Activation of extracellular signal-regulated kinases potentiates hemin toxicity in astrocyte cultures

Hemin is present in intracranial hematomas in high micromolar concentrations and is a potent, lipophilic oxidant. Growing evidence suggests that heme-mediated injury may contribute to the pathogenesis of CNS hemorrhage. Extracellular signal-regulated kinases (ERKs) are activated by oxidants in some cell types, and may alter cellular vulnerability to oxidative stress. In this study, the effect of hemin on ERK activation was investigated in cultured murine cortical astrocytes, and the consequence of this activation on cell viability was quantified. Hemin was rapidly taken up by astrocytes, and generated reactive oxygen species (ROS) within 30 min. Increased immunoreactivity of dually phosphorylated ERK1/2 was observed in hemin-treated cultures at 30-120 min, without change in total ERK. Surprisingly, ERK activation was not attenuated by concomitant treatment with antioxidants (U74500A or 1,10-phenanthroline) at concentrations that blocked ROS generation. Cell death commenced after 2 h of hemin exposure and was reduced by antioxidants and by the caspase inhibitor Z-VAD-FMK. Cytotoxicity was also attenuated by MEK inhibition with PD98059 or U0126 at concentrations that were sufficient to prevent ERK activation. Whereas the effect of Z-VAD-FMK on cell survival was transient, the effect of MEK inhibitors was long-lasting. MEK inhibitors had no effect on cellular hemin uptake or subsequent ROS generation. The present results suggest that hemin activates ERK in astrocytes via a mechanism that is independent of ROS generation. This activation sensitizes astrocytes to hemin-mediated oxidative injury.     

Shotgun cross-linking analysis for studying quaternary and tertiary protein structures

We developed a new approach that employs a novel computer algorithm for the sensitive and high-throughput analysis of tertiary and quaternary interaction sites from chemically cross-linked proteins or multi-protein complexes. First, we directly analyze the digests of the chemically cross-linked proteins using only high-accuracy LC-MS/MS data. We analyze these data using a computer algorithm, we term X!Link, to find cross-links between two peptides. Our algorithm is rapid, taking only a few seconds to analyze approximately 5000 MS/MS spectra. We applied this algorithm to analyze cross-linked sites generated chemically using the amino specific reagent, BS3, in both cytochrome c and the mitochondrial division dynamin mutant, Dnm1G385D, which exists as a stable homodimer. From cytochrome c, a well-established test protein, we identified a total of 31 cross-links, 21 interpeptide and 10 intrapeptide cross-links, in 257 MS/MS spectra from a single LC-MS/MS data set. The high sensitivity of this technique is indicated by the fact that all 19 lysines in cytochrome c were detected as a cross-link product and 33% of all the Lys pairs within 20 A were also observed as a cross-link. Analysis of the cross-linked dimeric form of Dnm1G385D identified a total of 46 cross-links, 38 interpeptide and 8 intrapeptide cross-links, in 98 MS/MS spectra in a single LC-MS/MS data set. These results represent the most abundant cross-links identified in a single protein or protein dimer to date. Statistical analysis suggests a 1% false discovery rate after optimization of filtering parameters. Further analysis of the cross-links identified using our approach indicates that careful manual inspection is important for the correct assignment of cross-linking sites when multiple cross-linkable sites or several similar sequences exist. In summary, we have developed a sensitive MS-based approach to identify peptide-peptide cross-links that does not require isotopic labeling or comparison with non-cross-linked controls, making it faster and simpler than current methodologies.     

Immunohistochemical localization of type D retrovirus serotype 1 in the digestive tract of rhesus monkeys with simian AIDS

Type D retrovirus infection of rhesus macaques (Macaca mulatta) shares many features with AIDS in man including gastrointestinal signs such as chronic diarrhea and wasting. In some humans and macaques afflicted with these signs and symptoms no etiology can be established. In this study immunohistochemistry was employed to localize D/1/California in the digestive tract of ten animals with simian AIDS. This revealed that both epithelial and lymphoid cells of the digestive tract are commonly infected by this immunosuppressive type D retrovirus.     

Increased abundance of native and non-native spiders with habitat fragmentation

Habitat fragmentation and invasive species often contribute to the decline of native taxa. Since the penetration of non‐native species into natural habitat may be facilitated by habitat fragmentation, it is important to examine how these two factors interact. Previous research documented that, in contrast to most other arthropod taxa, spiders increased in density and morphospecies richness with decreasing fragment area and increasing fragment age (time since insularization) in urban habitat fragments in San Diego County, California, USA. We tested whether a specific mechanism, an increase in non‐native species with fragmentation, is responsible for this pattern. We found that both native and non‐native taxa contributed to the pattern. Abundance of native spiders per pitfall trap sample increased significantly with decreasing fragment size (i.e. a negative density–area relationship) and abundance of non‐natives increased significantly with increasing fragment age. The proportion of non‐native individuals also increased significantly with age. One non‐native species, Oecobius navus, comprised the majority of non‐native individuals (82.2%) and a significant proportion of total individuals (25.1%). Richness of spider families per sample (family density) increased with fragment age due to an increase in the occurrence of non‐natives in older fragments, however, native family richness did not vary with age or area. Due to increasing dominance by non‐native and some native families, family evenness declined with decreasing fragment size and increasing fragment age. Native and non‐native abundance covaried positively arguing against strong negative interactions between the two groups. O. navus had a strong positive association with another common non‐native arthropod, the Argentine ant (Linepitheme humile), suggesting a possible direct interaction. In contrast, abundance of native spiders was negatively correlated with Argentine ant abundance. We hypothesize that fragmentation in this semiarid habitat increases productivity in smaller and older fragments enhancing the density of both native and non‐native taxa.     

Conservation of the expression of Dll, en, and wg in the eye-antennal imaginal disc of stalk-eyed flies

SUMMARY We studied the developmental basis of exaggerated eye span in two species of stalk-eyed flies ( Cyrtodiopsis dalmanni and Sphyracephala beccarri ). These flies have eyes laterally displaced at the end of eyestalks, and males have greatly exaggerated eye span, which they use as a sexual display. To investigate eye span development we have compared eye-antennal disc morphology and the expression of three key regulator genes of Drosophila head development, Distal-less ( Dll ) , engrailed ( en ), and wingless ( wg ), in the stalk-eyed flies and Drosophila . We found great similarity in the basic division of the disc into anterior-antennal and posterior-eye portions and in the general patterning of Dll, en , and wg . Unexpectedly, our results showed that although the eye and antenna are adjacent in adult stalk-eyed flies, their primordia are physically separated by the presence of an intervening region between the anterior and posterior portions of the disc. This region is absent from Drosophila eye-antennal discs. We chose two stalk-eyed fly species that differed in the degree of eyestalk exaggeration but surprisingly we found no corresponding difference in the size of the en-wg expression domains that mark the boundaries of the dorsal head capsule primordia. In summary, our expression data establish the regional identity of the eye-antennal disc and provide a framework from which to address the developmental genetics of hypercephaly.