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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.

This study examined whether chicken erythrocyte chromatin, whose great physicochemical stability is promoted by histone H5, has a critical electrolyte concentration (CEC) value comparable to that of other chromatin types with special condensed packing states but devoid of H5. The affinity for toluidine blue molecules under Mg2+ competitive binding action was thus investigated. The CEC value was found to be 0.12M, which is close to that of heterochromatin from mouse liver cells and to sperm chromatin with histone H1 variants. Acetic ethanol fixative was found to affect the CEC, possibly by influencing the chromatin packing state and DNA phosphate availability through dissociation of proteins from the DNA. The phenomenon of programmed cell death was considered to contribute to high CEC values by inducing special states of chromatin condensation.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:07:46Z No. of bitstreams: 1 WOSA1996VP96500007.pdf: 5197622 bytes, checksum: 707831360c936f41335438b46ec7a607 (MD5) Made available in DSpace on 2014-02-26T17:07:46Z (GMT). No. of bitstreams: 1 WOSA1996VP96500007.pdf: 5197622 bytes, checksum: 707831360c936f41335438b46ec7a607 (MD5) Previous issue date: 1996-01-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:23:53Z No. of bitstreams: 1 WOSA1996VP96500007.pdf: 5197622 bytes, checksum: 707831360c936f41335438b46ec7a607 (MD5) Made available in DSpace on 2014-05-20T15:23:53Z (GMT). No. of bitstreams: 1 WOSA1996VP96500007.pdf: 5197622 bytes, checksum: 707831360c936f41335438b46ec7a607 (MD5) Previous issue date: 1996-01-01 The pattern of availability of free DNA phosphates, and the kind of DNA-protein complex arrangement, both induced by nuclear basic proteins, and the richness in arginine residues in these proteins were investigated cytochemically and cytophysically in spermatozoa of the South-American Hylidae species, Hyla fuscovaria and Hyla biobeba. The aim was to demonstrate differences at the level of sperm histones in two species of Hyla until recently considered to be congeneric. The results indicated differences in the spermatozoal nuclear basic proteins and DNA-protein complexes when the two species were compared. The spermatozoa of Hyla biobeba were assumed to be likely to contain a Bloch's ''type 3'' protein type (intermediate sperm basic protein), similarly to Hyla species of North and Central America. on the other hand, the data obtained for the spermatozoa of Hyla fuscovaria indicated that they contain a protamine or protamine-like protein, differing from Hyla biobeba and Hyla species of North and Central America. It is suggested that the differences reported here may be genus-specific, since Hyla fuscovaria has recently been reclassified as Scinax fuscovaria based on parameters other than sperm histone types. These findings are in agreement with the general view of a wide variability in sperm nuclear proteins in the Anura group. UNICAMP,DEPT CELL BIOL,INST BIOL,BR-13083970 CAMPINAS,SP,BRAZIL UNESP,IBILCE,DEPT BIOL,BR-15054000 S JOSE RIO PR,SP,BRAZIL UNESP,IBILCE,DEPT BIOL,BR-15054000 S JOSE RIO PR,SP,BRAZIL

Changes in patterns of metachromatic staining due to RNA were followed by a critical electrolyte concentration method in culture cells during mitosis, and compared with AgNOR staining results and with published data especially referring to nucleolar/nuclear proteins. A deep metachromatic response was found in association with the chromosomes, radiating along the spindle structure and then becoming greatly reduced in nuclei at late telophase/early cytokinesis. This response distribution was not exactly coincident with the reported relocation of nucleolar/nuclear proteins or with a non-nucleolar RNA-containing layer during mitosis. The sites of RNA metachromasy also differed from those of Ag-NOR positive response. The results indicate that RNA most probably of nucleolar origin participates in the complex architecture which is assumed to protect the chromosomes, to store rRNA maturation factors, and to contribute to distribution of nucleolar/nuclear proteins and particular RNA types to daughter cells.

The discrimination of nuclear phenotypes of Feulgen-stained ras-transformed and nontransformed NIH 3T3 cells previously determined by image analysis using the scanning microspectrophotometry system, was investigated with the video system. A reasonable matching of the Feulgen-DNA values obtained with the two methods was demonstrated. Chromatin texture evaluated on the basis of the scatter diagram relating condensed chromatin relative areas vs. an average absorption ratio could also be assessed with the video system. The video image analysis system was effective in distinguishing nuclear phenotypes which differ in chromatin supraorganization in ras-transformed as well as in nontransformed NIH 3T3 cells, in distinguishing phenotypes not visually discernible from each other (transformed vs. nontransformed cells) and in discriminating among the cell lines used.

The autofluorescence of the Malpighian tubules of blood-sucking insects was studied microspectrofluorimetrically. Spectral emission profiles were determined for the naturally fluorescent substances present at the lumen of the proximal region and in the cytoplasm of the distal region of the organ. These were compared with the emission profiles exhibited by standards of fluorescent substances generally found in insect Malpighian tubules. The fluorescence detected at the proximal region was then concluded to be provided by uric acid whereas that of the distal region was attributed to kynurenine. The results did not vary as a function of the developmental stage of the insects. If pteridins are present in the tubules, they appear not to affect the fluorescence patterns determined in this work.

Critical electrolyte concentration (CEC) values were determined for DNA and DNA/Hl and DNA/protamine complexes under varying toluidine blue staining conditions and using Mg2+ as inorganic cation. CEC values were considered those molarities of MgCl2 at which green staining was detected microspectrophotometrically. Variation in the CEC values was found with respect to association of nuclear proteins with DNA and the nucleoprotein complex considered. The DNA filaments exhibited the highest range of CEC values, whereas the lowest CEC values were presented by the DNA/protamine complex, especially when the staining solutions were prepared in McIlvaine buffer. The method thus appears to be promising for further studies on differences among DNA/protein complexes in vitro or even in situ.