Separation of racemic bupivacaine using Simulated Moving Bed with mathematical model

Utilizing a Simulated Moving Bed (SMB), the conditions for separating bupivacaine enantiomers, a racemic local anesthetic, were determined by triangle theory and simulations using the gPROMS software and the Aspen Chromatography package. The SMB model in this study assumed an axial dispersion flow for the liquid phase and the intraparticle mass transfer was described in terms of a simple Linear Driving Force (LDF) approximation. The parameters (a andb) of the Langmuir Adsorption Isotherm (LAI) were determined using a dynamic method. In the optimal condition the calculated concentrations were confirmed by the experimental results. Values projected by the gPROMS software more closely matched experimental values than those projected by the Aspen Chromatography package.     

Huntingtin phosphorylation sites mapped by mass spectrometry. Modulation of cleavage and toxicity

Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing full-length myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development.     

Proteomic analysis of succinate dehydrogenase and ubiquinol-cytochrome c reductase (Complex II and III) isolated by immunoprecipitation from bovine and mouse heart mitochondria

The oxidative phosphorylation system (OXPHOS) consists of five multi-enzyme complexes, Complexes I-V, and is a key component of mitochondrial function relating to energy production, oxidative stress, cell signaling and apoptosis. Defects or a reduction in activity in various components that make up the OXPHOS enzymes can cause serious diseases, including neurodegenerative disease and various metabolic disorders. Our goal is to develop techniques that are capable of rapid and in-depth analysis of all five OXPHOS complexes. Here, we describe a mild, micro-scale immunoisolation and mass spectrometric/proteomic method for the characterization of Complex II (succinate dehydrogenase) and Complex III (ubiquinol-cytochrome c reductase) from bovine and rodent heart mitochondria. Extensive protein sequence coverage was obtained after immunocapture, 1D SDS PAGE separation and mass spectrometric analysis for a majority of the 4 and 11 subunits, respectively, that make up Complexes II and III. The identification of several posttranslational modifications, including the covalent FAD modification of flavoprotein subunit 1 from Complex II, was possible due to high mass spectrometric sequence coverage.     

Recovery of catechin compounds from Korean tea by solvent extraction

Catechin compounds from Korean green tea as potential sources of anticancer and antioxidant components were target materials in this work. The methodologies of solvent extraction and partition were utilized to recover catechin compounds from green tea. The optimum experimental condition was obtained by optimizing operating factors, such as, the extraction solvent, extraction time and operating temperature. After extracting the green tea with water at 80 degrees C for 40 min, the extract was partitioned with water/chloroform, which was best suited to remove caffeine impurity from the extract. Further, the resulting extract was partitioned water/ethyl acetate to deeply purify the catechin compounds of EGC, EC, EGCG and ECG. The experimental result in this work could be extended to preparative HPLC to obtain EGCG on commercial scale.     

Solid-phase extraction of tanshinones from Salvia Miltiorrhiza Bunge using ionic liquid-modified silica sorbents

New ionic liquid-modified silica sorbents were developed by the surface chemical modification of the commercial silica using synthesized ionic liquids. The obtained ionic liquid-modified particles were successfully used as a special sorbent in solid-phase extraction process to isolation of cryptotanshinone, tanshinone I and tanshinone IIA from Salvia Miltiorrhiza Bunge. Different washing and elution solvents such as water, methanol and methanol–acetic acid (90/10, v/v) were evaluated. A comparison of ionic liquid-modified silica cartridges and traditional silica cartridge show that higher recovery was observed using ionic liquid-modified silica sorbents. A quantitative analysis was conducted by high-performance liquid chromatography using a C₁₈ column (5 μm, 150 mm × 4.6 mm) with methanol–water (78:22, v/v, and containing 0.5% acetic acid) as a mobile phase. Good linearity was obtained from 0.5 × 10^?4 to 0.5 mg/mL (r² > 0.999) with the relative standard deviations less than 4.8%.     

Synthesis of 8-geranyloxypsoralen analogues and their evaluation as inhibitors of CYP3A4

Furanocoumarins have been shown to inhibit CYP3A4 in vitro with varying degrees of potency. In this study, we report the effects of a series of novel furanocoumarins based on the naturally occurring derivative 8-geranylepoxypsoralen which has been shown to be a more potent inhibitor of CYP3A4 than its 5-position-substituted counterpart bergamottin. Compounds were designed, synthesised and tested for their ability to inhibit CYP3A4 activity in human liver microsomes using testosterone as the marker substrate. Both the saturated and unsaturated phenolic furanocoumarin derivatives were found to be inactive. However, the 8-alkyloxy-furanocoumarin analogues were shown to inhibit CYP3A4 activity in a dose dependent manner, with IC(50) values ranging from 0.78+/-0.11 to 3.93+/-0.53 microM. The reduced furan derivative dihydro-8-geranyloxypsoralen showed a 4-fold decrease in inhibitory potency, suggesting that the furan moiety plays a role in the interaction between these compounds and CYP3A4.     

Structure of acacigenin-B,a novel triterpene ester isolated from Acacia concinna

The structure of acacigenin-B, a novel ester genin from the pods of Acacia concinna was established from its PMR and ¹³ C NMR spectra. It was identified as the 21-hydroxy ester of acacic acid; the esterifying acid was a hitherto unknown monoterpene acid of tetrahydrofuranoid structure. This appears to be the first report of a higher terpenoid forming an ester with a monoterpene acid     

Optimum separation condition of peptides in reversed-phase liquid chromatography

An efficient optimization method was suggested to separate biologically active peptides by RP-HPLC. In this work, the binary mobile phase of water and acetonitrile was used with the buffer of trifluoroacetic acid (TFA). The elution profiles were calculated by the plate theory based on the linear and quadratic equations of retention factor, lnk=A+BF, lnk=A+BF+CF(2), and F was the vol.% of acetonitrile. We modified the plate theory to calculate elution profile in both isocratic and gradient mode. From the final calculated results, the first mobile phase composition was water in 0.1% TFA/acetonitrile in 0.1% TFA, 81/19vol.%, then after 7-8 min, the second composition of mobile phase was linearly changed to 79/21vol.%, and finally after 8 min, it was kept at the isocratic mode. In the experimental conditions, the agreement between the experimental data and the calculated values was relatively good.     

Lysosome associated membrane protein 1 (Lamp1) traffics directly from the TGN to early endosomes

The precise trafficking routes followed by newly synthesized lysosomal membrane proteins after exit from the Golgi are unclear. To study these events we created a novel chimera (YAL) having a lumenal domain comprising two tyrosine sulfation motifs fused to avidin, and the transmembrane and cytoplasmic domains of lysosome associated membrane protein 1 (Lamp1). The newly synthesized protein rapidly transited from the trans- Golgi Network (TGN) to lysosomes (t(1/2) approximately 30 min after a lag of 15-20 min). However, labeled chimera was captured by biotinylated probes endocytosed for only 5 min, indicating that the initial site of entry into the endocytic pathway was early endosomes. Capture required export of YAL from the TGN, and endocytosis of the biotinylated reagent, and was essentially quantitative within 2 h of chase, suggesting that all molecules were following an identical route. There was no evidence of YAL trafficking via the cell surface. Fusion of TGN-derived vesicles with 5 min endosomes could be recapitulated in vitro, but neither late endosomes nor lysosomes could serve as acceptor compartments. This suggests that contrary to previous conclusions, most if not all newly synthesized Lamp1 traffics from the TGN to early endosomes prior to delivery to late endosomes and lysosomes.     

The effect of amino acid-modifying reagents on chloroplast protein import and the formation of early import intermediates

In order to identify functionally important amino acid residues in the chloroplast protein import machinery, chloroplasts were preincubated with amino-acid-modifying reagents and then allowed to import or form early import intermediates with precursor proteins. Incubation of chloroplasts with N-ethyl maleimide, diethyl pyrocarbonate, phenylglyoxal, 4,4'-di-isothiocyanatostilbene 2,2'-disulphonic acid (DIDS), dicyclohexylcarbodiimide (DCCD), and 1-ethyl- 3-dimethylaminopropylcarbodiimide (EDC) inhibited both import and formation of early import intermediates with precursor proteins by chloroplasts. This suggests that one or more of the binding components of the chloroplast protein import machinery contains functionally important solvent-exposed cysteine, histidine, arginine, and aspartate/glutamate residues, as well as functionally important lysine and aspartate/ glutamate residues in a hydrophobic environment.