胶粘香茶菜的化学成分

胶粘香茶菜(I.glutinosa C.Y.Wu et H.W.Li),为唇形科(Labiatae)香茶菜属(Isodon)植物,产于云南西北部及四川西南部,海拔2000-2300m的河谷两岸山坡砾石地或干燥灌丛中。从云南丽江产胶粘香茶菜中,已分离和鉴定了2种不同结构类型的二萜成分,为进一步比较不同地区产该种植物在化学成分上的差异,我们对大理苍山产胶粘香茶菜进行了研究。由2316g茎叶得149g提取物,然后经硅胶柱层析,依次用氯仿、氯仿-乙酸乙脂(8:2→4:6),乙酸乙酯梯度洗脱,除分离和鉴定了β-谷甾醇(β-sitosterol),胡萝卜甙(β-sitosterol-D-glucoside),乌苏酸(ursolic acid),山植酸(crataegolic acid)和丁二酸(butanedioic acid)外,还得到迄今从香茶菜属植物中分离得到的氧化程度最低的一个对映-贝壳杉烯型四环二萜类化合物:对映-贝壳杉烷-16β,17-二醇(ent—Kauran-16β,17-diol)(1)(2.8g)和具有抑制革兰氏阳性菌生长活性的松香烷(abitane)型二萜pisiferic acid(2)(1.2g)。pisiferic acid(2)系首次从该属植物中得到。     

叶穗香茶菜的二萜成分

从云南省中甸县产叶穗香茶菜[Rabdosia phyllostachys(Diels)Hara]叶中分得1个新二萜化合物,命名为叶穗香茶菜乙素(phyllostachysin B),经各项波谱数据确定,叶穗香茶菜乙素为6β,11β,14β—三羟基-15α-乙酰氧基-对映-贝壳杉-16-烯-20 醛-7-酮。另一个为已知化合物叶穗香茶菜甲素(phyllostachysin A)。     

细叶香茶菜中两个新的对映-贝壳杉烷二萜化合物

从云南中甸产细叶香茶菜(Isodon tenuifolia (W. W. Smith) Kudo)的地上部分分离得到6个化合物, 它们的结构通过波谱方法得到鉴定。其中化合物1和2为新的对映-贝壳杉烷二萜化合物, 即细叶香茶菜甲素(3β,6α,15β-trihydroxy-1α,7β-diacetoxy-11β,16β-epoxy-ent-kaurane) (1) 和细叶香茶菜乙素(1α,6α,11β-trihydroxy-3β,7β-diacetoxy-ent-kaur-16-en-15-one) (2)。     

细叶香茶菜中两个新的对映—贝壳杉烷二萜化合物

从云南中甸产细叶香茶菜 (Isodontenuifolia (W .W .Smith)Kudo)的地上部分分离得到 6个化合物 ,它们的结构通过波谱方法得到鉴定。其中化合物 1和 2为新的对映_贝壳杉烷二萜化合物 ,即细叶香茶菜甲素 (3β ,6α ,15 β_trihydroxy_1α ,7β_diacetoxy_11β ,16 β_epoxy_ent_kaurane) (1)和细叶香茶菜乙素 (1α,6α ,11β_trihydroxy_3β,7β_diacetoxy_ent_kaur_16_en_15_one) (2 )。     

近无毛灰岩香茶菜的研究

从云南省会泽县产近无毛灰岩香茶莱〔Rabdosia calcicola (Hand. -Mazz.) Hara var. subculva (Hand. -Mazz.) C. Y. Wu et H. W. Li〕叶中分得两个二萜化合物,一个为新化合物,命名为灰岩香茶菜甲素(calcieolin A),另一个为已知化合物维西香茶菜甲素(weisiensin A),经各项光谱数据和化学反应确定,灰岩香茶菜甲素为3β-羟基-1α,6α,7β,11β-四乙酰氧基-对映-贝壳杉-16-烯-15-酮(1);维西香茶菜甲素结构应为3β,6α-二羟基-1α,7β,11β-三乙酰氧基-对映—贝壳杉-16-烯-15酮(2)。     

紫萼香茶菜甲素的结构

从紫萼香茶菜叶的乙醚提取物中分得一个新的二萜化合物,命名为紫萼香茶菜甲素(rabdoforrestin A),根据波谱和化学证据,其结构为对映-11β-羟基-2α,3α,6β,7α-四乙酰氧基-16-贝壳杉烯-15-酮(ent-11β-hydroxy-2α,3α,6β,7α-tetraacetoxy-16-kauren-15-one)。另外还分离到已知黄酮化合物线蓟素(cirsilioi 4)。     

细锥香茶菜二萜的研究

从四川省峨眉山产细锥香茶菜[Rabdosia coetsa(Buch-Ham,ex,D.Don)Hara]乙醚抽提物中分得了一个新二萜化合物,命名为细锥香茶菜丁素(rabdocoetsin D),其结构经各项波谱数据和化学证据确定为1α,7β-二羟基-11β-乙酰氧基-对映-7β,20-环氧-贝壳杉-16-烯-15-酮(1)。另外还分离到3个已知二萜,细锥香茶菜乙素(rabdocoetsin B)、细锥香茶菜丙素(rabdocoetsin C)、瘿花香茶菜甲素(rosthorin A)。     

旱生香茶菜中三个新的对映-贝壳杉烷型二萜

从旱生香茶菜（ｌｓｏｄｏｎ ｘｅｒｏｐｈｉｌｕｓ）叶中分离得到３个新的对映－贝壳杉烷型二萜,旱生香茶菜素Ｇ,Ｈ和Ｌ０通过波谱方法鉴定它们的结构。     

疏花毛萼香茶菜中一新的对映-贝壳杉烷型二萜

从疏花毛萼香茶菜（Ｉｓｏｄｏｎ ｅｒｉｏｃａｌｙｘ ｖａｒ．ｌａｘｉｆｌｏｒａ）叶中分离得到一新的对映－贝壳杉烷型二萜,命名为疏花丁素（１）,通过波谱方法鉴定了它的结构。此外,还分离得到６个已知对映－贝壳杉烷型二萜化合物：疏花甲素（２）,毛萼晶Ａ－Ｃ（３－５）和Ｑ（６）,毛萼乙素（７）,以及ｃｉｒｓｉｍａｒｉｔｉｎ（８）和２α－羟基乌索酸（９）。     

兰萼丙素的结构

从北京地区产兰萼香茶菜[Rabdosia japonica (Burm. f.)Hara var.glaucocalyx(Maxim.)Hara]叶中分得三个对映-贝壳杉烯二萜化合物,其中二个为已知物兰萼甲素(2)和乙素(3),另一个为新化合物,命名为兰萼丙素,经光谱和化学方法证明,其结构为对映-7β,14α,15α-三羟基-16-贝壳杉烯-3-酮(1)。     

Direct methods and residue type specific isotope labeling in NMR structure determination and model-driven sequential assignment

Direct methods in NMR based structure determination start from an unassigned ensemble of unconnected gaseous hydrogen atoms. Under favorable conditions they can produce low resolution structures of proteins. Usually a prohibitively large number of NOEs is required, to solve a protein structure ab-initio, but even with a much smaller set of distance restraints low resolution models can be obtained which resemble a protein fold. One problem is that at such low resolution and in the absence of a force field it is impossible to distinguish the correct protein fold from its mirror image. In a hybrid approach these ambiguous models have the potential to aid in the process of sequential backbone chemical shift assignment when ¹³C^β and ¹³C′ shifts are not available for sensitivity reasons. Regardless of the overall fold they enhance the information content of the NOE spectra. These, combined with residue specific labeling and minimal triple-resonance data using ¹³C^α connectivity can provide almost complete sequential assignment. Strategies for residue type specific labeling with customized isotope labeling patterns are of great advantage in this context. Furthermore, this approach is to some extent error-tolerant with respect to data incompleteness, limited precision of the peak picking, and structural errors caused by misassignment of NOEs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Solution structure of monomeric BsaL, the type III secretion needle protein of Burkholderia pseudomallei

Many gram-negative bacteria that are important human pathogens possess type III secretion systems as part of their required virulence factor repertoire. During the establishment of infection, these pathogens coordinately assemble greater than 20 different proteins into a macromolecular structure that spans the bacterial inner and outer membranes and, in many respects, resembles and functions like a syringe. This type III secretion apparatus (TTSA) is used to inject proteins into a host cell's membrane and cytoplasm to subvert normal cellular processes. The external portion of the TTSA is a needle that is composed of a single type of protein that is polymerized in a helical fashion to form an elongated tube with a central channel of 2-3 nm in diameter. TTSA needle proteins from a variety of bacterial pathogens share sequence conservation; however, no atomic structure for any TTSA needle protein is yet available. Here, we report the structure of a TTSA needle protein called BsaL from Burkholderia pseudomallei determined by nuclear magnetic resonance (NMR) spectroscopy. The central part of the protein assumes a helix-turn-helix core domain with two well-defined alpha-helices that are joined by an ordered, four-residue linker. This forms a two-helix bundle that is stabilized by interhelix hydrophobic contacts. Residues that flank this presumably exposed core region are not completely disordered, but adopt a partial helical conformation. The atomic structure of BsaL and its sequence homology with other TTSA needle proteins suggest potentially unique structural dynamics that could be linked with a universal mechanism for control of type III secretion in diverse gram-negative bacterial pathogens.     

Solution structure of the chick TGFbeta type II receptor ligand-binding domain

The transforming growth factor beta (TGFbeta) signaling pathway influences cell proliferation, immune responses, and extracellular matrix reorganization throughout the vertebrate life cycle. The signaling cascade is initiated by ligand-binding to its cognate type II receptor. Here, we present the structure of the chick type II TGFbeta receptor determined by solution NMR methods. Distance and angular constraints were derived from 15N and 13C edited NMR experiments. Torsion angle dynamics was used throughout the structure calculations and refinement. The 20 final structures were energy minimized using the generalized Born solvent model. For these 20 structures, the average backbone root-mean-square distance from the average structure is below 0.6A. The overall fold of this 109-residue domain is conserved within the superfamily of these receptors. Chick receptors fully recognize and respond to human TGFbeta ligands despite only 60% identity at the sequence level. Comparison with the human TGFbeta receptor determined by X-ray crystallography reveals different conformations in several regions. Sequence divergence and crystal packing interactions under low pH conditions are likely causes. This solution structure identifies regions were structural changes, however subtle, may occur upon ligand-binding. We also identified two very well conserved molecular surfaces. One was found to bind ligand in the crystallized human TGFbeta3:TGFbeta type II receptor complex. The other, newly identified area can be the interaction site with type I and/or type III receptors of the TGFbeta signaling complex.     

The NMR structure of FliK, the trigger for the switch of substrate specificity in the flagellar type III secretion apparatus

The flagellar cytoplasmic protein FliK controls hook elongation by two successive events: by determining hook length and by stopping the supply of hook protein. These two distinct roles are assigned to different parts of FliK: the N-terminal half (FliK_N) determines length and the C-terminal half (FliK_C) switches secretion from the hook protein to the filament protein. The interaction of FliK_C with FlhB, the switchable secretion gate, triggers the switch. By NMR spectroscopy, we demonstrated that FliK is largely unstructured and determined the structure of a compact domain in FliK_C. The compact domain, denoted the FliK_C core domain, consists of two α-helices, a β-sheet with two parallel and two antiparallel strands, and several exposed loops. Based on the functional data obtained by a series of deletion mutants of the FliK_C core domain, we constructed a model of the complex between the FliK_C core domain and FlhB_C. The model suggested that one of the FliK_C loops has a high probability of interacting with the C-terminal domain of FlhB (FlhB_C) as the FliK molecule enters the secretion gate. We suggest that the autocleaved NPTH sequence in FlhB contacts loop 2 of FliK_C to trigger the switching event. This contact is sterically prevented when NPTH is not cleaved. Thus, the structure of FliK provides insight into the mechanism by which this bifunctional protein triggers a switch in the export of substrates.     

Immunoreactivity and conformation of the immunodominant domain of HTLV-I envelope surface glycoprotein

The envelope of the human retrovirus HTLV-I (humanT-cell leukemia virus type I), like those of otherretroviruses, plays an important role in viralinfection. One of the major immunodominant domains ofHTLV-I surface glycoprotein (gp46), inducing antibodyreactions in over 90% of infected individuals, isbounded by amino acids 175 and 199. As compared toHTLV-I prototype strain MT-2, few amino acidsubstitutions have been described in this region; themost frequently observed is the replacement of aproline by a serine at position 192. In order toinvestigate the antigenic impact of this variation, weanalysed the reactivity of synthetic peptides,harbouring either a proline or a serine residue,towards antibody containing HTLV-I positive sera inenzyme linked immunosorbent assays. The possibleinfluence of this amino acid substitution on theconformational behaviour has been examined by studyingthe solution structure of two model peptides(corresponding to the 175–199 region) usingtwo-dimensional ¹H NMR spectroscopy. The resultsof this work should allow us to find out whether thisamino acid substitution has to be taken into accountfor the design of a future peptide-based vaccineagainst HTLV-I infection.     

Immunoreactivity and conformation of the immunodominant domain of HTLV-I envelope surface glycoprotein

Summary The envelope of the human retrovirus HTLV-I (human T-cell leukemia virus type I), like those of other retroviruses, plays an important role in viral infection. One of the major immunodominant domains of HTLV-I surface glycoprotein (gp46), inducing antibody reactions in over 90% of infected individuals, is bounded by amino acids 175 and 199. As compared to HTLV-I prototype strain MT-2, few amino acid substitutions have been described in this region; the most frequently observed is the replacement of a proline by a serine at position 192. In order to investigate the antigenic impact of this variation, we analysed the reactivity of synthetic peptides, harbouring either a proline or a serine residue, towards antibody containing HTLV-I positive sera in enzyme linked immunosorbent assays. The possible influence of this amino acid substitution on the conformational behaviour has been examined by studying the solution structure of two model peptides (corresponding to the 175–199 region) using two-dimensional¹H NMR spectroscopy. The results of this work should allow us to find out whether this amino acid substitution has to be taken into account for the design of a future peptide-based vaccine against HTLV-I infection.     

Structural modification of acyl carrier protein by butyryl group

Fatty acid synthesis in bacteria is catalyzed by a set of individual enzymes known as the type II fatty acid synthase. Acyl carrier protein (ACP) shuttles the acyl intermediates between individual pathway enzymes. In this study, we determined the solution structures of three different forms of ACP, apo‐ACP, ACP, and butyryl‐ACP under identical experimental conditions. The structural studies revealed that attachment of butyryl acyl intermediate to ACP alters the conformation of ACP. This finding supports the more general notion that the attachment of different acyl intermediates alters the ACP structure to facilitate their recognition and turnover by the appropriate target enzymes.     

1H NMR metabonomics approach to the disease continuum of diabetic complications and premature death

Subtle metabolic changes precede and accompany chronic vascular complications, which are the primary causes of premature death in diabetes. To obtain a multimetabolite characterization of these high‐risk individuals, we measured proton nuclear magnetic resonance (¹H NMR) data from the serum of 613 patients with type I diabetes and a diverse spread of complications. We developed a new metabonomics framework to visualize and interpret the data and to link the metabolic profiles to the underlying diagnostic and biochemical variables. Our results indicate complex interactions between diabetic kidney disease, insulin resistance and the metabolic syndrome. We illustrate how a single ¹H NMR protocol is able to identify the polydiagnostic metabolite manifold of type I diabetes and how its alterations translate to clinical phenotypes, clustering of micro‐ and macrovascular complications, and mortality during several years of follow‐up. This work demonstrates the diffuse nature of complex vascular diseases and the limitations of single diagnostic biomarkers. However, it also promises cost‐effective solutions through high‐throughput analytics and advanced computational methods, as applied here in a case that is representative of the real clinical situation.