Iron uncouples oxidative phosphorylation in brain mitochondria isolated from vitamin E-deficient rats

Few, if any, studies have examined the effect of vitamin E deficiency on brain mitochondrial oxidative phosphorylation. The latter was studied using brain mitochondria isolated from control and vitamin E-deficient rats (13 months of deficiency) after exposure to iron, an inducer of oxidative stress. Mitochondria were treated with iron (2 to 50 microM) added as ferrous ammonium sulfate. Rates of state 3 and state 4 respiration, respiratory control ratios, and ADP/O ratios were not affected by vitamin E deficiency alone. However, iron uncoupled oxidative phosphorylation in vitamin E-deficient mitochondria, but not in controls. In vitamin E-deficient mitochondria, iron decreased ADP/O ratios and markedly stimulated state 4 respiration; iron had only a modest effect on these parameters in control mitochondria. Thus, vitamin E may have an important role in sustaining oxidative phosphorylation. Low concentrations of iron (2 to 5 microM) oxidized mitochondrial tocopherol that exists in two pools. The release of iron in brain may impair oxidative phosphorylation, which would be exacerbated by vitamin E deficiency. The results are important for understanding the pathogenesis of human brain disorders known to be associated with abnormalities in mitochondrial function as well as iron homeostasis (e.g., Parkinson's disease).     

Differential effects of Cbl isoforms on Egfr signaling in Drosophila

The Cbl family of proteins downregulate epidermal growth factor receptor (Egfr) signaling via receptor internalization and destruction. These proteins contain two functional domains, a RING finger domain with E3 ligase activity, and a proline rich domain mediating the formation of protein complexes. The Drosophila cbl gene encodes two isoforms, D-CblS and D-CblL. While both contain a RING finger domain, the proline rich domain is absent from D-CblS. We demonstrate that expression of either isoform is sufficient to rescue both the lethality of a D-cbl null mutant and the adult phenotypes characteristic of Egfr hyperactivation, suggesting that both isoforms downregulate Egfr signaling. Interestingly, targeted overexpression of D-CblL, but not D-CblS, results in phenotypes characteristic of reduced Egfr signaling and suppresses the effect of constitutive Egfr activation. The level of D-CblL was significantly correlated with the phenotypic severity of reduced Egfr signaling, suggesting that D-CblL controls the efficiency of downregulation of Egfr signaling. Furthermore, reduced dynamin function suppresses the effects of D-CblL overexpression in follicle cells, suggesting that D-CblL promotes internalization of activated receptors. D-CblL is detected in a punctate cytoplasmic pattern, whereas D-CblS is mainly localized at the follicle cell cortex. Therefore, D-CblS and D-CblL may downregulate Egfr through distinct mechanisms.     

A structural basis for Mg2+ homeostasis and the CorA translocation cycle

We describe the CorA Mg(2+) transporter homologue from Thermotoga maritima in complex with 12 divalent cations at 3.7 A resolution. One metal is found near the universally conserved GMN motif, apparently stabilized within the transmembrane region. This portion of the selectivity filter might discriminate between the size and preferred coordination geometry of hydrated substrates. CorA may further achieve specificity by requiring the sequential dehydration of substrates along the length of its approximately 55 A long pore. Ten metal sites identified within the cytoplasmic funnel domain are linked to long extensions of the pore helices and regulate the transport status of CorA. We have characterized this region as an intrinsic divalent cation sensor and provide evidence that it functions as a Mg(2+)-specific homeostatic molecular switch. A proteolytic protection assay, biophysical data, and comparison to a soluble domain structure from Archaeoglobus fulgidus have revealed the potential reaction coordinate for this diverse family of transport proteins.     

Design and performance of a 24-station high throughput microbioreactor

Two prototype 24-unit microbioreactors are presented and reviewed for their relative merits. The first used a standard 24-well plate as the template, while the second consisted of 24-discrete units. Both systems used non-invasive optical sensors to monitor pH and dissolved oxygen. The systems were used to cultivate Escherichia coli. Both designs had their merits and the results obtained are presented. In addition, dissolved oxygen control was demonstrated at the milliliter scale and 24 simultaneously monitored fermentations were successfully carried out. These results demonstrated high quality high throughput bioprocessing and provide important insights into operational parameters at small scale.     

Mapping novel traits by array-assisted bulk segregant analysis in Saccharomyces cerevisiae

We demonstrate a new method, microarray-assisted bulk segregant analysis, for mapping traits in yeast by genotyping pooled segregants. We apply a probabilistic model to the progeny of a single cross and as little as two microarray hybridizations to reliably map an auxotrophic marker, a Mendelian trait, and a major-effect quantitative trait locus.     

Glutamine administration ameliorates sepsis-induced kidney injury by downregulating the high-mobility group box protein-1-mediated pathway in mice

Acute kidney injury (AKI) is a severe complication of sepsis. High-mobility group box (HMGB)-1 was implicated as a late mediator of lethal systemic inflammation in sepsis. Since glutamine (GLN) was shown to have anti-inflammatory and antioxidant properties, we hypothesized that GLN administration may downregulate an HMGB-1-mediated pathway and thus ameliorate sepsis-induced AKI. Mice were randomly assigned to a normal group (NC), a septic saline group (SS), or a septic GLN group (SG). Sepsis was induced by cecal ligation and puncture (CLP). The SS group was injected with saline, and the SG group was given 0.75 g GLN/kg body wt once via a tail vein 1 h after CLP. Mice were killed 2, 6, and 24 h after CLP, and blood and kidneys of the animals were harvested for further analysis. The results showed that sepsis resulted in higher mRNA and/or protein expressions of kidney HMGB-1, toll-like receptor (TLR) 4, myeloid differentiation primary-response protein (MyD) 88, and receptor of advanced glycation end products (RAGE) compared with normal mice. Septic mice with GLN administration exhibited decreased HMGB-1, TLR4, RAGE, and phosphorylated NF-κB p65 protein expressions and reduced nitrotyrosine levels in kidney tissues. The histological findings showed that damage to the kidneys was less severe, and survival improved in the SG group. These results indicated that a single dose of GLN administered after the initiation of sepsis plays a prophylactic role in downregulating the expressions of HMGB-1-related mediators and decreasing oxidative stress in the kidneys, which may consequently have ameliorated AKI induced by sepsis.     

Ultrafiltration improves ELISA and Endopep MS analysis of botulinum neurotoxin type A in drinking water

The objective of this study was to adapt and evaluate two in vitro botulinum neurotoxin (BoNT) detection methods, including the Botulinum Toxin ELISA and the Endopep MS (a mass spectrometric-based endopeptidase method), for use with drinking water samples. The method detection limits (MDL) of the ELISA and Endopep MS were 260 pg/mL and 21 pg/mL of BoNT/A complex toxin, respectively. Since toxin could be present in water samples at highly dilute concentrations, large volume (100-L) samples of municipal tap water from five US municipalities having distinct water compositions were dechlorinated, spiked with 5 μg BoNT/A, and subjected to tangential-flow ultrafiltration (UF) using hollow fiber dialyzers. The recovery efficiency of BoNT/A using UF and quantified by ELISA ranged from 11% to 36% while efficiencies quantified by MS ranged from 26% to 55%. BoNT/A was shown to be stable in dechlorinated municipal tap water stored at 4°C for up to four weeks. In addition, toxin present in UF-concentrated water samples was also shown to be stable at 4°C for up to four weeks, allowing holding of samples prior to analysis. Finally, UF was used to concentrate a level of toxin (7 pg/mL) which is below the MDL for direct analysis by both ELISA and Endopep MS. Following UF, toxin was detectable in these samples using both in vitro analysis methods. These data demonstrate that UF-concentration of toxin from large volume water samples followed by use of existing analytical methods for detection of BoNT/A can be used in support of a monitoring program for contaminants in drinking water.