José F. Mesquita

Diabetes and mitochondrial oxidative stress: A study using heart mitochondria from the diabetic Goto-Kakizaki rat

Increasing evidence shows that the overproduction of reactive oxygen species, induced by diabetic hyperglycemia, contributes to the development of several cardiopathologies. The susceptibility of diabetic hearts to oxidative stress, induced in vitro by ADP-Fe2+ in mitochondria, was studied in 12-month-old Goto-Kakizaki rats, a model of non-insulin dependent diabetes mellitus, and normal (non-diabetic) Wistar rats. In terms of lipid peroxidation the oxidative damage was evaluated on heart mitochondria by measuring both the O2 consumption and the concentrations of thiobarbituric acid reactive substances. Diabetic rats display a more intense formation of thiobarbituric acid reactive substances and a higher O2 consumption than non-diabetic rats. The oxidative damage, assessed by electron microscopy, was followed by an extensive effect on the volume of diabetic heart mitochondria, as compared with control heart mitochondria. An increase in the susceptibility of diabetic heart mitochondria to oxidative stress can be explained by reduced levels of endogenous antioxidants, so we proceeded in determinating α-tocopherol, GSH and coenzyme Q content. Although no difference of α-tocopherol levels was found in diabetic rats as compared with control rat mitochondria, a significant reduction in GSH (21.5% reduction in diabetic rats) and coenzyme Q levels of diabetic rats was observed. The data suggest that a significant decrease of coenzyme Q9, a potent antioxidant involved in the elimination of mitochondria-generated reactive oxygen species, may be responsible for an increased susceptibility of diabetic heart mitochondria to oxidative damage.

Role of desensitization of AMPA receptors on the neuronal viability and on the [Ca2+]i changes in cultured rat hippocampal neurons

We investigated the role of desensitization of α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionate (AMPA) receptors on the neurotoxicity and on the [Ca2+]i changes induced by kainate or by AMPA in cultured rat hippocampal neurons. The neuronal viability was evaluated either by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, or by analysis of cell morphology. Short‐term exposure of the neurons to kainate or AMPA (30 min) was not toxic, but the exposure for 24 h to the excitotoxic drugs caused a concentration‐dependent neurotoxic effect which was prevented by LY 303070, a noncompetitive AMPA receptor antagonist. In the presence of cyclothiazide (CTZ), kainate or AMPA was toxic (30 min exposure), or the toxic effect was significantly enhanced (24 h exposure), but in this case LY 303070 did not completely protect the cells against kainate‐induced toxicity. The alterations in the [Ca2+]i caused by kainate or AMPA showed a great cell‐to‐cell variability. LY 303070 completely or partially inhibited the responses stimulated by kainate. CTZ differentially affected the responses evoked by kainate or AMPA. In the majority of hippocampal neurons, CTZ did not potentiate, or only slightly potentiated, the kainate‐stimulated responses but in 11% of neurons there was a great potentiation. In AMPA‐stimulated neurons, the responses were slightly or greatly potentiated in the majority of neurons, but not in all of them. The results show that AMPA and kainate may be toxic, depending on the time of exposure and on the blockade of the desensitization of the AMPA receptors. Overall, our results clearly show that there exist different populations of hippocampal neurons with different sensitivities to kainate, AMPA, CTZ and LY 303070. Moreover, the effects of CTZ on both [Ca2+]i alterations and neurotoxicity are not fully correlated.

Carrageenophytes of occidental Portuguese coast: 1-spectroscopic analysis in eight carrageenophytes from Buarcos bay

Infrared and Raman spectroscopic analysis of the carrageenan (alkaline extraction) in eight species (representing seven genera and four families) of Gigartinales, in different reproductive phases from Buarcos bay (Figueira da Foz, Portugal), were studied. Female gametophytes and non-fertile thalli samples of Chondrus crispus, Mastocarpus stellatus, Chondracanthus teedei var. lusitanicus, Gigartina pistillata and Chondracanthus acicularis present a kappa-carrageenan profile or varying degrees of a kappa-iota hybrid. The presence of kappa-iota hybrid carrageenan in C. teedei var. lusitanicus was confirmed by 13C NMR. The carrageenans extracted from Gymnogongrus crenulatus and Ahnfeltiopsis devoniensis are constituted mainly by iota-carrageenan but seasonal variations in the nature of carrageenans are present. lambda-Family carrageenans were found in tetrasporophytes of C. crispus, M. stellatus, C. teedei var. lusitanicus, C. acicularis and G. pistillata. Calliblepharis jubata presents carrageenans of iota-type in all reproductive stages.

Population studies and carrageenan properties of Chondracanthus teedei var. lusitanicus (Gigartinaceae, Rhodophyta)

Features of an intertidal population of Chondracanthus teedei var. lusitanicus, which occurs in sandy basins on rocky shores of part of the Portuguese coast (Buarcos, Figueira da Foz), were studied over one year. Biomass and plant size showed a small increase in early spring (April), a marked increase in early summer (June/July) and were at a minimum in late summer. There was generally more tetrasporophytes (4–32.5%) than female gametophytes (3–29%), which contrasts with other geographical regions where C. teedei populations have been studied, such as Brazil and France. However, non-fructified thalli predominated throughout the year. Phycocolloid extracts were compared for the various stages using spectroscopic methods (FTIR, FT-Raman, 1H- and 13C-NMR). These showed a hybrid carrageenan belonging to the lambda family in the tetrasporophyte and a hybrid kappa-iota-mu-nu carrageenan in the female gametophyte and non-fructified thalli. The average phycocolloid content was 34.9% dry weight, with a maximum of 43.6% in July. The combination of high available biomass and phycocolloid content makes this species a potentially important source of kappa/iota hybrid carrageenan in Portugal additional to the traditionally harvested carrageenophytes.

New electron microscope studies of Chloridella neglecta (Pascher & Geitler) Pascher (XantophyceaeEustigmatophyceae)

The human articular cartilage is a fundamental tissue in the function of the synovial articulation, has a complex arrangement of cells, as well as collagen fibrils and other elements of the matrix. Grafting has become an accepted method of treating specific clinical problems. Tansplantation of viable articular cartilage cryopreserved would seem to be promising, a means of preserving the donor material in a viable, functional state is highly desirable, but limited progress has been made with fragments of the articular cartilage cryopreserved. Previously, we developed a cryoprotect ion procedure for the c ryopreserva t ion of art icular cart i lage fragments. ( S. Tapia, Cryobiology 30 ( 6 ) : 628, 1993 ), recently we developed a new method for cartilage preservation : SETAMU. ( S. Tapia, Cryobiology 31 ( 6 ) : 5 8 2 , 1994). In the present study, we report the ultrastructural changes of the human articular cartilage cryopreserved, using SETAMU, method developed recently by our unity of cryobiology. The uttrastructural analysis the specimens were realized by transmission electron microscopy (T .E .M. ) , fixed by immersion in glutaraldehyde with ruthenium ( I I I ) hexamine trichtoride (R.H.T.) , and post-fixed in osmium tetroxide with R.H.T., that improved cellular and matrix preservation. Ours results show that SETAMU, play an important role for the cryopreservat ion of the human articular cartilage, preserving the ul t rastructure of the chondrocytes and the matrix. The good cryopreservation of the human cartilage is of particular importance in morphologic research and also in the study of reparation of articular surfaces lbr individuals clinically attain. L IGHT M I C R O S C O P Y A N D L T S E M S T U D I E S OF FUSARIUM R O O T ROT IN BEANS