Proposed model for the transcriptional activation of PltR and autoinduction of pyoluteorin on Plt biosynthetic operon in <i>P. aeruginosa</i> M18.

<p>(<b>A</b>)<b> </b> Thick arrows indicate activation, autoinduction, and positive regulation; thin arrows indicate biogenesis. P_L and P_R denote the promoters of <i>pltL</i> and <i>pltR</i>. P_X and P_Y are two uncharacterized promoters. The TSS is shown with +1. The LysR-type regulator PltR transcriptionally activates the <i>plt</i> operon expression by directly binding to the <i>pltLp</i> region at the indispensable palindromic <i>lys</i> box. PltR also functions as a candidate receptor of Plt. Plt, as a potential and nonessential cofactor of PltR, specifically induces the expression of the <i>pltL</i> promoter and thus the <i>plt</i> operon. (<b>B</b>) <b>The PltR protein comprise two domains:</b> the N-terminal LysR-family HTH (helix-turn-helix) DNA-binding domain and the C-terminal co-inducer binding domain (predicted by Pfam 25.0). aa, amino acids.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Detection of promoter activity in the intergenic region between divergently transcribed <i>pltR</i> and <i>pltLABCDEFG</i> genes using the <i>lacZ</i> reporter gene.

<p>(<b>A</b> and <b>B</b>) Promoter detection in the <i>pltR</i> direction. (A) Four fragments, R1 to R4, containing five putative promoters in the <i>pltR</i> direction (predicted by the NNPP promoter online prediction with a score cutoff 0.80) were fused with the promoterless <i>lacZ</i> gene on the plasmid pME6522. The R3 fragment includes two putative promoters. The putative +1 site indicates the putative TSS of the fifth predicted promoter. (B) β-Galactosidase expression (Miller Units) from above these <i>lacZ</i> fusion plasmids was assayed in both <i>E. coli</i> DH5α and <i>P. aeruginosa</i> M18 after 15 h of growth in KMB. The R4 fragment was detected to include a true promoter (shown by an asterisk). (<b>C</b> and <b>D</b>) Promoter detection in the <i>pltLABCDEFG</i> operon direction. (C) Five fragments, L1 to L5, which contain five putative promoters in the <i>pltL</i> direction, were respectively fused into the upstream of the promoterless <i>lacZ</i> gene on pME6522. Putative +1 indicates the putative TSS of the third predicted promoter. (D) β-Galactosidase expression from the above <i>lacZ</i> fusion recombined plasmids was measured in both DH5α and M18. The L1 fragment (marked with a triangle) exhibited a certain degree of promoter activity in DH5α, but not in M18. The other four fragments, L2 to L5, did not show any evident promoter activity. (<b>E</b> and <b>F</b>) Detection of promoter or its elements by prolongation analysis within the DNA fragment spanning from +10 to the <i>pltL</i> ATG, which was not predicted to carry promoters. (E) Four prolonging fragments based on L3, namely, L3–1 to L3–4, were respectively fused with the <i>lacZ</i> gene in pME6522. (F) The resulting <i>lacZ</i> fusion plasmids were assayed for β-galactosidase expressions in both DH5α and M18. The region covering +66 to +124 bp (relative to the putative +1 site) contained either a stronger promoter or its elements (marked with an asterisk). The <i>L3–3–lacZ</i> fusion expression significantly decreased, compared with that in the <i>L3–2–lacZ</i> fusion expression in <i>E. coli</i> DH5α, which implies that the promoter located at +66 to +124bp is not likely <i>pltLp</i> activated by PltR. Moreover, the region from +124 to +205 bp (marked with a triangle) possibly contains another promoter under the transcriptional activation of PltR. (<b>G</b> and <b>H</b>) Distinguishing <i>pltLp</i> activated by PltR from other promoters by deletion analysis. (G) Four fragments (L6 and its shortened derivative fragments, namely, L6–1 to L6–3), four continuously prolonged fragments based on L6–3 (L6-3-1 to L6-3-4), and the L6–4 fragment, were respectively cloned into pME6522. The L6 fragment was not predicted (score cutoff 0.60) to contain putative promoter regions. (H) The β-galactosidase expression from these recombinant <i>lacZ</i> reporter plasmids was analyzed in <i>E. coli</i> DH5α, <i>P. aeruginosa</i> M18, and the <i>pltR</i> mutant M18pltR. An intact promoter contained in the region from +66 to +124 (marked with an asterisk) was not controlled by PltR. The <i>pltLp</i> activated by PltR was located from +124 to +205 bp (marked with a triangle), relative to the putative +1 site.</p

Effects of <i>lys</i> box mutations on <i>pltLp</i> expression and the interaction between PltR and <i>pltLp</i>.

<p>(<b>A</b>) Construction map of the mutagenized <i>pltLp</i> with <i>lys</i> box mutations. Three <i>pltLp</i> mutant fragments, <i>pltLp-M2</i>, <i>-M4</i>, and <i>-M6</i>, respectively carried 2 bp, 4 bp, and 6 bp replacements in the <i>lys</i> box. The <i>pltLp</i> mutant M4-M was constructed by introducing a 4 bp complementary replacement into the <i>pltLp-M4</i> mutant. The above four <i>pltLp</i> mutagenized fragments were respectively cloned into pME6522. (<b>B</b>) β-galactosidase expression (Miller Units) from the above <i>lacZ</i> fusion plasmids was measured in the M18 strain. The <i>pltLp</i> expression gradually and significantly decreased, even entirely inhibited, with the opening of the <i>lys</i> box formed stem loop (Fig. 4A) by base substitutions. (<b>C</b>) The binding of PltR to <i>pltLp</i> was significantly weakened and even eliminated by the <i>lys</i> box mutations. <i>pltLp</i> and its derivative mutated fragments (0.5 nM) were respectively incubated with increasing amounts of PltR protein. The concentration of PltR was 0, 10, and 60 μM, respectively.</p

Development of Circulating Ultrasounic-Assisted Online Extraction Coupled to Countercurrent Chromatography and Centrifugal Partition Chromatography for Simultaneous Extraction and Isolation of Phytochemicals: Application to Ligusticum chuanxiong Hort

Circulating ultrasonic-assisted extraction (CUAE) was developed as a novel method for extraction of medicinal herbs, and the method was validated. In addition, the novel hyphenated technique comprising CUAE coupled with countercurrent chromatography (CCC) and centrifugal partition chromatography (CPC) was developed and applied to the continuous extraction and online isolation of chemical constituents from Ligusticum chuanxiong Hort. The optimum extraction parameters, including the extraction time of 30 min, extraction temperature of 45 °C, ultrasound power of 300 W, and the liquid–solid ratio of 10 mL/g were determined by response surface methodology. Furthermore, a schematic and the mechanism of online CUAE coupled with CCC and CPC were presented. Three lactones, levistolide A (52.2 mg), Z-ligustilide (48.3 mg), and wallichilide (118.2 mg), with respective purities of 95.8, 96.7, and 96.2%, were obtained from 500 g of the L. chuanxiong raw material using CUAE/CCC. In contrast, senkyunolide A (26.2 mg), levistolide A (34.2 mg), and wallichilide (95.1 mg), with respective purities of 96.2, 95.3, and 96.1%, were obtained from 500 g of the L. chuanxiong raw material by using CUAE/CPC employing the two-phase solvent system comprising <i>n</i>-hexane–ethyl acetate–methanol–water in a volume ratio of 4:3:4:2 (v/v). Compared with reference extraction methods, scientific and systematic extraction and isolation of natural products was achieved with the instrumental setup, and this system has great prospects for industrial application, where the combined use of CCC and CPC can enhance the separation efficiency

Mapping the TSSs and promoters of <i>pltR</i> and <i>pltL</i> genes through 5′RACE and the above <i>lacZ</i> reporter analysis (Fig. 1).

<p>P_R, the <i>pltR</i> promoter; P_L, the <i>pltLp</i> which needs to be specifically activated by PltR; P_X, a non-PltR controlled promoter closely neighboring the upstream of P_L; P_Y, another potential promoter silent in <i>P. aeruginosa</i> M18, but active in <i>E. coli</i> DH5α. The TSS (+1) of the <i>pltR</i> transcript was located at 63 bp upstream of the <i>pltR</i> translational start codon ATG, which is identical to the predicted +1 site. The TSS of the <i>plt</i> operon was located at 46 bp upstream of the <i>pltL</i> ATG. Closely linked to the <i>pltLp</i> with the P_x promoter, a palindromic <i>lys</i> box, including two 9 bp inverted complementary sequences separated by 4 bases, is boxed and highlighted by two head-to-head arrows.</p

Exogenous Plt specifically induced the <i>pltLp</i> promoter expression and thus enhanced the <i>plt</i> operon transcription.

<p>(<b>A</b> and <b>B</b>) β-galactosidase expression (Miller Units) from p6522–pltLp and p6522–L6-1, which respectively carry <i>pltLp</i> (−84 bp to +1) and its upstream P_x promoter (+66 bp to +124 bp), was assayed in the KMB without or with 10 μg mL⁻¹ exogenous Plt. The addition of 10 μg ml⁻¹ exogenous Plt significantly enhanced the <i>pltLp–lacZ</i> expression (A), but did not influence <i>P_X–lacZ</i> fusion expression (B) in the Plt-defective mutant M18pltB. (<b>C</b>) qRT-PCR analysis of the <i>pltA</i> transcript level in the strain M18pltB grown in the KMB without or with 10 μg ml⁻¹ exogenous Plt. The <i>pltA</i> transcript level was significantly enhanced by the addition of exogenous Plt.</p

APPLICATION OF HIGH-PERFORMANCE COUNTER-CURRENT CHROMATOGRAPHY AND MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY FOR RAPID ISOLATION OF LACTONES FROM <i>LIGUSTICUM CHUANXIONG</i> HORT.

<div><p>The root of <i>Ligusticum chuanxiong</i> Hort. is a well-known traditional Chinese medicine for treating headaches, ischemic stroke, anemia, and cerebral vascular disease. High-performance counter-current chromatography was applied to the isolation and purification of four lactones: 75.8 mg senkyunolide A, 3.5 mg levistolide A, 76.3 mg Z-ligustilide, and 0.8 mg wallichilide from 600 mg of the n-hexane extract of chuanxiong. Medium-pressure liquid chromatography was applied to the isolation and purification of one phthalide and two lactones: 2.9 mg chuanxingol, 11.3 mg senkyunolide A, and 20.1 mg Z-ligustilide from 800 mg of 60% ethanol extract of chuanxiong. The system composed of n-hexane–ethyl acetate–methanol–water in a volume ratio of 4:3:4:2 (<i>v/v</i>) was found to be optimum for HPCCC. The solvent system consisted of acetonitrile (A)−0.5% acetic acid (B) was used for MPLC, the binary gradient elution as follows: 0–40 min, 13%–100% A; and 40–50 min, 100% A. The target components separated by HPCCC and MPLC had higher purity determined by HPLC. The chemical structures of the target components were identified by electrospray ionization mass spectrometry (ESI-MS).</p> </div

Mechanism of fucoidan action.

<p>In ConA-induced acute liver injury, overexpression of TNF-α and IFN-γ activate the TRADD/TRAF2 and JAK2/STAT1 pathways by combining with TNFR and IFNGR to regulate apoptosis. On the one hand, the phosphorylated FADD cleaved procaspase 8 which mediated extrinsic apoptosis. On the other hand, Bid transfer was induced by cleaved caspase 8 to activate caspase 9 with Bax which mediated intrinsic apoptosis. Another cytokine, IFN-γ, promoted apoptosis by inhibiting Bcl-2 and Bcl-xL expression mediated by the JAK2/STAT1 pathway. Thus, fucoidan successfully inhibited the release of TNF-α and IFN-γ in damaged liver cells to attenuate both intrinsic and extrinsic apoptosis by reducing the phosphorylation of FADD and STAT1.</p

Effects of fucoidan on the activated TRADD signal pathway in ConA-induced acute liver injury.

<p>(A) The expression of TRADD and TRAF2 gene levels was measured by PCR. (B) The expression of TRADD, TRAF2, FADD and p-FADD protein levels was measured by western blot. The quantitative evaluation of p-FADD and FADD was determined by relative band density. (C) Immunohistochemistry was carried out to evaluate TRADD and p-FADD. Original magnifications: 200×. The above data are shown as means ± SD. *P<0.05 for ConA vs Fucoidan, ^#P<0.05 for ConA+Fucoidan (10, 25 and 50) vs ConA, ^P<0.05 for ConA+Fucoidan (25) vs ConA+Fucoidan (10), ⁺P<0.05 for ConA+Fucoidan (50) vs ConA+Fucoidan (25).</p