Highly potent and specific inhibitors of human renin

We have designed and synthesized a series of small peptides containing a perfluoroalkyl ketone group at the C-terminal position of the angiotensin I sequence as inhibitors of human renin. From this series of compounds, 8 and 10 showed strong inhibition of human renin (IC₅₀ = 3 × 10⁻⁹, 7 × 10⁻⁹ M, respectively). Compound 10 did not inhibit pepsin and cathepsin D at 10⁻⁴ M. Comparison of the IC₅₀ of compound 8 and compound 11 (8.7 × 10⁻⁷ M) demonstrated the marked effect of the perfluoropropyl group on the potency of inhibition on renin, presumably due to the strong electron-withdrawing effect causing the ketone in 8 to exist predominantly as the hydrate — thus mimicking the tetrahedral transition state during hydrolysis of the scissile Leu¹⁰—Val¹¹ amide bond.     

MTMR3基因1670C>T多态性与胃癌易感性的关系研究

目的:探讨MTMR3基因3'非翻译区(3'UTR)1670CT多态性与胃癌发病的关联。方法:通过病例对照研究,采用聚合酶链式反应-限制性片段长度多态性方法检测500例胃癌患者和502例正常对照者MTMR3基因1670CT位点的基因型,通过非条件Logistic回归,并校正了性别、年龄、吸烟、饮酒的影响,分析该位点与胃癌易感性的关系。结果:MTMR3基因1670CT基因型分布在病例组和对照组均符合Hardy-Weinberg平衡(P0.05)。经过年龄、性别、吸烟和饮酒状况校正的非条件logistic回归分析发现,与CC基因型患者相比,含有T等位基因的基因型(CT和TT)患者胃癌的发病风险增高(校正优势比=1.72,95%置信区间=1.36~2.16,P=3.99×10-5)。结论:MTMR3基因1670CT多态性位点与胃癌发病存在显著性关联,1670T可能是胃癌发生的一个遗传危险因素。     

Combining diagnostic test results to increase accuracy

When multiple diagnostic tests are performed on an individual or multiple disease markers are available it may be possible to combine the information to diagnose disease. We consider how to choose linear combinations of markers in order to optimize diagnostic accuracy. The accuracy index to be maximized is the area or partial area under the receiver operating characteristic (ROC) curve. We propose a distribution-free rank-based approach for optimizing the area under the ROC curve and compare it with logistic regression and with classic linear discriminant analysis (LDA). It has been shown that the latter method optimizes the area under the ROC curve when test results have a multivariate normal distribution for diseased and non-diseased populations. Simulation studies suggest that the proposed non-parametric method is efficient when data are multivariate normal.The distribution-free method is generalized to a smooth distribution-free approach to: (i) accommodate some reasonable smoothness assumptions; (ii) incorporate covariate effects; and (iii) yield optimized partial areas under the ROC curve. This latter feature is particularly important since it allows one to focus on a region of the ROC curve which is of most relevance to clinical practice. Neither logistic regression nor LDA necessarily maximize partial areas. The approaches are illustrated on two cancer datasets, one involving serum antigen markers for pancreatic cancer and the other involving longitudinal prostate specific antigen data.     

trans-Acting Arginine Residues in the AAA+ Chaperone ClpB Allosterically Regulate the Activity through Inter- and Intradomain Communication

The molecular chaperone ClpB/Hsp104, a member of the AAA+ superfamily (ATPases associated with various cellular activities), rescues proteins from the aggregated state in collaboration with the DnaK/Hsp70 chaperone system. ClpB/Hsp104 forms a hexameric, ring-shaped complex that functions as a tightly regulated, ATP-powered molecular disaggregation machine. Highly conserved and essential arginine residues, often called arginine fingers, are located at the subunit interfaces of the complex, which also harbor the catalytic sites. Several AAA+ proteins, including ClpB/Hsp104, possess a pair of such trans-acting arginines in the N-terminal nucleotide binding domain (NBD1), both of which were shown to be crucial for oligomerization and ATPase activity. Here, we present a mechanistic study elucidating the role of this conserved arginine pair. First, we found that the arginines couple nucleotide binding to oligomerization of NBD1, which is essential for the activity. Next, we designed a set of covalently linked, dimeric ClpB NBD1 variants, carrying single subunits deficient in either ATP binding or hydrolysis, to study allosteric regulation and intersubunit communication. Using this well defined environment of site-specifically modified, cross-linked AAA+ domains, we found that the conserved arginine pair mediates the cooperativity of ATP binding and hydrolysis in an allosteric fashion.     

Residue Leu940 Has a Crucial Role in the Linkage and Reaction Specificity of the Glucansucrase GTF180 of the Probiotic Bacterium Lactobacillus reuteri 180

Highly conserved glycoside hydrolase family 70 glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue Leu⁹⁴⁰ in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-ΔN in complex with maltose. Herein, we show that mutations in Leu⁹⁴⁰ of wild-type GTF180-ΔN all caused an increased percentage of (α1→6) linkages and a decreased percentage of (α1→3) linkages in the products. α-Glucans with potential different physicochemical properties (containing 67–100% of (α1→6) linkages) were produced by GTF180 and its Leu⁹⁴⁰ mutants. Mutant L940W was unable to form (α1→3) linkages and synthesized a smaller and linear glucan polysaccharide with only (α1→6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (α1→3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E, and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue Leu⁹⁴⁰ in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes.     

P2Y2R activation by nucleotides released from oxLDL-treated endothelial cells (ECs) mediates the interaction between ECs and immune cells through RAGE expression and reactive oxygen species production

Lipoprotein oxidation, inflammation, and immune responses involving the vascular endothelium and immune cells contribute to the pathogenesis of atherosclerosis. In an atherosclerotic animal model, P2Y₂ receptor (P2Y₂R) upregulation and stimulation were previously shown to induce intimal hyperplasia and increased intimal monocyte infiltration. Thus, we investigated the role of P2Y₂R in oxidized low-density lipoprotein (oxLDL)-mediated oxidative stress and the subsequent interaction between endothelial cells (ECs) and immune cells. The treatment of human ECs with oxLDL caused the rapid release of ATP (maximum after 5 min). ECs treated with oxLDL or the P2Y₂R agonists ATP/UTP for 1 h exhibited significant reactive oxygen species (ROS) production, but this effect was not observed in P2Y₂R siRNA-transfected ECs. In addition, oxLDL and ATP/UTP both induced RAGE expression, which was P2Y₂R dependent. Oxidized LDL- and ATP/UTP-mediated ROS production was diminished in RAGE siRNA-transfected ECs, suggesting that RAGE is an important mediator in P2Y₂R-mediated ROS production. Treatment with oxLDL for 24 h induced P2Y₂R expression in the human monocyte cell line THP-1 and increased THP-1 cell migration toward ECs. The addition of apyrase, an enzyme that hydrolyzes nucleotides, or diphenyleneiodonium (DPI), a well-known inhibitor of NADPH oxidase, significantly inhibited the increase in cell migration caused by oxLDL. P2Y₂R siRNA-transfected THP-1 cells did not migrate in response to oxLDL or ATP/UTP treatment, indicating a critical role for P2Y₂R and nucleotide release in oxLDL-induced monocyte migration. Last, oxLDL and ATP/UTP effectively increased ICAM-1 and VCAM-1 expression and the subsequent binding of THP-1 cells to ECs, which was inhibited by pretreatment with DPI or by siRNA against P2Y₂R or RAGE, suggesting that P2Y₂R is an important mediator in oxLDL-mediated monocyte adhesion to ECs through the regulation of ROS-dependent adhesion molecule expression in ECs. Taken together, our findings suggest that P2Y₂R could be a therapeutic target for the prevention of vascular disorders, including atherosclerosis.     

New evidence of ectomycorrhizal fungi in the Hawaiian Islands associated with the endemic host Pisonia sandwicensis (Nyctaginaceae)

Ectomycorrhizal plants and fungi are ubiquitous in mainland forests, but because of dispersal limitations are predicted to be less common on isolated islands. For instance, no native ectomycorrhizal plants or fungi have ever been reported from Hawaii, one of the most remote archipelagos on Earth. Members of the plant tribe Pisonieae are common on many islands, and prior evidence shows that some species associate with ectomycorrhizal fungi. However, until now, the Pisonieae species of Hawaii had yet to be examined for their mycorrhizal status. Here we sampled roots from members of the genus Pisonia growing on the Hawaiian islands of Oahu, Maui and Hawaii. We used molecular and microscopic techniques to categorize trees with respect to their mycorrhizal associations. We report that the Hawaiian endemic Pisonia sandwicensis forms ectomycorrhizas with at least five fungal operational taxonomic units (corresponding closely to species) belonging to four genera. We also report that this tree species is monophyletic with other ectomycorrhizal Pisonia species. We suggest that in light of the newly discovered Hawaiian ectomycorrhizal fungal community and other island ectomycorrhizal communities, dispersal limitations do not prevent the colonization of remote islands by at least some ectomycorrhizal fungi.     

Assessing Substitution Variation Across Sites in Grass Chloroplast DNA

We assess the similarity of base substitution processes, described by empirically derived 4 × 4 matrices, using chi-square homogeneity tests. Such significance analyses allow us to assess variation in sequence evolution across sites and we apply them to matrices derived from noncoding sites in different contexts in grass chloroplast DNA. We show that there is statistically significant variation in rates and patterns of mutation among noncoding sites in different contexts and then demonstrate a similar and significant influence of context on substitutions at fourfold degenerate sites of coding regions from grass chloroplast DNA. These results show that context has the same general effect on substitution bias in coding and noncoding DNA: the A+T content of flanking bases is correlated with rate of substitution, transition bias, and GC → AT pressure, while the number of flanking pyrimidines on a single strand is correlated with a mutational bias, or skew, toward pyrimidines. Despite the similarity in general trends, however, when we compare coding and noncoding matrices we find that there is a statistically significant difference between them even when we control for context. Most noticeably, fourfold degenerate sites in coding sequences are undergoing substitution at a higher rate and there are also significant differences in the relationship between pyrimidines skew and the number of flanking pyrimidines. Possible reasons for the differences between coding and noncoding sites are discussed. Furthermore, our analysis illustrates a simple statistical way for comparing substitution processes across sites allowing us to better study variation in evolutionary processes across a genome. [Reviewing Editor: Dr. Martin Kreitman]