Lower beta cell yield from donor pancreases after controlled circulatory death prevented by shortening acirculatory warm ischemia time and by using IGL-1 cold preservation solution.

Organs from donors after controlled circulatory death (DCD III) exhibit a higher risk for graft dysfunction due to an initial period of warm ischemia. This procurement condition can also affect the yield of beta cells in islet isolates from donor pancreases, and hence their use for transplantation. The present study uses data collected and generated by our Beta Cell Bank to compare the number of beta cells in isolates from DCD III (n = 141) with that from donors after brain death (DBD, n = 609), before and after culture, and examines the influence of donor and procurement variables. Beta cell number per DCD III-organ was significantly lower (58 x 106 versus 84 x 106 beta cells per DBD-organ; p < 0.001) but their purity (24% insulin positive cells) and insulin content (17 µg / 106 beta cells in DCD III-organs versus 19 µg / 106 beta cells in DBD-organs) were similar. Beta cell number correlated negatively with duration of acirculatory warm ischemia time above 10 min; for shorter acirculatory warm ischemia time, DCD III-organs did not exhibit a lower beta cell yield (74 x 106 beta cells). Use of Institut Georges Lopez-1 cold preservation solution instead of University of Wisconsin solution or histidine-tryptophan-ketoglutarate also protected against the loss in beta cell yield from DCD III-organs (86 x 106 for IGL-1 versus 54 x 106 and 65 x 106 beta cells respectively, p = 0.042). Multivariate analysis indicates that both limitation of acirculatory warm ischemia time and use of IGL-1 prevent the reduced beta cell yield in islet cell isolates from DCD III-organs.

Physiological aspects of raffinose family oligosaccharides in plants: protection against abiotic stress.

Abiotic stresses resulting from water deficit, high salinity or periods of drought adversely affect plant growth and development and represent major selective forces during plant evolution. The raffinose family oligosaccharides (RFOs) are synthesised from sucrose by the subsequent addition of activated galactinol moieties donated by galactinol. RFOs are characterised as compatible solutes involved in stress tolerance defence mechanisms, although evidence also suggests that they act as antioxidants, are part of carbon partitioning strategies and may serve as signals in response to stress. The key enzyme and regulatory point in RFO biosynthesis is galactinol synthase (GolS), and an increase of GolS in expression and activity is often associated with abiotic stress. It has also been shown that different GolS isoforms are expressed in response to different types of abiotic stress, suggesting that the timing and accumulation of RFOs are controlled for each abiotic stress. However, the accumulation of RFOs in response to stress is not universal and other functional roles have been suggested for RFOs, such as being part of a carbon storage mechanism. Transgenic Arabidopsis plants with increased galactinol and raffinose concentrations had better ROS scavenging capacity, while many sugars have been shown in vitro to have antioxidant activity, suggesting that RFOs may also act as antioxidants. The RFO pathway also interacts with other carbohydrate pathways, such as that of O-methyl inositol (OMI), which shows that the functional relevance of RFOs must not be seen in isolation to overall carbon re-allocation during stress responses.

Plant sugars are crucial players in the oxidative challenge during abiotic stress: extending the traditional concept.

Plants suffering from abiotic stress are commonly facing an enhanced accumulation of reactive oxygen species (ROS) with damaging as well as signalling effects at organellar and cellular levels. The outcome of an environmental challenge highly depends on the delicate balance between ROS production and scavenging by both enzymatic and metabolic antioxidants. However, this traditional classification is in need of renewal and reform, as it is becoming increasingly clear that soluble sugars such as disaccharides, raffinose family oligosaccharides and fructans--next to their associated metabolic enzymes--are strongly related to stress-induced ROS accumulation in plants. Therefore, this review aims at extending the current concept of antioxidants functioning during abiotic stress, with special focus on the emanate role of sugars as true ROS scavengers. Examples are given based on their cellular location, as different organelles seem to exploit distinct mechanisms. Moreover, the vacuole comes into the picture as important player in the ROS signalling network of plants. Elucidating the interplay between the mechanisms controlling ROS signalling during abiotic stress will facilitate the development of strategies to enhance crop tolerance to stressful environmental conditions.

Tightly regulated, beta-estradiol dose-dependent expression system for yeast.

We have refined the regulated expression of UASGAL1, 10-driven genes in yeast by modifying a vector encoding the beta-estradiol inducible activator, GAL4.ER.VP16 (GEV). The expression of GEV was placed under the regulation of the low-level, constitutive MRP7 promoter, and beta-estradiol-regulated expression was monitored by the expression of an integrated UASGAL10-lacZ reporter and by immunoblot analysis of a UASGAL1-regulated gene product. Target gene expression regulated by low levels of GEV has several advantages over the standard galactose-inducible expression systems. (i) Most importantly, the target gene expression is undetectable in the absence of hormone; (ii) target gene expression is beta-estradiol dose-dependent, and variable levels of target gene expression from low to several hundred-fold induction can be achieved; and (iii) induction or depletion studies can be conducted independent of carbon source in gal4 delta strains. In addition, any UASGAL1,10 expression construct can be used without modification of the target gene or many gal4 delta host strains, and GEV vectors are compatible with other inducible yeast expression systems. This method may be useful to researchers investigating the functions of essential genes, dominant negative mutants, mitochondrial genes, and viral, plant, and mammalian genes in yeast assay systems.