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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.

Valproic acid/sodium valproate (VPA), a drug originally prescribed as an anticonvulsant, has been widely reported to act on epigenetic marks by inducing histone acetylation, affecting the DNA and histone methylation status, and altering the expression of transcription factors, thus leading to modulation of gene expression. All these epigenetic changes have been associated with chromatin remodeling effects. The present minireview briefly reports the main effects of VPA on chromatin and image analysis and Fourier transform infrared (FTIR) microspectroscopy in association with molecular biology methodological approaches to investigate the VPA-induced changes in chromatin structure and at the higher-order supraorganizational level.

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

Made available in DSpace on 2020-12-12T01:23:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Constitutive heterochromatin typically exhibits low gene density and is commonly found adjacent or close to the nuclear periphery, in contrast to transcriptionally active genes concentrated in the innermost nuclear region. In Triatoma infestans cells, conspicuous constitutive heterochromatin forms deeply stained structures named chromocenters. However, to the best of our knowledge, no information exists regarding whether these chromocenters acquire a precise topology in the cell nuclei or whether their 18S rDNA, which is important for ribosome function, faces the nuclear center preferentially. In this work, the spatial distribution of fluorescent Feulgen-stained chromocenters and the distribution of their 18S rDNA was analyzed in Malpighian tubule cells of T. infestans using confocal microscopy. The chromocenters were shown to be spatially positioned relatively close to the nuclear periphery, though not adjacent to it. The variable distance between the chromocenters and the nuclear periphery suggests mobility of these bodies within the cell nuclei. The distribution of 18S rDNA at the edge of the chromocenters was not found to face the nuclear interior exclusively. Because the genome regions containing 18S rDNA in the chromocenters also face the nuclear periphery, the proximity of the chromocenters to this nuclear region is not assumed to be associated with overall gene silencing. Department of Structural and Functional Biology Institute of Biology University of Campinas (Unicamp), Rua Monteiro Lobato 255 Department of Biology Institute of Biosciences State University of S o Paulo (Unesp), Avenida 24-A, 1515 Superintendence for Control of Endemic Diseases (SUCEN), Rua Afonso Pessini, 86 Department of Biology Institute of Biosciences State University of S o Paulo (Unesp), Avenida 24-A, 1515

The larvae of the two distantly related nonsocial bees Ericrocis lata (Apidae) and Hesperapis (Carinapis) rhodocerata (Melittidae), which develop mostly under arid desert areas of North America, and that differ in that they either spin (E. lata) or do not spin (H. rhodocerata) protective cocoons before entering diapause, produce transparent films that cover the larval integument. To understand the nature of these films, their responses to topochemical tests and their characteristics when examined with fluorescence and high-performance polarization microscopy and microspectroscopy were studied. A positive staining by Sudan black B, birefringence of negative sign, and a Fourier transform-infrared (FT-IR) spectrum typical of lipids were detected for the integument covering of both species. The FT-IR signature, particularly, suggests a wax chemical composition for these lipid coverings, resembling the waxes that are used as construction materials in the honey cells produced by social bees. Considering the arid environmental conditions under which these larvae develop, we hypothesize that their covering films may have evolved as protection against water depletion. This hypothesis seems especially appropriate for H. rhodocerata larvae, which are capable of undergoing a long diapause period in the absence of a protective cocoon.

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.

The Feulgen reaction has been proposed by Robert Feulgen and Heinrich Rossenbeck for the identification of DNA nearly a hundred years ago. Since then, many other applications of this cytochemical/topochemical procedure at qualitative and quantitative level have been proposed in relation to DNA and its role in chromatin in human, animal and plant cells. In this article, we briefly review some fundamental aspects of the Feulgen reaction and current applications of such a method in studies of altered chromatin texture, including its association with or preceding changes in transcriptional activities and effect on epigenetic marks. Further perspectives on the use of the Feulgen reaction will depend of the proposal of innovative biological questions in which its reveals appropriate.

A previous study has not revealed the participation of a mucous component in the cocoon wall of the solitary bee, Lithurgus chrysurus, differing from the cocoon structure reported for many other bee species. However, uncertainty remains, because only the median and rear zones of this cocoon type have thus far been analyzed. Here, we studied the front zone of this cocoon, searching its components and their organization, to fill this knowledge gap. Topochemical assays, polarization microscopy and Fourier transform-infrared (FT-IR) microspectroscopy were used to study cross sections from L. chrysurus cocoon. Three main layers differing in structural organization were found to compose the cocoon wall. Silk fibroins were assumed to constitute the filamentous threads of the inner and outer layers and the laminar structure of the intermediate layer. Deduced from its topochemical properties and FT-IR spectral signature, a foamy material containing mucin glycoproteins and carboxylated acid glycosaminoglycans was found in the intermediate layer. FT-IR analysis using a Savitzky-Golay 2nd-derivative and absence of linear dichroism and birefringence phenomena suggest that a random-coil secondary structure predominates in the foam component. Co-existence of α-helical and β-sheet conformations is also hypothesized for the fibroin component of this cocoon. It is thus concluded that in addition to fibroin elements, a mucous component, likely contributed by a Malpighian tubule secretion, integrates the composition of the front zone of the cocoon wall of L. chrysurus. In addition, the FT-IR analysis of the inner layer silk of this cocoon suggests significant differences in comparison to the silk fibroins of the silkworm, and some minor spectral differences in comparison to published data on the honeybee silk, with respect to protein secondary structure.

Malpighian tubules of Triatoma infestans cultivated in vitro in presence of 0.05 mM sodium valproate (VPA) for 1-4 h had their nuclear phenotypes investigated regarding induction of chromatin remodeling. Under present conditions, although a few nuclei demonstrated heterochromatin decondensation, no overall differences resulted in comparison to tests in which live insects are injected with VPA.

Triatoma infestans, a vector of Chagas' disease, shows several particular cell biology characteristics, including the presence of conspicuous heterochromatic bodies (chromocenters) where DNA methylation has not been previously detected. Whether histone modifications contribute to the condensed state of these bodies has not yet been studied. Here, we investigated epigenetic modifications of histones H3 and H4 and presence of the non-histone heterochromatin protein (HP1-α) in the chromocenters and euchromatin of T. infestans cell nuclei, using immunocytochemistry. The effect of different concentrations of the histone deacetylase inhibitors valproic acid (VPA) and sodium butyrate (NaBt) on chromocenter condensation was visually examined; in VPA-treated specimens, this effect was also analyzed by image analysis. Trimethylated H3K9 signals, which were revealed in chromocenter and non-chromocenter areas, were strongest in chromocenters, whereas selected acetylated histone marks and mono- and dimethylated H3K9 and H4K20 signals were detected only in euchromatin. Weak trimethylated H4K20 signals and variable distribution of HP1-α were detected in chromocenters of part of the cellular population analyzed. Although specific VPA and NaBt treatment conditions affected the heterochromatin condensation pattern, they did not induce a decrease in survival and molting rates of the T. infestans nymphs. The VPA-induced chromatin remodeling was not accompanied by induction of H3K9 acetylation in chromocenters. Present findings regarding histone modifications and effects following VPA or NaBt treatments did not yet solve the question of which factors are responsible for maintenance of the condensed state of chromocenters in T. infestans. A possibility requiring further investigation remains on histone methylation marks and/or non-histone proteins.

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.

Made available in DSpace on 2018-12-11T16:54:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-08-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Panstrongylus megistus, a potential vector of Chagas disease, currently occupies a wider geographic distribution in Brazil than Triatoma infestans, another member of the hemipteran Reduviidae family and a vector of the same disease. A small heterochromatic body (chromocenter) formed by the Y chromosome is evident in the somatic cells of P. megistus, differing in size and chromosome type contribution from the well-studied chromocenters present in T. infestans. While the overall distribution of histone epigenetic marks differ when comparing the heterochromatin and euchromatin territories in T. infestans, no similar data have been established for other hemipteran reduviids, including P. megistus. In the present work, histone acetylation and methylation marks were investigated in cells of Malpighian tubules of P. megistus 5th instar nymphs using immunocytochemical assays and compared to previously published data for T. infestans. Although similarities between these species were found regarding absence of acetylated H3K9, H4K8 and H4K16, and H3K9me and H3K9me2 in the chromocenter, presence of these marks in euchromatin, and presence of H3K9me3 in the chromocenter, no intimate association of acetylated H4K8 and 18S rDNA was revealed in the chromocenter of P. megistus. The elevated abundance of H3K9me2 marks at the nuclear periphery in P. megistus cells, differing from data for T. infestans, is suggested to reflect differences in the interaction of lamina-associated chromatin domains with the nuclear lamina, methyl-transferase modulation and/or association with the last DNA endoreplication step in 5th instar nymphs, which is a matter for further investigation. Department of Structural and Functional Biology Institute of Biology University of Campinas (Unicamp) Department of Biology Institute of Biosciences State University of São Paulo (Unesp) Superintendence for Control of Endemic Diseases SUCEN Department of Genetics “Luiz de Queiroz” College of Agriculture (ESALQ) University of São Paulo (USP) Department of Morphology Institute of Biomedical Sciences Federal University of Uberlândia Department of Biology Institute of Biosciences State University of São Paulo (Unesp) CNPq: 132341/2010-7 CNPq: 304668/2014-1

The cocoon of Lithurgus chrysurus Fonscolobe, a univoltine species belonging to the megachilid subfamily Lithurginae, was studied using polarization microscopy and topochemical methods. The aim of the study was to establish the composition and structure of the cocoon wall of this bee in comparison with reported data for other species of this subfamily. The cocoon was found to be composed by macromolecularly oriented, positively birefringent silk protein layers that acquire a complex and thicker multilayered distribution at its rear zone. Although the composition and distribution of the silk threads in this species' cocoon was considered similar to the previously described patterns for other lithurgine bees, the absence of a mucous layer, typically produced by Malpighian tubules, differed from reported data for other species of the same subfamily.

Chromatin organization has been considered to play a major role on aging, by regulating DNA accessibility to transcription and repair machinery. Such organization can be modulated by epigenetic events, such as DNA methylation and histone post-translational modifications. Since changes on gene expression profiles have been described in aged neurons, our aim was to study the age-dependent relationship between structural and epigenetic alterations on chromatin of cortical neurons from mice. For this purpose, isolated neuronal nuclei from mice of two ages were studied by image analysis after cytochemistry, or assessed for chromatin accessibility by enzymatic digestion. Additionally, two epigenetic marks, for open and for densely packed chromatin fibers were quantified. Results indicate epigenetically driven alterations on chromatin organization of cortical neurons with advancing age, whose fibers seem to undergo redistribution and unpackaging. Since increased transcriptional activity is not characteristic of aged neurons, these loosened chromatin fibers may be associated with impaired genome stability, as well as with increased accessibility of repair machinery to a life span damaged DNA. J. Cell. Biochem. 115: 659–665, 2014. © 2013 Wiley Periodicals, Inc.

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.

Lysyl oxidases (LOXs) are enzymes that permit the covalent crosslinking of the component chains of collagen and elastin. These enzymes are present inside the nuclei of certain mammalian cells. Previous studies have proposed LOX binding to histone H1 in vitro, and histone H1 is known to control global chromatin compaction and mitotic chromosome architecture. Therefore, in the present study, we analyzed chromatin supraorganizational changes, mitotic abnormalities, mitotic indices and cell death ratios in COS-7 and NRK-49F cells with high and low lox expression levels, respectively. The objective was to support biochemical data of LOX-H1 interaction, by providing evidence of chromatin remodeling in vivo, under different lox expressions. Chromatin decondensation assessed by image analysis was observed in COS-7 cells with increased lox expression. This decondensation is suggested to be promoted by LOX actions on histone H1, which loosens the DNA-H1 complex. In NRK-49F cells transfected with antisense lox or subjected to treatment with beta-aminopropionitrile (BAPN), chromatin condensation and nuclear phenotypic variability were found, which may be due to reduced LOX-H1 interaction. When lox expression was increased in COS-7 cells, the frequency of irregular chromosome plates was not affected, but cell proliferation decreased and "cell death preceded by multinucleation" increased. In NRK-49F cells there was accelerated proliferation induced by transfection with the antisense lox, and confirmed when cells were treated with BAPN. Apoptosis increased in NRK-49F cells only with BAPN treatment whereas cell death preceded by multinucleation increased only after antisense lox transfection. The data presented herein regarding chromatin remodeling indirectly support the hypothesis that LOX binds to histone H1 in vivo. Cell proliferation in COS-7 and NRK-49F cells and cell death at least in COS-7 cells agree with predicted effects of LOX interference in these processes.

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.

Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation for these cell nuclei.

The argyrophylic staining of the nucleolar organizer regions (AgNOR positive response) in interphase nuclei is often related directly to the cellular demand for ribosome biogenesis and is considered of relevance in studies of tumor pathology. Transformation of human breast epithelial MCF-10A cells by the c-Ha-ras oncogene results in altered growth, invasiveness and tumorigenicity in nude mice. Since ras transformation may be associated with a more intense nucleolar activity, we examined the influence of transfection by the Ha-ras oncogene on AgNOR staining response in MCF-10A cells. Following assessment of the AgNOR response with video image analysis, the AgNOR-positive areas and the AgNOR area/nuclear area ratio, but not the number of AgNOR aggregates or dots per nucleus, were found to be much higher after ras transformation. A role of the Ha-ras transformation on the nucleolar activity of the MCF-10A is thus suggested as assessed by the AgNOR staining. Based on data in the literature, it is also hypothesized that a decreased wild-type p53 level, possibly promoted by the ras transformation, may be associated with the increased AgNOR response.

Forms of the protozoan of the Hepatozoon genus are detected free in the circulation and also within some of the erythrocytes of infected snakes. In healthy snakes, DNA fragmentation and cell death usually affect a few circulating erythrocytes in agreement with the long life span expected for these cells. In the present study we investigated whether infection by Hepatozoon spp. affected the incidence of DNA fragmentation and cell death in erythrocytes from the rattlesnake, Crotalus durissus terrificus. Methods such as the kinetics of Feulgen-DNA hydrolysis, and the TUNEL and comet assays, previously used for the study of chromatin organization and DNA fragmentation in erythrocytes of healthy snakes, were used. The results indicated that Hepatozoon spp. increased the DNA fragmentation and chromatin condensation typical of cell death in circulating erythrocytes of C. d. terrificus, including cells that do not harbour the parasite. The Hepatozoon infection is thus suggested to accelerate destruction of erythrocytes in the rattlesnake, not only affecting cells harbouring the parasite, but also in those without it.

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

Summary A variation of the Concanavalin A (Con-A)-peroxidase labelling method originally described by Kiernan [Localization of alpha- d -glucosyl and alpha- d -mannosyl groups of mucosubstances with Concanavalin A and horseradish peroxidase. Histochemistry 1975;44:39–45] was applied to unsectioned cell preparations, with an emphasis on the nuclear localization of glycoproteins. Mouse liver imprints and chicken blood smears fixed in acetic acid-ethanol solution were studied. Modifications of the method included using increased Con-A concentration, and a range of pH values for the Con-A solutions. The strongest Con-A labelling of both erythrocytes and hepatocytes was obtained after incubation with Con-A at pH 6.5 and with Con-A concentrations at least two-fold greater than those used for tissue sections. These conditions may alter the Con-A conformation, enabling the lectin molecule to enter the cell nucleus and bind to nuclear glycoproteins, thus allowing their localization and quantification.

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.

Sperm chromatin alteration is an important feature that can affect fertility of the male rabbit. This study compared toluidine blue staining with Feulgen reaction (as methods for evaluating chromatin alteration) and investigated the relationship between sperm morphology and chromatin alteration. Seven hundred rabbit ejaculates of animals with unknown fertility were used. Primary and secondary morphological sperm abnormalities were evaluated in semen smears with phase-contrast microscopy. Chromatin alterations were evaluated in semen smears stained with toluidine blue (pH 4.0 and 5.0) and with the Feulgen reaction. While the three methods were equally efficacious for identification of chromatin alterations, toluidine blue staining was more appropriate to characterize the intensity of chromatin alterations. The correlation between primary sperm defects and chromatin alteration was high and positive, suggesting that sperm chromatin structure affected sperm head morphology. The correlation between secondary sperm defects and chromatin alteration was also positive, but lower. The final chromatin compaction occurs in the epididymus, where secondary sperm defects originate. Therefore, the causes of secondary sperm defects could also intervene with final chromatin compaction. In summary, the toluidine blue stain was an effective means of evaluating the sperm chromatin alteration in rabbit spermatozoa.

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 frequency and responsiveness to the TUNEL assay were used to compare the DNA fragmentation associated with programmed cell death in erythrocytes of the circulating blood from chickens, pigeons, bullfrogs and tortoise species in which the life span and/or metabolic activities of these cells vary considerably. While most avian and adult bullfrog erythrocytes gave a positive response in this test, only 31% of the tortoise erythrocytes showed a positive TUNEL response, which was generally weak. The strongest positive response was seen in bullfrog erythrocytes. The positive results for avian erythrocytes may possibly be associated with their higher metabolic rate and body temperature while the lower response for tortoise erythrocytes may reflect the very long life span and lower metabolic rate of these cells. Based on previous literature reports, the results for the bullfrog are unlikely to be associated with the animal metabolic rate and do not necessarily indicate genomic inertness. Together with literature data showing divergent pathways for programmed cell death in erythrocytes, the differences in DNA fragmentation reported here indicate that further studies on vertebrate nucleate erythrocytes are required for a better understanding of cell death in these cells.

Nuclear image analysis of Feulgen-stained V79 fibroblasts after three days in culture was used to discriminate apoptotic cells and cells suspected to be undergoing apoptosis from control cells based on parameters such as the Feulgen-DNA content, degree of chromatin condensation and nuclear areas, in association with visual morphology. The fibroblasts were initially plated at a density of 10(5) cells/ml and incubated under optimal culture conditions without subculturing. Following confluency, the cells underwent contact inhibition apoptosis. Image analysis revealed three nuclear phenotypes which were defined in terms of their morphological characteristics and levels of chromatin condensation. A decrease in the amount of Feulgen-DNA was detected in apoptotic cells and in cells suspected of undergoing apoptosis. This decrease was assumed to indicate DNA loss. Image analysis procedures may therefore provide a useful tool for discriminating cells in the early stages of apoptosis.Análise de imagem de núcleos de fibroblastos V79 após três dias em cultura foi realizada em preparados submetidos à reação de Feulgen para discriminar células suspeitas de estarem em fases precoces da apoptose daquelas comprovadamente apoptóticas. Parâmetros tais como conteúdo de Feulgen-DNA, grau de condensação cromatínica e área nuclear foram estudados em associação com a morfologia estabelecida em termos visuais. Os fibroblastos foram inicialmente plaqueados numa densidade de 10(5) células/ml e encubados sob condições ótimas de cultura sem subcultura. Com a confluência, as células sofreram apoptose pela inibição por contato. A análise de imagem revelou três fenótipos nucleares definidos quanto a características morfológicas e níveis de condensação cromatínica. Foi detectado decréscimo no conteúdo de Feulgen-DNA das células apoptóticas mas também nas células suspeitas de apoptose. Admite-se que este decréscimo indique perda de DNA. A análise de imagem pode ser, portanto, uma ferramenta útil na discriminação de células mesmo em fases precoces da apoptose.

Increased amounts of chromatin condensation (i.e., localized areas of high DNA density, or chromatin higher order packing state) have been described in NIH 3T3 cells transformed with the Ha‐rasoncogene. The structural basis for this oncogene‐mediated alteration in nuclear organization is unknown. Since DNA methylation is likely to be involved in regulating the nucleosomal level of DNA packaging, we studied the role of DNA methylation in higher‐order chromatin organization induced by Ha‐ras. CpG‐methylated DNA content was estimated in “condensed” chromatin of Ha‐ras‐transformed NIH 3T3 cell lines which differ in ras expression and ras‐induced metastatic ability but present approximately the same values of “condensed” chromatin areas. The question posed was that if DNA methylation were involved with the chromatin higher‐order organization induced by Ha‐ras in these cell lines, the methylated DNA density in the “condensed” chromatin would also be the same. The DNA evaluation was performed by video image analysis in Feulgen‐stained cells previously subjected to treatment withMspI andHpaII restriction enzymes, which distinguish between methylated and non‐methylated DNA. The amount of methylated CpG sequences not digested byHpaII in “condensed” chromatin regions was found to vary in the studied ras‐transformed cell lines. DNA CpG methylation status is thus suggested not to be involved with the higher order chromatin condensation induced byrastransformation in the mentioned NIH 3T3 cell lines.

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.

"Pé-duro" (hard foot) is a rare breed of beef cattle of European (Bos taurus taurus) origin, originated in northern and northeastern Brazil. Y chromosome morphology, outer genital elements and other phenotypic characteristics were examined in 75 "pé-duro" bulls from the Empresa Brasileira de Pesquisa Agropecuária (Embrapa) herd in the Brazilian State of Piauí. The purpose was to investigate possible racial contamination with Zebu animals (Bos taurus indicus) in a cattle that has been considered closest to its European origin (B. t. taurus). The presence of both submetacentric and acrocentric Y chromosomes, typical of B. t. taurus and B. t. indicus, respectively, and the larger preputial sheath in bulls with an acrocentric Y chromosome indicated racial contamination of the "pé-duro" herd with Zebu cattle. Phenotypic parameters involving horn, dewlap, ear, chamfer, and coat color characteristics, indicative of apparent racial contamination, were not associated with acrocentric Y chromosome.Um plantel de touros "pé-duro", consistindo de 75 animais do núcleo da Embrapa envolvido com a preservação desse gado no Estado do Piauí, foi examinado quanto à morfologia do seu cromossomo Y, bem como em relação a elementos da genitália externa e outras características fenotípicas dos machos. O objetivo era investigar a contaminação racial por animais zebuínos (Bos taurus indicus) num gado bovino que tem sido considerado mais próximo de sua origem européia (Bos taurus taurus). Tanto a forma submetacêntrica quanto a forma acrocêntrica do cromossomo Y, típicas das sub-espécies B. t. taurus e B. t. indicus, respectivamente, bem como maior bainha prepucial nos espécimes portadores do cromossomo Y acrocêntrico, indicativa de contaminação racial por gado zebuíno, foram detectadas no rebanho "pé-duro" mantido no núcleo da Embrapa. Outras características fenotípicas analisadas que podem informar sobre a contaminação racial aparente não foram associadas ao tipo de cromossomo Y. A detecção de contaminação racial no estoque de touros "pé-duro" aqui analisado não invalida os esforços da Embrapa em preservar este gado de importância regional para o Nordeste do Brasil, porém quase extinto.

When transfected to benzo[a]pyrene (BP)‐transformed MCF‐10F human breast epithelial cells (BP1 cell line) the c‐Ha‐rasoncogene has proven to enhance the neoplastic changes initiated by exposure to BP, giving rise to an aggressive tumorigenic cell line, BP1‐Tras. We have previously demonstrated by image analysis that BP affects the DNA content and the chromatin supraorganization of MCF‐10F cells. Here Feulgen‐stained BP1‐Trascells were studied by image analysis in order to evaluate possible additional changes in DNA content and chromatin texture induced by insertion of therasoncogene. A high variability in DNA content also including polyploidy or near‐polyploidy, and an increase in the packing states of the chromatin which became still condensed in BP1 cells were found in BP1‐Trascells. The results differed from those reported for the BP1‐E1 cell line which is also an aggressive tumorigenic cell line, but was attained through progressive passages of BP‐transformed cells. It was demonstrated that different patterns of changes in DNA content and chromatin organization may be involved in equally aggressive tumorigenic BP‐transformed cell lines originated from the same cell line by different mechanisms.