The capacity of the -Proteobacterium Pseudomonas sp. strain P51, which degrades chlorinated benzenes, to metabolize 1,2,4-trichlorobenzene (TCB) under environmental conditions was tested by its release into two experimental systems. The first system consisted of laboratory scale microcosms which were operated with and without the addition of TCB and which were inoculated with sludge from a wastewater treatment plant. The second system consisted of a non sterile, water saturated soil column. We determined survival of strain P51 after its introduction and its ability to degrade TCB. The population dynamics was followed by selective plating and applying the polymerase chain reaction (PCR) to detect strain P51 and the chlorobenzene ( tcb) genes on catabolic plasmid pP51. The results showed a completely different behaviour of strain P51 in the two habitats under the applied conditions. In the soil column the P51 bacteria inoculated the entire area and their population reached 2 × 106 cells/g soil. The population remained active since TCB was degraded to concentrations below the detection limit of 30 g/l. In the sludge microcosms, the number of strain P51 cells immediately decreased from 4 × 107 cells/ml to 105 cells/ml over a period of 2 days after inoculation, and then the strain disappeared to levels below our detection limit (103–104 cells/ml). In the reactor without TCB the population of P51 maintained a stable value of 105 cells/ml during 8 days but then also decreased to levels below the detection limit. In addition, no significant TCB degradation was found in the sludge reactors. The influence of presence of TCB on maintenance of strain P51 in the two habitats is discussed. This work demonstrates the possibility to successfully apply preselected strains to degrade otherwise poorly degradable substances in complex mixed microbial communities. However, survival and activity may depend strongly on the type of system into which the strain is introduced. 相似文献
A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants. 相似文献
Four bacteriophages (C2, C2F, E3, and E16P) belonging to morphological group C3 and one belonging to morphological group A3 (E16B) were purified by deuterium oxide gradient centrifugation and cesium chloride gradient centrifugation. Morphological group C3 phages had a densityd=1.534–1.541 and group A3 phage (E16B) had a densityd=1.492 in CsCl. Phages of morphological group C3 isolated onEnterobacter sakazakii (C2, C2F) and onErwinia herbicola (E3, E16P) were compared withSalmonella newport phage 7-11 with respect to host-range, genome size, antigenic relatedness, and ultraviolet and heat susceptibility. Phages C2 and C2F could multiply inEnterobacter cloacae, E. sakazakii, Erwinia herbicola, E. rhapontici, andLevinea malonatica; whereas phages E3, E16P, and 7-11 could multiply on these same species and onEscherichia coli and severalSalmonella serotypes. Molecular weights of phage DNAs were determined to be 58×106 (C2), 60×106 (7-11), 67×106 (E3), and 39×106 (E16B).All studied phages of morphological group C3 (includingSalmonella newport phage 7-11) were neutralized by anti-phage C2 serum. Despite differences in neutralization kinetics and in ultraviolet and heat sensitivities, these phages of morphological group C3 constitute one phage species. Phage E16B (morphological group A3) had a host-range limited toEnterobacter cloacae, Erwinia herbicola, andE. rhapontici; it was antigenically unrelated to the preceding phage group C3, and showed ultraviolet and heat susceptibility close to that of coliphage T4. 相似文献
After stimulation with agonist, G protein coupled receptors (GPCR) undergo conformational changes that allow activation of G proteins to transduce the signal, followed by phosphorylation by kinases and arrestin binding to promote receptor internalization. Actual paradigm, based on a study of GPCR-A/rhodopsin family, suggests that a network of interactions between conserved residues located in transmembrane (TM) domains (mainly TM3, TM6 and TM7) is involved in the molecular switch leading to GPCR activation.
We evaluated in CHO cells expressing the VPAC1 receptor the role of the third transmembrane helix in agonist signalling by point mutation into Ala of the residues highly conserved in the secretin-family of receptors: Y224, N229, F230, W232, E236, G237, Y239, L240. N229A VPAC1 mutant was characterized by a decrease in both potency and efficacy of VIP stimulated adenylate cyclase activity, by the absence of agonist stimulated [Ca2+]i increase, by a preserved receptor recognition of agonists and antagonist and by a preserved sensitivity to GTP suggesting the importance of that residue for efficient G protein activation. N229D mutant was not expressed at the membrane, and the N229Q with a conserved mutation was less affected than the A mutant. Agonist stimulated phosphorylation and internalization of N229A and N229Q VPAC1 were unaffected. However, the re-expression of internalized mutant receptors, but not that of the wild type receptor, was rapidly reversed after VIP washing. Receptor phosphorylation, internalization and re-expression may be thus dissociated from G protein activation and linked to another active conformation that may influence its trafficking.
Mutation of that conserved amino acid in VPAC2 could be investigated only by a conservative mutation (N216Q) and led to a receptor with a low VIP stimulation of adenylate cyclase, receptor phosphorylation and internalization. This indicated the importance of the conserved N residue in the TM3 of that family of receptors. 相似文献
Six terrestrial ecosystems in the USA were exposed to elevated atmospheric CO(2) in single or multifactorial experiments for more than a decade to assess potential impacts. We retrospectively assessed soil bacterial community responses in all six-field experiments and found ecosystem-specific and common patterns of soil bacterial community response to elevated CO(2) . Soil bacterial composition differed greatly across the six ecosystems. No common effect of elevated atmospheric CO(2) on bacterial biomass, richness and community composition across all of the ecosystems was identified, although significant responses were detected in individual ecosystems. The most striking common trend across the sites was a decrease of up to 3.5-fold in the relative abundance of Acidobacteria Group 1 bacteria in soils exposed to elevated CO(2) or other climate factors. The Acidobacteria Group 1 response observed in exploratory 16S rRNA gene clone library surveys was validated in one ecosystem by 100-fold deeper sequencing and semi-quantitative PCR assays. Collectively, the 16S rRNA gene sequencing approach revealed influences of elevated CO(2) on multiple ecosystems. Although few common trends across the ecosystems were detected in the small surveys, the trends may be harbingers of more substantive changes in less abundant, more sensitive taxa that can only be detected by deeper surveys. Representative bacterial 16S rRNA gene clone sequences were deposited in GenBank with Accession No. JQ366086–JQ387568. 相似文献
Pregnane X receptor (PXR) agonism has been shown to affect multiple steps in both the synthesis and catabolism of HDL, but its integrated effect on HDL metabolism in vivo remains unclear. The aim of this study was to evaluate the net effect of PXR agonism on HDL metabolism in ApoE?3-Leiden (E3L) and E3L.CETP mice, well-established models for human-like lipoprotein metabolism. Female mice were fed a diet with increasing amounts of the potent PXR agonist 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN). In E3L and E3L.CETP mice, PCN increased liver lipids as well as plasma cholesterol and triglycerides. However, whereas PCN increased cholesterol contained in large HDL-1 particles in E3L mice, it dose-dependently decreased HDL-cholesterol in E3L.CETP mice, indicating that CETP expression dominates the effect of PCN on HDL metabolism. Analysis of the hepatic expression of genes involved in HDL metabolism showed that PCN decreased expression of genes involved in HDL synthesis (Abca1, Apoa1), maturation (Lcat, Pltp) and clearance (Sr-b1). The HDL-increasing effect of PCN, observed in E3L mice, is likely caused by a marked decrease in hepatic SR-BI protein expression, and completely reversed by CETP expression. We conclude that chronic PXR agonism dose-dependently reduces plasma HDL-cholesterol in the presence of CETP. 相似文献
Metabolism is a highly interconnected web of chemical reactions that power life. Though the stoichiometry of metabolism is well understood, the multidimensional aspects of metabolic regulation in time and space remain difficult to define, model and engineer. Complex metabolic conversions can be performed by multiple species working cooperatively and exchanging metabolites via structured networks of organisms and resources. Within cells, metabolism is spatially regulated via sequestration in subcellular compartments and through the assembly of multienzyme complexes. Metabolic engineering and synthetic biology have had success in engineering metabolism in the first and second dimensions, designing linear metabolic pathways and channeling metabolic flux. More recently, engineering of the third dimension has improved output of engineered pathways through isolation and organization of multicell and multienzyme complexes. This review highlights natural and synthetic examples of three-dimensional metabolism both inter- and intracellularly, offering tools and perspectives for biological design. 相似文献