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101.
Certain natural peptides and proteins of mammalian origin are able to bind and condense plasmid DNA, a prerequisite for the formation of transfection-competent complexes that facilitate nonviral gene delivery. Here we have generated recombinant derivatives of the human high-mobility group (HMG) protein HMGB2 and investigated their potential as novel protein-based transfection reagents. A truncated form of HMGB2 encompassing amino acids 1-186 of the molecule was expressed in Escherichia coli at high yield. This HMGB2186 protein purified from bacterial lysates was able to condense plasmid DNA in a concentration-dependent manner, and mediated gene delivery into different established tumor cell lines more efficiently than poly(l-lysine). By attaching, via gene fusion, additional functional domains such as the HIV-1 TAT protein transduction domain (TAT(PTD)-HMGB2186), the nuclear localization sequence of the simian virus 40 (SV40) large T-antigen (SV40(NLS)-HMGB2186), or the importin-beta-binding domain (IBB) of human importin-alpha (IBB-HMGB2186), chimeric fusion proteins were produced which displayed markedly improved transfection efficiency. Addition of chloroquine strongly enhanced gene transfer by all four HMGB2186 derivatives studied, indicating cellular uptake of protein-DNA complexes via endocytosis. The IBB-HMGB2186 molecule in the presence of the endosomolytic reagent was the most effective. Our results show that recombinant derivatives of human HMGB2 facilitate efficient nonviral gene delivery and may become useful reagents for applications in gene therapy. 相似文献
102.
Brandenburg K Garidel P Schromm AB Andrä J Kramer A Egmond M Wiese A 《European biophysics journal : EBJ》2005,34(1):28-41
Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein–lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria. 相似文献
103.
104.
Fate of Carbohydrates and Lignin during Composting and Mycelium Growth of Agaricus bisporus on Wheat Straw Based Compost 总被引:1,自引:0,他引:1
In wheat straw based composting, enabling growth of Agaricus bisporus mushrooms, it is unknown to which extent the carbohydrate-lignin matrix changes and how much is metabolized. In this paper we report yields and remaining structures of the major components. During the Phase II of composting 50% of both xylan and cellulose were metabolized by microbial activity, while lignin structures were unaltered. During A. bisporus’ mycelium growth (Phase III) carbohydrates were only slightly consumed and xylan was found to be partially degraded. At the same time, lignin was metabolized for 45% based on pyrolysis GC/MS. Remaining lignin was found to be modified by an increase in the ratio of syringyl (S) to guaiacyl (G) units from 0.5 to 0.7 during mycelium growth, while fewer decorations on the phenolic skeleton of both S and G units remained. 相似文献
105.
DOWNY MILDEW RESISTANT 6 and DMR6‐LIKE OXYGENASE 1 are partially redundant but distinct suppressors of immunity in Arabidopsis
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Tieme Zeilmaker Nora R. Ludwig Joyce Elberse Michael F. Seidl Lidija Berke Arjen Van Doorn Robert C. Schuurink Berend Snel Guido Van den Ackerveken 《The Plant journal : for cell and molecular biology》2015,81(2):210-222
Arabidopsis downy mildew resistant 6 (dmr6) mutants have lost their susceptibility to the downy mildew Hyaloperonospora arabidopsidis. Here we show that dmr6 is also resistant to the bacterium Pseudomonas syringae and the oomycete Phytophthora capsici. Resistance is accompanied by enhanced defense gene expression and elevated salicylic acid levels. The suppressive effect of the DMR6 oxygenase was confirmed in transgenic Arabidopsis lines overexpressing DMR6 that show enhanced susceptibility to H. arabidopsidis, P. capsici, and P. syringae. Phylogenetic analysis of the superfamily of 2‐oxoglutarate Fe(II)‐dependent oxygenases revealed a subgroup of DMR6‐LIKE OXYGENASEs (DLOs). Within Arabidopsis, DMR6 is most closely related to DLO1 and DLO2. Overexpression of DLO1 and DLO2 in the dmr6 mutant restored the susceptibility to downy mildew indicating that DLOs negatively affect defense, similar to DMR6. DLO1, but not DLO2, is co‐expressed with DMR6, showing strong activation during pathogen attack and following salicylic acid treatment. DMR6 and DLO1 differ in their spatial expression pattern in downy mildew‐infected Arabidopsis leaves; DMR6 is mostly expressed in cells that are in contact with hyphae and haustoria of H. arabidopsidis, while DLO1 is expressed mainly in the vascular tissues near infection sites. Strikingly, the dmr6‐3_dlo1 double mutant, that is completely resistant to H. arabidopsidis, showed a strong growth reduction that was associated with high levels of salicylic acid. We conclude that DMR6 and DLO1 redundantly suppress plant immunity, but also have distinct activities based on their differential localization of expression. 相似文献
106.
107.
Arjen van Ooyen Andrew Carnell Sander de Ridder Bernadetta Tarigan Huibert D. Mansvelder Fetsje Bijma Mathisca de Gunst Jaap van Pelt 《PloS one》2014,9(1)
Neuronal signal integration and information processing in cortical networks critically depend on the organization of synaptic connectivity. During development, neurons can form synaptic connections when their axonal and dendritic arborizations come within close proximity of each other. Although many signaling cues are thought to be involved in guiding neuronal extensions, the extent to which accidental appositions between axons and dendrites can already account for synaptic connectivity remains unclear. To investigate this, we generated a local network of cortical L2/3 neurons that grew out independently of each other and that were not guided by any extracellular cues. Synapses were formed when axonal and dendritic branches came by chance within a threshold distance of each other. Despite the absence of guidance cues, we found that the emerging synaptic connectivity showed a good agreement with available experimental data on spatial locations of synapses on dendrites and axons, number of synapses by which neurons are connected, connection probability between neurons, distance between connected neurons, and pattern of synaptic connectivity. The connectivity pattern had a small-world topology but was not scale free. Together, our results suggest that baseline synaptic connectivity in local cortical circuits may largely result from accidentally overlapping axonal and dendritic branches of independently outgrowing neurons. 相似文献
108.
109.
The gelation process of mixtures of γ-oryzanol and sitosterol structurants in sunflower oil was studied using light scattering,
rheology, and micro-scanning calorimetry (Micro-DSC). The relation between temperature and the critical aggregation concentration
(CAC) of tubule formation of γ-oryzanol and sitosterol was determined using these techniques. The temperature dependence of
the CAC was used to estimate the binding energy and enthalpic and entropic contribution to the tubular formation process.
The binding energy calculated at the corresponding temperatures and CACs were relatively low, in order of 2 RT (4.5 kJ mol−1), which is in accord with the reversibility of the tubular formation process. The formation of the tubules was associated
with negative (exothermic) enthalpy change (ΔH
0
) compared with positive entropy term (−T ΔS
0 >0), indicating that the aggregation into tubules is an enthalpy-driven process. The oryzanol–sitosterol ratio affected the
aggregation process; solutions with ratio of (60 oryzanol–40 sitosterol) started aggregation at higher temperature compared
with other ratios. 相似文献
110.
Lipid-accumulating fungi may be able to produce biodiesel precursors from agricultural wastes. As a first step in understanding
and evaluating their potential, a mathematical model was developed to describe growth, lipid accumulation and substrate consumption
of the oleaginous fungus Umbelopsis isabellina (also known as Mortierella isabellina) in submerged chemostat cultures. Key points of the model are: (1) if the C-source supply rate is limited, maintenance has
a higher priority than growth, which has a higher priority than lipid production; (2) the maximum specific lipid production
rate of the fungus is independent of the actual specific growth rate. Model parameters were obtained from chemostat cultures
of U. isabellina grown on mineral media with glucose and NH4
+. The model describes the results of chemostat cultures well for D > 0.04 h−1, but it has not been validated for lower dilution rates because of practical problems with the filamentous fungus. Further
validation using literature data for oleaginous yeasts is described in part II of this paper. Our model shows that not only
the C/N-ratio of the feed, but also the dilution rate highly influences the lipid yield in chemostat cultures. 相似文献