Large-scale preparation of thrombin from human plasma

Thrombin was prepared from human blood plasma (batch size 1200 L). First, prothrombin was isolated by the following separation techniques: cryoprecipitation, ion-exchange chromatography (diethyl aminoethyl, DEAE-IEX), heparin affinity chromatography, a second DEAE-IEX step, and immobilized metal-affinity chromatography (IMAC). Prothrombin was then activated to thrombin, which was purified by hydrophobic interaction chromatography (HIC) and concentrated by ultrafiltration. This process is cost-effective because a waste fraction can be used from one of the steps (heparin affinity chromatography) in the commercial production of plasma-derived Factor IX (FIX). The final thrombin preparation had a purity of approximately 75% (specific activity approximately 2400 IU/mg protein), which is sufficient for its intended purpose in a fibrin glue. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Factor X (FX) activity analysis, and analytical HIC were also used to characterize the thrombin. Three substantially different techniques were used to reduce any viral activity, namely: solvent/detergent (S/D) treatment, pasteurization, and virus filtration (nanofiltration). The manufacturing process presented here would be suitable for large-scale production of thrombin with a high degree of virus safety.     

Two-dimensional electrophoresis with cationic detergents: a powerful tool for the proteomic analysis of myelin proteins. Part 2: analytical aspects

The ability to analyze proteins in developing and damaged myelin will be crucial to improve our understanding of the mechanisms of myelinogenesis, dysmyelination, and demyelination. Comparative two-dimensional electrophoresis (2-DE) is a powerful approach to analyze these proteins. In part 1 of this study (see accompanying paper), a method for the 2-DE analysis of myelin proteins using the cationic detergents benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide; CTAB) was described. We obtained improved separation and found that 16-BAC is the most effective agent for separation in 2-DE of myelin proteins and that CTAB is the most effective agent for solubilization of myelin proteins. Here in part 2, major myelin proteins as well as membrane proteins with multitransmembrane domains were identified by mass spectrometry after 16-BAC/SDS-PAGE and CTAB/SDS-PAGE. In addition, a high-molecular-weight protein enriched in myelin fraction was identified as unconventional myosin ID using 16-BAC/SDS-PAGE, which had previously not been detected using immobilized pH gradient isoelectric focusing (IPG)/SDS-PAGE. From these results, we concluded that combinational analysis using IPG/SDS-PAGE, 16-BAC/SDS-PAGE, and CTAB/SDS-PAGE provides a powerful technique facilitating the proteomic analysis of myelin proteins in either developmental or pathological changes.     

Detergent binding explains anomalous SDS-PAGE migration of membrane proteins

Migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that does not correlate with formula molecular weights, termed “gel shifting,” appears to be common for membrane proteins but has yet to be conclusively explained. In the present work, we investigate the anomalous gel mobility of helical membrane proteins using a library of wild-type and mutant helix-loop-helix (“hairpin”) sequences derived from transmembrane segments 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR), including disease-phenotypic residue substitutions. We find that these hairpins migrate at rates of −10% to +30% vs. their actual formula weights on SDS-PAGE and load detergent at ratios ranging from 3.4–10 g SDS/g protein. We additionally demonstrate that mutant gel shifts strongly correlate with changes in hairpin SDS loading capacity (R² = 0.8), and with hairpin helicity (R² = 0.9), indicating that gel shift behavior originates in altered detergent binding. In some cases, this differential solvation by SDS may result from replacing protein-detergent contacts with protein-protein contacts, implying that detergent binding and folding are intimately linked. The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. The observed interdependence between hairpin migration, SDS aggregation number, and conformation additionally suggests that detergent binding may provide a rapid and economical screen for identifying membrane proteins with robust tertiary and/or quaternary structures.     

Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4

Although understanding of the olfactory system has progressed at the level of downstream receptor signaling and the wiring of olfactory neurons, the system remains poorly understood at the molecular level of the receptors and their interaction with and recognition of odorant ligands. The structure and functional mechanisms of these receptors still remain a tantalizing enigma, because numerous previous attempts at the large-scale production of functional olfactory receptors (ORs) have not been successful to date. To investigate the elusive biochemistry and molecular mechanisms of olfaction, we have developed a mammalian expression system for the large-scale production and purification of a functional OR protein in milligram quantities. Here, we report the study of human OR17-4 (hOR17-4) purified from a HEK293S tetracycline-inducible system. Scale-up of production yield was achieved through suspension culture in a bioreactor, which enabled the preparation of >10 mg of monomeric hOR17-4 receptor after immunoaffinity and size exclusion chromatography, with expression yields reaching 3 mg/L of culture medium. Several key post-translational modifications were identified using MS, and CD spectroscopy showed the receptor to be ≈50% α-helix, similar to other recently determined G protein-coupled receptor structures. Detergent-solubilized hOR17-4 specifically bound its known activating odorants lilial and floralozone in vitro, as measured by surface plasmon resonance. The hOR17-4 also recognized specific odorants in heterologous cells as determined by calcium ion mobilization. Our system is feasible for the production of large quantities of OR necessary for structural and functional analyses and research into OR biosensor devices.     

Reducing electrostatic charge on spacer devices and bronchodilator response

AIMS: Plastic spacers are widely used with pressurized metered dose inhalers (pMDI). Reducing electrostatic charge by washing spacers with detergent has been shown to greatly improve in vitro and in vivo drug delivery. We assessed whether this finding is associated with an improved bronchodilator response in adult asthmatics. METHODS: Twenty subjects (age 18-65 years) with a known bronchodilator response inhaled in random order salbutamol from a pMDI (Ventolin) through an untreated new spacer (Volumatic) and through a detergent washed spacer. Patients received the following doses of salbutamol via pMDI at 20 min intervals: 100 microg, 100 microg, 200 microg, 400 microg, 800 microg. Spirometry, heart rate and blood pressure were checked prior to each dose and 20 min after the last dose. RESULTS: There were no differences between baseline forced expiratory volume in 1 s (FEV1) using either spacer (2.61+/-0.56 and 2.52+/-0.45 l, untreated and treated with detergent, respectively; mean +/- s.d.). The provocation dose required to cause a clinically significant improvement of 10% in FEV1 (PD10) was significantly lower when the detergent treated spacer was used (1505 +/-1335 and 430+/-732 microg, untreated and treated, respectively, P<0.002). CONCLUSIONS: We have demonstrated an improvement in bronchodilator response, in adult asthmatics, after reducing the electrostatic charge in a spacer device by washing it with ordinary household detergent. This finding stresses the importance of an optimal choice of delivery device for asthma medication.     

Subcellular localization and detergent solubility of MVP17/rMAL,a lipid raft-associated protein in oligodendrocytes and myelin

Detergent-insoluble, glycosphingolipid-cholesterol-enriched microdomains (lipid rafts) have been implicated in both protein trafficking and signal transduction. Previously we identified in oligodendrocytes and myelin the lipid raft-associated, integral membrane protein myelin vesicular protein of 17 kDa (MVP17)/rMAL. Here we have examined the subcellular localization and/or detergent insolubility of native and recombinant MVP17/rMAL in transfected oligodendrocytes and COS-7 cells and purified myelin. Consistent with our previous report regarding the insolubility of MVP17/rMAL in the zwitterionic detergent 3-[(3-chloramidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS), MVP17/rMAL from purified myelin and oligodendrocytes in culture was mostly insoluble upon extraction at 4 degrees C with the non-ionic detergent Triton X-100 and floated to a low density in sucrose gradient ultracentrifugation, but became detergent soluble at 37 degrees C. Data obtained by immunofluorescence microscopy of the expression of epitope-tagged MVP17/rMAL transfected into oligodendrocytes and COS-7 cells were consistent with a model in which both the N- and C-termini of this protein face the cytoplasm. Mutational analysis identified domains of MVP17/rMAL important for its subcellular localization and for its detergent solubility profile. In particular, insertional mutagenesis of loop II prevented the insertion of the mutant protein into the plasma membrane of COS-7 cells and rendered it insoluble in TX-100. Expression of full-length constructs of MVP17/rMAL in COS-7 cells resulted in an enlargement of transfected COS-7 cells, consistent with a proposed role of rMAL in vesicular trafficking.     

食物中膳食纤维含量及其组成特点

用洗涤剂方法测定北京市34种常见食物中膳食纤维含量,包括中性洗涤剂纤维、酸性洗涤剂纤维、纤维素、半纤维素和木质素,以干样计,蔬菜膳食纤维含量与蔬菜种类有关,嫩茎、叶、苔、花类含量高,含淀粉较高的根茎类则低,其它根茎类居中。各类食物的膳食纤维的组成成分也不同,蔬菜、干豆类以纤维素为主,谷类绝大部分以半纤维素为主,除蒜苗外,所有样品中以木质素含量为最低。     

Inhibition of the ATP-sensitive potassium channel from mouse pancreatic β-cells by surfactants

1. We have used patch-clamp methods to study the effects of the detergents, Cremophor, Tween 80 and Triton X100 on the K_ATP channel in the pancreatic β-cell from mouse.
2. All three detergents blocked K_ATP channel activity with the following order of potency: Tween 80 (K_i<∼83 nM)>Triton X100 (K_i=350 nM)>Cremophor. In all cases the block was poorly reversible.
3. Single-channel studies suggested that at low doses, the detergents act as slow blockers of the K_ATP channel.
4. Unlike the block produced by tolbutamide, that produced by detergent was not affected by intracellular Mg²⁺-nucleotide, diazoxide or trypsin treatment, nor did it involve an acceleration of rundown or increase in ATP sensitivity of the chanel.
5. The detergents could block the pore-forming subunit, Kir6.2ΔC26, which can be expressed independently of SUR₁ (the regulatory subunit of the K_ATP channel). These data suggest that the detergents act on Kir6.2 and not SUR₁.
6. The detergents had no effect on another member of the inward rectifier family: Kir1.1a (ROMK1).
7. Voltage-dependent K-currents in the β-cell were reversibly blocked by the detergents with a far lower potency than that found for the K_ATP channel.
8. Like other insulin secretagogues that act by blocking the K_ATP channel, Cremophor elevated intracellular Ca²⁺ in single β-cells to levels that would be expected to elicit insulin secretion.
9. Given the role of the K_ATP channel in many physiological processes, we conclude that plasma borne detergent may have pharmacological actions mediated through blockage of the K_ATP channel

     

Different mechanisms of lysophosphatidylcholine-induced Ca mobilization in N2a mouse and SH-SY5Y human neuroblastoma cells

In mice, lysophosphatidylcholine (LPC) was found to be a physiological substrate of neuropathy target esterase, which is also bound by organophosphates that cause a delayed neuropathy in human and some animals. However, the mechanism responsible for causing the different symptoms in mice and humans that are exposed to neuropathic organophosphates still remains unknown. In the present study, we examined and compared the effect of exogenous LPC on intracellular Ca²⁺ overload in mouse N2a and human SH-SY5Y neuroblastoma cells. LPC caused an intracellular Ca²⁺ level ([Ca²⁺]_i) increase in both N2a and SH-SY5Y cells; moreover, the amplitude was higher in N2a cells than that in SH-SY5Y cells. Preincubation of the cells with verapamil, an L-type Ca²⁺ channel blocker, did not affect the LPC-induced Ca²⁺ increase in N2a cells, verapamil inhibited the response by 23% in SH-SY5Y cells. In Ca²⁺-free medium, LPC produced a significant [Ca²⁺]_i decrease in N2a cells, while it caused 64% of total [Ca²⁺]_i increase in SH-SY5Y cells. The results of a cell viability test suggest that N2a cells were more sensitive to LPC than were SH-SY5Y cells. These data suggested that the LPC-induced [Ca²⁺]_i increase was produced in each cell line through different mechanisms. In particular, the [Ca²⁺]_i increase occurred via entry through a permeabilized membrane in N2a cells, but through L-type Ca²⁺ channels as well as by Ca²⁺ release from intracellular Ca²⁺ stores in SH-SY5Y cells. Thus, the symptomatic differences of organophosphate-induced neurotoxicity between mice and humans are probably not related to the diverse amplitudes of intracellular Ca²⁺ overload produced by LPC. Moreover, the demyelination effect induced by LPC in mice may be a consequence of its detergent effect on membranes.