Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter⁻¹ acetate during fermentation of 114 g liter⁻¹ glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter⁻¹, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter⁻¹ and raised the ethanol yield to 7% above the wild-type level.     

A splice variant of the Drosophila vesicular monoamine transporter contains a conserved trafficking domain and functions in the storage of dopamine, serotonin, and octopamine

Vesicular monoamine transporters (VMATs) mediate the transport of dopamine (DA), serotonin (5HT), and other monoamines into secretory vesicles. The regulation of mammalian VMAT and the related vesicular acetylcholine transporter (VAChT) has been proposed to involve membrane trafficking, but the mechanisms remain unclear. To facilitate a genetic analysis of vesicular transporter function and regulation, we have cloned the Drosophila homolog of the vesicular monoamine transporter (dVMAT). We identify two mRNA splice variants (DVMAT-A and B) that differ at their C-terminus, the domain responsible for endocytosis of mammalian VMAT and VAChT. DVMAT-A contains trafficking motifs conserved in mammals but not C. elegans, and internalization assays indicate that the DVMAT-A C-terminus is involved in endocytosis. DVMAT-B contains a divergent C-terminal domain and is less efficiently internalized from the cell surface. Using in vitro transport assays, we show that DVMAT-A recognizes DA, 5HT, octopamine, tyramine, and histamine as substrates, and similar to mammalian VMAT homologs, is inhibited by the drug reserpine and the environmental toxins 2,2,4,5,6-pentachlorobiphenyl and heptachlor. We have developed a specific antiserum to DVMAT-A, and find that it localizes to dopaminergic and serotonergic neurons as well as octopaminergic, type II terminals at the neuromuscular junction. Surprisingly, DVMAT-A is co-expressed at type II terminals with the Drosophila vesicular glutamate transporter. Our data suggest that DVMAT-A functions as a vesicular transporter for DA, 5HT, and octopamine in vivo, and will provide a powerful invertebrate model for the study of transporter trafficking and regulation.     

Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.     

Alien plant species composition and associations with anthropogenic disturbance in North American forests

A probability-based sampling scheme was used to survey plant species composition in forests of 16 states in seven geopolitical regions of the United States (California, Colorado, Minnesota, and parts of the Pacific Northwest, Southeast, Mid-Atlantic, and Northeast) in 1994. The proportion of alien species relative to the total species number and to canopy cover in the ground stratum (0–0.6 m height) was evaluated in 279 plots. Visually evident anthropogenic disturbances (e.g., artificial regeneration, logging, prescribed burning, and grazing by livestock), if any, were recorded on each plot. In each of the seven regions we quantified (1) the percentage of the number of species and total cover comprised of alien species, (2) the difference in these percentages for disturbed and undisturbed plots, and (3) the origin or native range for the alien species.The percentage of alien species ranged from approximately 4.5% (Colorado) to approximately 13.2% (California). The percentage of alien species cover ranged from approximately 1.5% in Colorado to 25% in California. In five regions, species introduced from temperate Eurasia comprised the largest proportion of alien species and cover. In the Southeast, species introduced from far eastern and subtropical Asia dominated the alien flora. In the Mid-Atlantic, the majority of alien species was Eurasian and the majority of alien species cover consisted of far eastern and subtropical Asian species.The proportion of plots in which at least one alien species was recorded was significantly higher in disturbed than undisturbed plots in the Southeast and marginally significantly higher ($p=0.053$) in the Northeast. These results are consistent with other published studies that indicate that anthropogenic disturbance affects the structure and composition of both the ground stratum and upper canopy of forest habitats. In other regions, however, no significant differences were found.     

The MHC class I-like IgG receptor controls perinatal IgG transport,IgG homeostasis,and fate of IgG-Fc-coupled drugs

Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t(1/2) compared with Abs of other isotypes. Circumstantial evidence suggests that the MHC class I-related protein, the neonatal FcR (FcRn), is the FcR responsible for both in vivo functions. To understand the phenotypes imposed by FcRn, we produced and analyzed mice with a defective FcRn gene. The results provide direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis, essential for generating a potent IgG response to foreign Ags, and the basis of enhanced efficacy of Fc-IgG-based therapeutics. FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease, and improving drug efficacy.     

A Key Role of microRNA-29b for the Suppression of Colon Cancer Cell Migration by American Ginseng

Metastasis of colon cancer cells increases the risk of colon cancer mortality. We have recently shown that American ginseng prevents colon cancer, and a Hexane extract of American Ginseng (HAG) has particularly potent anti-inflammatory and anti-cancer properties. Dysregulated microRNA (miR) expression has been observed in several disease conditions including colon cancer. Using global miR expression profiling, we observed increased miR-29b in colon cancer cells following exposure to HAG. Since miR-29b plays a role in regulating the migration of cancer cells, we hypothesized that HAG induces miR-29b expression to target matrix metalloproteinase-2 (MMP-2) thereby suppressing the migration of colon cancer cells. Results are consistent with this hypothesis. Our study supports the understanding that targeting MMP-2 by miR-29b is a mechanism by which HAG suppresses the migration of colon cancer cells.     

Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

The melibiose permease of Salmonella typhimurium (MelB_St) catalyzes the stoichiometric symport of galactopyranoside with a cation (H⁺, Li⁺, or Na⁺) and is a prototype for Na⁺-coupled major facilitator superfamily (MFS) transporters presenting from bacteria to mammals. X-ray crystal structures of MelB_St have revealed the molecular recognition mechanism for sugar binding; however, understanding of the cation site and symport mechanism is still vague. To further investigate the transport mechanism and conformational dynamics of MelB_St, we generated a complete single-Cys library containing 476 unique mutants by placing a Cys at each position on a functional Cys-less background. Surprisingly, 105 mutants (22%) exhibit poor transport activities (<15% of Cys-less transport), although the expression levels of most mutants were comparable to that of the control. The affected positions are distributed throughout the protein. Helices I and X and transmembrane residues Asp and Tyr are most affected by cysteine replacement, while helix IX, the cytoplasmic middle-loop, and C-terminal tail are least affected. Single-Cys replacements at the major sugar-binding positions (K18, D19, D124, W128, R149, and W342) or at positions important for cation binding (D55, N58, D59, and T121) abolished the Na⁺-coupled active transport, as expected. We mapped 50 loss-of-function mutants outside of these substrate-binding sites that suffered from defects in protein expression/stability or conformational dynamics. This complete Cys-scanning mutagenesis study indicates that MelB_St is highly susceptible to single-Cys mutations, and this library will be a useful tool for further structural and functional studies to gain insights into the cation-coupled symport mechanism for Na⁺-coupled MFS transporters.     

Macroalgae and nutrients promote algal turf growth in the absence of herbivores

Coral Reefs - Closely cropped algal turfs are characteristic of healthy coral reefs, but unchecked growth can cause transitions into long sediment-laden turfs, which may be an alternative degraded...