Cytosolic calcium levels and stress induced y-amino butyrate synthesis in asparagus mesophyll cells

Numerous investigations have demonstrated large increases in y-amino butyrate (GABA) levels in response to a variety of stresses such as touch or cold shock (Wallace et ale 1984) Circumstantial evidence indicating a role of Ca2 + in these increases includes elevated Ca2+ levels in response to touch and cold shock (Knight et ale 1991), and the demonstration of a calmodulin binding domain on glutamate decarboxylase (GAD), the enzyme responsible for GABA synthesis (Baum et al 1993) In the present study the possible role of Ca2+ and calmodulin in stimulation of GAD and subsequent GABA accumulation was examined using asparagus mesophyll cells. Images of cells loaded with the Ca2+ indicator Fluo-3 revealed a rapid and transient increase in cytosolic Ca2+ in response to cold shock. GABA levels increased by 106% within 15 min. of cold shock. This increase was inhibited 70% by the calmodulin antagonist W7, and 42% by the Ca2+ channel blocker La3+.. Artificial elevation of intracellular Ca2+ by the Ca2+ionophore A23187 resulted in an 61% increase in GABA levels. Stimulation of GABA synthesis by ABA resulted in an 83% increase in GABA levels which was inhibited 55% by W7. These results support the hypothesis that cold shock stimulates Ca2+ entry into the cytosol of the cells which results in Ca2+/calmodulin mediated activation of GAD and consequent GABA synthesis.

The cardiovascular hemodynamic responses to various levels of orthostatic stress in children

The ability of the cardiovascular system to quickly and efficiently adapt to an orthostatic stress is vital for the human body to function on earth. The way in which the various aspects of the cardiovascular system work together to counteract an orthostatic stress has been previously quantified in the adult population. However, there are still many unknowns surrounding the topic of how the cardiovascular system functions to cope with this same stress in children. The purpose of this study was to describe the cardiovascular hemodynamic adaptations to various levels of orthostatic stress induced using a lower body negative pressure (LBNP) chamber in pre-pubertal boys. A secondary purpose was to determine indices of baroreceptor sensitivity (BRS) at both rest and during low levels of LBNP in this same pediatric sample. Finally, this study aimed to compare the relative responses to LBNP between the children and adults. To complete the study 20 healthy pre-pubertal boys and adult males (9.3 ± 1.1 and 23 ± 1.8 years of age respectively) were recruited and randomly exposed to three levels of LBNP (15, 20 and 25 mmHg). At rest and during the application of the LBNP heart rate (HR), manual and bcat-by-beat systolic (SBP), diastolic (DBP) and mean arterial blood pressure (MAP) were monitored continuously. Aortic diameter was measured at rest and peak aortic blood velocity (PV) was recorded continuously for at least I minute during each baseline and LBNP condition. From the raw data HR, stroke volume (SV), cardiac output (Q), total peripheral resistance (TPR), low frequency baroreceptor sensitivity (LF BRS), high frequency baroreceptor sensitivity (HF BRS) and LFIIIF ratio were calculated. At rest, llR wa'i higher and SBP, SV, Q and LF/HF ratio were lower in the children compared to the adult males (pgJ.05). In response to the increasing LEN!> IIR and TPR increased, and LF BRS. SV and Q decreased in the adult group (pSf).05). while the same levels of LBNP caused an increase in TPR and a decrease in SBP, SV and Q in the children (pSf).05). Although not significant, the LF/HF ratio in the adult group showed an increasing trend in response to increased negative pressure (p=O.088). As for resting BRS, there were no significant differences in LF or HF BRS between the children and the adults despite a tendency for both measures to be 18% lower in the children. Also the LF/HF ratio was almost significantly greater in the adults compared to the children (p=O.057). In addition, a comparison between the relative adult and child responses to LBNP yielded no significant group by level interactions. This result should be taken with caution though, as the low sample size and high measurement variability generated very low statistical power for this analysis. In conclusion, the results of this study suggest that the hemodynamic adaptations to an orthostatic stress were less pronounced in the prepubertal males, most likely due to an underdeveloped autonomic system. These results need to be strengthened by further research before any implications can be derived for health care purposes.

Lifespan, body size and oxygen : aerobic metabolism and oxidative stress in cultured fibroblasts /

The allometric scaling relationship observed between metabolic rate (MR) and species body mass can be partially explained by differences in cellular MR (Porter & Brand, 1995). Here, I studied cultured cell lines derived from ten mammalian species to determine whether cells propagated in an identical environment exhibited MR scaling. Oxidative and anaerobic metabolic parameters did not scale significantly with donor body mass in cultured cells, indicating the absence of an intrinsic MR setpoint. The rate of oxygen delivery has been proposed to limit cellular metabolic rates in larger organisms (West et al., 2002). As such cells were cultured under a variety of physiologically relevant oxygen tensions to investigate the effect of oxygen on cellular metabolic rates. Exposure to higher medium oxygen tensions resulted in increased metabolic rates in all cells. Higher MRs have the potential to produce more reactive oxygen species (ROS) which could cause genomic instability and thus reduced lifespan. Longer-lived species are more resistant to oxidative stress (Kapahi et al, 1999), which may be due to greater antioxidant and/or DNA repair capacities. This hypothesis was addressed by culturing primary dermal fibroblasts from eight mammalian species ranging in maximum lifespan from 5 to 120 years. Only the antioxidant manganese superoxide dismutases (MnSOD) positively scaled with species lifespan (p<0.01). Oxidative damage to DNA is primarily repaired by the base excision repair (BER) pathway. BER enzyme activities showed either no correlation or as in the case of polymerase p correlated, negatively with donor species (p<0.01 ). Typically, mammalian cells are cultured in a 20% O2 (atmospheric) environment, which is several-fold higher than cells experience in vivo. Therefore, the secondary aim of this study was to determine the effect of culturing mammalian cells at a more physiological oxygen tension (3%) on BER, and antioxidant, enzyme activities. Consistently, standard culture conditions induce higher antioxidant and DNA ba.se excision repair activities than are present under a more physiological oxygen concentration. Therefore, standard culture conditions are inappropriate for studies of oxidative stress-induced activities and species differences in fibroblast DNA BER repair capacities may represent differences in ability to respond to oxidative stress. An interesting outcome firom this study was that some inherent cellular properties are maintained in culture (i.e. stress responses) while others are not (i.e. MR).

The role of Stl1p in glycerol accumulation in osmotically stressed icewine yeast Saccharomyces cerevisiae K1 V1116

The high sugar concentration in Icewine juice exerts hyperosmotic stress in the wine yeast causing water loss and cell shrinkage. To counteract the dehydration, yeast synthesize and accumulate glycerol as an internal osmolyte. In a laboratory strain of S. cerevisiae, STLl encodes for Stllp, an H+ /glycerol symporter that is glucose inactivated, but induced upon hyperosmotic stress. STLl, was found to be a highly upregulated gene in Icewine fermenting cells and its expression was 25-fold greater than in yeast cells fermenting diluted Icewine juice, making it one of the most differentially expressed genes between the two fermentation conditions. In addition, Icewine fermenting cells showed a two-fold higher glycerol production in the wine compared to yeast fermenting diluted Icewine juice. We proposed that Stllp is (1) active during Icewine fermentation and is not glucose inactivated and (2) its activity contributes to the limited cell growth observed during Icewine fermentation as a result of the dissipation of the plasma membrane proton gradient. To measure the contribution ofStl1p in active glycerol transport (energy dependent) during Icewine fermentation, we first developed an Stllp-dependent (14C]glycerol uptake assay using a laboratory strain of S. cerevisiae (BY 4742 and LiSTLl) that was dependent on the plasma membrane proton gradient and therefore energy-dependent. Wine yeast K1-Vll16 was also shown to have this energy dependent glycerol uptake induced under salt stress. The expression of STLl and Stllp activity were compared between yeast cells harvested from Icewine and diluted Icewine fermentations. Northern blot analysis revealed that STLl was expressed in cells fermenting Icewine juice but not expressed under the diluted juice conditions. Glycerol uptake by cells fermenting Icewine juice was not significantly different than cells fermenting diluted Icewine juice on day 4 and day 7 of Vidal and Riesling fermentations respectively, despite encountering greater hyperosmotic stress. Furthermore, energy- dependent glycerol uptake was not detected under either fermentation conditions. Because our findings show that active glycerol uptake was not detected in yeast cells harvested from Icewine fermentation, it is likely that Stllp was glucose inactivated despite the hyperosmotic stress induced by the Icewine juice and therefore did not play a role in active glycerol uptake during Icewine fermentation.

Cellular Mechanisms of Resveratrol's Interaction with Mitochondrial Reactive Oxygen Species Metabolism

Resveratrol, a polyphenol found naturally in red wines, has attracted great interest in both the scientific community and the general public for its reported ability to protect against many of the diseases facing Western society today. While the purported health effects of resveratrol are well characterized, details of the cellular mechanisms that give rise to these observations are unclear. Here, the mitochondrial antioxidant enzyme Mn superoxide dismutase (MnSOD) was identified as a proximal target of resveratrol in vitro and in vivo. MnSOD protein and activity levels increase significantly in cultured cells treated with resveratrol, and in the brain tissue of mice given resveratrol in a high fat diet. Preventing the increase in MnSOD levels eliminates two of resveratrol’s more interesting effects in the context of human health: inhibition of proliferative cell growth and cytoprotection. Thus, the induction of MnSOD is a critical step in the molecular mechanism of resveratrol. Mitochondrial morphology is a malleable property that is capable of impeding cell cycle progression and conferring resistance against stress induced cell death. Using confocal microscopy and a novel ‘cell free’ fusion assay it was determined that concurrent with changes in MnSOD protein levels, resveratrol treatment leads to a more fused mitochondrial reticulum. This observation may be important to resveratrol’s ability to slow proliferative cell growth and confer cytoprotection. Resveratrol's biological activities, including the ability to increase MnSOD levels, are strikingly similar to what is observed with estrogen treatment. Resveratrol fails to increase MnSOD levels, slow proliferative cell growth and confer cytoprotection in the presence of an estrogen receptor antagonist. Resveratrol's effects can be replicated with the specific estrogen receptor beta agonist diarylpropionitrile, and are absent in myoblasts lacking estrogen receptor beta. Four compounds that are structurally similar to resveratrol and seven phytoestrogens predicted to bind to estrogen receptor beta were screened for their effects on MnSOD, proliferative growth rates and stress resistance in cultured mammalian cells. Several of these compounds were able to mimic the effects of resveratrol on MnSOD levels, proliferative cell growth and stress resistance in vitro. Thus, I hypothesize that resveratrol interacts with estrogen receptor beta to induce the upregulation of MnSOD, which in turn affects cell cycle progression and stress resistance. These results have important implications for the understanding of RES’s biological activities and potential applications to human health.