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141.
Jens Buchholz Andreas Schwentner Britta Brunnenkan Christina Gabris Simon Grimm Robert Gerstmeir Ralf Takors Bernhard J. Eikmanns Bastian Blombach 《Applied and environmental microbiology》2013,79(18):5566-5575
Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a YP/S of 0.24 mol per mol of glucose and a QP of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum
l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products. 相似文献
142.
Jessica Varela Villarreal Christina JungferUrsula Obst Thomas Schwartz 《Journal of microbiological methods》2013
Molecular techniques, such as polymerase chain reaction (PCR) and quantitative PCR (qPCR), are very sensitive, but may detect total DNA present in a sample, including extracellular DNA (eDNA) and DNA coming from live and dead cells. DNase I is an endonuclease that non-specifically cleaves single- and double-stranded DNA. This enzyme was tested in this study to analyze its capacity of digesting DNA coming from dead cells with damaged cell membranes, leaving DNA from living cells with intact cell membranes available for DNA-based methods. For this purpose, an optimized DNase I/Proteinase K (DNase/PK) protocol was developed. Intact Staphylococcus aureus cells, heat-killed Pseudomonas aeruginosa cells, free genomic DNA of Salmonella enterica, and a mixture of these targets were treated according to the developed DNase/PK protocol. In parallel, these samples were treated with propidium monoazide (PMA) as an already described assay for live-dead discrimination. Quantitative PCR and PCR-DGGE of the eubacterial 16S rDNA fragment were used to test the ability of the DNase/PK and PMA treatments to distinguish DNA coming from cells with intact cell membranes in the presence of DNA from dead cells and free genomic DNA. The methods were applied to three months old autochthonous drinking water biofilms from a pilot facility built at a German waterworks. Shifts in the DNA patterns observed after DGGE analysis demonstrated the applicability of DNase/PK as well as of the PMA treatment for natural biofilm investigation. However, the DNase/PK treatment demonstrated some practical advantages in comparison with the PMA treatment for live/dead discrimination of bacterial targets in drinking water systems. 相似文献
143.
Pawan K. Vohra Michael A. Thompson Venkatachalem Sathish Alexander Kiel Calvin Jerde Christina M. Pabelick Brij B. Singh Y.S. Prakash 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2013,1833(12):2953-2960
Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca2 + signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca2 + entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP–BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20 ng/ml, 48 h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10 μM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca2 + (EGTA; 1 mM) or intracellular Ca2 + (BAPTA; 5 μM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca2 + influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca2 + and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma. 相似文献
144.
Catherine Jarrett Mateja Lekic Christina L. Smith Carolina M. Pusec Karen L. Sweazea 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2013,183(7):959-967
For mammals, acetylcholine (ACh) promotes endothelium-dependent vasodilation primarily through nitric oxide (NO) and prostaglandin-mediated pathways, with varying reliance on endothelial-derived hyperpolarizing factors. Currently, no studies have been conducted on small systemic arteries from wild birds. We hypothesized that ACh-mediated vasodilation of isolated small arteries from mourning doves (Zenaida macroura) would likewise depend on endothelial-derived factors. Small resistance mesenteric and cranial tibial (c. tibial) arteries (80–150 μm, inner diameter) were cannulated and pre-constricted to 50 % of resting inner diameter with phenylephrine then exposed to increasing concentrations of ACh (10?9–10?5 M) or the NO donor, sodium nitroprusside (SNP; 10?12–10?3 M). For mesenteric arteries, ACh-mediated vasodilation was significantly blunted with the potassium channel antagonist tetraethylammonium chloride (TEA, 10 mM); whereas responses were only moderately impaired with endothelial disruption or inhibition of prostaglandins (indomethacin, 10 μM). In contrast, endothelial disruption as well as exposure to TEA largely abolished vasodilatory responses to ACh in c. tibial arteries while no effect of prostaglandin inhibition was observed. For both vascular beds, responses to ACh were moderately dependent on the NO signaling pathway. Inhibition of NO synthase had no impact, despite complete reversal of phenylephrine-mediated tone with SNP, whereas inhibition of soluble guanylate cyclase (sGC) caused minor impairments. Endothelium-independent vasodilation also relied on potassium channels. In summary, ACh-mediated vasodilation of mesenteric and c. tibial arteries occurs through the activation of potassium channels to induce hyperpolarization with moderate reliance on sGC. Prostaglandins likewise play a small role in the vasodilatory response to ACh in mesenteric arteries. 相似文献
145.
Lepanthes variabilis, a new species from Costa Rica, is described and illustrated. It is endemic to the northern part of the Talamanca mountain range, from 2750–2850 m. It is compared to the similar L. minutilabia, which also occurs in Costa Rica. 相似文献
146.
CD44 Receptor Unfolding Enhances Binding by Freeing Basic Amino Acids to Contact Carbohydrate Ligand
The extracellular carbohydrate-binding domain of the Type I transmembrane receptor CD44 is known to undergo affinity switching, where change in conformation leads to enhanced binding of its carbohydrate ligand hyaluronan. Separate x-ray crystallographic and NMR experiments have led to competing explanations, with the former supporting minor conformational changes at the binding site and the latter a major order-to-disorder unfolding transition distant from the binding site. Here, all-atom explicit-solvent molecular dynamics studies employing adaptive biasing force sampling revealed a substantial favorable free-energy change associated with contact formation between the Arg41 side chain and hyaluronan at the binding site, independent of whether the distant site was ordered or disordered. Analogous computational experiments on Arg41Ala mutants showed loss of this favorable free-energy change, consistent with existing experimental data. More provocatively, the simulation data revealed the molecular mechanism by which the order-to-disorder transition enhances hyaluronan binding: in the disordered state, a number of basic residues gain sufficient conformational freedom—lacking in the ordered state—to spontaneously form side-chain contacts with hyaluronan. Mutation of these residues to Ala had been known to decrease binding affinity, but there had previously been no structural explanation, given their lack of proximity to the carbohydrate-binding site in existing structures of the complex. 相似文献
147.
148.
149.
Pollen of the genus Passerina L. differs markedly from that of other southern African members of the Thymelaeaceae. Grains of most members of the Thymelaeaceae are characterised by a typical croton pattern, comprising rings of more or less trihedral sexine units mounted on an underlying reticulum of circular muri. In Passerina, however, the supratectal subunits are fused completely to form a continuous reticulum, which replaces the underlying reticulum. The reticulum in Passerina is therefore secondary in origin and not homologous with the basal reticulum of typical crotonoid grains in the family. The croton pattern has often been used as indication of a possible relationship between the Euphorbiaceae and Thymelaeaceae. Pollen of Passerina is adapted to anemophily. Grain sculpturing clearly demonstrates secondary derivation of a reticulate pattern from the typical croton pattern, through reduction, aggregation and fusion. Pollen of Passerina represents a climax of a continuum of variation in the exine of pollen in the Thymelaeaceae. As Passerina is considered phylogenetically advanced in the subfamily Thymelaeoideae, the subtribe Passerininae is raised to tribal rank, namely tribe Passerineae. 相似文献
150.
Christopher E. Cornwall Christopher D. Hepburn Christina M. McGraw Kim I. Currie Conrad A. Pilditch Keith A. Hunter Philip W. Boyd Catriona L. Hurd 《Proceedings. Biological sciences / The Royal Society》2013,280(1772)
Coastal ecosystems that are characterized by kelp forests encounter daily pH fluctuations, driven by photosynthesis and respiration, which are larger than pH changes owing to ocean acidification (OA) projected for surface ocean waters by 2100. We investigated whether mimicry of biologically mediated diurnal shifts in pH—based for the first time on pH time-series measurements within a kelp forest—would offset or amplify the negative effects of OA on calcifiers. In a 40-day laboratory experiment, the calcifying coralline macroalga, Arthrocardia corymbosa, was exposed to two mean pH treatments (8.05 or 7.65). For each mean, two experimental pH manipulations were applied. In one treatment, pH was held constant. In the second treatment, pH was manipulated around the mean (as a step-function), 0.4 pH units higher during daylight and 0.4 units lower during darkness to approximate diurnal fluctuations in a kelp forest. In all cases, growth rates were lower at a reduced mean pH, and fluctuations in pH acted additively to further reduce growth. Photosynthesis, recruitment and elemental composition did not change with pH, but δ13C increased at lower mean pH. Including environmental heterogeneity in experimental design will assist with a more accurate assessment of the responses of calcifiers to OA. 相似文献