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At least 991 records · Page 55Linked to original sources
PubMed1998

Sentinel lymph node biopsy: strategies for pathologic examination of the specimen.

BACKGROUND AND OBJECTIVES: Sentinel lymph node biopsy is a practical surgical procedure with high sensitivity for detection of metastases from breast carcinoma in the axilla. It offers economy by avoidance of axillary dissection in the majority of breast carcinoma patients who have negative sentinel node biopsies, and provides an opportunity to study axillary micrometastases with high efficiency in a small volume of tissue. METHODS: Sentinel node tissue is sliced at 2-mm intervals for fixation and paraffin embedding. Probabilities of finding spherical micrometastases of specific sizes randomly distributed in lymph nodes were calculated geometrically for several microsectioning plans. RESULTS AND CONCLUSIONS: Sentinel node tissue can studied by systematic serial sectioning technique designed to find metastases of given diameters with specific probabilities. A procedure whereby three microsections are prepared repeatedly at intervals of 250 microm appears to be practical. Two sections from each level can be examined by routine staining and the third by immunohistochemical stain; the latter is recommended particularly for infiltrating lobular carcinoma. This method will find metastases of 0.25-mm diameter with theoretical probability of 1, and metastases of 0.10-mm diameter with probability of 0.46, with reasonable costs. Metastases of these sizes are consequential and worth finding on biological and clinical grounds.

Axilla↗
PubMed1999

Improving the prognostic value of histopathological grading and clinical staging in renal cell carcinomas by means of computer-assisted microscopy.

The present work aims to refine prognosis in cases of renal cell carcinoma (RCC) by integrating a variety of parameters with the prognostic information provided by histopathological grading and clinical staging, carried out on a series of 97 RCCs. To this end, Feulgen-stained RCC cell nuclei were characterized by means of 38 variables describing nuclear DNA ploidy levels and morphology. All of these data were subjected to a principal components analysis. On the basis of this multivariate analysis, Fuhrman grade II was subdivided into grades II- and II+, and Fuhrman grade III into grade III- and III+. The same kind of subcategorization was performed in the case of the T2 and T3 clinical stages. The results show that the classification into grade II- and III- RCCs correspond to a more favourable prognosis than grade II+ and III+, to which shorter survival periods were attributable. Similar results were obtained for the subcategorization of the T2 and T3 clinical stages. Very simple biological characterizations of these grade- or stage-related RCC groups were obtained by means of a decision tree approach applied to the cytometry-generated variables. The resulting classification rules were validated on a new series of 18 patients and enabled very accurate predictions of survival.

Adult↗
PubMed2000

Micro-scale chromophore-assisted laser inactivation of nerve growth cone proteins.

Directed growth cone movement is crucial for the correct wiring of the nervous system. This movement is governed by the concerted actions of cell surface receptors, signaling proteins, cytoskeleton-associated molecules, and molecular motors. In order to investigate the molecular basis of growth cone motility, we applied a new technique to functionally inactivate proteins: micro-scale Chromophore-Assisted Laser Inactivation [Diamond et al. (1993) Neuron 11:409-421]. Micro-CALI uses laser light of 620 nm, focused through microscope optics into a 10-microm spot. The laser energy is targeted via specific Malachite green-labeled, non-function-blocking antibodies, that generate short-lived protein-damaging hydroxyl radicals [Liao et al. (1994) Proc Natl Acad Sci USA 91:2659-2663]. Micro-CALI mediates specific loss of protein function with unachieved spatial and temporal resolution. Combined with time-lapse video microscopy, it offers the possibility to induce and observe changes in growth cone dynamics on a real time base. We present here the effects of the acute and localized inactivation of selected growth cone molecules on growth cone behavior and morphology. Based on our observations, we propose specific roles for these proteins in growth cone motility and neurite outgrowth.

Actins↗
PubMed1997

Matrix-assisted laser desorption/ionization mass spectrometry of proteins extracted directly from sodium dodecyl sulphate-polyacrylamide gels.

A new strategy for the characterization of Coomassie Brilliant Blue stained SDS-PAGE separated proteins by UV-MALDI-MS is reported. The proteins are extracted directly from the polyacrylamide gel by treatment with an organic solvent mixture consisting of formic acid, acetonitrile, isopropanol and water in an ultrasonic bath. A fraction of the supernatant is then mixed directly with the matrix solution and measured by MALDI-MS. High quality spectra could be obtained from gels which were loaded with 6 pmol of myoglobin. Compared to other methods based on electroblotting or electroelution this method is much simpler and less time consuming. The sensitivity is higher than or comparable to the Coomassie Blue staining procedure for proteins up to about 25 kDa. Another advantage is that mass shifts due to charging effects of the membranes, which are common if membranes are mounted directly on the sample target, can be avoided. However, all proteins studied showed slightly higher masses than expected which reduces mass accuracy to 0.2-0.3%. This is presumably partly due to formylation of serine or threonine residues during incubation in formic acid. Gel electrophoresis induced modifications can contribute as well. The possibility of further characterizing the remaining part of the supernatant after extraction by means of proteolytic digestion is also demonstrated. The knowledge of both molecular weight of the whole protein and of the proteolytic fragments increases specificity for protein identification by searching in sequence databases.

Carbonic Anhydrases↗
PubMed2000

Analysis of missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation after in-gel tryptic digestion.

Peptide mass fingerprinting is a powerful tool for the identification of proteins. Trypsin is the most widely used enzyme for this purpose. Therefore, 104 protein digests from human Jurkat T cells and Mycobacterium were analyzed considering missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation. About 90% of the matched peptides with missed cleavage sites could be classified into three groups: (i) lysine and arginine with a neighbouring proline on the carboxy-terminal side, (ii) neighboring lysines/arginines, and (iii) lysines and arginines with an aspartic acid or glutamic acid residue on either the amino- or carboxy-terminal side. The first group is already accounted for by search programs. The number of missed cleavage sites can be increased without reducing the precision of the database search by taking the other two groups into consideration. Peptides with tryptophan were observed in non, singly (+16 Da) and doubly (+32 Da) oxidized forms. The higher oxidized form was only observed with lower intensity in the presence of the lower oxidized form. Peptides with N-terminal glutamine were found always as pyroglutamate (-17 Da), and in the majority of cases in pairs with unmodified glutamine. These data can be used for the refinement of protein searches by peptide mass fingerprinting.

Humans↗
PubMed1996

Toward a new method to in situ study of apoptosis and its relations with cell cycle.

Apoptosis or programmed cell death plays a key role in many biological processes particularly in oncology. The detection of apoptotic cells is crucial for the study of the phenomenon itself and its relation with the proliferative cell cycle. A new method to detect apoptosis in situ in Feulgen stained cells was developed, based on multiparametric analysis (factorial and decisional analysis), using 15 densitometric and textural parameters measured on a SAMBA 200 cell image processor. Six reference files corresponding to G1, S, and G2 phases and to apoptotic cells derived from these cell cycle phases were constructed. The projection of these files in the factorial principal plane formed distinct clusters. Using the decisional discriminant analysis, it was possible to ascertain the state (apoptosis or proliferative) and the phase for each cell of a population. The correct classification rate of this analysis was 0.9962. Determining the cell cycle phase from which each apoptotic cell comes, we are able to study the relation between apoptosis and the proliferative cell cycle. Moreover, the detection in situ allows us to study cell-cell interactions.

Apoptosis↗
PubMed1996

DNA-ploidy studies on cytological preparations from breast cancers by image analysis: comparison with Feulgen staining performed in destained Papanicolaou slides.

Image cytometric analysis of DNA ploidy status of 40 fresh primary breast carcinomas was performed in a comparative study on cytologic preparations stained directly by the Feulgen method and smears from the same tumors Feulgen-stained after Papanicolaou staining. There was a 0.82 overall concordance in diploid and aneuploid tumor classification and a good correlation (r = 0.736, P < 0.001) between the DNA indices determined by the two methods. Discordances are probably due to tumor heterogeneity. This study demonstrates that image cytometric DNA analysis on previously routine-stained cytologic preparations is feasible and reliable for pretreatment DNA analysis and suggests potential applications in retrospective studies with cytologic material from archives.

Adult↗
PubMed1998

Discrimination between chronic pancreatitis and pancreatic adenocarcinoma using artificial intelligence-related algorithms based on image cytometry-generated variables.

The incidence of pancreatic adenocarcinomas (PA) is increased in the setting of chronic pancreatitis. Distinguishing chronic pancreatitis from pancreatic adenocarcinomas is often difficult, and is based on routine brush cytological specimens provided during endoscopic retrograde cholangiopancreatography (ERCP). Reactive epithelial changes in chronic pancreatitis may appear similar to those of a well-differentiated cancer. Brush cytology specimens were obtained during ERCP from 49 patients with diseases for which the differential diagnosis included chronic pancreatitis and/or pancreatic adenocarcinoma Image cytometry was performed involving the assessment of between 200-400 Feulgen-stained nuclei per case; for each case, 40 quantitative cytometric variables were generated. Data analysis was performed using artificial intelligence methods of data classification that produced decision trees and production rule systems. Different classification models were produced for a subset of 34 patients. The best models were identified by the use of a sampling technique (leave-one-out), and were tested on the remaining 15 patients. These models were based on 5 of the 40 variables associated with a significant discriminatory function. Pancreatic adenocarcinoma was diagnosed in the training data set of 34 patients during a leave-one-out process with an estimated sensitivity of 91% and specificity of 87%. Both sensitivity and specificity were 80% in the independent test set of 15 patients. We conclude that inflammatory and malignant pancreatic epithelia exhibit distinct morphological features that can be distinguished by decision tree-based classifiers employing image-cytometric numerical data.

Adenocarcinoma↗
PubMed1998

Identification of early apoptosis in feulgen-stained cultured cells in situ by computerized image analysis.

A method is presented in which Feulgen-stained apoptotic cells are identified among cultured cells in situ using computerized image analysis of their nuclei. Images of either control (untreated) human lymphoblastoid cell nuclei or of similar cells treated with 3mM methyl methanesulfonate for 1 h, which induces apoptosis in 100% of the cells, were converted to a standard size (80 x 80 pixels) and Fourier transforms and boundary images based on 21 proportional mean gray-level thresholds obtained. The perimeter and the mean fractal dimension of the latter and 39 selected coefficients of the former were then obtained from which data were chosen heuristically and subjected to multivariate linear discrimination analysis. Eighty-two percent of a teaching set of 50 nuclei and 69% of a test set of 29 nuclei were correctly identified as apoptotic versus nonapoptotic by the computer, compared with 57% identified correctly by a panel of 7 pathologists. This study shows the feasibility of using this type of analysis to directly identify apoptotic cells in culture, and probably also in tissues, by direct observation using computerized imaging technology.

Apoptosis↗
PubMed2000

Improving accuracy in the grading of renal cell carcinoma by combining the quantitative description of chromatin pattern with the quantitative determination of cell kinetic parameters.

The determination of grade and stage in renal cell carcinomas (RCCs) often fails to predict the actual clinical outcome for individual patients. The aim of the present work was to investigate whether it is possible to significantly improve the prognostic accuracy of the grading system by using the combination of two independent computer-assisted microscopy techniques. The first technique relates to the quantitative description of morphonuclear and nuclear DNA content features by means of the image analysis of Feulgen-stained cell nuclei, and the second quantitatively characterizes tumor growth by means of different cell kinetic parameters. These parameters consist of a duplication of a time-related parameter determined by means of the technique of using silver-stained proteins in interphase nucleolar organizer regions (AgNOR), a proliferation index determined by means of MIB-1 immunohistochemistry, and an apoptotic index determined by means of the terminal dUTP nick end labeling technique. The prognostic value of these quantitative features was investigated in a series of 60 RCCs. The quantitative analysis of Feulgen-stained nuclei made it possible to identify subgroups of patients with significantly different prognoses in both grade II and grade III RCCs. We labeled the RCCs associated with the most favorable prognoses as grade II- and III- and those with the least favorable ones as grade II+ and III+. The two most important kinetic variables to identify patients with different clinical outcomes were the MIB-1 index and the mean AgNOR area in the MIB-1-positive cells. Three significantly different survival curves were obtained for the 53 grade II and III RCC patients. Our results show that conventional RCC grading can be significantly improved by the quantitative analysis of Feulgen-stained nuclei, by cell kinetic parameter determination, and, more importantly, by combining the proliferation index with the mean AgNOR area parameter.

Antigens, Nuclear↗
PubMed1999

Estrogenicity of bisphenol A and bisphenol A dimethacrylate in vitro.

Although pit and fissure sealants have been utilized extensively in dentistry as a way of preventing occlusal caries, results described by Olea et al. (1996) raised concerns about the safety of sealants and other resin-based dental materials due to the reported presence of bisphenol A (BPA) and its dimethacrylate ester (BPA-DM). Although the release of these compounds from dental materials has not been substantiated by two subsequent studies, we believed it was important to confirm or refute the report that BPA and BPA-DM have estrogenic activity in vitro. We grew breast cancer cells (MCF-7, T-47D, ZR-75-1) known to proliferate under estrogenic stimulation in phenol red-free DMEM containing human serum and concentrations of BPA or BPA-DM ranging from 10(-8)M to 5 x 10(-6)M. After 1 week, plates were harvested for crystal violet or sulforhodamine-B assays, and the optical densities of groups of treated cells were compared with values from control cells. At concentrations at or above 10(-6)M, both BPA and BPA-DM significantly increased cell proliferation (p < 0.05), comparable to the increase seen with 10(-9)M of estrogen. Flow cytometric methods demonstrated that these mitogenic effects occurred within 24 h of exposure to estrogen, BPA, or BPA-DM. The increase in DNA synthesis was analogous to that seen with estrogen stimulation. Thus, we confirmed that BPA and BPA-DM cause cell proliferation at micromolar concentrations that exceed the effective concentrations of estrogen by 1 to 10,000-fold.

Benzhydryl Compounds↗
PubMed1999

Focal adhesions and assessment of cytotoxicity.

Focal adhesions are highly ordered assemblies of transmembrane receptors, extracellular matrix proteins, and a large number of cytoplasmic proteins, including structural proteins, as well as tyrosine kinases, phosphatases, and their substrates. They are now accepted as a prime component of signal transduction. Because focal adhesions also play an important role in cell morphology and migration, it can be argued that their presence is indicative of healthy cells. This has been the reason for several research groups to conclude that biomaterials sustaining focal adhesion assembly are biocompatible. In this study we demonstrate that cells under cytotoxic stress may still be able to retain their focal adhesions. Human umbilical vein endothelial cells at passage 2 were exposed to nickel and zinc ion solutions ranging from 1 to 0.01 mM for 4 and 24 h. Cells were seeded on fibronectin precoated glass slides or in tissue culture quality 96-well plates. MTT conversion with 1 and 0.5 mM nickel and zinc was strongly depressed, indicating that these concentrations are cytotoxic. Proliferative activity was also affected by these concentrations. Cells exposed to zinc typically retracted and detached from the surface, whereas cells exposed to nickel remained on the surface without signs of retraction. Nevertheless, cells exposed to nickel were impaired to reach confluency, which was determined by cadherin-5 expression. All these data indicate that nickel ions at a sufficient concentration influence cells in a cytotoxic way. Despite this apparent cytotoxicity, focal adhesion distribution as visualized by immunofluorescence staining of vinculin was not affected. With zinc the morphological changes were accompanied by apparent fusion of focal adhesions during retraction and finally dissolution. These data indicate that the mere presence of focal adhesions does not allow a reliable statement about the functional status of a cell. On the other hand, when focal adhesions are affected it is an excellent monitor of disturbed cell function.

Cell Adhesion↗
PubMed1998

A chemical modification of myeloperoxidase and lactoperoxidase with 2-(O-methoxypolyethylene glycol)-4, 6-dichloro-s-triazine (activated PEG1).

The modification of myeloperoxidase and lactoperoxidase with 2-(O-methoxypolethylene glycol)-4, 6-dichloro-s-triazine, an activated polyethylene glycol (PEG1), was investigated. The modification caused a shift of the Soret band in the light absorption spectrum, from 430 nm to 418 nm in the case of myeloperoxidase (native ferric form), and from 412 nm to 406 nm in the case of lactoperoxidase (native ferric form). PEG1-modified myeloperoxidase and PEG1-modified lactoperoxidase both failed to bind with antiserum to the respective native enzyme, but both retained respectively 4.5 +/- 0.3 per cent (mean +/- SE, n = 5) and 0.6 +/- 0.2 per cent (mean +/- SE, n = 5) of the activities of peroxidation of the hydrogen donor o-methoxyphenol in comparison with the native enzyme, and 1.5 +/- 0.2 per cent (mean +/- SE, n = 5) and 1.2 +/- 0.2 per cent (mean +/- SE, n = 5) of the activities of destruction of fuchsin basic in the presence of hydrogen peroxide and a halide, bromide. The pH dependencies of the peroxidating activities were almost the same as those of the corresponding native enzymes, but both the optimal pHs of the reactions involving the destruction of fuchsin basic were shifted by approximately 1.0 pH unit toward neutral pH compared with the respective native enzymes.

Animals↗
PubMedDate not supplied

Mutagenicity of malachite green and leucomalachite green in in vitro tests.

The genotoxic potential of the fungicide malachite green (MG) and its reduced derivative leucomalachite green (LMG) was assessed in bacteria and mammalian cells using the standard Salmonella typhimurium/Ames and CHO/HGPRT tests. In vitro potential DNA damaging effects of MG and LMG were tested using the single-cell gel electrophoresis (Comet) assay on CHO cells. Malachite green was found to be extremely cytotoxic to bacteria and mammalian cells. It did not have any mutagenic activity, in any bacterial strains, in the presence or absence of metabolic activation for doses up to 10 microg per plate. In the CHO/HGPRT test, the mutagenic potential of MG could be evaluated only for very low concentrations ranging from 0.001 to 0.05 microg ml(-1) medium. When S9 fraction was added to the medium, the highest tested dose could be increased to 1 microg ml(-1). In these experimental conditions, MG did not increase the number of thioguanine-resistant mutants. Leucomalachite green was less toxic than MG to Salmonella typhimurium and did not have mutagenic activity in the Ames' test for doses up to 2000 microg per plate. It was also less cytotoxic than MG to CHO cells and was tested at doses ranging from 5 to 100 microg ml(-1). Overall results indicated that LMG was not mutagenic in the HGPRT test. In the Comet assay, MG induced DNA damage only at cytotoxic doses. Loss of cell viability was observed for doses of > or = 3 microg ml(-1), with parallel increase in DNA alterations as measured by the tail moment. After metabolic activation, however, DNA damage was observed at doses (15-20 microg ml(-1)) inducing only low cytotoxicity. In this case, the direct genotoxicity of MG metabolites could not be excluded. In the absence or presence of metabolic activation, LMG did not have any effect on cell viability or DNA damage for doses up to 500 microg ml(-1). This study indicates that LMG, which is the main residue found in fish tissues after treatment with MG, did not have any mutagenic or clastogenic effects in the experimental conditions used.

Aniline Compounds↗
PubMed1999

High-resolution electrophoretic analysis of rat parotid salivary proteins.

Silver staining and high-resolution electrophoretic methods have been used to compare the protein composition of rat parotid saliva evoked in response to (i) parasympathetic stimulation (including the nonadrenergic, noncholinergic, atropine-associated secretion), (ii) sympathetic stimulation, or (iii) the infusion of neuropeptides with secretagogue activity (substance P, calcitonin gene-related peptide, neuropeptide Y, or vasoactive intestinal peptide). The different stimuli influenced the protein concentration and flow rate of the evoked secretion but had little effect upon the protein composition of the saliva. In contrast to earlier studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining, the combination of silver staining and two-dimensional electrophoresis (2-DE) revealed many newly detected proteins. The results indicate that the protein composition of rat parotid saliva is more complex than previously reported but is unaffected by the mode of stimulation.

Animals↗
PubMed1986

Semi-automated diagnosis of cervical intra-epithelial neoplasia grade 2 by the measurement of acid labile DNA in cytologically normal nuclei.

Cytologically normal and abnormal nuclei in cervical smears from patients with cervical intra-epithelial neoplasia (CIN) grade 2 were assessed for their acid labile DNA content using the Feulgen technique. Semi-automated measurements were performed blind using a Vickers M86 microdensitometer (Vickers Instruments, Ltd., York, UK) interfaced to a Research Machines 380Z microcomputer (Research Machines, Ltd., Oxford, UK). They showed that significantly elevated levels of rapidly hydrolyzed DNA were present in both abnormal and normal looking cell fractions. Although the levels were lower in the nuclei of normal appearance, they were still significant over the early part of the hydrolysis profile. It is suggested that because these nuclei represent a more accessible fraction in CIN grade 2 smears they could prove extremely helpful in the cytodiagnosis of cervical neoplasms.

Cell Nucleus↗
PubMed1987

DNA distribution patterns in early gastric carcinomas. A Feulgen cytometric study of gastric brush smears.

DNA distribution patterns were studied by cytophotometry in 20 Feulgen-stained brush smears of early gastric carcinomas. Two different DNA distribution patterns, classified as predominantly diploid and aneuploid, were correlated with histologic data. Six of seven carcinomas of diffuse type were predominantly diploid. By contrast, 11 of 13 intestinal type carcinomas were aneuploid and two were diploid. Since the DNA distribution patterns recorded in this study were previously observed in advanced gastric carcinomas, this would suggest that the basic genetic make-up of the tumors does not change with tumor progression.

Adult↗