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Biomedical subjects

D G Winkler

Publications and source records attributed to D G Winkler.

16 recordsLinked to original sources
PubMed1995

Modification of Ser59 in the unique N-terminal region of tyrosine kinase p56lck regulates specificity of its Src homology 2 domain.

During T-cell activation, Ser59 in the unique N-terminal region of p56lck is phosphorylated. Mutation of Ser59 to Glu59 mimics Ser59 phosphorylation, and upon CD4 crosslinking, this mutant p56lck induces tyrosine phosphorylation of intracellular proteins distinct from those induced by wild-type p56lck. Mutant and wild-type p56lck have similar affinities for CD4 and similar kinase activities. In glutathione S-transferase fusion proteins, the p56lck Src homology 2 (SH2) domain with the SH3 domain and the unique N-terminal region (including Ser59) has a different binding specificity for phosphotyrosyl proteins than the SH2 domain alone. Either deletion of the unique N-terminal region or mutation of Ser59 to Glu59 in the fusion protein reverts the phosphotyrosyl protein binding specificity back to that of the SH2 domain alone. These results suggest that phosphorylation of Ser59 regulates the function of p56lck by controlling binding specificity of its SH2 domain.

Amino Acid Sequence↗
PubMed1991

Purification and initial characterization of the lymphoid-cell protein-tyrosine kinase p56lck from a baculovirus expression system.

The lymphocyte-specific protein-tyrosine kinase p56lck has been purified 90-fold to approximately 30% purity in 30% yield from a baculovirus expression system by a two-column purification procedure. At least two forms of p56lck were isolated, differing in the extent of phosphorylation and migrating as 56- and 59-kDa species on SDS/PAGE but as a single 56-kDa band after treatment with potato acid phosphatase. Autophosphorylation of purified p56lck occurred at a rate of 25 fmol/min to a maximum incorporation of approximately 2 mol of phosphate per mol of p56lck with tyrosine-394 (but not tyrosine-505) and other, unidentified tyrosine residue(s) being the major sites of phosphorylation in vitro. Phosphorylation of tyrosine-containing peptides was monitored using an automated HPLC system. Although peptide substrate Km values were in the 1-5 mM range, the Vmax for the 13-amino acid peptide RRLIEDAEYAARG (modified p60src autophosphorylation site) was 120 min-1 (350 min-1 when adjusted for p56lck purity), suggesting that the enzyme purified from recombinant baculovirus-infected Sf9 cells has a high catalytic turnover compared with other tyrosine kinases.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of a soluble catalytic fragment of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase from an Escherichia coli expression system.

A 350 amino acid soluble fragment of the intracellular catalytic domain of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase has been purified 17-fold to greater than 90% purity from an Escherichia coli expression vector in quantities sufficient for kinetic and structural characterization. To assess substrate specificity, phosphotyrosine peptides corresponding to autophosphorylation sites of the two major classes of tyrosine kinases have been synthesized. Thus 6-12-residue phosphotyrosine peptides of the insulin receptor and epidermal growth factor receptor kinase domains and of the autophosphorylation and C-terminal regulatory sites of p60src and p56lck have been analyzed for kcat and KM by using a nonradioactive chromogenic assay for Pi release. The catalytic domain of LAR PTPase shows kcat values of 20-70 s-1 for phosphotyrosine peptides and affinities that vary 150-fold from 27 microM to 4.1 mM.

Amino Acid Sequence↗
PubMed1984

Precision cell location and relocation techniques. An application for cell image analysis.

Two methods for precise cell location and relocation were developed at the Cell Image Analysis Laboratory, NASA, to increase the efficiency of the cell image acquisition process. The first method uses a Zeiss Zonax with a 10 micron scanning stage and automatically records cell coordinates on 5.25-inch floppy disks. The second method uses a Digital Positioning Device with a 10 micron scanning stage, and the cell coordinates displayed on light-emitting diodes (LEDs) are recorded manually. Because of its superior speed and automation, the Zonax was selected as the method of choice. By using a Zonax for off-line cell location and another Zonax for on-line cell relocation, more than three times the number of digitized images were collected in ten months than in the previous three years. Other beneficial applications of the cell location and relocation methods were realized in photomicrography, data base verification and cytopathology.

Computers↗
PubMed1983

Nuclear segmentation of bronchial epithelial cells by minimax and thresholding techniques. A comparison.

Two nuclear segmentation methods, Baky's minimax algorithm and thresholding, were compared on a sample of 879 atypical bronchial epithelial cells in sputum. Nuclear-cytoplasmic (N/C) ratios for all cells were determined by each segmentation method and compared to a visually determined value. Cells were categorized by atypia class (from metaplastic through malignant), by staining characteristics (orangeophilic and nonorangeophilic) and by method of digitization (either scanning microphotometry or video system). The method of digitization was confounded by subject differences. The results indicated that with most classes of atypia, N/C ratios determined by minimax were closer to the visually derived values than were those of thresholding, particularly with orangeophilic cells. Both methods become progressively less accurate, as compared to the visual procedure, as the degree of atypia increases.

Bronchi↗
PubMed1983

Classification of bronchial epithelial atypias by the atypia status index.

The atypia status index (ASI) is a categorization method of classifying digitized images of atypical bronchial epithelial cells in sputum. The ASI is defined as a linear composite of features linearly related to atypia stage. Over 200 features were examined for more than 3,000 cells that had been classified by atypia stage (squamous metaplasia, mild, moderate or severe atypia and malignant) and staining characteristic (orangeophilia and nonorangeophilia). We reduced the number of features by using a selection process to minimize redundancy. The feature weights were optimized via a least-squares procedure. The 14 features selected accounted for over 60% of the variation of atypia stage and produced ASI values that were within one atypia stage of the criterion classification for over 90% of the cells. The results are consistent with the hypothesis of a progressive pattern in bronchial epithelial atypia and indicate the feasibility of using image analysis for mass screening of premalignant atypias in sputum from subjects considered to be at high risk for lung cancer.

Adult↗
PubMed1981

Nuclear boundary detection algorithm based on a minimax derivative statistic for atypical bronchial squamous epithelial cells.

The thresholding approach in scene segmentation of digitized cell images was found to be unreliable for use with squamous epithelial cells characterized by chromatin condensation within the nucleus. It was observed that the nuclear boundaries of such cells were visually distinguishable. An algorithm was developed for detecting nuclear boundaries in digitized cell images. This algorithm is based on a minimax derivative statistic that has maximum values at nuclear boundaries and low values elsewhere. The statistical properties of the confusion matrix of cellular scene segmentation are outlined. A measurement and a test statistic for scene segmentation errors based on those properties are presented.

Bronchi↗
PubMed1981

Image analysis of atypical bronchial squamous epithelial cells. A quantitative and qualitative examination of squamous cell carcinogenesis.

Atypical bronchial squamous epithelial cells from cigarette smokers were digitized using a high-resolution microphotometric scanning system operating at a wavelength of 530 nm. All cells used for the purpose of developing a training set as well as those used as a test set were classified by a team of three cytopathologists. Reduction in the dimensionality of the feature space and cell classification were performed using a new system known as the atypia status index (ASI); this dual-purpose system will specifically classify cells into five distinct classes of progressive atypia as well as quantitate the degree of atypicality of each individual cell. The distribution of cellular ASI values was arranged from lowest to highest and plotted for each subject as a profile. The ASI range has well-defined intervals, representing the five different atypia classes used in the study (squamous metaplasia, mild atypia, moderate atypia, severe atypia and carcinoma). Subjects at different stages of carcinogenesis were found to have significantly different profiles (P less than 0.01). Changes in visual morphologic characteristics and their relation to changes in ASI values for the five different classes of atypia are presented.

Bronchi↗
PubMed1980

Aytpia status index of respiratory cells: a measurement for the detection and monitoring of neoplastic changes in squamous cell carcinogenesis.

A statistical procedure was developed for calculating an atypia status index (ASI) for cells from the bronchial mucosa. These indices represent the degree of abnormal changes in these cells and classify them as squamous metaplasia, mild atypia, moderate atypia, servere atypia or carcinoma. The classification accuracy of the procedure was more than 99% on trained data and was accomplished by minimizing the overlapping areas between the adjacent multivariate distributions of cell groups for selected features whose group means represent a monotonic function and for which the cell categories are distinguishable. The calculated ASI may reflect abnormal changes in the cell that may not be clearly evident visually. It appears that progression or reversal of bronchial epithelial atypia can be accurately monitored by studying the changes in the ASI, not only for preneoplasia but also for reactions to chemotherapy and various pulmonary infectious disease processes, such as influenza.

Adult↗
PubMed1980

Cell atypia profiles for bronchial epithelial cells: mathematical evaluation of sputum cellular atypia in squamous cell carcinogenesis of the lung.

Atypical bronchial epithelial cells from the sputum of male cigarette smokers were digitized by scanning microphotometry and analyzed by computerized image analysis techniques. A set of specific analytic features based on cell morphology was extracted and combined mathematically to be expressed as a single number (Atypia Status Index). This index represented the stages of the cellular atypia (squamous metaplasia; mild, moderate or severe atypia; or carcinoma) quantiatively as a single number varying between 0.0 and 5.5 The Atypia Status Index for each cell correlated highly (94% to 99%) with visual cytopathologic classification. The distributions of cells according to their indices were profiled for patients at various stages of carcinogenesis and used to classify patients relative to their degree of cell atypia. These results compared favorable to those of the clinical evaluation.

Bronchi↗
PubMed1977

Cytofluorograf detection of Plasmodium yoelii, Trypanosoma gambiense, and Trypanosoma equiperdum by laser excited fluorescence of stained rodent blood.

Samples of rat blood infected with Plasmodium yoelii (3% parasitized erythrocytes), Trypanosoma gambiense, or Trypanosoma equiperdum (greater than 50 trypanosomes per microscope field at 400 X) were fixed with 0.5% glutaraldehyde in phosphate buffered saline, then stained with acridine orange (AO) at 10(-4), 10(-5), or 10(-6) M for 0 to 15 min at 5 C or 25 C and/or ethidium bromide (EB) at 0.05 mg/ml for 20 min at 25 C. Stained cells were analyzed with a laser Cytofluorograf (Bio/Physics Systems, Inc.) to determine if parasites could be detected and differentiated from blood cells by their fluorescent characteristics. Samples of uninfected rat blood with and without leukocytes and P. yoelii-infected blood without leukocytes were treated similarly. In addition, suspensions of T. gambiense and T. equiperdum without all blood cells were stained with AO or EB and analyzed with the Cytofluorograf, as were mixed suspensions of both trypanosome species. EB- but not AO-stained P. yoelii-infected erythrocytes had fluorescent characteristics different from most blood cells. Neither AO- nor EB-stained T. gambiense or T. equiperdum could be differentiated from host blood cells or from each other. The results are discussed with respect to the use of laser flow systems in the detection and analysis of bloodstream dwelling protozoan parasites.

Acridines↗
PubMedDate not supplied

Application of cell-image analysis to the diagnosis of cellular atypias in sputum: a review.

The incidence of carcinoma of the lung continues to steadily rise, and attempts at early diagnosis to improve prognosis have not yet been rewarding. The goal of our research is to decrease the incidence of lung cancer by detecting premalignant bronchial dysplasias in individuals in whom development of lung cancer is potentially preventable. To achieve this, we have developed an atypia status index (ASI)--the assignment of numerical values to the various stages of atypical bronchial epithelial cells in sputum, and a cell atypia profile (CAP)--an ASI-generated scale of 200 such atypical bronchial epithelial cells in a single sputum specimen. Computerized cell-image analysis techniques and statistical data analysis are used to generate the ASIs and CAPs for each subject. This study is a step toward the development of an automated cell-image analysis system for mass screening of premalignant atypias in sputum of those considered at high risk for lung cancer (i.e., men and women of 40 yr of age and older, with more than 20 pack-yr of cigarette smoking).

Adult↗
PubMedDate not supplied

The application of visual cell profiles in the study of premalignant atypias in sputum.

Because the diagnosis of squamous-cell carcinoma of the lung by routine cytologic screening of sputum is often inconclusive, our laboratory is studying the use of cytomorphologic profiles as a reliable diagnostic aid. This study reports the analysis of the profiles of 75 subjects, both cigarette-smoking volunteers and hospitalized patients suspect for lung cancer. Twenty-five of the subjects had been classified as having squamous metaplasia, 25 as having atypias and 25 as having squamous-cell carcinoma. Four slides were made for each subject, with 100 random fields viewed on each slide. The frequency of free alveolar macrophages, metaplastic squamous cells, epithelial atypical cells and malignant squamous cells were noted for each field. The results indicated that, although there were large differences between individual profiles within each diagnostic category, there were significant differences between the average profiles for each diagnostic category. Furthermore, although there were differences in cell frequencies from slide to slide and within slides, the differences were constant across diagnostic categories. The results are supportive of the progressive-atypia hypothesis for squamous-cell carcinoma of the lung and provide a baseline for comparison with subsequent individual profiles.

Carcinoma↗
PubMedDate not supplied

Visual cell profiles for quantitation of premalignant cells in sputum: a preliminary report.

Visual cell profiles were used to analyze the distribution of atypical bronchial cells in sputum specimens from cigarette-smoking volunteers, cigarette-smoking asbestos workers and cigarette-smoking uranium miners. The preliminary results of these sputum visual cell profile studies have demonstrated distinctive distributions of bronchial cell atypias in progressive patterns of squamous metaplasia, mild, moderate and severe atypias and carcinoma, similar to those the authors have previously reported using cell image analysis techniques to determine an atypia status index (ASI). The information gained from this study will be helpful in further validating this ASI and subsequently achieving the ultimate goal of employing cell image analysis for the rapid and precise identification of premalignant atypias in sputum.

Age Factors↗