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A high throughput toxicity biosensor has been designed and constructed using recombinant Escherichia coli cells, containing stress specific promoters (recA, fabA, or katG) or constitutive promoters (lac) fused to luciferase genes originating from Vibrio fisheri. These genetically engineered cells were immobilized in 96 well plates. By optimizing cell immobilization conditions and the strains' response specificity to toxic chemicals, bioluminescent outputs decreased or increased dose-dependently upon adding test chemicals. However, to date the toxicity data obtained using this biosensor have not been compared with the results of other toxicity tests. Phenolics were chosen to evaluate the correlation between the LD50 and the EC50 (GC2) or EC120 (DPD2540) of Daphnia magna and E. coli, respectively. Toxicity data obtained from constitutive strains by bioluminescent level decrements were compared with the results from D. magna as a standard. LD50 values were used as parameters of D. magna toxicity and EC50 of EC120 values were used for the immobilized biosensor. In the DPD2540 test, phenolics, membrane damaging toxic chemicals, for testing immobilized stress specific bacterial strains trigger dose-dependant bioluminescence increase within specific concentration. Although the stress specific responsiveness from the strains could not be compared with D. magna's LD50 values, these responses offer additional information, such as upon the mode of toxic action in the sample, in addition to the cellular toxicity results as indicated by the EC50. This novel high throughput toxicity biosensor can be implemented to investigate the toxicity of any other soluble materials, and can be used as a standardization tool for the evaluation of toxicity. 相似文献
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The nature of bombesin-like immunoreactive peptides was studied in extracts of small cell carcinoma of the human lung. Three peaks, I, II and III, designated by their increasing retention times, were separated by reversed-phase high performance liquid chromatography (HPLC) with trifluoroacetic acid (TFA) as counter ion. None of the peaks corresponded to bombesin. Peak III was eluted at the same position as porcine gastrin releasing peptide (GRP) but was separated from it in another reversed-phase system using heptafluorobutyric acid (HFBA). Peak II material eluted in the position of bombesin in the HFBA system but not in the TFA system. The elution position of Peak I corresponded to that of the carboxyl terminal fragments of GRP, i.e. GRP18-27 and GRP19-27. This correspondence was observed in each of the reversed-phase and gel filtration systems used. The Peak III peptide was converted to peak I after incubation with trypsin. It was reasoned that this conversion could be one of the steps in the processing of bombesin-like peptides in human small cell carcinoma. 相似文献
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The genetically-engineeredEscherichia coli strain, DPD2540, which contains afabA::luxCDABE fusion gene, gives a bioluminescent output when membrane fatty acid synthesis is needed. For more practical application of
this strain in the field as biosensor, freeze-drying was adopted. A 12% sucrose solution with Luria-Bertani (LB) broth, as
determined by the viability after freeze-drying, was found to be the most effective composition for lyophilization solution
among various compositions tested. Rapid freezing with liquid nitrogen also gave the best viability after freeze-drying as
compared to samples frozen at −70°C and −20°C. The biosensing activities of the cells showed a greater sensitivity when the
cells from the exponential phase were freeze-dried. Finally, the optimum temperature for use of the freeze-dried cells in
the biosensor field was determined. 相似文献
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Magdalena A. Gutowska F. Melzner M. Langenbuch C. Bock G. Claireaux H. O. Pörtner 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2010,180(3):323-335
Acidification of ocean surface waters by anthropogenic carbon dioxide (CO2) emissions is a currently developing scenario that warrants a broadening of research foci in the study of acid–base physiology.
Recent studies working with environmentally relevant CO2 levels, indicate that some echinoderms and molluscs reduce metabolic rates, soft tissue growth and calcification during hypercapnic
exposure. In contrast to all prior invertebrate species studied so far, growth trials with the cuttlefish Sepia officinalis found no indication of reduced growth or calcification performance during long-term exposure to 0.6 kPa CO2. It is hypothesized that the differing sensitivities to elevated seawater pCO2 could be explained by taxa specific differences in acid–base regulatory capacity. In this study, we examined the acid–base
regulatory ability of S. officinalis in vivo, using a specially modified cannulation technique as well as 31P NMR spectroscopy. During acute exposure to 0.6 kPa CO2, S. officinalis rapidly increased its blood [HCO3
−] to 10.4 mM through active ion-transport processes, and partially compensated the hypercapnia induced respiratory acidosis.
A minor decrease in intracellular pH (pHi) and stable intracellular phosphagen levels indicated efficient pHi regulation. We conclude that S. officinalis is not only an efficient acid–base regulator, but is also able to do so without disturbing metabolic equilibria in characteristic
tissues or compromising aerobic capacities. The cuttlefish did not exhibit acute intolerance to hypercapnia that has been
hypothesized for more active cephalopod species (squid). Even though blood pH (pHe) remained 0.18 pH units below control values,
arterial O2 saturation was not compromised in S. officinalis because of the comparatively lower pH sensitivity of oxygen binding to its blood pigment. This raises questions concerning
the potentially broad range of sensitivity to changes in acid–base status amongst invertebrates, as well as to the underlying
mechanistic origins. Further studies are needed to better characterize the connection between acid–base status and animal
fitness in various marine species. 相似文献
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