Molecular Cloning and Expression Analysis of an Actin Gene from Chinese Licorice（Glycyrrhiza uralensis Fisch）

A PCR-based homologous cloning strategy was used to identify an actin gene from the roots of Chinese licorice(Glycyrrhiza uralensis Fisch). Results of sequence analysis indicate that a 1137 bp cDNA with an open reading frame encoding 377 amino acids,actin ortholog,GuActin,was successfully cloned and characterized(GenBank accession No. EU190972). Thus far,GuActin is the first actin of Chinese licorice that has been identified at a molecular level. Analysis by Northern blot shows that GuActin was expressed st...     

A novel neutral polysaccharide having activity on the reticuloendothelial system from the root of Glycyrrhiza uralensis

A neutral polysaccharide, named glycyrrhizan UC, was isolated from the root of Glycyrrhiza uralensis Fischer. It was homogeneous on electrophoresis and gel chromatography, and its molecular mass was estimated to be 69,000. Glycyrrhizan UC is composed of L-arabinose:D-galactose:D-glucose:L-rhamnose in the molar ratio of 10:30 27:1. Methylation analysis, carbon-13 nuclear magnetic resonance and periodate oxidation studies indicated its structural feature as an arabino-3,6-galacto-glucan type polysaccharide.     

Field survey of Glycyrrhiza plants in central Asia (2). Characterization of phenolics and their variation in the leaves of Glycyrrhiza plants collected in Kazakhstan

A new prenylated flavanone, licoleafol, and a new prenylated dihydrostilbene, uralstilbene, together with four known compounds, 8-dimethylallyleriodictyol, sophoraflavanone B, gancaonin R, and 6-dimethylallyleriodictyol, were isolated from the leaves of Glycyrrhiza uralensis collected in Kazakhstan. HPLC analysis of the leaves of Glycyrrhiza plants collected in Kazakhstan showed that both G. uralensis-specific and Glycyrrhiza glabra-specific compounds were detected in the leaves of the morphologically intermediate-type plants, suggesting that the intermediate-type plant is a hybrid of G. glabra and G. uralensis. In addition, HPLC profiles of leaf extracts from offspring of intermediate-type plants were divided into the three types: the G. uralensis type, G. glabra type, and the intermediate type. From these results, it appears likely that the intermediate-type plant back-crosses with G. glabra and G. uralensis to generate a G. glabra-type plant and a G. uralensis-type plant, respectively.     

马钱子与甘草配伍前后生物碱成分的变化规律

采用电喷雾质谱技术和高效液相色谱法分别对马钱子配伍甘草前后主要生物碱的变化规律进行了系统研究。实验结果表明,马钱子配伍甘草后其主要生物碱士的宁和马钱子碱的含量均有不同程度的降低,其中士的宁的含量下降显著。电喷雾质谱的实验结果与高效液相色谱的结果相吻合,为进一步阐明甘草解马钱子类药物毒性和马钱子合理配伍用药提供了科学的实验依据。     

乌拉尔甘草叶中氨基酸的反相高效液相色谱快速测定

用邻苯二甲醛柱前荧光衍生梯度洗脱的方法分析了乌拉尔甘草叶中14种氨基酸的含量，为利用甘草叶作为饲料提供了科学参考；该法操作简单，衍生反应迅速，灵敏度高，重复性好。     

An automated method of on-line extraction coupled with flow injection and capillary electrophoresis for phytochemical analysis

In this study, an automated system for phytochemical analysis was successfully fabricated for the first time in our laboratory. The system included on-line decocting, filtering, cooling, sample introducing, separation, and detection, which greatly simplified the sample preparation and shortened the analysis time. Samples from the decoction extract were drawn every 5 min through an on-line filter and a condenser pipe to the sample loop from which 20-μL samples were injected into the running buffer and transported into a split-flow interface coupling the flow injection and capillary electrophoresis systems. The separation of glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) took less than 5 min by using a 10 mM borate buffer (adjusted pH to 8.8) and +10 kV voltage. Calibration curves showed good linearity with correlation coefficients (R) more than 0.9991. The intra-day repeatabilities (n = 5, expressed as relative standard deviation) of the proposed system, obtained using GTA and GA standards, were 1.1% and 0.8% for migration time and 0.7% and 0.9% for peak area, respectively. The mean recoveries of GTA and GA in the off-line extract of Glycyrrhiza uralensis Fisch root were better than 99.0%. The limits of detection (signal-to-noise ratio = 3) of the proposed method were 6.2 μg/mL and 6.9 μg/mL for GTA and GA, respectively. The dynamic changes of GTA and GA on the decoction time were obtained during the on-line decoction process of Glycyrrhiza uralensis Fisch root.     

Molecular Characterization of N-Terminal Pro-sequence of Keratinase Ker P from Pseudomonas aeruginosa: Identification of Region with Chaperone Activity

In silico analysis of keratinase Ker P from Pseudomonas aeruginosa revealed that its full gene of 1,497 bp constituted of a 72-bp signal sequence along with a long 520 bp pro-sequence and 905 bp core region. Position specific multiple sequence alignment of Ker P protein with other distant proteases revealed high variability within their N-terminal regions while the core protein was considerably conserved. Ker P (F1) and its four N-terminal truncations (F2-F5) lacking 72, 177, 405, 507 bp, respectively, were cloned and constitutively expressed as extracellular protein in pEZZ-18 secretory vector with Escherichia coli HB101 as the expression host. Ker P F1, Ker P F2, Ker P F3 and Ker P F4 products were active whereas no keratinolytic activity was obtained in Ker P F5. Further analysis revealed that only 187 bp pro-sequence region is required for correct folding of the protein into its active conformation and, thus, has chaperone-like activity. Further, comparative biochemical characterization revealed that the full-length keratinase Ker P F1 was catalytically more efficient than the truncated forms. Among the truncated enzymes, keratinase Ker P F4 exhibited better thermostability than Ker P F2 with a t(1/2) of >1 h at 60 °C. It also had a higher V (max) and K (m) on casein as compared with Ker P F2. However, no significant variation was observed with respect to kinetics on synthetic substrates.     

Expression of aquaporin-5 and its regulation in skeletal muscle cells

The aquaporins constitute a family of homologous intrinsic membrane proteins that function as highly selective water channels and are highly expressed in tissues where rapid water movement across the cell membrane is required. Molecular mechanism of water transport through the plasma membrane of skeletal muscle is still not clear. This study was designed to identify aquaporin subtypes and their expression regulation in C2C12 cells, a mouse myoblastic cell line. RT-PCR, immunohistochemistry and Western blot analysis revealed that C2C12 cells express AQP5. AQP5 expression was increased by induction of C2C12 differentiation. Exposure of C2C12 cells to hypertonic solutions induced an increase in AQP5 expression and p38 kinase activation. However, a p38 kinase inhibitor failed to inhibit hyperosmolar induction of AQP5 expression in C2C12 cells. These data indicate that skeletal muscle cells express AQP5 protein and its expression is regulated by differentiation and hypertonic stress.     

Characterization of two polysaccharides having activity on the reticuloendothelial system from the root of Glycyrrhiza uralensis

Two polysaccharides, called glycyrrhizans UA and UB, were isolated from the root of Glycyrrhiza uralensis Fischer. They were homogeneous on electrophoresis and gel chromatography, and showed reticuloendothelial system-potentiating activity in a carbon clearance test. Glycyrrhizan UA is composed of L-arabinose: D-galactose: L-rhamnose: D-galacturonic acid in the molar ratio of 20:14:1:3, and glycyrrhizan UB is composed of L-arabinose: D-galactose: D-glucose: L-rhamnose: D-galacturonic acid in the molar ratio of 12:10:1:10:20, in addition to small amounts of O-acetyl groups and peptide moiety, respectively. About 10% (glycyrrhizan UA) and 35% (glycyrrhizan UB) of the D-galacturonic acid residues exist as the methyl esters. Methylation analysis, carbon-13 nuclear magnetic resonance and periodate oxidation studies indicated their structural features.     

The core structure and immunological activities of glycyrrhizan UA, the main polysaccharide from the root of Glycyrrhiza uralensis.

The controlled Smith degradation and limited hydrolysis of glycyrrhizan UA, the main phagocytosis-activating polysaccharide isolated from the root of Glycyrrhiza uralensis FISCHER, was performed. The reticuloendothelial system-potentiating, anti-complementary and alkaline phosphatase-inducing activities of glycyrrhizan UA and its degradation products were investigated. Methylation analyses of primary, secondary and tertiary Smith degradation products and of the limited hydrolysis product indicated that the core structural features of glycyrrhizan UA include a backbone chain composed of beta-1,3-linked D-galactose. All of the galactose units in the backbone carry side chains composed of mainly alpha-1,5-linked L-arabino-beta-1,6- or 1,3-linked D-galactose residues at position 6. Removal of the arabinosyl side chains caused a pronounced decrease in immunological activity.     

A novel biflavonoid from roots of Glycyrrhiza uralensis cultivated in China

A novel biflavonoid named licobichalcone was isolated from the roots of Glycyrrhiza uralensis cultivated in China, along with twelve known compounds, including five chalcones, two isoflavones, two flavanones, two flavones and one pterocarpan. Their structures were respectively elucidated on the basis of chemical and spectroscopic evidence.     

Preparative purification of glycyrrhizin extracted from the root of liquorice using high-speed counter-current chromatography

Glycyrrhizin is one of the main bioactive components in liquorice (Glycyrrhiza uralensis Fisch) which has recently been found to be highly active in inhibiting replication of the severe acute respiratory syndrome (SARS)-associated virus. The separation and purification of glycyrrhizin from a methanol-water (70:30 (v/v)) extract of liquorice roots was achieved using high-speed counter-current chromatography. The separation was performed at a preparative scale in a one-step separation with a two-phase solvent system composed of ethyl acetate-methanol-water (5:2:5 (v/v)). The lower phase was used as the mobile phase in the head-to-tail elution mode. The present method yielded 42.2 mg glycyrrhizin at 96.8% purity from 130 mg of the crude exact with 95.2% recovery as determined by HPLC analysis.     

Determination of liquiritigenin and isoliquiritigenin in Glycyrrhiza uralensis and its medicinal preparations by capillary electrophoresis with electrochemical detection

A simple, reliable, reproducible and sensitive method, based on capillary electrophoresis with electrochemical detection (ED), for the determination of liquiritigenin and isoliquiritigenin in Glycyrrhiza uralensis and its medicinal preparations was described. Operated in a wall-jet configuration, a 300 microm diameter carbon-disk electrode was used as the working electrode, which exhibits good responses at + 1000 mV (versus SCE) for the two analytes. Under the optimum conditions, the analytes were base-line separated within 8 min, and excellent linearity was obtained in the concentration range from 5.0 x 10(-4) to 1.0 x 10(-6) mol/l. The detection limit (S/N = 3) was 4.7 x 10(-7) and 2.9 x 10(-7) mol/l for liquiritigenin and isoliquiritigenin, respectively. This work provides a useful method for the analysis of traditional Chinese medicines.     

One step isolation and purification of liquiritigenin and isoliquiritigenin from Glycyrrhiza uralensis Risch. using high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale has been successfully applied to the separation of bioactive flavonoid compounds, liquiritigenin and isoliquiritigenin in one step from the crude extract of Glycyrrhiza uralensis Risch. The HSCCC was performed using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-acetonitrile-water (2:2:1:0.6:2, v/v). Yields of liquiritigenin (98.9% purity) and isoliquiritigenin (98.3% purity) obtained were 0.52% and 0.32%. Chemical structures of the purified liquiritigenin and isoliquiritigenin were identified by electrospray ionization-MS (ESI-MS) and NMR analysis.     

肠道病毒71型外壳蛋白VP1在大肠杆菌中的表达

将扩增得到的肠道病毒71型外壳蛋白VP1基因克隆到测序载体pGEM-T,测序验证该序列为目的片段后,将目的基因克隆到原核表达载体pGEX-5x-1中,转化大肠杆菌BL21,IPTG诱导表达,产物经SDS-PAGE分析和Western blot验证。结果表明,在经IPTG诱导的BL21中检测到分子量与预期大小相符的大约60 kDa的融合蛋白。利用表达产物作为抗原,对EV71感染病人阳性血清的检测初步证实,重组蛋白VP1可以作为检测EV71感染的检测用抗原。     

Effects of Aloe vera Flower Extract and Its Active Constituent Isoorientin on Skin Moisturization via Regulating Involucrin Expression: In Vitro and Molecular Docking Studies

Skin moisturization is very crucial for maintaining the flexibility, viscoelasticity, and differentiation of the epidermis and its deprivation causes several diseases from dry skin to dermatitis. Aloe vera, a miracle plant having diverse medicinal properties including skin moisturization effects. This study investigated for the first time the molecular mechanism targeting skin moisturization effects of the Aloe vera flower and its major active constituent. By treating human epidermal keratinocytes (HaCaT cells) with Aloe vera flower water extract (AFWE), we found that AFWE upregulated epidermal involucrin by activating the expression of protein kinase C, p38, and ERK 1/2. Additionally, it modulated filaggrin, increased aquaporin expression, and hyaluronan synthesis via a balanced regulation of HAS1 and HYAL1 protein. Similarly, it was able to protect UVB-induced photodamage. Western blot analysis, ELISA, and qRT- PCR were performed to evaluate various epidermal differentiation markers and moisturization-related factors on human epidermal keratinocytes (HaCaT cells). TLC and HPLC were used to detect and analyze the chemical constituents. Among them, we found that an active component of Aloe vera flower, isoorientin (IO) has a high binding affinity to all of its targeted proteins such as involucrin, PKC, P38, etc. through molecular docking assay. This study indicated that the Aloe vera flower and its active constituent, IO can be used as a prominent ingredient to enhance skin barrier function and improve its related pathologies.     

Effects of a chemical chaperone on genetic mutations in ��-galactosidase A in Korean patients with Fabry disease

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the gene encoding the α-galactosidase A (GLA) enzyme. We have identified 15 distinct mutations in the GLA gene in 13 unrelated patients with classic Fabry disease and 2 unrelated patients with atypical Fabry disease. Two of the identified mutations were novel (i.e., the D231G missense mutation and the L268delfsX1 deletion mutation). This study evaluated the effects of the chemical chaperones 1-deoxygalactonojirimycin (DGJ) on the function of GLA in vitro, in cells containing missense mutations in the GLA gene. Nine missense and a nonsense mutations, including one novel mutation were cloned into mammalian expression vectors. After transient expression in COS-7 cells, GLA enzyme activity and protein expression were analyzed using fluorescence spectrophotometry and Western blot analysis, respectively. DGJ enhanced GLA enzyme activity in the M42V, I91T, R112C and F113L mutants. Interestingly, the I91T and F113L mutations are associated with the atypical form of Fabry disease. However, DGJ treatment did not have any significant effect on the GLA enzyme activity and protein expression of other mutants, including C142W, D231G, D266N, and S297F. Of note, GLA enzyme activity was not detected in the novel mutant (i.e., D231G), although protein expression was similar to the wild type. In the absence of DGJ, the E66Q mutant had wild-type levels of GLA protein expression and approximately 40% GLA activity, indicating that E66Q is either a mild mutation or a functional single nucleotide polymorphism (SNP). Thus, the results of this study suggest that the chemical chaperone DGJ enhances GLA enzyme activity and protein expression in milder mutations associated with the atypical form of Fabry disease.     

口蹄疫病毒3ABC基因截短体在毕赤酵母中的表达及鉴定

将长为525 bp的口蹄疫病毒3ABC基因截短体克隆到毕赤酵母表达载体pPIC9K中, 构建了重组表达质粒pPIC9K-3ABCt. 用BglⅡ线性化后, 电转化毕赤酵母菌GS115, 经表型筛选, PCR鉴定, 获得阳性重组菌(GS115/pPIC9K-3ABCt). 然后进行诱导表达, 通过SDS-PAGE和Western blot鉴定表达产物. 结果表明, 重组菌株成功分泌表达了分子量为40000, 具有免疫反应活性, 且呈二聚体形式的目的蛋白. 在96 h时表达量达到最高峰, 占分泌总蛋白的18%, 达到23.4 mg/L. 为进一步研制口蹄疫免疫和感染动物鉴别诊断试剂奠定了基础.     

Beta-actin is not a reliable loading control in Western blot analysis

Beta-actin is often used as a loading control in Western blot analysis. We analyzed the ability of beta-actin-specific antibodies to recognize differences in protein loading. We found that, at higher total protein loads as required for the detection of low-abundance proteins, beta-actin-specific antibodies failed to distinguish differences in actin protein levels. Diluting the antibody working solution or changing the incubation time had little effect on this phenomenon. This shows that beta-Actin is not a reliable loading control in Western blot analysis. In general, it appeared that, at longer incubation times, antibodies seem to be less able to pick up differences in the level of its target protein.