1-Bromo-3-(1',1'-dimethylalkyl)-1-deoxy-Δ(8)-tetrahydrocannabinols: New selective ligands for the cannabinoid CB(2) receptor

Δ(8)-Tetrahydrocannabinol (26), 3-(1',1'-dimethylbutyl)- (12), 3-(1',1'-dimethylpentyl)- (13), 3-(1',1'-dimethylhexyl)- (14) and 3-(1',1'-dimethylheptyl)-Δ(8)-tetrahydrocannabinol (15) have been converted into the corresponding 1-bromo-1-deoxy-Δ(8)-tetrahydrocannabinols (25, 8-11). This was accomplished using a protocol developed in our laboratory in which the trifluoromethanesulfonate of a phenol undergoes palladium mediated coupling with pinacolborane. Reaction of this dioxaborolane with aqueous-methanolic copper(II) bromide provides the aryl bromide. The affinities of these bromo cannabinoids for the cannabinoid CB(1) and CB(2) receptors were determined. All of these compounds showed selectivity for the CB(2) receptor and one of them, 1-bromo-1-deoxy-3-(1',1'-dimethylhexyl)-Δ(8)-tetrahydrocannabinol (10), exhibits 52-fold selectivity for this receptor with good (28nM) affinity.     

Effect of the metal dilution on the thermal and light-induced spin transition in [FexMn1−x(bpp)2](NCSe)2: When T(LIESST) reaches T1/2

The thermal and light-induced spin transition in [Fe_xMn_1?x(bpp)₂](NCSe)₂ (bpp = 2,6-bis(pyrazol-3-yl)pyridine) has been investigated by magnetic susceptibility, photomagnetism and diffuse reflectivity measurements. These complexes display a thermal spin transition and exhibit the light-induced excited spin state trapping (LIESST) effect at low temperature. For each mixed-crystal system, the thermal spin transition temperature, T_1/2, and the relaxation temperature of the photo-induced high-spin state, T(LIESST), have been systematically determined. It appears that T_1/2 decreases with the metal dilution while T(LIESST) remains unchanged, suggesting that the two interconversion processes are controlled by different factors; i.e. the photomagnetic properties are governed at the molecular scale and the thermal spin crossover regime is affected by both the ligand field and crystal packing effects. For highly metal-diluted complex with x < 0.2, it is found that when T_1/2 reaches the T(LIESST), the complex remains HS on the whole range of temperature.     

Drosophila memory: will Orb(2) predict the future?

Genetically tractable organisms with relatively simple nervous systems offer a realistic platform to understand how and where memories are formed and stored in defined neural circuits. Recent work in Drosophila provides promise that this analysis may soon reach the resolution of identifiable synapses.     

Combination of Azidothymidine (AZT) and (E)-5-(2-Bromovinyl)-2′-deoxyuridine (BVDU) Inhibits the Replication of Herpes Simplex Virus Type 1 (HSV-1) and Type 2 (HSV-2) and Varicella Zoster Virus (VZV) Strains That Are Deficient in the Expression of the Viral Thymidine Kinase (tk)

Abstract

Combinations of high concentrations of AZT with BVDU, acyclovir (ACV) or ganciclovir (GCV) show antagonism against TK⁺ HSV-1, but not TK⁺ VZV strains, in cell cultures. When BVDU and AZT were used in combination against TK^? HSV-1, TK^? HSV-2 and TK^? VZV strains, a pronounced inhibition of viral replication was observed. This potentiating effect was not seen if AZT was combined with ACV or GCV.     

Feedback regulation of the α2(1) collagen gene via the Mek-Erk signaling pathway

The extracellular matrix (ECM) provides the microenvironment that is pivotal for cell growth, motility, attachment, and differentiation. Advances in cell culture techniques have led to the development of cell-derived ECM model systems that are more reflective of the in vivo architecture of the ECM in tissue. In this study, a fibroblast-derived ECM (fd-ECM) was used to study the feedback regulation of type I collagen synthesis in fibroblasts. Fibroblasts plated on a preformed fd-ECM showed a significant decrease in the production of type I collagen and pro-α2(1) collagen mRNA compared to cells grown in the absence of a matrix. Function-blocking antibodies showed that this downregulation of type I collagen gene expression is mediated via α2β1 integrin. The use of several kinase inhibitors and a dominant negative ras construct (N17Ras) showed that the matrix-mediated downregulation of COL1A2 occurs via Ras-dependent activation of the MEK/ERK signaling pathway. Deletion analysis of the COL1A2 promoter implicated the region between -375 and -107 as containing a potential matrix responsive element. The use of Sp1 siRNA demonstrated that Sp1 is an important mediator of this feedback inhibition. This study provides some new insights into the feedback regulation of COL1A2 gene expression.     

Comparative analysis of the sequences of the three collagen chains α1(I), α2 and α1(III): Functional and genetic aspects

The amino acid sequences of type I collagen containing α1(I) and α2 chains at a ratio of 2:1, and of type III collagen consisting of α1 (III) chains are known. A statistical analysis of the sequences of these α chains is presented. The inter-chain comparison showed a high level of homology between the three α chains. The interactive amino acids, such as the polar charged and part of the hydrophobic residues responsible for the assembly of the molecules, are strongly conserved. The intra-chain analysis revealed that the α chains are divided into four related D units, each with a length of 234 residues. Between the D units within a chain the polar residues show a higher variability than the hydrophobic amino acids.Besides the D units, other periodicities such as $\text{D}\text{3}$ (78 residues), $\text{D}\text{6}$ (39 residues), $\text{solD}\text{11}$ (21 residues) and $\text{solD}\text{13}$ (18 residues) were observed, particularly in α1 (I) and α1 (III). The D unit is a functional repeat that is formed by the interactive polar charged and hydrophobic residues and which determines the aggregation of the molecules. The $\text{solD}\text{3}$ unit is mainly pronounced by the non-interactive residues such as proline and alanine and appears to be a reminiscence of a primordial gene. The smaller periodic repeating units may be considered as additional genetic units or as structural units, which determine the triplehelical pitch and thus the lateral aggregation of the molecules.In contrast to α1 (I) and α1 (III), the α2 chain shows less regularity in its internal structure.     

Modeling GPCR active state conformations: the β(2)-adrenergic receptor

The recent publication of several G protein-coupled receptor (GPCR) structures has increased the information available for homology modeling inactive class A GPCRs. Moreover, the opsin crystal structure shows some active features. We have therefore combined information from these two sources to generate an extensively validated model of the active conformation of the β(2)-adrenergic receptor. Experimental information on fully active GPCRs from zinc binding studies, site-directed spin labeling, and other spectroscopic techniques has been used in molecular dynamics simulations. The observed conformational changes reside mainly in transmembrane helix 6 (TM6), with additional small but significant changes in TM5 and TM7. The active model has been validated by manual docking and is in agreement with a large amount of experimental work, including site-directed mutagenesis information. Virtual screening experiments show that the models are selective for β-adrenergic agonists over other GPCR ligands, for (R)- over (S)-β-hydroxy agonists and for β(2)-selective agonists over β(1)-selective agonists. The virtual screens reproduce interactions similar to those generated by manual docking. The C-terminal peptide from a model of the stimulatory G protein, readily docks into the active model in a similar manner to which the C-terminal peptide from transducin, docks into opsin, as shown in a recent opsin crystal structure. This GPCR-G protein model has been used to explain site-directed mutagenesis data on activation. The agreement with experiment suggests a robust model of an active state of the β(2)-adrenergic receptor has been produced. The methodology used here should be transferable to modeling the active state of other GPCRs.     

Selectively 13C-enriched DNA: Dynamics of the C1′-H1′ vector in d(CGCAAATTTGCG)2

Summary In order to examine the internal dynamic processes of the dodecamer d(CGCAAATTTGCG)₂, the ¹³C-enriched oligonucleotide has been synthesized. The three central thymines were selectively ¹³C-labeled at the C1′ position and their spin-lattice relaxation parameters R(C_Z), R(C_X,Y), R(H_Z→C_Z), R(2H_ZC_Z), R(2H_ZC_X,Y) and R(H _infZ ^supC ) were measured. Density functions were computed for two models of internal motions. Comparisons of the experimental data were made with the spin-lattice relaxation rates rather than with the density functions, whose values were altered by accumulation of the uncertainties of each relaxation rate measurement. The spin-lattice relaxation rates were computed with respect to the motions of the sugar around the C1′-N1 bond. A two-state jump model between the anti- and syn-conformations with P(anti)/P(syn)=91/9 or a restricted rotation model with Δχ=28° and an internal diffusion coefficient of 30×10⁷ s^-1 gave a good fit with the experimental data. Twist, tilt or roll base motions have little effect on ¹³C1′ NMR relaxation. Simulation of spin-relaxation rates with the data obtained at several temperatures between 7 and 32 °C, where the dodecamer is double stranded, shows that the internal motion amplitude is independent of the temperature within this range, as expected for internal motion. Using the strong correlation which exists in a B-DNA structure between the χ and δ angle, we suggest that the change in the glycosidic angle value should be indicative of a sugar puckering between the C1′-exo and C2′-endo conformations.     

MicroRNA-33a Mediates the Regulation of High Mobility Group AT-Hook 2 Gene (HMGA2) by Thyroid Transcription Factor 1 (TTF-1/NKX2–1)

In lung cancers, TTF-1 displays seemingly paradoxical activities. Although TTF-1 is amplified in primary human lung cancers, it inhibits primary lung tumors from metastasizing in a mouse model system. It was reported that the oncogenic proepithelial mesenchymal transition (EMT) high mobility group AT-hook 2 gene (HMGA2) mediates the antimetastatic function of TTF-1. To gain mechanistic insight into the metastasis-critical signaling axis of TTF-1 to HMGA2, we used both reverse and forward strategies and discovered that microRNA-33a (miR-33a) is under direct positive regulation of TTF-1. By chromatin immunoprecipitation, we determined that TTF-1 binds to the promoter of SREBF2, the host gene of miR-33a. The 3′-untranslated region (UTR) of HMGA2 contains three predicted binding sites of miR-33a. We showed that the first two highly conserved sites are conducive to HMGA2 repression by miR-33a, establishing HMGA2 as a genuine target of miR-33a. Functional studies revealed that enforced expression of miR-33a inhibits the motility of lung cancer cells, and this inhibition can be rescued by overexpression of the form of HMGA2 without the 3′-UTR, suggesting that TTF-1 keeps the prometastasis gene HMGA2 in check via up-regulating miR-33a. This study reports the first miRNAs directly regulated by TTF-1 and clarifies how TTF-1 controls HMGA2 expression. Moreover, the documented importance of SREBF2 and miR-33a in regulating cholesterol metabolism suggests that TTF-1 may be a modulator of cholesterol homeostasis in the lung. Future studies will be dedicated to understanding how miRNAs influence the oncogenic activity of TTF-1 and the role of TTF-1 in cholesterol metabolism.     

大肠杆菌44277(O2: K1) 中rmlB 基因功能

摘要: 【目的】确定rmlB 基因在大肠杆菌( O2: K1) L-型鼠李糖合成中的作用。【方法】将基因rmlB 进行原核表达并测定酶活; 用同源重组的方法将rmlB 基因敲除,分析表型变化,并运用质谱,以及核磁共振等手段分析脂多糖O 侧链的结构,以确定rmlB 在O 抗原合成中的作用。【结果】成功对rmlB 基因进行了表达并测定了重组蛋白的酶活,确定蛋白RmlB 具有dTDP-D-glucose 4,6-dehydratase 活性。成功构建了rmlB 基因缺失突变株,对突变株进行表型分析发现突变株的表型与野生株相比无变化。对突变株分析发现突变株中的O抗原仍含有L-型鼠李糖,说明在该菌株中可能存在RmlB 的同功能酶或者存在其它的L-型鼠李糖合成途径。【结论】rmlB 基因编码的蛋白具有dTDP-D-glucose 4,6-dehydratase 活性但此基因对于L-型鼠李糖的合成不是必需的。     

3-[(Imidazolidin-2-yl)imino]indazole ligands with selectivity for the α(2)-adrenoceptor compared to the imidazoline I(1) receptor

A series of 3-[(4,5-dihydroimidazolidin-2-yl)imino]indazoles has been synthesized as positional analogues of marsanidine, a highly selective ??₂-adrenoceptor ligand. Parent compound 4a and its 4-chloro (4c) and 4-methyl (4d) derivatives display ??₂-adrenoceptor affinity at nanomolar concentrations (K_i = 39.4, 15.9 and 22.6 nM, respectively) and relatively high ??₂/I₁ selectivity ratios of 82, 115 and 690, respectively. Evidence was obtained that these compounds act as partial agonists at ??_2A-adrenoceptors. Compound 4d with intrinsic activity comparable with that of marsanidine, but lower than that of clonidine, elicited pronounced cardiovascular effects in anesthetized rats at doses as low as 0.01 mg/kg iv     

人类生态学(八):野生动物管理(2)

栖息地遭受破坏和污染随着人类人口的增长和对自然资源需求的增加,人类一直在不断地砍伐森林、扩大耕地、建立城市、修筑道路、开采矿山、竖立井架、建设发电站等。热带雨林是保护生物遗传多样性最理想的地方,但地球上热带雨林的面积正在急剧缩小,顶极植被的破坏是对野生动物生存的最大威胁。每一种动物对于周围环境都有自己特定的忍受限度,栖息地的改变和破坏将使很多动物失去生存场所和必需的资源,造成这些动物的生存危机。虽然很多野生动     

行为生态学(十七):栖息地选择(2)

栖息地选择的遗传与获得据目前研究所知,动物对栖息地的选择具有一定的遗传性和后天获得性(即可借助于早期生活经验和学习而改进)。如果把两种动物饲养在同一环境中,发现它们对栖息地的选择有所不同,那么这种差异大都是由遗传所决定的。例如:把从未在自然植被中生活过的蓝山雀和煤山雀关在同一个鸟舍中,鸟舍内放有栎树枝和松树枝,观察记录表明,煤山雀大部分时间都停栖在松树枝上,而蓝山雀则主要停栖在栎树枝上。这种不同的选择同这两种山雀在自然状态下对栖息地的不同偏爱是一致的(蓝山雀偏爱阔叶林,而煤山雀偏爱针叶林)。     

人类生态学(七):野生动物管理(1)

地球上的生物已有大约40亿年的进化史,而人类的起源和进化大约只经历了400万年,约占整个生物进化史的1‰。人类是地球上最晚出现的一种智慧生物,人类起源于生物界,而又离不开生物界。在人类进化历史的绝大部分时期内,人类几乎完全依赖野生生物为生(靠狩猎和采集野生植物),只是在1万多年以前,当农业和畜牧业产生以后,人类才逐渐减少了对野生生物的依赖性(目前仍有许多国     

行为生态学(十六):栖息地选择(1)

栖息地选择是指动物对生活地点类型的选择或偏爱,显然,所有的动物都只能生活在环境中的一定空间范围之内。但是,每一种动物的现实分布状况是通过怎样的过程而完成的,目前生态学家还了解得很少,现在只知道同动物对栖息地的选择有关。栖息地选择常常可以产生深远影响。动物对一个特定栖息地的选择可使动物只生活在某一特定环境之中,这有利于动物的表现型的定向改造。因此,同一种动物对栖息地的不同选择往往会引起它们之间基因频率的差异,而不同种动物对栖息地的不同选择又往往能增加种     

Propylbenzmethylation at Val-1(α) markedly increases the tetramer stability of the PEGylated hemoglobin: A comparison with propylation at Val-1(α)

Background

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are potential pharmaceutical agents that can be used in surgery or emergency medicine. PEGylation can modulate the vasoactivity of Hb and is a widely used approach to develop HBOCs. However, PEGylation can significantly enhance the tetramer–dimer dissociation of Hb, which may perturb the structure of Hb and increase its observed adverse effect. Thus, it is necessary to increase the tetramer stability of the PEGylated Hb.

Methods

Propylbenzmethylation at Val-1(α) of HbA was carried out to stabilize the Hb tetramer. The propylbenzmethylated Hb at Val-1(α) (PrB-Hb) was used as the starting material for site-specific PEGylation at Cys-93(β) of Hb using maleimide PEG. Structural and functional properties, autoxidation rate and thermal stability of the resultant product (PEG-PrB-Hb) were measured.

Results

Propylbenzmethylation at Val-1(α) led to 25-fold and 24-fold decreases in the tetramer–dimer dissociation constant of HbA and PEG-Hb, respectively. The increased tetramer stability is due to the enhanced hydrophobicity of the area around Val-1(α) and the increased polar interaction of Hb upon propylbenzmethylation. Thus, the structural and functional properties of PEG-Hb were improved, and its autoxidation rate and thermal denaturation were decreased.

Conclusion

Propylbenzmethylation at Val-1(α) showed higher ability than propylation at Val-1(α) to improve the structural and functional properties and decrease the side effect of PEG-Hb.

General significance

Our study can facilitate the biotechnological development of stable PEGylated Hb as more advanced HBOC. Our study is also expected to improve the stability of the tetrameric or dimeric proteins (e.g., uric oxidase) by propylbenzmethylation at their N-terminus.