Chandra Srinivasan

Yeast Lacking Superoxide Dismutase(s) Show Elevated Levels of “Free Iron” as Measured by Whole Cell Electron Paramagnetic Resonance

A current hypothesis explaining the toxicity of superoxide anion in vivo is that it oxidizes exposed [4Fe-4S] clusters in certain vulnerable enzymes causing release of iron and enzyme inactivation. The resulting increased levels of “free iron” catalyze deleterious oxidative reactions in the cell. In this study, we used low temperature Fe(III) electron paramagnetic resonance (EPR) spectroscopy to monitor iron status in whole cells of the unicellular eukaryote, Saccharomyces cerevisiae. The experimental protocol involved treatment of the cells with desferrioxamine, a cell-permeant, Fe(III)-specific chelator, to promote oxidation of all of the “free iron” to the Fe(III) state wherein it is EPR-detectable. Using this method, a small amount of EPR-detectable iron was detected in the wild-type strain, whereas significantly elevated levels were found in strains lacking CuZn-superoxide dismutase (CuZn-SOD) (sod1Δ), Mn-SOD (sod2Δ), or both SODs, throughout their growth but particularly in stationary phase. The accumulation was suppressed by expression of wild-type human CuZn-SOD (in the sod1Δ mutant), by pmr1, a genetic suppressor of the sodΔ mutant phenotype (in thesod1Δsod2Δ double knockout strain), and by anaerobic growth. In wild-type cells, an increase in the EPR-detectable iron pool could be induced by treatment with paraquat, a redox-cycling drug that generates superoxide. Cells that were not pretreated with desferrioxamine had Fe(III) EPR signals that were equally as strong as those from treated cells, indicating that “free iron” accumulated in the ferric form in our strains in vivo. Our results indicate a relationship between superoxide stress and iron handling and support the above hypothesis for superoxide-related oxidative damage.

Delayed accumulation of intestinal coliform bacteria enhances life span and stress resistance in Caenorhabditis elegans fed respiratory deficient E. coli

BackgroundStudies with the nematode model Caenorhabditis elegans have identified conserved biochemical pathways that act to modulate life span. Life span can also be influenced by the composition of the intestinal microbiome, and C. elegans life span can be dramatically influenced by its diet of Escherichia coli. Although C. elegans is typically fed the standard OP50 strain of E. coli, nematodes fed E. coli strains rendered respiratory deficient, either due to a lack coenzyme Q or the absence of ATP synthase, show significant life span extension. Here we explore the mechanisms accounting for the enhanced nematode life span in response to these diets.ResultsThe intestinal load of E. coli was monitored by determination of worm-associated colony forming units (cfu/worm or coliform counts) as a function of age. The presence of GFP-expressing E. coli in the worm intestine was also monitored by fluorescence microscopy. Worms fed the standard OP50 E. coli strain have high cfu and GFP-labeled bacteria in their guts at the L4 larval stage, and show saturated coliform counts by day five of adulthood. In contrast, nematodes fed diets of respiratory deficient E. coli lacking coenzyme Q lived significantly longer and failed to accumulate bacteria within the lumen at early ages. Animals fed bacteria deficient in complex V showed intermediate coliform numbers and were not quite as long-lived. The results indicate that respiratory deficient Q-less E. coli are effectively degraded in the early adult worm, either at the pharynx or within the intestine, and do not accumulate in the intestinal tract until day ten of adulthood.ConclusionsThe findings of this study suggest that the nematodes fed the respiratory deficient E. coli diet live longer because the delay in bacterial colonization of the gut subjects the worms to less stress compared to worms fed the OP50 E. coli diet. This work suggests that bacterial respiration can act as a virulence factor, influencing the ability of bacteria to colonize and subsequently harm the animal host. Respiratory deficient bacteria may pose a useful model for probing probiotic relationships within the gut microbiome in higher organisms.

Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid.

Coq9 is a polypeptide subunit in a mitochondrial multi-subunit complex, termed the CoQ-synthome, required for biosynthesis of coenzyme Q (ubiquinone or Q). Deletion of COQ9 results in dissociation of the CoQ-synthome, but over-expression of Coq8 putative kinase stabilizes the CoQ-synthome in the coq9 null mutant and leads to the accumulation of two nitrogen-containing Q intermediates, imino-demethoxy-Q6 (IDMQ6) and 3-hexaprenyl-4-aminophenol (4-AP) when para-aminobenzoic acid (pABA) is provided as a ring precursor. To investigate whether Coq9 is responsible for deamination steps in Q biosynthesis, we utilized the yeast coq5-5 point mutant. The yeast coq5-5 point mutant is defective in the C-methyltransferase step of Q biosynthesis but retains normal steady-state levels of the Coq5 polypeptide. Here, we show that when high amounts of 13C6-pABA are provided, the coq5-5 mutant accumulates both 13C6-imino-demethyl-demethoxy-Q6 (13C6-IDDMQ6) and 13C6-demethyl-demethoxy-Q6 (13C6-DDMQ6). Deletion of COQ9 in the yeast coq5-5 mutant along with Coq8 over-expression and 13C6- pABA labeling leads to the absence of 13C6-DDMQ6, and the nitrogen-containing intermediates 13C6-4-AP and 13C6-IDDMQ6 persist. We describe a coq9 temperature-sensitive mutant and show that at the non-permissive temperature, steady-state polypeptide levels of Coq9-ts19 increased, while Coq4, Coq5, Coq6, and Coq7 decreased. The coq9-ts19 mutant had decreased Q6 content and increased levels of nitrogen-containing intermediates. These findings identify Coq9 as a multi-functional protein that is required for the function of Coq6 and Coq7 hydroxylases, for removal of the nitrogen substituent from pABA-derived Q intermediates, and is an essential component of the CoQ synthome.

Cognitive impairments following cranial irradiation can be mitigated by treatment with a tropomyosin receptor kinase B agonist.

Brain radiotherapy is frequently used successfully to treat brain tumors. However, radiotherapy is often associated with declines in short-term and long-term memory, learning ability, and verbal fluency. We previously identified a downregulation of the brain-derived neurotrophic factor (BDNF) following cranial irradiation in experimental animals. In the present study, we investigated whether targeting the BDNF high affinity receptor, tropomysin receptor kinase B (TrkB), could mitigate radiation-induced cognitive deficits. After irradiation, chronic treatment with a small molecule TrkB agonist, 7,8-dihydroxyflavone (DHF) in mice led to enhanced activation of TrkB and its downstream targets ERK and AKT, both important factors in neuronal development. DHF treatment significantly restored spatial, contextual, and working memory, and the positive effects persisted for at least 3months after completion of the treatment. Consistent with preservation of cognitive functions, chronic DHF treatment mitigated radiation-induced suppression of hippocampal neurogenesis. Spine density and major components of the excitatory synapses, including glutamate receptors and postsynaptic density protein 95 (PSD-95), were also maintained at normal levels by DHF treatment after irradiation. Taken together, our results show that chronic treatment with DHF after irradiation significantly mitigates radiation-induced cognitive defects. This is achieved most likely by preservation of hippocampal neurogenesis and synaptic plasticity.

Pesticide use among farmers in Mae Tha, Thailand: Perceptions of health risk as a determinant of practice

Human behavior can change through external influence. However, internal influences, such as individual perceptions, are more powerful in triggering long-term action, ultimately allowing for substantial changes in behavioral patterns. Applying the aforementioned idea to farming practices, the following study seeks to understand the possible association between risk perception and agro-chemical use patterns between conventional and organic farms, the former using chemical pesticides and fertilizers.

Superoxide Dismutase Family of Enzymes in Brain Neurogenesis and Radioprotection

The family of superoxide dismutases (SODs) have an almost ubiquitous presence in living organisms. Their importance in maintaining normal cellular functions and fate decisions has been demonstrated in a large number of studies since their discovery in 1968. Studies of pathological conditions resulting from naturally occurring SOD mutations in human populations or from altered SOD levels created in animal models further highlighted the critical roles of SODs in maintaining the redox balance for normal tissue functions. The production of adult-born neurons in the hippocampus is important for the neuronal plasticity of learning and memory, and yet the process is exquisitely sensitive to changes in the redox balance in its microenvironment. Consequently, mutant mice deficient in CuZnSOD or MnSOD showed altered progenitor cell differentiation towards the astroglial, instead of the neuronal, lineage, while EC-SOD deficient mice exhibited a significant reduction in the production of newborn neurons. Similarly, ionizing radiation, even at low doses, led to persistent perturbation of the tissue redox environment and suppression of hippocampal neurogenesis. The defects in hippocampal neurogenesis resulting from EC-SOD deficiency or ionizing irradiation also correlated with impairments in hippocampal-dependent functions of learning and memory. To correct the persistent redox imbalance following irradiation, enhanced EC-SOD levels in mature hippocampal neurons were achieved by transgenic approach. The transgenic mice were able to minimize irradiation-induced suppression of hippocampal neurogenesis and the associated cognitive defects, suggesting that SOD-based antioxidant supplementation may be an efficacious therapeutic approach to CNS defects following ionizing radiation therapy. The new generation Mn-containing porphyrins had been demonstrated to possess high SOD-like activities, and some of them were able to cross the blood brain barrier. Their radioprotective capacity had been demonstrated in a number of tissues in radiation animal models. Application of Mn porphyrins for the preservation of hippocampal neurogenesis and cognitive functions in experimental models will increase their potential relevance towards human radiation therapy of the CNS.