Training graduate students to be teachers.

Pedagogic education of graduate students, when and where it exists, is restricted to theoretical courses or to the participation of the students as teachers' assistants. This model is essentially reproductive and offers few opportunities for any significant curriculum innovation. To open an opportunity for novelty we have introduced a new approach in "Biochemistry Teaching", a course included in the Biochemistry Graduate Program of the Biochemistry Department (Universidade Estadual de Campinas and Universidade de São Paulo). The content of the course consists of a) choosing the theme, b) selecting and organizing the topics, c) preparing written material, d) establishing the methodological strategies, e) planning the evaluation tools and, finally, f) as teachers, conducting the course as an optional summer course for undergraduate students. During the first semester the graduate students establish general and specific educational objectives, select and organize contents, decide on the instructional strategies and plan evaluation tools. The contents are explored using a wide range of strategies, which include computer-aided instruction, laboratory classes, small group teaching, a few lectures and round table discussions. The graduate students also organize printed class notes to be used by the undergraduate students. Finally, as a group, they teach the summer course. In the three versions already developed, the themes chosen were Biochemistry of Exercise (UNICAMP), Biochemistry of Nutrition (UNICAMP) and Molecular Biology of Plants (USP). In all cases the number of registrations greatly exceeded the number of places and a selection had to be made. The evaluation of the experience by both graduate and undergraduate students was very positive. Graduate students considered this experience to be unique and recommended it to their schoolmates; the undergraduate students benefited from a more flexible curriculum (more options) and gave very high scores to both the courses and the teachers.

The permeability transition pore opening in intact mitochondria and submitochondrial particles.

The mitochondrial permeability transition was investigated under both oxidative and nonoxidative conditions. It was observed that dithiothreitol (DTT) was able to inhibit the permeability transition only when an oxidant, t-butylhydroperoxide, was used. Although cyclosporin A (CsA) showed also a partial protective effect under these conditions, it progressively lost its ability as the oxidant concentration was increased. Indeed, CsA and ADP were very effective under nonoxidative conditions where Ca2+ and Pi were used to induce the permeability transition, and no effect of DTT was observed. These results suggest that the Ca(2+)-dependent permeability transition pore opening is not directly dependent of dithiol oxidation. It was also shown here that CsA, independent of the presence of ADP, was able to restore the mitochondrial membrane electrical potential (delta psi) after the Ca(2+)-induced collapse. Moreover, carboxyatractyloside (CAT) did not prevent the effect of CsA, even when previously added, although it completely abolished the protective effect of ADP, indicating the participation of the ADP/ATP carrier on this process. The data with submitochondrial particles, besides providing further support to the existence of two distinct binding sites for Ca2+ in the mitochondrial inner membrane, with opposite effects on the pore opening probability, demonstrated, for the first time, that very low Ca2+ concentrations induced the permeability transition pore (PTP) opening in submitochondrial particles, an event fully prevented by CsA. The existence of such CsA-sensitive Ca(2+)-induced pore in submitochondrial particles also suggests that matrix cyclophilin is probably not the mediator of this process.

Involvement of the ADP/ATP carrier in permeabilization processes of the inner mitochondrial membrane.

The effect of different agents on inner-mitochondrial-membrane permeabilization and lipoperoxidation induced by Ca2+ and the pyridine-nucleotide oxidant t-butylhydroperoxide or inorganic phosphate was investigated. Comparing the protection conferred by ADP, a substrate of the ADP/ATP carrier, dithiothreitol, a disulfide reductant and butylhydroxytoluene, a radical scavenger, it was found that ADP was always the most effective against mitochondrial damage, when present in the incubation medium from the beginning. Moreover, carboxyatractyloside, a specific inhibitor of the ADP/ATP carrier, abolished completely the protective effect of ADP on both the lipoperoxidation and mitochondrial swelling processes. Experiments where deenergized mitochondria were previously incubated with Ca2+ showed a decrease in the content of active ADP/ATP carrier, indicating a direct involvement of this protein in the formation of a non-specific Ca(2+)-dependent pore. Our results also eliminate the possibility of an attack of oxygen radicals on lipids or proteins of the mitochondrial membrane as the primary event triggering the permeability transition of the inner mitochondrial membrane.