis none of the markers can given independent prognostic information over and above that provided by histological evaluation.

Antibodies↗
PubMed1990

Immunohistological distribution of the secretory endometrial protein, 'pregnancy-associated endometrial alpha 2-globulin', a glycosylated beta-lactoglobulin homologue, in the human fetus and adult employing monoclonal antibodies.

We have previously demonstrated that pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG), the human glycosylated beta-lactoglobulin homologue (HG-BLG), is quantitatively the major secretory soluble protein product of the secretory endometrium during the latter half of the menstrual cycle and decidua spongiosa of the gestational endometrium during early pregnancy, and is principally localized to the glandular epithelium. In the present study employing monoclonal antibodies in immunohistological techniques, the distribution and localization has been examined in normal and pathological tissues of the adult and first-trimester fetus. No significant staining for alpha 2-PEG was detected in any non-reproduction-associated tissue in the normal adult nor any tissue in the fetus. In the adult, most intense staining was associated with the endometrial glandular epithelium in the uterus or in ectopic sites in patients with endometriosis. During the menstrual cycle and pregnancy, appearance of alpha 2-PEG in endometriosis was strongly linked with its appearance in uterine endometrial tissue, suggesting that endometriotic tissue exhibited competence to respond to the same hormonal milieu required to induce synthesis in the uterine endometrium. Localization to the mucosal epithelium of the Fallopian tube was consistent with synthesis of alpha 2-PEG, albeit at low levels, and staining at this site reflected fluctuations of staining within the uterus. Of the pathological specimens examined, staining was only detected in a proportion of ovarian carcinomas. No staining was detected in the mammary gland, a site of beta-lactoglobulin synthesis, whether obtained during pregnancy or lactation.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1990

Immunohistological distribution of the secretory endometrial protein, 'pregnancy-associated endometrial alpha 2-globulin', a glycosylated beta-lactoglobulin homologue, in the human fetus and adult employing monoclonal antibodies.

We have previously demonstrated that pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG), the human glycosylated beta-lactoglobulin homologue (HG-BLG), is quantitatively the major secretory soluble protein product of the secretory endometrium during the latter half of the menstrual cycle and decidua spongiosa of the gestational endometrium during early pregnancy, and is principally localized to the glandular epithelium. In the present study employing monoclonal antibodies in immunohistological techniques, the distribution and localization has been examined in normal and pathological tissues of the adult and first-trimester fetus. No significant staining for alpha 2-PEG was detected in any nonreproduction-associated tissue in the normal adult nor any tissue in the fetus. In the adult, most intense staining was associated with the endometrial glandular epithelium in the uterus or in ectopic sites in patients with endometriosis. During the menstrual cycle and pregnancy, appearance of alpha 2-PEG in endometriosis was strongly linked with its appearance in uterine endometrial tissue, suggesting that endometriotic tissue exhibited competence to respond to the same hormonal milieu required to induce synthesis in the uterine endometrium. Localization to the mucosal epithelium of the Fallopian tube was consistent with synthesis of alpha 2-PEG, albeit at low levels, and staining at this site reflected fluctuations of staining within the uterus. Of the pathological specimens examined, staining was only detected in a proportion of ovarian carcinomas. No staining was detected in the mammary gland, a site of beta-lactoglobulin synthesis, whether obtained during pregnancy or lactation.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1990

Human 'pregnancy-associated endometrial alpha 1-globulin', a 32 kDa insulin-like growth factor-binding protein: immunohistological distribution and localization in the adult and fetus.

We have previously shown that pregnancy-associated alpha 1-globulin, a small molecular weight (32 kDa) insulin-like growth factor-binding protein (IGF-BP), is quantitatively the major secretory protein product of the decidualized endometrium during human pregnancy and is localized principally in the decidual cell. In the present study, employing monoclonal antibodies in immunohistological techniques, the distribution and localization of IGF-BP has been examined in normal and pathological tissues of the adult and first trimester fetus. In the adult, most intense reactivity was associated with endometrial stroma and their derived decidual cells in the uterus or in ectopic sites in patients with endometriosis. During the menstrual cycle, the appearance of IGF-BP in endometriotic tissue was linked with its appearance in uterine endometrial tissue. The only other adult cells where significant staining was detected was in the luteal cells of the corpus luteum. Production of the protein was not a feature of carcinomas. In the fetus, the protein was localized in lymphoid-myeloid progenitor cells and hepatocytes of the liver and at lower levels in testicular Leydig cells and adenocortical cells. These observations suggest highly specific tissue expression of this protein and support a specialized role for this protein in progenitor cells of the lymphomyeloid system, in certain steroid hormone-producing cells and in the decidual cell in pregnancy.

Endometrium↗
PubMed1989

Stabilization of microtubules by tubulin-GDP-Pi subunits.

Microtubule dynamic instability has been accounted for by assuming that tubulin subunits at microtubule ends differ from the tubulin-GDP subunits that constitute the bulk of the microtubule. It has been suggested that this heterogeneity results because ends contain tubulin subunits that have not yet hydrolyzed an associated GTP molecule. Alternatively, in a recent model it was proposed that ends contain tubulin-GDP-Pi subunits from which Pi has not yet dissociated. The models differ in their predicted response to added ligands: because GDP in subunits in microtubules does not exchange with nucleotide in solution, the heterogeneity from a tubulin-GTP cap will not be eliminated by added GTP; however, the dissociability of Pi in tubulin-GDP-Pi subunits will allow a heterogeneity resulting from a tubulin-GDP-Pi cap to be eliminated by added excess Pi. Elimination of the heterogeneity is expected to be manifested by an elimination of dynamic instability behavior. Using video microscopy to study the kinetic behavior of individual microtubules under reaction conditions where dynamic instability is the dominant mechanism for microtubule length changes, we have determined the effects of 0.167 M Pi on the rate of subunit addition in the elongation phase, the rate of subunit dissociation in the rapid shortening phase, and the rates of the phase transitions from elongation to rapid shortening and from rapid shortening to growing. Since 0.167 M Pi did not decrease the subunit dissociation rate in the rapid shortening phase or the rate of the phase transition from growing to rapid shortening, our results provide no support for the hypothesis that tubulin-GDP-Pi subunits are responsible for dynamic instability behavior of microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

An immunohistochemical and in situ hybridization study of c-myc and c-erbB-2 expression in primary human breast carcinomas.

In previous studies of the expression and organization of proto-oncogenes in human breast a significant correlation has been found between amplification of c-myc and c-erbB-2 genes in carcinomas and poor short-term prognosis. Gene expression was estimated by analysis of total RNA from tissues, and similarly assessment of gene organization relied upon extraction of DNA from tissues. The present study has compared the expression of c-myc and c-erbB-2 mRNA as determined by in situ hybridization, and c-myc and c-erbB-2 protein expression detected by immunohistochemistry in a group of carcinomas for which there was knowledge of genomic organization and/or expression. Formalin-fixed, paraffin-embedded tissues of 38 carcinomas were assessed for the presence of c-myc protein, and 13 of these were examined for c-myc mRNA by in situ hybridization. Similarly processed tissue from 14 tumours was tested for c-erbB-2 protein using the antiserum 21N and ten of these carcinomas studied for c-erbB-2 mRNA localization. There was a good correlation between gene amplification, the presence of c-erbB-2 protein and mRNA: both the latter were detected in six of the seven carcinomas with an amplification but in none without. For some carcinomas there was a good correlation between c-myc protein and mRNA levels. Three carcinomas with gene amplification had a lower percentage of cells with detectable protein than showed hybridization for mRNA. Other carcinomas had a lower level of mRNA expression than protein. Neither approach could predict which carcinomas had amplification of the c-myc gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗
PubMed1989

EEG discriminant analyses of mild head trauma.

Measures of EEG power spectral analyses were obtained from a total of 608 mild head trauma patients and 108 age-matched normal subjects. A training-set discriminant function was first developed from 264 mild head-injured patients and 83 age-matched controls yielding an overall discriminant classification accuracy of 94.8%. The first independent cross-validation of the discriminant function using 130 mild head trauma patients and 21 age-matched normals yielded a discriminant classification accuracy of 96.2% for the trauma patients and 90.5% for the normals. A second independent cross-validation of the discriminant function using 51 patients and measures of test-retest reliability from 93 patients yielded classification accuracies ranging between 77.8% and 92.3%. A third independent cross-validation of 70 mild head-injured patients tested at a different location with a different EEG computer system yielded a discriminant accuracy of 92.8%. The discriminating EEG power spectral analyses indicated 3 classes of neurophysiological variables which are attributable to mechanical head injury: (1) increased coherence and decreased phase in frontal and frontal-temporal regions; (2) decreased power differences between anterior and posterior cortical regions; and (3) reduced alpha power in posterior cortical regions.

Adolescent↗
PubMed1989

Analysis of glycoproteins released from benign and malignant human breast: changes in size and fucosylation with malignancy.

Radiolabelled glycoproteins released into media from benign and malignant human breast tissue after 48 h organ culture have been analysed using SDS polyacrylamide gel electrophoresis and fluorography. Major differences were identified between benign and malignant tissues: (i) a considerably higher incidence of glycoproteins in the molecular weight range 210-280 kDa detected from carcinomas than benign samples, with incidence being greater in poorly differentiated tumours; (ii) fucosylation of these glycoproteins released from carcinomas but not benign breast; (iii) heterogeneity, particularly of fucosylation, between the carcinomas but consistency amongst benign breast. A glycoprotein of MW 230 kDa was of particular interest since it was not detected from any benign samples but was present in 11 of 17 carcinomas, when it was almost always fucosylated. This could prove to be a useful tumour marker.

Adenofibroma↗
PubMed1989

The use of lectins in histopathology.

Lectins are proteins and glycoproteins extracted predominantly from plants which have the capacity to bind sugars specifically. This property makes them of interest for histopathology since they will bind to saccharides forming parts of glycoproteins and glycolipids of tissue constituents. Lectins have and can be used as reagents for mucin histochemistry, to identify specific cells, in the recognition of glycoprotein alterations in disease states, in studies of infectious diseases, and in the assessment of glycoconjugate alterations occurring with malignancy. They can be used for both light microscopic and ultrastructural localisation and various methods are available. It is important though, to consider the nature of the glycoconjugates under study and select lectins appropriately because of their varying specificities and binding characteristics. A panel of lectins should be used to study a particular configuration. Care should be taken with tissue fixation and processing. It must be remembered that an open and critical mind should be kept concerning interpretation of results. At the present time lectins have a limited value diagnostically, but the binding of Ulex europeus agglutinin to endothelium is certainly of value.

Animals↗