Molecular epidemiological studies on genetic predispositions to squamous cell carcinoma of the head and neck

Two molecular epidemiological studies were conducted to examine associations between genetic variation and risk of squamous cell carcinoma of the head and neck (SCCHN). In the first study, we hypothesized that genetic variation in p53 response elements (REs) may play roles in the etiology of SCCHN. We selected and genotyped five polymorphic p53 REs as well as a most frequently studied p53 codon 72 (Arg72Pro, rs1042522) polymorphism in 1,100 non-Hispanic White SCCHN patients and 1,122 age-and sex-matched cancer-free controls recruited at The University of Texas M. D. Anderson Cancer Center. In multivariate logistic regression analysis with adjustment for age, sex, smoking and drinking status, marital status and education level, we observed that the EOMES rs3806624 CC genotype had a significant effect of protection against SCCHN risk (adjusted odds ratio= 0.79, 95% confidence interval =0.64–0.98), compared with the -838TT+CT genotypes. Moreover, a significantly increased risk associated with the combined genotypes of p53 codon 72CC and EOMES -838TT+CT was observed, especially in the subgroup of non-oropharyneal cancer patients. The values of false-positive report probability were also calculated for significant findings. In the second study, we assessed the association between SCCHN risk and four potential regulatory single nucleotide polymorphisms (SNPs) of DEC1 (deleted in esophageal cancer 1) gene, a candidate tumor suppressor gene for esophageal cancer. After adjustment for age, sex, and smoking and drinking status, the variant -606CC (i.e., -249CC) homozygotes had a significantly reduced SCCHN risk (adjusted odds ratio = 0.71, 95% confidence interval = 0.52–0.99), compared with the -606TT homozygotes. Stratification analyses showed that a reduced risk associated with the -606CC genotype was more pronounced in subgroups of non-smokers, non-drinkers, younger subjects (defined as ≤ 57 years), carriers of TP53 Arg/Arg (rs1042522) genotype, patients with oropharyngeal cancer or late-stage SCCHN. Further in silico analysis revealed that the -249 T-to-C change led to a gain of a transcription factor binding site. Additional functional analysis showed that the -249T-to-C change significantly enhanced transcriptional activity of the DEC1 promoter and the DNA-protein binding activity. We conclude that the DEC1 promoter -249 T>C (rs2012775) polymorphism is functional, modulating susceptibility to SCCHN among non-Hispanic Whites. Additional large-scale, preferably population-based studies are needed to validate our findings.^

Induction of synthetic lethality in mutant KRAS cells for non-small cell lung cancers chemoprevention and therapy

Lung cancer is the leading cause of cancer death in both men and women in the United States and worldwide. Despite improvement in treatment strategies, the 5-year survival rate of lung cancer patients remains low. Thus, effective chemoprevention and treatment approaches are sorely needed. Mutations and activation of KRAS occur frequently in tobacco users and the early stage of development of non-small cell lung cancers (NSCLC). So they are thought to be the primary driver for lung carcinogenesis. My work showed that KRAS mutations and activations modulated the expression of TNF-related apoptosis-inducing ligand (TRAIL) receptors by up-regulating death receptors and down-regulating decoy receptors. In addition, we showed that KRAS suppresses cellular FADD-like IL-1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP) expression through activation of ERK/MAPK-mediated activation of c-MYC which means the mutant KRAS cells could be specifically targeted via TRAIL induced apoptosis. The expression level of Inhibitors of Apoptosis Proteins (IAPs) in mutant KRAS cells is usually high which could be overcome by the second mitochondria-derived activator of caspases (Smac) mimetic. So the combination of TRAIL and Smac mimetic induced the synthetic lethal reaction specifically in the mutant-KRAS cells but not in normal lung cells and wild-type KRAS lung cancer cells. Therefore, a synthetic lethal interaction among TRAIL, Smac mimetic and KRAS mutations could be used as an approach for chemoprevention and treatment of NSCLC with KRAS mutations. Further data in animal experiments showed that short-term, intermittent treatment with TRAIL and Smac mimetic induced apoptosis in mutant KRAS cells and reduced tumor burden in a KRAS-induced pre-malignancy model and mutant KRAS NSCLC xenograft models. These results show the great potential benefit of a selective therapeutic approach for the chemoprevention and treatment of NSCLC with KRAS mutations.

The transcriptional regulation of the {\it neu\/} gene: Examples of activation, repression, and cell-specific expression

The neu oncogene encodes a growth factor receptor-like protein, p185, with an intrinsic tyrosine kinase activity. A single point mutation, an A to T transversion resulting in an amino acid substitution from valine to glutamic acid, in the transmembrane domain of the rat neu gene was found to be responsible for the transforming and tumorigenic phenotype of the cells that carry it. In contrast, the human proto-neu oncogene is frequently amplified in tumors and cell lines derived from tumors and the human neu gene overexpression/amplification in breast and ovarian cancers is known to correlate with poor patient prognosis. Examples of the human neu gene overexpression in the absence of gene amplification have been observed, which may suggest the significant role of the transcriptional and/or post-transcriptional control of the neu gene in the oncogenic process. However, little is known about the transcriptional mechanisms which regulate the neu gene expression. In this study, three examples are presented to demonstrate the positive and negative control of the neu gene expression.^ First, by using band shift assays and methylation interference analyses, I have identified a specific protein-binding sequence, AAGATAAAACC ($-$466 to $-$456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.^ Second, it has been reported that the E3 region of adenovirus 5 induces down-regulation of epidermal growth factor (EGF) receptor through endocytosis. I found that the human neu gene product, p185, (an EGF receptor-related protein) is also down-regulated by adenovirus 5, but via a different mechanism. I demonstrate that the adenovirus E1a gene is responsible for the repression of the human neu gene at the transcriptional level.^ Third, a differential expression of the neu gene has been found in two cell model systems: between the mouse fibroblast Swiss-Webster 3T3 (SW3T3) and its variant NR-6 cells; and between the mouse liver tumor cell line, Hep1-a, and the mouse pancreas tumor cell line, 266-6. Both NR-6 and 266-6 cell lines are not able to express the neu gene product, p185. I demonstrate that, in both cases, the transcriptional repression of the neu gene may account for the lack of the p185 expression in these two cell lines. ^

Aberrant regulation of the Src -FAK signaling pathway in the human colon adenocarcinoma cell line HT-29

c-Src, a protein tyrosine kinase (PTK) the specific activity of which is increased $>$20-fold in $\sim$80% of colon tumors and colon tumor cell lines, plays a role in both growth regulation and tumorigenicity of colon tumor cells. To examine the effect of increased c-Src specific activity on colon tumor cells, coumarin-derived tyrosine analog PTK inhibitors were assessed in a standard colon tumor cell line, HT-29. Of the nine compounds tested for inhibiting c-Src activity in a standard immune complex kinase assay from c-Src precipitated from HT-29 cells, the 7,8-dihydroxy-containing compounds daphnetin and fraxetin were most effective, with IC$\sb{50}$s of 0.6 $\pm$ 0.2 mM and 0.6 $\pm$ 0.3 mM, respectively. Treatment of HT-29 cells with daphnetin resulted in inhibition of cell growth in a dose-dependent manner. In contrast, scopoletin, a relatively poor Src inhibitor in vitro, did not inhibit HT-29 cell growth in the concentration range tested. In daphnetin treated cells, a dose-dependent decrease of c-Src activity paralleling cell growth inhibition was also observed; the IC$\sb{50}$ was 0.3 $\pm$ 0.1 mM for c-Src autophosphorylation. In contrast, the IC$\sb{50}$ for c-Src protein level was $>$ 0.6 mM, indicating that the effects of daphnetin were primarily an enzymatic activity of c-Src, rather than protein level in HT-29 cells. These results are the first to demonstrate that c-Src specific activity regulates colon tumor cell growth.^ To elucidate the signaling pathways activated by c-Src in colon tumor cells, the Src family substrate FAK, which has been shown to play a role in both extracellular matrix-dependent cell growth and survival, was examined. Coprecipitation assays showed Src-FAK association in detergent insoluble fractions of both attached and detached HT-29 cells, indicating that Src-FAK association in HT-29 cells is stable and, unlike untransformed cells, not dependent on cell-substratum contact. FAK also coprecipitated with Grb2, an adaptor protein also playing a role in cell proliferation and survival, in both attached and detached HT-29 cells, suggesting that a Src-FAK-Grb2-mediated signaling pathway(s) in HT-29 cells is/are constitutively activated.^ FAK was also analyzed in c-src antisense HT-29 clones AS15 and AS33 in which c-Src is specifically reduced by transfection of an antisense expression vector. FAK protein level is unexpectedly decreased in both AS15 and AS33 cells by 5-fold and 1.5-fold compared to HT-29, respectively, corresponding with the decreased expression of c-Src observed in these cells. FAK protein level was not decreased compared to parental in the c-src "sense" clone S8. Northern blot analyses showed decreased FAK mRNA levels compared to parental in AS15 and AS33, correlating with decreased FAK protein level, indicating that FAK activity in the antisense cells is regulated, at least in part, by altering FAK expression, and that this regulation is Src dependent. Because FAK has been implicated in anoikis, the ability of c-src antisense cells to survive in the absence of cell-substratum contact was examined. Decreased cell survival is seen in both AS15 and AS33, correlating with the decreases in c-Src and FAK levels and tumorigenicity in these cells. These results suggest that at least one mechanism by which activation of c-Src contributes to tumorigenic phenotype of colon tumor cells is by aberrantly promoting a survival signal through unregulated Src-FAK-Grb2 complexes. (Abstract shortened by UMI.) ^

Transcriptional regulation of cell lineage-specific expression of the murine type I collagen genes and of osteoblast differentiation

The aim of my project is to examine the mechanisms of cell lineage-specific transcriptional regulation of the two type I collagen genes by characterizing critical cis-acting elements and trans-acting factors. I hypothesize that the transcription factors that are involved in the cell lineage-specific expression of these genes may have a larger essential role in cell lineage commitment and differentiation. I first examined the proximal promoters of the proα1(I) and the proα2(I) collagen genes for cell type-specific DNA-protein interactions, using in vitro DNaseI and in vivo DMS footprinting. These experiments demonstrated that the cis-acting elements in these promoters are accessible to ubiquitous DNA-binding proteins in fibroblasts that express these genes, but not in other cells that do not express these genes. I speculate that in type I collagen-expressing cells, cell type-specific enhancer elements facilitate binding of ubiquitous proteins to the proximal promoters of these genes. Subsequently, examination of the upstream promoter of the proα(I) collagen gene by transgenic mice experiments delineated a 117 bp sequence (-1656 to -1540 bp) as the minimum element required for osteoblast-specific expression. This 117 bp element contained two segments that appeared to have different functions: (1) the A-segment, which was necessary to obtain osteoblast-specific expression and (2) the C-segment, which was dispensable for osteoblast-specific expression, but was necessary to obtain high-level expression. In experiments to identify trans-acting factors that bind to the 117 bp element, I have demonstrated that the cell lineage-restricted homeodomain proteins, Dlx2, Dlx5 and mHOX, bound to the A-segment and that the ubiquitous transcription factor, Sp1, bound to the C-segment of this element. These results suggested a model where the binding of cell lineage-restricted proteins to the A-segment and of ubiquitous proteins to the C-segment of the 117 bp element of the proα1 (I) collagen gene activated this gene in osteoblasts. These results, combined with additional evidence that Dlx2, Dlx5 and mHOX are probably involved in osteoblast differentiation, support my hypothesis that the transcription factors involved in osteoblast-specific expression of type I collagen genes may have essential role in osteoblast lineage commitment and differentiation. ^

Axl -Gas6 signaling counteracts adenovirus type 5 E1A-mediated cell growth suppression and proapoptotic activity

The adenovirus type 5 E1A (abbreviated E1A) has previously been known as an immortalization oncogene because E1A is required for transforming oncogenes, such as ras and E1B, to transform cells in primary cultures. However, E1A has also been shown to downregulate the overexpression of the Her-2/neu oncogene, resulting in suppression of transformation and tumorigenesis induced by that oncogene. In addition, E1A is able to promote apoptosis induced by anticancer drugs, irradiation, and serum deprivation. Many tyrosine kinases, such as the EGF receptor, Her-2/Neu, Src, and Axl are known to play a role in oncogenic signals in transformed cells. To study the mechanism underlying the E1A-mediated tumor-suppressing function, we exploited a modified tyrosine kinase profile assay (Proc. Natl. Acad. Sci, 93, 5958–5962, 1996) to identify potential tyrosine kinases regulated by E1A. RT-PCR products were synthesized with two degenerate primers derived from the conserved motifs of various tyrosine kinases. A tyrosine kinase downregulated by E1A was identified as Axl by analyzing the Alu I-digested RT-PCR products. We isolated the DNA fragment of interest, and found that E1A negatively regulated the expression of the transforming receptor tyrosine kinase Axl at the transcriptional level. To study whether downregulation of the Axl receptor is involved in E1A-mediated growth suppression, we transfected axl cDNA into E1A-expressing cells (ip1-E1A) to establish cells that overexpressed Axl (ip1-E1A-Axl). The Axl ligand Gas6 triggered a greater mitogenic effect in these ip1-E1A-Axl cells than in the control cells ip1-E1A and protected the Axl-expressing cells from serum deprivation-induced apoptosis. Further study showed that Akt is required for Axl-Gas6 signaling to prevent ip1-E1A-Axl cells from serum deprivation-induced apoptosis. These results indicate that downregulation of the Axl receptor by E1A is involved in E1A-mediated growth suppression and E1A-induced apoptosis, and thereby contributes to E1A's anti-tumor activities. ^

The role of Ski/Sno oncoproteins as negative regulators of the TGF-beta/SMAD signaling pathway in B -cell non -Hodgkin's lymphoma (NHL-B)

Non-Hodgkin's lymphomas are common tumors of the human immune system, primarily of B cell lineage (NHL-B). Negative growth regulation in the B cell lineage is mediated primarily through the TGF-β/SMAD signaling pathway that regulates a variety of tumor suppressor genes. Ski was originally identified as a transforming oncoprotein, whereas SnoN is an isoform of the Sno protein that shares a large region of homology with Ski. In this study, we show that Ski/SnoN are endogenously over-expressed both in patients' lymphoma cells and NHL-B cell lines. Exogenous TGF-β1 treatment induces down-regulation of Ski and SnoN oncoprotein expression in an NHL-B cell line, implying that Ski and SnoN modulate the TGF-β signaling pathway and are involved in cell growth regulation. Furthermore, we have developed an NHL-B cell line (DB) that has a null mutation in TGF-β receptor type II. In this mutant cell line, Ski/SnoN proteins are not down-regulated in response to TGF-β1 treatment, suggesting that downregulation of Ski and SnoN proteins in NHL-B require an intact functional TGF-β signaling pathway Resting normal B cells do not express Ski until activated by antigens and exogenous cytokines, whereas a low level of SnoN is also present in peripheral blood Go B cells. In contrast, autonomously growing NHL-B cells over-express Ski and SnoN, implying that Ski and SnoN are important cell cycle regulators. To further investigate a possible link between reduction of the Ski protein level and growth inhibition, Ski antisense oligodeoxynucleotides were transfected into NHL-B cells. The Ski protein level was found to decrease to less than 40%, resulting in restoring the effect of TGF-β and leading to cell growth inhibition and G1 cell cycle arrest. Co-immunoprecipitation experiments demonstrated that Ski associates with Smad4 in the nucleus, strongly suggesting that over-expression of the nuclear protein Ski and/or SnoN negatively regulates the TGF-β pathway, possibly by modulating Smad-mediated tumor suppressor gene expression. Together, in NHL-B, the TGF-β/SMAD growth inhibitory pathway is usually intact, but over-expression of the Ski and/or SnoN, which binds to Smad4, abrogates the negative regulatory effects of TGF-β/SMAD in lymphoma cell growth and potentiates the growth potential of neoplastic B cells. ^

Long-term Evaluation of Hybrid, Nitrogen, and Potassium Interactions in Continuous Corn

A long-term experiment was established in 2009 to study continuous corn responses to potassium (K), nitrogen (N), and hybrid rootworm resistance. Previous research suggested a need for this study. A long-term trial conducted until 2001 at the ISU Northern Research Farm showed that the maximum corn yield level and the N rate that maximized yield was higher when K was optimal or greater. In contrast, the relative yield response to N and the N rate that maximized yield were similar for soil-test phosphorus (P) levels ranging from very low to very high. Other studies have shown that rootworm resistance often increases yield compared with untreated susceptible hybrids. Also, that rootworm resistance does not consistently affect the K rate that maximizes yield, but increases K removal because of the higher yield levels. Therefore, this new study evaluates possible interactions between rootworm resistance and N and K fertilization in corn.

High-resolution marine record of sediment core MD99-2343 in the Balearic Sea

The IMAGES core MD99-2343, recovered from a sediment drift north of the island of Minorca, in the north-western Mediterranean Sea, holds a high-resolution sequence that is perfectly suited to study the oscillations of the overturning system of the Western Mediterranean Deep Water (WMDW). Detailed analysis of grain-size and bulk geochemical composition reveals the sensitivity of this region to climate changes at both orbital and centennial-millennial temporal scales during the last 50 kyr. The dominant orbital pattern in the K/Al record indicates that sediment supply to the basin was controlled by the insolation evolution at 40°N, which forced changes in the fluvial regime, with more efficient sediment transport during insolation maxima. This orbital control also modulated the long-term pattern of the WMDW intensity as illustrated by the silt/clay ratio. However, deep convection was particularly sensitive to climatic changes at shorter time-scales, i.e. to centennial-millennial glacial and Holocene oscillations that are well documented by all the paleocurrent intensity proxies (Si/Al, Ti/Al and silt/clay ratios). Benthic isotopic records (d13C and d18O) show a Dansgaard-Oeschger (D-O) pattern of variability of WMDW properties, which can be associated with changing intensities of the deep currents system. The most prominent reduction on the WMDW overturning was caused by the post-glacial sea level rise. Three main scenarios of WMDW overturning are revealed: a strong mode during D-O Stadials, a weak mode during D-O Interstadials and an intermediate mode during cooling transitions. In addition, D-O Stadials associated with Heinrich events (HEs) have a very distinct signature as the strong mode of circulation, typical for the other D-O Stadials, was never reached during HE due to the surface freshening induced by the inflowing polar waters. Consequently, the WMDW overturning system oscillated around the intermediate mode of circulation during HE. Though surface conditions were more stable during the Holocene, the WMDW overturning cell still reacted synchronously to short-lived events, as shown by increments in the planktonic d18O record, triggering quick reinforcements of the deep water circulation. Overall, these results highlight the sensitivity of the WMDW to rapid climate change which in the recent past were likely induced by oceanographic and atmospheric reorganizations in the North Atlantic region.

The photo-physiological response of a model cnidarian-dinoflagellate symbiosis to CO2-induced acidification at the cellular level

We measured the relationship between CO2-induced seawater acidification, photo-physiological performance and intracellular pH (pHi) in a model cnidarian-dinoflagellate symbiosis - the sea anemone Aiptasia sp. -under ambient (289.94 ± 12.54 µatm), intermediate (687.40 ± 25.10 µatm) and high (1459.92 ± 65.51 µatm) CO2 conditions. These treatments represented current CO2 levels, in addition to CO2 stabilisation scenarios IV and VI provided by the Intergovernmental Panel on Climate Change (IPCC). Anemones were exposed to each treatment for two months and sampled at regular intervals. At each time-point we measured a series of physiological responses: maximum dark-adapted fluorescent yield of PSII (Fv/Fm), gross photosynthetic rate, respiration rate, symbiont population density, and light-adapted pHi of both the dinoflagellate symbiont and isolated host anemone cell. We observed increases in all but one photo-physiological parameter (Pgross:R ratio). At the cellular level, increases in light-adapted symbiont pHi were observed under both intermediate and high CO2 treatments, relative to control conditions (pHi 7.35 and 7.46 versus pHi 7.25, respectively). The response of light-adapted host pHi was more complex, however, with no change observed under the intermediate CO2 treatment, but a 0.3 pH-unit increase under the high CO2 treatment (pHi 7.19 and 7.48, respectively). This difference is likely a result of a disproportionate increase in photosynthesis relative to respiration at the higher CO2 concentration. Our results suggest that, rather than causing cellular acidosis, the addition of CO2 will enhance photosynthetic performance, enabling both the symbiont and host cell to withstand predicted ocean acidification scenarios.

Response of the calcifying coccolithophore Emiliania huxleyi to low pH/high pCO2: from physiology to molecular level, 2011

The emergence of ocean acidification as a significant threat to calcifying organisms in marine ecosystems creates a pressing need to understand the physiological and molecular mechanisms by which calcification is affected by environmental parameters. We report here, for the first time, changes in gene expression induced by variations in pH/pCO2 in the widespread and abundant coccolithophore Emiliania huxleyi. Batch cultures were subjected to increased partial pressure of CO2 (pCO2; i.e. decreased pH), and the changes in expression of four functional gene classes directly or indirectly related to calcification were investigated. Increased pCO2 did not affect the calcification rate and only carbonic anhydrase transcripts exhibited a significant down-regulation. Our observation that elevated pCO2 induces only limited changes in the transcription of several transporters of calcium and bicarbonate gives new significant elements to understand cellular mechanisms underlying the early response of E. huxleyi to CO2-driven ocean acidification.

Hydrochemistry, water level, and discharge of water from Samoylov Island, Lena Delta, northeastern Siberia, in 2008

Although ponds make up roughly half of the total area of surface water in permafrost landscapes, their relevance to carbon dioxide emissions on a landscape scale has, to date, remained largely unknown. We have therefore investigated the inflows and outflows of dissolved organic and inorganic carbon from lakes, ponds, and outlets on Samoylov Island, in the Lena Delta of northeastern Siberia in September 2008, together with their carbon dioxide emissions. Outgassing of carbon dioxide (CO2) from these ponds and lakes, which cover 25% of Samoylov Island, was found to account for between 74 and 81% of the calculated net landscape-scale CO2 emissions of 0.2-1.1 g C/m**2/d during September 2008, of which 28-43% was from ponds and 27-46% from lakes. The lateral export of dissolved carbon was negligible compared to the gaseous emissions due to the small volumes of runoff. The concentrations of dissolved inorganic carbon in the ponds were found to triple during freezeback, highlighting their importance for temporary carbon storage between the time of carbon production and its emission as CO2. If ponds are ignored the total summer emissions of CO2-C from water bodies of the islands within the entire Lena Delta (0.7-1.3 Tg) are underestimated by between 35 and 62%.

Nanotechnology for more efficient photovoltaics: the quantum dot intermediate band solar cell

The intermediate band solar cell [1] has been proposed as a concept able to substantially enhance the efficiency limit of an ordinary single junction solar cell. If a band permitted for electrons is inserted within the forbidden band of a semiconductor then a novel path for photo generation is open: electron hole pairs may be formed by the successive absorption of two sub band gap photons using the intermediate band (IB) as a stepping stone. While the increase of the photovoltaic (PV) current is not a big achievement —it suffices to reduce the band gap— the achievement of this extra current at high voltage is the key of the IB concept. In ordinary cells the voltage is limited by the band gap so that reducing it would also reduce the band gap. In the intermediate band solar cell the high voltage is produced when the IB is permitted to have a Quasi Fermi Level (QFL) different from those of the Conduction Band (CB) and the Valence Band (VB). For it the cell must be properly isolated from the external contacts, which is achieved by putting the IB material between two n- and p-type ordinary semiconductors [2]. Efficiency thermodynamic limit of 63% is obtained for the IB solar cell1 vs. the 40% obtained [3] for ordinary single junction solar cells. Detailed information about the IB solar cells can be found elsewhere [4].

Implication of the oep16-1 mutation in a flu-independent, singlet oxygen-regulated cell death pathway in Arabidopsis thaliana

Singlet oxygen is a prominent form of reactive oxygen species in higher plants. It is easily formed from molecular oxygen by triplet–triplet interchange with excited porphyrin species. Evidence has been obtained from studies on the flu mutant of Arabidopsis thaliana of a genetically determined cell death pathway that involves differential changes at the transcriptome level. Here we report on a different cell death pathway that can be deduced from the analysis of oep16 mutants of A. thaliana. Pure lines of four independent OEP16-deficient mutants with different cell death properties were isolated. Two of the mutants overproduced free protochlorophyllide (Pchlide) in the dark because of defects in import of NADPH:Pchlide oxidoreductase A (pPORA) and died after illumination. The other two mutants avoided excess Pchlide accumulation. Using pulse labeling and polysome profiling studies we show that translation is a major site of cell death regulation in flu and oep16 plants. flu plants respond to photooxidative stress triggered by singlet oxygen by reprogramming their translation toward synthesis of key enzymes involved in jasmonic acid synthesis and stress proteins. In contrast, those oep16 mutants that were prone to photooxidative damage were unable to respond in this way. Together, our results show that translation is differentially affected in the flu and oep16 mutants in response to singlet oxygen.

A common mechanism of defective channel trafficking underlying DFNA2 hearing loss result in different cell surface expression levels of KCNQ4 mutants

KCNQ4 mutations underlie DFNA2, a subtype of autosomal dominant hearing loss. We had previously identified the pore-region p.G296S mutation that impaired channel activity in two manners: it greatly reduced surface expression and abolished channel function. Moreover, G296S mutant exerted a strong dominant-negative effect on potassium currents by reducing the channel expression at the cell surface representing the first study to identify a trafficking-dependent dominant mechanism for the loss of KCNQ4 channel function in DFNA2. Here, we have investigated the pathogenic mechanism associated with all the described KCNQ4 mutations (F182L, W242X, E260K, D262V, L274H, W276S, L281S, G285C, G285S and G321S) that are located in different domains of the channel protein. F182L mutant showed a wild type-like cell-surface distribution in transiently transfected NIH3T3 fibroblasts and the recorded currents in Xenopus oocytes resembled those of the wild-type. The remaining KCNQ4 mutants abolished potassium currents, but displayed distinct levels of defective cell-surface expression in NIH3T3 as quantified by flow citometry. Co-localization studies revealed these mutants were retained in the ER, unless W242X, which showed a clear co-localization with Golgi apparatus. Interestingly, this mutation results in a truncated KCNQ4 protein at the S5 transmembrane domain, before the pore region, that escapes the protein quality control in the ER but does not reach the cell surface at normal levels. Currently we are investigating the trafficking behaviour and electrophysiological properties of several KCNQ4 truncated proteins artificially generated in order to identify specific motifs involved in channel retention/exportation. Altogether, our results indicate that a defect in KCNQ4 trafficking is the common mechanism underlying DFNA2