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Biomedical subjects

D M Barnes

Publications and source records attributed to D M Barnes.

At least 19 recordsLinked to original sources

Abnormal expression of wild type p53 protein in normal cells of a cancer family patient.

Mutations in the p53 gene are the commonest specific genetic change in human cancer. In normal tissues, p53 protein is present in such low quantities that it is not readily detectable by immunochemical techniques. However, in many tumour cells large amounts of p53 protein accumulate and can be seen by simple immunohistochemical staining; this is generally attributed to the accumulation of stabilised, mutant protein. We have found a mother and daughter, who both have a history of breast cancer, who show strong immunohistochemical staining of p53 in most of their normal epithelial and mesenchymal cells. Their family has a history of multiple cancers developing at an early age. Detailed protein analysis and gene sequencing of material obtained from cultured cells, grown from a skin biopsy taken from the daughter, suggest that her cells contained large quantities of normal (unmutated) p53. We suggest that this phenotype defines a new inherited cancer susceptibility syndrome that is distinct from the germ-line mutations in p53 found in some Li-Fraumeni families. This new syndrome affects p53 tumour suppressor function through an indirect mechanism that stabilises normal p53. It remains to be established whether this mechanism also contributes to the accumulation of p53 in sporadic cancers.

Adult

Source of amino acids for tRNA acylation. Implications for measurement of protein synthesis.

Estimates of protein-synthesis rates using radioisotopes require accurate measurement of the specific radioactivity of the label in protein and in the precursor pool over time. Although the extracellular and intracellular pools of amino acids are easiest to sample, the tRNA pool is the direct precursor and is the appropriate pool for sampling. To test if the intracellular or extracellular pools reflect the tRNA specific radioactivity, a chicken macrophage cell line was incubated in medium containing either 0.23 mM-leucine and 14.5 microCi of [3H]leucine (tracer dose) or 2.3 microM-leucine plus 145.0 microCi of [3H]leucine (flooding dose). At both leucine levels, the tRNA specific radioactivity reached a plateau quickly, but did not equilibrate with either the extracellular or intracellular specific radioactivity within 30 min, and remained closer to that of protein. In a second experiment, proteins in chicken macrophages were labelled with [3H]leucine for 2 days. Labelling medium was removed, and the cells were washed free of residual free [3H]leucine and incubated with medium containing either 0.23 mM- or 2.3 mM-leucine (unlabelled). The specific radioactivity of leucyl-tRNA leucine reached a plateau within 2 min and remained considerably closer to that in the protein than that in intracellular or extracellular pools for at least 60 min. These results suggest that amino acids from protein degradation are a primary source for charging tRNA. When protein-synthesis rates are estimated by label incorporation, use of extracellular or intracellular specific-radioactivity values result in a marked underestimation.

Acylation

KiS1--a novel monoclonal antibody which recognizes proliferating cells: evaluation of its relationship to prognosis in mammary carcinoma.

Immunohistochemical staining with a novel monoclonal antibody, KiS1, which recognizes a cell cycle-associated antigen, was investigated in 142 cases of stage I and II invasive breast carcinoma. KiS1 staining indices were compared with disease-free interval, overall survival, and post-relapse survival. Using a semi-quantitative method of assessment, we found that tumours with a high level of staining (34/142, 24 per cent) had a significantly worse prognosis than those with a low level of staining (108/142, 76 per cent). Significant correlations were found between KiS1 staining and disease-free interval (P < 0.001), overall survival (P < 0.001), and post-relapse survival (P = 0.008). A more time-consuming, quantitative method of assessment gave similar results. Cox multivariate analysis showed these results to be independent of nodal status, histological type, and grade of tumour (P = 0.01). We conclude that KiS1 is a valuable new antibody which affords useful prognostic information in breast carcinoma. As it can be used in formalin-fixed, paraffin-embedded material, it may be of particular use in the study of small lesions such as those identified in the Breast Cancer Screening Programme.

Adult

Flow cytometric characterisation of proliferating cell nuclear antigen using the monoclonal antibody PC10.

Anti-PCNA antibodies have aroused considerable interest recently as potential immunohistochemical markers of proliferation for use on clinical samples. PC10 is a monoclonal antibody which has been shown to recognise its epitope on formalin-fixed, paraffin-embedded, archival material. However, whilst PC10 gives the expected labelling pattern for growth fraction in normal tissues and some tumours, discrepant results have been obtained, for example, in carcinoma of the breast. By means of flow cytometry, we have attempted to characterise the different staining patterns that can be obtained with PC10. Intact fixed cells from proliferative mammalian cultures show 100% labelling, consistent with a growth fraction estimate. In contrast, detergent-extracted nuclei show S-phase specific staining. Nuclei extracted by treatment of fixed cells with pepsin show a different staining pattern again, with many G1 cells weakly stained and staining intensity increasing through S-phase into G2. The results demonstrate that multiparametric flow cytometry can define the cell populations which label with proliferation-related antibodies, such as PC10, under a variety of experimental conditions.

Animals

Overexpression of the c-erbB-2 oncoprotein: why does this occur more frequently in ductal carcinoma in situ than in invasive mammary carcinoma and is this of prognostic significance?

Overexpression of c-erbB-2 occurs in 60% of in situ and 25% of infiltrating ductal carcinomas. We have previously found very strong associations between immunohistochemical staining for c-erbB-2 and histological pattern and nuclear size in ductal carcinoma in situ (DCIS) and less strong correlation with proliferative activity. In a further study of infiltrating ductal carcinomas we have found that, in addition to tumours arising from c-erbB-2 positive, large celled, rapidly proliferating, comedo carcinomas and c-erbB-2 negative small celled cribriform/micropapillary carcinomas with a low proliferative rate, there is a third group of c-erbB-2 negative tumours with large nuclei and variable proliferative activity. These latter tumours are not seen in pure DCIS suggesting that they have a very transient in situ stage. Therefore, although in pure DCIS c-erbB-2 positively appears to be associated with tumours with a greater invasive potential, and c-erbB-2 negativity with tumours having a more favourable prognosis, the latter is not necessarily true in infiltrating disease.

Breast Neoplasms

Expression of the ERBB3 gene product in breast cancer.

Abnormalities of the EGF receptor and/or the related ERBB2 receptor occur in a significant proportion of cases of human breast cancer and are important influences in the behaviour of this tumour type. In this report we demonstrate by nucleic acid analysis and immunohistochemistry that the recently recognised third member of this gene family, ERBB3, shows a wide range of expression in breast cancer, and shows stronger immunoreactivity than that observed in normal tissue in 43 out of 195 cases (22%) of primary breast cancer. Overexpression of ERBB3 appears to result from increased levels of gene transcription since in none of the cell lines or primary cancers analysed did we find evidence of gene amplification. High expression of ERBB3 is positively associated with the presence of lymph node metastases, but there was no demonstrable relationship with patient survival in this series.

Blotting, Southern

DNA-damaging and transcription-terminating lesions induced by AF64A in vitro.

Although immediate cholinergic deficits produced by AF64A can be explained adequately by inhibition of enzymes involved in acetylcholine metabolism, the structural similarity of AF64A to a number of DNA-damaging antitumor agents suggested that the observed long-term cholinergic deficits may involve damage to the cell's informational molecules. This study was initiated to determine if AF64A can damage DNA and prematurely terminate RNA transcription in vitro, and to produce cytotoxic and DNA damaging effects in cells exposed to the drug in vivo. The ability of AF64A to produce N-7 guanine alkylations in DNA in vitro was assessed using a modified Maxam and Gilbert DNA sequencing technique, and the ability of AF64A to terminate RNA transcription was assessed by an in vitro RNA transcription system. AF64A was capable of producing extensive dose-dependent N-7 guanine alkylations in DNA fragments exposed to AF64A in vitro, although no sequence specificity of AF64A attack could be discerned. Furthermore, AF64A was able to produce RNA transcription-terminating lesions in vitro, also in a dose-dependent fashion. Transcription of AF64A-damaged DNA resulted in RNA molecules terminated not at every alkylated guanine, but at various discrete sites along the DNA template. AF64A was also found to be cytotoxic in a dose-dependent manner in cultured mouse leukemia L1210 cells. The induced cytotoxicity was accompanied by DNA lesions which were detected as DNA single strand breaks using the DNA alkaline elution technique. The results of these experiments support the hypothesis that AF64A may alter the structure and function of cellular DNA and may help explain the observed long-term cholinergic deficits.

Animals

Clinical evaluation of castable ceramic veneers.

Laboratory fabricated veneering of discolored or malformed anterior teeth is becoming a widely used technique. Etched Dicor and Dicor Plus ceramic veneers cemented with Dicor Light Activated Cement with Fluoride is one technique available to the practitioner. The purpose of this study was to evaluate the clinical effectiveness of castable ceramic veneers and the cementation system. Forty-three Dicor and 18 Dicor Plus veneers were placed in 12 patients. An intraenamel preparation was completed with a bullet-nosed diamond bur in an ultra-speed handpiece with air-water spray, and a polyvinylsiloxane impression was made. All veneers were fabricated on dies by the same laboratory technician and placed by one practitioner. The restorations were examined by two evaluators at baseline, 3 months, and 1 year, using modified Ryge criteria.

Ceramics

Analysis of p53 expression in human tumours: an antibody raised against human p53 expressed in Escherichia coli.

A cDNA encoding the complete normal human p53 protein was expressed in Escherichia coli using an expression system based on the bacteriophage T7 promoter. The cDNA was adapted so that the full-length protein was produced without fusion to any other sequence. Large amounts of the protein were isolated and the purified protein used to produce very high titre polyclonal antibodies to p53. These new antibodies permit the sensitive detection of p53 and p53 complexes in ELISA and immunoblotting assays. Most importantly, they also permit the detection of p53 in archival tumour material that has been conventionally fixed in formalin and embedded in paraffin wax. Using this reagent we have found that aberrant expression of p53 is a frequent feature of human breast cancer. We are able to recognise six different classes of p53 expression pattern that may be of help in the subclassification of breast tumours.

Antibodies

Relationship between c-erbB-2 immunoreactivity and thymidine labelling index in breast carcinoma in situ.

The relationship between the presence of the c-erbB-2 protein, demonstrated immunohistochemically with antibody 21N, and thymidine labelling index (TLI) has been studied in the in situ component of 70 cases of carcinoma of the breast. A significant association was found between high TLI and positive staining. Twenty of the 70 cases stained (29%) and of those that were stained 75% had a high TLI; 43% of those with a high TLI stained positively but only 14% of those with a low TLI stained positively. Strong correlations were seen between nuclear size and histological pattern and both 21N staining and TLI. The majority of carcinomas of comedo pattern with large nuclei were 21N positive and had a high TLI, whilst those with small nuclei and a predominantly cribriform/micropapillary appearance did not stain and had a low TLI. In tumours of mixed histological pattern there was less concordance between staining and TLI. The significance of these findings in relation to the biological behaviour of these tumours and their clinical management is discussed.

Breast Neoplasms

Computer image processing and automatic counting and measuring of fouling organisms.

The purpose of this study was to optimize the counting and measuring of marine fouling organisms on experimental panels using automated computer techniques. A script was developed that reduced the time required for the counting and measuring of marine fouling tubeworms by at least one order of magnitude over manual counts, with an error of five percent or less. Small, distinct tubeworms can be successfully counted by computer using a correction factor. Measurements of percentage cover or area should be made with larger tubeworms that have overlapping tubes.

Animals

c-erbB-2 protein overexpression in breast cancer is a risk factor in patients with involved and uninvolved lymph nodes.

The c-erbB-2 gene is overexpressed in about 20% of human breast cancers. Four hundred and eighty-three cases previously examined by immunohistochemical staining for c-erbB-2 expression were analysed to assess the risk associated with the elevated protein expression. Oncoprotein expression was correlated with increasing tumour grade but not with oestrogen receptor status, nodal involvement, tumour size or age. There was an increased risk of relapse and death associated with c-erbB-2 expression irrespective of nodal involvement. This marker thus appears to be a significant prognostic factor in the early as well as the late stages of breast cancer.

Adult

The relationship between c-erbB-2 expression, S-phase fraction and prognosis in breast cancer.

The relationship between c-erbB-2 gene expression (assessed immunohistochemically), S-phase fraction (SPF) and prognosis has been analysed in 172 women with primary breast cancer. c-erbB-2 staining was independent of age, tumour size, number of nodes involved, tumour grade and DNA ploidy, but was more common in oestrogen receptor (ER) negative tumours (P = 0.02) and progesterone receptor (PgR) negative tumours (P = 0.03). A weak correlation between c-erbB-2 staining and SPF was observed (r = 0.18). Amongst women with node negative disease, SPF was significantly related to relapse free survival (RFS, P = 0.04) while c-erbB-2 staining was not (P = 0.2). In contrast, both SPF (P = 0.002) and c-erbB-2 staining (P = 0.016) provided significant prognostic information on RFS for women with node positive disease. Multivariate analysis showed that c-erbB-2 staining and SPF gave independent information on RFS for women with node positive disease.

Breast Neoplasms

A 5- and 8-year clinical evaluation of a posterior composite resin.

Class I and II posterior composite resin (Ful-Fil) restorations were placed and were clinically evaluated, according to the US Public Health Service criteria, at baseline, 6 months, 5 years, and 8 years. At 5 years/8 years, alpha scores were assigned to the following percentages of restorations: 16%/31% for color match; 65%/62% for marginal discoloration; 74%/54% for cavosurface marginal discoloration; 100%/100% for axial contour; and 81%/65% for anatomic form. At 5 years/8 years, 56%/33% of Class II restorations had tight proximal contacts; 45%/44% had light contacts; and 0%/22% had no contacts. Transient postoperative sensitivity was reported in 6% of the restorations. Indirect evaluation indicated that there was an average of 64 +/- 35 microns of wear at baseline (because of overfinishing), 105 +/- 67 microns at 6 months, 217 +/- 76 microns at 5 years, and 253 +/- 82 microns at 8 years. At 5 years, three of the restorations, and at 8 years, four additional restorations, were clinically unacceptable because of recurrent caries and/or excessive wear. Final success rates of 90% and 77% at 5 and 8 years, respectively, were calculated.

Adult

Reproductive and neonatal losses associated with possible encephalomyocarditis virus infection in pigs.

A possible infection with encephalomyocarditis virus was investigated on two Minnesota pig farms which experienced an increase in stillborn and mummified fetuses, high pre-weaning mortality and reduced farrowing rates. The monthly averages for the numbers of piglets born dead per litter on farms A and B reached 4-6 and 3-6, the pre-weaning mortalities 50 per cent and 31 per cent, and the farrowing rates 52 per cent and 63 per cent, respectively. Serological and histopathological examinations supported a diagnosis of infection with encephalomyocarditis virus, but attempts to isolate the virus failed. Specific antibody to the virus was detected in both fetal and neonatal sera collected from abnormal litters. The predominant histopathological finding was myocarditis consisting of focal or diffuse mononuclear cell infiltration. The detection of specific antibody, and the myocardial lesions in stillborn fetuses, suggested that the problems were associated with infection by encephalomyocarditis virus.

Abortion, Veterinary