Isolation and Characterization of Two Furan-side Photoadducts of 7-Methylpyrido[3,4-c] Psoralen to the Sugar Moiety of 2-Deoxyadenosine†

Abstract. The UVA-mediated photoreaction of the monofunctional 7-methylpyrido[3,4-c]psoralen (MePyPs) with 2′-deoxy-adenosine was investigated in the dry state. Two main 7-methylpyrido[3,4-c]psoralen monoadducts to 2′-deoxy-adenosine were isolated by HPLC and characterized by soft ionization mass spectrometry (fast atom bombardment) and extensive ¹H NMR analyses including correlation spectroscopy, total correlation spectroscopy and nuclear Overhauser effect measurements. The two unusual photoadducts exhibit a covalent bond between the C-4′ of the furan moiety of the psoralen on one hand and either the C-l' or the C-4′ of the furanose ring of the purine nucleoside on the other hand. The furan-side monoadducts appear to be specific for both 2′-deoxy-adenosine and MePyPs. They are likely to result from the recombination of the 4′,5′-dihydrofuran-4′-yl radical of MePyPs with 2′-deoxyadenosyl carbon-centered sugar radicals at either the 1′ or the 5′ position.     

Subsynaptic Distribution,Lipid Raft Targeting and G Protein-Dependent Signalling of the Type 1 Cannabinoid Receptor in Synaptosomes from the Mouse Hippocampus and Frontal Cortex

Numerous studies have investigated the roles of the type 1 cannabinoid receptor (CB1) in glutamatergic and GABAergic neurons. Here, we used the cell-type-specific CB1 rescue model in mice to gain insight into the organizational principles of plasma membrane targeting and Gαi/o protein signalling of the CB1 receptor at excitatory and inhibitory terminals of the frontal cortex and hippocampus. By applying biochemical fractionation techniques and Western blot analyses to synaptosomal membranes, we explored the subsynaptic distribution (pre-, post-, and extra-synaptic) and CB1 receptor compartmentalization into lipid and non-lipid raft plasma membrane microdomains and the signalling properties. These data infer that the plasma membrane partitioning of the CB1 receptor and its functional coupling to Gαi/o proteins are not biased towards the cell type of CB1 receptor rescue. The extent of the canonical Gαi/o protein-dependent CB1 receptor signalling correlated with the abundance of CB1 receptor in the respective cell type (glutamatergic versus GABAergic neurons) both in frontal cortical and hippocampal synaptosomes. In summary, our results provide an updated view of the functional coupling of the CB1 receptor to Gαi/o proteins at excitatory and inhibitory terminals and substantiate the utility of the CB1 rescue model in studying endocannabinoid physiology at the subcellular level.     

Fluorescence Imaging of Single-Copy DNA Sequences within the Human Genome Using PNA-Directed Padlock Probe Assembly

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Highlights? PNA-directed assembly of the labeling complex on duplex DNA ? Padlock probes combined with rolling circle amplification for signal amplification ? Multitarget visualization within the human genome with high sequence selectivity     

扶正丹中补骨脂素含量的测定方法

采用含０．５％羰甲基纤维素钠硅胶Ｇ薄层色谱分离和ＣＳ－９３０荧光扫描法测定扶正丹中有脂素的含量,不受处方中其他成分的干扰,方法灵敏,特异性强,操作简便,结果可靠,可作为质量控制的定理标准。该法线性回归方程为Ｙ＝４２６１６．８６Ｘ＋１１３０１．９１,ｒ＝０．９９６,加标回收率的１０１．６％,同一样品点样６次测得的ＲＳＤ为２．２５％。     

Photochemical and Photobiological Studies of a Furonaphthopyranone as a Benzo-spaced Psoralen Analog in Cell-free and Cellular DNA

Abstract— Photobiological activities of the benzo-spaced psoralen analog furonaphthopyranone 3 have been investigated in cell-free and cellular DNA. The molecular geometry parameters of 3 suggest that it should not form interstrand crosslinks with DNA. With cell-free DNA no evidence for crosslinking but also not for monoadduct formation was obtained; rather, the unnatural furocoumarin 3 induces oxidative DNA modifications under near-UVA irradiation. The enzymatic assay of the photosensitized damage in cell-free PM2 DNA revealed the significant formation of lesions sensitive to formamidopyrimidine DNA glyco-sylase (Fpg protein). In the photooxidation of calf thymus DNA by the furonaphthopyranone 3, 0.29±0.02% 8-oxo-7,8-dihydroguanine (8-oxoGua) was observed. With 2'-deoxyguanosine (dGuo), the guanidine-releasing photooxidation products oxazolone and oxoimidazolidine were formed predominately, while 8-oxodGuo and 4-HO-8-oxodGuo were obtained in minor amounts. The lack of a significant D₂O effect in the photooxidation of DNA and dGuo reveals that singlet oxygen (type II process) plays a minor role; control experiments with tert -butanol and mannitol confirm the absence of hydroxyl radicals as oxidizing species. The furonaphthopyranone 3 (E_red= -1.93±0.03V) should act in its singlet-excited state as electron acceptor for the photooxidation of dGuo (δG_ET ca – kcal/mol), which corroborates photoinduced electron transfer (type I) as a major DNA-oxidizing mechanism. A comet assay in Chinese hamster ovary (CHO) AS52 cells demonstrated that the psoralen analog 3 damages cellular DNA upon near-UVA irradiation; however, no photosensitized mutagenicity was observed in CHO AS52 cell cultures     

用荧光法研究补骨脂素与异补骨脂素的药代动力学

补骨脂素和异补骨脂素是补骨脂的主要有效成分。为了研究补骨脂素和异补骨脂素的药物代谢动力学,本文采用胶束荧光法对补骨脂素和异补骨脂素在兔体内的血药浓度进行２４ｈ监测,求出系列药动学参数。结果表明：补骨脂素的Ｋａ为３．２２５１ｈ－１,ｔ１／２（α）为 ０．１８４９ｈ,ｔ１／２（β）为１０．７０６５ｈ,Ｔ为０．８２００ｈ,ＡＵＣ为２１．６１０４ｍｇ·ｈ／Ｌ;异补骨脂素的Ｋａ为４．４３２９ｈ－１,ｔ１／２（Ｋａ）为０．１５６４ｈ,ｔ１／２（Ｋｅ）为２．１７７７ｈ,Ｔ为１．０９５５ｈ,ＡＵＣ为７．２４１８ｍｇ·ｈ／Ｌ。     

Targeting the proangiogenic VEGF-VEGFR protein-protein interface with drug-like compounds by in silico and in vitro screening

Protein-protein interactions play a central role in medicine, and their modulation with small organic compounds remains an enormous challenge. Because it has been noted that the macromolecular complexes modulated to date have a relatively pronounced binding cavity at the interface, we decided to perform screening experiments over the vascular endothelial growth factor receptor (VEGFR), a validated target for antiangiogenic treatments with a very flat interface. We focused the study on the VEGFR-1 D2 domain, and 20 active compounds were identified. These small compounds contained a (3-carboxy-2-ureido)thiophen unit and had IC₅₀ values in the low micromolar range. The most potent compound inhibited the VEGF-induced VEGFR-1 transduction pathways. Our findings suggest that our best hit may be a promising scaffold to probe this macromolecular complex and for the development of treatments of VEGFR-1-dependent diseases.     

Synthesis and Photooxygenation of Linear and Angular Furocoumarin Derivatives as a Hydroxyl Radical Source: Psoralen,Pseudopsoralen, Isopseudopsoralen,and Allopsoralen

Synthesis of linear and angular furocoumarins with new skeleton structure of potential photobiological feature interest was carried out through Williamson reaction of hydroxycoumarins with 3‐chloro‐2‐butanone followed by cyclization with polyphosphoric acid or by heating in a strongly alkaline solution. The photooxygenation reactions of synthesized furocoumarins were performed in chloroform and in the presence of tetraphenylporphyrin as singlet oxygen sensitizer (¹O₂). The photooxygenation reactions afforded the photocleaved product through [2 + 2] cycloaddition and the photooxygenated products through ene reaction and [4 + 2] cycloaddition. The photoproducts were isolated and fully characterized by spectral analyses.     

Biosynthesis of Psoralen: Mechanism of a Cytochrome P450 Catalyzed Oxidative Bond Cleavage

In contrast to the stepwise oxidative dealkylation of steroids [Eq. (1)], (+)‐marmesin ( 1 ) is directly converted by the psoralen synthase (a cytochrome P450) into the phototoxic furanocoumarin psoralen ( 2 ) and acetone. The dealkylation proceeds as a syn elimination.     

Inactivation of Trypanosoma cruzi Trypomastigote Forms in Blood Components with a Psoralen and Ultraviolet A Light

Inactivation of the blood-borne parasite Trypanosoma cruzi by UVA and 4'-aminomethyl-4,5',8-trimethylpsor-alen (AMT) was studied in the blood components fresh frozen plasma (FFP) and platelet concentrate (PC). The AMT was utilized at a concentration of 50 μg/mL and the inactivation procedure included the flavonoid rutin (at 0.35 mM), a quencher of type I and type II photo-reactants, which we have previously found to maintain platelet integrity during this treatment regimen. Within both FFP and PC, complete inactivation of the infective form of T. cruzi , the trypomastigote, was achieved at a UVA (320–400 nm radiation) fluence of 4.2 J/cm². We note that while the infectivity of the parasite is eliminated at 4.2 J/cm^Z the trypomastigote motility continues for at least 16 h post-treatment and is inhibited only after much higher light doses. Isolation of total DNA from the parasite cells after treatment in the presence of ³H-AMT indicated that at the lethal UVA fluence about 0.5 AMT adducts per kilobase pairs occurred. These results suggest that this psoralen plus UVA methodology, which shows promise in enhancing the viral safety of PC, may in addition eliminate bloodborne T. cruzi , the causative agent of Chagas disease.     

Dependence of Fluorescence Properties of Compounds from Psoralen,Angelicin, and Carbazole Series on the Character of Their Terminal Substituents

Absorption, luminescence excitation and emission spectra of nine compounds from 4,8,4'-trimethylpsoralen, 4,4'-dimethylangelicin, and 1,2,3,4-tetrahydrocarbazole series with various terminal substituents were studied in water and 2-propanol. Proceeding from the data obtained in the present and also some previous studies we have formulated the main rules concerning a general mechanism of changes in the fluorescence characteristics of DNA-specific dyes depending on their chemical structure, substrate properties, and measurement medium.     

Transfer Chemical Potentials,Solubility and Reactivity of Psoralen and 8-Hydroxypsoralen in Binary Aqueous Methanol Mixtures

Solubilities are reported for psoralen and 8-hydroxypsoralen in binary aqueous mixtures at ambient pressure and 298 K, with organic cosolvent methanol. Gibb's energies of transfer of there two compounds are calculated from their solubilities. Transfer chemical potentials of both compounds are stabilised by increasing prapartion of organic cosolvent because of their markedly hydrophobic character. Basic hydrolysis of psoralen and 8-hydroxypsoralen in aqueous methanol mixtures was examines kinetically, yielding Gibb's energies of activation. Derived transfer parameters are used in analysis of kinetic data to describe the influence of solvent composition on initial and transition states. Derived transfer chemical potentials for a single ion such as hydroxide ion lead to an interesting comparison of the effects of extrathermodynamic assumptions, Wells estimates and TATB assumption, on the initial and transition states. The analysis comfirm the important role played by the choice of extrathermodynamic assumption in determining the overall kinetic pattern. We describe misgivings concerning procedures used by wells to derive transfer chemical potential for single ions.     

人类基因组和DNA序列分析新进展

0世纪 90年代世界各国纷纷将人类基因组研究列为国家重大研究项目。很多人认为这是当代也可能是整个时代最重要的科学事业[1] ,因为它是全面揭开人类自身奥秘的划时代研究 ,对人类的生命和生存具有极其重大而深远的影响。1 990年美国制定了世界上最庞大的人类基因组项目 (Ｈｕｍａｎｇｅｎｏｍｅｐｒｏｊｅｃｔ) ,计划在 1 5年内投资 30亿美元 ,完成人类基因组全部脱氧核糖核酸 (ＤＮＡ)测序、定位及遗传学研究。由于计划庞大 ,人们称其为“人的阿波罗”计划。英国、日本和德国都有较大的人类基因组研究项目。我国在北京和上海分别建立了人…     

Construction, Expression and Characterization of a Chimeric Protein Targeting Carcinoembryonic Antigen in Lung Cancer

The carcinoembryonic antigen(CEA) is an oncofetal glycoprotein known as an important clinical tumor marker and is overexpressed in several types of tumors, including colorectal and lung carcinomas. We constructed a chimeric protein that exhibits both specific binding and immune stimulating activities, by fusing staphylococcal enterotoxin A(SEA) to the C-terminus of an anti-CEA single-chain disulfide-stabilized Fv(scdsFv) antibody (single-chain-C-terminus/SEA, SC-C/SEA). The SC-C/SEA protein was expressed in...     

鼠肝星状细胞体内与体外激活的显微拉曼光谱

采用密度梯度离心法从肝组织中分离、提纯肝星状细胞, 进行常规细胞鉴定后, 通过体外培养诱导肝星状细胞体外活化, 在不同的时间点上进行原位拉曼光谱表征; 通过一次性腹腔注射CCl4诱导鼠急性肝损伤, 取不同的时间点的肝损伤组织做拉曼光谱表征, 并以肝组织的光谱变化来间接反映肝星状细胞的体内活化. 结果表明, 用拉曼光谱能快速、 灵敏地监测肝星状细胞体内和体外活化过程中的分子变化, 可为肝纤维化的早期诊断提供依据.     

Targeting the hemozoin synthesis pathway for new antimalarial drug discovery: technologies for in vitro beta-hematin formation assay

Clinical manifestations of malaria primarily result from proliferation of the parasite within the hosts' erythrocytes. During this process, hemoglobin is utilized as the predominant source of nutrition. The malaria parasite digests hemoglobin within the digestive vacuole through a sequential metabolic process involving multiple proteases. Massive degradation of hemoglobin generates large amount of toxic heme. Malaria parasite, however, has evolved a distinct mechanism for detoxification of heme through its conversion into an insoluble crystalline pigment, known as hemozoin. Hemozoin is identical to beta-hematin, which is constituted of cyclic heme dimers arranged in an ordered crystalline structure through intermolecular hydrogen bonding. The exact mechanism of biogenesis of hemozoin in malaria is still obscure and is the subject of intense debate. Hemozoin synthesis is an indispensable process for the parasite and is the target for action of several known antimalarials. The pathway has therefore attracted significant interest for new antimalarial drug discovery research. Formation of beta-hematin may be achieved in vitro under specific chemical and physiochemical conditions through a biocrystallization process. Based on these methods several experimental approaches have been described for the assay of formation of beta-hematin in vitro and screening of compounds as inhibitors of hemozoin synthesis. These assays are primarily based on differential solubility and spectral characteristics of monomeric heme and beta-hematin. Different factors viz., the malaria parasite lysate, lipids extracts, preformed beta-hematin, malarial histidine rich protein II and some unsaturated lipids have been employed for promoting beta-hematin formation in these assays. The assays based on spectrophotometric quantification of beta-hematin or incorporation of (14)C-heme yield reproducible results and have been applied to high throughput screening. Several novel antimalarial pharmacophores have been discovered through these assays.     

Evaluating cell migration in vitro by the method based on cell patterning within microfluidic channels

Cell migration is an early-stage and critical step for cancer metastasis. The most common approach to monitor this process is wound-healing assay. However, this traditional method has some unavoidable limitations. We observed that simply scratching the monolayer of cultured cells might cause local cell damage around the injury line. The cells along the scratched border seemed to be irritated and exhibited abnormal distribution of cytoskeleton reassembly with protruding "cell islands" and "pseudopodia" during wound healing, which might potentially affect the assessment of cell migration behavior. Herein, we applied a microfluidic device that mechanically constrained cells seeded in a designed pattern inside microchannels, and monitored cell movement in a way of mimicking the natural microenvironment of cancerous tissues. We illustrated the capacity of this simple method to probe cellular migration behaviors and to screen some biological active agents that reflected in their influence on cellular motility.