新疆胀果甘草幼苗耐盐性及对NaCl胁迫的离子响应

以盐碱荒漠草甸药用植物胀果甘草(Glycyrrhiza inflata)为材料, 采用水培法研究了盐处理(50、100、200、300 mmol·L^-1NaCl) 28天后幼苗株高、生物量、含水量、根粗、甘草酸含量和不同器官的离子含量及离子的选择吸收、运输能力, 并对丙二醛、脯氨酸含量进行测定, 以确定其耐盐范围及耐盐方式。结果表明, 低盐浓度对胀果甘草幼苗生长无显著影响, 只有较高盐浓度(≥200 mmol·L^-1 NaCl)使幼苗总生物量、株高、甘草酸含量显著降低; 根据耐盐系数与盐浓度的拟合方程, 确定适宜幼苗生长的盐浓度范围为0-278.17 mmol·L^-1。随盐浓度上升, 植株选择性吸收K⁺、Ca²⁺、Mg²⁺, 而抑制Na⁺进入体内, 幼苗对进入植株体内的Na⁺在不同盐浓度下采取了不同的分配策略, 低盐浓度下(0-100 mmol·L^-1), 植株体内Na⁺主要积累在根中, 避免了叶中Na⁺的过多积累, 其盐适应机制以耐盐方式为主; 高盐浓度下(≥200 mmol·L^-1 NaCl), Na⁺主要积累在下部叶, 并通过叶片脱落的方式带走体内的盐分, 其盐适应机制以避盐方式为主。盐胁迫下, 幼苗能促进K⁺而抑制Na⁺向上部叶的运输, 使上部叶拒Na喜K, 维持了较高的K⁺/Na⁺比值, 有利于幼苗生长; 同时, 地下根系能通过积累Ca²⁺、Mg²⁺和合成脯氨酸、甘草酸, 以提高渗透调节能力, 缓解Na⁺毒害, 使根的生长不受影响, 有利于保证幼苗在盐环境中吸收维持生长的必要养分, 这是胀果甘草幼苗具有较强耐盐性的原因。以上结果说明, 胀果甘草幼苗通过对盐离子的吸收和运输调控、离子区域化和渗透调节, 以耐盐和避盐两种方式适应盐碱荒漠环境。     

甘草根中甘草酸的免疫组织化学定位研究

应用免疫组织化学定位和HPLC分析方法,对人工种植乌拉尔甘草根中的甘草酸分布和积累变化规律进行分析,以阐明药用甘草根在发育过程中甘草酸的积累变化规律,探讨甘草药材质量形成的内在机制。结果表明:(1)甘草酸主要分布在根的次生韧皮部薄壁细胞和维管射线细胞中,且主要积累在这些细胞的细胞壁上。(2)HPLC分析显示,主根的韧皮部中甘草酸含量最高,其次是木质部,根皮中含量最少。(3)甘草酸在主根中的含量随着生长年限的增加而提高,2、3年生甘草根中甘草酸含量上升较快,3年后甘草酸的含量达到3.66%,超过了国家药典规定的甘草酸含量标准。     

The effect of bile salts on human vascular endothelial cells

The uptake and release of radiochromium from adult human vascular endothelial cells in culture was employed to determine the relative toxicity of different bile salts. Endothelial cells after pre-incubation with 51Cr for 18 h were incubated with bile salts for 24 h and percentage chromium release was taken as a measure of toxicity to cells. Lithocholic acid (LC) (potassium salt) was cytotoxic at concentrations greater than 50 microM. However, LC glucuronide, sulfate and the beta-epimer were progressively less toxic with toxicity seen at concentrations of 60, 110 and 180 microM, respectively. The greatest cytotoxic effect was observed with glycolithocholic acid (GLC) (potassium salt) which was toxic at every concentration tested (20-200 microM). Sulfation abolished the toxic effect of GLC. At the concentrations employed for the assay (between 20 and 240 microM) GLC sulfate (disodium salt), taurolithocholic acid sulfate (disodium salt), cholic acid (sodium salt), glycocholic acid (sodium salt), deoxycholic acid (sodium salt) and ursodeoxycholic acid (sodium salt) were not cytotoxic. The 51Cr release cytotoxicity assay was validated with lactate dehydrogenase leakage from endothelial cells with a good correlation (r = 0.87). These data confirm in a human cellular system that LC and its conjugates were the most toxic of the bile salts tested and explains its pathophysiological importance in hepatobiliary disease. It also suggests that biotransformation by either sulfation or beta-epimerisation of bile salts especially of LC, as occurs in patients with intrahepatic or extrahepatic biliary obstruction or severe cholestasis, is hepatoprotective.     

New retention mechanism of mononucleotides with buffer concentrations in ion-suppressing RP-HPLC

HPLC separation of ionic samples tends to be more complicated and difficult to understand than that of non-ionic compounds. On the other hand, band spacing is much more easily manipulated for ionic than for neutral samples. Ion-suppressing RP-HPLC method was used with organic modifier and aqueous buffer solution. In this work, five mononucleotides of cytidine-5-monophosphate (5′-CMP) disodium salt, uridine-5-monophosphate disodium salt (5′-UMP), guanosine-5-monophosphate disodium salt (5′-GMP), inosine-5-monophosphate disodium salt (5′-IMP), and adenosine-5-monophosphate disodium salt (5′-AMP) were examined. Acetic acid and sodium phosphate were used as buffers, and methanol as an organic modifier. A new relationship between the retention factor and the buffer concentration at a fixed modifier content (5% of methanol) could be expressed by fol|lowing:k=(k ₋₁+k ₀ (K _B/K _S)C _B ^a)/(1+(K _B/K _S)C _B ^a), whereC _B was the concentration of buffer. Using this relationship, the calculated values closely matched the experimental data. The derived relationship showed that as the buffer concentration increased, the retention factor approached a certain value, and this was buffer dependent.     

Copy number variations of functional genes influence contents of glycyrrhizic acid in Glycyrrhiza uralensis

Glycyrrhiza uralensis is a widely used Chinese herb and glycyrrhizic acid is believed to be its marker compound. Three key enzymes, 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), squalene synthase (SQS) and beta-amyrin synthase (β-AS), are involved in the glycyrrhizic acid biosynthetic pathway. In this paper, the relationship between copy number variations (CNVs) of HMGR, SQS and β-AS genes and the content levels of glycyrrhizic acid in G. uralensis were investigated. CNVs of the 62 G. uralensis samples from different origins were determined by real-time PCR and their glycyrrhizic acid contents were analyzed by HPLC. The real-time PCR results showed that the copy numbers of HMGR, SQS1 and β-AS in the 62 G. uralensis samples were either one copy or two copies. According to the copy number patterns of HMGR, SQS1 and β-AS, the 62 G. uralensis samples can be divided into six groups. Among the six groups, group B with two copies of HMGR, one copy of SQS1 and β-AS contained relatively higher contents of glycyrrhizic acid. The accumulation of glycyrrhizic acid was lower in the group C with two copies of β-AS, one copy of SQS1 and HMGR. The results of this work may provide a basis for enhancing the accumulation of glycyrrhizic acid in cultivars of G. uralensis.     

HPLC法测定病毒性疫苗中EDTA二钠残余量

实验中用高效液相色谱法测定病毒性疫苗中乙二胺四乙酸二钠残余量。EDTA二钠与FeCl3反应生成的络合物NaFeEDTA在257nm波长处有明显吸收,以C18柱将它与其余组分分开后,用外标法建立五级校正曲线可测定制品中EDTA-2Na含量。该方法准确,快速,可用于检测病毒性疫苗中残余乙二胺四乙酸二钠。     

Aqueous two-phase extraction for downstream processing of amyloglucosidase

A polymer/salt aqueous two-phase system has been successfully employed for separation and purification of amyloglucosidase. The effects of system pH, molecular weight of polymer and composition of the two-phase system on amyloglucosidase partition behaviour in polyethylene glycol (PEG 4000, 6000)/disodium hydrogen phosphate were investigated. Experimental data are explained based on Kim's theoretical model for the prediction of biomolecule partitioning in a PEG/salt system.     

芍药甘草汤“酸甘化阴”配伍的化学内涵研究初探

目的：探讨芍药与甘草“酸甘化阴”配伍的化学内涵。方法：①薄层色谱法对比分析芍药甘草配伍前后不同极性提取部位化学成分的变化。②反相高效液相色谱法对芍药与甘草配伍前后主要化学成分芍药苷、甘草酸进行含量测定并比较煎出率。结果：①芍药与甘草配伍前后水提液的不同极性溶剂提取部位进行薄层色谱定性分析结果表明,总体成分在配伍前后无明显变化,未发现新物质的产生;②高效液相检测结果表明,芍药配伍甘草能够促进芍药苷、甘草酸的煎出。结论：芍药配伍甘草不产生新的物质,但对其中一些主要化学成分的煎出有一定的影响。     

甘草酸对巨噬细胞抗绵羊肺炎支原体感染的调控作用研究

为观察甘草酸在小鼠巨噬细胞系RAW264.7抗绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)感染中的作用,实验利用CCK-8细胞活性检测找到最佳甘草酸处理浓度;检测甘草酸对受MO感染的巨噬细胞活性的影响。流式细胞仪检测甘草酸对RAW264.7巨噬细胞生长周期的影响;ELISA检测甘草酸对受MO感染的巨噬细胞分泌TNF-α的影响;Western blot检测细胞凋亡因子Bax、Bad的表达情况。RT-PCR检测凋亡和自噬相关基因的表达情况。结果显示,浓度为12μmol/L的甘草酸显著升高RAW264.7的活性(P=0.012 9),且处于G1期的细胞数减少,G2期的细胞数增加。甘草酸(12μmol/L)可提高受MO感染的RAW264.7的增殖率(P=0.034 0),培养上清中TNF-α含量升高(P=0.015 2),巨噬细胞中促凋亡蛋白Bax表达量增加,但基因caspase 3和caspase 9的表达量显著下调(P<0.000 1),自噬相关基因Atg 7和Beclin 1表达量显著升高(P<0.000 1)。结果提示在MO感染巨噬细胞引起免疫抑制的情况下,甘草酸可通过促增殖、抑凋亡、促进TNF-α的表达、增加自噬来起到免疫调控作用。     

Transformation of glycyrrhizinic acid. XV. Synthesis of triterpene saponins with monosaccharide residues, attached through complex ester bonds

Triterpene saponins, glycoside analogues of glycyrrhizic acid with a modified carbohydrate chain containing monosaccharide residues attached through ester bonds, were synthesized. To this end, peracetylated glycyrrhizic acid or its 30-methyl ester were glycosylated by 2,3,4,6-tetra-O-acetyl-alpha-D-gluco-or-alpha-D-galactopyranosyl bromide in dichloroethane in the presence of Ag2CO3. Glycerrhetinic acid saponin with D-Galp residues exhibited a higher antiulcer activity than glycyrrhizic acid in rats at a dose of 25 mg/kg. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Composition and structure of nucleolar skeleton (nucleolar matrix)

Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6–7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fibrillarin are two major protein components of nucleolar skeleton of HeLa cells.     

Solution properties of sulfated monohydroxy bile salts. Relative insolubility of the disodium salt of glycolithocholate sulfate

Physical-chemical properties of the major sulfated monohydroxy bile salts of man are described. In general, the sulfates are significantly more water-soluble than the non-sulfated species as a result of lower critical micellar temperatures, high aqueous monomeric solubilities and critical micellar concentrations. Nevertheless, at 37 degrees C the disodium salt of glycolithocholate sulfate, the major monohydroxy bile salt of man is not more soluble than its non-sulfated form. Since aqueous solubility correlates inversely with the cholestatic potential of bile salts, our results suggest that this sulfate may be potentially hepatoxic. Micellar solubility of phosphatidylcholine and cholesterol by the majority of non-sulfated and sulfated monohydroxy bile salts is slight. Nonetheless, phosphatidylcholine is very well solubilized by taurolithocholate sulfate but cholesterol solubility is not increased appreciably. Cholesterol saturation in model bile systems of taurochenodeoxycholate and phosphatidylcholine is impaired by the addition of sulfated lithocholate conjugates but with physiological bile salt compositions this reduction is not significant.     

Composition and structure of nucleolar skeleton (nucleolar matrix)-Actin and fibrillarin are two main protein components of nucleolar skeleton

Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6–7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fibrillarin are two major protein components of nucleolar skeleton of HeLa cells.     

Circadian rhythms in Neurospora crassa: a clock mutant, prd-1, is altered in membrane fatty acid composition

The fatty acid compositions of the phospholipids of Neurospora crassa mutants with altered periods were determined to test the possibility that some of these mutants might have altered membrane composition. In liquid shaker culture in constant light the bd (band) strain, which has a normal period (21.6 h), exhibited a growth-dependent increase in linoleic acid content and a decrease in linolenic acid content during early log phase growth. By late log phase, fatty acid composition was essentially constant. The phospholipid fatty acid compositions of bd strains containing mutations at the frq (frequency) and chr (chrono) loci were indistinguishable from that of the bd strain under the conditions used. However, a bd strain containing a mutation at the prd-1 (period) locus, as well as prd-1 segregants from a cross of this strain to a bd strain, had altered patterns of growth-dependent fatty acid composition; linoleic and linolenic acid contents changed more slow than in the bd strain and continued to change throughout growth. In addition, the fatty acid composition of a bd prd-1 strain on solid medium differed from that of the bd strain. It is proposed that the prd-1 mutation leads to altered membrane homeostasis, which in turn affects circadian rhythmicity because some or all components of the rhythm-generating system are membrane-localized.     

The Chemical Constitution of Rubrofusarin

Alkaline degradation of rubrofusarin and nor-rubrofusarin were studied; nor-rubrofusarin readily underwent hydrolysis to give a tetrahydroxynaphthalenc, acetone, and acetic acid; whereas, rubrofusarin, after prolonged time of hydrolysis, yielded a β-methoxytrihydroxynaphthalene instead of the naphthol. Physical and chemical studies revealed that the naphthol is 1,3,6,8-tetrahydroxynaphthalene and it has been confirmed by the synthesis from chromotropic acid (disodium salt). Thus, evidently, rubrofusarin has a naphthalene nucleus to which a methoxyl group is attached at β-position. The formation, on the hydrolysis, of acetone and acetic acid, along with the naphthol, indicates the presence of 2-methyl-γ-pyrone structure in rubrofusarin.     

Evaluation of the double‐disk synergy test for New Delhi metallo‐β‐lactamase‐1 and other metallo‐β‐lactamase producing gram‐negative bacteria by using metal‐ethylenediaminetetraacetic acid complexes

New Delhi metallo‐β‐lactamase‐1 (NDM‐1), one of the metallo‐β‐lactamases (MBLs), has been identified from clinical isolates worldwide. Rapid detection of NDM‐1 producers is necessary to prevent their dissemination. Seven types of EDTA complexes were evaluated as MBL inhibitors in double‐disk synergy tests (DDSTs), resulting in detection of the first isolate of NDM‐1‐producing Escherichia coli (NDM‐1 Dok01) in Japan. NDM‐1 Dok01 was detected when EDTA magnesium disodium salt tetrahydrate (Mg‐EDTA), EDTA calcium disodium salt dihydrate, EDTA cobalt disodium salt tetrahydrate and EDTA copper disodium salt tetrahydrate were used as MBL inhibitors. The sensitivity and specificity of DDSTs using Mg‐EDTA for 75 MBL producers and 25 non‐MBL producers were 96.0% and 100%, respectively. These findings indicate that the DDST method using Mg‐EDTA can detect MBL‐producing strains, including NDM‐1 producers.     

Detection of superoxide radicals in intact heart mitochondria by spin trapping

Tiron (4,5-dihydroxybenzene-1,3-disulfonic acid, disodium salt) has been used as a spin trap to detect superoxide radicals produced by the rat heart mitochondria. In the presence of the oxidative phosphorylation uncoupler carbonyl cyanide (3-chlorophenyl)-hydrazone the superoxide production rate increases.     

In vitro stimulation of chick brain lipid peroxidation by aluminium, and effects of tiron, EDTA and some antioxidants.

In vitro effect of aluminium (Al) on Fe(2+)-induced lipid peroxidation (LPX) in various subcellular fractions from the cerebral hemispheres (CH) of 7- and 30-day old chicks was studied. Stimulation of Fe(2+)-induced LPX by Al was observed to be the highest in microsomal fraction. The magnitude of elevation of Fe(2+)-induced LPX in various subcellular fractions of brain showed age related variation. Of the six chemicals tested for their influence on Al-induced lipid peroxidation, both doses of 1,2-dihydroxybenzene-3,5-disulphonic acid disodium salt (Tiron), ethylene diamine tetra acetic acid disodium salt (EDTA), and ascorbic acid prevented the Al-induced LPX in crude homogenates of the CH, whereas only at a higher dose inhibition by 1,4-diazabicyclo (2.2.2.) octan (DABCO) was observed. On the contrary, mannitol and dimethyl sulfoxide did not inhibit the induction of LPX by Al in crude homogenate. The effect of test chemicals on Al-induced LPX in both the ages of chick tissue was almost similar. The results suggest that Al further augments Fe(2+)-induced LPX in various compartments of the cell due to generation of free radicals. The results also showed that Tiron, EDTA and antioxidants, such as ascorbic acid and DABCO can prevent LPX induced by Al.     

Drug discovery for DNA break repair system by screening from TCM database and molecular dynamics approach

In search for novel anti-tumour agents targeting non-homologous end joining pathway, we conducted a virtual screening using traditional Chinese medicine (TCM) on Ku heterodimer which plays an important role on DNA break repair system. Docking results gave glycyrrhizic acid and macedonoside C as potential TCM candidates. Both glycyrrhizic acid and macedonoside C were docked onto the Ku heterodimer with high-predicted binding affinities as evidenced in their Dock Score. These two ligands also interact with key residues which have been previously shown to influence Ku DNA-binding affinity. Both compounds show consistent hydrogen bonding interactions with key residues throughout the 10- ns dynamics simulation run. Furthermore, glycyrrhizic acid and macedonoside C are able to form additional hydrogen bond interactions with positively charged Ku heterodimer surface. Such interactions strengthen the binding interaction of the top two TCM compounds with Ku heterodimer. Glycyrrhizic acid and macedonoside C are products of licorice (Glycyrrhiza glabra), which is a commonly used herb in TCM formulations. Overall, we report glycyrrhizic acid and macedonoside C as potential anti-tumour agents.