System ASC amino acid transporter-2 (ASCT2) was previously demonstrated to be essential for human hepatoma cell growth and survival, as its silencing via inducible antisense RNA expression results in complete apoptosis within 48 h by a mechanism that transcends its role in amino acid delivery. To gain mechanistic insights into the reliance of cancerous liver cells on ASCT2, the aim of this study was to determine the early consequences of its silencing on the growth and survival signaling that presage apoptosis. Induced antisense ASCT2 RNA in SK-Hep1 cells led to >90% suppression of ASCT2 mRNA by 6 h and inhibition of mammalian target-of-rapamycin (mTOR)/raptor (mTOR complex-1; mTORC1) signaling by 8 h, as manifested by diminished p70 ribosomal protein S6 kinase-1 and eukaryotic initiation factor-4E (eIF4E) binding protein-1 phosphorylation, while protein synthesis rates declined by nearly 50% despite no measurable decreases in the cap binding protein eIF4G or cellular ribosomal protein content. Depressed mTORC1 signaling occurred before detectable reduction in ASCT2 activity but coincided with a 30% decline in total cellular ASCT2 protein. By 12 h after ASCT2 silencing, further decrements were observed in protein synthesis rates and ASCT2 protein and activity, each by approximately 50%, while signaling from mTOR/rictor (mTOR complex-2; mTORC2) was stimulated as indexed by enhanced phosphorylation of the Akt/PKB kinase on serine-473 and of its proapoptotic substrate Bad on serine-136. These results suggest that ASCT2 silencing inhibits mTORC1 signaling to the translational machinery followed by an mTORC2-initiated survival response, establishing a link between amino acid transporter expression and mTOR function.

Glutathione S-transferases (GSTs) are ubiquitous enzymes that catalyze the conjugation of toxic xenobiotics and oxidatively produced compounds to reduced glutathione, which facilitates their metabolism, sequestration, or removal. We report here that soybean (Glycine max) root nodules contain at least 14 forms of GST, with GST9 being most prevalent, as measured by both real-time reverse transcription-polymerase chain reaction and identification of peptides in glutathione-affinity purified extracts. GST8 was prevalent in stems and uninfected roots, whereas GST2/10 prevailed in leaves. Purified, recombinant GSTs were shown to have wide-ranging kinetic properties, suggesting that the suite of GSTs could provide physiological flexibility to deal with numerous stresses. Levels of GST9 increased with aging, suggesting a role related to senescence. RNA interference studies of nodules on composite plants showed that a down-regulation of GST9 led to a decrease in nitrogenase (acetylene reduction) activity and an increase in oxidatively damaged proteins. These findings indicate that GSTs are abundant in nodules and likely function to provide antioxidant defenses that are critical to support nitrogen fixation.

Nodulation is the result of a symbiosis between legumes and rhizobial bacteria in soil. This symbiosis is mutually beneficial, with the bacteria providing a source of nitrogen to the host while the plant supplies carbon to the symbiont. Nodule development is a complex process that is tightly regulated in the host plant cell through networks of gene expression. In order to examine this regulation in detail, a library of quantitative reverse transcription-polymerase chain reaction primer sets was developed for a large number of soybean (Glycine max) putative regulatory genes available in the current expressed sequence tag collection. This library contained primers specific to soybean transcription factor genes as well as genes involved in chromatin modification and translational regulation. Using this library, we analyzed the expression of this gene set during nodule development. A large number of genes were found to be differentially expressed, especially at the later stages of nodule development when active nitrogen fixation was occurring. Expression of these putative regulatory genes was also analyzed in response to the addition of nitrate as a nitrogen source. This comparative analysis identified genes that may be specifically involved in nitrogen assimilation, metabolism, and the maintenance of active nodules. To address this possibility, the expression of one such candidate was studied in more detail by expressing in soybean roots promoter β-glucuronidase and green fluorescent protein fusions. This gene, named Control of Nodule Development (CND), encoded a Myb transcription factor gene. When the CND gene was silenced, nodulation was reduced. These results, associated with a strong expression of the CND gene in the vascular tissues, suggest a role for CND in controlling soybean nodulation.

Understanding how to display e ectively uncertain information has become increasingly important as decision aids can provide operators with situational estimates and their associated uncertainty. The paper describes two studies in which degraded or blended icons were used to convey uncertainty regarding the identity of a radar contact as hostile or friendly. A classi®cation study ®rst showed that participants could sort, order and rank icons from ®ve sets intended to represent di erent levels of uncertainty. Three icon sets were selected for further study in an experiment in which participants had to identify the status of contacts as either hostile or friendly. Contacts and probabilistic estimates of their identities were depicted on a simulated radar screen in one of three ways: with degraded icons and probabilities, with non-degraded icons and probabilities and with degraded icons only. Results showed that participants using displays with only degraded icons performed better on some measures and as well on other measures, than the other tested conditions. These results are signi®cant because they indicate both that people can understand uncertainty conveyed through such a manner and thus that the use of distorted or degraded images may be a viable alternative to convey situational uncertainty.

Historically, secondary clarifiers have contributed to instability in plant performance. The flocculator-clarifier development has demonstrated in a variety of plant situations that stable secondary process operation is achievable. Consistently low effluent SS levels (average values of 10 mg/l) are realistically achievable. This performance enhancement has been obtained at lower costs than for conventional designs. The clarifier incorporates some special design features including a large flocculator centerwell to incorporate dispersed solids into settleable floc. Other features include rapid sludge removal, inboard weir placement and deeper sidewater depths than conventionally used (5.5 to 6m).

Understanding how to effectively display uncertain information has become increasingly important as decision aids are able to provide operators with situational estimates and their associated uncertainty. This paper describes an experiment in which degraded icons were used to convey uncertainty. Results indicated similar performance using degraded images or numeric probabilities, suggesting that the use of degraded images may be a viable method for displaying uncertainty.

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Amino acid transporters ASCT2 and LAT1 are coordinately enhanced in a broad array of human cancers where they associate in the plasma membrane and are proposed to work cooperatively to activate mTOR signaling through a tertiary-active transport mechanism involving glutamine (ASCT2) and essential branch-chain amino acids (LAT1). Silencing of ASCT2 expression in liver cancer cells has been shown to induce apoptosis and inhibit mTOR signaling while Myc expression has been implicated in promoting ASCT2 and LAT1 expression as well as a glutamine-intensive metabolic profile typical of many cancers, which helps support the switch to aerobic glycolysis (Warburg effect). In particular, ASCT2 and LAT1 are nearly undetectable in normal liver but are enhanced in hepatocellular carcinoma (HCC). To test the relationship between amino acid transporters, myc expression, metabolism and mTOR, we examined the expression and function of ASCT2 and LAT1 as a function of N-myc and C-myc status, reliance on glycolysis for growth and sensitivity to rapamycin (mTOR) in a broad panel of 14 human HCC lines (7 Group I (epithelial) and 7 Group II (mesenchymal)). We hypothesized that transporter expression is proportional to myc expression, and that glycolytic reliance and both transporters are enhanced in the more aggressive Group II HCC lines. Our results show that all human HCC lines are N-myc-, ASCT2- and LAT1-positive, and variably c-myc positive, with no clear correlation between specific myc isoform and transporter expression levels. In general, LAT1 protein expression was uniformly high in all mesenchymal HCC lines, but more variable in epithelial HCC, while BCH-inibitable leucine uptake rates displayed a wide array of values in both HCC groups; the same was true for ASCT2 protein and glutamine uptake activities. Treatment of HCC cells for 24 hours with ASCT2 and LAT1 substrate inhibitors GPNA and BCH, respectively, failed to inhibit mTOR signaling as indexed by 4EBP1 phosphorylation. Inhibition of glycolysis and growth with 3-bromopyruvate was generally more pronounced in epithelial HCC, but variably evident in both HCC groups. Rapamycin-dependent growth inhibition was variable between HCC lines and showed no preferential epithelial or mesenchymal HCC efficacy. Collectively, the results suggest that N-myc, ASCT2 and LAT1 are ubiquitously expressed in both epithelial and mesenchymal human HCC cells, where they help drive growth in cells variably reliant on mTOR signaling and glycolysis. Thus, both transporters remain the subject of ongoing investigation as targeted therapies for HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5148. doi:1538-7445.AM2012-5148