Acclimation of Two Tomato Species to High Atmospheric CO(2): I. Sugar and Starch Concentrations

Lycopersicon esculentum Mill. cv Vedettos and Lycopersicon chmielewskii Rick, LA 1028, were exposed to two CO₂ concentrations (330 or 900 microliters per liter) for 10 weeks. Tomato plants grown at 900 microliters per liter contained more starch and more sugars than the control. However, we found no significant accumulation of starch and sugars in the young leaves of L. esculentum exposed to high CO₂. Carbon exchange rates were significantly higher in CO₂-enriched plants for the first few weeks of treatment but thereafter decreased as tomato plants acclimated to high atmospheric CO₂. This indicates that the long-term decline of photosynthetic efficiency of leaf 5 cannot be attributed to an accumulation of sugar and/or starch. The average concentration of starch in leaves 5 and 9 was always higher in L. esculentum than in L. chmielewskii (151.7% higher). A higher proportion of photosynthates was directed into starch for L. esculentum than for L. chmielewskii. However, these characteristics did not improve the long-term photosynthetic efficiency of L. chmielewskii grown at high CO₂ when compared with L. esculentum. The chloroplasts of tomato plants exposed to the higher CO₂ concentration exhibited a marked accumulation of starch. The results reported here suggest that starch and/or sugar accumulation under high CO₂ cannot entirely explain the loss of photosynthetic efficiency of high CO₂-grown plants.     

Quiescent human diploid fibroblasts. Common mechanism for inhibition of DNA replication in density-inhibited and serum-deprived cells

The mechanism for cessation of proliferation in density-inhibited quiescent human diploid fibroblasts (HDF) and serum-deprived quiescent HDF was compared in two ways. Density-inhibited HDF were fused to either replicating HDF or SV40-transformed HDF and DNA synthesis was measured in the resulting heterokaryons. DNA synthesis was inhibited in the replicating HDF nuclei in heterokaryons in a way that suggested that entry into S phase was blocked, but ongoing DNA synthesis was not inhibited. In contrast, DNA synthesis was induced in the quiescent nuclei in heterokaryons formed with SV40-transformed HDF. Previous experiments had shown that serum-deprived HDF also behave in this way in heterokaryons. To test this similarity further, we examined the inhibitory activity of cell membranes prepared from both types of quiescent HDF. We found that both types of quiescent HDF contain DNA synthesis-inhibitory activity that is (1) effective on replicating HDF; (2) ineffective on SV40-transformed HDF; (3) sensitive to heat and trypsin. Thus, these results support the hypothesis that both density-inhibited HDF and serum-deprived HDF share a common mechanism for arrest in G1 phase. They also suggest that a membrane-bound protein plays a role in the inhibition of DNA synthesis in quiescent HDF.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Prdx1 inhibits tumorigenesis via regulating PTEN/AKT activity

It is widely accepted that reactive oxygen species (ROS) promote tumorigenesis. However, the exact mechanisms are still unclear. As mice lacking the peroxidase peroxiredoxin1 (Prdx1) produce more cellular ROS and die prematurely of cancer, they offer an ideal model system to study ROS‐induced tumorigenesis. Prdx1 ablation increased the susceptibility to Ras‐induced breast cancer. We, therefore, investigated the role of Prdx1 in regulating oncogenic Ras effector pathways. We found Akt hyperactive in fibroblasts and mammary epithelial cells lacking Prdx1. Investigating the nature of such elevated Akt activation established a novel role for Prdx1 as a safeguard for the lipid phosphatase activity of PTEN, which is essential for its tumour suppressive function. We found binding of the peroxidase Prdx1 to PTEN essential for protecting PTEN from oxidation‐induced inactivation. Along those lines, Prdx1 tumour suppression of Ras‐ or ErbB‐2‐induced transformation was mediated mainly via PTEN.     

Early alterations in mitochondrial reserve capacity; a means to predict subsequent photoreceptor cell death

Although genetic and environmental factors contribute to neurodegenerative disease, the underlying etiology common to many diseases might be based on metabolic demand. Mitochondria are the main producer of ATP, but are also the major source of reactive oxygen species. Under normal conditions, these oxidants are neutralized; however, under environmental insult or genetic susceptibility conditions, oxidative stress may exceed cellular antioxidant capacities, leading to degeneration. We tested the hypothesis that loss in mitochondrial reserve capacity plays a causative role in neuronal degeneration and chose a cone photoreceptor cell line as our model. 661W cells were exposed to agents that mimic oxidant stress or calcium overload. Real-time changes in cellular metabolism were assessed using the multi-well Seahorse Biosciences XF24 analyzer that measures oxygen consumption (OCR) and extracellular acidification rates (ECAR). Cellular stress resulted in an early loss of mitochondrial reserve capacity, without affecting basal respiration; and ECAR was increased, representing a compensatory shift of ATP productions toward glycolysis. The degree of change in energy metabolism was correlated with the amount of subsequent cell death 24-hours post-treatment, the concentration-dependent loss in mitochondrial reserve capacity correlated with the number of live cells. Our data suggested first, that loss in mitochondrial reserve capacity is a major contributor in disease pathogenesis; and second, that the XF24 assay might represent a useful surrogate assay amenable to the screening of agents that protect against loss of mitochondrial reserve capacity. In future experiments, we will explore these concepts for the development of neuroprotective agents.     

Characterization of functionally distinct mitochondrial subpopulations

Mitochondrial stress results in changes in mitochondrial function, morphology and homeostasis (biogenesis, fission/fusion, mitophagy) and may lead to changes in mitochondrial subpopulations. While flow cytometric techniques have been developed to quantify features of individual mitochondria related to volume, Ca²⁺ concentration, mtDNA content, respiratory capacity and oxidative damage, less information is available concerning the identification and characterization of mitochondrial subpopulations, particularly in epithelial cells. Mitochondria from rabbit kidneys were stained with molecular probes for cardiolipin content (nonyl acridine orange, NAO) and membrane potential (tetramethylrhodamine, TMRM) and analyzed using flow cytometry. We validated that side scatter was a better indicator of volume and that as side scatter (SSC) decreased mitochondrial volume increased. Furthermore, those mitochondria with the highest NAO content had greater side scattering and were most highly charged. Mitochondria with average NAO content were of average side scattering and maintained an intermediate charge. Those mitochondria with low NAO content had minimal side scattering and exhibited minimal charge. Upon titration with the uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), it was found that the high NAO content subpopulations were more resistant to uncoupling than lower NAO content populations. Ca²⁺-induced swelling of mitochondria was evaluated using probability binning (PB) analyses of SSC. Interestingly, only 30 % of the mitochondria showed changes in response to Ca²⁺, which was blocked by cyclosporine A. In addition, the small, high NAO content mitochondria swelled differentially in response to Ca²⁺ over time. Our results demonstrate that flow cytometry can be used to identify mitochondrial subpopulations based on high, mid and low NAO content and/or volume/complexity. These subpopulations showed differences in membrane potential, volume, and responses to uncoupling and Ca²⁺-induced swelling.