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Biomedical subjects

M L Mello

Publications and source records attributed to M L Mello.

At least 37 records · Page 2Linked to original sources
PubMed1994

Quantitative evaluation of AgNOR in liver cells by high-resolution image cytometry.

This study is aimed at determining the relationship between polyploidy and the amount of nucleolar organiser region (AgNOR) positive dots or aggregates using high resolution image analysis. Liver imprints from mice in which hepatocyte polyploidy is very well documented and related to variation in nuclear area were used as a model for this investigation. A technical variant of the AgNOR method using a Triton X-100 treatment was developed for removal of some proteins from the cytoplasm, which produced a clearer and cleaner background. Feulgen-stained preparations were used to detect the association of various ploidy degrees with their respective values of nuclear area and to subsequently furnish a basis for the association of the nuclear areas in AgNOR-stained cell preparations with different ploidy levels. A high correlation between nuclear and AgNOR-stained areas was revealed and was demonstrated to be much higher than the correlation between nuclear area and the number of AgNOR positive dots or aggregates. The use of two different thresholds for segmentation of the grey levels of the AgNOR-stained material demonstrated the importance of the appropriate decision to obtain the best results to be associated with polyploidy in terms of the real biological event involved, the nuclear area being in this case correlated to Feulgen-DNA values.

Animals↗
PubMed1994

Image analysis of Feulgen-stained transformed NIH 3T3 cells differing in p21 expression and ras-induced metastatic ability.

Nuclear phenotypes of clonal and polyclonal T24 H-ras-transformed NIH 3T3 cells differing in p21 expression and experimental metastatic ability were studied in Feulgen-stained preparations by image analysis. The objective was to determine if these cells varied in their degree of chromatin condensation, as previously reported with cell transformation, or in any other chromatin texture property highlighted by image analysis parameters. The majority of nuclei in all of these ras-transformed cells exhibited increased levels of chromatin condensation, independent of ras levels or metastatic properties. This chromatin texture characteristic was assumed to be related to the transformed phenotype. No significant changes in chromatin supraorganization that could be correlated directly with ras levels or metastatic ability were found, with the exception of an increased frequency of a relatively rare phenotype in highly metastatic cells. This phenotype was characterized by an extreme contrast in packing state between condensed and noncondensed chromatin. It is suggested that ras transformation results in alterations in chromatin structure but that there is not a simple relationship between ras p21 expression levels or ras-induced metastatic ability and the degree of these changes.

3T3 Cells↗
PubMed1992

Interference image analysis of heat-shocked HeLa cells.

HeLa cells were studied with the interference microscope 1 h after heat shock at temperatures of 40 degrees C and 43 degrees C and also under conditions of recovery from the shock. The aim was to investigate changes in patterns of cellular dry mass distribution with the heat shock, based on variation of interference colours in interphase cells. A change in concentration and distribution of a partly high-salt-resistant material in the nuclear and perinuclear regions of the cells was found to be induced by the heat shock at 43 degrees C, a situation which reverted to control under recovery conditions. A similar interference image response was obtained for the heat shock assay at 40 degrees C, but it was detected only during the 4 h recovery period, suggesting that it could have been elicited later. The material induced by the heat shock and visualized by the analysis of interference images is assumed to be a part of the nuclear matrix-intermediate filament cell fraction.

Cell Nucleolus↗
PubMed1992

Image analysis of Feulgen-stained NIH/3T3 cells transformed with DNA of 4-nitroquinoline 1-oxide treated human breast epithelial cells.

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

3T3 Cells↗
PubMed1991

[Survival and occurrence of molting in Triatoma infestans Klug (Hemiptera, Reduviidae) after temperature shock].

Survival and molting occurrence were studied in specimens of Triatoma infestans over 30 days after temperature shocks. Hyperthermal and hypothermal shocks could be found to affect both survival and molting incidence as a function of temperature and period of the development phase and sex of the specimens. Considering the various test conditions, the shock at 0 degree C for 12 h was found to elicit the most deleterious effect, whereas shocks at 40 degrees C and 0 degree C even for 1 h are interpreted as affecting the hormonal balance which controls molting. Cases of a rise in post-shock survival are suggested to have been favored by heat-shock protein action.

Animals↗
PubMed1990

Endopolyploidy in follicle epithelial cells around the nurse chamber during oogenesis in Chrysomya putoria (Diptera, Calliphoridae).

Feulgen-DNA values and nuclear areas were evaluated microspectrophotometrically for epithelial cells of the ovarian follicle during oogenesis in Chrysomya putoria. The aim was to investigate whether polyploidization occurred in the cells surrounding the nurse chamber and/or in those around the oocyte as well as whether different DNA amounts were found regarding the cell types considered. Four DNA endoreduplicative cycles could be demonstrated for the epithelial cells regardless of their localization on the follicle, during oogenesis. A small percentage of epithelial cells reaches a 32C degree. The nuclear area, however, did not increase at the same rate in cells covering the oocyte as in those covering the nurse chamber, in some of the oogenesis stages. The meaning of endopolyploidy for these cells is discussed, considering reports on relevance of secretory activities and their maintenance in polyploid cell systems.

Animals↗
PubMed1989

Critical electrolyte concentration of the heterochromatin and euchromatin of Triatoma infestans.

The binding of toluidine blue molecules under Mg2+ competitive staining conditions was investigated in chromocentres and the euchromatin of single- and multi-chromocentred nuclei of Triatoma infestans Malpighian tubule cells. It was demonstrated that the chromocentre of single-chromocentred nuclei exhibited the largest critical electrolyte concentration (CEC) value (0.4 M), followed by the chromocentres of multi-chromocentred nuclei (0.3 M) and the euchromatin (0.2 M). The differences in CEC values were assumed to be due to differences in availability of free DNA phosphates and in packing states of the DNA-protein complexes of these chromatin types. Differences in chromatin supra-organization were evident for the chromocentral heterochromatin of single vs multi-chromocentred nuclei. This was also valid for the chromocentral heterochromatin in some multi-chromocentred nuclei, when one of the heterochromatic bodies was especially larger than the others.

Animals↗
PubMed1989

Nuclear changes induced in NIH/3T3 cells by transfection and tumorigenesis.

Discriminants such as nuclear phenotypes, nuclear areas and perimeters, multinucleation, nuclear fusion and micronucleation were investigated in NIH/3T3 cells transfected with the whole genomic DNA of T24 and MCF-7 cells and with the ras oncogene of T24 cells, and primary cultures of tumours induced in nude mice by injection of transfected cells. The observations were carried out in cell preparations stained with 0.025% toluidine blue solution at pH 4.0. An increase in chromatin condensation involving euchromatin areas was detected in transfected cells and in cells derived from tumours. The pattern of condensation varied with the type of DNA transfected to the recipient cells. Based upon the enhanced expression of nuclear phenotypes exhibiting clumps of condensed chromatin in tumour-derived cells, it was concluded that chromatin condensation is associated with cell transformation, which is also manifested by multinucleation, nuclear fusion and micronucleation.

Animals↗
PubMed1989

Critical electrolyte concentration of heterochromatin and euchromatin in cells of starved animals.

The critical electrolyte concentration (CEC) of chromatin, considered to be the salt concentration at which the phenomenon of metachromasy is completely abolished, when using toluidine blue as the cationic dye and Mg2+ ions as inorganic cations, was determined for the euchromatin and chromocentral heterochromatin of cells from starved animals. The objective was to determine variations in CEC induced by the starvation stress, which could be related to structural alterations and/or to the composition of these chromatin types. The cells investigated were from Triatoma infestans Malpighian tubules and mouse liver. It was found that the CEC values were affected both by fasting and by refeeding, the effect varying according to chromatin type and the cell system under consideration.

Animals↗
PubMed1987

Polyploidization of the BTC-32 cell line from Triatoma infestans (Hemiptera, Reduviidae).

The cytophotometric evaluation of the Feulgen-DNA content of the BTC-32 cells at passage 160 after 6 days of growth demonstrated that polyploidy is relatively frequent in this cell line. 4C values were assumed to pertain to diploid nuclei at the S or G2 phases but also to polyploid nuclei at the G1 phase. Polyploidy in 4C nuclei is assumed to be attained by endomitosis. However, there are morphological indications that polyploidization in cells with an 8C-128C Feulgen-DNA content could result from cellular and nuclear fusions, possibly mediated by viral particles present in the cell culture. Micronucleation was also frequent; it was assumed to be promoted by viral action or deficiency in some culture medium nutrient. These nuclear characteristics should be considered when using the BTC-32 cell line for monitoring the action of infective agents or their products.

Animals↗
PubMed1986

Lability to acid hydrolysis in some different DNA-protein complexes of spermatozoa.

The Feulgen hydrolysis kinetics was investigated in spermatozoa with different composition in DNA-protein complexes. The species used were: Bos taurus (arginine rich nuclear protein also containing cystine residues), Pichroplus bergi, Triatoma infestans (arginine-rich nuclear protein), Lytechinus variegatus and Apis mellifera (lysine-rich nuclear protein). The spermatozoa were subjected to Feulgen's reaction, after varying the fixative type and the hydrolysis times. Feulgen-DNA values were obtained with an automatic scanning cytophotometric procedure. Differences were demonstrated in the hydrolysis kinetics as a function of differences in composition of the DNA-protein complexes being present in the spermatozoon chromatin. Differences in the profiles of Feulgen hydrolysis curves, having for basis the fixation, were rather clear for bull, grasshopper, and blood-sucking insect spermatozoa than for the sea-urchin and bee spermatozoa. The different hydrolysis kinetics of chromatin of blood-sucking insect spermatozoa compared to that of grasshopper, sea-urchin, and bee sperm cells suggests that, although the first 2 materials contain an arginine-rich "germinative" protein and the latter 2 ones contain a lysine-rich protein, these differ to each other. The DNA depurination was obtained more quickly for T. infestans (20 min) and P. bergi (10 min) spermatozoa when they were fixed in the ethanol-acetic acid (EA) mixture. Morphologically anomalous bull spermatozoa fixed in the EA mixture presented a quicker depurination (30 min) as compared to the normal cells (1 h). The fast lability to acid hydrolysis in the abnormal cells is certainly due to anomalies in their basic nuclear "germinative" protein. In the formalin fixed materials the maximal depurination was obtained earlier in bull spermatozoa (30 min) followed by sperm cells of P. bergi, T. infestans, L. variegatus (all of them one-hour hydrolysis) and finally Apis mellifera (2 h hydrolysis). The presence of secondary peaks at the descending branch of the hydrolysis curves of grasshopper and sea-urchin spermatozoa, indicates for these, more than 1 kind of apurinic-acid protein complexes. The spermatozoa bearing arginine-and/or cystine-rich nuclear protein contain a more easily soluble apurinic acid protein complex. Due to the differences in hydrolysis kinetics of chromatin in spermatozoa containing different nuclear "germinative" proteins, this cellular type does not appear indicated as a haploid control for evaluation of Feulgen-DNA contents of diploid and polyploid somatic cells.

Animals↗
PubMed1986

Investigation of hycanthone binding to DNA in chromatin with different supra-organization, composition, and function.

Hycanthone is a drug used for the clinical treatment of schistosomiasis, although inducing chromosomes breaks and mutagenesis when it enters the DNA double helix. Based on visual observation of characteristics of basophilia and anisotropy after toluidine blue staining, a preferential binding of hycanthone to heterochromatin could be demonstrated for the nuclei of the Malpighian tubules of Triatoma infestans. In other cellular systems like cattle kidney cells in culture, plant cells, and mouse lymphocytes, the drug could be demonstrated to bind heterochromatin and euchromatin, irrespective of the packing state of the latter. When penetrating the various heterochromatin types (with the exception of T. infestans), the drug induced a chromatin loosening that could favor incidence of chromatin breaks. The variation of hycanthone binding to DNA in different cell types is possibly related to differences in composition, stereo-arrangement and stability of the DNA-protein complexes involved.

Animals↗
PubMed1986

Number of chromosomes, Feulgen-DNA content, and nuclear phenotypes in domestic and wild specimens of Panstrongylus megistus.

Specimens of Panstrongylus megistus with different levels of adaptation to a domestic way of life had some of their nuclear and chromosomal characteristics compared, in an attempt to differentiate them by cytological means. The number of chromosomes in male testes was found not to differ. The nuclear frequency and phenotypes of the Malpighian tubules were also found to be the same. On the other hand, Feulgen-DNA values of metaphase chromosomes in insects from a population with a sylvatic distribution were found to be larger than those in specimens from a domestic population. The differences in Feulgen-DNA values, however, may not represent real differences in DNA content, but may possibly be due to differences in DNA-protein complexes.

Analysis of Variance↗
PubMed1983

Cytochemical properties of euchromatin and heterochromatin.

Two classic cytochemical tests, the Feulgen-Schiff reaction and Toluidine Blue basophilia, have been employed for investigating the differential characteristics of heterochromatin and euchromatin. Differences have been detected in the Feulgen hydrolysis kinetics, the Feulgen absorption spectrum, the image analysis of Feulgen-stained material, and the binding of Toluidine Blue under ordinary and Mg2+ competitive staining conditions. The differences are assumed to be a function of the composition and stereo-arrangement of the DNA and DNA-protein complexes present in these chromatin types and are possibly associated with physiological activities whose whole meaning is far from being clear. Differences in optical retardations in Toluidine Blue-stained material were also found. These are interpreted as being due to chromatin packing state and selective removal of histones promoted by the acetic acid-ethanol fixative.

Animals↗