ASA法测定细胞色素P4502D6酶缺陷等位基因CYP2D6E

目的 :建立细胞色素P4 50 2D6(CYP2D6)第 3 0 2 3位A→C突变造成CYP2D6酶活性缺陷的等位基因CYP2D6E的测定方法。方法 :利用等位基因特异扩增法 (ASA)为基本原理 ,设计两对引物分别扩增野生型等位基因和突变型等位基因。结果 :经 3 96例测定 ,发现 2例CYP2D6E与CYP2D6B的异突变型纯合子 ,其表现型均为慢代谢者。阳性对照说明本法重复性好 ,阴性对照显示本法无污染问题。结论 :本法比PCR -RFLP法更为快捷、更少污染。

鲫鱼HindⅢ高重复DNA序列的分子克隆(英文)

基因组DNA高重复序列的研究有助于解释许多重要的生命现象 ,如基因调节、基因转座、基因进化等 ,还可以用于进行种群的遗传分析。鱼类的DNA高重复序列研究资料较少 ,曾在鲤科鱼类发现HindⅢ高重复序列家族。本研究用HindⅢ内切酶消化 ,从鲫鱼 (Carassiusauratusauratus)基因组DNA也克隆出一种独特的高重复序列。序列测定揭示该重复序列长度为 175bp ,在单倍体基因组的拷贝数为 1× 10 5。鲫鱼HindⅢ高重复序列与鲫鱼属 (Carassius)其它同类已知的高重复序列存在某种程度的变异 。

灵芝的生物活性组分及其抑制人体外周血淋巴细胞繁殖的研究(英文)

传统药用灵芝主要是用来医治肝病,肾炎,高血压,高血脂,关节炎,神经衰落,失眠,支气管炎,哮喘,胃溃疡,动脉硬化和糖尿病等。另外,灵芝的一些组分还有一些新发现的功效,如对肝细胞瘤的细胞毒害作用,抑制血小板的凝聚作用,消除紧张,降低胆固醇,阻止组胺的释放和抗艾滋病毒等。尽管在这种真菌中发现了很多中活性物质,如多糖、蛋白质、矿物质和甾醇等,但三萜系化合物是主要的活性成分。在过去的30年中发现120多种三萜系化合物,有必要对这些物质的活性及其结果作出总结。根据羊毛甾烷类三萜系化合物的结构和它们已知的生物学和药学功效可以分为10个类型,本文就此做了一些阐述。有关灵芝促进人体免疫能力的报道还不多。为了阐明灵芝的免疫抑制力的机制,自子实体中的萃取的甲醇抽取物可以分成6个组分,并把它们逐个加到人体血液外围单核细胞(PBMCs)中,发现在无粘附性细胞中,它们可以在细胞繁殖和生长时捕获细胞,但在粘附性的细胞上活性较差,用氯仿萃取物的活性更强,这种活性物质进一步硅胶柱纯化后,可分为7个亚组分。它们当中,GLE1,具有最高的迁移值(Rf值),在一定浓度下可以抑制细胞繁殖,该组分也可以抑制人体异源的淋巴细胞培养物(MLS)。用ELISA检测培养液上清液中分泌的白细胞素-2(IL-2)。

Lipophilic Enhance of Vitamin C Protective Activity Against Free-radical-induced Damage in vivo

A group of experimental rats under free radical damage are given various amounts of a lipophilic vitamin C(ascorbyl-6-laurate, VC-12), and its parent compound, vitamin C, respectively. It has been found that the effects of 1 12 mmol/kg VC-12 on decreasing triglyceride(TG), total cholesterol(TC), low density lipoprotein cholesterol (LDL-c) and lipid peroxide(LPO), and increasing high density lipoprotein cholesterol(HDL-c) and superoxide dismutase(SOD) are similar to those of 2 27 mmol/kg vitamin C. In addition, VC-12(1 12 mmol/kg) can increase the prostacycline(PGI 2) and decrease the thromboxane(TXA 2) even better than vitamin C(2 27 mmol/kg). The above facts demonstrate that the antioxidative activity of VC-12 is higher than twice that of vitamin C. So, ascorbyl-6-laurate may be a novel antioxidant drug against free radical damage.

Modulation of GdCl_3 and Angelica Sinensis polysaccharides on differentially expressed genes in liver of hepatic immunological injury mice by cDNA microarray

AIM: To study the modulating effect of GdCl3 and Angelica Sinensis polysaccharides (ASP) on differentially expressed genes in liver of hepatic immunological mice by cDNA microarray.METHODS: Hepatic immunological injury was induced by lipopolysaccharide (LPS ip, 0.2 mg.kg-1) in bacillus calmetteguerin (BCG ip, 1 mg.kg-1) primed mice; A single dose of 20 mg.kg-1 GdCl3 was simultaneously pretreated and 30 mg.kg-1 ASP (ig, qd×7 d) was administrated when the BCG+LPS was primed. The mice were sacrificed at the end of the 7th day after ip LPS for 6 h and the liver was removed quickly. The PCR products of 512 genes were spotted onto a chemical material-coated glass plate in array. The DNAs were fixed to the glass plate after series of treatments. The total RNAs were isolated from the liver tissue, and were purified to mRNAs by Oligotex.Both mRNAs from the normal liver tissue and the liver tissue from the mice with hepatic immunological injury or that pretreated with GdCl3 or ASP were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP to prepare the hybridization probes. The mixed probes were hybridized to the cDNA microarray. After highstringent washing, the cDNA microarray was scanned for fluorescent signals and showed differences between the two tissues.RESULTS: Among the 512 target genes, 18 differed in liver tissue of hepatic immunological injury mice, and 6 differed in those pretreated by ASP, 7 differed in those pretreated by GdCl3.CONCLUSION: cDNA microarray technique is effective in screening the differentially expressed genes between two different kinds of tissue. Further analysis of those obtained genes will be helpful to understand the molecular mechanism of hepatic immunological injury and to study the intervention of drug. Both ASP and GdCl3 can decrease the number of the differentially expressed genes in liver tissue of mice with hepatic immunological injury.

Effect of planting density on plant growth and camptothecin content of Camptotheca acuminata seedlings

C. acuminata seedlings cultivated in greenhouse were transplanted into the fields with 5 designed planting densities (11, 16, 25,44 and 100 plants·m^-2) in May of 2004 and were harvested in the middle of September of 2004. The seedling growth indexes including plant height and crown width, biomass allocation, camptothecin (CPT) content and CPT yield of different organs (young leaf, old leaf, stem,and root) were studied. For the 5 selected planting densities, the plant biomass, height, crown width, and total leaf area of C. acuminata seedlings all showed highest values at the planting density of 25 plants ·m^-2. CPT content in young leaves was higher than that in other organs of seedlings and presented an obvious change with the variation of planting densities and with the highest value at density of 100plants·m-2, while for other organs no significant variation in CPT content was found with change of planting density. The accumulation of CPT was enhanced significantly at the planting density of 25 plants·m^-2. It is concluded that for the purpose to get raw materials with more CPT from C. acuminata, the optimal planting density of C. acuminata seedlings should be designed as 25 plants·m^-2.

Binding activity of H-Ras is necessary for in vivo inhibition of ASK1 activity

H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and transmitting extracellular signals from growth factor receptors to Raf, leading to the propagation of signals to modulate a serious of cellular survival events. Apoptosis signal-regulating kinasel (ASK1) serves as a general mediator of cell death because it is responsive to a variety of death signals. In this study, we found that H-Ras interacted with ASK1 to cause the inhibition of both ASK1 activity and ASK1-induced apoptosis in vivo, which was reversed only partially by addition of RafS621A, an antagonist of Raf, whereas MEK inhibitor, PD98059, and PI3K inhibitor, LY294002, did not disturb the inhibitory effect of H-Ras on ASK-1-induced apoptosis. Furthermore, by means of immunoprecipitate and kinase assays, we demonstrated that the interaction between H-Ras and ASK1 as well as the inhibition of ASK1 activity were dependent on the binding activity of H-Ras. These results suggest that a novel mechanism may be involved in H-Rasmediated cell survival in addition to the well established MEK/ERK and PI3K/Akt kinase-dependent enhancement of cell survival.

A New Synthetic Route for Preparation of 2-Chloro-6-fluoro- benzonitrile and 2-Chloro-6-fluorobenzoic Acid

An effective synthetic route for preparation of 2-chloro-6-fluorobenzonitrile, 2-chloro-6-fluorobenzamide and 2-chloro-6-fluorobenzoic acid has been described. It includes diazotization, fluorination, ammoxidation and hydrolysis reactions.

Influence of Kupffer cells on hepatic signal transduction as demonstrated by second messengers and nuclear transcription factors

AIM: To understand the influence of Kupffer cell (KC) on signal transduction pathways in the liver.METHODS: To decrease selectively the number and function of KC, Kunming mice were ip injected with a single dose of gadolinium chloride (GdCl3, 20 mg.kg-1), the time-effect relationship assessment was performed after 1 d, 3 d and 6 d. sALT, sGST, liver glycogen content, phagocytic index,and expression of CD68 were assessed as the indexes of hepatotoxicity and functions of KC respectively, and morphology of KC was observed with transmission electron microscopy. Furthermore, cAMP, PGE2 level, nitric oxide(NO)content, and mRNA expression of NFkappaBp65, Erk1, STAT1 were examined.RESULTS: GdCl3 could selectively cause apoptosis of KC and obvious reduction of KC's activity, but no hepatotoxicity was observed. One day after KC blockade, NO, PGE2, cAMP contents in the liver were reduced 21.0 %, 6.94-fold, 8.3 %,respectively, and mRNA expression of NFkappaBp65 was decreased 3.0-fold. The change tendency of NO, PGE2,and cAMP contents and mRNA expression of NFkappaBp65 were concomitant with recovery of the functions of KC.The contents of NO, PEG2, cAMP were increased when the functions of KC was recovered. However, all of the changes could not return to the normal level except NO content after 6 d Gdcl3 treatment. No obvious changes were found in STAT1 and Erk1 mRNA expression in the present study.CONCLUSION: Hepatic NO, PGE2, cAMP level and mRNA expression of NFkappaBp65 are closely related with the status of KC. It suggests that KC may play an important role in the cell to cell signal transduction in the liver.

A New Labdane Diterpenoid from the Bark of Larix olgensis Henry var. koreana Nakai

A new diterpenoid was isolated from the bark of Larix olgensis Henry var. koreana Nakai. Its structure was elucidated as 5S, 9S, 10R-labda-8 (17), 13-diene-6a, 15-diol l by spectroscopic methods.

A Fast Determination of DNA Mutation Induced by Ultraviolet Radiation

Electrophoresis, chromatography, immunoassay, sequencing and other time consuming ap-proaches have been developed to determine DNA base mismatching, oxidative lesion or strand breaks. Sometimes,however, only qualitative information is enough to decide whether mutation has happened to DNA and its extent.Convolution spectrometry (CS), a new technique to discover ultrafme difference on ultraviolet (UV) absorption ofdifferent substances, is originally employed to find out any subtle mutation of DNA induced by UV radiation. Muta-tive DNA is compared with ego criteria based on the spectra of the former DNA, any difference is quantitatively ex-pressed by dispersion (5). Visible changes cannot be observed on second -derivative spectra until the mutation gets 5up to 11.48%. Dimethyl sulfoxide is an intensifier of UV 254 nm induced DNA mutation and protector at 365 nm,which is simply confirmed by increasing and decreasing 5. Every convolution procedure takes less than 1 min. Convolution spectrometry provides a fast, simple, sensitive and inexpensive alternative to determine DNA mutation, andto screen anti-mutational medicines.

Modulation of Kupffer cells on hepatic drug metabolism

AIM: To observe the effects of Kupffer cells on hepatic drug metabolic enzymes.METHODS: Kunming mice were ip injected with GdCI310,20, 40 mg/kg to decrease the number and block the function of kupffer cells selectively. The contents of drug metabolic enzymes, cytochrome P450, NADPH-cytochrom C redutase(NADPH-C), aniline hydroxylase (ANH), aminopyrine N-demethylase (AMD), erythromycin N-demethylase (EMD),and glutathione s-transferase (mGST) in hepatic microsome and S9-GSTpi, S9-GST in supernatant of 9 000 g were accessed 1 d after the injection. The time course of alteration of drug metabolic enzymes was observed on d 1, 3, and 6 treated with a single dose GdCI3. Mice were treated with Ange/ica sinensis polysaccharides (ASP) of 30, 60, 120 mg/kg, ig, qd x6 d, respectively and the same assays were performed.RESULTS: P450 content and NADPH-C, ANH, AMD, and EMD activities were obviously reduced 1 d after Kupffer cell blockade. However, mGST and S9-GST activities were significantly increased. But no relationship was observed between GdCI3 dosage and enzyme activities. With single dose GdCI3 treatment, P450 content, NADPH-C, and ANH activities were further decreased following Kupffer cell blockade lasted for 6 d, by 35.7%, 50.3%, 36.5% after 3 d, and 57.9%, 57.9%, 63.2% after 6 d, respectively. On the contrary, AMD, EMD, mGST, and S9-GST activities were raised by 36.5%, 71.9%, 23.1%, 35.7% after 3 d,and 155%, 182%, 21.5%, 33.7% after 6 d, respectively.Furthermore, the activities of drug metabolic enzymes were markedly increased after 30 mg/kg ASP treatment,and decreased significantly after 120 mg/kg ASP treatment.No change in activity of S9-GSTpi was observed in the present study.CONCLUSION: Kupffer cells play an important role in the modulation of drug metabolic enzymes. The changes of drug metabolic enzyme activities depend on the time of kupffer cell blockade and on the degree of Kupffer cells activated. A low concentration of ASP increases the activities of drug metabolic enzymes, but a high concentration of ASP decreases the activities of drug metabolic enzymes.

Synthesis and Crystal Structure of (4S)-5-(2-Methoxy-2-oxoethylamino)-5-oxo- 4-(3,4,5-trimethoxybenzamido) Pentanoic Acid

The crystal structure of (4S)-5-(2-methoxy-2-oxoethylamino)-5-oxo-4-(3,4,5- trimethoxybenzamido) pentanoic acid 5 (C18H24N2O9, Mr = 412.39) has been determined by single- crystal X-ray diffraction analysis. The crystal belongs to monoclinic, space group P21/c with a = 27.665(9), b = 5.1444(16), c = 13.907(4) ?, β = 98.401(5)o, V = 1958.0(11) ?3, Z = 4, Dc = 1.399 g/cm3, μ = 0.113 mm-1, F(000) = 872, R = 0.0606 and wR = 0.1405 (I > 2σ(I)). The results con- firmed that 5 could be assigned to the tautomeric form. The intermolecular hydrogen bonds between O(5)–H(5)…O(7), N(2)–H(2)…O(6) and N(1)–H(1)…O(4) have been observed.

New triterpenoid saponins from bulbs of Bolbostemma paniculatum

Nine new triterpenoid saponins were isolated from the bulbs of Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae): 7beta,18,20,26-tetrahydroxy-(20S)-dammar-24 E-en-3-O-alpha-L-(3-acetyl)arabinopyranosyl-(1-->2)-beta-D-glucopyranoside, 7beta,18,20,26-tetrahydroxy-(20S)-dammar-24E-en-3-O-alpha-L-(4-acetyl)arabinopyranosyl-(1-->2)-beta-D-glucopyranoside, 7beta,18,20,26-tetrahydroxy-(20S)-dammar-24E-en-3-O-alpha-L-arabinopyranosyl-(1-->2)-beta-D-(6-acetyl)glucopyranoside, 7beta,20,26-trihydroxy-(20S)-dammar-24E-en-3-O-alpha-L-arabinopyranosyl-(1-->2)-beta-D-glucopyranoside, 7beta,20,26-trihydroxy-(20S)-dammar-24E-en-3-O-alpha-L-(3-acetyl)arabinopyranosyl-(1-->2)-beta-D-glucopyranoside, 7beta,20,26-trihydroxy-(20S)-dammar-24E-en-3-O-alpha-L-(4-acetyl)arabinopyranosyl-(1-->2)-beta-D-glucopyranoside, 7beta,20,26-trihydroxy-8-formyl-(20S)-dammar-24E-en-3-O-alpha-L-(3-acetyl)arabinopyranosyl-(1-->2)-beta-D-glucopyranoside, 7beta,20,26-trihydroxy-8-formyl-(20S)-dammar-24E-en-3- O-alpha-L-(4-acetyl)arabinopyranosyl-(1-->2)-beta-D-glucopyranoside and 6’-O-palmitoyltubeimoside Ⅰ. In addition, four known triterpenoid saponins: tubeimoside Ⅰ, tubeimoside Ⅱ, tubeimoside Ⅲ and tubeimoside Ⅳ were isolated. The structures of the above compounds were elucidated based on spectroscopic studies, and the configuration of C-20 of tubeimoside Ⅳ was revised as S rather than R as reported in previous literature. The compounds were tested for their antiviral activity.

Sub-critical Column and Capillary Chromatography with Water as Mobile Phase and Flame Ionization Detection

A sub-critical chromatography (SubWC) with water as mobile phase and FID detection system is employed to separate several alcohols with high or medium polarity, with pure water as the eluent. The flow rate gets up to 50 μ1-min^-1 for packed column (1 mm i.d.) and 70 μ1-min^-1 for capillary (50 μm i.d.). Increasing the temperatureup to 140℃, together with temperature programming, markedly improves the separation and peak shapes within short analysis time. Sub-critical state is guaranteed.

Aged garlic extract and its components protect cultured rat hippocampal neurons from amyloid β—protein—in—duced neuronal death

Aged garlic extract and its components such as S-allyl-L-cysteine (SAC) and sllixin have been shown to possess various biological effects including neurotrophic activity.We characterized the neuronal death induced by amyloid β-protein (Aβ),4-hydroxynoenal (HNE),tunicamycin(TM),and trophic factor-deprivation (TFD),and ivestigated whether these garlic compounds could prevent this in cultured PC12 cells and rat hippocampal neurons.Treatment with SAC protected these cells against Aβ- and TM-induced neuronal death.SAC also attenuated the processing of procaspase-12 induced by Aβ25-35 or TM.In contrast,allixin and its analogue,DHP,afforded no protection against Aβ-induced cell death.SAC afforded no protection against HNE- and TFD-induced cell death,which has been shown to be mediated by caspase-3 dependent pathway.These results suggest that SAC protect against the neuronal cell death that is triggered by ER dysfunction.

Synthesis of β-L-2',3'-Dideoxy-2'-fluoro-3'-hydroxy-methylarabinofuranosyl Pyrimidine Nucleosides

Dideoxy-2'-fluoro-3'-hydroxymethylarabinofuranosylthymine 10 and cytosine 12 were synthesized from L-xylose and were found to be inactive against HIV-1 in acutely infected lymphocytes.