Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies

High level expression of recombinant human tumour necrosis factor β (rh TNF-β) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rh TNF-β from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm^?3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.     

On the design of multiple key hashing files for concurrent orthogonal range retrieval between two disks

This paper concerns the following problem: given a set of multi-attribute records, a fixed number of buckets and a two-disk system, arrange the records into the buckets and then store the buckets between the disks in such a way that, over all possible orthogonal range queries (ORQs), the disk access concurrency is maximized. We shall adopt the multiple key hashing (MKH) method for arranging records into buckets and use the disk modulo (DM) allocation method for storing buckets onto disks. Since the DM allocation method has been shown to be superior to any other allocation methods for allocating an MKH file onto a two-disk system for answering ORQs, the real issue is knowing how to determine an optimal way for organizing the records into buckets based upon the MKH concept.

A performance formula that can be used to evaluate the average response time, over all possible ORQs, of an MKH file in a two-disk system using the DM allocation method is first presented. Based upon this formula, it is shown that our design problem is related to a notoriously difficult problem, namely the Prime Number Problem. Then a performance lower bound and an efficient algorithm for designing optimal MKH files in certain cases are presented. It is pointed out that in some cases the optimal MKH file for ORQs in a two-disk system using the DM allocation method is identical to the optimal MKH file for ORQs in a single-disk system and the optimal average response time in a two-disk system is slightly greater than one half of that in a single-disk system.     

Influence of nitrate, ascorbic acid, and nitrate reductase microorganisms on N-nitrosamine formation during Korean-style soysauce fermentation.

Traditional Korean soysauce samples were collected from households in Chinju, Gyeongnam, Korea and analysed for volatile N-nitrosamines. Five of 24 samples contained NDMA (range = 1.6-10.4 micrograms/l) which was the only volatile N-nitroso compound found. Soysauce made from well water contained NDMA more often (4 of 6 samples) than soysauce made from tap water (1 of 18). This suggests that the water source is a determinate in the NDMA content of soysauce, probably due to a higher nitrate content of well water. The source of salt used did not clearly influence NDMA content. Soysauce was prepared in the laboratory using traditional methods but with 0 to 400 mg/l nitrate and in some cases made 6.5 to 65 mM in ascorbic acid and fermented for 120 days. The NDMA content of the samples was positively correlated with increasing nitrate concentration. Nitrate at 400 mg/l resulted in an NDMA content of 203 micrograms/l. Ascorbic acid substantially inhibited NDMA formation. All samples contained large numbers of nitrate reductase-containing organisms (greater than 1 x 10(7) CFU/ml).     

Effect of storage temperature on phenolics stability in hawthorn (Crataegus pinnatifida var. major) fruits and a hawthorn drink

The stability of five major phenolics, namely (−)-epicatechin (EC), procyanidin B₂ (PC-B₂), chlorogenic acid (ChA), hyperoside (HP) and isoquercitrin (IQ), in hawthorn fruits and a canned hawthorn drink were evaluated during 6 months of storage in the dark at three different temperatures (4, 23 and 40 °C). HPLC with a diode-array detector was used to determine the contents of the individual compounds. The results showed that the studied phenolics in the hawthorn fruits and the drink were both stable at 4 °C and relatively unstable at 23 and 40 °C with varied extents of degradation. At room temperature (23 °C), marked degradations of EC and PC-B₂ were observed in both the fruits and the drink with around 50% and 30% decrease after a 6-month storage, respectively. A more significant decrease of the phenolics was observed at 40 °C, especially for EC and PC-B₂, which were almost completely degraded after a 6-month storage. HP, IQ and ChA were relatively stable at 23 °C, but unstable at 40 °C. Therefore, low-temperature storage is recommended for maintaining the quality and efficacy of hawthorn fruits and its preparations.     

Kinetics of AuSn4 migration in lead-free solders

The relatively fast diffusion of Au atoms in eutectic PbSn matrix is considered one of the contributing factors to the Au embrittlement problem. In this study, we further investigated the Au embrittlement problem in high-Sn solders. Experimentally, Sn3.5Ag (wt.%) spheres with 500-μm diameter were soldered over the Au/Ni soldering pads. It was found that some of the AuSn₄ needles that formed after reflow inside the solder migrated back to the solder/pad interface during thermal aging. However, the migration kinetics in high-Sn solders was slower compared to that in eutectic PbSn. The difference in migration kinetics of AuSn₄ in eutectic PbSn and SnAg was ascribed to the difference in the magnitudes of the Au flux and the Ni flux. In eutectic PbSn, the Au flux was much greater than that of the Ni flux, and the Au and Ni flux were in the same order of magnitude in eutectic SnAg. The relative magnitude of the Au and Ni flux changed in eutectic PbSn and SnAg because the homologous temperatures of PbSn and SnAg were different.     

Organic‐acid‐catalyzed sol–gel route for preparing poly(methyl methacrylate)–silica hybrid materials

In this study, a series of organic–inorganic hybrid sol–gel materials consisting of a poly(methyl methacrylate) (PMMA) matrix and dispersed silica (SiO₂) particles were successfully prepared through an organic‐acid‐catalyzed sol–gel route with N‐methyl‐2‐pyrrolidone as the mixing solvent. The as‐synthesized PMMA–SiO₂ nanocomposites were subsequently characterized with Fourier transform infrared spectroscopy and transmission electron microscopy. The solid phase of organic camphor sulfonic acid was employed to catalyze the hydrolysis and condensation (i.e., sol–gel reactions) of tetraethyl orthosilicate in the PMMA matrix. The formation of the hybrid membranes was beneficial for the physical properties at low SiO₂ loadings, especially for enhanced mechanical strength and gas barrier properties, in comparison with the neat PMMA. The effects of material composition on the thermal stability, thermal conductivity, mechanical strength, molecular permeability, optical clarity, and surface morphology of the as‐prepared hybrid PMMA–SiO₂ nanocomposites in the form of membranes were investigated with thermogravimetric analysis, differential scanning calorimetry, dynamic mechanical analysis, gas permeability analysis, ultraviolet–visible transmission spectroscopy, and atomic force microscopy, respectively. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Restraining the associations of anthracene fluorophore by chemically linking to poly(methyl methacrylate)

Associations (dimer or aggregate) of anthracene (An) fluorophores tend to interrupt the monomer emission and reduce the quantum yield (Φ_PL); therefore, poly(methyl methacrylate) (PMMA) chain was used in this study to chemically link to anthracene and to block the mutual associations among the anthracene fluorophores. With this aim, the target polymers were prepared by anionic polymerizations with 9,10‐dibromoanthracene/s‐butyllithium as initiating system to proceed polymerizations of methyl methacrylate (MMA) directly or in the presence of 1,1‐diphenylethylene (DPE). Use of DPE before addition of MMA produces stable initiating anionic species and avoids potential side reactions during polymerization; however, it also introduces four β‐phenylene rings around the central anthracene ring, which interfere with the corresponding emission pattern and reduce the Φ_PL (32%) value due to potential interactions between phenylene rings and anthracene. On the contrast, polymerization without participation of DPE results in polymer with central anthracene ring directly connected to two PMMA chains, which gives clean vibronic emission pattern with limited association emissions and enhanced Φ_PL (52%) value. Physical blending of anthracene by PMMA is less efficient to restrain the associations and results in a film of lower Φ_PL (20%). © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008