PROSIT,AN ONLINE SERVICE TO CALCULATE PSEUDOROTATIONAL PARAMETERS OF NUCLEOSIDES AND NUCLEOTIDES

The online service PROSIT (Pseudo-Rotational Online Service and Interactive Tool) is a free service available at http://cactus.nci.nih.gov/prosit/ that performs pseudorotational analysis of nucleosides(tides). PROSIT reads the 3D coordinates of nucleosides and returns the pseudorotational phase angle P, puckering amplitude ν_max, and other related information. As examples, the sugar conformations in a parallel-stranded guanine tetraplex and a four-way Holliday junction are presented here.     

Accumulation of antioxidants in apricot fruit through ripening: characterization of a genotype with enhanced functional properties

Two apricot genotypes, 'Gonci magyarkajszi' and 'Preventa' were assayed at three ripening stages for flesh color indices (L*, a*, b*, C* and Ho), contents of total phenolics and vitamin C, and both water- and lipid-soluble antioxidant capacities (ferric reducing antioxidant power; 2,2'-diphenyl-1-picrylhydrazyl scavenging activity; total radical scavenging activity; and Photochem lipid-soluble antioxidant capacity) to compare their dynamics in the accumulation of antioxidant compounds and capacities through ripening. The increase in a*, b* and C* and decrease in Ho during ripening represented a color shift from green to yellow and orange due to carotenoid accumulation. Parallel to carotenoid accumulation, contents of total phenolics and vitamin C and antioxidant capacity increased significantly (p < 0.05) from unripe to fully ripe fruits. More phenolics and vitamin C accumulated in fully ripe fruits of 'Preventa' than 'G?nci magyarkajszi'. The accumulation patterns of these compounds were different: while the vitamin C contents in unripe fruit of 'Preventa' and 'G?nci magyarkajszi' were identical (approx. 6 mg/100 g fresh weight), unripe 'Preventa' contained even more phenolics (approx. 12 mmolGA/l) than fully ripe 'G?nci magyarkajszi' (8 mmolGA/l). Our results confirm that fully ripe 'Preventa' fruits are characterized by outstanding functional properties due to the increased accumulation of vitamin C and phenolics throughout the ripening process.     

A random variance model for detection of differential gene expression in small microarray experiments

MOTIVATION: Microarray techniques provide a valuable way of characterizing the molecular nature of disease. Unfortunately expense and limited specimen availability often lead to studies with small sample sizes. This makes accurate estimation of variability difficult, since variance estimates made on a gene by gene basis will have few degrees of freedom, and the assumption that all genes share equal variance is unlikely to be true. RESULTS: We propose a model by which the within gene variances are drawn from an inverse gamma distribution, whose parameters are estimated across all genes. This results in a test statistic that is a minor variation of those used in standard linear models. We demonstrate that the model assumptions are valid on experimental data, and that the model has more power than standard tests to pick up large changes in expression, while not increasing the rate of false positives. AVAILABILITY: This method is incorporated into BRB-ArrayTools version 3.0 (http://linus.nci.nih.gov/BRB-ArrayTools.html). SUPPLEMENTARY MATERIAL: ftp://linus.nci.nih.gov/pub/techreport/RVM_supplement.pdf     

caCORE: a common infrastructure for cancer informatics

MOTIVATION:Sites with substantive bioinformatics operations are challenged to build data processing and delivery infrastructure that provides reliable access and enables data integration. Locally generated data must be processed and stored such that relationships to external data sources can be presented. Consistency and comparability across data sets requires annotation with controlled vocabularies and, further, metadata standards for data representation. Programmatic access to the processed data should be supported to ensure the maximum possible value is extracted. Confronted with these challenges at the National Cancer Institute Center for Bioinformatics, we decided to develop a robust infrastructure for data management and integration that supports advanced biomedical applications. RESULTS: We have developed an interconnected set of software and services called caCORE. Enterprise Vocabulary Services (EVS) provide controlled vocabulary, dictionary and thesaurus services. The Cancer Data Standards Repository (caDSR) provides a metadata registry for common data elements. Cancer Bioinformatics Infrastructure Objects (caBIO) implements an object-oriented model of the biomedical domain and provides Java, Simple Object Access Protocol and HTTP-XML application programming interfaces. caCORE has been used to develop scientific applications that bring together data from distinct genomic and clinical science sources. AVAILABILITY: caCORE downloads and web interfaces can be accessed from links on the caCORE web site (http://ncicb.nci.nih.gov/core). caBIO software is distributed under an open source license that permits unrestricted academic and commercial use. Vocabulary and metadata content in the EVS and caDSR, respectively, is similarly unrestricted, and is available through web applications and FTP downloads. SUPPLEMENTARY INFORMATION: http://ncicb.nci.nih.gov/core/publications contains links to the caBIO 1.0 class diagram and the caCORE 1.0 Technical Guide, which provide detailed information on the present caCORE architecture, data sources and APIs. Updated information appears on a regular basis on the caCORE web site (http://ncicb.nci.nih.gov/core).     

PathVisio-MIM: PathVisio plugin for creating and editing Molecular Interaction Maps (MIMs)

MOTIVATION: A plugin for the Java-based PathVisio pathway editor has been developed to help users draw diagrams of bioregulatory networks according to the Molecular Interaction Map (MIM) notation. Together with the core PathVisio application, this plugin presents a simple to use and cross-platform application for the construction of complex MIM diagrams with the ability to annotate diagram elements with comments, literature references and links to external databases. This tool extends the capabilities of the PathVisio pathway editor by providing both MIM-specific glyphs and support for a MIM-specific markup language file format for exchange with other MIM-compatible tools and diagram validation. AVAILABILITY: The PathVisio-MIM plugin is freely available and works with versions of PathVisio 2.0.11 and later on Windows, Mac OS X and Linux. Information about MIM notation and the MIMML format is available at http://discover.nci.nih.gov/mim. The plugin, along with diagram examples, instructions and Java source code, may be downloaded at http://discover.nci.nih.gov/mim/mim_pathvisio.html.     

Intrinsic conformational properties of deoxyribonucleosides: implicated role for cytosine in the equilibrium among the A, B, and Z forms of DNA.

Structural properties of biomolecules are dictated by their intrinsic conformational energetics in combination with environmental contributions. Calculations using high-level ab initio methods on the deoxyribonucleosides have been performed to investigate the influence of base on the intrinsic conformational energetics of nucleosides. Energy minima in the north and south ranges of the deoxyribose pseudorotation surfaces have been located, allowing characterization of the influence of base on the structures and energy differences between those minima. With all bases, chi values associated with the south energy minimum are lower than in canonical B-DNA, while chi values associated with the north energy minimum are close to those in canonical A-DNA. In deoxycytidine, chi adopts an A-DNA conformation in both the north and south energy minima. Energy differences between the A and B conformations of the nucleosides are <0.5 kcal/mol in the present calculations, except with deoxycytidine, where the A form is favored by 2.3 kcal/mol, leading the intrinsic conformational energetics of GC basepairs to favor the A form of DNA by 1.5 kcal/mol as compared with AT pairs. This indicates that the intrinsic conformational properties of cytosine at the nucleoside level contribute to the A form of DNA containing predominately GC-rich sequences. In the context of a B versus Z DNA equilibrium, deoxycytidine favors the Z form over the B form by 1.6 kcal/mol as compared with deoxythymidine, suggesting that the intrinsic conformational properties of cytosine also contribute to GC-rich sequences occurring in Z DNA with a higher frequency than AT-rich sequences. Results show that the east pseudorotation energy barrier involves a decrease in the furanose amplitude and is systematically lower than the inversion barrier, with the energy differences influenced by the base. Energy barriers going from the south (B form) sugar pucker to the east pseudorotation barrier are lower in pyrimidines as compared with purines, indicating that the intrinsic conformational properties associated with base may also influence the sugar pseudorotational population distribution seen in DNA crystal structures and the kinetics of B to A transitions. The present work provides evidence that base composition, in addition to base sequence, can influence DNA conformation.     

Conformational study of trinucleoside tetraphosphate d(pCpGpCp): Transition of right-handed form to left-handed form

Using the semiempirical potential functions, conformational energies of the model compounds DMP^?, d(pCp), d(pGp), and d(pCpGpCp) are calculated, and the B → Z transition is discussed along the pseudorotational path of the sugar ring. For dimethylmonophosphate anion, DMP^?, the energy contour map is presented and the stabilities of the phosphodiester conformations discussed. For the sugar ring without the base attached, the minimum energies for each sugar-puckering form are calculated along the pseudorotational path. The energy barrier of the interconversion between the C(3′)-endo form and the C(2′)-endo form is calculated to be about 2.0 kcal/mol. From the conformational energy calculations of the interconversions of mononucleoside diphosphates, d(pCp) and d(pGp), between the C(2′)-endo conformer and the C(3′)-endo conformer, the purine sugar segment is known to be more convertible than the pyrimidine sugar segment. The results also support the finding that the pseudorotational transition occurred with the O(1′)-endo form more easily than with the O(1′)-exo form. Based on the results of conformational studies of DMP^?, d(pCp), and d(pGp), a topological transition of the handedness of the model compound, d(pCpGpCp), is studied. The left-handed Z-form is found to be less stable by about 8.5 kcal/mol than is the right-handed B-form. The energy barrier of the Z → B transition is calculated to be about 17.4 kcal/mol. The contributions of the electrostatic and nonbonded energies to the energy barrier are discussed in connection with the conformation changes of the model compound, d(pCpGpCp).     

caGrid: design and implementation of the core architecture of the cancer biomedical informatics grid

MOTIVATION: The complexity of cancer is prompting researchers to find new ways to synthesize information from diverse data sources and to carry out coordinated research efforts that span multiple institutions. There is a need for standard applications, common data models, and software infrastructure to enable more efficient access to and sharing of distributed computational resources in cancer research. To address this need the National Cancer Institute (NCI) has initiated a national-scale effort, called the cancer Biomedical Informatics Grid (caBIGtrade mark), to develop a federation of interoperable research information systems. RESULTS: At the heart of the caBIG approach to federated interoperability effort is a Grid middleware infrastructure, called caGrid. In this paper we describe the caGrid framework and its current implementation, caGrid version 0.5. caGrid is a model-driven and service-oriented architecture that synthesizes and extends a number of technologies to provide a standardized framework for the advertising, discovery, and invocation of data and analytical resources. We expect caGrid to greatly facilitate the launch and ongoing management of coordinated cancer research studies involving multiple institutions, to provide the ability to manage and securely share information and analytic resources, and to spur a new generation of research applications that empower researchers to take a more integrative, trans-domain approach to data mining and analysis. AVAILABILITY: The caGrid version 0.5 release can be downloaded from https://cabig.nci.nih.gov/workspaces/Architecture/caGrid/. The operational test bed Grid can be accessed through the client included in the release, or through the caGrid-browser web application http://cagrid-browser.nci.nih.gov.     

VISDA: an open-source caBIG analytical tool for data clustering and beyond

SUMMARY: VISDA (Visual Statistical Data Analyzer) is a caBIG analytical tool for cluster modeling, visualization and discovery that has met silver-level compatibility under the caBIG initiative. Being statistically principled and visually interfaced, VISDA exploits both hierarchical statistics modeling and human gift for pattern recognition to allow a progressive yet interactive discovery of hidden clusters within high dimensional and complex biomedical datasets. The distinctive features of VISDA are particularly useful for users across the cancer research and broader research communities to analyze complex biological data. AVAILABILITY: http://gforge.nci.nih.gov/projects/visda/     

Crystal structure and conformation of 5-fluorouridine: conformational preferences for 5-fluorinated pyranosides

Crystals of 5-fluorouridine (5FUrd) have unit cell dimensions a = 7.716(1), b = 5.861(2), c = 13.041(1)A, alpha = gamma = 90 degrees, beta = 96.70 degrees (1), space group P2(1), Z = 2, rho obs = 1.56 gm/c.c and rho calc = 1574 gm/c.c The crystal structure was determined with diffractometric data and refined to a final reliability index of 0.042 for the observed 2205 reflections (I > or = 3sigma). The nucleoside has the anti conformation [chi = 53.1(4) degrees] with the furanose ring in the favorite C2'-endo conformation. The conformation across the sugar exocyclic bond is g+, with values of 49.1(4) and -69.3(4) degrees for phi(theta c) and phi (infinity) respectively. The pseudorotational amplitude tau(m) is 34.5 (2) with a phase angle of 171.6(4) degrees. The crystal structure is stabilized by a network of N-H...O and O-H...O involving the N3 of the uracil base and the sugar 03' and 02' as donors and the 02 and 04 of the uracil base and 03' oxygen as acceptors respectively. Fluorine is neither involved in the hydrogen bonding nor in the stacking interactions. Our studies of several 5-fluorinated nucleosides show the following preferred conformational features: 1) the most favored anti conformation for the nucleoside [chi varies from -20 to + 60 degrees] 2) an inverse correlation between the glycosyl bond distance and the chi angle 3) a wide variation of conformations of the sugar ranging froni C2'-endo through C3'-endo to C4'-exo 4) the preferred g+ across the exocyclic C4'-C5' bond and 5) no role for the fluorine atom in the hydrogen bonding or base stacking interactions.     

Computational studies of polynucleotide flexibility.

Details of polynucleotide flexibility may be probed through a combination of semiempirical potential energy calculations and statistical mechanical analyses. The pseudorotational motions of the furanose and the long-range correlated rotations of the chain backbone are described briefly here.     

Determination of urinary nucleosides by direct injection and coupled-column high-performance liquid chromatography

A coupled-column liquid chromatographic method for the direct analysis of 14 urinary nucleosides is described. Efficient on-line clean-up and concentration of 14 nucleosides from urine samples were obtained by using a boronic acid-substituted silica column (40 mm x 4.0 mm I.D.) as the first column (Col-1) and a Hypersil ODS2 column (250 mm x 4.6 mm I.D.) as the second column (Col-2). The mobile phases applied consisted of 0.25 mol/L ammonium acetate (pH 8.5) on Col-1, and of 25 mmol/L potassium dihydrogen phosphate (pH 4.5) on Col-2, respectively. Determination of urinary nucleosides was performed on Col-2 column by using a linear gradient elution comprising 25 mmol/L potassium dihydrogen phosphate (pH 4.5) and methanol-water (60:40, v/v) with UV detection at 260 nm. Urinary nucleosides analysis can be carried out by this procedure in 50 min requiring only pH adjustment and the protein precipitation by centrifugation of urine samples. Calibration plots of 14 standard nucleosides showed excellent linearity (r > 0.995) and the limits of detection were at micromolar levels. Both of intra- and inter-day precisions of the method were better than 6.6% for direct determination of 14 nucleosides. The validated method was applied to quantify 14 nucleosides in 20 normal urines to establish reference ranges.     

2'-O,4'-C-ethylene-bridged nucleic acids (ENA): highly nuclease-resistant and thermodynamically stable oligonucleotides for antisense drug.

To develop antisense oligonucleotides, novel nucleosides, 2'-O,4'-C-ethylene nucleosides and their corresponding phosphoramidites, were synthesized as building blocks. The 1H NMR analysis showed that the 2'-O,4'-C-ethylene linkage of these nucleosides restricts the sugar puckering to the N-conformation as well as the linkage of 2'-O,4'-C-methylene nucleosides which are known as bridged nucleic acids (BNA) or locked nucleic acids (LNA). The ethylene-bridged nucleic acids (ENA) showed a high binding affinity for the complementary RNA strand (DeltaT(m)=+5.2 degrees C/modification) and were more nuclease-resistant than natural DNA and BNA/LNA. These results indicate that ENA have better properties as antisense oligonucleotides than BNA/LNA.     

Thymidine uptake in bacteria: the effect of purine nucleosides.

The kinetics of thymidine uptake by Escherichia coli and Bacillus subtilis cells in the presence of adenine and guanine nucleosides was investigated. The initial concentration of thymidine in the growth medium was 0.35 microng/ml while the initial concentration of purine nucleosides ranged from 25 to 250 microng/ml. Adenine nucleosides when present at a concentration more than 50 microng/ml strongly inhibit thymidine uptake by the bacteria. The duration of the inhibition depends on the initial concentration of adenine nucleoside in the growth medium. At an initial concentration of deoxyadenosine (or adenosine) of 250 microng/ml the time of inhibition of thymidine uptake was about 60 min. During this period thymidine is almost completely preserved from the action of bacterial thymidine phosphorylase. Guanine nucleosides (guanosine or deoxyguanosine) do not markedly inhibit thymidine uptake by bacteria even at a concentration of 250 microng/ml. It is shown that they do protect thymidine from the phosphorolytic action of the thymidine phosphorylase although much less effectively than adenine nucleosides. It is suggested that some areas in the bacterial membrane where thymidine phosphorylase is located are not available to guanine nucleosides.     

Urinary Nucleosides as Biomarkers of Breast,Colon, Lung,and Gastric Cancer in Taiwanese

Urinary nucleosides are associated with many types of cancer. In this study, six targeted urinary nucleosides, namely adenosine, cytidine, 3-methylcytidine, 1-methyladenosine, inosine, and 2-deoxyguanosine, were chosen to evaluate their role as biomarkers of four different types of cancer: lung cancer, gastric cancer, colon cancer, and breast cancer. Urine samples were purified using solid-phase extraction (SPE) and then analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The Mann-Whitney U test and Principal Component Analysis (PCA) were used to compare differences in urinary nucleosides between patients with one of four types of cancer and healthy controls. The diagnostic sensitivity of single nucleosides for different types of cancer ranged from 14% to 69%. In contrast, the diagnostic sensitivity of a set of six nucleosides ranged from 37% to 69%. The false-positive identification rate associated with the set of six nucleosides in urine was less than 2% compared with that of less than 5% for a single nucleoside. Furthermore, combining the set of six urinary nucleosides with carcinoembryonic antigen improved the diagnostic sensitivity for colon cancer. In summary, the study show that a set of six targeted nucleosides is a good diagnostic marker for breast and colon cancers but not for lung and gastric cancers.     

Synthesis and properties of 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) as effective antisense oligonucleotides

Novel bicyclo nucleosides, 2'-O,4'-C-ethylene nucleosides and 2'-O,4'-C-propylene nucleosides, were synthesized as building blocks for antisense oligonucleotides to further optimize the 2'-O,4'-C-methylene-linkage of bridged nucleic acids (2',4'-BNA) or locked nucleic acids (LNA). Both the 2'-O,4'-C-ethylene- and propylene-linkage within these nucleosides restrict the sugar puckering to the N-conformation of RNA as do 2',4'-BNA/LNA. Furthermore, ethylene-bridged nucleic acids (ENA) having 2'-O,4'-C-ethylene nucleosides had considerably increased the affinity to complementary RNA, and were as high as that of 2',4'-BNA/LNA (DeltaT(m)=+3 approximately 5 degrees C per modification). On the other hand, addition of 2'-O,4'-C-propylene modifications in oligonucleotides led to a decrease in the affinity to complementary RNA. As for the stability against nucleases, incorporation of one 2'-O,4'-C-ethylene or one 2'-O,4'-C-propylene nucleoside into oligonucleotides considerably increased their resistance against exonucleases to an extent greater than 2',4'-BNA/LNA. These results indicate that ENA is more suitable as an antisense oligonucleotide and is expected to have better antisense activity than 2',4'-BNA/LNA.     

灵芝孢子粉中核苷类成分分析

本文利用高效液相色谱方法（HPLC）同时对灵芝孢子粉中的15种核苷类成分的含量进行测定。采用Ultimate AQ-C₁₈（4.6mm×250mm,5μm）色谱柱,以甲醇和水为流动相进行梯度洗脱,流速1.0mL/min,检测波长259nm,柱温30℃,进样量10μL。方法学考察结果表明,该方法准确度高,稳定性、精密度、重现性好,适用于灵芝孢子粉中核苷类成分的测定分析。运用建立的方法对不同破壁时间、不同采收时期龙泉、奉化、大别山、黄山4个产区的灵芝孢子粉中的15种核苷类成分的含量进行测定。结果表明破壁处理对灵芝孢子粉中核苷类成分提取率的影响不大,不同产地的灵芝孢子粉中核苷类成分的组成和含量具有显著差异,且孢子粉中的核苷含量随着产粉时间的延长有所增加。各待测样品中均含有胞嘧啶、尿苷、腺嘌呤、鸟苷、腺苷等成分,其中尿苷、鸟苷、腺苷3种核苷的含量占总量的比例在待测样品中均达到70%以上,为灵芝孢子粉中的主要核苷类成分。     

Freezing tolerance of chickpea: biochemical and molecular changes at vegetative stage

The aim of this study was to find a correlation between the freezing tolerance of three chickpea (Cicer arietinum L.) cultivars (?nci, I??k-05, and Sar?-98) and their wild relative C. echinospermum and physiological responses. Chickpea plants (15-d-old) were subjected to cold acclimation (CA) (10 °C for 7 d), freezing (-3 or -5 °C for 2 h), and subsequent rewarming (25 °C for 7 d). In two separate experiments with three replications, we determined growth, water status, photosystem 2 photochemical activity, photosynthetic pigments, H₂O₂, malondialdehyde, and proline content, relative leakage ratio, antioxidant enzyme activities, and gene expressions in cultivars different in freezing tolerance. Freezing temperatures adversely affected all the physiological parameters of all cultivars. Rewarming did not lead to complete recovery. The cultivar ?nci was more tolerant to the freezing temperatures than others.