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Retinoblastoma (Rb) protein is a tumor suppressor that is dysregulated in a majority of human cancers. Rb functions to inhibit cell cycle progression in part by directly disabling the E2F family of cell cycle-promoting transcription factors. Because the de novo synthesis of multiple glutamine-derived anabolic precursors is required for cell cycle progression, we hypothesized that Rb also may directly regulate proteins involved in glutamine metabolism. We examined glutamine metabolism in mouse embryonic fibroblasts (MEFs) isolated from mice that have triple knock-outs (TKO) of all three Rb family members (Rb-1, Rbl1 and Rbl2) and found that loss of global Rb function caused a marked increase in 13 C-glutamine uptake and incorporation into glutamate and tricarboxylic acid cycle (TCA) intermediates in part via upregulated expression of the glutamine transporter ASCT2 and the activity of glutaminase 1 (GLS1). The Rb-controlled transcription factor E2F-3 altered glutamine uptake by direct regulation of ASCT2 mRNA and protein expression, and E2F-3 was observed to associate with the ASCT2 promoter. We next examined the functional consequences of the observed increase in glutamine uptake and utilization and found that glutamine exposure potently increased oxygen consumption, whereas glutamine deprivation selectively decreased ATP concentration in the Rb TKO MEFs but not the wild-type (WT) MEFs. In addition, TKO MEFs exhibited elevated production of glutathione from exogenous glutamine and had increased expression of gamma-glutamylcysteine ligase relative to WT MEFs. Importantly, this metabolic shift towards glutamine utilization was required for the proliferation of Rb TKO MEFs but not for the proliferation of the WT MEFs. Last, addition of the TCA cycle intermediate α-ketoglutarate to the Rb TKO MEFs reversed the inhibitory effects of glutamine deprivation on ATP, GSH levels and viability. Taken together, these studies demonstrate that the Rb/E2F cascade directly regulates a major energetic and anabolic pathway that is required for neoplastic growth.

Choline is an essential anabolic substrate for the synthesis of phospholipids. Choline kinase phosphorylates choline to phosphocholine that serves as a precursor for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the synthesis of lipid signaling molecules. Nuclear magnetic resonance (NMR)-based metabolomic studies of human tumors have identified a marked increase in the intracellular concentration of phosphocholine relative to normal tissues. We postulated that the observed intracellular pooling of phosphocholine may be required to sustain the production of the pleiotropic lipid second messenger, phosphatidic acid. Phosphatidic acid is generated from the cleavage of phosphatidylcholine by phospholipase D2 and is a key activator of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT survival signaling pathways. In this study we show that the steady-state concentration of phosphocholine is increased by the ectopic expression of oncogenic H-Ras V12 in immortalized human bronchial epithelial cells. We then find that small interfering RNA (siRNA) silencing of choline kinase expression in transformed HeLa cells completely abrogates the high concentration of phosphocholine, which in turn decreases phosphatidylcholine, phosphatidic acid and signaling through the MAPK and PI3K/AKT pathways. This simultaneous reduction in survival signaling markedly decreases the anchorage-independent survival of HeLa cells in soft agar and in athymic mice. Last, we confirm the relative importance of phosphatidic acid for this pro-survival effect as phosphatidic acid supplementation fully restores MAPK signaling and partially rescues HeLa cells from choline kinase inhibition. Taken together, these data indicate that the pooling of phosphocholine in cancer cells may be required to provide a ready supply of phosphatidic acid necessary for the feed-forward amplification of cancer survival signaling pathways.

Neoplastic cells transport large amounts of glucose in order to produce anabolic precursors and energy within the inhospitable environment of a tumor. The ras signaling pathway is activated in several cancers and has been found to stimulate glycolytic flux to lactate. Glycolysis is regulated by ras via the activity of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFK2/FBPase), which modulate the intracellular concentration of the allosteric glycolytic activator, fructose-2,6-bisphosphate (F2,6BP). We report herein that sequential immortalization and ras- transformation of mouse fibroblasts or human bronchial epithelial cells paradoxically decreases the intracellular concentration of F2,6BP. This marked reduction in the intracellular concentration of F2,6BP sensitizes transformed cells to the antimetabolic effects of PFK2/FBPase inhibition. Moreover, despite co-expression of all four mRNA species (PFKFB1-4), heterozygotic genomic deletion of the inducible PFKFB3 gene in ras -transformed mouse lung fibroblasts suppresses F2,6BP production, glycolytic flux to lactate, and growth as soft agar colonies or tumors in athymic mice. These data indicate that the PFKFB3 protein product may serve as an essential downstream metabolic mediator of oncogenic ras , and we propose that pharmacologic inhibition of this enzyme should selectively suppress the high rate of glycolysis and growth by cancer cells.

The tolerance of germinating rice seedlings to anaerobiosis cannot be fully accounted for by ethanolic fermentation alone. Nitrate metabolism (nitrate reduction to $\\mathrm{N}{\\mathrm{H}}_{4}^{+}$ plus its subsequent assimilation) may provide an additional sink mechanism for excess protons and NADH produced during anaerobiosis. To follow the fate of nitrate, 15N-labelled nitrate and ammonium incorporation in aerobic and anaerobic rice coleoptiles was examined using 15N-edited 1H NMR and gas chromatography-mass spectrometry methods. After 22 h of treatments, protein-free Ala, Glu, Gln, and γ-amino-butyrate were the main 15N-labelled products for both nitrate and ammonium-treated anaerobic rice coleoptiles, with Gln, Glu, and Ala being the most enriched. The total amount of 15N label incorporation into Ala and GAB increased significantly in response to anaerobiosis. The 15N-labelling pattern of Glu and Gln suggests that the GS/GOGAT system was primarily involved in ammonium assimilation whereas Glu dehydrogenase may play a role in nitrate assimilation. 15N incorporation into protein-derived amino acids was also significant and was more substantial in anaerobic than in aerobic rice coleoptiles, which indicate that protein biosynthesis remained active in anaerobic rice coleoptiles. Thus, anaerobic assimilation of inorganic N into amino acids, particularly Ala and Glu/GAB, may serve to supplement ethanolic fermentation in sustaining glycolysis and energy production in rice coleoptiles.

Cross-saturation experiments have been shown to give accurate information regarding the interacting surfaces in protein-protein and protein-RNA complexes. The rate of magnetization transfer depends on a number of factors including geometry, spin topology, and effective correlation times. To assess the influence of these variables on such experiments, and to determine the range of applicability of the technique, we have simulated the time-course of magnetization transfer across the interface in a variety of protein-nucleic acid complexes (434 Cro, SRY, MetJ and U1A), each having a different binding geometry. The simulations have been carried out primarily to provide information about the experimentally accessible targets for selective saturation, such as the anomeric protons and the imino protons of the nucleic acid. Saturation of either of these groups of signals leads to partial excitation throughout the nucleic acid molecule, and the resulting transfer of saturation to the labelled protein moiety can be readily detected by the reduction in intensity of particular peaks in the HSQC spectrum of the protein. The simulations show that information can be obtained about the residues in contact with the nucleic acid without any need for deuteration. Experimental cross-saturation data have been obtained from the Mbp1-DNA complex and interpreted in conjunction with the simulations to map out the binding surface in detail.

The 1H- and 13C-NMR spectra of antifreeze glycoprotein fractions 1–5 from Antarctic cod have been assigned, and the dynamics have been measured using 13C relaxation at two temperatures. The chemical shifts and absence of non-sequential 1H- 1H NOEs are inconsistent with a folded, compact structure. 13C relaxation measurements show that the protein has no significant long-range order, and that the local correlation times are adequately described by a random coil model. Hydroxyl protons of the sugar residues were observed at low temperature, and the presence of exchange-mediated ROEs to the sugar indicate extensive hydration. The conformational properties of AFGP1–5 are compared with those of the previously examined 14-mer analog AFGP8, which contains proline residues in place of some alanine residues (Lane, A. N., L. M. Hays, R. E. Feeney, L. M. Crowe, and J. H. Crowe. 1998. Protein Sci. 7:1555–1563). The infrared (IR) spectra of AFGP8 and AFGP1–5 in the amide I region are quite different. The presence of a wide distribution of backbone torsion angles in AFGP1–5 leads to a rich spectrum of frequencies in the IR spectrum, as interconversion among conformational states is slow on the IR frequency time scale. However, these transitions are fast on the NMR chemical shift time scales. The restricted motions for AFGP8 may imply a narrower distribution of possible ø, ψ angles, as is observed in the IR spectrum. This has significance for attempts to quantify secondary structures of proteins by IR in the presence of extensive loops.

Activated leukocyte cell adhesion molecule (ALCAM) plays an important role in cancer via its homotypical and heterotypical interactions with ALCAM or other proteins and can also mediate cell-cell interactions. The present study investigated the expression of ALCAM in relation to epithelial-to-mesenchymal transition (EMT) markers and its downstream signal proteins including Ezrin-Moesin-Radixin (ERM), in clinical colon cancer and in the progression of the disease. Expression of ALCAM was determined in a clinical colon cancer cohort and assessed against the clinical pathological factors and outcome, together with the expression patterns of the ERM family and EMT markers. ALCAM protein was detected using immunohistochemistry. Cell line models, with ALCAM knock-down and over-expression, were established and used to test cells' responses to drugs. Tumours from patients who had distant metastasis and died of colon cancer had low levels of ALCAM. Dukes B and C tumours also had lower ALCAM expression than Dukes A tumours. Patients with high levels of ALCAM had a significantly longer overall and disease-free survival than those with lower ALCAM levels (p=0.040 and p=0.044). ALCAM is not only significantly correlated with SNAI1 and TWIST, also positively correlated with SNAI2. ALCAM enhanced the adhesiveness of colorectal cancer, an effect inhibited by both sALCAM and SRC inhibitors. Finally, high ALCAM expression rendered cells resistant, especially to 5-fluorouracil. Reduced expression of ALCAM in colon cancer is an indicator of disease progression and a poor prognostic indicator for patient's survival. However, ALCAM can enhance the adhesion ability of cancer cells and render them resistant to chemotherapy drugs.

Shade coffee is a well-studied cultivation strategy that creates habitat for tropical birds while also maintaining agricultural yield. Although there is a general consensus that shade coffee is more “bird-friendly” than a sun coffee monoculture, little work has investigated the effects of specific shade tree species on insectivorous bird diversity. This study involved avian foraging observations, mist-netting data, temperature loggers, and arthropod sampling to investigate bottom-up effects of two shade tree taxa - native Cordia sp. and introduced Grevillea robusta - on insectivorous bird communities in central Kenya. Results indicate that foliage-dwelling arthropod abundance, and the richness and overall abundance of foraging birds were all higher on Cordia than on Grevillea. Furthermore, multivariate analyses of the bird community indicate a significant difference in community composition between the canopies of the two tree species, though the communities of birds using the coffee understorey under these shade trees were similar. In addition, both shade trees buffered temperatures in coffee, and temperatures under Cordia were marginally cooler than under Grevillea. These results suggest that native Cordia trees on East African shade coffee farms may be better at mitigating habitat loss and attracting insectivorous birds that could promote ecosystem services. Identifying differences in prey abundance and preferences in bird foraging behaviour not only fills basic gaps in our understanding of the ecology of East African coffee farms, it also aids in developing region-specific information to optimize functional diversity, ecosystem services, and the conservation of birds in agricultural landscapes.

Mbp1 is a transcription factor involved in the regulation of the cell cycle in yeast. The N-terminus of this protein contains a DNA binding domain that includes a winged helix-turn-helix motif. The C-terminal 24 residues of this domain (the 'tail') are disordered in the crystal state, but are important for DNA binding. We have measured 15N NMR relaxation rates at 11.75 and 14.1 T to determine the dynamics of the free protein and in its complex with a specific DNA duplex. The dynamics data were quantitatively analysed using both spectral density mapping and the Lipari-Szabo formalism including the effects of chemical exchange and rotational anisotropy. A detailed analysis has been made of the effect of anisotropy, exchange and experimental precision on the recovered motional parameters. The backbone NH relaxation is affected by motions on a variety of time scales from millisecond to tens of picoseconds. The relaxation data show a structured core of 100 residues corresponding to that observed in the crystal state. Within the core of the protein, two regions on either side of the putative recognition helix (helix B) show slow (ca. 0.2 ms) conformational exchange dynamics that are quenched upon DNA binding. The C-terminal 24 residues are generally more dynamic than in the core. However, in the free protein, a stretch of approximately 8 residues in the middle of the tail show relaxation behaviour similar to that in the core, indicating a structured region. NOEs between Ala 114 in this structured part of the tail and residues in the N-terminal beta strand of the core of the protein demonstrate that the tail folds back onto the core of the protein. In the complex with DNA, the structured part of the tail extends by ca. 3 residues. These data provide a framework for understanding the biochemical data on the mechanism and specificity of DNA binding.