Organochlorine Pesticides in Chlorioallantoic Membranes of Morelet\u27s Crocodile Eggs from Belize

Recent studies examined the utility of the chorioallantoic membrane (CAM) as a nonlethal, noninvasive indicator of environmental contaminant exposure in oviparous wildlife. The CAM is a highly vascularized extraembryonic membrane that functions as a site for respiration, nutrient transport, and waste storage during embryonic development. After hatching, the CAM is usually discarded with the eggshell and can be used for chemical residue analysis. Chorioallantoic membranes have been used successfully to examine contaminant exposure and predict chemical concentrations in multiple species of birds and reptiles. In this study, we examined organochlorine (OC) pesticide concentrations in CAMs from eggs of Morelet\u27s crocodiles (Crocodylus moreletii) from northern Belize. Multiple OCs were detected in crocodile CAMs, including aldrin, dieldrin, endrin, dichlorodiphenyltrichloroethane, dichlorodiphenyldichloroethane, dichlorodiphenyldichloroethylene (DDE), heptachlor, lindane, and methoxychlor. Number and concentrations of OC compounds in CAMs were variable. The most prevalent contaminant detected was DDE, which occurred in 69% of CAMs, with concentrations ranging from 0.3 parts per billion (ppb) to 17.0 ppb. The OC burdens in crocodile CAMs confirm contamination of eggs and suggest exposure in embryos and maternal females. These results further support the use of CAMs as qualitative indicators of OC exposure in oviparous wildlife. The efficacy of this sampling technique in the field will depend on the logistics and cost associated with CAM collection and the specific life history traits of the wildlife species

Low levels of nucleotide diversity in Crocodylus moreletii and evidence of hybridization with C. acutus

Examinations of both population genetic structure and the processes that lead to such structure in crocodilians have been initiated in several species in response to a call by the IUCN Crocodile Specialist Group. A recent study used microsatellite markers to characterize Morelet\u27s crocodile (Crocodylus moreletii) populations in north-central Belize and presented evidence for isolation by distance. To further investigate this hypothesis, we sequenced a portion of the mitochondrial control region for representative animals after including samples from additional locales in Belize, Guatemala and Mexico. While there is limited evidence of subdivision involving other locales, we found that most of the differentiation among populations of C. moreletii can be attributed to animals collected from a single locale in Belize, Banana Bank Lagoon. Furthermore, mitochondrial DNA sequence analysis showed that animals from this and certain other locales display a haplotype characteristic of the American crocodile, C. acutus, rather than C. moreletii. We interpret this as evidence of hybridization between the two species and comment on how these new data have influenced our interpretation of previous findings. We also find very low levels of nucleotide diversity in C. moreletii haplotypes and provide evidence for a low rate of substitution in the crocodilian mitochondrial control region. Finally, the conservation implications of these findings are discussed

Cohesin Proteins Promote Ribosomal RNA Production and Protein Translation in Yeast and Human Cells

Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. The human cohesinopathy diseases are thought to stem not from defects in chromosome segregation but from gene expression. The role of cohesin in gene expression is not well understood. We used budding yeast strains bearing mutations analogous to the human cohesinopathy disease alleles under control of their native promoter to study gene expression. These mutations do not significantly affect chromosome segregation. Transcriptional profiling reveals that many targets of the transcriptional activator Gcn4 are induced in the eco1-W216G mutant background. The upregulation of Gcn4 was observed in many cohesin mutants, and this observation suggested protein translation was reduced. We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and 35S-methionine incorporation, all of which indicate a deficit in protein translation. Metabolic labeling shows that the eco1-W216G and smc1-Q843Δ mutants produce less ribosomal RNA, which is expected to constrain ribosome biogenesis. Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged. Similar defects in rRNA production and protein translation are observed in a human Roberts syndrome cell line. In addition, cohesion is defective specifically at the rDNA locus in the eco1-W216G mutant, as has been previously reported for Roberts syndrome. Collectively, our data suggest that cohesin proteins normally facilitate production of ribosomal RNA and protein translation, and this is one way they can influence gene expression. Reduced translational capacity could contribute to the human cohesinopathies

Rapamycin Response in Tumorigenic and Non-Tumorigenic Hepatic Cell Lines

The mTOR inhibitor rapamycin has anti-tumor activity across a variety of human cancers, including hepatocellular carcinoma. However, resistance to its growth inhibitory effects is common. We hypothesized that hepatic cell lines with varying rapamycin responsiveness would show common characteristics accounting for resistance to the drug.We profiled a total of 13 cell lines for rapamycin-induced growth inhibition. The non-tumorigenic rat liver epithelial cell line WB-F344 was highly sensitive while the tumorigenic WB311 cell line, originally derived from the WB-F344 line, was highly resistant. The other 11 cell lines showed a wide range of sensitivities. Rapamycin induced inhibition of cyclin E-dependent kinase activity in some cell lines, but the ability to do so did not correlate with sensitivity. Inhibition of cyclin E-dependent kinase activity was related to incorporation of p27(Kip1) into cyclin E-containing complexes in some but not all cell lines. Similarly, sensitivity of global protein synthesis to rapamycin did not correlate with its anti-proliferative effect. However, rapamycin potently inhibited phosphorylation of two key substrates, ribosomal protein S6 and 4E-BP1, in all cases, indicating that the locus of rapamycin resistance was downstream from inhibition of mTOR Complex 1. Microarray analysis did not disclose a unifying mechanism for rapamycin resistance, although the glycolytic pathway was downregulated in all four cell lines studied.We conclude that the mechanisms of rapamycin resistance in hepatic cells involve alterations of signaling downstream from mTOR and that the mechanisms are highly heterogeneous, thus predicting that maintaining or promoting sensitivity will be highly challenging

Better Regional Ocean Observing Through Cross-National Cooperation: A Case Study From the Northeast Pacific

The ocean knows no political borders. Ocean processes, like summertime wind-driven upwelling, stretch thousands of kilometers along the Northeast Pacific (NEP) coast. This upwelling drives marine ecosystem productivity and is modulated by weather systems and seasonal to interdecadal ocean-atmosphere variability. Major ocean currents in the NEP transport water properties such as heat, fresh water, nutrients, dissolved oxygen, pCO2, and pH close to the shore. The eastward North Pacific Current bifurcates offshore in the NEP, delivering open-ocean signals south into the California Current and north into the Gulf of Alaska. There is a large and growing number of NEP ocean observing elements operated by government agencies, Native American Tribes, First Nations groups, not-for-profit organizations, and private entities. Observing elements include moored and mobile platforms, shipboard repeat cruises, as well as land-based and estuarine stations. A wide range of multidisciplinary ocean sensors are deployed to track, for example, upwelling, downwelling, ocean productivity, harmful algal blooms, ocean acidification and hypoxia, seismic activity and tsunami wave propagation. Data delivery to shore and observatory controls are done through satellite and cell phone communication, and via seafloor cables. Remote sensing from satellites and land-based coastal radar provide broader spatial coverage, while numerical circulation and biogeochemical modeling complement ocean observing efforts. Models span from the deep ocean into the inland Salish Sea and estuaries. NEP ocean observing systems are used to understand regional processes and, together with numerical models, provide ocean forecasts. By sharing data, experiences and lessons learned, the regional ocean observatory is better than the sum of its parts

Examination of genetic structure in wild populations of Morelet's crocodile (Crocodylus moreletii) in Belize, Central America

Biologists agree that both environmental and genetic factors contribute to species survival (Haig 1998; Partridge and Bmford 1994). Previous work has demonstrated the significance of short-term demographic and environmental threats to populations, as well as the important role of genetic diversity in species viability (Mace et al. 1996). Determining the genetic structure of populations of endangered species is a way to optimize conservation programs (Saltonstall et al. 1998; Avise 1996; Moritz 1994). Little is known about the biology of Morelet's crocodile, a freshwater species found in the lowlands of Central America. As a top predator, this species plays a vital role in the marshland ecosystem. In this project, I have investigated the genetics of several wild populations of Morelet's crocodiles in Belize. Microsatellites, tandemly repeated segments of DNA, were accessed using primers developed for other Crocodylus species. Nine polymorphic microsatellite loci were examined to determine the genetic structure of populations, as well as estimate the mode of gene flow exhibited in this taxa. Analysis of molecular variance, average heterozygosities, Nm and genetic subdivision estimates (FST, RsT and d\i ), suggest frequent movement of individuals between populations. The data are consistent in supporting an isolation by distance and island model of gene flow. Furthermore, I found two contiguous populations to be an important source of variation for outlying smaller populations. This study is part of a broad body of continuing work that will help build the foundation for future conservation programs involving Morelet's crocodiles