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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.

Optical anisotropy properties and infrared spectra of collagen and DNA as individual entities have been widely described. However, they have not been studied in collagen-DNA complex biofilms. We investigated whether collagen type I and DNA birefringence evaluated using polarization microscopy, and the vibrational patterns of their chemical groups identified in Fourier transform-infrared (FT-IR) spectra are retained in collagen-DNA complexes. Collagen gel rods extruded into a DNA saline solution and films dried from drops using mixtures of a 10-μL (14 μg) collagen solution and a 50-μL (305 μg) DNA solution and of a 50-μL (70 μg) collagen solution and a 10-μL (61 μg) DNA solution were used. Changes in the collagen fibers' stereoarrangement occurred in the gel rods interacting with the DNA. Even when the DNA concentration (305 μg) was significantly higher than the collagen concentration (14 μg) and the DNA played the main role in establishing the overall oriented geometry in the collagen-DNA films (intertwisted fibrillary structure and negative birefringence), some collagen markers were present in the FT-IR spectra of the complex. When collagen and DNA concentrations were similar, DNA denaturation may occur. Present observations suggest formation of collagen-DNA complexes even if highly organized collagen macromolecules are offered to DNA.

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

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.

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 control of DNA packaging has been reported to be dependent on an ordered liquid-crystalline state. However, the textural characteristics that are typical of crystals and that resemble mesophases have not been reported for highly polymerized or even shorter types of DNA filaments under in vitro conditions that favor crystallization. Because DNA crystals are expected to exhibit particular textural optical anisotropies, pure and highly polymerized calf thymus DNA and simpler λ phage DNA were crystallized from solution drops and were analyzed using high-performance polarization microscopy with and without differential interference contrast (DIC) optics. Both types of DNA formed crystals that exhibited chiral supramolecular textures resembling the twist-grain boundary (TGB) columnar mesophases described for liquid crystals and exhibited intrinsic negative birefringence. To the best of our knowledge, this is the first observation using polarization/interference optics of pure DNA crystals that have TGB columnar mesophase-like textural characteristics. A comparison of the crystals formed from the highly polymerized calf thymus DNA and those formed from the shorter phage DNA strands revealed textural differences. Compared to the phage DNA crystals, the crystals of highly polymerized thymus DNA exhibited a more intertwisted columnar distribution and a fibrous texture between their columnar structures. In addition, a form birefringence phenomenon was detected only in the thymus DNA crystals. These characteristics are presumed to reflect the higher level of supramolecular order, self-assembly and chirality in highly polymerized calf thymus DNA crystals relative to that of crystals formed from the simpler, shorter, λ phage DNA. The higher-order supramolecular organization revealed here for in vitro DNA preparations raises the possibility that this structure could also occur, possibly to a smaller degree, during DNA self-aggregation under specific in vivo conditions. Whether the DNA crystal properties presently described play a role in the establishment of higher-order levels of hierarchical chromatin structure and consequently in chromatin physiology, should be further investigated.

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

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.

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.

Summary Optical anisotropy as dispersion of birefringence (DB) (birefringence studied for light of different wavelengths) and linear dichroism (LD) (selective absorption of polarized light) in stained substrates reflects their macromolecular orientation states. Birefringence interference colors of alcian blue (AB)-stained glycosaminoglycans (GAG) and glycoconjugates observed with polarization microscopy have been found to vary, although their staining characteristics under unpolarized light are practically the same. We investigated the optical anisotropy of GAG-AB and some glycoconjugate-AB complexes used as standards, to provide a basis for interpreting results for AB-stained materials in situ. Filamentous preparations of hyaluronic acid (HA), chondroitinsulfates, proteoglycans, and a mucus sulfoglycoconjugate were studied. Anomalous DB (birefringence sign changing with the wavelength of the incident light) was generally observed, but LD was seen only in the AB–HA complex. LD simultaneous to anomalous DB characteristics on the AB–HA complex were assumed to be caused by a maximally oriented helical conformation of the HA. For the other AB–GAG studied, the optical anisotropic characteristics were suggestive of some degree of folding of their chains into a tertiary structure. The profiles of the anomalous DB curves for the AB-stained sulfoglycoconjugate differed from those of the other materials, probably due to different organization of its dye-binding sites.

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.

Esta é uma proposta de representação teatral como um recurso didático para aulas de Genética e Biologia Celular e com a finalidade de esclarecer, aprofundar e fixar os conhecimentos sobre as características diferenciais dos elementos moleculares da cromatina e dos cromossomos, a organização espacial e de funcionamento desses mesmos elementos.