The aim of this study is to investigate the effect of isofraxidin, which is a major component of Acanthopanax senticosus Harms, on cell growth and the expression of genes encoding IL-1β, IL-6, MMP-1, and COX-2. For this purpose, a human synovial sarcoma cell line SW982 was used. Isofraxidin slightly inhibited the growth of SW982 cells at 450μM, but significantly suppressed the gene expression of IL-1β, IL-6, MMP-1, and COX-2 at 135μM as well as at 450μM. In addition, Isofraxidin supressed IL-6 production. These results suggest that isofraxidin exhibits anti-inflammatory effects.

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In this paper, we describe a technique to concentrate and separate low-abundant proteins in human plasma. A human plasma sample was fractionated by ammonium sulfate and the precipitate obtained at 35% saturated ammonium sulfate was dialyzed and subjected to isoelectric focusing employing an agarose slab gel (55mm×73mm×3mm, 1% agarose). The IEF gel was cut into strips and each strip was transferred on the top of an SDS-containing column gel (5% T, 5% C polyacrylamide gel) prepared in a Pasture pipette. After SDS electrophoresis, each pipette column gel was transferred onto a top of a micro slab gel (38mm×38mm×1mm, 8-17% T, 5% C polyacrylamide gradient gel) and second SDS gel electrophoresis was run. If the step of pipette electrophoresis was deleted, the proteins appeared as horizontal faint bands on the slab gel. By using the three-step electrophoresis, the low-abundant proteins were detected as densely stained spots on the slab gel with 10-25 fold higher concentration of corresponding proteins than in the case of two-step electrophoresis. The spots on the slab gels after the three-step electrophoresis have been subjected to protein identification by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and peptide sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS). The results showed the advantages of the present technique for the structural analysis of low-abundant proteins in human plasma.

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To study the functional cooperativity due to allosteric regulation in E.coli aspartate transcarbamoylase, two mutations (Lys60Glu & Lys94Glu) were introduced in the regulatory chain. This was done in order to disable the domain from binding the nucleotide effectors and at the same time would bring sufficient charge change on the surface of the protein molecule to be separated by ion-exchange chromatography. The mutant dimers along with the wild type dimers were used to reconstitute the holoenzyme hybrids. Due to different combinations four different types of hybrids were formed apparently exhibiting similar binding behavior to ion-exchange column. To purify these hybrid enzymes to homogeneity, preparative electrophoresis was successfully employed. The enzymes thus purified were assayed for comparison.

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Protein sample for two-dimensional electrophoresis (2-DE) is conventionally extracted in a buffer containing urea and non-ionic detergent. However, poorly soluble proteins are hardly introduced to the first-dimensional isoelectric focusing (IEF) under the conventional denaturing condition. Moreover, some kinds of tissues and organs contain extraordinary amount of proteases and phosphatases, which reduce the reproducibility in two-dimensional protein profiling. Various kinds of detergents have been tested to solve the problem, and consequently sodium dodecyl sulfate (SDS) is realized as the best detergent because of its high solubilizing capacity. However, sample solution containing a high concentration of SDS can not be directly applicable to 2-DE because the anionic detergent disturbs the formation of stable pH gradient in the first-dimensional IEF. Therefore, it has been considered that buffer exchange is necessary to reduce SDS concentration in the sample solutions. And then, acetone and trichloroacetic acid (TCA) have been adopted for precipitation of proteins in SDS-containing samples. However, some of proteins precipitated in acetone or TCA are hardly re-solubilized in a 2-DE sample buffer. In this paper, ultra-filtration (UF) membrane was used for the buffer exchange without protein insolubilization. Consequently, proteins of high molecular weight were successfully detected on 2-DE gels when proteins were extracted in the SDS-containing buffer and treated with UF membrane to reduce the concentration of SDS prior to IEF. These results indicate that the protein extraction in SDS-containing buffer and subsequent buffer exchange using UF membrane are useful for 2-DE of poorly soluble proteins.

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