A critical electrolyte concentration (CEC) method based on binding of toluidine blue molecules under Mg2+ competitive conditions has been proposed for discriminating differences in DNA-protein complexes in vitro (8) and associated with different states of chromatin supra-organization (1, 4). In the method proposed by Vidal and Mello (8), Mg2+ ions are added to the experimental staining solutions and the Mg2+ concentration at which metachromasy is prevented has been considered to be the CEC value.When studying variants of this technique, it was found that ribonucleoprotein complexes could be metachromatically highlighted after the CEC value of DNA-protein complexes was attained. This report describes a variant of the CEC technique in which the nucleolus is significantly discriminated. MATERIALS AND METHODSRoot tip cells of Allium ceps, salivary gland cells of Drosophila aracataca, Balb/3T3 cells, and NIH/3T3 cells transformed by transfection with DNA from carcinogen-treated human cells (5) were used.All the materials were fixed in an ethanol-acetic acid (3 : 1, v/v) mixture for 1 min, with the exception of the onion root tips, which lasted 15 min in the fixative. The glands of D, aracataca were squashed in a drop of 45% acetic acid after fixation, and frozen for removal of the coverslips, after which the preparations were rinsed in 70% ethanol and air dried.In the other materials, fixation was immediately followed by a 5-min bath in 70% ethanol. The onion root tips were subsequently processed for paraffin wax embedding and cut into 12-pm thick sections. The cultured cells were air dried immediately after the 70% ethanol treatment.Staining was performed with a 0.025% toluidine blue (Merck) solution in McIlvaine buffer at pH 4. 1, for 15 min. Immediately after staining, the preparations were treated with 0.02 M to 0.10 M aqueous MgC12 solutions for 15 min, rapidly rinsed in distilled water (5s), air dried, cleared in xylene and mounted in Canada balsam.Some preparations were treated with a 0.1 % RNase III (Sigma) aqueous solution at 37°C for 1 hr prior to staining.Spectral absorption curves were obtained for the nucleolus and the chromatin of the onion cell nuclei in order to detect their absorption peak wavelengths, and consequently, metachromasy loss at the chromatin level. Microspectrophotometry was carried out with Zeiss equipment (EMI 6256 photomultiplier, photometer 01, Planapo 100/1.25 objective, optovar 2, measuring diaphragm diameter of 0.

. et al. Sobrevivência e ocorrência de muda em Triatoma infestans Klug (Hemiptera, Reduviidae) após choque de temperatura. Rev. Saúde públ., S. Paulo, 25: 1991. A sobrevivência e a ocorrência de mudas em espécimes de Triatoma infestans foram estudados num perío-do de 30 dias após choques de temperatura. Foi demonstrado que choques hipertérmico e hipotérmico interferem nesses processos, na dependência da temperatura do choque, tempo de sua duração, fase de desenvolvimento e sexo dos espécimes. Dentre as situações experimentais utilizadas, o choque a 0°C por 12 h pareceu produzir a ação mais deletéria, porém choques a 40°C e 0°C mesmo por uma hora são admitidos como afetando o desenvolvimento hormonal que controla a muda. Casos de aumento de sobrevivência pós-choque são sugeridos como tendo sido favorecidos por ação de proteínas de choque térmico.

In this study, we examined cell survival and cell death in response to heat shock in an insect organ composed of highly polyploid cells no longer capable of cell division. For this, the frequency of nuclear phenotypes in Feulgen-stained Malpighian tubules of the blood-sucking insect, Triatoma infestans, was analyzed at various times after a short heat shock with or without subsequent moderate fasting. Cell death DNA fragmentation was studied immunocytochemically. Normal phenotypes and phenotypes indicative of cell survival (heterochromatin decondensation, nuclear fusion) and death (apoptosis, necrosis) were observed, especially in heat-shocked specimens. While the number of total and normal nuclei decreased following heat shock, the frequency of apoptosis increased during a short period (7 days) after heat shock. During a 30-day period following heat shock, the frequency of necrosis in fasted but not in fully nourished nymphs increased simultaneously with a decrease in the frequency of apoptosis. This finding suggests that the stress promoted by heat shock, but not that associated with heat shock plus fasting, can be dealt with by the apoptosis program. When considering the forms of cell survival, heterochromatin decondensation was more relevant in fully nourished nymphs, whereas nuclear and cell fusions were more important in fasted specimens. The forms of cell survival and cell death reported here may have protected the organ from damage by the stressing agents. In cells with no induction or accumulation of heat-shock proteins, cell death and the forms of cell survival observed here were the probable consequence.

In this study, we examined cell survival and cell death in response to heat shock in an insect organ composed of highly polyploid cells no longer capable of cell division. For this, the frequency of nuclear phenotypes in Feulgen-stained Malpighian tubules of the blood-sucking insect, Triatoma infestans, was analyzed at various times after a short heat shock with or without subsequent moderate fasting. Cell death DNA fragmentation was studied immunocytochemically. Normal phenotypes and phenotypes indicative of cell survival (heterochromatin decondensation, nuclear fusion) and death (apoptosis, necrosis) were observed, especially in heat-shocked specimens. While the number of total and normal nuclei decreased following heat shock, the frequency of apoptosis increased during a short period (7 days) after heat shock. During a 30-day period following heat shock, the frequency of necrosis in fasted but not in fully nourished nymphs increased simultaneously with a decrease in the frequency of apoptosis. This finding suggests that the stress promoted by heat shock, but not that associated with heat shock plus fasting, can be dealt with by the apoptosis program. When considering the forms of cell survival, heterochromatin decondensation was more relevant in fully nourished nymphs, whereas nuclear and cell fusions were more important in fasted specimens. The forms of cell survival and cell death reported here may have protected the organ from damage by the stressing agents. In cells with no induction or accumulation of heat-shock proteins, cell death and the forms of cell survival observed here were the probable consequence.

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