The conversion of light to chemical energy by the process of photosynthesis is localized to the thylakoid membrane network in plant chloroplasts. Although several pathways have been described that target proteins into and across the thylakoids, little is known about the origin of this membrane system or how the lipid backbone of the thylakoids is transported and fused with the target membrane. Thylakoid biogenesis and maintenance seem to involve the flow of membrane elements via vesicular transport. Here we show by mutational analysis that deletion of a single gene called VIPP1 (vesicle-inducing protein in plastids 1) is deleterious to thylakoid membrane formation. Although VIPP1 is a hydrophilic protein it is found in both the inner envelope and the thylakoid membranes. In VIPP1 deletion mutants vesicle formation is abolished. We propose that VIPP1 is essential for the maintenance of thylakoids by a transport pathway not previously recognized.

ResearchThe lipid content and fatty acid composition of fresh immature and in vitro matured bovine oocytes cultured in media with or without serum, and also those of frozen-thawed immature oocytes were analysed. All oocytes were ranked (A or B) on the basis of their cytoplasmic quality. Fatty acid composition (mol %; w/w) in the total lipid fraction was analysed by gas chromatography. Triglyceride, total cholesterol, phospholipid (phosphocholine-containing phospholipid) and non-esterified fatty acid contents of immature and in vitro matured oocytes were determined using lipid analysis kits. Phosphocholine-containing phospholipid and non-esterified fatty acid contents were determined in frozen-thawed immature bovine oocytes. Palmitic acid was the most abundant fatty acid in immature oocytes (A: 35%, B: 36%), and in in vitro matured oocytes cultured in the medium containing serum (A: 36%, B: 35%) or polyvinyl alcohol (A: 33%, B: 36%). Oleic acid was the second most abundant fatty acid in all A ranked oocytes, whereas stearic acid was the second most abundant fatty acid in all B ranked oocytes. There were significant differences (P < 0.05) in linoleic and arachidonic acid fractions between A and B ranked immature oocytes. In vitro matured oocytes had significantly (P < 0.05) lower proportions of linoleic and arachidonic acids, and significantly (P < 0.01) lower contents of triglyceride and total cholesterol compared with those of immature oocytes. The fatty acid composition of in vitro matured oocytes cultured in medium containing fetal calf serum or polyvinyl alcohol was similar, but significant differences (P < 0.01) in triglyceride and the total cholesterol content were observed. There was a significant decrease (P < 0.05) in the arachidonic acid proportion in frozen-thawed immature oocytes compared with that in fresh immature oocytes. In addition, significant (P < 0.05) decreases in both phospholipid (15.8-10.6 pmol) and non-esterified fatty acid (11.0-4.1 pmol) were found in frozen-thawed immature oocytes. The results indicate that lipids are available for use as an energy source for maturation and that serum lipids are incorporated into the oocyte cytoplasm during in vitro maturation. The changes in the lipid content (mainly phospholipid) and fatty acid composition were also observed in frozen-thawed immature oocytes. The study indicates that the alteration of fatty acid composition in bovine oocytes might improve maturation and cryopreservation.

Oxidized low density lipoprotein (oxLDL) induces apoptosis in vascular cells. To elucidate the mechanisms involved in this apoptosis, we studied the apoptosisinducing activity in lipid fractions of oxLDL and the roles of two common mechanisms, ceramide generation and the activation of caspases, in apoptosis in human umbilical vein endothelial cells treated with oxLDL. We also studied the effects of antioxidants and cholesterol. oxLDL induced endothelial apoptosis in a time-and dosedependent fashion. Apoptosis-inducing activity was recovered in the neutral lipid fraction of oxLDL. Various oxysterols in this fraction induced endothelial apoptosis. Neither the phospholipid fraction nor its component lysophosphatidylcholine induced apoptosis. ox-LDL induced ceramide accumulation temporarily at 15 min in a dose-dependent fashion. Two inhibitors of acid sphinogomyelinase inhibited both the increase in ceramide and the apoptosis induced by oxLDL. Furthermore, a membrane-permeable ceramide (C 2 -ceramide) induced endothelial apoptosis. These findings demonstrated that ceramide generation by acid sphingomyelinase is indispensable for the endothelial apoptosis induced by oxLDL. Inhibitors of both caspase-1 and caspase-3 inhibited the apoptosis, suggesting that oxLDL induced apoptosis by activating these cysteine proteases. The antioxidants butylated hydroxytoluene and superoxide dismutase but not catalase inhibited the apoptosis induced by oxLDL or 25-hydroxycholesterol. This suggests not only that superoxide plays an important role but also that a critical interaction between oxLDL and the cell takes place on the outer surface of the membrane, because superoxide dismutase is not membrane-permeable. Exogenous cholesterol also inhibited the apoptosis. Our study demonstrated that neutral lipids in oxLDL induce endothelial apoptosis by activating membrane sphingomyelinase in a superoxidedependent manner, as well as by activating caspases.

We investigated the digestion of cerebrosides of plant origin prepared from maize, focusing especially on the digestive fates of trans-4, cis-8- and trans-4, trans-8-sphingadienine, which are common in higher plants. In the small intestinal mucosa and cecal contents of rats, the cerebrosidase activity at pH 5.2 toward the glucosyl linkage in maize cerebrosides (glucosylceramides) was similar to that in cerebrosides of mammalian origin. Similarly, the ceramidase activity toward the amide linkage in ceramides prepared from maize cerebrosides at pH 7.0 was the same as that toward ceramides of mammalian origin. In addition, maize cerebrosides were hydrolyzed to ceramide and free sphingoid bases in the digestive tract of rats after oral administration. To further evaluate the uptake by enterocytes of 4,8-sphingadienine, we used differentiated Caco-2 cells, derived from human colonic carcinoma, as a model of intestinal epithelial cells. The accumulation of sphingoid bases in Caco-2 cells incubated with each isomer of sphingadienine was lower than that after incubation with sphingosine (P < 0.05). Verapamil, a P-glycoprotein inhibitor, increased the accumulation of each sphingadienine but not of sphingosine, suggesting that the efflux of sphingadienine of plant origin, but not sphingosine of mammalian origin, was affected by P-glycoprotein. The digestibility of maize cerebrosides appears similar to that of cerebrosides of mammalian origin, but the metabolic fate of sphingoid bases of plant origin within enterocytes differs from that of sphingosine. Isomers of 4,8-sphingadienine degraded from dietary plant cerebrosides appear to be poorly absorbed from the digestive tract.

In this study, the tolerance to salt stress of the photosynthetic machinery was examined in relation to the effects of the genetic enhancement of the unsaturation of fatty acids in membrane lipids in wild-type and desA ϩ cells of Synechococcus sp. PCC 7942. Wild-type cells synthesized saturated and mono-unsaturated fatty acids, whereas desA ϩ cells, which had been transformed with the desA gene for the ⌬12 acyl-lipid desaturase of Synechocystis sp. PCC 6803, also synthesized diunsaturated fatty acids. Incubation of wild-type and desA ϩ cells with 0.5 m NaCl resulted in the rapid loss of the activities of photosystem I, photosystem II, and the Na ϩ /H ϩ antiport system both in light and in darkness. However, desA ϩ cells were more tolerant to salt stress and osmotic stress than the wild-type cells. The extent of the recovery of the various photosynthetic activities from the effects of 0.5 m NaCl was much greater in desA ϩ cells than in wild-type cells. The photosystem II activity of thylakoid membranes from desA ϩ cells was more resistant to 0.5 m NaCl than that of membranes from wild-type cells. These results demonstrated that the genetically engineered increase in unsaturation of fatty acids in membrane lipids significantly enhanced the tolerance of the photosynthetic machinery to salt stress. The enhanced tolerance was due both to the increased resistance of the photosynthetic machinery to the salt-induced damage and to the increased ability of desA ϩ cells to repair the photosynthetic and Na ϩ /H ϩ antiport systems.Salt stress is one of the main environmental factors that limit the growth and productivity of plants and micro-organisms. We have been investigating the mechanisms of the hyperosmotic stress-induced and the salt stress-induced inactivation of the photosynthetic machinery, focussing on the oxygen-evolving machinery of the photosystem II complex, which is the system that is most susceptible to such environmental stress in Synechococcus sp. PCC 7942 (hereafter Synechococcus; Allakhverdiev et al., 2000aAllakhverdiev et al., , 2000b. Hyperosmotic stress due to 1.0 m sorbitol induces the efflux of water through water channels and reduces the volume of cells by more than 50%. This loss of water from the cytosol might be expected to increase the intracellular concentration of salts, and it leads to the rapid but reversible inactivation of the oxygenevolving machinery (Allakhverdiev et al., 2000b).Salt stress due to 0.5 m NaCl has both osmotic and ionic effects (Allakhverdiev et al., 2000a). The osmotic effect due to 0.5 m NaCl is not as strong as the effect of 1.0 m sorbitol and inactivates reversibly the oxygen-evolving machinery. The ionic effect of 0.5 m NaCl is caused by the influx of Na ϩ ions through K ϩ (Na ϩ ) channels and the resultant increase in the intracellular concentration of Na ϩ ions and counterpart anions that are mostly Cl Ϫ ions (Allakhverdiev et al., 2000a). These changes result in the irreversible inactivation of the oxygen-evolving machinery. As a consequence salt stress appears t...