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PubMed2002

Simian virus 40 large T antigen and two independent T-antigen segments sensitize cells to apoptosis following genotoxic damage.

The simian virus 40 (SV40) large tumor (T) antigen is sufficient to transform cells in cultures and induce tumors in experimental animals. Transformation of primary cells in cultures requires both overcoming growth arrest by stimulating the cell cycle and blocking cell death activities presumably activated by oncogene-mediated hyperproliferation signals. The study presented here examined the ability of specific regions and activities of T antigen to modulate apoptosis in cells treated with the genotoxic agent 5-fluorouracil (5-FU). The results showed that the expression of full-length T antigen rendered rat embryo fibroblasts (REF) sensitive to 5-FU-induced apoptosis. Thus, neither the p53-binding region nor the Bcl-2 homology region of T antigen was sufficient to prevent cell death induced by the DNA-damaging agent. T-antigen-mediated sensitization occurred independently of retinoblastoma protein or p53 and p300 binding. An N-terminal segment containing the first 127 T-antigen amino acids (T1-127) was sufficient to sensitize cells. A C-terminal segment consisting of T-antigen amino acids 251 to 708 (T251-708) also sensitized cells to 5-FU-induced apoptosis. This sensitization did not occur when T251-708 was targeted to the nucleus by inclusion of the SV40 nuclear localization signal. The introduction of mutations into the T-antigen J domain resulted in mutation-specific and variable inhibition of apoptosis. This result suggested that either the structural or the functional integrity of the J domain is required to sensitize cells to apoptosis. Treatment of REF or REF expressing full-length T antigen, an N-terminal segment, or T251-708 resulted in increased expression of the p53-responsive MDM2 gene; apoptosis occurred through a p53-dependent pathway, as p53-null cells expressing these T antigens were resistant to 5-FU-induced apoptosis. Possible mechanisms involved in sensitizing cells to a p53-dependent apoptosis pathway in spite of the ability of T antigen to bind and inactivate the transcriptional transactivating activity of p53 are discussed.

Animals↗
PubMed2004

A semantic approach to segmentation of overlapping objects.

OBJECTIVES: This paper aims at introducing a novel approach for segmentation of overlapping objects and at demonstrating its applicability to medical images. METHODS: This work details a novel approach enhancing the known theory of full-segmentation of an image into regions by lifting it to a semantic segmentation into objects. Our theory allows the formal description of partitioning an image into regions on the first level and allowing the occurrence of overlaps and occlusions of objects on a second, semantic level. Possible applications for the use of this 'semantical segmentation' are the analysis of radiographs and micrographs. We demonstrate our approach by the example of segmentation and separation of overlapping cervical cells and cell clusters on a set of 787 image pairs of registered PAP- and DAPI-stained micrographs. The semantical cell segmentation yielding areas of cell plasmas and nuclei are compared to a manual segmentation of the same images, where 2212 cells have been labeled. A direct comparison of over and under-segmentation between the two segmentation sets yields a mean difference value of 10.15% for the nuclei and 10.80% for the plasma. RESULTS: Using the proposed theory of semantical segmentation of images in combination with adequate models of the image contents, our approach allows identifying, separating and distinguishing several overlapping, occluding objects in medical images. Applying the proposed theory to the application of cervical cell segmentation from overlapping cell clusters and aggregates, it can be seen that it is possible to formally describe the complex image contents. CONCLUSIONS: The proposed method of semantical segmentation is a mighty tool and under the assumption of the subtractive transparency model can be used in different medical image processing applications such as radiology and microscopy. By using alternative models to solve the ambiguities attached to overlaps and occlusions, further fields of application can be addressed.

Algorithms↗
PubMedDate not supplied

Fundamental study of automatic cyto-screening for uterine cancer. II. Segmentation of cells and computer simulation.

In images of Papanicolaou stained cells 64 gray levels have been differentiated by scanning densitomery. One of the two peaks in a differential histogram indicates the threshold of the cytoplasm, the other that of the nucleus. The two modes indicate where in the digitized image of good segmentation of the cell from its background and the nucleus from the cytoplasm can be accomplished.

Cell Nucleus↗
PubMed1986

Abdominal segmentation, pole cell formation, and embryonic polarity require the localized activity of oskar, a maternal gene in Drosophila.

Embryos derived from oskar females lack pole cells and the specialized pole plasm including polar granules. In addition, the abdominal region remains unsegmented and eventually dies. Transplantation of cytoplasm from normal embryos into mutant embryos reveals that osk-dependent activity is strictly localized at the posterior pole and has three distinct functions. In mutant embryos the activity will normalize pole cell formation when transplanted into the posterior pole and abdominal segmentation after transplantation to a more anterior, the prospective abdominal, region. Furthermore, osk activity can provoke the formation of a second "posterior center" at the anterior. The participation of the osk product in the establishment of a source of morphogenetic activity in the posterior pole plasm is discussed.

Abdomen↗
PubMed1985

Plasma membrane structure at the axon hillock, initial segment and cell body of frog dorsal root ganglion cells.

Analysis of the plasmalemma of frog dorsal root ganglion cells by freeze-fracture demonstrates regional differences in the distribution of intramembranous particles. Although P-face particles are distributed rather uniformly, the E-face particle concentration at the cell body (approximately 300 micron -2) is much lower than that at the axon hillock (approximately 900 micron -2), proximal initial segment (approximately 1000 micron -2), or intermediate portion of the initial segment (approximately 800 micron -2). The particle concentrations in the latter regions approach that at the node of Ranvier and, moreover, particle size analysis reveals that the E-face particles, like those at the node, include a large number that are 10 nm or more in diameter. Thin sections reveal patches of a dense undercoating on the cytoplasmic surface of the axolemma in some regions of the initial segment but not the axon hillock. It is concluded from these results that the axon hillock and the initial segment of dorsal root ganglion cells have some of the structural characteristics of the node of Ranvier.

Animals↗
PubMed1998

Cadherin-defined segments and parasagittal cell ribbons in the developing chicken cerebellum.

In the developing chicken cerebellar cortex, three cadherins (Cad6B, Cad7, and R-cadherin) are expressed in distinct parasagittal segments that are separated from each other by ribbons of migrating interneurons and granule cells which express R-cadherin and Cad7, respectively. The segment/ribbon pattern is respected by the expression of other types of molecules, such as engrailed-2 and SC1/BEN/DM-GRASP. The cadherin-defined segments contain young Purkinje cells which are connected to underlying nuclear zones expressing the same cadherin, thereby forming parasagittal cortico-nuclear zones of topographically organized connections. In addition, R-cadherin-positive mossy fiber terminals display a periodic pattern in the internal granular layer. In this layer, Cad7 and R-cadherin are associated with synaptic complexes. These results suggest that cadherins play a pivotal role in the formation of functional cerebellar architecture by providing a three-dimensional scaffold of adhesive information.

Animals↗
PubMed1977

High resolution analysis of cervical cells--a progress report.

This paper presents preliminary results of research toward the development of a high resolution analysis stage for a dual resolution image processing-based prescreening device for cervical cytology. Experiments using both manual and automatic methods for cell segmentation are described. In both cases, 1500 cervical cells were analyzed and classified as normal or abnormal (dysplastic or malignant) using a minimum Mahalanobis distance classifier with eight subclasses of normal cells, and five subclasses of abnormal cells. With manual segmentation, false positive and false negative error rates of 2.98 and 7.73% were obtained. Similar experiments using automatic cell segmentation methods yielded false positive and false negative error rates of 3.90 and 11.56%, respectively. In both cases, independent training and testing data were used.

Autoanalysis↗
PubMed1999

Automated identification of stained cells in tissue sections using digital image analysis.

OBJECTIVE: To develop a novel automated image analysis system to differentiate immunohistochemically stained cells from background. STUDY DESIGN: Cell segmentation was performed by applying global thresholding algorithms to find an approximate threshold at which cells could be separated from background followed by a novel refinement algorithm to erode edge pixels of the region. To separate overlapping cells, a new decomposition method was developed that uses both semantic knowledge and high-level relational information. Both the cell segmentation and separation methods were evaluated on images of stained tissue sections and the manually outlined cell areas and numbers compared to the computed. RESULTS: Macrophage areas computed at the first stage by Otsu's algorithm did not differ significantly (P = .07) from those traced manually, while the areas computed by Kittler's and Kurita's algorithms did not agree (P < .01). Both Otsu's and Kurita's algorithms performed well when combined with edge pixel erosion. Kittler's algorithm proved unsuccessful even with edge erosion. Comparison of the computed and manually determined cell numbers showed a significant correlation, and regression analysis resulted in the unity curve. CONCLUSION: A combination of global thresholding and a novel edge erosion technique allowed identification of immunohistochemically stained macrophages; the computed cell areas agreed with the manual results.

Aorta↗
PubMed1994

Risk assessment of d-limonene: an example of male rat-specific renal tumorigens.

The naturally occurring food constituent d-limonene has been found to cause tumors at high doses only in the kidney of the male rat in association with the development of hyaline droplet nephropathy. In contrast, neither kidney tumors nor the associated nephropathy have been found in female rats or mice at much higher doses. Adult male rats produce large quantities of a specific low-molecular-weight protein in the liver, which is known as alpha 2U-globulin (alpha 2U-g). With administration of sufficient doses of d-limonene to male rats, this protein has been found to accumulate excessively in the P2 segment cells of renal proximal tubules, resulting in hyaline droplet formation as a manifestation of protein overload. Hyaline droplet accumulation is the first stage in a unique sequence of nephropathic lesions (also known as alpha 2U-g nephropathy), including granular casts in the outer medulla and linear mineralization in the papilla. The mechanism underlying protein accumulation appears to be the reversible binding of chemical to alpha 2U-g with subsequent prolongation of its half-life in the tubule cell. In the case of d-limonene, the minor metabolite d-limonene-1,2-oxide has been shown to be the primary chemical species that binds reversibly to alpha 2U-g, impeding the normal process of lysosomal proteinase degradation of alpha 2U-g. The ensuing nephropathy is associated with a sustained increase in compensatory renal tubule cell proliferation, which provides the putative mechanistic link with renal tumor formation possibly through tumor promotion of spontaneously initiated cells or enhanced spontaneous mutagenesis. This proposed mechanism has been supported by additional information, including negative genotoxicity tests for d-limonene and its oxide metabolites, experimentally verified tumor promotion, and enhanced cell proliferation primarily in P2 segment tubule cells in male F344 rats, but no such effects in the alpha 2U-g-deficient NBR rat. The mechanism of d-limonene tumor development does not appear to be possible in humans since neither the quantity nor the type of protein that binds d-limonene or d-limonene-1,2-oxide is present. The deduction that the renal tumors induced in male rats are not relevant to human carcinogenicity in the hazard evaluation step of risk assessment completes the evaluation of human risk for d-limonene. Consequently, it can be concluded that d-limonene does not pose any carcinogenic or nephrotoxic risk to humans.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1998

Automated segmentation of cell nuclei in fine needle aspirates of the breast.

OBJECTIVE: To achieve a classifier of breast lesions to discriminate between benign and malignant cases of cytologic smears with automated segmentation image analysis techniques. STUDY DESIGN: The techniques were applied to images of epithelial cell nuclei from cytologic smears obtained by fine needle aspiration. The images of the nuclei were taken from 95 cases of malignant lesions and 47 benign (approximately 25 nuclei per case), and 28 nuclear variables were measured. The data were analyzed by a double methodology, discriminant analysis and classification and regression trees (CART), to determine which provided the best results. RESULTS: CART selected the SD of the nuclear area with correct classification of 85.1% of benign and 94.7% malignant aspirates. Discriminant analysis selected the group of variables formed by axis lengths, SD of the longest axis, sphericity and variance of gray levels, with results similar to those of CART. CONCLUSION: Automated segmentation image analysis techniques were effective, and the classifier was quick, simple and efficacious in malignant-benign discrimination.

Algorithms↗
PubMed2006

Distribution of vitamin D3 receptor in the epididymal region of roosters (Gallus domesticus) is cell and segment specific.

Vitamin D3 is a steroid hormone well known by its role in maintaining calcium homeostasis, however this hormone may also participate in other biological functions, including control of reproductive processes. The vitamin D3 action is mediated by the vitamin D3 receptor (VDR). VDR is widely distributed in the rodent reproductive tract, however the occurrence of VDR and the role of the vitamin D3 in the avian reproductive tract remain unknown. The aim of the present study was to investigate the expression and cellular distribution of VDR in the epididymal region of roosters. VDR expression was investigated by Western blotting analysis and the tissue distribution of the receptor was determined by immunohistochemistry. The Western blotting assay revealed a major VDR protein band of 61kDa in the epididymal region of rooster. Nuclear VDR expression was found in all segments of the epididymal region, namely rete testis, efferent ductules, connecting ducts and epididymal ducts. Nonciliated cells of the distal efferent ductules showed the highest levels of VDR expression, followed by the proximal efferent ductules and rete testis. The connecting and epididymal ducts showed less intense VDR immunostaining. The differential VDR expression in the epididymal region segments reveals that several extratesticular ducts may be target for vitamin D3 action and suggests that vitamin D3 may have a regional-specific function, such as calcium transport, that is modulated through VDR activity.

Animals↗
PubMed1976

Hybridization of mRNA from adenovirus-transformed cells to segments of the adenovirus genome.

Cytoplasmic viral-specific RNA from the 8617 line of adenovirus type 2 (Ad2) transformed rat cells was hybridized to DNA fragments derived either by Eco-RI or Sma-I restriction endonucleases. Three discrete classes of viral RNA (26S, 20S, and 15S) were detected by hybridization of total cytoplasmic RNA with the fragment, and all of these RNA molecules also hybridized to the much smaller Sma-E fragment of Ad2 DNA. Poly (A)-containing RNA (exposed to 90% formamide) contained only the 20S and 15S mRNA species, suggesting that the 26S RNA might be an aggregated species. Viral RNA of the same size derived from the same regions of the genome has not been detected in lytically infected cells, suggesting some change in the pattern of transcription or RNA processing of the Ad2 DNA in the integrated state.

Adenoviridae↗
PubMed1994

Coenzyme A independent acylation of phosphatidylethanolamine in isolated bovine rod cell outer segments.

The acylation of membrane phospholipids in isolated bovine rod outer segments (ROS) by exogenous, radiolabeled palmitic acid (16:0) was assayed, using [14C]palmitoyl-coenzyme A (16:0-CoA), [3H]16:0 alone and [3H]16:0 with added ATP and CoA. Both [14C]16:0-CoA and [3H]16:0 with added ATP and CoA are incorporated predominantly into phosphatidylcholine (PC), whereas [3H]16:0 without added ATP and CoA is incorporated almost exclusively into phosphatidylethanolamine (PE). Without added ATP and CoA in the incubation medium, [3H]16:0 is esterified predominantly to the 2 position of PE, but with added ATP and CoA, it is esterified to both the 1 and 2 positions; the rates of esterification of [3H]16:0 to the 2 position of PE under these two conditions are similar. These results suggest that 16:0 is esterified to the 2 position of PE of ROS membranes by an acyl transferase mechanism that does not utilize an acyl-CoA intermediate. The product of this reaction is likely to be a molecular species of PE with 16:0 at the 2 position and a polyunsaturated fatty acid at the 1 position.

Acyltransferases↗