Catabolic regulation analysis of Escherichia coli and its crp, mlc, mgsA, pgi and ptsG mutants

<p>Abstract</p> <p>Background</p> <p>Most bacteria can use various compounds as carbon sources. These carbon sources can be either co-metabolized or sequentially metabolized, where the latter phenomenon typically occurs as catabolite repression. From the practical application point of view of utilizing lignocellulose for the production of biofuels etc., it is strongly desirable to ferment all sugars obtained by hydrolysis from lignocellulosic materials, where simultaneous consumption of sugars would benefit the formation of bioproducts. However, most organisms consume glucose prior to consumption of other carbon sources, and exhibit diauxic growth. It has been shown by fermentation experiments that simultaneous consumption of sugars can be attained by <it>ptsG, mgsA </it>mutants etc., but its mechanism has not been well understood. It is strongly desirable to understand the mechanism of metabolic regulation for catabolite regulation to improve the performance of fermentation.</p> <p>Results</p> <p>In order to make clear the catabolic regulation mechanism, several continuous cultures were conducted at different dilution rates of 0.2, 0.4, 0.6 and 0.7 h^-1using wild type <it>Escherichia coli</it>. The result indicates that the transcript levels of global regulators such as <it>crp, cra, mlc </it>and <it>rpoS </it>decreased, while those of <it>fadR, iclR, soxR/S </it>increased as the dilution rate increased. These affected the metabolic pathway genes, which in turn affected fermentation result where the specific glucose uptake rate, the specific acetate formation rate, and the specific CO₂evolution rate (CER) were increased as the dilution rate was increased. This was confirmed by the ¹³C-flux analysis. In order to make clear the catabolite regulation, the effect of <it>crp </it>gene knockout (Δ<it>crp</it>) and crp enhancement (<it>crp⁺</it>) as well as <it>mlc, mgsA, pgi </it>and <it>ptsG </it>gene knockout on the metabolism was then investigated by the continuous culture at the dilution rate of 0.2 h^-1and by some batch cultures. In the case of Δ<it>crp </it>(and also Δ<it>mlc</it>) mutant, TCA cycle and glyoxylate were repressed, which caused acetate accumulation. In the case of <it>crp⁺</it>mutant, glycolysis, TCA cycle, and gluconeogenesis were activated, and simultaneous consumption of multiple carbon sources can be attained, but the glucose consumption rate became less due to repression of <it>ptsG </it>and <it>ptsH </it>by the activation of Mlc. Simultaneous consumption of multiple carbon sources could be attained by <it>mgsA, pgi</it>, and <it>ptsG </it>mutants due to increase in <it>crp </it>as well as <it>cyaA</it>, while glucose consumption rate became lower.</p> <p>Conclusions</p> <p>The transcriptional catabolite regulation mechanism was made clear for the wild type <it>E. coli</it>, and its <it>crp, mlc, ptsG, pgi, and mgsA </it>gene knockout mutants. The results indicate that catabolite repression can be relaxed and <it>crp </it>as well as <it>cyaA </it>can be increased by <it>crp⁺, mgsA, pgi</it>, and <it>ptsG </it>mutants, and thus simultaneous consumption of multiple carbon sources including glucose can be made, whereas the glucose uptake rate became lower as compared to wild type due to inactivation of <it>ptsG </it>in all the mutants considered.</p

Identification of Zebrafish Fxyd11a Protein that is Highly Expressed in Ion-Transporting Epithelium of the Gill and Skin and its Possible Role in Ion Homeostasis

FXYD proteins, small single-transmembrane proteins, have been proposed to be auxiliary regulatory subunits of Na+–K+-ATPase and have recently been implied in ion osmoregulation of teleost fish. In freshwater (FW) fish, numerous ions are actively taken up through mitochondrion-rich cells (MRCs) of the gill and skin epithelia, using the Na+ electrochemical gradient generated by Na+–K+-ATPase. In the present study, to understand the molecular mechanism for the regulation of Na+–K+-ATPase in MRCs of FW fish, we sought to identify FXYD proteins expressed in MRCs of zebrafish. Reverse-transcriptase PCR studies of adult zebrafish tissues revealed that, out of eight fxyd genes found in zebrafish database, only zebrafish fxyd11 (zfxyd11) mRNA exhibited a gill-specific expression. Double immunofluorescence staining showed that zFxyd11 is abundantly expressed in MRCs rich in Na+–K+-ATPase (NaK-MRCs) but not in those rich in vacuolar-type H+-transporting ATPase. An in situ proximity ligation assay demonstrated its close association with Na+–K+-ATPase in NaK-MRCs. The zfxyd11 mRNA expression was detectable at 1 day postfertilization, and its expression levels in the whole larvae and adult gills were regulated in response to changes in environmental ionic concentrations. Furthermore, knockdown of zFxyd11 resulted in a significant increase in the number of Na+–K+-ATPase–positive cells in the larval skin. These results suggest that zFxyd11 may regulate the transport ability of NaK-MRCs by modulating Na+–K+-ATPase activity, and may be involved in the regulation of body fluid and electrolyte homeostasis

Molecular-beam epitaxial growth of a far-infrared transparent electrode for extrinsic Germanium photoconductors

We have evaluated the optical and electrical properties of a far-infrared (IR) transparent electrode for extrinsic germanium (Ge) photoconductors at 4 K, which was fabricated by molecular beam epitaxy (MBE). As a far-IR transparent electrode, an aluminum (Al)-doped Ge layer is formed at well-optimized doping concentration and layer thickness in terms of the three requirements: high far-IR transmittance, low resistivity, and excellent ohmic contact. The Al-doped Ge layer has the far-IR transmittance of >95 % within the wavelength range of 40--200 microns, while low resistivity (~5 ohm-cm) and ohmic contact are ensured at 4 K. We demonstrate the applicability of the MBE technology in fabricating the far-IR transparent electrode satisfying the above requirements.Comment: 18 pages, 7 figures, accepted for publication in the PAS

Characterization of stability of benchmark organic photovoltaic films after proton and electron bombardments

Organic solar cells have attractive potential for space applications as they have very high specific power (power generated per weight) and ultra-high flexibility (to reduce stowed volume). However, one critical issue is whether they are stable under the harsh space environment, particularly their stability under high energy, high flux, electron and proton bombardment. In this paper, the stability of benchmark organic photovoltaic layers under proton bombardment (150 keV with a fluence of 1 × 1012/cm2) and electron bombardment (1 MeV with a fluence of 1 × 1013/cm2) under vacuum is investigated. Raman spectroscopy, photoluminescence spectroscopy, and optical reflectance spectroscopy are applied to study their chemical/structural, photo-chemical/morphological, and optical stability after the bombardments. The results show that all the benchmark organic photovoltaic films are stable under the radiation, implying that organic solar cells could be feasible for space applications

Molecular Identification of t(w5): Vps52 Promotes Pluripotential Cell Differentiation Through Cell-Cell Interactions

After implantation, pluripotent epiblasts are converted to embryonic ectoderm through cell-cell interactions that significantly change the transcriptional and epigenetic networks. An entree to understanding this vital developmental transition is the t(w5) mutation of the mouse t complex. This mutation produces highly specific defects in the embryonic ectoderm before gastrulation, leading to death of the embryonic ectoderm. Using a positional cloning approach, we have now identified the mutated gene, completing a decades-long search. The gene, vacuolar protein sorting 52 (Vps52), is a mouse homolog of yeast VPS52 that is involved in the retrograde trafficking of endosomes. Our data suggest that Vps52 acts in extraembryonic tissues to support the growth and differentiation of embryonic ectoderm via cell-cell interactions. It is also required in the formation of embryonic structures at a later stage of development, revealing hitherto unknown functions of Vps52 in the development of a multicellular organism.NSFCellular and Molecular Biolog

Aberrant Glycogen Synthase Kinase 3β Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

Background and Purpose: The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer. Methods: Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined. Results: Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts. Conclusion: The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer. © 2013 Kitano et al

Delphi Method Consensus-Based Identification of Primary Trauma Care Skills Required for General Surgeons in Japan

Purpose General surgeons at regional hospitals should have the primary trauma care skills necessary to treat critically ill trauma patients to withstand transfer. This study was conducted to identify a consensus on primary trauma care skills for general surgeons. Methods An initial list of acute care surgical skills was compiled, and revised by six trauma experts (acute care surgeons); 33 skills were nominated for inclusion in the Delphi consensus survey. Participants (councilors of the Japanese Society for Acute Care Surgery) were presented with the list of 33 trauma care skills and were asked (using web-based software) to rate how strongly they agreed or disagreed (using a 5-point Likert scale) with the necessity of each skill for a general surgeon. The reliability of consensus was predefined as Cronbach’s α ≥ 0.8, and trauma care skills were considered as primarily required when rated 4 (agree) or 5 (strongly agree) by ≥ 80% participants. Results There were 117 trauma care specialists contacted to participate in the Delphi consensus survey panel. In the 1st round, 85 specialists participated (response rate: 72.6%). In the 2nd round, 66 specialists participated (response rate: 77.6%). Consensus was achieved after two rounds, reliability using Cronbach’s α was 0.94, and 34 items were identified as primary trauma care skills needed by general surgeons. Conclusion A consensus-based list of trauma care skills required by general surgeons was developed. This list can facilitate the development of a new trauma training course which has been optimized for general surgeons