Physiological Responses to Acute Silver Exposure in the Freshwater Crayfish (\u3cem\u3eCambarus diogenes diogenes\u3c/em\u3e)—A Model Invertebrate?

Adult crayfish (Cambarus diogenes diogenes) exposed to 8.41 ± 0.17 μg silver/L (19.4% as Ag+) in moderately hard freshwater under flow-through conditions for 96 h exhibited ionoregulatory disturbance, elevated metabolic ammonia (Tamm) production and substantial silver accumulation in the gills, hemolymph, and hepatopancreas. The ionoregulatory disturbance included both a generally reduced unidirectional Na1 influx and an increased unidirectional Na+ efflux, leading to a substantial net loss of Na+ from the silver-exposed crayfish. The Na+ uptake in silver-exposed crayfish differed overall from controls, while the increased Na+ efflux recovered to control values 48 h into the 96 h of exposure. The general inhibition of Na+ uptake could be explained by a reduced sodium/potassium-adenosine triphosphatase (Na/K-ATPase) activity in terminally obtained gill samples from the silver exposed crayfish. The silver-induced effect on Na+ uptake and loss translated to reduced hemolymph Na+ concentrations but not significantly reduced hemolymph Cl- concentrations. Hemolymph Tamm and Tamm efflux both increased in silver-exposed crayfish, indicating an increased metabolic Tamm production. The present study demonstrates that the toxic mechanism of waterborne silver exposure in freshwater crayfish resembles that of freshwater teleost fish. The crayfish might therefore be a useful model system for extending current environmental regulatory strategies, currently based on teleost fish, to invertebrates

The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin.

Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites

Seasonal and interannual variability of North American isoprene emissions as determined by formaldehyde column measurements from space

Formaldehyde (HCHO) columns measured from space by solar UV backscatter allow mapping of reactive hydrocarbon emissions. The principal contributor to these emissions during the growing season is the biogenic hydrocarbon isoprene, which is of great importance for driving regional and global tropospheric chemistry. We present seven years (1995-2001) of HCHO column data for North America from the Global Ozone Monitoring Experiment (GOME), and show that the general seasonal and interannual variability of these data is consistent with knowledge of isoprene emission. There are some significant regional discrepancies with the seasonal patterns predicted from current isoprene emission models, and we suggest that these may reflect flaws in the models. The interannual variability of HCHO columns observed by GOME appears to follow the interannual variability of surface temperature, as expected from current isoprene emission models

De-novo transcriptome assembly for gene identification, analysis, annotation, and molecular marker discovery in Onobrychis viciifolia

Background Sainfoin (Onobrychis viciifolia) is a highly nutritious tannin-containing forage legume. In the diet of ruminants sainfoin can have anti-parasitic effects and reduce methane emissions under in vitro conditions. Many of these benefits have been attributed to condensed tannins or proanthocyanidins in sainfoin. A combination of increased use of industrially produced nitrogen fertilizer, issues with establishment and productivity in the first year and more reliable alternatives, such as red clover led to a decline in the use of sainfoin since the middle of the last century. In recent years there has been a resurgence of interest in sainfoin due to its potential beneficial nutraceutical and environmental attributes. However, genomic resources are scarce, thus hampering progress in genetic analysis and improvement. To address this we have used next generation RNA sequencing technology to obtain the first transcriptome of sainfoin. We used the library to identify gene-based simple sequence repeats (SSRs) and potential single nucleotide polymorphisms (SNPs). Results One genotype from each of five sainfoin accessions was sequenced. Paired-end (PE) sequences were generated from cDNA libraries of RNA extracted from 7 day old seedlings. A combined assembly of 92,772 transcripts was produced de novo using the Trinity programme. About 18,000 transcripts were annotated with at least one GO (gene ontology) term. A total of 63 transcripts were annotated as involved in the tannin biosynthesis pathway. We identified 3786 potential SSRs. SNPs were identified by mapping the reads of the individual assemblies against the combined assembly. After stringent filtering a total of 77,000 putative SNPs were identified. A phylogenetic analysis of single copy number genes showed that sainfoin was most closely related to red clover and Medicago truncatula, while Lotus japonicus, bean and soybean are more distant relatives. Conclusions This work describes the first transcriptome assembly in sainfoin. The 92 K transcripts provide a rich source of SNP and SSR polymorphisms for future use in genetic studies of this crop. Annotation of genes involved in the condensed tannin biosynthesis pathway has provided the basis for further studies of the genetic control of this important trait in sainfoin

Annexin A8 identifies a subpopulation of transiently quiescent c-kit positive luminal progenitor cells of the ductal mammary epithelium

We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8’s association with this breast cancer subgroup we established ANXA8’s cellular distribution in the mammary gland and ANXA8’s effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (−ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ~15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G0/G1 arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8’s association with their specific cells of origin