Varying the Unsaturation in N 4,N 9-Dioctadecanoyl Spermines: Nonviral Lipopolyamine Vectors for More Efficient Plasmid DNA Formulation

Purpose The aim of the study is to analyze the effect of varying the degree of unsaturation in synthesized N⁴,N⁹-dioctadecanoyl spermines on DNA condensation and then to compare their transfection efficiency in cell culture. Methods The N⁴,N⁹-di-C18 lipopolyamines—saturated (stearoyl), C9-cis- (oleoyl), and C9,12-di-cis- (linoleoyl)—were synthesized from the naturally occurring polyamine spermine. The ability of these novel compounds to condense DNA and form nanoparticles was studied using ethidium bromide fluorescence quenching and nanoparticle characterization techniques. Transfection efficiency was studied in several primary skin cells (FEK4, FCP4, FCP5, FCP7, and FCP8) and in an immortalized cancer cell line (HtTA) and was compared with the commercially available nonliposomal transfection formulation Transfectam^? (dioctadecylamidoglycyl spermine), which also contains two saturated C18 lipid chains. Results N⁴,N⁹-Dilinoleoyl spermine (C18, di-cis-9,12) is efficient at circular plasmid DNA (pEGFP) condensation and gives the most effective transfection in a series of primary skin cells and cancer cell lines at low charge ratios of 5.5 (± ammonium/phosphate). Conclusions The dienoic fatty acyl spermine conjugate N⁴,N⁹-dilinoleoyl spermine efficiently condenses DNA and achieves the highest transfection levels among the studied lipopolyamines in cultured cells.     

Lipopolyamine-Mediated Single Nanoparticle Formation of Calf Thymus DNA Analyzed by Fluorescence Correlation Spectroscopy

Purpose The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N⁴,N⁹-dioleoylspermine and N¹-cholesteryl spermine carbamate. Methods Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen (PG) to allow fluorescence intensity fluctuation measurement and analysis. Results N⁴,N⁹-Dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 × 10⁻¹² m²/s and particle number (PN) = 0.7 [at ammonium/phosphate (N/P) charge ratio=1.0–1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N¹-Cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5–2.5) with D = 1.3 × 10⁻¹² m²/s and PN value of 5.2. N⁴,N⁹-Dioleoylspermine is a more efficient DNA-condensing agent than N¹-cholesteryl spermine carbamate. Conclusions FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.     

Effect of N-nitro-l-arginine on shock induced by endotoxin and by platelet activating factor in dogs

When N^G-nitro-l-arginine, a nitric oxide synthase inhibitor, administration was started 5 min prior to shock induction in anesthetized dogs, a partial restoration was observed in endotoxin-induced shock and a complete recovery in platelet activating factor (PAF)-induced shock. When N^G-nitro-l-arginine infusion was started 5 min after shock induction, no significant recovery was observed in endotoxin-induced shock and a complete recovery in PAF-induced shock. These data indicate that enhanced production of nitric oxide by vascular endothelial cells may contribute to endotoxin- or PAF-induced shock and also that some mediators including inducible nitric oxide synthase and/or cellular damage might be involved in endotoxin-induced shock.     

N-Nitro-L-arginine protects against ischaemia-induced increases in nitric oxide and hippocampal neuro-degeneration in the gerbil

To assess the effects of the nitric oxide synthase inhibitor N^G-Nitro-L-arginine on behavioural, biochemical and histological changes following global ischaemia, the Mongolian gerbil was used. Ischaemia was induced by bilateral carotid occlusion for 5 min. N^G-Nitro-L-arginine was administered i.p. at either 1 or 10 mg/kg 30 min, 6, 24, and 48 h after surgery. 5 min bilateral carotid occluded animals were hyperactive 24, 48 and 72 h after surgery. N^G-Nitro-L-arginine caused some attenuation in this hyperactivity. The activity of nitric oxide synthase was increased in the cerebellum, brain stem, striatum, cerebral cortex and hippocampus of 5 min bilateral carotoid occluded animals. N^G-Nitro-L-arginine reversed the increase in nitric oxide synthase activity in all brain regions. Extensive neuronal death was observed in the CA₁ layer of the hippocampus in 5 min bilateral carotid occluded animals 96 h after surgery. N^G-Nitro-L-arginine significantly protected against the neuronal death of cells in the CA₁ layer.     

A novel compound N,N-(Z)-N-(E)-tri-p-coumaroylspermidine isolated from Carthamus tinctorius L. and acting by serotonin transporter inhibition

Safflower, the dry flower of Carthamus tinctorius L., has long been applied for empirically treating cerebral ischemia and depression in traditional Chinese medicine. Pathogenesis of major depression involves monoaminergic transmission. The present study assessed whether safflower or its isolate would be effective in functionally regulating monoamine transporter using in vitro screening cell lines. We discovered that safflower insoluble fraction significantly inhibited serotonin uptake in Chinese hamster ovary cells stably expressing serotonin transporter (i.e. S6 cells). This fraction went through an activity-guided isolation and an active ingredient was obtained, which was subsequently elucidated as a novel coumaroylspermidine analog N¹,N⁵-(Z)-N¹⁰-(E)-tri-p-coumaroylspermidine using NMR techniques. Pharmacologically, this compound potently and selectively inhibited serotonin uptake in S6 cells or in synaptosomes, with IC₅₀ of 0.74 ± 0.15 µM for S6 cells or 1.07 ± 0.23 µM for synaptosomes and with a reversible competitive property for the 5HT-uptake inhibition. The potency of it for 5HT uptake was weaker than that of fluoxetine whereas efficacy generally similar for both. Animals treated with this testing compound showed a significant decrease in synaptosomal 5HT uptake capacity. Thus, N¹,N⁵-(Z)-N¹⁰-(E)-tri-p-coumaroylspermidine is a novel serotonin transporter inhibitor, which could improve neuropsychological disorders through regulating serotoninergic transmission.     

<Emphasis Type="Italic">N</Emphasis><Superscript>4</Superscript>,<Emphasis Type="Italic">N</Emphasis><Superscript>9</Superscript>-Dioleoyl Spermine Is a Novel Nonviral Lipopolyamine Vector for Plasmid DNA Formulation

Purpose To study the effect of synthesized N⁴,N⁹-dioleoyl spermine on DNA condensation and then measure its transfection efficiency in cell culture.Methods The lipopolyamine was synthesized from the naturally occurring polyamine spermine. The ability of this novel compound to condense DNA was studied using ethidium bromide fluorescence quenching and light scattering assays. Transfection efficiency was studied in primary skin cells (FEK4) and in an immortalized cancer cell line (HtTA), and compared with the commercially available transfection formulations Lipofectin and Lipofectamine.Results The synthesized N⁴,N⁹-dioleoyl spermine formula is efficient at condensing calf thymus and circular plasmid DNA and effectively transfects both primary skin cells and cancer cell lines at low charge ratios of (+/– ammonium/phosphate) 2.5.Conclusions N⁴,N⁹-Dioleoyl spermine condenses DNA and achieves high transfection levels in cultured cells.     

Constitutive nitric oxide modulates the injurious actions of vasopressin on rat intestinal microcirculation in acute endotoxaemia

The administration of the nitric oxide (NO) synthase inhibitor, N^G-nitro-l-arginine methyl ester (L-NAME, 5 mg/kg s.c.) concurrently with Escherichia coli endotoxin (3 mg/kg i.v.) increased vascular permeability and caused mucosal damage in the rat intestine 1 h later. The vasopressin V₁ receptor antagonist, [Mca¹,Tyr(Me)²,Arg⁸]vasopressin (0.01–0.2 μg/kg s.c., 15 min before endotoxin) dose-dependently reduced this damage. These results suggest a beneficial role of NO, counteracting the injurious vascular actions of endogenous vasopressin, in maintaining intestinal mucosal integrity in acute endotoxaemic states.     

Flavonoids from the whole plants ofOrostachys japonicus

From the whole plants ofOrostachys japonicus, kaempferol, quercetin, astragalin, quercitrin, isoquercitrin, cynaroside, afzelin, 3-O-α-L-rhamnosyl-7-O-β-D-glucosyl kaempferol, and 3,7-di-O-β-D-glucosyl kaempferol were isolated and characterized by spectral data.     

High grazer toxicity of [D-Asp,(E)-Dhb]microcystin-RR of Planktothrix rubescens as compared to different microcystins

Planktothrix rubescens, the dominant cyanobacterium in Lake Zürich, is generally considered to be toxic to zooplankton. The major toxin was determined by NMR spectroscopy and chemical analysis to be [D-Asp³,(E)-Dhb⁷]microcystin-RR. The compound was isolated in high purity, and its 24-h acute grazer toxicity was compared with microcystin-LR, microcystin-RR, microcystin-YR, and nodularin using a Thamnocephalus platyurus bioassay. Based on LC₅₀ values [D-Asp³,(E)-Dhb⁷]microcystin-RR was the most toxic microcystin tested. Nodularin was slightly more toxic under the conditions of the assay. The large number of individuals available for the grazer bioassay allowed the determination of dose-response curves of the different microcystins. These curves showed marked differences in their steepness. Microcystin-RR, which had nearly the same LC₅₀ as microcystin-LR and microcystin-YR, exhibited a very flat dose-response curve. This flat curve indicates that, for some individuals, lower concentrations of this microcystin are much more toxic than are the other two microcystins. Mortality of 100% requires much higher concentrations of microcystin-RR, indicating the resistance of some animals to the toxin. The purified [D-Asp³,(E)-Dhb⁷]microcystin-RR exhibited a higher molar absorption coefficient determined by quantitative amino acid analysis than the coefficients generally used for other microcystins. This observation has consequences for the risk assessment for microcystins and makes a structural determination of microcystins an absolute requirement. The presence of the dehydrobutyrine residue may be the reason for the higher specific toxicity of [D-Asp³,(E)-Dhb⁷]microcystin-RR when compared to the N-methyldehydroalanine-containing microcystins.     

VKORC1-3673G>A、CYP2C9*3、CYP4F2 rs2108622和CYP2C19*2基因多态性对中国汉族房颤患者华法林维持剂量的影响

目的 探讨VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622和CYP2C19^*2位点基因多态性对中国汉族房颤患者华法林维持剂量的影响。方法 收集107例服用华法林达维持剂量的汉族房颤患者的血样和临床相关资料,应用PCR-RFLP法检测VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622和CYP2C19^*2基因型,采用独立样本t检验分析基因型与华法林维持剂量的相关性。多元线性回归建立给药模型,探讨基因多态性对华法林维持剂量的影响。结果 VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622基因多态性和患者年龄、体质量能解释45.2%的华法林维持剂量差异。CYP2C19^*2基因多态性对本研究人群华法林维持剂量无影响。结论 VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622基因多态性显著影响中国汉族房颤患者的华法林维持剂量。     

7α-和7β-甲基-10β,17β-二乙酰氧基-△4-雌甾烯-3酮的抗早孕作用

7α-和7β-甲基-10β,17β-二乙酰氧基-△⁴-雌甾烯-3酮(简称7α-和7β-甲-乙氧雌酮)对小鼠抗早孕ED₅₀分别为1.6和5.5 mg/kg。7α-甲-乙氧雌酮在大鼠也有抗早孕作用并使血浆孕酮浓度降低,应用10 μg/ml浓度能抑制离体妊娠大鼠卵巢孕酮合成。7α-和7β-甲-乙氧雌酮与兔子宫胞浆雌二醇受体的相对结合亲和力(RBA)分别为10.8和1.5,与孕酮受体的RBA均<1.7α-和7β-甲-乙氧雌酮都有较弱的雌激素和抗雌激素活性。     

Endothelium-derived nitric oxide partially mediates salbutamol-induced vasodilatations

This study examined the ability of salbutamol (selective β₂-adrenoceptor agonist) to cause endothelium-dependent relaxation in rat aortic rings and depressor response in conscious rats. Salbutamol (0.01–100 μM) concentration dependently relaxed preconstricted aortic rings. The relaxant response was partially attenuated by either mechanical removal of the endothelium or treatment with N^G-nitro-l-arginine methyl ester (L-NAME, 100 μM). In conscious rats, either i.v. infused phenylephrine (5 μg/kg per min) or i.v. bolus injected L-NAME (12.8 mg/kg), but not the vehicle, caused similar sustained increases in mean arterial pressure (MAP). I.v. infused salbutamol (2–128 μg/kg per min, each dose for 5 min) dose dependently decreased MAP in vehicle-treated rats; the depressor responses were potentiated by hypertension induced by phenylephrine. In contrast, the magnitudes of the depressor response to salbutamol in L-NAME-treated rats were less than those in rats pretreated with phenylephrine or the vehicle. I.v. bolus injections of salbutamol (0.25–16 μg/kg) also caused dose-dependent and transient decreases in MAP in vehicle-treated rats. The magnitude but not the duration of the depressor response to salbutamol was less in rats treated with L-NAME, compared to those in rats given phenylephrine or the vehicle. These results suggest that endothelium-derived nitric oxide is partially involved in β₂-adrenoceptor-mediated vasodilatation.     

Effects of glibenclamide and tetracaine on 86Rb+ fluxes in mouse pancreatic β-cells

Summary Potassium transport was measured in -cell-rich islets from ob/ob-mice using the K⁺-analogue ⁸⁶Rb⁺. Both tetracaine (0.1 mM) and glibenclamide (0.1 M) reduced the oubain-resistant ⁸⁶Rb⁺ influx but did not significantly affect the oubain-sensitive portion (Na⁺/K⁺ pump). Tetracaine (0.5–1 mM) or glibenclamide (0.2 mM) decreased the ⁸⁶Rb⁺ equilibrium content and glibenclamide (1 M) transiently reduced the ⁸⁶Rb⁺ efflux rate but 0.1 mM tetracaine had only a slight effect on this flux rate. The results suggest that a change in ouabain-resistant (passive) K⁺ fluxes, but not the Na⁺/K⁺ pump, is involved in stimulation of insulin secretion by glibenclamide and tetracaine. Both drugs may exert similar effects on the -cell plasma membrane.     

多色探针熔解曲线法在卡马西平不良反应相关基因检测中的临床评价

目的 对多色探针熔解曲线法（multicolor melting curve analysis,MMCA）用于卡马西平不良反应相关的HLA-B^*15：02基因检测进行临床评价。方法 收集厦门市中心血站1 147份厦门地区无偿献血者的外周静脉血样本,经基因DNA提取后,按双盲对照试验,分别应用MMCA法和HLA-SBT测序法对各样本进行HLA-B^*15：02基因检测,比较2种检测方法的符合率。对于检测结果不一致的标本,采用第三方Sanger测序和电泳验证,计算总符合率。结果 采用MMCA法共检出77份HLA-B^*15：02阳性标本,1 070份HLA-B^*15：02阴性标本。采用HLA-SBT测序法共检出74份HLA-B^*15：02阳性标本,1 070份HLA-B^*15：02阴性标本,以及3份无明确的分型信息的标本（仅显示为B^*15：VG-B^*15：CYS型）。该3份标本经Sanger测序以及电泳验证,确认为HLA-B^*15：02阳性标本。因此,MMCA法检测HLA-B^*15：02基因的阳性检出率为100%（77/77）,阴性检出率为100%（1 070/1 070）,总符合率为100%（1 147/1 147）。此外,在1 147份临床标本中共检出77份阳性结果,HLA-B^*15：02的携带率为6.7%（77/1147）,这与文献报道的数据基本一致。结论 MMCA法用于HLA-B^*15：02基因的检测,具有简便、快速、灵敏度高、特异性强等优点,可应用于卡马西平不良反应相关的HLA-B^*15：02基因的临床辅助检测。     

11α-羟基炔雌醚和11α-甲氧基炔雌醚的合成及其抗生育作用

为了寻找抗生育药物,从△⁹⁽¹¹⁾-雌素酮开始,经环戊醚化、缩酮化、硼氢化-氧化、甲基化、脱缩酮、乙炔化等反应合成了11α-羟基炔雌醚、11α-甲氧基炔雌醚。目的化合物及相应中间体的结构依据波谱分析等确定。小鼠实验表明这两个化合物不具明显抗生育活性。     

北京鸭红细胞膜β肾上腺素受体放射配基结合测定法

本文以[³H]dihydroalprenolol([³H]DHA)为放射配基。对北京鸭红细胞(RBC)膜β受体进行了系统研究。结果表明,[³H]DHA与北京鸭RBC结合具有饱和性,肾上腺素能激动剂与[³H]DHA竞争结合的强弱顺序与它们的生物活性一致,提示结合有结构特异性和立体特异性,[³H]DHA与鸭RBC结合迅速、可逆。可见北京鸭RBC膜存在β受体,用于放射配基结合测定有取材方便,受体较丰富,膜蛋白得率高,[³H]DHA非特异结合低和易于保存等优点,可以代替火鸡RBC。     

S-adenosyl- -methionine N-ole-1-oyltaurate: pharmacokinetic of the orally administered salt in rats

A pharmacokinetic study based on the distribution of radioactivity from the double labelled S-adenosyl- -methionine (SAM) has been carried out by oral administration of the liposoluble stable salt [methyl-¹⁴C, 8-³H]SAM N-ole-1-oyltaurate to rats. The SAM sulfate p-toluensulfonate salt, the only SAM salt at present commercialized as drug, was chosen as reference compound to have a comparative pharmacokinetic analysis. The metabolism of the SAM is characterised by a differential use of the two labelled moieties by the various organs, liver being the most active in metabolizing the sulfonium compound with a preferential uptake of the methyl-¹⁴C fragment. The radioactivity detected after the administration of [methyl-¹⁴C, 8-³H]SAM N-ole-1-oyltaurate is, in all the organs examined, two times higher than the [methyl-¹⁴C, 8-³H]SAM sulfate p-toluensulfonate compound, attesting that the liposoluble [methyl-¹⁴C, 8-³H]SAM N-ole-1-oyltaurate is provided with better bioavailability.     

吡喹酮对日本血吸虫雄虫的Ca2+,Mg2+,K+与Na+的含量及45Ca2+在虫体内分布的影响

日本血吸虫雄虫在4℃或37℃的HBS及无⁴⁵Ca²⁺的HBS中经吡喹酮1或30μg/ml作用0.5～2h后,未见虫的Ca²⁺,Mg²⁺含量有明显变化,但除4℃的HBS组外,余2组虫的K⁺含量明显减少,而虫的Na⁺含量的增加则不明显。在含30 mM Mg²⁺的HBS中,雄虫经吡喹酮作用1h后,虫的Mg²⁺含量明显增加。在37℃的HBS中,血吸虫雄虫经吡喹酮1μg/ml作用5～60min后,虫的皮层胞质中的⁴⁵²⁺含量的百分比较各相应对照组的明显减少,而虫体肌肉的则相反。在4℃的HBS或无⁴⁵Ca²⁺的HBS中,吡喹酮对⁴⁵²⁺在虫体内的分布无明显影响。