Nuclei of lymphocytes of the T- and B-cell lines during normal embryogenesis and after treatment with hydrocortisone (morphometric and probabilistic-statistical parameters)

Lymphoid cells of the thymus and of the Fabricius bursa have been studied in 18-day-old chick embryos, normal and after injection of hydrocortisone on the 11th day of embryogenesis. By means of optical-structural computer analysis, the complex of morphometric and probability-statistic parameters of the nuclei in the lymphocytes are estimated: area of the nuclei, optical density of chromatin, asymmetry coefficient and variance. Normal T-lymphocytes possess less density of the nuclei, greater optical density of chromatin, greater values of negative asymmetry. The complex of these parameters can be used for identification of visually similar lymphoid cells of T- and B-lines. Under hydrocortisone effect structural changes of the nuclei in the thymus and Fabricius bursa lymphocytes of the chick embryo are uniform: increase in the area of the nuclei, decrease in optical density of chromatin, the asymmetry coefficient becomes positive.     

Chromatin texture in high grade prostatic intraepithelial neoplasia and early invasive carcinoma

OBJECTIVE: To evaluate individual nuclei from high grade prostatic intraepithelial neoplasia (PIN) lesions with early invasive carcinoma foci in the area of microinvasion and in the gland in which the microinvasion originated. STUDY DESIGN: High-resolution, digitized images of nuclei from defined locations were recorded and segmented, and karyometric variables were computed. These included a set of 93 features, which form a nuclear signature characterizing the spatial and statistical distribution of the nuclear chromatin. Nuclei in the glandular epithelium were recorded sequentially, along the basal cell layer, at increasing distances from the point of microinvasion and by random selection in the region of microinvasion. RESULTS: At a distance > 60 nuclear locations from the point of microinvasion, the nuclear signatures corresponded to those seen in high grade PIN. Between 40 and 20 nuclear locations removed from the microinvasion focus the signatures began to change gradually until at a distance of 15-5 locations they strongly resembled the signatures seen in adenocarcinoma. The total optical density decreased to values seen in adenocarcinoma, and the nuclear chromatin had finer granularity. While nuclei in high grade PIN followed a widely dispersed total optical density distribution suggestive of wide-ranging aneuploidy, the nuclei in the region of microinvasion exhibited a less dispersed and bimodal total optical density distribution. CONCLUSION: The chromatin texture signatures showed a clear trend: there was an obvious attenuation as the measured nuclei approached the microinvasion area. The decrease in total optical density at the microinvasion might suggest the emergence of one or two clones that can be responsible for the invasive phenotype.     

Change of chromatin morphology during the cell cycle detected by means of automated image analysis

Mouse embryo fibroblasts growing asynchronously in vitro stained with Feulgen method and their nuclear chromatin was analysed by means of the image analysing computer Quantimet 720D. Cells with 2C, 3C and 4C content of DNA were considered as being in G₁, middle S and G₂ phase of cell cycle, respectively. It was found that the projected area of nuclei increases during the cell cycle and that the mean optical density of chromatin increases from G₁ through S to G₂ phase. The curves showing the areas of chromatin at different optical density thresholds are different for cells in G₁, S and G₂ phase. The results demonstrate cyclic changes in chromatin morphology in the interphase nuclei during the cell cycle.     

Karyometric features in nuclei near colonic adenocarcinoma. Statistical analysis.

The normal mucosa adjacent to colonic adenocarcinoma (marginal or transitional mucosa) has been shown to have subtle alterations of architecture, surface glycoproteins and proliferative activity. To evaluate possible changes in nuclear configurations in this marginal mucosa, a large set of cytometric features was evaluated using a computer-assisted video analysis system. Preliminary statistical analysis of the measurements identified six nuclear features useful for discriminating marginal mucosa nuclei from normal (control) mucosa nuclei: total optical density (OD), nuclear area, chromatin texture (from gray value cooccurrence matrix), chromatin coarseness, average OD of nuclear staining and peripheral tendency of the chromatin in the nucleus. An analysis of variance revealed that both patient-to-patient and gland-to-gland variation would limit the usefulness of any one feature as a screening tool. As a group, however, these six features should be investigated further as markers of preneoplastic changes in histologically normal-appearing mucosa.     

Influence of tumor drug resistance phenotype on the dynamics of cisplatin-induced changes of rat peripheral lymphocyte chromatin structure in Guerin's carcinoma

The correlation between the tumor drug sensitivity and the degree of lymphocyte interphase nuclei chromatin damages induced by cisplatin in rats was found using sensitive and resistant to cisplatin variants of Guerin's carcinoma. Increased optical density of lymphocyte chromatin in first minutes after cisplatin injection both in rats without Guerin's carcinoma and with sensitive to cisplatin variants of this tumor was observed. Lymphocyte chromatin structure remains unchanged in rats with cisplatin resistant carcinoma. Normal blood cells are suppose to change their sensitiveness to cisplatin under the humoral influence of the growing tumor in according with its phenotype.     

Chromatin organization in the rat hypothalamus during early development.

The organization of chromatin in neuronal and glial nuclei isolated from different brain regions of rats during development was studied by digestion of nuclei with micrococcal nuclease. A short chromatin repeat length (approx. 176 base-pairs compared with that of glial nuclei from foetal cerebral cortex (approx. 200 base-pairs) was present in hypothalamic neurons throughout the ages studied, which was similar to the repeat length of cortical neurons from 7- and 25-day-old animals (approx. 174 base-pairs). Whereas in cortical neurons the chromatin repeat length shortened from approx. 200 base-pairs in the foetus to approx. 174 base-pairs in the first postnatal week, the short chromatin repeat length of hypothalamic neurons was already present 2 days before birth, indicating that hypothalamic neurons differentiate earlier than cortical neurons during brain development.     

Protein chain initiation in vitro by liver cell components from DMNA-treated rats.

The mechanism of inhibition of protein synthesis in rat liver after dimethylnitrosamine (DMNA) administration was studied at the level of peptide-chain initiation by use of initiation-dependent amino acid incorporating systems. Ribosomal monomers, poly(A)-concontaining loss of acticity due to the DMNA treatment. The poly(A) RNA from monosomes and polysomes, and crude initiation factors from microsomes were prepared 2 h after a single dose of DMNA (75 mg/kg), and their activities in the production of new protein chains determined under conditions of nearly linear response. Monosomes and crude initiation factors from DMNA-treated rats were at least as active as those from controls. Preparations of poly(A)-containing RNA had a consistently higher template activity when prepared from polysomes instead of monosomes. However, in neither case was there any ltaining RNA was methylated by DMNA to about the same extent as the 18S and 28S rRNA. The methylation was consistently somewhat higher in the RNA preparations from monosomes than in those from polysomes.     

Cytophotometric evaluation of the transition of cells from the G0 phase to the G1 phase of the cell cycle

Feulgen stained nuclei of PHA-stimulated human blood lymphocytes were used for cytophotometric chromatin pattern analysis. Similar distributions of low optical density values indicating the predominance of diffuse chromatin were obtained for G1, S and G2 cells. Condensed chromatin was predominant in G0 and M nuclei. Integral versus average optical densities scatter plots analyses permitted one to distinguish cells undergoing different phases of cell cycle including G0 and G1.     

Effect of hypophysectomy on liver nuclear ribonucleic acid synthesis in aging rats.

Changes in RNA synthesis in liver nuclei were observed at different ages and after hypophysectomy and hormone replacement in female Sprague-Dawley rats. As determined by the incorporation of [3H]UMP into an acid-insoluble product, RNA synthesis decreased by about 75% in intact rats from 6 months to 24 months of age. This decline with age was not observed in liver nuclei from 24-month-old rats that had been hypophysectomized at 12 months and maintained on a minimal hormone-replacement therapy. Thyroid hormones and somatotropin (growth hormone) had an additive effect on RNA synthesis in liver nuclei from these hypophysectomized rats. The same hormones had no significant effect on intact, age-matched rats. With advancing age, nuclei of intact rats had an increase in the pool of free RNA polymerase and an apparent decrease in the enzyme activity bound to nuclear chromatin. There was no change in total enzyme with age. In hypophysectomized, hormone-treated rats, free RNA polymerase activity decreased and chromatin-bound activity increased. There was no difference in total nuclear RNA polymerase activity between operated or intact rats. However, the ratio of the bound to the free activity was different. These results suggest that the ability of RNA polymerase to bind to chromatin may be involved in the age-related decrease in liver nuclear RNA synthesis of intact rats.     

Incorporation of 1-(14)C linoleic acid in rat liver nuclei and chromatin fractions

The incorporation of 1-(14)C linoleic acid in several chromatin fractions of rat liver nuclei was investigated using two different procedures: (1) rat liver nuclei were incubated with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid. After 40 min at 37 degrees C the chromatin obtained by sonication of nuclei suspended in 0.25 M sucrose was fractionated by differential sedimentation; (2) chromatin fractions obtained by differential sedimentation were incubated separately with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid 40 min at 37 degrees C in order to characterize the fatty acid incorporation in isolated chromatin. A comparative study of the incorporation of 1-(14)C linoleic acid in microsomes and nuclei isolated from rat liver is also presented for the purpose of comparison. Linoleic acid was incorporated into nuclear lipids as well as in chromatin fractions. The fatty acid incorporation was stimulated considerably in the acylation system when compared to control, it appears to be highly dependent on the state of condensation of chromatin, being barely detectable in the lowest density fraction. The major proportion of 1-(14)C linoleic acid was found in phospholipids and in a lesser proportion it remained esterified to triglycerides and cholesteryl esters. The distribution of radioactivity in different classes of phospholipids present in microsomes and nuclei isolated from rat liver, showed a similar profile of distribution. The major proportion of radioactivity, approximately 50% was found in phosphatidylcholine and in a lesser proportion in sphingomyelin, phosphatidylserine and phosphatidylethanolamine. When chromatin fractions were incubated separately, it was observed that the major proportion of 1-(14)C linoleic acid in phospholipids was found in heavy chromatin fractions whereas low density chromatin fraction only incorporated in a lesser proportion.     

Effects of ubiquinone-9 on lipids of nuclei and chromatin of the rat liver under continuous irradiation]

The influence of continuous gamma irradiation on the lipids of nuclei and chromatin of rat liver at a dose-rate of 0,129 Gy/day for 155 days (a total dose of 20 Gy) and by feeding of ubiquinone-9 has been studied. The amount of phosphatidylcholine with phosphatidylserine and phosphatidyl-ethanolamine in liver nuclei of irradiated rats was found to increase. Ubiquinone-9 had a normalizing effect. A decrease of cardiolipin was observed in the liver chromatin of irradiated rats. The amount of free fatty acids had a tendency to decrease in homogenate, nuclei and liver chromatin of irradiated rats. Ubiquinone was found to increase the amount of free fatty acids up to the control level. The amount of cholesterol in nuclei was increased after irradiation and that in chromatin tended to rise. Ubiquinone-9 significantly decreased the amount of cholesterol in nuclei and chromatin of irradiated rats.     

Isolation and characterization of nuclei from Neurospora crassa.

A procedure was developed for isolating nuclei from either the conidial or germinated conidial growth phase of Neurospora crassa. A frozen conidial suspension was lysed by passage through a French pressure cell, and the nuclei were freed from the broken cells by repeated homogenization in an Omni-Mixer. Pure nuclei were obtained from the crude nuclear fraction by density banding in a Ludox gradient. The final nuclear yield was 20 to 30%. The nuclei had a deoxyribonucleic acid (DNA):ribonucleic acid (RNA):protein ratio of 1:3.5:7 and were active in RNA synthesis. The nuclei, stained with the DNA stain 4,6-diamidino-2-phenylindole, appeared under fluorescence microscopy as bright blue spheres, 1 micron in diameter, essentially free from cytoplasmic attachments. Chromatin extracted from the nuclei in a 70 to 75% yield by dissociation with 2 M sodium chloride and 5 M urea had a DNA:RNA:protein ratio of 1:1.05:1.7. Chromatin reconstituted from this preparation exhibited a level of RNA polymerase template activity lower than that of pure Neurospora DNA, but the maximum level of reconstitution obtained was only 10%. Fractionation of Neurospora chromatin on hydroxylapatite separated the histones from the chromatin acidic proteins. The normal complement of histone proteins was present in both the reconstituted and dissociated chromatin preparations. The acidic protein fraction exhibited a variety of bands on sodium dodecyl sulfate gel electrophoresis ranging in molecular weight from 15,000 to 70,000. The gel pattern was much more complex for total dissociated chromatin than for reconstituted chromatin.     

Standardization of the Feulgen reaction: the influence of chromatin condensation on the kinetics of acid hydrolysis.

The purpose of the present study was to investigate the influence of chromatin compactness on the kinetics of acid hydrolysis in the Feulgen reaction in cytology. Tissue imprints of rabbit liver, of human bronchial carcinoma and of human blood smears, fixed with alcohol, formaldehyde or with B?hm's solution with and without prior air drying, were stained with a standardized pararosanilin-Feulgen reagent. The time for hydrolysis varied between 7.5 and 120 min. The integrated optical density (IOD) of the cell nuclei was measured with an image analyzer (IBAS 2000). Cells with condensed chromatin (lymphocytes, small cell carcinoma, formaldehyde fixed cells) showed a slow increase of staining intensity and late plateau phase as compared with cells with decondensed chromatin. DNA in condensed nuclei was less susceptible to acid hydrolysis. The degree of chromatin compactness which determines the sensitivity of DNA to hydrolysis is influenced by the type of fixation, cell type and by the functional status of the cell. The conclusion is that Feulgen staining intensities of cells with different degrees of chromatin compactness cannot be compared unless measured in the respective plateau phases of the relevant hydrolysis curves which must be determined individually for each cell type.     

Nuclear DNA variability withinPisum sativum (Leguminosae): Cytophotometric analyses

The cultivars or experimental lines ofPisum sativum were analyzed cytophotometrically for nuclear DNA content of early prophases after ethanol-acetic acid fixation. Wide variability was found (from 3.93 to 5.07pg per haploid nucleus). This result was confirmed by the cytophotometric analysis of interphase nuclei isolated from leaf tissues fixed in formalin. Analysis of interphase nuclei at different thresholds of optical density showed that certain chromatin fractions are involved in the variations.     

Growth hormone and drug metabolism. Acute effects on nuclear ribonucleic acid polymerase activity and chromatin.

Adult male rats, subjected either to sham operation or to hypophysectomy and adrenalectomy were maintained for 10 days before treatment with growth hormone. Results of the acute effects of growth hormone on the rat liver nuclear RNA polymerase I (nucleolar) and II (nucleoplasmic) activities as well as the chromatin template capacity were then studied and compared with the growth-hormone effects on the drug metabolism described in the preceding paper (Wilson & Spelsberg, 1976). 2. Conditions for isolation and storage of nuclei for maintenance of optimal polymerase activities are described. It is verified that the assays for polymerase activities require a DNA template, all four nucleoside triphosphates, and a bivalent cation, and that the acid-insoluble radioactive product represents RNA. Proof is presented that under high-salt conditions DNA-like RNA (polymerase II) is synthesized, and that under low-salt conditions in the presence of alpha-amanitin, rRNA (polymerase I) is synthesized. 3. In the livers of hypophysectomized/adrenalectomized rats, growth hormone increases the activity of both RNA polymerase enzymes and the chromatin template capacity within 1h after treatment. The effects last for 12h in the case of polymerase II but for only 6h in the case of polymerase I. Sham-operated rats respond to growth hormone in a manner somewhat similar to that shown by hypophysectomized/adrenalectomized rats. These results, which demonstrate an enhancement of RNA polymerase I activity in response to growth hormone, support those from other laboratories. 4. Growth-hormone enhancement of the chromatin template capacity in the liver of hypophysectomized/adrenalectomized rats contrasts with previous reports. The growth-hormone-induced de-repression of the chromatin DNA could represent the basis of the growth-hormone-induced enhancement of RNA polymerase II activity in the hypophysectomized/adrenalectomized rats, although some effect of growth-hormone on the polymerase enzymes is still suggested.     

Evaluation of the DNA-repair host-mediated assay. I. Induction of repairable DNA damage in E. coli cells recovered from liver, spleen, lungs, kidneys, and the blood stream of mice treated with methylating carcinogens

The DNA-repair host-mediated assay was further calibrated by determining the genotoxic activities of 4 methylating carcinogens, namely, dimethylnitrosamine (DMNA), 1,2-dimethylhydrazine (SDMH), methyl nitrosourea (MNU) and methyl methanesulphonate (MMS) in various organs of treated mice. The ranking of the animal-mediated genotoxic activities of the compounds was compared with that obtained in DNA repair assays performed in vitro. The differential survival of strain E. coli K-12/343/113 and of its DNA-repair-deficient derivatives recA, polA and uvrB/recA, served as a measure of genotoxic potency. In the in vitro assays and at equimolar exposure concentrations, MMS and MNU are the most active chemicals, followed by DMNA, which shows a slight genotoxic effect only in the presence of mouse liver homogenate; SDMH has no activity under these conditions. In the host-mediated assays, the order of genotoxic potency of the compounds was quite different: those carcinogens which require mammalian metabolic activation, namely, DMNA and SDMH, show strong effects in liver and blood, a lesser effect in the lungs and kidneys and the least effect in the spleen. The activity of MNU, a directly acting compound, is similar in all organs investigated, but it is clearly lower than that of DMNA and SDMH. MMS, also a directly acting carcinogen, causes some (barely significant) effect at the highest dose tested. A similar order of potency was observed when the compounds were tested in intrasanguineous host-mediated assays with gene mutation as an endpoint. DMNA and SDMH induce comparable frequencies of L-valine-resistant mutants in E. coli K-12/343/113 recovered from liver and spleen of treated mice, the effect in the liver being the strongest. MNU is mutagenic only at a higher dose, while MMS shows no effect. The results are discussed with respect to the literature data on organ-specific DNA adduct formation induced by the compounds. It is concluded that qualitatively there is a good correlation between the degree of genotoxic activity found in the DNA repair host-mediated assay and DNA adduct formation in the animal's own cells. This is exemplified by the finding that the relative order of genotoxic activity of the 4 methylating agents in bacteria recovered from various organs (DMNA approximately equal to SDMH greater than MNU greater than MMS) is reflected by the same order of magnitude in DNA alkylation in corresponding mammalian organs. Quantitatively, the indirectly acting agents DMNA and SDMH seem to induce fewer genotoxic effects in bacteria present in the liver than would be expected on the basis of DNA-adduct formation data.     

Isolation and purification of myotube and myoblast nuclei from cultures of embryonic chick skeletal muscle.

Methods are described for the preparation of purified myotubes from embryonic chick skeletal muscle cultures and the preparation of purified nuclei from both myotubes and myoblasts. Myotubes are released from the culture dish by digestion of their collagen substratum with collagenase, and purified by sucrose density gradient sedimentation. Nuclei are prepared from the isolated myotubes by controlled homogenization in Ca²⁺-free medium and sedimentation through 2.1 M sucrose. Nuclei are prepared from cultured myoblasts in a similar fashion, with the inclusion of the non-ionic detergent NP-40 in the homogenization medium and sedimentation through 2.4 M sucrose. Phase contrast microscopic examination showed that the nuclear preparations are free of visible cytoplasmic contamination, and are morphologically similar to nuclei observed in situ. Biochemical assays (protein/DNA and $\text{RNA}\text{DNA}$ ratios) confirm the purity of the nuclear preparations. Both nuclear preparations have been used to prepare purified chromatin which has spectral and chemical properties similar to those reported for chromatin purified directly from several chick tissues.     

The variation with age of the structure of chromatin in three cell types from rat liver.

The organization of chromatin in three rat liver nuclear populations, namely diploid stromal, diploid parenchymal, and tetraploid parenchymal nuclei, which were separated by zonal centrifugation, was studied by digestion with micrococcal nuclease and pancreatic deoxyribonuclease in 3-week-old rats in which the parenchymal cells contain diploid nuclei and in 2-and 4-month-old rats with a high proportion of tetraploid nuclei. Digestion by micrococcal nuclease allowed the estimation of DNA-repeat length in chromatin. Parenchymal nuclei have shorter repeat length than stromal nuclei and DNA-repeat length increases with the age in all three nuclei populations. The kinetics of digestion by micrococcal nuclease showed that nuclei with shorter repeat length are more sensitive to micrococcal nuclease and that the sensitivity of chromatin decreases with age for all the types of nuclei in this study. The kinetics of digestion by pancreatic deoxyribonuclease showed that sensitivity of chromatin is related to the repeat length and that the sensitivity decreases with the ages.