Anti-oxidative hormetic effects of cellular autophagy induced by nutrient deprivation in a molluscan animal model.

This investigation using a molluscan animal model tested the hypothesis that experimentally induced lysosomal autophagy protects against oxidative cell injury. Induction of augmented lysosomal autophagy has previously been implicated in this protective process. Four treatment groups of blue mussels (Mytilus galloprovincialis) were used: Group 1 (fed - control), Group 2 (fasted), Group 3 (copper + fed) and Group 4 (copper + fasted). Groups 2 and 4 were fasted in order to trigger autophagy; and samples of hepatopancreas (liver analogue or digestive gland) from all 4 groups were taken at 3, 6 and 15 days. Treatment with copper provided a positive reference for oxidative stress: Groups 3 and 4 were treated with copper (10 µg Cu2+/animal/day) for three days only. Oxidative damage and cellular injury in hepatopancreatic digestive cells was found to decrease in Group 2 (fasted) compared to Group 1 (fed - control). Group 3 (fed + copper) showed clear evidence of oxidative stress and cell injury, as well as induction of antioxidant activities. Group 4 (copper + fasted) had a reduced uptake of copper and toxicity of copper was also reduced, compared with Group 3. It was concluded that augmented autophagy had a hormetic cytoprotective anti-oxidant effect.

Oxidative stress, lysosomal damage and dysfunctional autophagy in molluscan hepatopancreas (digestive gland) induced by chemical contaminants.

Autophagy is a highly conserved evolutionary survival or defence process that enables cells and organisms to survive periods of environmental stress by breaking down cellular organelles and macromolecules in autolysosomes to provide a supply of nutrients for cell maintenance. However, autophagy is also a part of normal cellular physiology that facilitates the turnover of cellular constituents under normal conditions: it can be readily augmented by mild environmental stress; but becomes dysfunctional with severe oxidative stress leading to cellular pathology. The molluscan hepatopancreas or digestive gland provides a versatile and environmentally relevant model to investigate lysosomal autophagy and stress-induced dysfunctional autophagy. This latter process has been implicated in many animal and human disease conditions, including degenerative and neurodegenerative diseases, as well as obesity related conditions. Many environmental pollutants have also been found to induce dysfunctional autophagy in molluscan hepatopancreatic digestive cells, and in this study, the marine blue mussel Mytilus galloprovincialis was exposed for 7 days to: 0.1 µM, 1 µM and 10 µM concentrations of fluoranthene and phenanthrene (PAHs); chlorpyrifos and malathion (organophosphorus compounds); atrazine (triazine herbicide); copper (transition metal) and dodecylbenzene sulphonic acid (LAS, surfactant). The marine snail or periwinkle, Littorina littorea, was also exposed to phenanthrene, chlorpyrifos and copper. Indices of oxidative stress, cell injury and dysfunctional autophagy were measured (i.e., lysosomal membrane stability, protein carbonyls, lipofuscin, and lysosomal accumulation of lipid or lipidosis). Evidence of oxidative stress, based on the elevation of lipofuscin and protein carbonyls, was found for all compounds tested; with chlorpyrifos being the most toxic to both species. Dysfunctional autophagy was induced by all of the compounds tested in both species, except for atrazine in mussels. This failure of normal autophagy was consistently associated with oxidative stress. Autophagic dysfunction is an important emerging feature in the aetiology of many disease conditions in animals and humans; and an explanatory conceptual mechanistic model has been developed for dysregulation of autophagy in response to oxidative stress.

A novel BRD4-NUT fusion in an undifferentiated sinonasal tumor highlights alternative splicing as a contributing oncogenic factor in NUT midline carcinoma.

NUT midline carcinoma (NMC) is a fatal cancer that arises in various tissues along the upper midline of the body. The defining molecular feature of NMC is a chromosomal translocation that joins (in the majority of cases) the nuclear testis gene NUT (NUTM1) to the bromodomain protein family member 4 (BRD4) and thereby creating a fusion oncogene that disrupts cellular differentiation and drives the disease. In this study, we report the case of an adolescent NMC patient presenting with severe facial pain, proptosis and visual impairment due to a mass arising from the ethmoid sinus that invaded the right orbit and frontal lobe. Treatment involved radical resection, including exenteration of the affected eye with the view to consolidate treatment with radiation therapy; however, the patient experienced rapid tumor progression and passed away 79 days post resection. Molecular analysis of the tumor tissue identified a novel in-frame BRD4-NUT transcript, with BRD4 exon 15 fused to the last 124 nucleotides of NUT exon 2 (BRD4-NUT ex15:ex2Δnt1-585). The partial deletion of NUT exon 2 was attributed to a mid-exonic genomic breakpoint and the subsequent activation of a cryptic splice site further downstream within the exon. Inhibition of the canonical 3' acceptor splice site of NUT intron 1 in cell lines expressing the most common NMC fusion transcripts (PER-403, BRD4-NUT ex11:ex2; PER-624, BRD4-NUT ex15:ex2) induced alternative splicing from the same cryptic splice site as identified in the patient. Detection of low levels of an in-frame BRD4-NUT ex11:ex2Δnt1-585 transcript in PER-403 confirmed endogenous splicing from this alternative exon 2 splice site. Although further studies are necessary to assess the clinical relevance of the increasing number of variant fusions described in NMC, the findings presented in this case identify alternative splicing as a mechanism that contributes to this pathogenic complexity.

A pre-clinical model of resistance to induction therapy in pediatric acute lymphoblastic leukemia.

Relapse and acquired drug resistance in T-cell acute lymphoblastic leukemia (T-ALL) remains a significant clinical problem. This study was designed to establish a preclinical model of resistance to induction therapy in childhood T-ALL to examine the emergence of drug resistance and identify novel therapies. Patient-derived T-ALL xenografts in immune-deficient (non-obese diabetic/severe combined immunodeficient) mice were exposed to a four-drug combination of vincristine, dexamethasone (DEX), L-asparaginase and daunorubicin (VXLD). 'Relapse' xenografts were characterized by responses to drugs, changes in gene expression profiles and Connectivity Map (CMap) prediction of strategies to reverse drug resistance. Two of four xenografts developed ex vivo and in vivo drug resistance. Both resistant lines showed altered lipid and cholesterol metabolism, yet they had a distinct drug resistance pattern. CMap analyses reinforced these features, identifying the cholesterol pathway inhibitor simvastatin (SVT) as a potential therapy to overcome resistance. Combined ex vivo with DEX, SVT was significantly synergistic, yet when administered in vivo with VXLD it did not delay leukemia progression. Synergy of SVT with established chemotherapy may depend on higher drug doses than are tolerable in this model. Taken together, we have developed a clinically relevant in vivo model of T-ALL suitable to examine the emergence of drug resistance and to identify novel therapies.

Comparative drug screening in NUT midline carcinoma.

BACKGROUND: The NUT midline carcinoma (NMC) is a rare but fatal cancer for which systematic testing of therapy options has never been performed. METHODS: On the basis of disease biology, we compared the efficacy of the CDK9 inhibitor flavopiridol (FP) with a panel of anticancer agents in NMC cell lines and mouse xenografts. RESULTS: In vitro anthracyclines, topoisomerase inhibitors, and microtubule poisons were among the most cytotoxic drug classes for NMC cells, while efficacy of the bromodomain inhibitor JQ1 varied considerably between lines carrying different BRD4 (bromodomain-containing protein 4)-NUT (nuclear protein in testis) translocations. Efficacy of FP was comparable to vincristine and doxorubicin, drugs that have been previously used in NMC patients. All three compounds showed significantly better activity than etoposide and vorinostat, agents that have also been used in NMC patients. Statins and antimetabolites demonstrated intermediate single-agent efficacy. In vivo, vincristine significantly inhibited tumour growth in two different NMC xenografts. Flavopiridol in vivo was significantly effective in one of the two NMC xenograft lines, demonstrating the biological heterogeneity of this disease. CONCLUSIONS: These results demonstrate that FP may be of benefit to a subset of patients with NMC, and warrant a continued emphasis on microtubule inhibitors, anthracyclines, and topoisomerase inhibitors as effective drug classes in this disease.

Novel BRD4-NUT fusion isoforms increase the pathogenic complexity in NUT midline carcinoma.

Nuclear protein in testis (NUT)-midline carcinoma (NMC) is a rare, aggressive disease typically presenting with a single t(15;19) translocation that results in the generation of a bromodomain-containing protein 4 (BRD4)-NUT fusion. PER-624 is a cell line generated from an NMC patient with an unusually complex karyotype that gave no initial indication of the involvement of the NUT locus. Analysis of PER-624 next-generation transcriptome sequencing (RNA-Seq) using the algorithm FusionFinder identified a novel transcript in which Exon 15 of BRD4 was fused to Exon 2 of NUT, therefore differing from all published NMC fusion transcripts. The three additional exons contained in the PER-624 fusion encode a series of polyproline repeats, with one predicted to form a helix. In the NMC cell line PER-403, we identified the 'standard' NMC fusion and two novel isoforms. Knockdown by small interfering RNA in either cell line resulted in decreased proliferation, increased cell size and expression of cytokeratins consistent with epithelial differentiation. These data demonstrate that the novel BRD4-NUT fusion in PER-624 encodes a functional protein that is central to the oncogenic mechanism in these cells. Genomic PCR indicated that in both PER-624 and PER-403, the translocation fuses an intron of BRD4 to a region upstream of the NUT coding sequence. Thus, the generation of BRD4-NUT fusion transcripts through post-translocation RNA-splicing appears to be a common feature of these carcinomas that has not previously been appreciated, with the mechanism facilitating the expression of alternative isoforms of the fusion. Finally, ectopic expression of wild-type NUT, a protein normally restricted to the testis, could be demonstrated in PER-403, indicating additional pathways for aberrant cell signaling in NMC. This study contributes to our understanding of the genetic diversity of NMC, an important step towards finding therapeutic targets for a disease that is refractory to current treatments.

Impact of elevated levels of CO2 on animal mediated ecosystem function: the modification of sediment nutrient fluxes by burrowing urchins.

A mesocosm experiment was conducted to quantify the relationships between the presence and body size of two burrowing heart urchins (Brissopsis lyrifera and Echinocardium cordatum) and rates of sediment nutrient flux. Furthermore, the impact of seawater acidification on these relationships was determined during this 40-day exposure experiment. Using carbon dioxide (CO2) gas, seawater was acidified to pHNBS 7.6, 7.2 or 6.8. Control treatments were maintained in natural seawater (pH≈8.0). Under normocapnic conditions, burrowing urchins were seen to reduce the sediment uptake of nitrite or nitrate whilst enhancing the release of silicate and phosphate. In acidified (hypercapnic) treatments, the biological control of biogeochemical cycles by urchins was significantly affected, probably through the combined impacts of high CO2 on nitrifying bacteria, benthic algae and urchin behaviour. This study highlights the importance of considering biological interactions when predicting the consequences of seawater acidification on ecosystem function.

Integration of biochemical, histochemical and toxicogenomic indices for the assessment of health status of mussels from the Tamar Estuary, U.K.

The aim of this study was to examine whether a combination of biochemical, histopathological and toxicogenomic data could be used as a valuable tool for the assessment of biological risk associated with pollutants within the Tamar River and Estuary, S.W. England, U.K. Accordingly, biochemical and histopathological biomarkers (protein carbonyls, lipofuscin, neutral lipids, lysosomal stability [N-acetyl-ß-hexosaminidase and neutral red], lysosomal volume, ferric reducing antioxidant power [FRAP] and malonaldehyde [MDA]) and gene expression profiles were assessed in 5 sites from the Tamar River and Estuary (Neal Point, Town Quay, Wilcove, Cremyll Ferry and Whitsand; and a reference site, Trebarwith Strand, N. Cornwall). PAHs were measured in mussel tissue and sediment and metals were measured in mussel tissue only. Data from the biomarkers was integrated into a Mussel Expert System (MES) model to produce a simple assessment of mussel stress. Clear gradients of mussel toxicity were identified by the biomarkers (with the exception of neutral lipids) with the highest impacted animals found furthest up the Tamar, whilst the MES was unable to identify a gradient of effect. Gene expression profiles also indicated a gradient of stress with the greatest number of significantly up- or down- regulated genes found at the uppermost 2 sites. The MES did, however, determine that mussels from all sites, except the reference site, were highly stressed; a conclusion that could not be inferred from the biomarker data alone. It is concluded that the MES is a valuable tool that permits integration and interpretation of complex sets of biomarker data by identifying the biological meaning of biomarker changes.

Glucocorticoid resistance in T-lineage acute lymphoblastic leukaemia is associated with a proliferative metabolism.

Glucocorticoids (GCs) are among the most important drugs for acute lymphoblastic leukaemia (ALL), yet despite their clinical importance, the exact mechanisms involved in GC cytotoxicity and the development of resistance remain uncertain. We examined the baseline profile of a panel of T-ALL cell lines to determine factors that contribute to GC resistance without prior drug selection. Transcriptional profiling indicated GC resistance in T-ALL is associated with a proliferative phenotype involving upregulation of glycolysis, oxidative phosphorylation, cholesterol biosynthesis and glutamate metabolism, increased growth rates and activation of PI3K/AKT/mTOR and MYC signalling pathways. Importantly, the presence of these transcriptional signatures in primary ALL specimens significantly predicted patient outcome. We conclude that in lymphocytes the activation of bioenergetic pathways required for proliferation may suppress the apoptotic potential and offset the metabolic crisis initiated by GC signalling. It is likely that the link between GC resistance and proliferation in T-ALL has not been fully appreciated to date because such effects would be masked in the context of current multiagent therapies. The data also provide the first evidence that altered expression of wild-type MLL may contribute to GC-resistant phenotypes. Our findings warrant the continued development of selective metabolic inhibitors for the treatment of ALL.

Evolution of chemical species during electrodeposition of uranium for alpha spectrometry by the Hallstadius method.

The morphology and composition of uranium alpha sources with co-deposited platinum have been investigated by scanning electron microscopy (SEM) with energy dispersive X-ray fluorescence analysis (EDX), X-ray photoelectron spectroscopy (XPS) and X-ray absorption fine structure (XAFS) studies. Combined SEM and EDX measurements reveal the effect of porous platinum on the morphology of the sources which in turn affects their alpha-spectral resolution. The XPS analysis suggests that the presence of platinum initially increases the concentration of hydroxyl species in the deposits, which then act as centres for subsequent preferential uranium precipitation. XPS and XAFS analysis also provide for first time an indication of oxidation states of uranium present in the sources prepared by the Hallstadius method. These results are in line with Hansen's theory of electrodeposition of actinides.

Authenticity and drug resistance in a panel of acute lymphoblastic leukaemia cell lines.

Cell lines are important models for drug resistance in acute lymphoblastic leukaemia (ALL), but are often criticised as being unrepresentative of primary disease. There are also doubts regarding the authenticity of many lines. We have characterised a panel of ALL cell lines for growth and drug resistance and compared data with that published for primary patient specimens. In contrast to the convention that cell lines are highly proliferative, those established in our laboratory grow at rates similar to estimates of leukaemic cells in vivo (doubling time 53-442 h). Authenticity was confirmed by genetic fingerprinting, which also demonstrated the potential stability of long-term cultures. In vitro glucocorticoid resistance correlated well with that measured ex vivo, but all lines were significantly more sensitive to vincristine than primary specimens. Sensitivity to methotrexate was inversely correlated to that of glucocorticoids and L-asparaginase, indicating possible reciprocity in resistance mechanisms. A cell line identified as highly methotrexate resistant (IC50 > 8000-fold higher than other lines) was derived from a patient receiving escalating doses of the drug, indicating in vivo selection of resistance as a cause of relapse. Many of these lines are suitable as models to study naturally occurring resistance phenotypes in paediatric ALL.

Altered glucose metabolism in childhood pre-B acute lymphoblastic leukaemia.

The cells of solid tumours are known to have an altered metabolism, with high rates of glucose uptake and glycolysis, which results in the excessive production of lactate. To date there has been no definitive research documenting metabolic changes in acute lymphoblastic leukaemia (ALL) cells. In order to investigate whether ALL cells have an altered metabolism, we initially compared the transcriptional profiles of 22 specimens from paediatric patients diagnosed with ALL to five CD34+ specimens isolated from bone marrow, which was verified in an independent cohort of 101 specimens. Profiling revealed the upregulation of genes facilitating glycolysis in the ALL specimens compared to the CD34+ specimens, while those involved in the tricarboxylic acid cycle were downregulated. Functional studies supported the microarray findings threefold: (1) higher expression of the glucose transport protein glucose transporter 1 in ALL compared to CD34+ specimens, (2) the excessive production of lactate in ALL cell lines and (3) sensitivity of ALL cell lines to the glycolysis inhibitor 2-deoxy-D-glucose. While metabolic alterations have been well documented in solid tumours, this is the first study to provide direct evidence for the existence of metabolic changes in the leukaemic cells of ALL patients. The finding offers new options for targeted therapy for ALL patients.

The role of Sb(5+) in the structure of Sb(2)O(3)-B(2)O(3) binary glasses--an NMR and Mössbauer spectroscopy study.

Glasses of general formula xSb(2)O(3) (1-x)B(2)O(3) (0

Tracing the origin of the RACTK1 K(+) channel.

Potassium secretion by the kidney is vital for the maintenance of K(+) homeostasis. RACTK1, a putative inwardly rectifying potassium channel cloned from cultured rabbit collecting duct cells, has been proposed to play a role in this process. However, the lack of homology with any other cloned potassium channel and the inability to reproduce the results across different laboratories has brought into question the existence of RACTK1. Recently, it has been suggested that RACTK1 is a contamination from Escherichia coli. In this work we add conclusive evidence supporting the bacterial origin of RACTK1. Using both genomic PCR and RT-PCR we were unable to detect RACTK1 in a number of mammalian species. In addition sequencing of RACTK1 cDNA confirmed a complete homology between RACTK1 and a region of E. coli genomic DNA. Finally, a hypothesis on how RACTK1 could have been generated from a contamination by E. coli genomic DNA is presented.

Stable, polarised, functional expression of Kir1.1b channel protein in Madin-Darby canine kidney cell line.

1. The family of Kir1.1 (ROMK) channel proteins constitute a secretory pathway for potassium in principal cells of cortical collecting duct and thick ascending limb of Henle's loop. Mutations in Kir1.1 account for some types of Bartter's syndrome. 2. Here we report that stable transfection of Kir1.1b (ROMK2) in Madin-Darby canine kidney (MDCK) cell line results in expression of inwardly rectifying K+ currents and transmonolayer electrical and transport properties appropriate to Kir1.1 function. When grown on permeable supports, transfected monolayers secreted K+ into the apical solution. This secretion was inhibited by application of barium to the apical membrane, or by reduction in expression temperature from 37 to 26 C. However, whole-cell voltage clamp electrophysiology showed that K+ conductance was higher in cells expressing Kir1.1b at 26C. 3. To investigate this further, Kir1.1b was tagged with (EGFP), a modification that did not affect channel activity. Protein synthesis was inhibited with cycloheximide. Spectrofluorimetry was used to compare protein degradation at 37 and 26 C. The increased level of Kir1.1b at the plasma membrane at 26 C was due to an increase in protein stability. 4. Confocal microscopic investigation of EGFP-Kir1. 1b fluorescence in transfected cells showed that the channel protein was targeted to the apical domain of the cell. 5. These results demonstrate that Kir1.1b is capable of appropriate trafficking and function in MDCK cell lines at physiological temperatures. In addition, expression of Kir1.1b in MDCK cell lines provides a useful and convenient tool for the study of functional activity and targeting of secretory K+ channels.

Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia.

Many pathogens causing diarrhea do so by modulating ion transport in the gut. Respiratory pathogens are similarly associated with disturbances of fluid balance in the respiratory tract, although it is not known whether they too act by altering epithelial ion transport. Here we show that influenza virus A/PR/8/34 inhibits the amiloride-sensitive Na(+) current across mouse tracheal epithelium with a half-time of about 60 min. We further show that the inhibitory effect of the influenza virus is caused by the binding of viral hemagglutinin to a cell-surface receptor, which then activates phospholipase C and protein kinase C. Given the importance of epithelial Na(+) channels in controlling the amount of fluid in the respiratory tract, we suggest that down-regulation of Na(+) channels induced by influenza virus may play a role in the fluid transport abnormalities that are associated with influenza infections.

Histopathology of the skin of UV-B irradiated sole (Solea solea) and turbot (Scophthalmus maximus) larvae.

Larval stages of two economically important flatfish, the sole (Solea solea) and turbot (Scophthalmus maximus) were exposed to ambient and elevated levels of UV-B. Sole larvae, which naturally occur in the plankton in early spring, demonstrated skin lesions at elevated levels of UV-B. Histopathology of the sole revealed cellular changes in the integument, characteristic of sunburn damage, with a reduction in the size of mucus-secreting cells and an increased epidermal thickening, especially at the highest doses of UV-B (2.15 KJ bio eff/m2). Pigmentation in the sole is restricted to a few isolated melanocytes. The integrity of the heavily pigmented skin of turbot appeared to be unaffected by comparable doses of UV-B. Both species have protective mechanisms, which minimize the effects of naturally-occurring levels of UV-B. However, sole appear to be poorly adapted to accommodate any further increase in solar radiation.

Expression of sulphonylurea receptor protein in mouse kidney.

The sulphonylurea receptor (SUR) is the site of action for sulphonylurea derivatives such as glibenclamide, which are widely used as oral hypoglycaemic agents. Sulphonylureas have also been shown to affect urine flow and salt excretion by the kidney; therefore, the use of these drugs may have important implications for the pharmacological manipulation of renal salt handling. The purpose of the present investigation was to increase our understanding of the possible role of SUR in the regulation of renal function by determining the distribution of SUR isoforms within mouse kidney. Immunostaining with anti-SUR antisera revealed specific staining of SUR2B in distal nephron segments of mouse kidney. A diffuse, low level staining was observed in proximal tubules in the inner cortical region. No evidence was found for the presence of SUR2B in intra-renal blood vessels. Reverse-transcription polymerase chain reaction and Western blotting experiments indicated that SUR2B is the only known isoform expressed. These data demonstrate that SUR2B in mouse kidney is expressed in tubule regions that are critical in determining renal salt excretion.

Splicing of a retained intron within ROMK K+ channel RNA generates a novel set of isoforms in rat kidney.

The renal outer medulla K+ channel (ROMK) family of K+ channels may constitute a major pathway for K+ secretion in the distal nephron. To date, four main isoforms of this gene have been identified in the rat that differ only in their NH2-terminal amino acids and that share a common "core exon" that determines the remaining protein sequence. Using RT-PCR, we have identified a new set of ROMK isoforms in rat kidney that are generated by the deletion of a region within the ROMK core sequence that is identifiable as a typical mammalian intron. This splicing event was shown to be reproducible in vitro by detection of deleted ROMK mRNA in Madin-Darby canine kidney (MDCK) cells stably transfected with the gene for ROMK2. Translation of the deletion variant of ROMK2 was confirmed in vitro and visualized in MDCK cells following transient transfection with an enhanced green fluorescent protein tag. The deletion in this core region is predicted to generate hydrophilic proteins that are approximately one-third of the size of native ROMK and lack membrane-spanning domains.