Evidence of repetitive patterns of chromatin distribution in cell nuclei of rat liver

Samples of rat livers were fixed in glutaraldehyde, contrasted en bloc with phosphotungstic acid, embedded in an epoxy resin and serially sectioned. The study of three-dimensional models of 20 complete nuclei shows that all of them share some general features: they have more than one nucleolus (2-4), an irregular layer of compact chromatin adjacent to the nuclear membrane and well-delimited clumps of chromatin both in the nuclear sap and surrounding the nucleoli. A space of 8 sections containing the central nucleolus and a lateral one was studied in detail. In this space, 8 clumps of compact chromatin were found in 17 nuclei and 9 clumps in the other 3 nuclei. No other number of clumps was found in those zones. In all the nuclei studied the compact chromatin surrounding the central nucleolus contacts the nuclear envelope. This contact takes place in a region almost diametrically opposed to the lateral nucleolus in 13 nuclei. In 7 nuclei, these structures were at angles between 50 and 125 degrees. These results support the existence of nonrandom repetitive patterns of chromatin distribution in liver cells.     

Characterization of chromatin distribution in cell nuclei

In this paper we develop four measures to describe the distribution of nuclear chromatin. These measures attempt to describe in an objective and meaningful way the heterogeneity, granularity, condensation, and margination of chromatin in cell nuclei. Starting with a high-resolution digitized image of a cell where the nuclear pixels have been identified, the four measures may be rapidly estimated. The range of each is derived and the interpretation of the measures in the context of chromatin compaction and distribution is developed. Implementation issues such as sampling density, thresholding and subsequent pre-processing, and algorithmic complexity are discussed.     

Isolation of nuclei for chromatin analysis in fission yeast.

The methods available for analysis of the chromatin of Schizosaccharomyces pombe are time consuming (>8 h) and/or result in some degradation of the chromatin. Here we report an optimised method for the preparation of spheroplasts and the isolation of nuclei which takes <25 min and is suitable for analysis of chromatin structure by micrococcal nuclease, restriction endonuclease or by immunoprecipitation.     

Age-related changes in chromatin of liver cell nuclei of different ploidity.

A comparison of the Feulgen hydrolysis curves and the chromatin compactness of the liver cell nuclei of young and old rats was made. It was found that the rate of DNA depurination and chromatin compactness are higher in the liver cell nuclei of old rats, both in di-and tetraploidal cells. The effect of fixation upon the course of the hydrolysis curves is discussed.     

Cytochemical analysis of the chromatin of liver cell nuclei and nuclei of oval cells by means of the Feulgen hydrolysis curve in rats treated by thioacetamide

The present study is an analysis of the Feulgen hydrolysis characteristics in nuclei of liver cells and oval cells in rats treated by thioacetamide (TAA) and of liver cells in control rats. The curves show a double-peaked pattern. A slower hydrolysis is noted in the first peak after TAA treatment. This suggests an alteration of the acid-labile part of the chromatin. The curve obtained in the oval cells is different from the one in the liver cells. The implications of these differences are discussed with respect to development of cholangiocarcinomas.     

Texture analysis of fluorescence lifetime images of AT- and GC-rich regions in nuclei.

We used intensity and fluorescence lifetime microscopy (FLIM) of 3T3 nuclei to investigate the existence of AT-rich and GC-rich regions of the nuclear DNA. Hoechst 33258 (Ho) and 7-aminoactinomycin D (7-AAD) were used as fluorescence probes specific for AT and GC base pairs, respectively. YOYO-1 (Yo) was used as a dye that displays distinct fluorescence lifetimes when bound to AT or GC base pairs. We combined fluorescence imaging of Ho and 7-AAD with time-resolved measurements of Yo and took advantage of an additional information content of the time-resolved fluorescence. Because a single nucleus could not be stained and measured with all three dyes, we used texture analysis to compare the spatial distribution of AT-rich and GC-rich DNA in 100 nuclei in different phases of the cell cycle. The fluorescence intensity-based analysis of Ho- or 7-AAD-stained images indicates increased number and larger size of the DNA condensation centers in the G2/M-phases compared to G0/1-phases. The lifetime-based study of Yo-stained images suggests spatial separation of the AT- or GC-rich DNA regions in the G2/M-phase. Texture analysis of fluorescence intensity and lifetime images was used to quantitatively study the spatial change of condensation and separation of AT- and GC-rich DNA during the cell cycle.     

Three dimensional analysis of histone methylation patterns in normal and tumor cell nuclei

Histone modifications represent an important epigenetic mechanism for the organization of higher order chromatin structure and gene regulation. Methylation of position-specific lysine residues in the histone H3 and H4 amino termini has linked with the formation of constitutive and facultative heterochromatin as well as with specifically repressed single gene loci. Using an antibody, directed against dimethylated lysine 9 of histone H3 and several other lysine methylation sites, we visualized the nuclear distribution pattern of chromatin flagged by these methylated lysines in 3D preserved nuclei of normal and malignant cell types. Optical confocal serial sections were used for a quantitative evaluation. We demonstrate distinct differences of these histone methylation patterns among nuclei of different cell types after exit of the cell cycle. Changes in the pattern formation were also observed during the cell cycle. Our data suggest an important role of methylated histones in the reestablishment of higher order chromatin arrangements during telophase/early G1. Cell type specific histone methylation patterns are possibly casually involved in the formation of cell type specific heterochromatin compartments, composed of (peri)centromeric regions and chromosomal subregions from neighboring chromosomes territories, which contain silent genes.     

3D cell nuclei segmentation based on gradient flow tracking

Background  

Reliable segmentation of cell nuclei from three dimensional (3D) microscopic images is an important task in many biological studies. We present a novel, fully automated method for the segmentation of cell nuclei from 3D microscopic images. It was designed specifically to segment nuclei in images where the nuclei are closely juxtaposed or touching each other. The segmentation approach has three stages: 1) a gradient diffusion procedure, 2) gradient flow tracking and grouping, and 3) local adaptive thresholding.     

HAMLET interacts with histones and chromatin in tumor cell nuclei

HAMLET is a folding variant of human alpha-lactalbumin in an active complex with oleic acid. HAMLET selectively enters tumor cells, accumulates in their nuclei and induces apoptosis-like cell death. This study examined the interactions of HAMLET with nuclear constituents and identified histones as targets. HAMLET was found to bind histone H3 strongly and to lesser extent histones H4 and H2B. The specificity of these interactions was confirmed using BIAcore technology and chromatin assembly assays. In vivo in tumor cells, HAMLET co-localized with histones and perturbed the chromatin structure; HAMLET was found associated with chromatin in an insoluble nuclear fraction resistant to salt extraction. In vitro, HAMLET bound strongly to histones and impaired their deposition on DNA. We conclude that HAMLET interacts with histones and chromatin in tumor cell nuclei and propose that this interaction locks the cells into the death pathway by irreversibly disrupting chromatin organization.     

Symmetry analysis of cell nuclei

An algorithm is described that is used to analyze the two-dimensional spatial symmetry of cell nuclei. The method provides two symmetry features: the symmetry index (SI), which estimates the precise spatial symmetry of a given chromatin component, Cn, and the quadrant symmetry index (QSI), which estimates the number of quadrants being occupied by Cn. A previous analysis is used to show that age-related change in Malpighian tubule nuclei from the adult housefly is associated with significant alterations in the spatial symmetry of low-, medium-, and high-density chromatin components (LDC, MDC, HDC). This included a seven-fold increase in the spatial symmetry of HDC and a shift in the symmetry profile (from highest to lowest degree of symmetry) from LDC-MDC-HDC to MDC-LDC-HDC. The increased spatial symmetry of HDC suggests that it occurs at new nuclear sites as the fly ages and that these sites are distributed over approximately 60% of the chromosome population.     

Action of neocarzinostatin on cell nuclei: release of specific chromatin

We report the first complete sequence of a P450 monoxygenase cytochrome. The P450_CAM from $\text{Pseudomonas}\text{putida}$ is a single polypeptide of 412 residues as determined from the isolated tryptic, clostripain, CNBr, and mild acid cleavage fragments. Significant molecular features, including secondary structure, are discussed.     

Three-dimensional visualization and quantitative analysis of cervical cell nuclei with confocal laser scanning microscopy

OBJECTIVE: To develop a method for the acquisition and processing of 3-dimensional images based on confocal laser scanning microscopy for the purpose of 3-dimensional visualization and quantitative analysis of cell nuclei. STUDY DESIGN: A contour-based surface rendering method was used, and volume rendering was implemented according to the basic volume rendering pipeline. To extract quantitative features, a 3-dimensional labeling method based on slice information was used. After applying the labeling algorithm, the measurements for 3-dimensional quantitative analysis of nuclei were extracted: nuclear volume, surface area and spherical shape factor. We compared the 3-dimensional features of normal and abnormal cervical cell nuclei. RESULTS: Comparison of the size of 3-dimensional cervical cell nuclei between normal and abnormal revealed a statistically significant difference. The proposed method could overcome the limitation inherent in 2-dimensional analysis and could become a way of improving the accuracy and reproducibility of quantification of cell nuclei. CONCLUSION: Three-dimensional visualization and quantification of cell nuclei provide valuable medical information that can lead to a more objective diagnosis.     

Markovian analysis of cervical cell images.

Markovian analysis is a method to measure optical texture based on gray-level transition probabilities in digitized images. Experiments are described that investigate that classification performance of parameters generated by Markovian analysis. Results using Markov texture parameters show that the selection of a Markov step size strongly affects classification error rates and the number of parameters required to achieve the maximum correct classification rates. Markov texture parameters are shown to achieve high rates of correct classification in discriminating images of normal from abnormal cervical cell nuclei.     

High resolution segmentation of cervical cells.

A major problem in the automation of cervical cytology screening is the segmentation of cell images. This paper presents the present status of the work on that problem at the University of Uppsala. A dual resolution system is used. Suspect malignant cells are located at 4 mu resolution. Each such cell is rescanned at 0.5 mu resolution at two different wavelengths, 530 and 570 nm. The nucleus and the cytoplasm are isolated each by two independent methods. For the nucleus adaptive thresholding in the histogram of the 570 nm image and a contouring in a radially transformed version of that image is used. For the cytoplasm a two dimensional thresholding in the 2D histogram and a contouring in a radially transformed version of the 530 nm image is used. If the two nuclear masks agree the surrounding area is checked for disturbing objects. If also the cytoplasm masks agree and are without disturbing objects the whole cell is accepted. The result of the cytoplasm masks agree and are without disturbing objects the whole cell is accepted. The result of the segmentation is thus three categories; free cells, free nuclei and rejected objects. The shape of the objects belonging to the former two categories is checked and irregularly shaped ones are rejected as probably consisting of several overlapping nuclei. Cells passing also this test are classified as normal or malignant. The experience from using this algorithm is discussed and areas for further research are pointed out.     

Texture segregation in chromatic element-arrangement patterns.

An element-arrangement pattern is composed of two types of elements arranged differently in different regions of a pattern. Rapid texture segregation depends on spontaneously discriminating the difference in the arrangement of the elements. Five experiments investigated the perceived segregation of patterns composed of two types of squares arranged in vertical stripes in the top and bottom regions and in a checkerboard arrangement in the middle region. The squares were either equal in luminance and different in hue or equal in hue and different in luminance. The rated similarities of the two hues in a pattern failed to predict perceived segregation. For a given background luminance, the perceived segregation was predicted by the square-root of the sum of the squares of the differences in the outputs of the L - M + S and L + M - S opponent channels, where L, M, and S were the cone contrasts of the long-, medium-, and short-wavelength receptors. The perceived similarity of the two hues in a pattern was not affected by the background luminance but was a function of cone excitations instead. For patterns differing in hue and equal in luminance, perceived segregation was an inverse function of the background luminance. A white background decreased the perceived segregation, but a black background did not. The effect of background luminance was not on the discrimination of the individual hues. The two hues making up a texture pattern were clearly distinguishable on a white background. A white background interfered with the discrimination of the vertical and diagonal columns of squares that distinguished the texture regions. For patterns differing in luminance and equal in hue, black and white backgrounds decreased the perceived segregation. The results indicate that adapting to an achromatic luminance distant from the luminance of the squares increased the Weber threshold for discriminating luminance differences, but did not increase the Weber threshold for discriminating hue differences. The experiments also revealed that luminance was the primary factor affecting perceived segregation and that perceived brightness is secondary. The results are consistent with the hypothesis that perceived segregation in element-arrangement patterns is primarily a function of the differences in the outputs of relatively early filtering mechanisms that encode pattern differences prior to the specification of the element shapes and their properties.     

A comparison of the digestion of nuclei and chromatin by staphylococcal nuclease.

We have followed the kinetics of staphylococcal nuclease digestion of duck reticulocyte nuclei and chromatin from early stages to the digestion limit. We confirm that partial digestion of nuclei produces discrete DNA bands which are multiples of a monomer, 185 base pairs in length. The multimers are shown to be precursors of the monomer, which is next digested to a homogeneous, 140 base pair fragment. This fragment in turn gives rise to an array of nuclear limit digest DNA bands, which is almost identical with the limit digest pattern of isolated chromatin. As in the case of chromatin, half the DNA of nuclei is acid soluble at this limit. While the DNA limit digest patterns of nuclei and chromatin are similar, the large multimeric structures present as intermediates in nuclear digestion are absent in chromatin digestion. Alternate methods of chromatin gel preparation appear to leave more of the higher order structure intact, as measured by the production of these multimeric bands. Our results are consistent with the "beads on a string" model of chromatin proposed by others.