Near ultraviolet light inactivation of dihydroxyacid dehydratase in Escherichia coli

The effects of near ultraviolet (NUV) light on a NUV chromophore-containing oxidant-sensitive enzyme, dihydroxyacid dehydratase (DHAD), were measured in seven strains of Escherichia coli. The strains differed in production of the oxidant-defense enzymes, superoxide dismutases (Fe-SOD and Mn-SOD), and catalases HPI and HPII. With the stress of aerobic growth but without NUV exposure, the strains lacking either Fe or Mn SOD or both SODs had 57%, 25%, and 12%, respectively, of the DHAD-specific activity of the parent (K12) strain. Under the same conditions, the catalase strains that were wild type, overproducing, and deficient had comparable DHAD-specific activities. When aerobic cultures were exposed for 30 min to NUV with a fluence of 216 J/m²/s at 310–400 nm, the percentage decreases in DHAD-specific activities were similar (ranging from 75% to 89%) in strains with none, either, or both SODs missing, and in the catalase-overproducing strain. However, the decreases were only 58% and 52% in the strain with catalase missing and in its parent, respectively. The NUV-induced loss of DHAD enzyme activity was not accompanied by any detectable loss of the DHAD protein as measured by polyclonal antibody to DHAD.     

Electrical impedance of white spruce shoots in relation to pressure-volume analysis and free sugar content

Electrical impedance measurements made on white spruce, Picea glauca (Moench) Voss, stems were related to shoot free sugar contents and to osmotic, turgor and water potential. During seasonal dormancy induction, there were commensurate increases in free sugar contents, osmotic potential at full turgor and impedance which resulted in linear relationships among these variables. When measured over the course of laboratory drying, impedance increased curvilinearly with decreasing relative water content. There was a linear increase in impedance with decreasing water potential, with a break point coincident with the turgor loss point, possibly attributed to disruption to current flow through broken plasmodesmatal connections between adjacent cells. This technique offers a non-destructive method to measure tissue free sugar content, and therefore, short- and long-term shifts in parameters historically derived from pressure-volume analysis.     

Endothelin-1 stimulates catalase activity through the PKCδ-mediated phosphorylation of serine 167

Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and in lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H₂O₂) levels. These changes correlated with an increase in serine-phosphorylated catalase. Using the inhibitory peptide δV1.1, this phosphorylation was shown to be protein kinase Cδ (PKCδ) dependent. Mass spectrometry identified serine 167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from Escherichia coli or transiently transfected COS-7 cells demonstrated that S167D catalase had an increased ability to degrade H₂O₂ compared to the wild-type enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist tezosentan. S167 is located on the dimeric interface, suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel filtration to examine the multimeric structure of recombinant wild-type and S167D catalase. We found that recombinant wild-type catalase was present as a mixture of monomers and dimers, whereas S167D catalase was primarily tetrameric. Further, the incubation of wild-type catalase with PKCδ was sufficient to convert wild-type catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity.     

Critical role of mitochondrial dysfunction and impaired mitophagy in diabetic nephropathy

Mitochondrial dynamics play a critical role in deciding the fate of a cell under normal and diseased condition. Recent surge of studies indicate their regulatory role in meeting energy demands in renal cells making them critical entities in the progression of diabetic nephropathy. Diabetes is remarkably associated with abnormal fuel metabolism, a basis for free radical generation, which if left unchecked may devastate the mitochondria structurally and functionally. Impaired mitochondrial function and their aberrant accumulation have been known to be involved in the manifestation of diabetic nephropathy, indicating perturbed balance of mitochondrial dynamics, and mitochondrial turnover. Mitochondrial dynamics emphasize the critical role of mitochondrial fission proteins such as mitochondrial fission 1, dynamin-related protein 1 and mitochondrial fission factor and fusion proteins including mitofusin-1, mitofusin-2 and optic atrophy 1. Clearance of dysfunctional mitochondria is aided by translocation of autophagy machinery to the impaired mitochondria and subsequent activation of mitophagy regulating proteins PTEN-induced putative kinase 1 and Parkin, for which mitochondrial fission is a prior event. In this review, we discuss recent progression in our understanding of the molecular mechanisms targeting reactive oxygen species mediated alterations in mitochondrial energetics, mitophagy related disorders, impaired glucose transport, tubular atrophy, and renal cell death. The molecular cross talks linking autophagy and renoprotection through an intervention of 5′-AMP-activated protein kinase, mammalian target of rapamycin, and SIRT1 factors are also highlighted here, as in-depth exploration of these pathways may help in deriving therapeutic strategies for managing diabetes provoked end-stage renal disease.     

Chronic ethanol ingestion induces oxidative kidney injury through taurine-inhibitable inflammation

Chronic ethanol ingestion mildly damages liver through oxidative stress and lipid oxidation, which is ameliorated by dietary supplementation with the anti-inflammatory β-amino acid taurine. Kidney, like liver, expresses cytochrome P450 2E1 that catabolizes ethanol with free radical formation, and so also may be damaged by ethanol catabolism. Sudden loss of kidney function, and not liver disease itself, foreshadows mortality in patients with alcoholic hepatitis [J. Altamirano, Clin. Gastroenterol. Hepatol. 2012, 10:65]. We found that ethanol ingestion in the Lieber-deCarli rat model increased kidney lipid oxidation, 4-hydroxynonenal protein adduction, and oxidatively truncated phospholipids that attract and activate leukocytes. Chronic ethanol ingestion increased myeloperoxidase-expressing cells in kidney and induced an inflammatory cell infiltrate. Apoptotic terminal deoxynucleotidyl transferase nick-end labeling-positive cells and active caspase-3 increased in kidney after ethanol ingestion, with reduced filtration with increased circulating blood urea nitrogen (BUN) and creatinine. These events were accompanied by release of albumin, myeloperoxidase, and the acute kidney injury biomarkers kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin, and cystatin c into urine. Taurine sequesters HOCl from myeloperoxidase of activated leukocytes, and taurine supplementation reduced renal lipid oxidation, reduced leukocyte infiltration, and reduced the increase in myeloperoxidase-positive cells during ethanol feeding. Taurine supplementation also normalized circulating BUN and creatinine levels and suppressed enhanced myeloperoxidase, albumin, KIM-1, and cystatin c in urine. Thus, chronic ethanol ingestion oxidatively damages kidney lipids and proteins, damages renal function, and induces acute kidney injury through an inflammatory cell infiltrate. The anti-inflammatory nutraceutical taurine effectively interrupts this ethanol-induced inflammatory cycle in kidney.     

Rutin decreases lipopolysaccharide-induced acute lung injury via inhibition of oxidative stress and the MAPK–NF-κB pathway

Acute lung injury (ALI) is a serious disease with unacceptably high mortality and morbidity rates. Up to now, no effective therapeutic strategy for ALI has been established. Rutin, quercetin-3-rhamnosyl glucoside, expresses a wide range of biological activities and pharmacological effects, such as anti-inflammatory, antihypertensive, anticarcinogenic, vasoprotective, and cardioprotective activities. Pretreatment with rutin inhibited not only histopathological changes in lung tissues but also infiltration of polymorphonuclear granulocytes into bronchoalveolar lavage fluid in lipopolysaccharide (LPS)-induced ALI. In addition, LPS-induced inflammatory responses, including increased secretion of proinflammatory cytokines and lipid peroxidation, were inhibited by rutin in a concentration-dependent manner. Furthermore, rutin suppressed phosphorylation of NF-κB and MAPK and degradation of IκB, an NF-κB inhibitor. Decreased activities of antioxidative enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and heme oxygenase-1 caused by LPS were reversed by rutin. At the same time, we found that ALI amelioration by chelation of extracellular metal ions with rutin is more efficacious than with deferoxamine. These results indicate that the protective mechanism of rutin is through inhibition of MAPK–NF-κB activation and upregulation of antioxidative enzymes.     

Cystine Deprivation Induces Oligodendroglial Death: Rescue by Free Radical Scavengers and by a Diffusible Glial Factor

Abstract: In this study we examined the effect on oligodendroglial survival of exogenous cystine deprivation. Oligodendroglia isolated from mixed glial primary cultures derived from brains of 1-day-old rats, and then grown for 3 days, were markedly dependent on extracellular cystine for survival. The EC₅₀ values for cystine for a 24-h exposure ranged from 2 to 65 µ M . After 6 h of cystine deprivation, the cellular glutathione level decreased to 21 ± 13% of the control. Free radical scavengers (α-tocopherol, ascorbate, idebenone, and N-tert -butyl-α-phenylnitrone) were protective against cystine deprivation but had no effect on the glutathione level. An iron chelator, desferrioxamine mesylate, also was protective. These findings suggest that intracellular hydroxyl radicals are important for this toxicity. In contrast to the observations in 3-day-old cultures, the dependence on exogenous cystine for cell viability was not observed consistently in oligodendroglia cultured for 6 days before the onset of cystine deprivation. Several observations suggested that this loss of cystine dependence was due to a diffusible factor. Sensitivity to the toxicity of cystine deprivation in day 6 cultures increased as the volume of medium was increased from 0.3 to 2 ml. Furthermore, preincubation of cystine-depleted medium with astrocyte cultures eliminated the toxicity of the cystine deprivation. HPLC assay of the conditioned cystine-depleted medium showed no significant change in cystine or cysteine concentration. We conclude that oligodendroglia are highly susceptible to cystine deprivation in day 3 cultures and that this susceptibility is due to the accumulation of intracellular free radicals in the setting of glutathione depletion. The resistance of day 6 oligodendroglial cultures is caused at least in part by a diffusible factor.     

Nitrate induced iron deficiency chlorosis in Juncus acutiflorus

Chlorosis caused by iron deficiency is commonly associated with high bicarbonate levels in the soil. However, in rare cases such chlorosis has been observed in soils with high nitrate levels. In a dutch rich-fen, chlorosis has been noted in stands of Juncus acutiflorus at locations where groundwater containing high levels of nitrate reached the surface. Experiments revealed that the chlorosis could be attributed to iron deficiency although iron levels in the shoots were well above the known physiological threshold values for iron deficiency. It is postulated that increased nitrate assimilation leads to an increased apoplastic pH and to a concomitant immobilisation of iron and/or lower iron (III) reduction. Moreover free amino acid levels were markedly higher in the iron deficient plants in the field. It was found, however, that the percentage of nitrogen present as free amino acids was not influenced directly by low iron levels but mainly by the C/N ratios in the shoots. Nowadays, nitrate concentrations in ground water as high 1000 µM are no longer an exception in the Netherlands. We propose that strongly increased nitrate inputs may cause iron stress in natural vegetations, especially in wet habitats.