Search

Because the liver plays a major role in metabolic homeostasis and secretion of clotting factors and inflammatory innate immune proteins, there is interest in understanding the mechanisms of hepatic cell activation under hyperglycaemia and whether this can be attenuated pharmacologically. We have previously shown that hyperglycaemia stimulates major changes in chromatin organization and metabolism in hepatocytes, and that the histone deacetylase inhibitor valproic acid (VPA) is able to reverse some of these metabolic changes. In this study, we have used RNA-sequencing (RNA-seq) to investigate how VPA influences gene expression in hepatocytes. Interesting, we observed that VPA attenuates hyperglycaemia-induced activation of complement and coagulation cascade genes. We also observe that many of the gene activation events coincide with changes to histone acetylation at the promoter of these genes indicating that epigenetic regulation is involved in VPA action.

Valproic acid/sodium valproate (VPA) constitutes a widely prescribed drug for the treatment of seizure disorders and is a well-known epigenetic agent, inducing the acetylation of histones and affecting the methylation status of DNA and histones, with consequences on gene expression. Because this drug has been recently reported to exert affinity for histone H1, and to a minor degree for DNA, in this work, we investigated a possible interaction of sodium valproate with DNA and histones H1 and H3 using high-performance polarization microscopy and Fourier-transform infrared (FTIR) microspectroscopy. The preparations under examination consisted of hemispheres resulting from drop-casting samples containing VPA-DNA and VPA-histone mixtures. The results indicated that VPA may interact with DNA and histones, inducing changes in the textural superstructure and molecular order of the DNA possibly through van der Waals forces, and in histone H1 and H3 conformations, probably as a result of electrostatic binding between the drug and protein amino acid residues. These results contribute to a better understanding of the pharmacological potential of VPA. The precise sites and mechanisms involved in these interactions would certainly benefit from investigations provided by complementary methodologies.

Sodium valproate/valproic acid (VPA), a histone deacetylase inhibitor, and 5-aza-2-deoxycytidine (5-aza-CdR), a DNA methyltransferase 1 (DNMT1) inhibitor, induce DNA demethylation in several cell types. In HeLa cells, although VPA leads to decreased DNA 5-methylcytosine (5mC) levels, the demethylation pathway involved in this effect is not fully understood. We investigated this process using flow cytometry, ELISA, immunocytochemistry, Western blotting and RT-qPCR in G1 phase-arrested and proliferative HeLa cells compared to the presumably passive demethylation promoted by 5-aza-CdR. The results revealed that VPA acts predominantly on active DNA demethylation because it induced TET2 gene and protein overexpression, decreased 5mC abundance, and increased 5-hydroxy-methylcytosine (5hmC) abundance, in both G1-arrested and proliferative cells. However, because VPA caused decreased DNMT1 gene expression levels, it may also act on the passive demethylation pathway. 5-aza-CdR attenuated DNMT1 gene expression levels but increased TET2 and 5hmC abundance in replicating cells, although it did not affect the gene expression of TETs at any stage of the cell cycle. Therefore, 5-aza-CdR may also function in the active pathway. Because VPA reduces DNA methylation levels in non-replicating HeLa cells, it could be tested as a candidate for the therapeutic reversal of DNA methylation in cells in which cell division is arrested.

Made available in DSpace on 2021-06-25T10:22:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Sodium valproate (VPA) is a classic anticonvulsive, a histone deacetylase inhibitor, and a chromatin remodeling inducer. When injected into specimens of Triatoma infestans, a vector of Chagas disease, VPA affects the chromatin supraorganization of chromocenter heterochromatin in only a few cells of the Malpighian tubules. To test whether this result was explained by the inaccessibility of all of the organ's cells to the drug, we investigated the nuclear phenotypes and global acetylation of lysine 9 in histone H3 (H3K9ac) in Malpighian tubules cultivated in vitro for 1–24 h in the presence of 0.05 mM–1 mM VPA. The present results revealed that the chromatin decondensation event in the chromocenter body, which was detected only under low VPA concentrations up to a 4-h treatment, was not frequent during organ culture, similar to the results for injected insects. Cultivation of T. infestans Malpighian tubules in vitro for 24 h revealed inadequate for cell preservation even in the absence of the drug. Immunofluorescence signals for H3K9ac following VPA treatment showed a slightly increased intensity in the euchromatin, but were never detected in the chromocenter bodies, except with great intensity at their periphery, where the 18S rDNA is located. In conclusion, when VPA affects the chromocenter heterochromatin in this animal cell model, it occurs through a pathway that excludes a classic global H3K9ac mark. Investigation of nonhistone proteins associated with histone methylation marks is still required to further explain the differential response of T. infestans chromatin to VPA. Department of Structural and Functional Biology Institute of Biology University of Campinas (Unicamp) Superintendence for Control of Endemic Diseases (SUCEN) School of Pharmaceutical Sciences São Paulo State University (Unesp) School of Pharmaceutical Sciences São Paulo State University (Unesp) CNPq: 304797/2019-7 CNPq: 307398/2018-8

The frequency of polyploid nuclei in the aging human heart is in sharp contrast with that in the human liver. An inverse pattern exists between the mouse heart and liver cells. Ploidy degrees in mouse hepatocytes under hyperglycemic conditions are elevated to higher levels than those in aged hepatocytes. In this study, image analysis cytometry was used to investigate the effect of diabetes and aging on Feulgen-DNA quantities, ploidy degrees, nuclear shapes and chromatin texture in mouse cardiomyocytes compared to previously reported data for mouse hepatocytes. Adult, non-obese diabetic (NOD) hyperglycemic and normoglycemic females and 56-week-old normoglycemic BALB/c females were used. A small percentage (∼7%) of the cardiomyocyte nuclei in severely hyperglycemic NOD adult mice possessed higher ploidy values than those in the 8-week-old normoglycemic mice. Surprisingly, the Feulgen-DNA values and the frequency of nuclei belonging to the 4C and 8C ploidy classes were even higher (∼6%) in normoglycemic NOD specimens than in age-matched hyperglycemic NOD specimens. Additionally, a pronounced elongated nuclear shape was observed especially in adult normoglycemic NOD mice. In conclusion, NOD mice, irrespective of their glycemic level, exhibit a moderate increase in ploidy degrees within cardiomyocyte nuclei during the adult lifetime. As expected, aging did not affect the Feulgen-DNA values and the ploidy degrees of cardiomyocytes in BALB/c mice. The differences in ploidy degrees and chromatin textures such as absorbance variability and entropy, between adult NOD and aged BALB/c mice are consistent with other reports, indicating dissimilarities in chromatin functions between diabetes and aging.

Diabetes is a complex multifactorial disorder characterized by chronic hyperglycemia due to impaired insulin secretion. Recent observations suggest that the complexity of the disease cannot be entirely accounted for genetic predisposition and a compelling argument for an epigenetic component is rapidly emerging. The use of histone deacetylase inhibitor (HDACi) in clinical setting is an emerging area of investigation. In this study, we have aimed to understand and compare the response of hepatocyte chromatin to valproic acid (VPA) and trichostatin A (TSA) treatments under normoglycemic or hyperglycemic conditions to expand our knowledge about the consequences of HDACi treatment in a diabetes cell model. Under normoglycemic conditions, these treatments promoted chromatin remodeling, as assessed by image analysis and H3K9ac and H3K9me2 abundance. Simultaneously, H3K9ac marks shifted to the nuclear periphery accompanied by HP1 dissociation from the heterochromatin and a G1 cell cycle arrest. More striking changes in the cell cycle progression and mitotic ratios required drastic treatment. Under hyperglycemic conditions, high glucose per se promoted chromatin changes similar to those promoted by VPA and TSA. Nonetheless, these results were not intensified in cells treated with HDACis under hyperglycemic conditions. Despite the absence of morphological changes being promoted, HDACi treatment seems to confer a physiological meaning, ameliorating the cellular hyperglycemic state through reduction of glucose production. These observations allow us to conclude that the glucose level to which the hepatocytes are subjected affects how chromatin responds to HDACi and their action under high-glucose environment might not reflect on chromatin remodeling. J. Cell. Physiol. 231: 2257-2265, 2016. © 2016 Wiley Periodicals, Inc.

Toluidine blue (TB) staining either alone or in association with other methodologies has the potential to answer a variety of biological questions regarding the human, animal and plant tissues or cells. In this brief review, we not only report the primary use of TB to detect the anionic substrates and availability of their binding sites, but also unveil the resulting applications of TB staining in biological research. Among these applications, the uses of TB staining to identify the changes in chromatin DNA-protein complexes, nucleolus location, and extracellular matrix proteoglycan complexes associated with different physiological and pathological events are described. The usefulness of TB staining to monitor the effects elicited by environmental insults on chromatin and intercalation of drugs into the DNA is also included.

Atherothrombosis remains the leading cause of morbidity and mortality in patients diagnosed with diabetes mellitus, but the molecular mechanisms underpinning this remain unresolved. As the liver plays a major role in metabolic homeostasis and secretion of clotting factors and inflammatory innate immune proteins, there is an interest in understanding the mechanisms of hepatic cell activation under hyperglycemia and whether this can be attenuated pharmacologically. We have previously shown that hyperglycemia stimulates major changes in chromatin organisation and metabolism in hepatocytes, and that the histone deacetylase inhibitor valproic acid (VPA; IUPAC: 2-propylpentanoic acid) is able to reverse some of these metabolic changes. In this study, we used deep transcriptome sequencing to show that VPA attenuates hyperglycemia-induced activation of complement and coagulation cascade genes. These findings reveal a novel mechanism of VPA protection against hyperglycemia, which might improve the therapeutic approaches for diabetes.

Entheses are specialized biological structures that functionally anchor tendons to bones. The complexity, mechanical characteristics and properties of the entheses, particularly those related to exercise, mechanical load and pathologies, have been extensively analyzed; however, the macromolecular organization of the enthesis fibers, as assessed by polarization microscopy, has not yet been investigated. Morphological and optical anisotropy characteristics, such as birefringence, linear dichroism (LD) and differential interference contrast (DIC-PLM) properties, are thus analyzed in this study of a healthy adult mouse calcaneal tendon-bone enthesis. The molecular and supramolecular order of collagen and GAGs was determined for the collagen bundles of this enthesis. Based on a birefringence plot pattern as well as on metachromasy and linear dichroism after toluidine blue staining at pH 4.0, a similarity between the calcaneal tendon-bone enthesis and cartilage during ossification may be assumed. This similarity is assumed to favor the adequacy of this enthesis to support a compressive load. Considering that the collagen-proteoglycan complexes and the enthesis fibers themselves have a chiral nature, these structures could be acting via reciprocal signaling with the cellular environment of the enthesis.

Changes in polyploidization, chromatin supraorganization, and chromatin accessibility were investigated in hepatocytes collected from adult, nonobese diabetic (NOD) mice with increasing hyperglycemia and compared with adult normoglycemic controls and 56-week-old normoglycemic BALB/c mice. Our goal was to determine the changes in ploidy degrees and chromatin characteristics in mouse hepatocytes that are associated with insulin-dependent diabetes and to detect similarities in these aspects with those verified with aging, with greater accuracy than previous studies. Image analysis of Feulgen-stained nuclei revealed changes in ploidy degrees and chromatin supraorganization. Chromatin accessibility was assessed with micrococcal nuclease (MNase) digestion. Increased polyploidy was associated with increasing levels of glycemia, and this trend toward polyploidy was found even under normoglycemic conditions in NOD mice. Although high degrees of ploidy were also detected in aged BALB/c mice, the magnitude of polyploidy was not the same magnitude as that in the diabetic mice. While there was increased homogeneity of chromatin packaging with increasing polyploidy under conditions of severe hyperglycemia (and even under conditions of normoglycemia) in NOD mice, an inverse relationship was observed in aged BALB/c mice. Chromatin accessibility to MNase increased under severe hyperglycemia and advanced age, but it was much higher in the diabetic mice. In conclusion, although similarities in polyploidy were observed between the hepatocytes from increasingly hyperglycemic adult mice and those from normoglycemic aged mice, the relationship between chromatin remodeling and increases in ploidy degrees was not the same between the hepatocytes of these two groups. These findings demonstrate that strict similarities between diabetes and aging are not always true at the cellular level. This discordance is likely due to differences in the metabolic state of mouse hepatocytes during aging and diabetic conditions consequent to specificities in their gene regulatory programs.

Early pregnancy and multiparity are known to reduce the risk of women to develop breast cancer at menopause. Based on the knowledge that the differentiation of the breast induced by the hormones of pregnancy plays a major role in this protection, this work was performed with the purpose of identifying what differentiation-associated molecular changes persist in the breast until menopause. Core needle biopsies (CNB) obtained from the breast of 42 nulliparous (NP) and 71 parous (P) postmenopausal women were analyzed in morphology, immunocytochemistry and gene expression. Whereas in the NP breast, nuclei of epithelial cells were large and euchromatic, in the P breast they were small and hyperchromatic, showing strong methylation of histone 3 at lysine 9 and 27. Transcriptomic analysis performed using Affymetrix HG_U133 oligonucleotide arrays revealed that in CNB of the P breast, there were 267 upregulated probesets that comprised genes controlling chromatin organization, transcription regulation, splicing machinery, mRNA processing and noncoding elements including XIST. We concluded that the differentiation process induced by pregnancy is centered in chromatin remodeling and in the mRNA processing reactome, both of which emerge as important regulatory pathways. These are indicative of a safeguard step that maintains the fidelity of the transcription process, becoming the ultimate mechanism mediating the protection of the breast conferred by full-term pregnancy.

This study aimed to contribute to our knowledge of the parasite-vector interaction associated with Trypanosoma cruzi (Chagas, 1909) infection in Panstrongylus megistus (Burmeister, 1835), an important vector of Chagas' disease in Brazil. The prevalence and intensity of T. cruzi infection, the incidence of metacyclogenesis and the frequency of nuclear phenotypes in Malpighian tubules were investigated in nymphs of P. megistus, reared at 28 °C and subjected to heat shock (40 °C, 1 hour) two days after infection with T. cruzi II (Y strain). Following the 45-day post-infection period, the frequency of epimastigotes was much higher than that of trypomastigotes in both heat-shocked and non-shocked insects, and the prevalence of infection was not altered by heat shock. Fewer epimastigotes and trypomastigotes were found in the infected insects subjected to the heat shock, indicating that the multiplication and metacyclogenesis of the parasites were affected by the stress. In infected specimens heat shock promoted an increased frequency of cell nuclei with heterochromatin decondensation, a cell survival response to stress, and did not affect insect survival. The effects of infection and heat shock, especially on the multiplication and metacyclogenesis of T. cruzi, and the observed resistance to heat shock developed by P. megistus nymphs are suggestive that they should be considered when adequate conditions for rearing these infected insects in the laboratory are pursued.

Chromatin supraorganization and extensibility and nuclear glycoprotein content have been reported to change in hepatocytes from mice during development and aging, as well as under starvation and refeeding conditions. In non-obese diabetic (NOD) mice, the expression of insulin-dependent diabetes may be accompanied by metabolic changes in the liver. These changes are likely to be similar to those involved in the aging processes of non-diabetic animals. Therefore, we hypothesized that the chromatin organization, as well as the physical properties and compositions of hepatocyte nuclei would also be affected in NOD mice in the same way as those in aged non-diabetic mice. Nuclear image parameters were evaluated by image analysis of Feulgen-stained preparations. Chromatin extensibility in response to gravity was observed with polarized light after lysis and toluidine blue staining. The Con-A response of nuclear glycoproteins was evaluated with scanning microspectrophotometry. These characteristics were assessed using hepatocyte imprints from female NOD mice after a 28-day period of diabetes expression. Observations and measurements were made in comparison to healthy BALB/c mice. Total RNA amounts were determined for livers of NOD and BALB/c mice. Enhanced polyploidy levels, a decrease in chromatin higher-order packing states, an increased frequency of extended chromatin fiber formation, and deeper Con-A-responsive chromatin areas were observed in the hepatocytes of the NOD mice expressing insulin-dependent diabetes. Reduced amounts of total RNA were also found in the livers of these mice. Our findings for NOD mice expressing insulin-dependent diabetes are consistent with previously reported data for old-aged mice of the inbred strain A/Uni and may reflect changes in transcriptional activities associated with the stressful physiological demands on the liver during the expression of diabetes.

The immortalized human breast epithelial MCF-10F cell line, although estrogen receptor alpha negative, develops cell proliferating activities and invasiveness indicative of neoplastic transformation, after treatment with 17-beta-estradiol (E-2). These effects are similar to those produced by benzo[a]pyrene (BP). Since we have previously reported changes in the nuclear parameters accompanying BP-induced tumorigenesis in MCF-10F cells, we have examined whether similar alterations occur in E-2-treated cells. We therefore studied DNA amounts and other nuclear parameters in Feulgen-stained MCF-10F cells after treatment with various concentrations of E-2, BP, the estrogen antagonist ICI 182,780, and E-2 in the presence of ICI 182,780. E-2 caused a certain loss of DNA and changes in the nuclear size and chromatin supraorganization of MCF-10F cells. Many of these changes were similar to those produced by BP and were indicative of neoplastic transformation. More intense chromatin remodelling was seen with 70 nM E-2. Since these changes were not abrogated totally or partially by ICI 182,780, the neoplastic transformation of MCF-10F cells stimulated by E-2 involved a process that was independent of estrogen alpha-receptors. The changes produced by ICI 182,780 alone were attributed to effects other than its well-known anti-estrogenic activity.

Background: Chromatin supraorganization and extensibility, which lead to the formation of extended chromatin fibers (ECF), are affected by starvation and refeeding in adult mouse hepatocytes. It is expected that they could also change with mouse development and aging. Methods: Methods used involved topochemistry, image analysis, microspectrophotometry, gravity action, and polarization microscopy. Results: Increased nuclear areas and Feulgen-DNA amounts with advancing hepatocyte polyploidy were found with development and aging. A slightly less packed chromatin with more heterogeneously distributed condensation levels was detected in young and old mice. Con-A responsiveness was almost absent in young mice but very deep in aged mice. ECFs formed from nuclei of adult and aged mice but not from nuclei of young mice. The frequency of ECF formation with the long lysis protocol increased with aging. Conclusions: In young mice, a less packed chromatin state may be associated with more intense gene activity, thus increasing the DNA-nuclear matrix interactions, and inhibiting ECF formation. Reduced DNA-nuclear matrix interactions besides defects in heterochromatin formation may induce higher ECF formation and chromatin unpackaging in old mice. We suggest that differences in Con-A staining relate to different gene activity with advancing development and aging. © 2007 International Society for Analytical Cytology

Changes in DNA/chromatin structure, ploidy degrees and cell death possibly caused by oxidative stress during the insulin-dependent diabetes have been reported for different cell types. However, all these studies have been carried in streptozotocin-induced diabetic rats or mice and showed contradictory results. In this work, nuclear phenotypes and DNA fragmentation were investigated in fibroblasts from mice spontaneously developing insulin- dependent diabetes (NOD) and compared with healthy (BALB/C) mice. Geometric, densitometric and textural pa- rameters obtained for Feulgen-stained nuclei by image analysis were used to define nuclear phenotypes. Significant differences were observed for nuclear sizes and for densitometric and textural parameters of the tendon nuclei. Op- tical density, Feulgen-DNA values, transmittance variability per nucleus and nuclear entropy values were signifi- cantly higher in the NOD mice. The Feulgen-DNA amounts for the NOD and BALB/C mice were found to be dis- tributed into several doubling Feulgen-DNA classes. The frequency of nuclei with the smallest Feulgen-DNA amounts, which may represent DNA fragmentation and loss, was lower in fibroblasts of the NOD mice (2.3%) in comparison to the BALB/C mice (38%). In contrast, the frequency of polyploid nuclei in NOD mice was higher (24.5%) than that in BALB/C mice (1.9%). Based on optical density, transmittance variability per nucleus, and nu- clear entropy data, a larger contrast between highly and less densely packed states, was demonstrated for the fibrob- lasts of the NOD mice. Maybe the deeper condensation of the highly packed chromatin evident in NOD fibroblasts is related to silencing of some genes involved with changes in tendon supraorganization with the diabetes.

The effect of fasting and of fasting followed by refeeding and heat shock was studied in Malpighian tu- bules of fourth instar nymphs of the blood-sucking hemipteran, Panstrongylus megistus (Burmeister). The aim was to detect different frequencies of nuclear changes (apoptosis, necrosis, heterochromatin decondensation) under conditions assumed to be stressful in blood-sucking hemipterans. The insects were fasted for up to 90 days at 28 o C and their survival was followed daily. Groups of nymphs were separated each month, with part of the group being refed and the other part kept fasting. Insects in each of these subgroups received either a heat shock at 40 o C for 1 h or were maintained at 28 o C (control for heat shock). The Malpighian tubules were removed one and seven days af- ter each assay and subjected to the Feulgen reaction for identification and counting of the various nuclear pheno- types. Insect survival was high (90%) even after 40 days of starvation but decreased thereafter. Necrosis rather than apoptosis, increased with fasting. Feeding after fasting increased the frequency of apoptosis but not of necrosis. The short heat shock as used here did not additionally affect the responses induced by fasting and refeeding. P. megistus nymphs could withstand relatively long periods of fasting although individual variation in the mean length of cell survival had been found especially after a three-month fasting. The results related to cell necrosis suggest that part of the Malpighian tubule cells may not have developed highly efficient mechanisms for dealing with fasting. For those cells resistant to fasting, feeding subsequent to fasting acted only as a mild stressing agent and heat shock was well tolerated. The ability of P. megistus nymphs to withstand and recover from periods of inadequate or poor nu- trition inclusive in association to a short heat shock as demonstrated here is certainly an important adaptation for the survival of the species.

Background: How much DNA remains in mouse hepatocyte nuclei after extended chromatin fiber (ECF) formation or whether this content varies within the nuclear population is not known. This information could be relevant to understanding chromatin extensibility as related to chromatin organization, possibly associated with variable nuclear activities in hepatocytes. Methods: A protocol for ECF formation under the gravity action, image analysis of Feulgen-stained unfixed mouse hepatocyte remnants, and DAPI fluorescence were used. Results: Areas, shape, Feulgen-DNA amounts, and chromatin texture were affected in unfixed, lysed nuclei. The Feulgen-DNA values in nuclear remnants represented ∼37% of the content in fixed, nonlysed nuclei in terms of median values; the coefficient of variation of Feulgen-DNA values in the nuclear remnants was much higher than those in controls. Enhancement in DAPI fluorescence was evident in chromocenters of the fixed nuclei and in remnants and some ECF granules of the unfixed, lysed nuclei. Conclusions: The DNA content of the nuclear remnants was much more variable than that assumed from known variability in hepatocyte ploidy degrees. The variable constraint to chromatin extrusion from hepatocyte nuclei is hypothesized to depend on variable chromatin organization with possible involvement of nuclear matrix association, transcriptional activities, and AT-rich DNA-containing heterochromatin. © 2006 International Society for Analytical Cytology

Fluoro-Jade (FJ) and Fluoro-Jade B (FJB) are fluorescein derivatives currently used to stain brain cells under degeneration. In this study, we investigated the FJ staining of nondegenerating cells in embryonic and neonatal rat brain and retina. In embryonic rat brain (embryonic day 15; E15), very intense staining of cells was observed. The number of FJ-stained cells and the intensity of staining decreased with increasing in animal age, being almost absent by postnatal day 16 (P16). Only a few cells in neonatal rat brain were in the process of cell death, as verified by the TUNEL technique. The FJ-stained cells in neonatal brain were positive for the neuronal marker neuronal nuclei antigen (NeuN). In retina, FJ stained mainly cells from the ganglion cell layer at P2 and the neuroblastic layer at P2 and P6. In contrast to FJ, FJB did not stain nondegenerating cells in embryonic and neonatal rats. These results show that in addition to staining degenerating brain cells, FJ also stains nondegenerating central nervous system cells in embryonic and neonatal stages.

Background A relation between the changes in DNA content and chromatin supra-organization and the expression of gradual steps of tumorigenesis has been assessed by image analysis in human breast epithelial cells (MCF-10F) treated with benzo[a]pyrene (BP) (cell lines BP1, BP1-E, BP1-Tras, and others). Methods Because abnormal chromosomes 11 and 17 have been associated with neoplastic progression in BP-transformed MCF-10F cells, image analysis of Feulgen-stained tumorigenic BP1-E cells with the microcell-mediated chromosome transfer of normal chromosomes 11 and 17 was carried out. Results A tendency of DNA amount distribution and nuclear size restoration to values typical of non-transformed MCF-10F cells was demonstrated, especially after the transfer of chromosome 17. No reversion in chromatin texture was found after the transfer of chromosome 11 or 17. Conclusions Although the presence of a normal chromosome 17 should be considered among the necessary steps for tumorigenic human breast epithelial cells to recover their normality, a more complex genome balance is required for the entire nuclear chromatin of these cells to recover its totally normal supra-organization and expression. Cytometry Part A 52A:70–76, 2003. © 2003 Wiley-Liss, Inc.

Prostatic lesions in Brazilian patients with benign prostatic hyperplasia (BPH, 26 cases) or adenocarcinoma (AC, 25 cases) were compared by qualitative microscopy and morphometric analysis. In 12 cases of BPH, prostate regions with no histological alterations were considered as controls (Ct). Archival material consisted of formalin-fixed, paraffin-embedded specimens obtained from prostatic transurethral resection and radical prostatectomy. Haematoxylin/eosin (HE)- stained sections were used to estimate the nuclear areas, perimeters and form factor values. HE-stained sections from AC specimens were also used for Gleason grading. BPH, AC and Ct could be discriminated by their nuclear areas and nuclear perimeters, but not by the nuclear form factor parameter. No significant differences were found when the AC data were compared using the combined version or the predominant grade version of the Gleason score (p = 0.8380 for nuclear area; p = 0.6076 for nuclear perimeter; p = 0.9202 for nuclear form fa...

The nuclear phenotypes of Malpighian tubule cells in fifth instar nymphs of Triatoma infestans, one of the most important vectors of Chagas disease, were studied following sequential shocks at 0 degrees C, separated by intervals of 8 h and 24 h at 30 degrees C, under conditions of moderate fasting and full nourishment. The insects pertained to colonies reared in the laboratory and originated from domestic specimens collected in the Brazilian states of São Paulo (north) and Minas Gerais (south). Since nuclear phenotypes in this species are affected by single cold shocks, it was expected that these phenotypes could also be changed by sequential shocks. Nuclear phenotypes indicative of mechanisms of cell survival (nuclear fusion and heterochromatin decondensation) and cell death (apoptosis and necrosis) were observed concomitantly in all the conditions tested. Nuclear fusion and heterochromatin decondensation were not found relevant for the presumed acquisition of the cold-hardening response in T. infestans. The decreased frequency of apoptosis and necrosis following sequential cold shocks including under fasting conditions, indicated that tolerance to sequential cold shocks occurred in T. infestans of the mentioned origin.

Thermal shocks induce changes in the nuclear phenotypes that correspond to survival (heterochromatin decondensation, nuclear fusion) or death (apoptosis, necrosis) responses in the Malpighian tubules of Panstrongylus megistus. Since thermal tolerance increased survival and molting rate in this species following sequential shocks, we investigated whether changes in nuclear phenotypes accompanied the insect survival response to sequential thermal shocks. Fifth instar nymphs were subjected to a single heat (35 or 40 degrees C, 1 h) or cold (5 or 0 degrees C, 1 h) shock and then subjected to a second shock for 12 h at 40 or 0 degrees C, respectively, after 8, 18, 24 and 72 h at 28 degrees C (control temperature). As with specimen survival, sequential heat and cold shocks induced changes in frequency of the mentioned nuclear phenotypes although their patterns differed. The heat shock tolerance involved decrease in apoptosis simultaneous to increase in cell survival responses. Sequential cold shocks did not involve cell/nuclear fusion and even elicited increase in necrosis with advancing time after shocks. The temperatures of 40 and 0 degrees C were more effective than the temperatures of 35 and 5 degrees C in eliciting the heat and cold shock tolerances, respectively, as shown by cytological analysis of the nuclear phenotypes. It is concluded that different sequential thermal shocks can trigger different mechanisms of cellular protection against stress in P. megistus, favoring the insect to adapt to various ecotopes.

Valproic acid (VPA), a well-known histone deacetylase inhibitor, has been reported to affect the DNA methylation status in addition to inducing histone hyperacetylation in several cell types. In HeLa cells, VPA promotes histone acetylation and chromatin remodeling. However, DNA demethylation was not checked in this cell model for standing effects longer than those provided by histone acetylation, which is a rapid and transient phenomenon. Demonstration of VPA-induced DNA demethylation in HeLa cells would contribute to understanding the effect of VPA on an aggressive tumor cell line. In the present work, DNA demethylation in VPA-treated HeLa cells was assessed by image analysis of chromatin texture, the abundance of 5-methylcytosine (5mC) immunofluorescence signals and Fourier transform-infrared (FT-IR) microspectroscopy centered on spectral regions related to the vibration of–CH3 groups. Image analysis indicated that increased chromatin unpacking promoted by a 4-h-treatment with 1.0 mM VPA persisted for 24 h in the absence of the drug, suggesting the occurrence of DNA demethylation that was confirmed by decreased 5mC immunofluorescence signals. FT-IR spectra of DNA samples from 1 mM or 20 mM VPA-treated cells subjected to a peak fitting analysis of the spectral window for–CH3 stretching vibrations showed decreased vibrations and energy of these groups as a function of the decreased abundance of 5mC induced by increased VPA concentrations. Only the 20 mM-VPA treatment caused an increase in the ratio of -CH3 bending vibrations evaluated at 1375 cm-1 in relation to in-plane vibrations of overall cytosines evaluated at 1492 cm-1. CH3 stretching vibrations showed to be more sensitive than–CH3 bending vibrations, as detected with FT-IR microspectroscopy, for studies aiming to associate vibrational spectroscopy and changes in DNA 5mC abundance.

Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase inhibitors (HDACIs) with epigenetic activity that affect chromatin supra-organization, nuclear architecture, and cellular proliferation, particularly in tumor cells. In this study, chromatin remodeling with effects extending to heterochromatic areas was investigated by image analysis in non-transformed NIH 3T3 cells treated for different periods with different doses of VPA and TSA under conditions that indicated no loss of cell viability. Image analysis revealed chromatin decondensation that affected not only euchromatin but also heterochromatin, concomitant with a decreased activity of histone deacetylases and a general increase in histone H3 acetylation. Heterochromatin protein 1-α (HP1-α), identified immunocytochemically, was depleted from the pericentromeric heterochromatin following exposure to both HDACIs. Drastic changes affecting cell proliferation and micronucleation but not alteration in CCND2 expression and in ratios of Bcl-2/Bax expression and cell death occurred following a 48-h exposure of the NIH 3T3 cells particularly in response to higher doses of VPA. Our results demonstrated that even low doses of VPA (0.05 mM) and TSA (10 ng/ml) treatments for 1 h can affect chromatin structure, including that of the heterochromatin areas, in non-transformed cells. HP1-α depletion, probably related to histone demethylation at H3K9me3, in addition to the effect of VPA and TSA on histone H3 acetylation, is induced on NIH 3T3 cells. Despite these facts, alterations in cell proliferation and micronucleation, possibly depending on mitotic spindle defects, require a longer exposure to higher doses of VPA and TSA. J. Cell. Biochem. 115: 1937–1947, 2014. © 2014 Wiley Periodicals, Inc.

Background Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. Methodology/Principal Findings The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. Conclusions/Significance Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas –CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of –CH3 groups. Other spectral differences were found at 1700–1500 cm−1 and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.

In this study, we examined cell survival and cell death in response to heat shock in an insect organ composed of highly polyploid cells no longer capable of cell division. For this, the frequency of nuclear phenotypes in Feulgen-stained Malpighian tubules of the blood-sucking insect, Triatoma infestans, was analyzed at various times after a short heat shock with or without subsequent moderate fasting. Cell death DNA fragmentation was studied immunocytochemically. Normal phenotypes and phenotypes indicative of cell survival (heterochromatin decondensation, nuclear fusion) and death (apoptosis, necrosis) were observed, especially in heat-shocked specimens. While the number of total and normal nuclei decreased following heat shock, the frequency of apoptosis increased during a short period (7 days) after heat shock. During a 30-day period following heat shock, the frequency of necrosis in fasted but not in fully nourished nymphs increased simultaneously with a decrease in the frequency of apoptosis. This finding suggests that the stress promoted by heat shock, but not that associated with heat shock plus fasting, can be dealt with by the apoptosis program. When considering the forms of cell survival, heterochromatin decondensation was more relevant in fully nourished nymphs, whereas nuclear and cell fusions were more important in fasted specimens. The forms of cell survival and cell death reported here may have protected the organ from damage by the stressing agents. In cells with no induction or accumulation of heat-shock proteins, cell death and the forms of cell survival observed here were the probable consequence.Key words: heat shock, fasting, apoptosis, necrosis, cell survival, Triatoma infestans.

The nuclear phenotypes of Malpighian tubule epithelial cells of male nymphs of the blood-sucking insect, Panstrongylus megistus, subjected to short- and long-duration heat shocks at 40ºC were analyzed immediately after the shock and 10 and 30 days later. Normal nuclei with a usual heterochromatic body as well as phenotypes indicative of survival (unravelled heterochromatin, giants) and death (apoptosis, necrosis) responses were observed in control and treated specimens. However, all nuclear phenotypes, except the normal ones, were more frequent in shocked specimens. Similarly altered phenotypes have also been reported in Triatoma infestans following heat shock, although at different frequencies. The frequency of the various nuclear phenotypes observed in this study suggests that the forms of cell survival observed were not sufficient or efficient enough to protect all of the Malpighian tubule cells from the deleterious effects of stress. In agreement with studies on P. megistus survival following heat shock, only long-duration shock produced strongly deleterious effects.

The nuclear phenotypes of Malpighian tubule epithelial cells of 5th instar male nymphs of the blood-sucking insect Panstrongylus megistus were studied immediately after a short (1 h) cold shock at 0 degrees C, and 10 and 30 days later. The objective was to compare the responses to a cold shock with those known to occur after hyperthermia in order to provide insight into the cellular effect of cold in this species. Nuclei which usually exhibited a conspicuous Y chromosome chromocenter were the most frequent phenotype in control and treated specimens. Phenotypes in which the heterochromatin was unravelled, or in which there was nuclear fusion or cell death were more abundant in the shocked specimens. Most of the changes detected have also been found in heat-shocked nymphs, except for nuclear fusion which generates giant nuclei and which appeared to be less effective or necessary than that elicited after heat shock. Since other studies showed that a short cold shock does not affect the survival of more than 14% of 5th instar nymphs of P. megistus with domestic habit and can induce tolerance to a prolonged cold shock, heat shock proteins proteins are probably the best candidates for effective protection of the cells and the insects from drastic damage caused by low temperature shocks.

DNA content and chromatin supraorganization defined in terms of patterns of chromatin texture were studied by image analysis in Feulgen-stained human breast MCF-10F epithelial cells expressing different stages of tumorigenic progression after treatment with benzo[a]pyrene (BP). Nontransformed MCF-10F, nontumorigenic transformed BP1, and tumorigenic BP1-E1 cell lines were analyzed. A relationship between changes in DNA content and chromatin texture and the expression of different stages of tumorigenesis were researched in interphase nuclei. A significant decrease in Feulgen–DNA amounts and absorbing areas suggesting aneuploidy, and first detected in nontumorigenic transformed BP1 cells, became more relevant in the tumorigenic cell line BP1-E1. Simultaneously, an increase in optical densities and a change in related parameters, demonstrating increased chromatin higher order packing states, occurred in transformed BP1 cells and extended over larger chromatin areas in tumorigenic BP1-E1 cells. The results indicate loss of DNA and a change in chromatin higher order packing states accompanying the expression of different stages of the in vitro tumorigenesis process in BP-transformed human breast epithelial cells. It is suspected that the gradually acquired chromatin “condensation” supraorganization pattern is associated with activated transcription activities previously suggested for these cells.

In normal aging, nuclear sirtuin levels decline and specific histone acetylation increases. In diabetic mice, Sirt1 and Sirt6 levels increase, which may induce gluconeogenesis through the PGC-1α pathway and DNA repair of the oxidative damage caused by hyperglycemia., Hyperglycemia, like aging, induces chromatin remodeling in mouse hepatocytes in comparison to normoglycemia and younger age, respectively. Changes in glucose metabolism also affect the action and expression of sirtuins, promoting changes in chromatin conformation and dynamics. Here we investigate the abundance and activity of the nuclear sirtuins Sirt1, Sirt6, and Sirt7 in mouse hepatocytes in association with specific histone acetylation, DNA damage, and the activation of nucleolar organizing regions (NORs) in hyperglycemic nonobese diabetic (NOD) and old normoglycemic BALB/c mouse strains. Higher levels of Sirt1 and PGC-1α and increased expression of gluconeogenesis pathway genes are found in the hyperglycemic NOD mice. Increased Sirt6 abundance is found in the hyperglycemic NOD mice, which might increase DNA damage repair. With aging, lower Sirt1 abundance and activity, increased acetylated histone modifications and Sirt7 levels, and NOR methylation are found. Thus, whereas in normal aging cell metabolism is reduced, in the diabetic mice a compensatory mechanism may elevate Sirt1 and Sirt6 levels, increasing gluconeogenesis and DNA repair from the oxidative damage caused by hyperglycemia. Therefore understanding the regulation of epigenetic factors in diabetes and aging is crucial for the development of new therapeutic approaches that could prevent diseases and improve quality of life.

Background Birefringence can reveal much of the morphology, molecular order, heterogeneity of fiber orientation, and nonlinear optical properties of biopolymers such as collagen. However, the detailed characterization of skin collagen fibers using optical anisotropy methods remains elusive. A clear understanding of collagen fiber organization in skin tissues may be important in the interpretation of their structural-functional relationships under normal and pathological conditions. In this study, fiber orientation in collagen bundles (CBs) and their supramolecular organization were examined in rat skin using polarization microscopy and image analysis. Methodology/Principal Findings Image variations with rotation of the microscope stage and selection of the in-depth focus plane were investigated in unstained sections of varying thicknesses from rat skin fragments. Total birefringence (image analysis) and form and intrinsic birefringence (Senarmont’s method) were estimated. Based on the birefringent images, CBs were found to contain intercrossing points with a twisted helical distribution of collagen fibers (chiral elements) and frequently presented circular structures. Collagen fibers were observed to extend from the surface level to deeper planes, creating a 3D-network of oriented intertwined CBs. At least three levels of birefringent brilliance intensity were revealed by image analysis, indicating a heterogeneous spatial organization of the CBs. Slight differences in optical retardations were found for CBs immersed in some of the fluids used in a comparison of 170- and 240-day old rats. Conclusion/Significance Polarization microscopy studies provide detailed high-quality structural information on rat skin CBs. A 3D-network structure based on image analysis and birefringence compensation for collagen fibers is suggested for CBs. Form and intrinsic birefringence evaluation can reveal differences in the rat skin associated with age at the levels of collagen fiber crystallinity and macromolecular organization. These findings may inspire future studies of the feedback mechanisms by which spatial, bioelectrical and biomechanical information is transmitted from CBs to skin cells.

Summary Changes in chromatin order with cell transformation were studied in terms of optical anisotropy (birefringence) in toluidine blue-stained NIH 3T3 and Balb/3T3 cells. The transformed NIH 3T3 cell lines used were obtained by transfection with the T24 cell H- ras oncogene and the whole genomic DNA of MCF-7 human breast carcinoma cells. C2PO and PAP2 cell lines were used as representatives of Ha- ras -transformed NIH 3T3 cells with poor and high metastatic ability, respectively. The chromatin of all cells examined exhibited metachromasy and a birefringence of greenish-yellow interference color. The highest birefringence intensity was found in the chromatin granules of the most frequent nuclear phenotype of ras - and MCF-7 DNA-transformed cells, with the exception of the cells with high metastatic ability, in which a faint birefringence was observed. The differences in chromatin birefringence intensity are assumed to indicate differences in chromatin stereoarrangement with cell transformation. In the case of the highly tumorigenic and/or highly metastatic transformants the faint birefringence is assumed to be associated with the heterogeneous and complex physiological processes that may require a relatively less ordered arrangement of the chromatin.

Rat ear cartilage was studied using Fourier transform-infrared (FT-IR) microspectroscopy to expand the current knowledge which has been established for relatively more complex cartilage types. Comparison of the FT-IR spectra of the ear cartilage extracellular matrix (ECM) with published data on articular cartilage, collagen II and 4-chondroitin-sulfate standards, as well as of collagen type I-containing dermal collagen bundles (CBs) with collagen type II, was performed. Ear cartilage ECM glycosaminoglycans (GAGs) were revealed histochemically and as a reduction in ECM FT-IR spectral band heights (1140-820 cm-1) after testicular hyaluronidase digestion. Although ear cartilage is less complex than articular cartilage, it contains ECM components with a macromolecular orientation as revealed using polarization microscopy. Collagen type II and GAGs, which play a structural role in the stereo-arrangement of the ear cartilage, contribute to its FT-IR spectrum. Similar to articular cartilage, ear cartilage showed that proteoglycans add a contribution to the collagen amide I spectral region, a finding that does not recommend this region for collagen type II quantification purposes. In contrast to articular cartilage, the symmetric stretching vibration of -SO3- groups at 1064 cm-1 appeared under-represented in the FT-IR spectral profile of ear cartilage. Because the band corresponding to the asymmetric stretching vibration of -SO3- groups (1236-1225 cm-1) overlapped with that of amide III bands, it is not recommended for evaluation of the -SO3- contribution to the FT-IR spectrum of the ear cartilage ECM. Instead, a peak (or shoulder) at 1027-1016 cm-1 could be better considered for this intent. Amide I/amide II ratios as calculated here and data from the literature suggest that protein complexes of the ear cartilage ECM are arranged with a lower helical conformation compared to pure collagen II. The present results could motivate further studies on this tissue under pathological or experimental states involving ear cartilage.

Mg(2+)-dependent ATPases were investigated in Malpighian tubules of the blood-sucking insect, Triatoma infestans, with cytochemical procedures for light and electron microscopy. The aim was to establish patterns of enzyme occurrence in the blood-sucking insect under control rearing conditions for further comparisons with animals subjected to the action of stress factors. Enzyme activity was found in laminated "concretions" present in distal cells, in edges of urate crystals at the lumen of the proximal region of tubules, in the basement membrane of proximal cells, and variously distributed in plasmalemma invaginations of both distal and proximal cells. Presence of ATPases in the "concretions" and urate crystals is presumed to be due to engulfment of other ATPase-containing components during formation of these structures. Cytochemical reactivity in the basement membrane and plasmalemma invaginations is assumed to be involved with active transport of waste molecules from and to hemolymph and differs as a function of the Malpighian tubule region. This paper provides a basic understanding of the enzyme occurrence in the blood sucking insects, and can be used as a pattern for comparative means of the staining patterns among Triatominae species.

Ethosuximide is a T-type Ca2+ channel blocker that has been used as an anticonvulsant to treat absence seizures. Because no data have yet been reported on the cytotoxicity, genotoxicity and cell death effects of this drug, we have investigated ethosuximide-treated human lymphocytes with Fenech's CBMN (cytochalasin-blocked micronucleus cytome) assay and single-cell gel electrophoresis (comet assay). The tests allowed us to examine micronucleus formation, the proliferative status of the viable cells, nuclear blebbing, nucleoplasmic bridge formation, cell death (CBMN assay) and DNA damage (comet assay). The lymphocytes were treated for 24 h with 25, 50 or 100 μg/ml ethosuximide. The cells used for the CBMN assay were examined either immediately or 24 h after the cytochalasin blockade. For the comet assay, cells were examined immediately or 22 h after 2 h ethosuximide treatment. The results indicate that 25 and 50 μg/ml ethosuximide did not induce micronucleus formation, nuclear abnormalities, cell death or DNA damage, nor did they affect cell proliferation, suggesting no cytotoxic or genotoxic effects under such experimental conditions. However, under treatment with 100 μg/ml ethosuximide, an increase in micronucleus formation and nuclear abnormalities, but a decrease in cell proliferation and in DNA damage, and no change in the cell death ratio, were detected. Although apparently contradictory, the data obtained with the 100 μg/ml concentration may indicate that induction of cytotoxicity and genotoxicity are not to be disregarded when considering this drug concentration. The mechanisms underlying the cellular response to ethosuximide remain to be explored.

Collagen fiber structure and organization have been found to vary in different tendon types. Differences have been reported in the FT-IR spectra of the amide I band of collagen-containing structures. In the present study, the FT-IR spectral characteristics of the amide I band of the bovine flexor tendon and the extended rat tail tendon were compared by using the diamond attenuated total reflectance technique. The objective was to associate FT-IR spectral characteristics in tendons with their different collagen fiber supraorganization and biomechanical properties. Nylon 6 and poly-L-lysine were used as polyamide models. Each of these materials was found to exhibit molecular order and crystallinity, as revealed by their birefringence. The following FT-IR parameters were evaluated: amide I band profile, absorption peaks and areas, and the 1655 cm⁻¹/1690 cm⁻¹ absorbance ratio. The amide I area and the 1655 cm⁻¹/1690 cm⁻¹ absorbance ratio were significantly higher for the bovine flexor tendon, indicating that its collagen fibers are richer in pyridinoline-type cross-linking, proline and/or hydroxyproline and H-bonding, and that these fibers are more packed and supraorganizationally ordered than those in the rat tail tendon. This conclusion is additionally supported by differences in collagen solubility and biochemical/biomechanical properties of the tendons.

Transcribed sequences have been suggested to be associated with the nuclear matrix, differing from non-transcribing sequences, which have been reported to be contained in DNA loops. However, although a dozen of genes have their expression level affected by aging, data on chromatin-nuclear matrix interactions under this physiological condition are still scarce. In the present study, liver imprints from young, adult and old mice were subjected to FISH (fluorescence in situ hybridization) for 45S rDNA and telomeric sequences, with or without a lysis treatment to produce extended chromatin fibres. There was an increased amount of 45S rDNA sequences located in DNA loops as the animals grow older, while telomeric sequences were always observed in DNA loops irrespective of the animal age. We assume that active rRNA genes associate with the nuclear matrix, while DNA loops contain silent sequences. Transcription of each 45S rDNA repeat unit is suggested to be dependent on its interaction with the nuclear matrix.

Studies of chromatin extensibility have revealed the flow of chromatin and DNA from cell nuclei and chromosomes in response to gravity or mechanical stretch following lysis by hypertonic saline and detergent solutions. Since this phenomenon was first reported, the technical methods by which extended chromatin fibers (ECFs) may be analyzed have been improved. These methods include topochemical assays, fluorescence in situ hybridization, immunofluorescence, electron microscopy, and polarization microscopy. Chromatin and DNA "halos" also have been studied in materials subjected to lysis, especially in a horizontal position or after cytocentrifugation. The analysis of ECF formation is useful not only as a tool for detecting the positioning of certain DNA signals on chromatin filaments, but also for describing diverse DNA-protein associations that may be related to varying transcriptional activities and chromatin supraorganization. A brief review of the methods and applications of ECF formation is presented here. We focus on light microscopy studies of ECF formation in mouse hepatocytes under different chromatin supraorganization and physiological conditions and in sperm cells with different DNA-protein complexes.

The nuclear phenotypes of Feulgen-stained NIH/3T3 cells transformed with 4-nitroquinoline 1-oxide (4NQO) treated, human breast epithelial cell (HBEC) DNA were studied by scanning microspectrophotometry and image analysis and compared with data obtained for nontransformed cells and for NIH/3T3 cells under ras oncogene transfecting situations. The Feulgen–DNA content of the individual nuclei (NQ1, NQ2, and NQ3 phenotypes) of the transformed cells was found not to be deeply affected, although presence of chromatin structures resembling double minutes could be verified in part of the metaphases of the transformed cells. On the other hand, the chromatin supraorganization of these cells showed some changes involving increased (NQ2, NQ3) or decreased (NQ1) levels of condensation. The changes in chromatin packing states, however, were of small magnitude compared with those reported for NIH/3T3 cells transfected with a c-H-ras oncogene or an N-ras-containing MCF-7 cell DNA. It was assumed that the transformation of the NIH/3T3 cells is not always necessarily accompanied by high levels of chromatin condensation. The transformation of the NIH/3T3 cells induced by the 4NQO-treated HBEC DNA and particularly the changes in chromatin condensation in these transformed cells could not be attributed merely to a ras activation elicited by the carcinogen. It is suggested that a more complex transforming mechanism is involved, probably owing to the fact that a whole genomic DNA of the 4NQO-treated HBEC has been used for transfection.Key words: cell transformation, 4-nitroquinoline 1-oxide, NIH/3T3 cell assay, human breast epithelial cells, image analysis.

Made available in DSpace on 2019-09-12T16:25:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2009 Este trabalho é parte de uma investigação sobre o potencial citotóxico e genotóxico da água do Rio Paraíba do Sul (Brasil) utilizando raízes de Allium cepa. Foi analisado um parâmetro anátomo-morfológico (crescimento das raízes), bem como o índice mitótico e a frequência de micronúcleos. Aleatoriamente oito bulbos foram submetidos aos tratamentos de 24 a 120 horas com água do Rio proveniente dos pontos de coleta das cidades de Tremembé e Aparecida nos anos de 2005 e 2006. O comprimento das raízes de cada bulbo foi acompanhado diariamente ao longo do experimento. O índice mitótico (IM) e a frequência de micronúcleos (MN) foram determinados pela análise de 2.000 células por raiz, sendo utilizadas 3-5 raízes de outros bulbos (7-10). Somente as raízes tratadas com amostras de água coletadas em 2005 na cidade de Tremembé, apresentaram decréscimo no comprimento das raízes quando comparadas com o controle. Entretanto, foi observada redução do IM nas raízes tratadas com água de ambos os pontos de coleta no mesmo ano. Considerando os dados de crescimento de raiz e especialmente IM, um potencial citotóxico é sugerido para a água do Rio Paraíba do Sul em Tremembé e Aparecida, no ano de 2005. Por outro lado, para este mesmo ano, a frequência de micronúcleos não foi alterada; assim, a genotoxicidade não foi assumida para a água do Rio nos pontos mencionados. This work investigated the cytotoxic and genotoxic potential of water from the River Paraíba do Sul (Brazil) using Allium cepa roots. An anatomo-morphological parameter (root length), mitotic indices, and frequency of micronuclei were analysed. Eight bulbs were chosen at random for treatment for 24 to 120 hours with the River water collected in the years of 2005 and 2006 from sites in the cities of Tremembé and Aparecida (São Paulo state, Brazil). Daily measurements of the length of the roots grown from each bulb were carried out throughout the experiment. Mitotic index (MI) and frequency of micronuclei (MN) were determined for 2000 cells per root, using 3-5 root tips from other bulbs (7-10). Only in the roots treated with samples of the River water collected in 2005 in Tremembé city was there a decrease in the root length growth compared to the respective control. However, a reduction in MI values was verified for both sites analysed for that year. Considering the data involving root length growth and especially MI values, a cytotoxic potential is suggested for the water of the River Paraíba do Sul at Tremembé and Aparecida, in the year of 2005. On the other hand, since in this year the MN frequency was not affected with the river water treatments, genotoxicity is not assumed for the river water sampled at the aforementioned places. [Barbério, A.; Voltolini, JC.] Universidade de Taubaté, Brazil Barbério, A.; Barros, L.; Mello, MLS.] Universidade Estadual de Campinas, Brazil

The structural and supraorganizational arrangement of the chordae tendineae components is very important for a better understanding of their morphophysiological relationships. This study aims to evaluate the degree of statistical variability of the distribution and orientation of collagen fibers and their undulations (crimps), in porcine chordae tendineae. Polarization microscopy, in association with image analysis, was used for the analysis of birefringent images and detection of surface plots, Fast Fourier transforms, and form birefringence curve profiles. A marked variability in the collagen fiber's twisted and intertwined orientation was found not only along the long axis of the chordae tendineae but also in their 3-D structure, including that of the crimp structures. Crimp was demonstrated to not represent a homogeneous distribution of bands; the best methods to quantify its variability were the Fast Fourier transform and the line profile extended along the long axis of the chordae tendineae. Collagen fiber rings, considered to possibly protecting the integrity of the chordae tendineae, were observed to wrap around them. A statistically helical structure is assumed for the supraorganization of the collagen fibers in the chordae tendineae, which is suggested here to be a chiral body.

Optical anisotropic characteristics investigated by polarization microscopy have been valuable for the study of the oriented organization of collagen fibers in tendons. However, topographic differences in supramolecular organization of collagen fibers along extensive areas in tendons have not yet been described. Here, the statistical variability of the oriented organization of collagen fibers along extensive areas (10(5)-10(6)microm(2)) of 7-microm thick unstained sections of rat calcaneal tendons were studied by assessing their birefringence with polarization microscopy and image analysis, and the periodic frequency distribution in their birefringent images with the fast Fourier transform (FFT). Various levels of birefringence intensity were determined principally by image analysis procedures, and periodicity in the birefringent images was revealed by FFT line profile and spectrum image patterns. Present results support the idea of a helical distribution for collagen bundles along the tendon long axis, and of a statistical architecture for the rat calcaneal tendons in terms of variability in the oriented distribution of their collagen fibers.

Feulgen–DNA content, nuclear phenotypes, and levels of chromatin condensation were evaluated by image analysis in NIH/3T3 cells transformed with the c-H-ras oncogene of T24 cells. Three nuclear phenotypes, differing from those of untransformed control cells and defined in terms of patterns of chromatin condensation, were demonstrated microspectrophotometrically for the tumor cells. Polyploidy could only be observed in nuclei with extensive and deeply stained areas covered with condensed chromatin, i.e., only in a small fraction of the tumor cell nuclear population. The increased chromatin condensation that appeared with cell transformation affected the euchromatin zones. The image analysis provided data that, compared with those obtained in other situations involving cell transformation, could be relevant to the understanding of changes in chromatin supraorganization related to tumorigenesis and to tumor cell diagnosis.Key words: cell transformation, c-H-ras oncogene, NIH/3T3 cells, image analysis, Feulgen staining.

Treatment of the human breast epithelial cells MCF-10F with 17-beta-estradiol (E2) induces transformation and tumorigenesis. E2-transformed MCF-10F cells are known to exhibit progressive loss of ductulogenesis, and invasive and tumorigenic phenotypes. Although DNA amounts and chromatin supraorganization change in E2-transformed MCF cells, no comparative study has yet been undertaken in the resulting cells selected for aggressive invasiveness (C5) and tumor generation in a heterologous host. The aim of this study was thus to determine whether changes in Feulgen-DNA content and chromatin supraorganization are involved during E2-induced transformation and tumorigenesis of the MCF-10F cells. Image analysis was performed for nontransformed and E2-transformed MCF cells, highly invasive cells (C5), and for cell lines (C5-A6-T6 and C5-A8-T8) derived from tumors generated by injection of C5 cells in SCID mice. A decrease in Feulgen-DNA amounts and nuclear sizes induced by E2 treatment was accented with selection of the highly invasive tumorigenesis potential. However, in the tumor-derived cells a high variability in cellular phenotypes resulted inclusively in near-polyploidy. Significant changes in textural parameters, including nuclear entropy, indicated chromatin structural remodeling with advancing tumorigenesis. An increased variability in the degree of chromatin packing states in the E2-transformed MCF cells is followed by reduction in chromatin condensation and in contrast between condensed and noncondensed chromatin in the highly invasive C5 cells and tumor-derived cell lines. Studies on epigenetic mechanisms involving DNA methylation and/or the histone code would contribute to a better interpretation of the chromatin supraorganization changes reported herein.

Background The effect of 48 h of starvation and of 48 h of refeeding subsequent to starvation on chromatin supraorganization and extensibility was studied in hepatocytes from adult mice. Methods Methods used involved topochemical assays, image analysis, gravity action, and polarization microscopy. Results Starvation increased the chromatin packing states, especially in areas of noncondensed chromatin, and induced drastic decreases in concanavalin A reactivity due to nuclear matrix glycoproteins and the frequency of nuclei with chromatin extensibility under gravity. Changes in chromatin packing state were accompanied by shifts of nuclear areas of part of the nuclear population to smaller values but did not affect the respective Feulgen-DNA amounts except for a few nuclei. The extent of chromatin unpackaging, but not of frequency of nuclei with formation of extended chromatin fibers, in starved mice that were refed was greater than in well-fed controls. Refeeding induced increase in Feulgen-DNA amounts and regain and redistribution of concanavalin A–reactive nuclear glycoproteins. However, the duration of refeeding used was probably insufficient to reestablish the stereo arrangement of the chromatin-nuclear matrix and to restore chromatin fluidity to the level seen in well-fed mice. Conclusions The changes in the liver cell nuclei associated with starvation and refeeding of adult mice involved chromatin supraorganization, hepatocyte proliferation (refeeding), and the loss, regain, and redistribution of nuclear proteins, especially nuclear matrix components, related to chromatin organization and extensibility. These changes are suggested as favoring the silencing and reactivation of transcriptional activities, depending on the organism's nutritional state. © 2005 Wiley-Liss, Inc.

The incidence of apoptosis and nuclear instability, including the incidence of catastrophic death, were investigated in benzo[a]pyrene (BP)-transformed human breast epithelial cells (BP1-E cell line) after microcell-mediated transfer of chromosomes 11 and 17. Since the introduction of normal chromosomes 11 and 17 into tumorigenic human breast BP1-E cells reverts some of these cells’ characteristics (especially those affected by microsatellite instabilities and loss of heterozygosity) to those of parental non-transformed MCF-10F cells, it was expected that the cell death rates would also be affected by this treatment. The transfer of the mentioned chromosomes, especially chromosome 17, to tumorigenic BP1-E cells increased the apoptotic ratios and decreased the nuclear instability ratios, thus showing that the microsatellite instability and loss of heterozygosity induced by BP in these chromosomes of MCF-10F cells affect the control of cell death mechanisms. © 2003 Elsevier B.V. All rights reserved.

The survival rate of domestic male and female adult Panstrongylus megistus was studied after sequential heat and cold shocks in order to investigate shock tolerance compared to that previously reported for nymphs. Sequential shocks were such that a milder shock (0°C, 5°C, 35°C, or 40°C for 1 h) preceded a severe one (0°C or 40°C for 12 h), separated by intervals of 8, 18, 24, and 72 h at 28°C (control temperature). The preliminary thermal shock induced tolerance to the more severe one, although tolerance intensity depended on the initial shock temperature and the interval between treatments. Despite the observed tolerance, the survival rate for insects subjected to both shocks decreased when compared to that of individuals subjected to a single mild shock. When tolerance differed with sex, females showed greater values than males. In contrast to the response detected in nymphs, for which higher heat tolerance values were sustained for intervals of up to 24 h (preliminary shock, 35ºC) or even longer (preliminary shock, 40ºC) between sequential shocks, significant values were verified in adults only for shock intervals of up to 8 h (preliminary shock, 40ºC). While findings for nymphs exhibited considerable cold-shock tolerance under conditions in which preliminary shocks were given at 5ºC or 0ºC and the periods between shocks were up to 72 h long, the adults were shown to be capable of acquiring a substancial tolerance response to a more severe cold shock only when the preliminary shock was given at 0ºC and shock interval surpassed 18 h. It is assumed that the mechanisms involved in the cellular protection of P. megistus under sequential temperature shocks (heat shock protein action?) may loose effectiveness with insect development. A taxa de sobrevivência de alados domiciliares machos e fêmeas de Panstrongylus megistus foi analisada, após choques seqüenciais quentes e frios, a fim de investigar aquisição de termotolerância em comparação a dados já reportados para ninfas. Os insetos foram submetidos a choques térmicos seqüenciais, sendo que um choque mais suave (0°C, 5°C, 35°C ou 40°C por 1 h) precedeu o choque mais severo (0°C ou 40°C por 12 h). Estes foram separados por intervalos de 8, 18, 24 e 72 h a 28°C (temperatura-controle). Demonstrou-se que o choque térmico preliminar pode induzir tolerância ao mais severo, embora a intensidade de tolerância seja dependente da temperatura inicial do choque e do intervalo entre os tratamentos. Apesar da tolerância observada, a taxa de sobrevivência diminuiu em insetos submetidos a ambos os choques quando comparados àqueles espécimes submetidos a um único choque suave. Quando a tolerância diferiu com o sexo, as fêmeas apresentaram tolerância maior. Diferentemente da resposta observada em ninfas, para as quais a tolerância a choques quentes se estende até quando o período entre os choques é de 24 h (choque inicial a 35ºC) ou mais (choque inicial a 40ºC), taxa expressiva de tolerância nos adultos foi verificada apenas em condições de até 8 h de intervalo entre os choques, sendo o choque inicial dado a 40ºC. Também diferindo do que acontece às ninfas, cuja tolerância ao choque frio se expressa em todas as condições semelhantemente estudadas, nos adultos observou-se expressiva tolerância ao choque frio apenas quando o choque inicial foi dado a 0ºC e o tempo entre choques seguidos ultrapassou 18 h. Presume-se que em P. megistus os mecanismos que desempenham papel na proteção celular a choques seqüenciais de temperatura (proteínas de choque?) possam perder a eficácia com o desenvolvimento do inseto.