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981.
Inhibition of starch synthesis results in overproduction of lipids in Chlamydomonas reinhardtii 总被引:1,自引:0,他引:1
Yantao Li Danxiang Han Guangrong Hu Milton Sommerfeld Qiang Hu 《Biotechnology and bioengineering》2010,107(2):258-268
Starch and neutral lipids are two major carbon storage compounds in many microalgae and plants. Lipids are more energy rich and have often been used as food and fuel feedstocks. Genetic engineering of the lipid biosynthesis pathway to overproduce lipid has achieved only limited success. We hypothesize that through blocking the competing pathway to produce starch, overproduction of neutral lipid may be achieved. This hypothesis was tested using the green microalga Chlamydomonas reinhardtii and its low starch and starchless mutants. We discovered that a dramatic increase in neutral lipid content and the neutral lipid/total lipid ratio occurred among the mutants under high light and nitrogen starvation. BAFJ5, one of the mutants defective in the small subunit of ADP‐glucose pyrophosphorylase, accumulated neutral and total lipid of up to 32.6% and 46.4% of dry weight (DW) or 8‐ and 3.5‐fold higher, respectively, than the wild‐type. These results confirmed the feasibility of increasing lipid production through redirecting photosynthetically assimilated carbon away from starch synthesis to neutral lipid synthesis. However, some growth impairment was observed in the low starch and starchless mutants, possibly due to altered energy partitioning in PSII, with more excitation energy dissipated as heat and less to photochemical conversion. This study demonstrated that biomass and lipid production by the selected mutants can be improved by physiological manipulation. Biotechnol. Bioeng. 2010;107: 258–268. © 2010 Wiley Periodicals, Inc. 相似文献
982.
Tatyana B. Eronina Natalia A. Chebotareva Sergey Yu. Kleymenov Svetlana G. Roman Valentina F. Makeeva Boris I. Kurganov 《Biopolymers》2010,93(11):986-993
The study of the kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscles by dynamic light scattering at 48°C showed that 2‐hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) accelerated the aggregation process and induced the formation of the larger protein aggregates. The reason of the accelerating effect of HP‐β‐CD is destabilization of the protein molecule under action of HP‐β‐CD. This conclusion was supported by the data on differential scanning calorimetry and the kinetic data on thermal inactivation of Phb. It is assumed that destabilization of the Phb molecule is due to preferential binding of HP‐β‐CD to intermediates of protein unfolding in comparison with the original native state. The conclusion regarding the ability of the native Phb for binding of HP‐β‐CD was substantiated by the data on the enzyme inhibition by HP‐β‐CD. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 986–993, 2010. 相似文献
983.
Natalia Gomez Abigail R. Vinson Jun Ouyang Mary D.H. Nguyen Xiaoying‐Nancy Chen Vikas K. Sharma Inn H. Yuk 《Biotechnology and bioengineering》2010,105(4):748-760
THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species—Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) production. In studies covering 12 THIOMABs produced by 66 stable cell lines, increased free LC polypeptide expression—evaluated as the ratio of mRNA encoding for LC to the mRNA encoding for heavy chain (HC)—correlated with increased 3LC levels. The amino acid sequence of the THIOMAB molecule also impacted its susceptibility to 3LC formation: hydrophilic LC polypeptides showed elevated 3LC levels. Finally, increased GSH production—evaluated as the ratio of the cell‐specific production rate of GSH (qGSH) to the cell‐specific production rate of THIOMAB (qp)—corresponded to decreased 3LC levels. In time‐lapse studies, changes in extracellular 3LC levels during cell culture corresponded to changes in mRNA LC/HC ratio and qGSH/qp ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high qGSH/qp ratio yielded the lowest levels of 3LC. These findings provide us with factors to consider in selecting a cell line to produce THIOMABs with minimal levels of the 3LC impurity. Biotechnol. Bioeng. 2010. 105: 748–760. © 2009 Wiley Periodicals, Inc. 相似文献
984.
Abolfazl Barzegar Ali A. Moosavi-Movahedi Saman Hosseini Ashtiani 《Carbohydrate research》2010,345(2):243-249
The chaperone action of α-cyclodextrin (α-CyD), based on providing beneficial microenvironment of hydrophobic nanocavity to form molecular complex with alcohol dehydrogenase (ADH) was examined by experimental and computational techniques. The results of UV-vis and dynamic light scattering (DLS) indicated that the chaperone-like activity of α-CyD depends on molecular complex formation between α-CyD and ADH, which caused to decrease the amount and size of polymerized molecules. Computational calculations of molecular dynamic (MD) simulations and blind docking (BD) demonstrated that α-CyD acts as an artificial chaperone because of its high affinity to the region of ADH’s two chains interface. The hydrophobic nanocavity of α-CyD has the ability to form inclusion complex due to the presence of phenyl ring of aromatic phenylalanine (Phe) residue in the dimeric intersection area. Delocalization of ADH subunits, which causes the exposure of Phe110, takes part in the enzyme polymerization and has proven to be beneficial for aggregation inhibition and solubility enhancement within the host α-CyD-nanocavity. 相似文献
985.
The multisolute osmotic virial equation is the only multisolute thermodynamic solution theory that has been derived from first principles and can make predictions of multisolute solution behaviour in the absence of multisolute solution data. Other solution theories either (i) include simplifying assumptions that do not take into account the interactions between different types of solute molecules or (ii) require fitting to multisolute data to obtain empirical parameters. The osmotic virial coefficients, which are obtained from single-solute data, can be used to make predictions of multisolute solution osmolality. The osmotic virial coefficients for a range of solutes of interest in cryobiology are provided in this paper, for use with concentration units of both molality and mole fraction, along with an explanation of the background and theory necessary to implement the multisolute osmotic virial equation. 相似文献
986.
Anita Balakrishnan Adam T. Stearns Peter J. Park Jonathan M. Dreyfuss David B. Rhoads Ali Tavakkolizadeh 《Experimental cell research》2010,316(20):3512-3521
Background and aims
The intestine exhibits profound diurnal rhythms in function and morphology, in part due to changes in enterocyte proliferation. The regulatory mechanisms behind these rhythms remain largely unknown. We hypothesized that microRNAs are involved in mediating these rhythms, and studied the role of microRNAs specifically in modulating intestinal proliferation.Methods
Diurnal rhythmicity of microRNAs in rat jejunum was analyzed by microarrays and validated by qPCR. Temporal expression of diurnally rhythmic mir-16 was further quantified in intestinal crypts, villi, and smooth muscle using laser capture microdissection and qPCR. Morphological changes in rat jejunum were assessed by histology and proliferation by immunostaining for bromodeoxyuridine. In IEC-6 cells stably overexpressing mir-16, proliferation was assessed by cell counting and MTS assay, cell cycle progression and apoptosis by flow cytometry, and cell cycle gene expression by qPCR and immunoblotting.Results
mir-16 peaked 6 hours after light onset (HALO 6) with diurnal changes restricted to crypts. Crypt depth and villus height peaked at HALO 13-14 in antiphase to mir-16. Overexpression of mir-16 in IEC-6 cells suppressed specific G1/S regulators (cyclins D1-3, cyclin E1 and cyclin-dependent kinase 6) and produced G1 arrest. Protein expression of these genes exhibited diurnal rhythmicity in rat jejunum, peaking between HALO 11 and 17 in antiphase to mir-16.Conclusions
This is the first report of circadian rhythmicity of specific microRNAs in rat jejunum. Our data provide a link between anti-proliferative mir-16 and the intestinal proliferation rhythm and point to mir-16 as an important regulator of proliferation in jejunal crypts. This function may be essential to match proliferation and absorptive capacity with nutrient availability. 相似文献987.
Helene Knævelsrud 《FEBS letters》2010,584(12):2635-31696
Ubiquitinated protein aggregates are hallmarks of a range of human diseases, including neurodegenerative, liver and muscle disorders. These protein aggregates are typically positive for the autophagy receptor p62. Whereas the ubiquitin-proteasome system (UPS) degrades shortlived and misfolded ubiquitinated proteins that are small enough to enter the narrow pore of the barrel-shaped proteasome, the lysosomal pathway of autophagy can degrade larger structures including entire organelles or protein aggregates. This degradation requires autophagy receptors that link the cargo with the molecular machinery of autophagy and is enhanced by certain posttranslational modifications of the cargo. In this review we focus on how autophagy clears aggregate-prone proteins and the relevance of this process to protein aggregate associated diseases. 相似文献
988.
Hideaki Shimada 《FEBS letters》2010,584(13):2827-2832
Lysophosphatidic acid (LPA), an inflammatory mediator that is elevated in multiple inflammatory diseases, is a potent activator of Rho kinase (ROCK) signaling and of chemokine production in endothelial cells. In this study, LPA activated ROCK, p38, JNK and NF-κB pathways and induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) mRNA and protein expression in human endothelial cells. We mapped signaling events downstream of ROCK, driving chemokine production. In summary, MCP-1 production was partly regulated by ROCK acting upstream of p38 and JNK and mediated downstream by NF-κB. IL-8 production was largely driven by ROCK through p38 and JNK activation, but with no involvement of NF-κB. 相似文献
989.
Light chain-associated (AL) amyloidosis is characterized by dominant fibril deposition of the variable domain (VL) of an immunoglobulin light chain, and thus its constant domain (CL) has been considered not to be amyloidogenic. We examined the in vitro fibril formation of the isolated CL in comparison with β2-microglobulin (β2-m), an immunoglobulin domain-like amyloidogenic protein responsible for dialysis-related amyloidosis. Two methods useful for β2-m at neutral pH also induced amyloid fibrils of CL, which were monitored by thioflavin-T binding and electron microscopy (EM). These results suggest that CL plays an important role, more than previously assumed, in the development of AL-amyloidosis. 相似文献
990.
To examine the role of two light chains (LCs) of the myosin II on Ca2+ regulation, we produced hybrid heavy meromyosin (HMM) having LCs from Physarum and/or scallop myosin using the smooth muscle myosin heavy chain. Ca2+ inhibited motility and ATPase activity of hybrid HMMs with LCs from Physarum myosin but activated those of hybrid HMM with LCs from scallop myosin, indicating an active role of LCs. ATPase activity of hybrid HMMs with LCs from different species showed the same effect by Ca2+ even though they did not support motility. Our results suggest that communication between the original combinations of LC is important for the motor function. 相似文献