Single-step protein purification by back flush in ion exchange chromatography

Ion exchange chromatography, one of the major procedures for protein purification, seldom provides single-step purification due to a lack of specific affinity. In this work, a novel and simple method called “back flush” (i.e., reversing the flow direction of elution relative to that of sample loading) was developed to achieve single-step purification on an ion exchanger. Tips for the conditions and operation by back flush are presented. Our study demonstrates, for the first time, the feasibility and dramatic improvement for protein purification by the back-flush method.     

Pseudomonas exotoxin A-epidermal growth factor (EGF) mutant chimeric protein as an indicator for identifying amino acid residues important in EGF-receptor interaction.

Epidermal growth factor (EGF) was fused to the carboxyl end of a modified pseudomonas exotoxin A that has its toxin binding domain deleted. This chimeric toxin designated as PE(delta Ia)-EGF kills A431 cells through the EGF receptor-mediated pathway. In this study, we used a random mutagenesis approach to make point mutations on EGF, followed by replacing the wild type EGF in PE(delta Ia)-EGF with these EGF mutants. We have constructed 14 different PE(delta Ia)-EGFmutants, and examined their EGF receptor binding activity as well as their cytotoxicity to A431 cells. Our results showed that individual mutations of Val19 to Glu and Val34 to Asp in the EGF domain of PE(delta Ia)-EGFmutants resulted in an increase in the binding affinity to EGF receptor and cytotoxicity to A431 cells. On the other hand, individual mutations of His16 to Asp and Gly18 to Ala in the EGF domain of PE(delta Ia)-EGFmutants lead to a decrease in the binding affinity to EGF receptor and cytotoxicity to A431 cells. In addition, mutations of any of the cysteine residues of EGF in PE(delta Ia)-EGFmutants resulted in the loss of their binding activity to EGF receptor and a corresponding loss of their cytotoxicity. This study indicates that the cytotoxicity of PE(delta Ia)-EGFmutant to EGF receptor-bearing cells may be used as an indicator to screen mutations of EGF important in EGF-receptor interactions.     

Microbial processes and temperature in Chesapeake Bay: current relationships and potential impacts of regional warming

The importance of temperature in regulating physiological processes is without question; however, the interpretation of the relationship between temperature and ecological data is much more complicated. Consequently, it is difficult to decide how the nature of the temperature response terms should be included in models used to predict responses of microbial processes to increasing regional temperature. This analysis compiles several years of data from a research programme conducted in Chesapeake Bay, in an effort to examine how individual microbial processes ? as well as the balance between autotrophy and heterotrophy ? have responded to temperature, and to predict changes in microbial trophic state based on realistic increases in global temperature. The upper boundary on all of the pelagic microbial rate processes that were measured could be described remarkably well as a linear function of temperature, although there was substantial scatter in the data. Pelagic microbial rate processes (e.g. phytoplankton production, respiration, bacterial productivity) showed a remarkably constrained range of Q₁₀ values from 1.7 to 3.4. The one notable exception to this was nitrogen uptake in the North and Mid Bay, which exhibited Q₁₀ values < 1.0. Proxies for phytoplankton biomass (e.g. chlorophyll) were largely independent of temperature while bacterial abundance was significantly related to temperature and was found to have a Q₁₀ of 1.88. Using these individual temperature responses, the balance of autotrophy and heterotrophy was assessed by calculating the community photosynthesis to respiration (P:R), NH₄⁺ uptake to regeneration (U:R) and phytoplankton to bacterial productivity (PP:BP) ratios for current conditions (all ratios) and for a 2 and 5 °C temperature increase (NH₄⁺ U:R excluded). The NH₄⁺ U:R ratio stayed remarkable constant at ～1 over the entire temperature range supporting the importance of regenerative processes to nitrogen availability even during periods of heavy allochthonous inputs. These elevated temperature calculations for P:R and PP:BP suggest that the magnitude of autotrophic production during the spring bloom may decrease with increased regional temperature and, as a consequence, the Chesapeake Bay might become net heterotrophic on an annual timescale. These calculations should be considered with caution, but nonetheless demonstrate that the impact of increasing temperature on the balance of autotrophic and heterotrophic processes needs to be researched further.     

A three-dimensional model of the upper tracheobronchial tree

A new model of the upper tracheobronchial tree is proposed to account for the three-dimensional nature of the airway system. In addition to the tube length, the tube diameter, and the branching angle, the model includes information on the orientation angle of each tube relative to its parent tube. The orientation angle, defined as the angle between two successive bifurcations, is useful for calculating the gravitational inclination of each tube. The information on orientation angle is further used to construct a binary coding system for identifying individual tubes in the airway tree. The proposed model is asymmetrical, but the same principles can be readily used to construct a symmetrical one.     

Effects of increasing nutrient supply and omnivorous feeding on the size spectrum slope: a size-based nutrient-phytoplankton-zooplankton model

Size spectrum slope has been considered as an indicator for system productivity and size-dependent trophic interactions in plankton communities. We investigated the effect of new nutrient flux (N _in ) and feeding breadth of zooplankton (σ²) on spectral slopes of the plankton community using a size-structured nutrient-phytoplankton-zooplankton model. We studied two types of plankton systems (1) at a steady state and (2) with fluctuating plankton populations. For the steady state systems, the spectral slopes increased with N _in and σ². In contrast, for the systems in fluctuation, spectral slopes constructed from long-term average plankton populations decreased with increasing N _in and σ². In addition to spectral slopes constructed from long-term average population densities, we investigated slope variability in systems with fluctuating plankton populations. The variability of spectral slopes was found to increase with N _in and σ², indicating that spectral slope variation is large in a eutrophic system or when zooplankton feed on a wide size range of prey. Empirically, because N _in is difficult to measure, nutrient concentration is investigated instead. Our model predicts that the spectral slope is positively correlated with nutrient concentration due to the accumulated mesozooplankton biomass, which was confirmed by our study in plankton community in a subtropical reservoir in Taiwan.     

Summer profundal hypoxia determines the coupling of methanotrophic production and the pelagic food web in a subtropical reservoir

相似文献   

Elevation of Soluble Guanylate Cyclase Suppresses Proliferation and Survival of Human Breast Cancer Cells

Nitric oxide (NO) is an essential signaling molecule in biological systems. Soluble guanylate cyclase (sGC), composing of α1 and β1 subunit, is the receptor for NO. Using radioimmunoassay, we discovered that activation of sGC by treatment with bradykinin or sodium nitroprusside (SNP) is impaired in MCF-7 and MDA-MB-231 breast cancer cells as compared to normal breast epithelial 184A1 cells. The 184A1 cells expressed both sGC α1 and sGCβ1 mRNAs. However, levels of sGCβ1 mRNAs were relatively lower in MCF-7 cells while both mRNA of sGC subunits were absent in MDA-MB-231 cells. Treatment with DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) increased mRNA levels of both sGCα1 and sGCβ1 in MDA-MB-231 cells but only sGCβ1 mRNAs in MCF-7 cells. The 5-aza-dC treatment increased the SNP-induced cGMP production in MCF-7 and MDA-MB-231, but not in 184A1 cells. Bisulfite sequencing revealed that the promoter of sGCα1 in MDA-MB-231 cells and promoter of sGCβ1 in MCF-7 cells were methylated. Promoter hypermethylation of sGCα1 and sGCβ1 was found in 1 out of 10 breast cancer patients. Over-expression of both sGC subunits in MDA-MB-231 cells induced apoptosis and growth inhibition in vitro as well as reduced tumor incidence and tumor growth rate of MDA-MB-231 xenografts in nude mice. Elevation of sGC reduced protein abundance of Bcl-2, Bcl-xL, Cdc2, Cdc25A, Cyclin B1, Cyclin D1, Cdk6, c-Myc, and Skp2 while increased protein expression of p53. Our study demonstrated that down-regulation of sGC, partially due to promoter methylation, provides growth and survival advantage in human breast cancer cells.     

Caspase-8 mutations associated with head and neck cancer differentially retain functional properties related to TRAIL-induced apoptosis and cytokine induction

The cysteine protease, caspase-8, undergoes dimerization, processing, and activation following stimulation of cells with death ligands such as TRAIL, and mediates TRAIL induction of the extrinsic apoptosis pathway. In addition, caspase-8 mediates TRAIL-induced activation of NF-κB and upregulation of immunosuppressive chemokines/cytokines, via a mechanism independent of caspase-8 catalytic activity. The gene encoding procaspase-8 is mutated in 10% of human head and neck squamous cell carcinomas (HNSCCs). Despite a paucity of experimental evidence, HNSCC-associated caspase-8 mutations are commonly assumed to be loss of function. To investigate their functional properties and phenotypic effects, 18 HNSCC-associated caspase-8 mutants were expressed in doxycycline-inducible fashion in cell line models wherein the endogenous wild-type caspase-8 was deleted. We observed that 5/8 mutants in the amino-terminal prodomain, but 0/10 mutants in the carboxyl-terminal catalytic region, retained an ability to mediate TRAIL-induced apoptosis. Caspase-8 proteins with mutations in the prodomain were defective in dimerization, whereas all ten of the catalytic region mutants efficiently dimerized, revealing an inverse relationship between dimerization and apoptosis induction for the mutant proteins. Roughly half (3/8) of the prodomain mutants and 9/10 of the catalytic region mutants retained the ability to mediate TRAIL induction of immunosuppressive CXCL1, IL-6, or IL-8. Doxycycline-induced expression of wild-type caspase-8 or a representative mutant led to an increased percentage of T and NKT cells in syngeneic HNSCC xenograft tumors. These findings demonstrate that HNSCC-associated caspase-8 mutants retain properties that may influence TRAIL-mediated apoptosis and cytokine induction, as well as the composition of the tumor microenvironment.Subject terms: Medical research, Preclinical research