Cystic fibrosis transmembrane regulator missing the first four transmembrane segments increases wild type and DeltaF508 processing

We previously generated an adenoassociated viral gene therapy vector, rAAV-Delta264 cystic fibrosis transmembrane conductance regulator (CFTR), missing the first four transmembrane domains of CFTR. When infected into monkey lungs, Delta264 CFTR increased the levels of endogenous wild type CFTR protein. To understand this process, we transfected Delta264 CFTR plasmid cDNA into COS7 cells, and we noted that protein expression from the truncation mutant is barely detectable when compared with wild type or DeltaF508 CFTR. Delta264 CFTR protein expression increases dramatically when cells are treated with proteasome inhibitors. Cycloheximide experiments show that Delta264 CFTR is degraded faster than DeltaF508 CFTR. VCP and HDAC6, two proteins involved in retrograde translocation from endoplasmic reticulum to cytosol for proteasomal and aggresomal degradation, coimmunoprecipitate with Delta264 CFTR. In cotransfection studies in COS7 cells and in transfection of Delta264 CFTR into cells stably expressing wild type and DeltaF508 CFTR, Delta264 CFTR increases wild type CFTR protein and increases levels of maturation of immature band B to mature band C of DeltaF508 CFTR. Thus the adenoassociated viral vector, rAAV-Delta264 CFTR, is a highly promising cystic fibrosis gene therapy vector because it increases the amount of mature band C protein both from wild type and DeltaF508 CFTR and associates with key elements in quality control mechanism of CFTR.     

Activation of the oxygen-sensing signal cascade prevents mitochondrial injury after mouse liver ischemia-reperfusion

The mitochondrial permeability transition (MPT) plays an important role in hepatocyte death caused by ischemia-reperfusion (IR). This study investigated whether activation of the cellular oxygen-sensing signal cascade by prolyl hydroxylase inhibitors (PHI) protects against the MPT after hepatic IR. Ethyl 3,4-dihyroxybenzoate (EDHB, 100 mg/kg ip), a PHI, increased mouse hepatic hypoxia-inducible factor-1alpha and heme oxygenase-1 (HO-1). EDHB-treated and untreated mice were subjected to 1 h of warm ischemia to approximately 70% of the liver followed by reperfusion. Mitochondrial polarization, cell death, and the MPT were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. EDHB largely blunted alanine aminotransferase (ALT) release and necrosis after reperfusion. In vehicle-treated mice at 2 h after reperfusion, viable cells with depolarized mitochondria were 72%, and dead cells were 2%, indicating that depolarization preceded necrosis. Mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. NIM811, a specific inhibitor of the MPT, blocked mitochondrial depolarization after IR, further confirming that mitochondrial depolarization was due to MPT onset. EDHB decreased mitochondrial depolarization to 16% and prevented the MPT. Tin protoporphyrin (10 micromol/kg sc), an HO-1 inhibitor, partially abrogated protection by EDHB against ALT release, necrosis, and mitochondrial depolarization. In conclusion, IR causes the MPT and mitochondrial dysfunction, leading to hepatocellular death. PHI prevents MPT onset and liver damage through an effect mediated partially by HO-1.     

pdx-1 function is specifically required in embryonic beta cells to generate appropriate numbers of endocrine cell types and maintain glucose homeostasis

The pdx1 gene is essential for pancreatic organogenesis in humans and mice; pdx1 mutations have been identified in human diabetic patients. Specific inactivation of pdx1 in adult beta cells revealed that this gene is required for maintenance of mature beta cell function. In the following study, a Cre-lox strategy was used to remove pdx1 function specifically from embryonic beta cells beginning at late-gestation, prior to islet formation. Animals in which pdx1 is lost in insulin-producing cells during embryogenesis had elevated blood glucose levels at birth and were overtly diabetic by weaning. Neonatal and adult mutant islets showed a dramatic reduction in the number of insulin(+) cells and an increase in both glucagon(+) and somatostatin(+) cells. Lineage tracing revealed that excess glucagon(+) and somatostatin(+) cells did not arise by interconversion of endocrine cell types. Examination of mutant islets revealed a decrease in proliferation of insulin-producing cells just before birth and a concomitant increase in proliferation of glucagon-producing cells. We propose that pdx1 is required for proliferation and function of the beta cells generated at late gestation, and that one function of normal beta cells is to inhibit the proliferation of other islet cell types, resulting in the appropriate numbers of the different endocrine cell types.     

Pdx-1 and Ptf1a concurrently determine fate specification of pancreatic multipotent progenitor cells

The pancreas is derived from a pool of multipotent progenitor cells (MPCs) that co-express Pdx-1 and Ptf1a. To more precisely define how the individual and combined loss of Pdx-1 and Ptf1a affects pancreatic MPC specification and differentiation we derived and studied mice bearing a novel Ptf1a^YFP allele. While the expression of Pdx-1 and Ptf1a in pancreatic MPCs coincides between E9.5 and 12.5 the developmental phenotypes of Pdx-1 null and Pdx-1; Ptf1a double null mice are indistinguishable, and an early pancreatic bud is formed in both cases. This finding indicates that Pdx-1 is required in the foregut endoderm prior to Ptf1a for pancreatic MPC specification. We also found that Ptf1a is neither required for specification of Ngn3-positive endocrine progenitors nor differentiation of mature β-cells. In the absence of Pdx-1 Ngn3-positive cells were not observed after E9.5. Thus, in contrast to the deletion of Ptf1a, the loss of Pdx-1 precludes the sustained Ngn3-based derivation of endocrine progenitors from pancreatic MPCs. Taken together, these studies indicate that Pdx-1 and Ptf1a have distinct but interdependent functions during pancreatic MPC specification.     

Effective population size and tests of neutrality at cytoplasmic genes in Arabidopsis.

Cytoplasmic genomes typically lack recombination, implying that genetic hitch-hiking could be a predominant force structuring nucleotide polymorphism in the chloroplast and mitochondria. We test this hypothesis by analysing nucleotide polymorphism data at 28 loci across the chloroplast and mitochondria of the outcrossing plant Arabidopsis lyrata, and compare patterns with multiple nuclear loci, and the highly selfing Arabidopsis thaliana. The maximum likelihood estimate of the ratio of effective population size at cytoplasmic relative to nuclear genes in A. lyrata does not depart from the neutral expectation of 0.5. Similarly, the ratio of effective size in A. thaliana is close to unity, the neutral expectation for a highly selfing species. The results are thus consistent with neutral organelle polymorphism in these species or with comparable effects of hitch-hiking in both cytoplasmic and nuclear genes, in contrast to the results of recent studies on gynodioecious taxa. The four-gamete test and composite likelihood estimation provide evidence for very low levels of recombination in the organelles of A. lyrata, although permutation tests do not suggest that adjacent polymorphic sites are more closely linked than more distant sites across the two genomes, suggesting that mutation hotspots or very low rates of gene conversion could explain the data.     

Integrating economic input–output life cycle assessment with risk assessment for a screening-level analysis

Goal, Scope, and Background  The paper describes the integration of the economic input–output life cycle assessment (EIO-LCA) model and the environmental fate and transport model (CHEMGL) with a risk assessment tool. Utilizing the EIO-LCA, instead of a traditional LCA, enables a rapid, screening-level analysis of an emerging chemical of concern, decabromodiphenyl ether (DecaBDE). The risk assessment in this study is evaluated based on the mass of chemical released, estimated concentrations, exposure, and chemical toxicity. Methods  The relative risk from ten economic sectors identified within the EIO-LCA model, 55 chemicals utilized in those sectors and DecaBDE along with four potential DecaBDE breakdown products, were evaluated for the life cycle stages and exposure pathways. The relative risk (expressed as toluene equivalents) of the different chemicals, sectors, and life cycle stages were compared to assess those representing the greatest overall relative risks to humans (via inhalation and ingestion) and fish. Results  The greatest overall risk to human health resulted from the manufacturing and production stages. For fish, the manufacturing stage represented virtually all of the risk. Of the 56 chemicals evaluated, DecaBDE represented the majority of the total risk to humans. However, DecaBDE posed the least risk compared to its potential breakdown products. Discussion  The risk to humans from ingestion, which represented the greatest risk, from the production, manufacturing, and consumption stages can be controlled and reduced through various safety precautions in the workplace. Additionally, the increasing concentration of DecaBDE in anaerobic compartments represents a threat to humans and fish via the higher risk DecaBDE breakdown products. Conclusions  Overall, the manufacturing and production life cycle stages pose the greatest risk to humans and fish. The sediment compartment received the highest DecaBDE concentration for the production, manufacturing, and consumption stages. This case study demonstrates that the integrated EIO-LCA with risk assessment is suitable for screening-level analysis of emerging chemicals due to rapid life cycle inventory analysis. Recommendations  The production and manufacturing stages allow for greater industry control and government regulation, compared to the consumption stage, because there are fewer point sources. This integrated life cycle methodology may allow chemical designers to evaluate each stage and assess areas where risks can be minimized.     

Nonselective oviposition by a fastidious insect: the laboratory host range of the melaleuca gall midge Lophodiplosis trifida (Diptera: Cecidomyiidae)

Invasion by the Australian paperbark, Melaleuca quinquenervia, has degraded large areas of south Florida wetlands. Restoration of these wetlands requires the removal of expansive monocultures of this large tree while simultaneously curtailing its spread. Management strategies developed by federal and state agencies include biological control to halt the spread of this species and to prevent reinfestation of cleared areas. This requires biological agents able to reduce flowering, seed production, and growth while increasing mortality of seedlings and saplings. Two of the three introduced agents (Oxyops vitiosa Pascoe and Boreioglycaspis melaleucae Moore) partially meet these needs but outcomes are not spatially or temporally consistent. Thus, additional agents are needed. The bud-gall fly Fergusonina turneri Taylor, with its mutualistic nematode Fergusobia quinquenerviae Davies and Giblin-Davis, is actively being released but has not established. A fourth promising agent, the gall midge Lophodiplosis trifida Gagné, manifested an extremely narrow host range during laboratory testing. Oviposition was indiscriminant in caged environments. Small, incipient, unilocular galls were initiated on Melaleuca viminalis, but larval development ensued only on M. quinquenervia. The unilocular galls on M. viminalis did not grow and produced no adult flies. As a result, M. viminalis test plants suffered only minor cosmetic damage. Observations from both Australia and Florida attest to the ability of this midge to impede M. quinquenervia growth and kill small plants. Thus, L. trifida is safe to release and will likely contribute to management objectives for control of this pernicious wetland invader.     

Mortality risks in patients with constitutional autosomal chromosome deletions in Britain: a cohort study

Constitutional chromosome deletions result in wide ranging morbidity and often fatality. Information about risks and causes of death in these patients is important for counselling, and may illuminate the functions of the part of the chromosome deleted. There have been no cohort studies analysing mortality risks in persons with specific deletions compared with general population rates. We therefore conducted a cohort study following cause-specific mortality in 2,561 patients with autosomal chromosome deletions diagnosed by light microscopy or fluorescence in situ hybridisation at cytogenetic laboratories across Britain, 1965–2002. The commonest deletions were of 22q (544 patients), 15q (460) and 7q (210) and the least common 19q (0) and 20q (2). The prevalence of visible deletions of different chromosome arms was significantly inversely correlated with gene density of the arm (p < 0.001). Mortality was 11-fold raised in the cohort compared with the general population (standardised mortality ratio = 11.4 (95% confidence interval 10.0–12.8)), was significantly raised for each deletion with ≥25 subjects in the study, and had a lower confidence limit >10 for deletions of 1p, 1q, 3p, 4p, 5q and 22q. Overall, 29% of deaths were due to congenital anomalies; significantly raised mortality occurred also from many other causes, varying by chromosome and arm of deletion. The data imply that viability of foetuses with visible chromosome deletions may be inversely related to gene density, and that all visible and fluorescence in situ hybridisation-detectable deletions lead to much raised mortality, but the extent and causes of mortality vary according to the specific deletion. The UK Clinical Cytogenetics Group comprises: The above, plus Paul J Batstone (Inverness Genetics Laboratory), Thomas Spencer (NE London Regional Cytogenetics Laboratory, Great Ormond Street Hospital), Teresa Davies (Bristol Genetics Laboratory), Valerie Davison (Birmingham Genetics Laboratory), Zoe Docherty (SE Thames, Guy’s Hospital Genetics Laboratory), David P. Duckett (Leicestershire Genetics Centre), Margaret Fitchett (Oxford Genetics Laboratory), Alison Fordyce (MRC Human Genetics Unit, Edinburgh); Lorraine Gaunt (Manchester Genetics Laboratory), Elizabeth Grace (Edinburgh Genetics Laboratory), Peter Howard (Liverpool Genetics Laboratory), Gordon W. Lowther (Glasgow Genetics Laboratory), Christine Maliszewska (Dundee Genetics Laboratory), Edna L. Maltby (Sheffield Genetics Laboratory), Kevin P. Ocraft (Nottingham Genetics Laboratory); Selwyn Roberts (Wales Genetics Laboratory), Kim K. Smith (Cambridge Genetics Laboratory), Gordon S. Stephen (Aberdeen Genetics Laboratory), John W. Taylor (SW Thames, St George’s Hospital Genetics Laboratory), Catherine S. Waters (NW Thames, Northwick Park Hospital Genetics Laboratory), Jeffery Williams (Leeds Genetics Laboratory), John Wolstenholme (Newcastle Genetics Laboratory), Sheila Youings (Wessex Regional Genetics Laboratory).     

O(2)-sensing signal cascade: clamping of O(2) respiration, reduced ATP utilization, and inducible fumarate respiration

These studies explore the consequences of activating the prolyl hydroxylase (PHD) O(2)-sensing pathway in spontaneously twitching neonatal cardiomyocytes. Full activation of the PHD pathway was achieved using the broad-spectrum PHD inhibitor (PHI) dimethyloxaloylglycine (DMOG). PHI treatment of cardiomyocytes caused an 85% decrease in O(2) consumption and a 300% increase in lactic acid production under basal conditions. This indicates a approximately 75% decrease in ATP turnover rate, inasmuch as the increased ATP generation by glycolysis is inadequate to compensate for the lower respiration. To determine the extent to which decreased ATP turnover underlies the suppressed O(2) consumption, mitochondria were uncoupled with 2,4-dinitrophenol. We were surprised to find that 2,4-dinitrophenol failed to increase O(2) consumption by PHI-treated cells, indicating that electron transport chain activity, rather than ATP turnover rate, limits respiration in PHI-treated cardiomyocytes. Silencing of hypoxia-inducible factor-1alpha (HIF-1alpha) expression restored the ability of uncoupled PHI-treated myocytes to increase O(2) consumption; however, basal O(2) uptake rates remained low because of the unabated suppression of cellular ATP consumption. Thus it appears that respiration is actively "clamped" through an HIF-dependent mechanism, whereas HIF-independent mechanisms are responsible for downregulation of ATP consumption. In addition, we find that PHD pathway activation enables mitochondria to utilize fumarate as a terminal electron acceptor when cytochrome c oxidase is inactive. The source of fumarate for this unusual respiration is derived from aspartate via the purine nucleotide cycle. In sum, these studies show that the O(2)-sensing pathway is sufficient to actively "clamp" O(2) consumption and independently suppress cellular ATP consumption. The PHD pathway also enables the mitochondria to utilize fumarate for respiration.