细菌产生的蔗糖磷酸化酶的筛选及其特性

蔗糖磷酸化酶(SPase)能催化蔗糖磷酸化,生成α-D葡萄糖-1-磷酸脂(G-l-P)和D-果糖,此催化反应是一个可逆反应.该酶是一种葡糖基转移酶,能转移蔗糖葡糖基和G-l-P到P_1以及包括D-果糖在内的一些糖.已有报道来源于肠膜明串珠菌ATCC12291是以糖、酒精和酚的化合物作为糖基受体.除来源于肠膜明串珠菌的蔗糖磷酸化酶外,其它蔗糖磷酸化酶已用于P_1的酶促实验与各种酶促萄糖基化的作用中.     

改造糖基侧链合成新型抗生素的研究进展

［摘要］放线菌抗生素核心结构普遍存在糖基化,糖基在抗生素药理作用的发挥中起到了非常重要的作用。随着抗生素糖基合成基因簇研究的深入,运用现代基因工程技术,借助糖基转移酶的底物灵活性,已实现了多种抗生素糖基侧链的改造,进而合成新结构的抗生素。文中详细介绍了目前抗生素糖基侧链改造的主要方法,以及运用基因工程技术对糖类合成途径进行调控以产生新型糖基化衍生物的研究进展。     

尿苷二磷酸葡萄糖醛酸转移酶基因多态性对药物代谢影响的研究进展

由尿苷二磷酸葡萄糖醛酸转移酶(UDP-glucuronosyltransferase,UGT)催化完成的葡萄糖醛酸结合反应是生物体内重要的Ⅱ相代谢途径,它与毒性或活性物质结合形成葡萄糖醛酸苷,将内源性、外源性化合物通过胆汁或肾脏排出体外。UGT是一个超家族酶,因主要利用UDP-尿苷二磷酸葡糖醛酸为糖基供体而得名。人类UCT广泛分布于体内的各种组织,包括肾、脑、皮肤、肠、脾、胸腺、心脏等,其中以     

山姜素在人肝微粒体的葡萄糖醛酸化反应研究

目的研究体外肝微粒体孵育体系中山姜素的葡萄糖醛酸化代谢情况,鉴定参与山姜素葡萄糖醛酸化代谢的UGT亚型。方法用体外肝微粒体孵育体系,用HPLC-UV检测方法,检测山姜素的葡萄糖醛酸化代谢情况。将代谢产物进行纯化后,用质谱(MS)和核磁共振(NMR)法进一步鉴定其结构。用商业化重组表达的UGT单酶,鉴定代谢产物的结构和归属可能参与山姜素葡萄糖醛酸化代谢反应的葡萄糖醛酸转移酶(UGTs)亚型。结果山姜素葡萄糖醛酸代谢产生一个代谢产物,经结构鉴定为山姜素-氧-单葡萄糖醛酸化产物。人肝微粒体代谢山姜素的动力学行为,符合米方程且动力学参数:Vmax=(101.9±3.0)nmol·min-1·mg-1·pro,Km=(40.6±3.6)μmol·L-1。UGT1A1、UGT1A3、UGT1A9和UGT2B15均参与了山姜素的葡萄糖醛酸化反应。结论山姜素在人肝微粒体孵育体系中会被代谢成为一个单葡萄糖醛酸化产物,且归属了参与的UGT酶。     

金催化甘露糖-b-(1→6)-甘露糖-b-(1→6)-葡萄糖三糖的合成

目的 利用金催化立体选择性构建b-甘露糖苷键的方法高效合成甘露糖-b-(1→6)-甘露糖-b-(1→6)-葡萄糖三糖。方法 以4,6-O-苄叉保护的甘露糖邻己炔基苯甲酸酯3为供体,在Au(I) 复合物和银盐催化下,与葡萄糖受体4反应,得到二糖产物5;二糖5在Bu₂BOTf/BH_3.THF条件下,将苄叉选择性还原开环至6-羟基,得到二糖受体6,进而再与糖基供体3反应,得到三糖2。 结果 分别以90%的产率、b/a = 14.2/1的选择性得到二糖5和88%的产率、b/a = 15.9/1的选择性得到三糖2。     

葡萄糖-β-(1→4)-半乳糖二糖片段的合成

目的 利用化学方法高效合成葡萄糖-β-(1→4)-半乳糖二糖片段。方法 以4,6位苄叉保护的葡萄糖硫苷11为原料通过4步反应以39%的产率合成了硫苷供体15 ,再以TMSOTf与NIS作为促进剂,硫苷供体15与低活性的半乳糖受体18进行糖苷化反应合成葡萄糖-β-(1→4)-半乳糖二糖2。 结果 糖苷化反应以81%的产率得到葡萄糖-β-(1→4)-半乳糖二糖2, 为新型海洋天然产物Ancorinoside A的合成提供物质基础和技术保障。     

娑罗子提取物酶解产物的化学成分研究

目的制备新的七叶皂苷衍生物。方法采用β-葡萄糖苷酶对娑罗子提取物进行生物转化,采用大孔吸附树脂、反相硅胶柱色谱等分离方法对酶解产物进行分离纯化,根据MS、NMR数据,对得到的单体化合物进行了结构鉴定。结果 4个化合物分别鉴定为21β-O-巴豆酰基-22α-O-乙酰基原七叶皂苷元-3β-O-[β-D-葡萄糖基(1→2)]-β-D-葡萄糖醛酸苷(1)、21β-O-当归酰基-22α-O-乙酰基原七叶皂苷元-3β-O-[β-D-葡萄糖基(1→2)]-β-D-葡萄糖醛酸苷(2)、21β-O-巴豆酰基-28-O-乙酰基原七叶皂苷元-3β-O-[β-D-葡萄糖基(1→2)]-β-D-葡萄糖醛酸苷(3)、21β-O-当归酰基-28-O-乙酰基原七叶皂苷元-3β-O-[β-D-葡萄糖基(1→2)]-β-D-葡萄糖醛酸苷(4)。分别命名为七叶皂苷Ie、七叶皂苷If、异七叶皂苷Ie和异七叶皂苷If。结论 4个化合物均为新化合物。     

针对糖基化进行抗肿瘤药物开发的研究进展

糖基化是由糖基转移酶调控的产生糖与其他糖、蛋白质或脂类的糖苷连接的酶促过程，是各种生物过程的基础。异常糖基化是许多癌症的共同特征，在肿瘤的发生发展中起着至关重要的作用，是抗肿瘤研究的重要靶标。本文首先对糖基化、糖基转移酶和肿瘤相关糖类抗原进行简单介绍，主要针对糖基化过程进行抗肿瘤药物开发的研究现状进行了综述和展望。     

环糊精包合物在药剂学上的应用进展

<正> 环糊精(cyclodextrin)简称CD或YD,是由淀粉经环糊精糖基转移酶酶解而得,是以α-1,4键联结成的环状低聚糖化合物。环合的葡萄糖分子数主要有6、7、8个三种,分别称为α-、β-和γ-环糊精,亦有少量8个以上葡萄糖环合物。环糊精的衍生物近年来也有较多的报道。     

用于非酶糖化抑制剂实验大鼠模型比较研究

蛋白质、脂质与核酸等与葡萄糖及其他还原糖可在非酶条件下,自发进行非酶糖化反应,产生稳定、结构多样的共价产物——晚期糖基化终末产物(Advanced glycation end     

Enhancement of platinum-drug cytotoxicity in a human head and neck squamous cell carcinoma line and its platinum-resistant variant by liposomal amphotericin B and phospholipase A2-II.

Platinum drugs comprise one of the main classes of chemotherapy drugs that can induce remissions in various solid tumors. Although tumors often regress on treatment with cis-diamminedichloroplatinum II (cisplatin) or cis-diammine-1,1-cyclobutane dicarboxylate platinum II (carboplatin), they usually relapse as a drug-resistant tumor. Most mechanisms of platinum resistance could be overcome by increasing the amount of drug that is accumulated by tumor cells. Amphotericin B (Amph B) is efficient at increasing platinum drug uptake, but because of nephrotoxicity associated with extended usage, and the potential for synergistic nephrotoxicity when used with platinum drugs, Amph B has not been used clinically for this purpose. A liposomal preparation of Amph B (LipoAmph B), which is substantially less nephrotoxic, was studied for its ability to enhance platinum-drug toxicity to a human oral squamous cell carcinoma line, HN-5a, and its carboplatin-resistant variant, 5a/carbo-15a, in which cisplatin accumulation was reduced by approximately 40%. Amph B at 10 microg/ml enhanced cisplatin accumulation by approximately 100% in both cell lines, enhancing cytotoxicity of the drugs by 35 to 60%, and completely reversed resistance to both cisplatin and carboplatin. LipoAmph B in the presence of phospholipase A(2)-II (PLA2-II) was able to enhance cisplatin and carboplatin cytotoxicity as effectively as free Amph B in both cell lines. At optimal concentrations, LipoAmph B plus PLA2-II enhanced drug uptake sufficiently to abolish resistance in the platinum-resistant line. Because PLA2-II is elevated in some tumor microenvironments and in plasma of ill patients, LipoAmph B has potential clinical usefulness as a modulator of platinum-drug efficacy.     

Can the ”state-dependency” hypothesis explain prevention of amphetamine sensitization in rats by NMDA receptor antagonists?

 Many laboratories have reported that coadministration of N-methyl-d-aspartate (NMDA) receptor antagonists with psychomotor stimulants prevents the development of behavioral sensitization and therefore concluded that NMDA receptor transmission is necessary for sensitization. According to an alternative ”state-dependency” interpretation, NMDA receptor antagonists do not prevent sensitization. Rather, they become a conditioned stimulus for the sensitized response, i.e., it is only elicited in response to combined administration of the NMDA receptor antagonist and the stimulant. This hypothesis is supported by progressive augmentation of the locomotor response to the drug combination during the induction phase, and expression of sensitization when challenged with the combination but not the stimulant alone. To test this hypothesis, rats were treated during a 6-day induction phase with amphetamine (Amph) alone or in combination with the competitive NMDA receptor antagonist CGS 19755 (10 mg/kg) or the non-competitive NMDA receptor antagonist MK-801 (0.05, 0.1 and 0.25 mg/kg). When CGS 19755 was coadministered with Amph, there was no progressive augmentation of response to the drug combination. When challenged with Amph alone, rats did not exhibit the biphasic pattern of locomotor activity characteristic of Amph sensitization. No sensitization of stereotyped behaviors was evident, although the ambulatory response was greater than that exhibited by naive rats. Results with MK-801 were complex, but progressive augmentation of response to the drug combination appeared to in part reflect sensitization to MK-801 and could be dissociated from the ability of MK-801 to prevent the development of sensitization as assessed by response to challenge with Amph alone. Many of these findings are inconsistent with predictions of the ”state-dependency” hypothesis. Moreover, the ability of NMDA receptor antagonists to prevent biochemical and electrophysiological correlates of sensitization is difficult to reconcile with the idea that sensitization develops in the presence of NMDA receptor blockade but cannot be expressed. Together, these findings suggest that the ability of NMDA receptor antagonists to prevent Amph sensitization reflects a requirement for NMDA receptor transmission during its induction. Received: 29 January 1998 / Final version: 23 July 1998     

Low-dose dietary chlorophyll inhibits multi-organ carcinogenesis in the rainbow trout.

We recently reported that chlorophyll (Chl) strongly inhibits aflatoxin B(1) preneoplasia biomarkers in rats when administered by co-gavage (Simonich et al., 2007. Natural chlorophyll inhibits aflatoxin B1-induced multi-organ carcinogenesis in the rat. Carcinogenesis 28, 1294-1302.). The present study extends this by examining the effects of dietary Chl on tumor development, using rainbow trout to explore ubiquity of mechanism. Duplicate groups of 140 trout were fed diet containing 224 ppm dibenzo[a,l]pyrene (DBP) alone, or with 1000-6000 ppm Chl, for 4 weeks. DBP induced high tumor incidences in liver (51%) and stomach (56%), whereas Chl co-fed at 2000, 4000 or 6000 ppm reduced incidences in stomach (to 29%, 23% and 19%, resp., P<0.005) and liver (to 21%, 28% and 26%, resp., P<0.0005). Chlorophyllin (CHL) at 2000 ppm gave similar protection. Chl complexed with DBP in vitro (2Chl:DBP, K(d1)=4.44+/-0.46 microM, K(d2)=3.30+/-0.18 microM), as did CHL (K(d1)=1.38+/-0.32 microM, K(d2)=1.17+/-0.05 microM), possibly explaining their ability to inhibit DBP uptake into the liver by 61-63% (P<0.001). This is the first demonstration that dietary Chl can reduce tumorigenesis in any whole animal model, and that it may do so by a simple, species-independent mechanism.     

Cancer chemoprevention by dietary chlorophylls: a 12,000-animal dose-dose matrix biomarker and tumor study

Recent pilot studies found natural chlorophyll (Chl) to inhibit carcinogen uptake and tumorigenesis in rodent and fish models, and to alter uptake and biodistribution of trace (14)C-aflatoxin B1 in human volunteers. The present study extends these promising findings, using a dose-dose matrix design to examine Chl-mediated effects on dibenzo(def,p)chrysene (DBC)-induced DNA adduct formation, tumor incidence, tumor multiplicity, and changes in gene regulation in the trout. The dose-dose matrix design employed an initial 12,360 rainbow trout, which were treated with 0-4000ppm dietary Chl along with 0-225ppm DBC for up to 4weeks. Dietary DBC was found to induce dose-responsive changes in gene expression that were abolished by Chl co-treatment, whereas Chl alone had no effect on the same genes. Chl co-treatment provided a dose-responsive reduction in total DBC-DNA adducts without altering relative adduct intensities along the chromatographic profile. In animals receiving DBC alone, liver tumor incidence (as logit) and tumor multiplicity were linear in DBC dose (as log) up to their maximum-effect dose, and declined thereafter. Chl co-treatment substantially inhibited incidence and multiplicity at DBC doses up to their maximum-effect dose. These results show that Chl concentrations encountered in Chl-rich green vegetables can provide substantial cancer chemoprotection, and suggest that they do so by reducing carcinogen bioavailability. However, at DBC doses above the optima, Chl co-treatments failed to inhibit tumor incidence and significantly enhanced multiplicity. This finding questions the human relevance of chemoprevention studies carried out at high carcinogen doses that are not proven to lie within a linear, or at least monotonic, endpoint dose-response range.     

Development of novel lyophilized mixed micelle amphotericin B formulation for treatment of systemic fungal infection

The purpose of the study was to develop a stable, controlled release Amphotericin B (Amph B) lyophilized mixed micelle (MM) formulation using hydrogenated soya phosphatidylcholine (HSPC) and bile salts in monomeric form and evaluate it for therapeutic performance and side effects. The MM formulations of Amph B were prepared using sodium deoxycholate (NDC)/sodium taurocholate (NTC)/sodium cholate (NC), and HSPC. The optimization of bile salt: HSPC ratio in the MM formulation was done using 2(4) factorial designs. MM formulations were lyophilized using sucrose as a cryoprotectant and analyzed for per cent micelle yield, per cent drug loading and per cent entrapment efficiency. Comparative in vitro diffusion studies, hemolytic activity, and minimum inhibitory concentration (MIC) of developed MM formulations and marketed formulation (Fungizone) were evaluated using cellophane membrane, human red blood cells and Candida albicans respectively. In vivo studies of MM formulations were also carried out on Candida albicans infected white albino rats and compared with Fungizone. The optimized molar ratio of bile salt: HSPC was found to be 8:11. Among all MM formulations prepared, NDC/ HSPC formulation found to have maximum per cent drug loading (4.96+/-0.8%), per cent entrapment efficiency (93.2+/-1.5%) and per cent micelle yield (96.4+/-1.4%). The in vitro drug diffusion studies of developed MM formulations show close to zero-order diffusion kinetics. All MM formulations show improved therapeutic index and reduced side effects compared to reference formulation Fungizone. The NDC/HSPC MM formulation was found to have least hemolytic activity, MIC and mortality rate at all dosage levels. Improved antifungal activity and reduced toxicity of Amph B solubilized in MM may be due to higher cellular uptake of the drug by fungal cells of infected tissues from MM formulations. Hence, Amph B MM formulation could be a safe and effective viable alternative in the treatment of systemic fungal infections.     

Differential effects on prepulse inhibition of withdrawal from two different repeated administration schedules of amphetamine

In this study, rats were tested in behavioural paradigms relevant to schizophrenia during withdrawal from two different administration schedules of amphetamine (Amph). One of the escalating administration schedules, which has been employed in previous studies, consisted of three daily injections for 6 d with increasing dosages from 1 to 5 mg/kg Amph (Esc-5) and was compared to a hitherto never examined escalating administration schedule [three injections per day for 6 d escalating from 1 to 8 mg/kg Amph (Esc-8)]. Control animals received an equivalent volume of saline (Sal) injections according to the same schedule. Whereas rats treated with Esc-5, as reported before, failed to show an effect on prepulse inhibition (PPI), the Esc-8-treated rats exhibited a long-lasting disruption of PPI in a drug-free state on days 6, 13 and 55 of withdrawal. The Amph-pretreated animals demonstrated a similar magnitude of behavioural sensitization following an Amph challenge on withdrawal day 58 irrespective of the administration schedule. To evaluate if the withdrawal from the two Amph schedules led to a change in brain monoamine levels, a subgroup of animals was neurochemically examined in post-mortem for eight parameters in seven brain regions on withdrawal day 55. Withdrawal from the Esc-8 schedule induced reduced dopamine levels in the caudate putamen. Only this neurochemical finding and the PPI attenuation differentiated the Esc-8 animals from the Esc-5 and Sal animals. These data suggest that, based on the endogenous sensitization hypothesis of schizophrenia, the persistent disruption of PPI observed in animals withdrawn from Esc-8 can be used as a valid animal model of specific symptoms of schizophrenic patients.     

Uridine diphosphate sugar-selective conjugation of an aldose reductase inhibitor (AS-3201) by UDP-glucuronosyltransferase 2B subfamily in human liver microsomes

N-Glucosidation is known as a major metabolic reaction for barbiturates in humans. However, the enzyme(s) involved in this N-glucosidation has not been clarified yet. Thus, to clarify the enzyme(s) involved in the N-glucosidation in human liver microsomes, we investigated the N-glucosyltransferase activity in recombinant UDP-glucuronosyltransferases (UGTs) using AS-3201, an aldose reductase inhibitor, as a substrate. AS-3201 was found to be biotransformed to both N-glucoside and N-glucuronide in human liver microsomes. The N-glucosyltransferase activities were detectable with multiple UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT2B4, UGT2B7, and UGT2B15). In contrast, the N-glucuronyltransferase activities for the same substrate were seen with UGT1A (UGT1A1, UGT1A3, UGT1A4, and UGT1A9) but not UGT2B isoforms. We then determined the relative activity factor of each recombinant UGT and estimated the contribution of each UGT isoform to the N-glucosidation in human liver microsomes. The results showed that UGT2B isoforms mainly contribute to AS-3201 N-glucosidation in human liver microsomes. In addition, the activity of AS-3201 N-glucosyltransferase significantly correlated with that of amobarbital N-glucosyltransferase in microsomes from sixteen human livers (r=0.964, P<0.01), indicating that UGT2B isoforms were also involved in the barbiturate N-glucosidation in humans. The findings of this study clearly show that UGT2B specifically utilizes UDP-glucose but not UDP-glucuronic acid as a sugar donor for the conjugation of AS-3201 in human liver microsomes.     

Adolescent amphetamine exposure elicits dose-specific effects on monoaminergic neurotransmission and behaviour in adulthood

Despite the growing non-medical consumption of amphetamine (Amph) during adolescence, its long-term neurobiological and behavioural effects have remained largely unexplored. The present research sought to characterize the behavioural profile and electrophysiological properties of midbrain monoaminergic neurons in adult rodents after Amph exposure during adolescence. Adolescent rats were administered vehicle, 0.5, 1.5, or 5.0 mg/kg.d Amph from postnatal day (PND) 30-50. At adulthood (PND 70), rats were tested in an open-field test (OFT) and elevated plus maze (EPM), paralleled by in-vivo extracellular recordings of serotonin (5-HT), dopamine (DA) and norepinephrine (NE) neurons from the dorsal raphe nucleus, ventral tegmental area, and locus coeruleus, respectively. 5-HT firing in adulthood was increased in rats that had received Amph (1.5 mg/kg.d) during adolescence. At this regimen, DA firing activity was increased, but not NE firing. Conversely, the highest Amph dose regimen (5.0 mg/kg.d) enhanced NE firing, but not DA or 5-HT firing rates. In the OFT, Amph (1.5 mg/kg.d) significantly increased the total distance travelled, while the other doses were ineffective. In the EPM, all three Amph doses increased time spent in the open arms and central platform, as well as the number of stretch-attend postures made. Repeated adolescent exposure to Amph differentially augments monoaminergic neuronal firing in a dose-specific fashion in adulthood, with corresponding alterations in locomotion, risk assessment (stretch-attend postures and central platform occupancy) and risk-taking behaviours (open-arm exploration). Thus, adolescent Amph exposure induces long-lasting neurophysiological alterations that may have implications for drug-seeking behaviour in the future.     

[3H]dopamine release by d-amphetamine from striatal synaptosomes of reserpinized rats

The injection of reserpine, 5 mg/kg i.p. (ipRes), the regimen employed by a majority of investigators, results in synaptosomal and vesicular preparations which are incompletely reserpinized as determined by [³H]dopamine ([³H]DA) accumulation. Reserpine administered by the subcutaneous route, 5 mg/kg (scRes), appears to produce complete reserpinization. Release of [³H]DA by d-amphetamine (Amph) was observed from striatal synaptosomes prepared both from normal rats and those pretreated with reserpine intraperitoneally but not from those injected subcutaneously. In the more completely reserpinized scRes synaptosomes, so little [³H]DA had accumulated that release by Amph was not measurable, indicating that if a labile, reserpine-resistant, extravesicular DA storage pool releasable by Amph is present under these conditions, it must be extremely small. In scRes monoamine oxidase (MAO)-inhibited preparations, Amph released preloaded [³H]DA located in the cytosol in the absence of functional vesicles. Although Chromatographic analysis of the superfusate from ipRes striatal synaptosomes showed that significant amounts of preloaded [³H]DA were released by Amph, the level of dihydroxyphenylacetic acid was not increased over controls, indicating that Amph releases only DA and not its metabolite and is also acting as a MAO inhibitor. No [³H]DA could be released by Amph from superfused hyposmotically shocked normal or ipRes synaptosomes, suggesting that an intact membrane is required for Amph-induced release.     

The combined effects of cocaine and amphetamine on primary postnatal rat heart cell cultures

Recent reports demonstrated that perinatal exposure to cocaine (Coc) and amphetamines (Amph) predisposed the infant to adverse cardiovascular consequences. Dose- and time-dependent effects of Coc and Amph on postnatal rat myocardial cell cultures are described. Contractile activity, morphology, lactate dehydrogenase (LDH) release, MTT formazan production, and neutral red (NR) retention were determined. No contractile activity was observed in cultures treated with the highest drug doses. After 24 h, the percentage of areas exhibiting contractile activity was decreased in cultures exposed to the lowest doses of both drugs. When Coc and Amph were combined, beating rates were significantly altered. Morphologic alterations were observed in all treatment groups. LDH release occurred in cultures exposed to the highest doses of both drugs. No significant differences were observed for MTT or NR. These data demonstrate that Coc and Amph doses ≥ 1 × 10⁻⁵M induce adverse effects on morphology and contractile activity of postnatal myocardial cell cultures.