Effect of high oxygen on placental function in short-term explant cultures

Ex situ culture of human gestational tissues has been routinely used as a model to investigate tissue function. The objective of this study was to determine the effect of varying oxygen concentrations on human term placental explants over a 24-h time period. Specifically, the effect of incubating placental explants in oxygen concentrations of 8%, 21% or 95% on tissue viability, metabolism and cell death was measured by assessing glucose consumption, lactate production, release of lactate dehydrogenase, parathyroid hormone-related protein (PTHrP), tumour necrosis factor-alpha (TNF-α) and 8-isoprostane, immunoreactivity for cleaved-caspase-9 and immunohistochemistry for the caspase-3-cleaved cytokeratin-18 neoepitope, M30. Exposure to higher oxygen concentrations significantly increased the rates of glucose consumption and lactate production. Apoptosis was significantly increased under conditions of higher oxygen as evidenced by increased M30 in placental explant sections. Similarly, hyperoxia significantly increased the releases of PTHrP, TNF-α and 8-isoprostane. Thus, incubation of placental explants with oxygen concentrations of 95% and, to a lesser extent, 21% oxygen was associated with the modulation of multiple cellular response pathways including those associated with tissue viability and cell death. These data are consistent with the hypothesis that hyperoxia activates pathways and mechanisms involved in cellular metabolism, necrosis and apoptosis, thereby shifting the balance from a steady state towards cell death.     

E-cadherin interactions regulate beta-cell proliferation in islet-like structures.

Islet function is dependent on cells within the islet interacting with each other. E-cadherin (ECAD) mediates Ca(2+)-dependent homophilic cell adhesion between b-cells within islets and has been identified as a tumour suppressor. We generated clones of the MIN6 beta-cell line that stably over- (S) and under-express (alphaS) ECAD. Modified expression of ECAD was confirmed by quantitative RT-PCR, immunoblotting and immunocytochemistry. Preproinsulin mRNA, insulin content and basal rates of insulin secretion were higher in S cells compared to aS and control (V) cells. However, stimulated insulin secretory responses were unaffected by ECAD expression levels. ECAD expression did affect proliferation, with enhanced ECAD expression being associated with reduced proliferation and vice versa. Formation of islet-like structures was associated with a significant reduction in proliferation of V and S cells but not alphaS cells. These data suggest that ECAD expression levels do not modulate insulin secretory function but are consistent with a role for ECAD in the regulation of beta-cell proliferation.     

Differential regulation of imprinting in the murine embryo and placenta by the Dlk1-Dio3 imprinting control region

Genomic imprinting is an epigenetic mechanism controlling parental-origin-specific gene expression. Perturbing the parental origin of the distal portion of mouse chromosome 12 causes alterations in the dosage of imprinted genes resulting in embryonic lethality and developmental abnormalities of both embryo and placenta. A 1 Mb imprinted domain identified on distal chromosome 12 contains three paternally expressed protein-coding genes and multiple non-coding RNA genes, including snoRNAs and microRNAs, expressed from the maternally inherited chromosome. An intergenic, parental-origin-specific differentially methylated region, the IG-DMR, which is unmethylated on the maternally inherited chromosome, is necessary for the repression of the paternally expressed protein-coding genes and for activation of the maternally expressed non-coding RNAs: its absence causes the maternal chromosome to behave like the paternally inherited one. Here, we characterise the developmental consequences of this epigenotype switch and compare these with phenotypes associated with paternal uniparental disomy of mouse chromosome 12. The results show that the embryonic defects described for uniparental disomy embryos can be attributed to this one cluster of imprinted genes on distal chromosome 12 and that these defects alone, and not the mutant placenta, can cause prenatal lethality. In the placenta, the absence of the IG-DMR has no phenotypic consequence. Loss of repression of the protein-coding genes occurs but the non-coding RNAs are not repressed on the maternally inherited chromosome. This indicates that the mechanism of action of the IG-DMR is different in the embryo and the placenta and suggests that the epigenetic control of imprinting differs in these two lineages.     

Proteomic analysis of high-grade dysplastic cervical cells obtained from ThinPrep slides using laser capture microdissection and mass spectrometry

The purpose of this discovery phase study was to identify candidate protein biomarkers for high-grade dysplastic cervical cells using mass spectrometry. Laser Capture Microdissection (LCM) was utilized to isolate high-grade dysplastic and normal cells from ThinPrep slides prepared from cervical cytological specimens. Following cell capture, samples were solubilized and proteins separated by gel electrophoresis in preparation for enzymatic digestion and liquid chromatography mass spectrometry analysis (LC-MS). Processed samples were subsequently analyzed using a linear ion trap coupled with a Fourier transform mass spectrometer (LTQ-FT MS). It was determined that both PreservCyt Solution and ThinPrep Pap Stain (Cytyc Corporation) were compatible with the sample processing and LC-MS analysis. In total, from 9 normal and 9 abnormal cervical cytological specimens, more than 1000 unique proteins were identified with high confidence, based on approximately 12,000 captured cells per specimen. Quantitative protein differences between HSIL (High-Grade Squamous Intraepithelial Lesion) and NILM (Negative for Intraepithelial Lesions or Malignancy) samples were determined by comparing the intensities of the representative (label-free) peptide ions. More than 200 proteins were found to exhibit a 3-fold difference in protein level. Interestingly, significant up-regulation of nuclear and mitochondrial proteins in HSIL specimens was noted. In several cases, the increased protein abundance observed in high-grade cells, as determined by quantitative LC-MS, was validated by immunocytochemical methods using ThinPrep cervical specimens. With the study of additional clinical specimens, the differential abundance of proteins in high-grade dysplastic cells versus morphologically normal cervical cells may lead to validated novel biomarkers for cervical disease.     

Testing assumptions of central place foraging theory: a study of Adélie penguins Pygoscelis adeliae in the Ross Sea

We investigated central place foraging (CPF) in the context of optimal foraging theory in Adélie penguins Pygoscelis adeliae of the southern Ross Sea by using satellite tracking and time‐depth recorders to explore foraging at two spatio‐temporal scales: within the day‐to‐day (sub‐mesoscale: single foraging trip, 10s of km²) and the entire breeding season (mesoscale: trips by multiple individuals across the collective foraging area, 100s of km²). Specifically, we examine whether three basic assumptions of the Orians–Pearson CPF model, shown to occur in other CPF species, are met: 1) within a patch, the rate of prey acquisition declines with time spent in that patch; 2) food is distributed in discrete patches and is not available between those patches; and 3) CPF species have knowledge of the potential (or average, at least) feeding rate within their universe of patches, and use this knowledge to determine their foraging strategy when planning or engaging in a foraging trip. We found that prey consumption rates did not decline with time spent in patches, and penguins foraged to some degree most of the time when at sea. Food availability, as measured by foraging dive rate, appeared to be predictable within the same day at the same location, but predictability broke down after 2 d at distances > 10 km away. We conclude that the assumptions of the Orians–Pearson CPF model are not a good fit to the circumstances of Ross Sea penguins, which clearly are central place foragers.     

Population genetic diversity and structure within and among disjunct populations of <Emphasis Type="Italic">Alnus maritima</Emphasis> (seaside alder) using microsatellites

Small, isolated populations are prone to genetic drift and high levels of inbreeding that can threaten their long-term survival. Alnus maritima persists exclusively in three groups of small, highly disjunct, regional populations in the Delmarva Peninsula, Georgia, and Oklahoma. Trees in the three regions are recognized as separate subspecies. Microsatellite markers were used to measure fine-scale population genetic diversity and structure (1) within and among regions and (2) within and among populations in each region. Compared to a previous study utilizing allozymes, microsatellite data show higher levels of variation, lower levels of inbreeding, but similar levels of genetic differentiation among regions. Significant genetic differentiation was detected among regions and among distinct populations within regions. Genetic differentiation was significantly correlated with geographic distance among regional populations, but not among populations within regions. Populations, therefore, likely represent fragments of formerly extensive networks of populations that have decayed and retracted due to competition with other species better adapted to the shadier habitats of late-succession environments. The unique genetic features of populations within different regions should be considered as part of future conservation efforts.