Analysis of the DNA Fourier transform-infrared microspectroscopic signature using an all-reflecting objective.

The Fourier transform-infrared (FT-IR) signature of dry samples of DNA and DNA-polypeptide complexes, as studied by IR microspectroscopy using a diamond attenuated total reflection (ATR) objective, has revealed important discriminatory characteristics relative to the PO2(-) vibrational stretchings. However, DNA IR marks that provide information on the sample's richness in hydrogen bonds have not been resolved in the spectral profiles obtained with this objective. Here we investigated the performance of an "all reflecting objective" (ARO) for analysis of the FT-IR signal of hydrogen bonds in DNA samples differing in base richness types (salmon testis vs calf thymus). The results obtained using the ARO indicate prominent band peaks at the spectral region representative of the vibration of nitrogenous base hydrogen bonds and of NH and NH2 groups. The band areas at this spectral region differ in agreement with the DNA base richness type when using the ARO. A peak assigned to adenine was more evident in the AT-rich salmon DNA using either the ARO or the ATR objective. It is concluded that, for the discrimination of DNA IR hydrogen bond vibrations associated with varying base type proportions, the use of an ARO is recommended.

Enhanced peripheral nerve regeneration by the combination of a polycaprolactone tubular prosthesis and a scaffold of collagen with supramolecular organization.

The purpose of this study was to investigate the influence of implanting collagen with a supramolecular organization on peripheral nerve regeneration, using the sciatic nerve tubulization technique. For this purpose, adult female Sprague Dawley rats were divided into five groups: (1) TP - sciatic nerve repaired with empty polyethylene tubular prothesis (n = 10), (2) TPCL - nerve repair with empty polycaprolactone (PCL) tubing (n = 8), (3) TPCLF - repair with PCL tubing filled with an implant of collagen with a supramolecular organization (n = 10), (4) AG - animals that received a peripheral nerve autograft (n = 8), and (5) Normal nerves (n = 8). The results were assessed by quantification of the regenerated fibers, nerve morphometry, and transmission electron microscopy, 60 days after surgery. Immunohistochemistry and polarization microscopy were also used to analyze the regenerated nerve structure and cellular elements. The results showed that the AG group presented a larger number of regenerated axons. However, the TPCL and TPCLF groups presented more compact regenerated fibers with a morphometric profile closer to normal, both at the tube midpoint and 2 mm distal to the prosthesis. These findings were reinforced by polarization microscopy, which indicated a better collagen/axons suprastructural organization in the TPCLF derived samples. In addition, the immunohistochemical results obtained using the antibody anti-p75NTR as a Schwann cell reactivity marker demonstrated that the Schwann cells were more reactive during the regenerative process in the TPCLF group as compared to the TPCL group and the normal sciatic nerve. Altogether, the results of this study indicated that the implant of collagen with a supramolecular organization positively influenced and stimulated the regeneration process through the nerve gap, resulting in the formation of a better morphologically arranged tissue.

Pregnancy-induced chromatin remodeling in the breast of postmenopausal women.

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.

Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug.

Heterochromatin bodies in single- and multichromocentered interphase cell nuclei of Triatoma infestans, a vector of Chagas disease, have been suggested to contain AT-rich DNA, based on their positive response to Q-banding and Hoechst 33248 treatment. No information exists on whether GC-rich DNA is also present in these nuclei and whether it plays a role on chromatin condensation. Considering that methodologies more precise than those previously used to determine DNA base composition in situ are currently available, and that the spatial distribution of chromatin areas differing in composition in interphase cell nuclei of different species is a matter of interest, the localization of AT- and GC-rich DNA in T. infestans nuclei is revisited here. The methodologies used included DAPI/AMD and CMA(3)/Distamycin differential staining, Feulgen-DNA image analysis following Msp I and Hpa II enzymatic digestion, 5-methylcytidine immunodetection, AgNOR response, confocal microscopy, and the 5-aza-2'-deoxycytidine (5-AZA) demethylation assay. The results identified the presence of AT-rich/GC-poor DNA in chromocenters and evenly distributed AT and GC sequences in euchromatin. A GC-rich DNA zone encircling the chromocenters was also found but it could not be associated with NOR regions. To corroborate the DNA AT-richness in T. infestans nuclei, bioinformatic analyses were also performed. Methylated cytosine was evident at some points of the chromocenters' edge in single- and multichromocentered nuclei and at the euchromatin of multichromocentered nuclei and could be transiently affected by the 5-AZA treatment. The present results suggest that in the particular case of chromocenters of the hemipteran T. infestans, cytosine methylation is not a relevant factor involved in chromatin condensation.

Entropy of Feulgen-stained 17-beta-estradiol-transformed human breast epithelial cells as assessed by restriction enzymes and image analysis.

MCF-10F human breast epithelial cells when transformed with 17-beta-estradiol (E2) give rise to highly invasive C5 cells that generate adenocarcinomas in SCID mice. From these tumors, cell lines such as C5-A6-T6 and C5-A8-T8 have been derived. Variable patterns of chromatin supraorganization have been demonstrated for these cells during the transformation/tumorigenesis progress, when assessing chromatin entropy by image analysis in Feulgen-stained preparations. Since epigenetic dysregulation might contribute to the chromatin textural repatterning in transformed MCF-10F cells, the association of the variable chromatin packing states with global DNA methylation was investigated in these cells after their treatment with restriction enzymes followed by Feulgen staining and chromatin entropy evaluation by image analysis. The results indicate that although -CmCGG- sequences may affect chromatin supraorganization in some of the analyzed cell types (perhaps due to localized hypermethylation), not all the chromatin condensation patterns in these cells with transformation and/or tumorigenesis are associated with DNA methylation (e.g. E2 cells). Chromatin supraorganization remodeling in C5-A6-T6 and C5-A8-T8 cells may be attained by different mechanisms, with C5-A6-T6 chromatin packing states perhaps being associated with local DNA hypermethylation or other epigenetic factors, and C5-A8-T8 likely being associated with global DNA hypomethylation, as reported in the literature for other cell types. Thus, we assume that a variable epigenetic modulation affecting the higher-order packing states of chromatin in the estrogen-transformed MCF-10F cell model could be evident with the chromatin entropy study by image analysis.

Structural and biochemical analysis of the effect of immobilization followed by stretching on the calcaneal tendon of rats.

Little is known about the stretching effects on the biochemical and morphological features of tendons submitted to a long period of immobilization. Our purpose was to evaluate the response of rat tendons to stretching procedures after immobilization. The animals were separated into five experimental groups: GI--control of immobilized and euthanized animals; GII--immobilized and euthanized animals; GIII--control of immobilized animals and afterward stretched or allowed free cage activity; GIV--immobilized and stretched animals; and GV--immobilized and allowed free cage activity. Analysis in SDS-PAGE showed no remarkable differences among the groups, but a prominent collagen band was observed in GV, as compared to GIV and the control group, both in the compression and tension regions. Hydroxyproline content was highest in the compression region of GII. No differences among the groups were observed in the tension region. In regard to the concentration of noncollagenous proteins, differences were detected only in the tension region, where larger concentrations were found in the GII. When GII and GIV were compared, highest values were found in the GII. A more abundant presence of sulfated glycosaminoglycans, especially chondroitin sulfate, was detected in GIV, at the compression region of tendons. The presence of dermatan sulfate was outstanding in the compression and tension regions of the GII and GV groups. In the Ponceau SS stained sections, analyzed under polarization microscopy, GII exhibited the highest disorganization of the collagen bundles, partially recovered after stretching or with only remobilization. Our results indicate that a revision in the stretching procedures, in terms of duration and periodicity of the sessions, could benefit the efficiency of the stretching in cases of previous immobilization of tendons.

Image analysis of the AgNOR response in ras-transformed human breast epithelial cells.

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.

17-beta-estradiol affects nuclear image properties in MCF-10F human breast epithelial cells with tumorigenesis.

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

Proteoglycan synthesis by human corneal explants submitted to laser in situ keratomileusis (LASIK).

PURPOSE: To evaluate the acute effects of laser in situ keratomileusis (LASIK) upon the synthesis of proteoglycans (PGs) and collagen fibril organization in human corneal explants. METHODS: Human corneas that had been rejected for transplants were obtained at Banco de Olhos of Hospital São Paulo. For each eye pair, one cornea was submitted to refractive surgery, and the other was used as its matched control. After surgery, the corneas were excised from the eyes and immediately placed in a Ham F-12 nutrient mixture containing (35)S-sulfate for the metabolic labeling of PGs. After 24 h incubation, PGs were extracted and identified by a combination of agarose gel electrophoresis and enzymatic degradation with protease and specific glycosaminoglycan lyases. Histopathological and birefringence analysis were performed in fixed tissue slices. RESULTS: A marked decrease in (35)S-sulfate incorporation in PGs was observed in corneal explants that received LASIK, especially concerning dermatan sulfate-PGs, with keratan sulfate- and heparan sulfate-PG synthesis reduced to a lower degree. Only low molecular weight PGs were present in the corneas, both before and 24 h after LASIK. No sign of wound healing processes were observed, but a marked change in corneal birefringence was seen following LASIK treatment. CONCLUSIONS: Laser application led to decreased PG biosynthesis in human corneal explants, with marked changes in the collagen fibril organization, as revealed by changes in the tissue birefringence.

DNA content and chromatin texture of human breast epithelial cells transformed with 17-beta-estradiol and the estrogen antagonist ICI 182,780 as assessed by image analysis.

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.

Nucleus image properties assessed by video image analysis in mouse hepatocytes under a short lysis for extended chromatin fiber formation.

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.

Chromatin supraorganization, DNA fragmentation, and cell death in snake erythrocytes.

In nucleate erythrocytes of several vertebrate groups, the frequency and intensity of DNA fragmentation associated with programmed cell death vary considerably. Although hemoglobin efficiency may be related to erythrocyte life span, and hemoglobin types and erythrocyte life spans are assumed to vary in reptiles, no data on DNA fragmentation and chromatin organization as related to cell death exist for snakes. In the present study, chromatin supraorganization, DNA fragmentation, and cell death were investigated in four snake species (Crotalus durissus terrificus, Bothrops jararaca, Bothrops alternatus, and Bothrops neuwiedii), which differ in their geographical distribution and habitats, by using image analysis of Feulgen hydrolysis kinetics, the TUNEL assay, single-cell gel electrophoresis, and transmission electron microscopy. Relatively few circulating erythrocytes were found to be simultaneously committed to cell death, although there was some variation among the snake species. Conspicuous nuclear and cytoplasmic organelles suggestive of metabolic activity were seen ultrastructurally in most snake erythrocytes. The DNA of the snake erythrocyte chromatin was much more resistant to Feulgen acid hydrolysis (DNA depurination and breakdown) than that of young adult bullfrog erythrocytes, which had a high frequency and intensity of DNA fragmentation. Of the species studied, B. neuwiedii and C. d. terrificus showed the greatest resistance to Feulgen acid hydrolysis and to the DNA fragmentation, revealed by the TUNEL assay. Although B. neuwiedii also showed the lowest frequency of cells with more damaged DNA in the single-cell gel electrophoresis assay, C. d. terrificus had the highest frequency of damaged cells, possibly because of the abundance of alkaline-sensitive DNA sites. The results for DNA fragmentation and cell death in erythrocytes of B. jararaca and B. alternatus generally differed from those for C. d. terrificus and B. neuwiedii and may reflect differences in the biology of these species selected under different geographical habitats. The differences in erythrocyte cell biology reported here may be related to hemoglobin variants selected in the mentioned snake species and that would lead the cells to different resistances to unfavorable environmental conditions.

Electrophoretic karyotype analysis of Crinipellis perniciosa, the causal agent of witches' broom disease of Theobroma cacao.

Pulse-field gel electrophoresis (PFGE) was used to determine the genome size and characterize karyotypic differences in isolates of the cacao biotype of Crinipellis perniciosa (C-biotype). The karyotype analysis of four isolates from Brazil revealed that this biotype could be divided into two genotypes: one presenting six chromosomal bands and the other presenting eight. The size of the chromosomes ranged from 2.7 to 5.3 Mb. The different genotypes correlate with telomere-based PCR analysis. The isolates with six chromosomal bands had two that appeared to be doublets, as shown by densitometric analysis, indicating that the haploid chromosome number for this biotype is eight. The size of the haploid genomes was estimated at approximately 30 Mb by both PFGE and Feulgen-image analysis. DNA hybridization revealed that the rDNA sequences are clustered on a single chromosome and these sequences were located on different chromosomes in an isolate dependent manner. This is the first report of genome size and chromosomal polymorphism for the C-biotype of C. perniciosa.

Optical anisotropy of a pig tendon under compression.

The proximal region of the superficial digital flexor tendon of pigs passes under the tibiotarsal joint, where it is subjected to compressional and tensional forces. This region was divided into a surface portion (sp), which is in direct contact with the bone and into a deep portion (dp), which is the layer opposite the articulating surface. The purpose of this work was to analyse the distribution and organisation of the collagen bundles and proteoglycans in the extracellular matrix in sp and dp. Toluidine-blue-stained sections were analysed under a polarising microscope. Strong basophilia and metachromasia were observed in sp, demonstrating accumulation of proteoglycan in a region bearing compression, but the intensity was reduced the further layers were from the bone. Linear dichroism confirmed that the glycosaminoglycan molecules were disposed predominantly parallel to the longest axis of the collagen fibrils. Birefringence analysis showed a higher molecular order and aggregation of the collagen bundles in areas where the tension was more prominent. The crimp pattern was more regular in dp than in sp, probably as a requirement for tendon stretching. The optical anisotropy exhibited by the collagen bundles also confirmed the helical organisation of the collagen bundles in the tendon. Hyaluronidase digestion caused a decrease in the basophilia, but this was not eliminated, supporting the idea that in the matrix, proteoglycans are not completely available to the enzyme action.

A propósito de um novo "sling" pubovaginal absorvível: experiência inicial / A new absobable pubovaginal sling: Preliminary experience

A utilizaçao de material autólogo na criaçao de "Slings" pubovaginais tem sido a conduta de escolha no tratamento dos casos complexos de incotinência urinária aos esforços (IUE). Esta preferência em relaçao aos materiais sintético deve-se em grande parte a duas preocupações básicas, ou seja, aumento de incidência de infecçoes e a erosao da uretra. Por outro lado, a autilizaçao de slings sintéticos possibilita a realizaçao de uma grande cirurgia, com características minimamente invasivos, simplifica o procedimento e reduz o tempo operatório. Os autores descrevem a experiência inicial com a utilizaçao de um "sling" de colágeno altamente purificado, que por ser biocompatível e absorvível associa as características do material autólogo com as vantagens do material sintético. Este sling foi utilizado em 10 pacientes portadores de IUE complexa. Nao houve infecçao, rejeiçao e tampouco reaçoes alérgicas locais. O seguimento variou de 3 a 15 meses com seguimento médio de 6 meses com 80 por cento de cura. Estes resultados sugerem que o sling de colágeno pode ser útil no tratamento de IUE, caso os bons resultados obtidos até o momento se mostrem duradouros