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Find video protocols related to scientific articles indexed in Pubmed.
Proteomic profiling of Botrytis cinerea conidial germination.
Arch. Microbiol.
PUBLISHED: 08-21-2014
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Botrytis cinerea is one of the most relevant plant pathogenic fungi. The first step during its infection process is the germination of the conidia. Here, we report on the first proteome analysis during the germination of B. cinerea conidia, where 204 spots showed significant differences in their accumulation between ungerminated and germinated conidia by two-dimensional polyacrylamide gel electrophoresis and qPCR. The identified proteins were grouped by gene ontology revealing that the infective tools are mainly preformed inside the ungerminated conidia allowing a quick fungal development at the early stages of conidial germination. From 118 identified spots, several virulence factors have been identified while proteins, such as mannitol-1-phosphate dehydrogenase, 6,7-dimethyl-8-ribityllumazine synthase or uracil phosphoribosyltransferase, have been disclosed as a new potential virulence factors in botrytis whose role in pathogenicity needs to be studied to gain new insights about the role of these proteins as therapeutic targets and virulence factors.
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Cytotoxic constituents from the Vietnamese fungus Xylaria schweinitzii.
Nat Prod Commun
PUBLISHED: 07-17-2014
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Two new fungal pigments named schweinitzins A and B (1-2), together with (S)-torosachrysone-8-O-methyl ether (3) and emodin-6,8-di-O-methyl ether (4) have been isolated from the methanolic extract of the fruit bodies of Xylaria schweinitzii (Xylariaceae) collected in Cuc Phuong national park, Ninh Binh province, Vietnam, by silica gel column chromatography and preparative HPLC. Their structures were elucidated by spectroscopic analysis such as IR, UV-Vis, 2D NMR and FT-ICR-MS. In addition, two compounds (1 and 3) showed strong cytotoxicity against all four cancer cell lines, KB (a human epidermal carcinoma), MCF7 (human breast carcinoma), SK-LU-I (human lung carcinoma) and HepG2 (hepatocellular carcinoma).
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Penarines A-F, (nor-)sesquiterpene carboxylic acids from Hygrophorus penarius (Basidiomycetes).
Phytochemistry
PUBLISHED: 07-07-2014
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Five sesquiterpene carboxylic acids (1-5) and one nor-sesquiterpene carboxylic acid (6) of the very rare ventricosane type, named penarines A-F, were isolated from fruiting bodies of the basidiomycete Hygrophorus penarius (Hygrophoraceae). This is the first report of (nor)-sesquiterpenes isolated from basidiocarps of the family Hygrophoraceae. Their structures were elucidated on the basis of extensive 1D ((1)H, (13)C) and 2D (HSQC, HMBC, COSY, ROESY) NMR spectroscopic analyses as well as high-resolution mass spectrometry studies. Additionally, the only known member of this rare type of sesquiterpenes, ventricos-7(13)-ene (7), could be identified via headspace GC-MS analysis in a fruiting body of H. penarius. Compounds 1-6 were devoid of remarkable antifungal activity against Cladosporium cucumerinum. Additionally, the cytotoxic activities of compounds 1 and 2 were evaluated against the human prostate cancer cell line PC-3 and the colon cancer cell line HT-29 showing no significant cytotoxic activity.
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Explaining the success of Kogelnik's coupled-wave theory by means of perturbation analysis: discussion.
J Opt Soc Am A Opt Image Sci Vis
PUBLISHED: 07-01-2014
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The problem of diffraction of an electromagnetic wave by a thick hologram grating can be solved by the famous Kogelnik's coupled-wave theory (CWT) to a very high degree of accuracy. We confirm this finding by comparing the CWT and the exact result for a typical example and propose an explanation in terms of perturbation theory. To this end we formulate the problem of diffraction as a matrix problem following similar well-known approaches, especially rigorous coupled-wave theory (RCWT). We allow for a complex permittivity modulation and a possible phase shift between refractive index and absorption grating and explicitly incorporate appropriate boundary conditions. The problem is solved numerically exact for the specific case of a planar unslanted grating and a set of realistic values of the material's parameters and experimental conditions. Analogously, the same problem is solved for a two-dimensional truncation of the underlying matrix that would correspond to a CWT approximation but without the usual further approximations. We verify a close coincidence of both results even in the off-Bragg region and explain this result by means of a perturbation analysis of the underlying matrix problem. Moreover, the CWT is found not only to coincide with the perturbational approximation in the in-Bragg and the extreme off-Bragg cases, but also to interpolate between these extremal regimes.
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Metabolite profiling and fingerprinting of Suillus species (Basidiomycetes) by electrospray mass spectrometry.
Eur J Mass Spectrom (Chichester, Eng)
PUBLISHED: 06-03-2014
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The genus Suillus is known for the occurrence of a series of prenylated phenols and boviquinones. The extracts of four different Suillus species [S. bovinus, S. granulatus, S. tridentinus and S.variegatus) were investigated by using rapid ultra-performance Liquid chromatography/electrospray ionization mass spectrometry (UPLC/ESI-MS) and direct infusion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS). While direct infusion ESI-FT-ICR mass spectra give a fast overview concerning the elemental compositions of the compounds and, therefore, hints to the main metabolites, UPLC/ESI-tandem mass spectrometry is shown to be a useful tool for their identification. A principal component analysis (PCA) and hierarchical cluster analysis (HCA) based on the UPLC/ESI-MS clearly showed that the metabolite profiles can be used not only for the identification and classification of such fungi but also as a sophisticated and powerful tool for the chemotaxonomy of fungi. Furthermore, a clear discrimination of various types of biological samples (fruiting bodies versus mycelial cultures) is also possible. The orthogonal partial least squares (OPLS) two-class models of both UPLC/ESI-MS and ESI-FT-ICR-MS possess a clear differentiation of two compared Suillus species representing the between class variation and the within class variation. Based on generated S-plots and Loading plots, statistically significant metabolites could be identified as potential biomarker for one species.
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Global, regional, and national levels of neonatal, infant, and under-5 mortality during 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013.
Haidong Wang, Chelsea A Liddell, Matthew M Coates, Meghan D Mooney, Carly E Levitz, Austin E Schumacher, Henry Apfel, Marissa Iannarone, Bryan Phillips, Katherine T Lofgren, Logan Sandar, Rob E Dorrington, Ivo Rakovac, Troy A Jacobs, Xiaofeng Liang, Maigeng Zhou, Jun Zhu, Gonghuan Yang, Yanping Wang, Shiwei Liu, Yichong Li, Ayse Abbasoglu Ozgoren, Semaw Ferede Abera, Ibrahim Abubakar, Tom Achoki, Ademola Adelekan, Zanfina Ademi, Zewdie Aderaw Alemu, Peter J Allen, Mohammad AbdulAziz AlMazroa, Elena Alvarez, Adansi A Amankwaa, Azmeraw T Amare, Walid Ammar, Palwasha Anwari, Solveig Argeseanu Cunningham, Majed Masoud Asad, Reza Assadi, Amitava Banerjee, Sanjay Basu, Neeraj Bedi, Tolesa Bekele, Michelle L Bell, Zulfiqar Bhutta, Jed D Blore, Berrak Bora Basara, Soufiane Boufous, Nicholas Breitborde, Nigel G Bruce, Linh Ngoc Bui, Jonathan R Carapetis, Rosario Cárdenas, David O Carpenter, Valeria Caso, Ruben Estanislao Castro, Ferrán Catalá-López, Alanur Cavlin, Xuan Che, Peggy Pei-Chia Chiang, Rajiv Chowdhury, Costas A Christophi, Ting-Wu Chuang, Massimo Cirillo, Iuri da Costa Leite, Karen J Courville, Lalit Dandona, Rakhi Dandona, Adrian Davis, Anand Dayama, Kebede Deribe, Samath D Dharmaratne, Mukesh K Dherani, Ugur Dilmen, Eric L Ding, Karen M Edmond, Sergei Petrovich Ermakov, Farshad Farzadfar, Seyed-Mohammad Fereshtehnejad, Daniel Obadare Fijabi, Nataliya Foigt, Mohammad H Forouzanfar, Ana C Garcia, Johanna M Geleijnse, Bradford D Gessner, Ketevan Goginashvili, Philimon Gona, Atsushi Goto, Hebe N Gouda, Mark A Green, Karen Fern Greenwell, Harish Chander Gugnani, Rahul Gupta, Randah Ribhi Hamadeh, Mouhanad Hammami, Hilda L Harb, Simon Hay, Mohammad T Hedayati, H Dean Hosgood, Damian G Hoy, Bulat T Idrisov, Farhad Islami, Samaya Ismayilova, Vivekanand Jha, Guohong Jiang, Jost B Jonas, Knud Juel, Edmond Kato Kabagambe, Dhruv S Kazi, André Pascal Kengne, Maia Kereselidze, Yousef Saleh Khader, Shams Eldin Ali Hassan Khalifa, Young-Ho Khang, Daniel Kim, Yohannes Kinfu, Jonas M Kinge, Yoshihiro Kokubo, Soewarta Kosen, Barthélemy Kuate Defo, G Anil Kumar, Kaushalendra Kumar, Ravi B Kumar, Taavi Lai, Qing Lan, Anders Larsson, Jong-Tae Lee, Mall Leinsalu, Stephen S Lim, Steven E Lipshultz, Giancarlo Logroscino, Paulo A Lotufo, Raimundas Lunevicius, Ronan Anthony Lyons, Stefan Ma, Abbas Ali Mahdi, Melvin Barrientos Marzan, Mohammad Taufiq Mashal, Tasara T Mazorodze, John J McGrath, Ziad A Memish, Walter Mendoza, George A Mensah, Atte Meretoja, Ted R Miller, Edward J Mills, Karzan Abdulmuhsin Mohammad, Ali H Mokdad, Lorenzo Monasta, Marcella Montico, Ami R Moore, Joanna Moschandreas, William T Msemburi, Ulrich O Mueller, Magdalena M Muszyńska, Mohsen Naghavi, Kovin S Naidoo, K M Venkat Narayan, Chakib Nejjari, Marie Ng, Jean de Dieu Ngirabega, Mark J Nieuwenhuijsen, Luke Nyakarahuka, Takayoshi Ohkubo, Saad B Omer, Angel J Paternina Caicedo, Victoria Pillay-van Wyk, Dan Pope, Farshad Pourmalek, Dorairaj Prabhakaran, Sajjad U R Rahman, Saleem M Rana, Robert Quentin Reilly, David Rojas-Rueda, Luca Ronfani, Lesley Rushton, Mohammad Yahya Saeedi, Joshua A Salomon, Uchechukwu Sampson, Itamar S Santos, Monika Sawhney, Jürgen C Schmidt, Marina Shakh-Nazarova, Jun She, Sara Sheikhbahaei, Kenji Shibuya, Hwashin Hyun Shin, Kawkab Shishani, Ivy Shiue, Inga Dora Sigfusdottir, Jasvinder A Singh, Vegard Skirbekk, Karen Sliwa, Sergey S Soshnikov, Luciano A Sposato, Vasiliki Kalliopi Stathopoulou, Konstantinos Stroumpoulis, Karen M Tabb, Roberto Tchio Talongwa, Carolina Maria Teixeira, Abdullah Sulieman Terkawi, Alan J Thomson, Andrew L Thorne-Lyman, Hideaki Toyoshima, Zacharie Tsala Dimbuene, Parfait Uwaliraye, Selen Begüm Uzun, Tommi J Vasankari, Ana Maria Nogales Vasconcelos, Vasiliy Victorovich Vlassov, Stein Emil Vollset, Stephen Waller, Xia Wan, Scott Weichenthal, Elisabete Weiderpass, Robert G Weintraub, Ronny Westerman, James D Wilkinson, Hywel C Williams, Yang C Yang, Gokalp Kadri Yentur, Paul Yip, Naohiro Yonemoto, Mustafa Younis, Chuanhua Yu, Kim Yun Jin, Maysaa El Sayed Zaki, Shankuan Zhu, Theo Vos, Alan D Lopez, Christopher J L Murray.
Lancet
PUBLISHED: 05-02-2014
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Remarkable financial and political efforts have been focused on the reduction of child mortality during the past few decades. Timely measurements of levels and trends in under-5 mortality are important to assess progress towards the Millennium Development Goal 4 (MDG 4) target of reduction of child mortality by two thirds from 1990 to 2015, and to identify models of success.
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Rare biscoumarin derivatives and flavonoids from Hypericum riparium.
Phytochemistry
PUBLISHED: 03-05-2014
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Hypericum riparium A. Chev. is a Cameroonian medicinal plant belonging to the family Guttiferae. Chemical investigation of the methanol extract of the stem bark of H. riparium led to the isolation of four natural products, 7,7'-dihydroxy-6,6'-biscoumarin (1), 7,7'-dihydroxy-8,8'-biscoumarin (2), 7-methoxy-6,7'-dicoumarinyl ether (3), 2'-hydroxy-5'-(7?-methoxycoumarin-6?-yl)-4'-methoxyphenylpropanoic acid (4), together with one known 7,7'-dimethoxy-6,6'-biscoumarin (5), two flavones, 2'-methoxyflavone (6) and 3'-methoxy flavone (7), and two steroids, stigmast-4-en-3-one (8) and ergosta-4,6,8,22-tetraen-3-one (9). In addition, tetradecanoic acid (10), n-pentadecanoic acid (11), hexadecanoic acid (12), cis-10-heptadecenoic acid (13), octadecanoic acid (14) campesterol (15), stigmasterol (16), ?-sitosterol (17), stigmastanol (18), ?-eudesmol (19), 1-hexadecanol (20), and 1-octadecanol (21) were identified by GC-MS analysis. Compound 4 consists of a phenylpropanoic acid derivative fused with a coumarin unit, while compounds 2 and 3 are rare members of C8-C8' and C7-O-C6 linked biscoumarins. Their structures were elucidated by UV, IR, extensive 1D- and 2D-NMR experiments and electrospray (ESI) high resolution mass spectrometry (MS) including detailed MS/MS studies. This is the first report on the isolation of biscoumarins from the genus Hypericum, although simple coumarin derivatives have been reported from this genus in the literature. The cytotoxic activities of compounds 2-5 were evaluated against the human prostate cancer cell line PC-3 and the colon cancer cell line HT-29. They do not exhibit any significant cytotoxic activity.
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Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases.
Nat Commun
PUBLISHED: 01-14-2014
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Generation of mouse models by introducing transgenes using homologous recombination is critical for understanding fundamental biology and pathology of human diseases. Here we investigate whether artificial transcription activator-like effector nucleases (TALENs)-powerful tools that induce DNA double-strand breaks at specific genomic locations-can be combined with a targeting vector to induce homologous recombination for the introduction of a transgene in embryonic stem cells and fertilized murine oocytes. We describe the generation of a conditional mouse model using TALENs, which introduce double-strand breaks at the genomic locus of the special AT-rich sequence-binding protein-1 in combination with a large 14.4?kb targeting template vector. We report successful germline transmission of this allele and demonstrate its recombination in primary cells in the presence of Cre-recombinase. These results suggest that TALEN-assisted induction of DNA double-strand breaks can facilitate homologous recombination of complex targeting constructs directly in oocytes.
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Aperiodic CrSc multilayer mirrors for attosecond water window pulses.
Opt Express
PUBLISHED: 10-10-2013
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Extending single attosecond pulse technology from currently sub-200 eV to the so called water window spectral range may enable for the first time the unique investigation of ultrafast electronic processes within the core states of bio-molecules as proteins or other organic materials. Aperiodic multilayer mirrors serve as key components to shape these attosecond pulses with a high degree of freedom and enable tailored short pulse pump-probe experiments. Here, we report on chirped CrSc multilayer mirrors, fabricated by ion beam deposition with sub-angstrom precision, designed for attosecond pulse shaping in the water window spectral range.
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Evaluation of biological and chemical insect repellents and their potential adverse effects.
Parasitol. Res.
PUBLISHED: 09-16-2013
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Plant extracts, particularly plant oils, had been used and were still in use as repellents against mosquitoes. Some of them (e.g., lavender, geraniol, and citriodiol) have been notified by the European Commission as active substances to be used in repellents, which are categorized as biocides in product type 19. In the literature, it is known that these substances must be added to repellent products in high concentrations (e.g., 20 % and more) in order to reach repellent efficacy. Therefore, the question arose whether they also have repellent effects if they were added as fragrances at low concentrations of 0.25 or 1 % to registered active substances in order to obtain a better scent of this product. In the present study, the repellent effects of 0.25 and 1 % additions of 15 plant extracts (citronellol, cinerol, citral, menthol, linalyl acetate, Eucalyptus citriodora, Eucalyptus globulus, Cymbopogon nardus, lilac, sandalwood, Vitex agnus castus, rosewood, lavender, geraniol, and paramenthan diol) when exposed on skin to hungry Aedes aegypti mosquitoes. These experiments showed that there was no repellent effect in any of these compounds even when the test was done already 10 min after distributing any of the compounds onto the hands of volunteers. These experiments have proven that these 15 compounds do not produce repellent effects as long as they are used in low concentrations of 0.25 or 1 % as fragrances to ameliorate the odor of a notified repellent that is brought onto the skin.
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Evaluation of ethnomedical claim III: anti-hyperglycemic activities of Gongronema latifolium root and stem.
J Diabetes
PUBLISHED: 05-28-2013
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The insulinotropic activity of the combined root and stem of Gongronema latifolium (Asclepiadaceae) was evaluated to justify its African ethnomedicinal use in the management of diabetes.
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Observations of ejecta clouds produced by impacts onto Saturns rings.
Science
PUBLISHED: 04-27-2013
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We report observations of dusty clouds in Saturns rings, which we interpret as resulting from impacts onto the rings that occurred between 1 and 50 hours before the clouds were observed. The largest of these clouds was observed twice; its brightness and cant angle evolved in a manner consistent with this hypothesis. Several arguments suggest that these clouds cannot be due to the primary impact of one solid meteoroid onto the rings, but rather are due to the impact of a compact stream of Saturn-orbiting material derived from previous breakup of a meteoroid. The responsible interplanetary meteoroids were initially between 1 centimeter and several meters in size, and their influx rate is consistent with the sparse prior knowledge of smaller meteoroids in the outer solar system.
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Novel candidate genes influencing natural variation in potato tuber cold sweetening identified by comparative proteomics and association mapping.
BMC Plant Biol.
PUBLISHED: 03-22-2013
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Higher plants evolved various strategies to adapt to chilling conditions. Among other transcriptional and metabolic responses to cold temperatures plants accumulate a range of solutes including sugars. The accumulation of the reducing sugars glucose and fructose in mature potato tubers during exposure to cold temperatures is referred to as cold induced sweetening (CIS). The molecular basis of CIS in potato tubers is of interest not only in basic research on plant adaptation to environmental stress but also in applied research, since high amounts of reducing sugars affect negatively the quality of processed food products such as potato chips. CIS-tolerance varies considerably among potato cultivars. Our objective was to identify by an unbiased approach genes and cellular processes influencing natural variation of tuber sugar content before and during cold storage in potato cultivars used in breeding programs. We compared by two-dimensional polyacrylamide gel electrophoresis the tuber proteomes of cultivars highly diverse for CIS. DNA polymorphisms in genomic sequences encoding differentially expressed proteins were tested for association with tuber starch content, starch yield and processing quality.
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Dynamics of amino acid utilization in Phaeobacter inhibens DSM 17395.
Proteomics
PUBLISHED: 02-06-2013
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Time-resolved utilization of multiple amino acids by Phaeobacter inhibens DSM 17395 was studied during growth with casamino acids. The 15 detected amino acids could be grouped according to depletion rate into four different categories, i.e. from rapid (category I) to nondepletion (category IV). Upon entry into stationary growth phase, amino acids of category I (e.g. glutamate) were (almost) completely depleted, while those of categories II (e.g. leucine) and III (e.g. serine) were further consumed at varying rates and to different extents. Thus, cultures entered stationary growth phase despite the ample presence of organic nutrients, i.e. under nonlimiting conditions. Integrated proteomic and metabolomic analysis identified 1747 proteins and 94 intracellular metabolites. Of these, 180 proteins and 86 metabolites displayed altered abundance levels during growth. Most strikingly, abundance and activity profiles of alanine dehydrogenase concomitantly increased with the onset of enhanced alanine utilization during transition into stationary growth phase. Most enzymes of amino acid and central metabolism, however, displayed unaltered abundances across exponential and stationary growth phases. In contrast, metabolites of the Entner-Doudoroff pathway and gluconeogenesis as well as cellular fatty acids increased markedly in abundance in early stationary growth phase.
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Adaptation of Phaeobacter inhibens DSM 17395 to growth with complex nutrients.
Proteomics
PUBLISHED: 02-06-2013
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Phaeobacter inhibens DSM 17395, a member of the Roseobacter clade, was studied for its adaptive strategies to complex and excess nutrient supply, here mimicked by cultivation with Marine Broth (MB). During growth in process-controlled fermenters, P. inhibens DSM 17395 grew faster (3.6-fold higher ?max ) and reached higher optical densities (2.2-fold) with MB medium, as compared to the reference condition of glucose-containing mineral medium. Apparently, in the presence of MB medium, metabolism was tuned to maximize growth rate at the expense of efficiency. Comprehensive proteomic analysis of cells harvested at ½ ODmax identified 1783 (2D DIGE, membrane and extracellular protein-enriched fractions, shotgun) different proteins (50.5% coverage), 315 (based on 2D DIGE) of which displayed differential abundance profiles. Moreover, 145 different metabolites (intra- and extracellular combined) were identified, almost all of which (140) showed abundance changes. During growth with MB medium, P. inhibens DSM 17395 specifically formed the various proteins required for utilization of phospholipids and several amino acids, as well as for gluconeogenesis. Metabolic tuning on amino acid utilization is also reflected by massive discharge of urea to dispose the cell of excess ammonia. Apparently, P. inhibens DSM 17395 modulated its metabolism to simultaneously utilize diverse substrates from the complex nutrient supply.
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Large-scale proteomic analysis of the grapevine leaf apoplastic fluid reveals mainly stress-related proteins and cell wall modifying enzymes.
BMC Plant Biol.
PUBLISHED: 01-31-2013
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The extracellular space or apoplast forms a path through the whole plant and acts as an interface with the environment. The apoplast is composed of plant cell wall and space within which apoplastic fluid provides a means of delivering molecules and facilitates intercellular communications. However, the apoplastic fluid extraction from in planta systems remains challenging and this is particularly true for grapevine (Vitis vinifera L.), a worldwide-cultivated fruit plant. Large-scale proteomic analysis reveals the protein content of the grapevine leaf apoplastic fluid and the free interactive proteome map considerably facilitates the study of the grapevine proteome.
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Proteomic analysis of conidia germination in Colletotrichum acutatum.
Arch. Microbiol.
PUBLISHED: 01-14-2013
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Colletotrichum acutatum is an important phytopathogenic fungus causing anthracnose in commercially important fruit crops, such as strawberry. The conidia produced by the fungus are survival structures which play a key role in host infection and fungal propagation. Despite its relevance to the fungal life cycle, conidial biology has not been extensively investigated. Here, we provide the first proteomic description of the conidial germination in C. acutatum by comparing the proteomic profiles of ungerminated and germinated conidia. Using two-dimensional electrophoresis combined with MALDI-TOF/TOF mass spectrometry, we have identified 365 proteins in 354 spots, which represent 245 unique proteins, including some proteins with key functions in pathogenesis. All these proteins have been classified according to their molecular function and their involvement in biological processes, including cellular energy production, oxidative metabolism, stress, fatty acid synthesis, protein synthesis, and folding. This report constitutes the first comprehensive study of protein expression during the early stage of the C. acutatum conidial germination. It advances our understanding of the molecular mechanisms involved in the conidial germination process, and provides a useful basis for the further characterization of proteins involved in fungal biology and fungus life cycles.
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Alkaloids from Papaver coreanum.
Nat Prod Commun
PUBLISHED: 12-23-2011
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The alkaloid pattern of the endemic plant Papaver coreanum Nakai (Papaveraceae) was determined for the first time. Eight alkaloids could be identified by LC/ESI-MS/MS and high-resolution mass spectrometry. Among them, protopine and allocryptopine represent the main components. Besides norsanguinarine, sanguinarine, dihydrosanguinarine, oxysanguinarine, lincangenine, and cryptopine, some other trace alkaloids were found whose structures remain unknown.
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Analysis of natural variation of the potato tuber proteome reveals novel candidate genes for tuber bruising.
J. Proteome Res.
PUBLISHED: 11-22-2011
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Potato (Solanum tuberosum) presents a challenging organism for the genetic and molecular dissection of complex traits due to its tetraploidy and high heterozygosity. One such complex trait of high agronomic interest is the tuber susceptibility to bruising upon mechanical impact, which involves an enzymatic browning reaction. We have compared the tuber proteome of two groups of 10 potato cultivars differing in bruising susceptibility to (i) identify de novo proteins that contribute to bruising, based on differential protein expression, and (ii) validate these proteins by combining proteomics with association genetics. The comparison of 20 potato varieties yields insight into the high natural variation of tuber protein patterns due to genetic background. Seven genes or gene families were found that were both differentially expressed on the protein level between groups and for which DNA polymorphisms were associated with the investigated traits. A putative class III lipase was identified as a novel factor contributing to the natural variation of bruising. Additionally, tuber proteome changes triggered by mechanical impact, within and between groups, were monitored over time. Differentially expressed proteins were found, notably lipases, patatins, and annexins, showing remarkable time-dependent protein variation.
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Mining the Active Proteome of Arabidopsis thaliana.
Front Plant Sci
PUBLISHED: 09-12-2011
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Assigning functions to the >30,000 proteins encoded by the Arabidopsis genome is a challenging task of the Arabidopsis Functional Genomics Network. Although genome-wide technologies like proteomics and transcriptomics have generated a wealth of information that significantly accelerated gene annotation, protein activities are poorly predicted by transcript or protein levels as protein activities are post-translationally regulated. To directly display protein activities in Arabidopsis proteomes, we developed and applied activity-based protein profiling (ABPP). ABPP is based on the use of small molecule probes that react with the catalytic residues of distinct protein classes in an activity-dependent manner. Labeled proteins are separated and detected from proteins gels and purified and identified by mass spectrometry. Using probes of six different chemotypes we have displayed activities of 76 Arabidopsis proteins. These proteins represent over 10 different protein classes that contain over 250 Arabidopsis proteins, including cysteine, serine, and metalloproteases, lipases, acyltransferases, and the proteasome. We have developed methods for identification of in vivo labeled proteins using click chemistry and for in vivo imaging with fluorescent probes. In vivo labeling has revealed additional protein activities and unexpected subcellular activities of the proteasome. Labeling of extracts displayed several differential activities, e.g., of the proteasome during immune response and methylesterases during infection. These studies illustrate the power of ABPP to display the functional proteome and testify to a successful interdisciplinary collaboration involving chemical biology, organic chemistry, and proteomics.
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Modulated spin waves and robust quasi-solitons in classical Heisenberg rings.
J Phys Condens Matter
PUBLISHED: 09-08-2011
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We investigate the dynamical behavior of finite rings of classical spin vectors interacting via nearest-neighbor isotropic exchange in an external magnetic field. Our approach is to utilize the solutions of a continuum version of the discrete spin equations of motion (EOM) which we derive by assuming continuous modulations of spin wave solutions of the EOM for discrete spins. This continuum EOM reduces to the Landau-Lifshitz equation in a particular limiting regime. The usefulness of the continuum EOM is demonstrated by the fact that the time-evolved numerical solutions of the discrete spin EOM closely track the corresponding time-evolved solutions of the continuum equation. It is of special interest that our continuum EOM possesses soliton solutions, and we find that these characteristics are also exhibited by the corresponding solutions of the discrete EOM. The robustness of solitons is demonstrated by considering cases where initial states are truncated versions of soliton states and by numerical simulations of the discrete EOM equations when the spins are coupled to a heat bath at finite temperatures.
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Modified metal-oxide affinity enrichment combined with 2D-PAGE and analysis of phosphoproteomes.
Methods Mol. Biol.
PUBLISHED: 08-13-2011
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Protein phosphorylation is a dynamic process of widespread regulatory significance. Phosphoproteomics attempts to provide a global view of this process during biological processes, but the approach is generally limited by the low relative amounts of phosphoproteins in biological samples. Although mass spectrometry (MS)-based technologies exist for the in-depth characterization of protein phosphorylation, these techniques are typically highly focused, have low throughput, and generally require special equipment and expertise. These specialized techniques are best used to support hypotheses generated by an initial broad-based survey, like the one described here. In this chapter, we outline a 2D gel-based phosphoproteomic methodology based on relatively inexpensive materials and basic, widely available MS technology. The goal is to provide a preparative and analytical laboratory framework that can generate the samples and hypotheses for phosphoproteomic MS, as well as a set of tools for biologically relevant phosphoproteomics for investigators who do not have ready access to phospo-MS technology. The combination of 2D gel-compatible metal-oxide affinity chromatography (MOAC)-based phosphoprotein enrichment and phospho-specific staining provides both the sensitivity necessary to make low-level phosphoproteins observable and identifiable, and the twofold phospho-selectivity to support their identity as phosphoproteins. An on-blot dephosphorylation assay for verifying the phospho-specificity of the enrichment method is also described here and provides a general tool for the validation of protein phosphorylation.
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Restenosis after microsurgical non-patch carotid endarterectomy in 586 patients.
Acta Neurochir (Wien)
PUBLISHED: 08-08-2011
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Carotid endarterectomy (CEA) reduces the risk of stroke in patients with symptomatic (>50%) and asymptomatic (>60%) carotid artery stenosis. Here we report the midterm results of a microsurgical non-patch technique and compare these findings to those in the literature.
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Assessment of tinnitus-related impairments and disabilities using the German THI-12: sensitivity and stability of the scale over time.
Int J Audiol
PUBLISHED: 07-15-2011
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To evaluate the reliability, dimensionality, predictive validity, construct validity, and sensitivity to change of the THI-12 total and sub-scales as diagnostic aids to describe and quantify tinnitus-evoked reactions and evaluate treatment efficacy.
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One-bond and two-bond J couplings help annotate protein secondary-structure motifs: J-coupling indexing applied to human endoplasmic reticulum protein ERp18.
Proteins
PUBLISHED: 05-28-2011
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NMR coupling constants, both direct one-bond ((1)J) and geminal two-bond ((2)J), are employed to analyze the protein secondary structure of human oxidized ERp18. Coupling constants collected and evaluated for the 18 kDa protein comprise 1268 values of (1)J(C?H?), (1)J(C?C?), (1)J(C?C), (1)J(CN), (1)J(NC?), (1)J(N) (HN), (2)J(C?N), (2)J(HNC?), (2)J(CHN), and (2)J(H?C). Comparison with (1)J and (2)J data from reference proteins and pattern analysis on a per-residue basis permitted main-chain ?,? torsion-angle combinations of many of the 149 amino-acid residues in ERp18 to be narrowed to particular secondary-structure motifs. J-coupling indexing is here being developed on statistical criteria and used to devise a ternary grid for interpreting patterns of relative values of J. To account for the influence of the varying substituent pattern in different amino-acid sidechains, a table of residue-type specific threshold values was compiled for discriminating small, medium, and large categories of J. For the 15-residue insertion that distinguishes the ERp18 fold from that of thioredoxin, the J-coupling data hint at a succession of five isolated Type-I ? turns at progressively shorter sequence intervals, in agreement with the crystal structure.
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The effect of plasma chemical oxidation of titanium alloy on bone-implant contact in rats.
Biomaterials
PUBLISHED: 05-23-2011
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Many different technologies have been used to enhance osseointegration in orthopaedic and dental implant surgery. Hydroxyapatite coatings, pure or in combination with growth factors or bisphosphonates, showed improved osseointegration of titanium alloy implants. We choose a different approach to enhance osseointegration: plasma chemical oxidation was used to modify the surface of titanium alloy implants. This technique converts the nm-thin natural occurring titanium oxide layer on an implant to a 4 ?m thick ceramic coating (TiOB surface). Bioactive TiOB surfaces have a macroporous structure and were loaded with calcium and phosphorus, while bioinert TiOB surfaces are smooth. A rat tibial model with bilateral placement of titanium alloy implants was employed to analyze the bone response to TiOB surfaces in vivo. 64 rats were randomly assigned to four groups of implants: (1) titanium alloy (control), (2) titanium alloy, type III anodization, (3) bioinert TiOB surface and (4) bioactive TiOB surface. Mechanical fixation, peri-implant-bone area and bone contact were evaluated by pull-out tests and histology at three and eight weeks. Shear strength and bone contact at eight weeks were significantly increased in the bioactive TiOB group compared to all other groups. The results of plasma chemical oxidation in a rat model showed that the bioactive TiOB surface has a positive effect on implant anchorage by enhancing the bone-implant contact in normal bone.
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Understanding the interaction of Lipoarabinomannan with membrane mimetic architectures.
Tuberculosis (Edinb)
PUBLISHED: 05-10-2011
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Lipoarabinomannan (LAM) is a critical virulence factor in the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. LAM is secreted in urine and serum from infected patients and is being studied as a potential diagnostic indicator for the disease. Herein, we present a novel ultra-sensitive and specific detection strategy for monomeric LAM based on its amphiphilic nature and consequent interaction with supported lipid bilayers. Our strategy involves the capture of LAM on waveguides functionalized with membrane mimetic architectures, followed by detection with a fluorescently labeled polyclonal antibody. This approach offers ultra-sensitive detection of lipoarabinomannan (10 fM, within 15 min) and may be extended to other amphiphilic markers. We also show that chemical deacylation of LAM completely abrogates its association with the supported lipid bilayers. The loss of signal using the waveguide assay for deacylated LAM, as well as atomic force microscopy (AFM) images that show no change in height upon addition of deacylated LAM support this hypothesis. Mass spectrometry of chemically deacylated LAM indicates the presence of LAM-specific carbohydrate chains, which maintain antigenicity in immunoassays. Further, we have developed the first three-dimensional structural model of mannose-capped LAM that provides insights into the orientation of LAM on supported lipid bilayers.
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Ovicidal effects of a neem seed extract preparation on eggs of body and head lice.
Parasitol. Res.
PUBLISHED: 03-24-2011
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The eggs (nits) of head and body lice (Pediculus humanus capitis, Pediculus humanus corporis) were incubated for 5, 10, 15, 20, 30 or 45 min into a neem seed extract contained in a fine shampoo formulation (e.g. Wash Away® Louse), which is known for its significant killing effects of larvae and adults of head lice. The aim of the study was to test whether the developmental stages inside the eggs are also killed after the incubation into the shampoo. It was found that an incubation time of only 5 min was sufficient to prohibit any hatching of larvae, whilst 93 ± 4% of the larvae in the untreated controls of body lice hatched respectively about 76% of the controls in the case of head lice. Apparently, the neem-based shampoo blocked the aeropyles of the eggs, thus preventing the embryos of both races of lice from accessing oxygen and from releasing carbon dioxide. Thus, this product offers a complete cure from head lice upon a single treatment, if the lice (motile stages, eggs) are fully covered for about 10 min.
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Improved accuracy in measuring one-bond and two-bond (15)N, (13)C (?) coupling constants in proteins by double-inphase/antiphase (DIPAP) spectroscopy.
J. Biomol. NMR
PUBLISHED: 02-28-2011
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An extension to HN(CO-?/?-N,C(?)-J)-TROSY (Permi and Annila in J Biomol NMR 16:221-227, 2000) is proposed that permits the simultaneous determination of the four coupling constants (1) J (N(i)C?(i)), (2) J (HN(i)C?(i)), (2) J (C?(i-1)N(i)), and (3) J (C?(i-1)HN(i)) in (15)N,(13)C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the (2) J (C?N) coupling as inphase and antiphase splitting (IPAP), we here record four subspectra that exhibit all combinations of inphase and antiphase splittings possible with respect to both (2) J (C?N) and (1) J (NC?) (DIPAP). Complementary sign patterns in the different spectrum constituents overdetermine the coupling constants which can thus be extracted at higher accuracy than is possible with the original experiment. Fully exploiting data redundance, simultaneous 2D lineshape fitting of the E.COSY multiplet tilts in all four subspectra provides all coupling constants at ultimate precision. Cross-correlation and differential-relaxation effects were taken into account in the evaluation procedure. By applying a four-point Fourier transform, the set of spectra is reversibly interconverted between DIPAP and spin-state representations. Methods are exemplified using proteins of various size.
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Freezing of rat tibiae at -20°c does not affect the mechanical properties of intramedullary bone/implant-interface: brief report.
Open Orthop J
PUBLISHED: 02-19-2011
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The effects of freezing-thawing cycles on intramedullary bone-implant interfaces have been studied in a rat model in mechanical pull-out tests. IMPLANTS: Twenty TiAl6V4 rods (Ø 0.8 mm, length 10 mm) implanted in rat tibiae
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Furanocoumarins from Dorsteniafoetida.
Phytochemistry
PUBLISHED: 02-01-2011
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The linear furanocoumarins 5-(2,3-epoxy-3-methyl-butoxy)-chalepensin, 5-methoxy-3-(3-methyl-2,3-dihydroxybutyl)-psoralen-diacetate (7), 5-methoxy-3-[3-(?-d-glucopyranosyloxy)-2-acetyloxy-3-methyl-butyl]-psoralen and 5-(3-methyl-2,3-dihydroxybutyloxy)-3-[3-(?-d-glucopyranosyloxy)-2-hydroxy-3-methyl-butyl]-psoralen, and the coumarin derivative 7-hydroxy-5-methoxy-6-carboxymethyl-3-[3-(?-d-glucopyranosyloxy)-2-hydroxy-3-methyl-butyl]-coumarin were isolated from the leaves of Dorstenia foetida (Moraceae) along with the known compounds psoralen, bergapten, isopimpinellin, phellopterin, 5-methoxychalepensin and turbinatocoumarin. Further furanocoumarins were characterized by ESI-MS/MS investigations. The nonpolar extracts of D. foetida exhibit antifungal, antibacterial and cytotoxic activity, however, no anthelminthic activity.
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Chemoenzymatic synthesis of diverse thiohydroximates from glucosinolate-utilizing enzymes from Helix pomatia and Caldicellulosiruptor saccharolyticus.
Biotechnol. Lett.
PUBLISHED: 01-26-2011
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Thiohydroximates comprise a diverse class of compounds important in both biological and industrial chemistry. Their syntheses are generally limited to simple alkyl and aryl compounds with few stereocenters and a narrow range of functional groups. We hypothesized that sequential action of two recombinant enzymes, a sulfatase from Helix pomatia and a ?-O-glucosidase from Caldicellulosiruptor saccharolyticus, on glucosinolates would allow synthesis of thiohydroximates from a structurally broad array of abundant precursors. We report successful synthesis of thiohydroximates of varied chemical classes, including from homochiral compounds of demonstrated biological activity. The chemoenzymatic synthetic route reported here should allow access to many, if not all, of the thiohydroximate core structures of the ~200 known naturally occurring glucosinolates. The enrichment of this group for compounds with possible pharmacological potential is discussed.
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Immunomagnetic separation and quantification of butyrylcholinesterase nerve agent adducts in human serum.
Anal. Chem.
PUBLISHED: 07-13-2010
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A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 microL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilution-tandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agent-exposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure.
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Ampullosine, a new isoquinoline alkaloid from Sepedonium ampullosporum (Ascomycetes).
Nat Prod Commun
PUBLISHED: 07-10-2010
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A new isoquinoline alkaloid, ampullosine (3-methyl-isoquinoline-6-carboxylic acid, 1), was isolated from Sepedonium ampullosporum and characterized by spectroscopic analysis and chemical reactions. This compound is responsible for the deep yellow color of the culture fluid of this species. Moreover, the known compounds sepedonin (2) and anhydrosepedonin (3) were detected. Twelve strains belonging to eight species of Sepedonium have been screened for these three metabolites by LC/ESI-SRM (selected reaction monitoring). Ampullosine (1) could be detected in almost all species in Sepedonium, but not in the phylogenetically more distant species S. brunneum and S. tulasneanum. Anhydrosepedonin (3) showed antifungal activity against the phytopathogenic fungus Cladosporium cucumerinum.
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Synthesis, characterization and reactivity of carbohydrate platinum(IV) complexes with thioglycoside ligands.
Dalton Trans
PUBLISHED: 06-01-2010
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Reactions of fac-[PtMe3(4,4-R2bpy)(Me2CO)][BF4] (R = H, 1a; tBu, 1b) and fac-[PtMe3(OAc-kappa2O,O)(Me2CO)] (2), respectively, with thioglycosides containing thioethyl (ch-SEt) and thioimidate (ch-STaz, Taz = thiazoline-2-yl) anomeric groups led to the formation of the carbohydrate platinum(IV) complexes fac-[PtMe3(4,4-R2bpy)(ch*)][BF4] (ch* = ch-SEt, 8-14; ch-STaz, 15-23) and fac-[PtMe3(OAc-kappa2O,O)(ch*)] (ch* = ch-SEt, 24-28; ch-STaz = 29-35), respectively. NMR (1H, 13C, 195Pt) spectroscopic investigations and a single-crystal X-ray diffraction analysis of 19 (ch-STaz = 2-thiazolinyl 2,3,4,6-tetra-O-benzoyl-1-thio-beta-D-galactopyranose) revealed the S coordination of the ch-SEt glycosides and the N coordination of the ch-STaz glycosides. Furthermore, X-ray structure analyses of the two decomposition products fac-[PtMe3(bpy)(STazH-kappaS)][BF4] (21a) and 1,6-anhydro-2,3,4-tri-O-benzoyl-beta-D-glucopyranose (23a), where a cleavage of the anomeric C-S bond had occurred in both cases, gave rise to the assumption that this decomposition was mediated due to coordination of the thioglycosides to the high electrophilic platinum(IV) atom, in non-strictly dried solutions. Reactions of fac-[PtMe3(Me2CO)3][BF4] (3) with ch-SEt as well as with ch-SPT and ch-Sbpy thioglycosides (PT = 4-(pyridine-2-yl)-thiazole-2-yl; bpy = 2,2-bipyridine-6-yl), having N,S and N,N heteroaryl anomeric groups, respectively, led to the formation of platinum(IV) complexes of the type fac-[PtMe3(ch*)][BF4] (ch* = ch-SEt, 36-40, ch-SPT 42-44, ch-Sbpy 45, 46). The thioglycosides were found to be coordinated in a tridentate kappaS,kappa2O,O, kappaS,kappaN,kappaO and kappaS,kappa2N,N coordination mode, respectively. Analogous reactions with ch-STaz ligands succeeded for 2-thiazolinyl 2,3,4-tri-O-benzyl-6-O-(2,2-bipyridine-6-yl)-1-thio-beta-D-glucopyranoside (5h) resulting in fac-[PtMe3(ch-STaz)][BF4] (41, ch-STaz = 5h), having a kappa3N,N,Ncoordinated thioglycoside ligand.
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2-DE proteomic approach to the Botrytis cinerea secretome induced with different carbon sources and plant-based elicitors.
Proteomics
PUBLISHED: 04-09-2010
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Botrytis cinerea is a phytopathogenic fungus infecting a number of crops (tomatoes, grapes and strawberries), which has been adopted as a model system in molecular phytopathology. B. cinerea uses a wide variety of infection strategies, which are mediated by a set of genes/proteins called pathogenicity/virulence factors. Many of these factors have been described as secreted proteins, and thus the study of this sub-proteome, the secretome, under changing circumstances can help us to understand the roles of these factors, possibly revealing new loci for the fight against the pathogen. A 2-DE, MALDI TOF/TOF-based approach has been developed to establish the proteins secreted to culture media supplemented with different carbon sources and plant-based elicitors (in this study: glucose, cellulose, starch, pectin and tomato cell walls). Secreted proteins were obtained from the culture media by deoxycholate-trichloroacetic acid/phenol extraction, and 76 spots were identified, yielding 95 positive hits that correspond to 56 unique proteins, including several known virulence factors (i.e. pectin methyl esterases, xylanases and proteases). The observed increases in secretion of proteins with established virulence-related functions indicate that this in vitro-induction/proteome-mining approach is a promising strategy for discovering new pathogenicity factors and dissecting infection mechanisms in a discrete fashion.
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Profiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus).
Phytochemistry
PUBLISHED: 02-18-2010
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A dsRNAi approach silencing a key enzyme of sinapate ester biosynthesis (UDP-glucose:sinapate glucosyltransferase, encoded by the UGT84A9 gene) in oilseed rape (Brassica napus) seeds was performed to reduce the anti-nutritive properties of the seeds by lowering the content of the major seed component sinapine (sinapoylcholine) and various minor sinapate esters. The transgenic seeds have been produced so far to the T6 generation and revealed a steady suppression of sinapate ester accumulation. HPLC analysis of the wild-type and transgenic seeds revealed, as in the previous generations, marked alterations of the sinapate ester pattern of the transformed seeds. Besides strong reduction of the amount of the known sinapate esters, HPLC analysis revealed unexpectedly the appearance of several minor hitherto unknown rapeseed constituents. These compounds were isolated and identified by mass spectrometric and NMR spectroscopic analyses. Structures of 11 components were elucidated to be 4-O-glucosides of syringate, caffeyl alcohol and its 7,8-dihydro derivative as well as of sinapate and sinapine, along with sinapoylated kaempferol glycosides, a hexoside of a cyclic spermidine alkaloid and a sinapine derivative with an ether-bridge to a C(6)-C(3)-unit. These results indicate a strong impact of the transgenic approach on the metabolic network of phenylpropanoids in B. napus seeds. Silencing of UGT84A9 gene expression disrupt the metabolic flow through sinapoylglucose and alters the amounts and nature of the phenylpropanoid endproducts.
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Why do health systems matter? Exploring links between health systems and HIV response: a case study from Russia.
Health Policy Plan
PUBLISHED: 02-08-2010
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Studies on the relevance of stronger health systems to the success of vertical programmes has focused mainly on developing countries with fragile infrastructures and limited human resources. Research in middle-income, and particularly post-Soviet, settings has been scarce. This article examines the relationships between health system characteristics and the HIV response in Russia, the country which towards the end of the Soviet period had the worlds highest ratios of doctors and hospital beds to population and yet struggled to address the growing threat of HIV/AIDS.
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Correlation of (2)J couplings with protein secondary structure.
Proteins
PUBLISHED: 02-05-2010
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Geminal two-bond couplings ((2)J) in proteins were analyzed in terms of correlation with protein secondary structure. NMR coupling constants measured and evaluated for a total six proteins comprise 3999 values of (2)J(CalphaN), (2)J(CHN), (2)J(HNCalpha), (2)J(CCalpha), (2)J(HalphaC), (2)J(HalphaCalpha), (2)J(CbetaC), (2)J(NHalpha), (2)J(NCbeta), and (2)J(NC), encompassing an aggregate 969 amino-acid residues. A seamless chain of pattern comparisons across the spectrum datasets recorded allowed the absolute signs of all (2)J coupling constants studied to be retrieved. Grouped by their mediating nucleus, C, N or C(alpha), (2)J couplings related to C and N depend significantly on phi,psi torsion-angle combinations. beta turn types I, I, II and II, especially, can be distinguished on the basis of relative-value patterns of (2)J(CalphaN), (2)J(HNCalpha), (2)J(CHN), and (2)J(HalphaC). These coupling types also depend on planar or tetrahedral bond angles, whereas such dependences seem insignificant for other types. (2)J(HalphaCbeta) appears to depend on amino-acid type only, showing negligible correlation with torsion-angle geometry. Owing to its unusual properties, (2)J(CalphaN) can be considered a "one-bond" rather than two-bond interaction, the allylic analog of (1)J(NCalpha), as it were. Of all protein J coupling types, (2)J(CalphaN) exhibits the strongest dependence on molecular conformation, and among the (2)J types, (2)J(HNCalpha) comes second in terms of significance, yet was hitherto barely attended to in protein structure work.
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Proteasome activity profiling: a simple, robust and versatile method revealing subunit-selective inhibitors and cytoplasmic, defense-induced proteasome activities.
Plant J.
PUBLISHED: 12-23-2009
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The proteasome plays essential roles in nearly all biological processes in plant defense and development, yet simple methods for displaying proteasome activities in extracts and living tissues are not available to plant science. Here, we introduce an easy and robust method to simultaneously display the activities of all three catalytic proteasome subunits in plant extracts or living plant tissues. The method is based on a membrane-permeable, small-molecule fluorescent probe that irreversibly reacts with the catalytic site of the proteasome catalytic subunits in an activity-dependent manner. Activities can be quantified from fluorescent protein gels and used to study proteasome activities in vitro and in vivo. We demonstrate that proteasome catalytic subunits can be selectively inhibited by aldehyde-based inhibitors, including the notorious caspase-3 inhibitor DEVD. Furthermore, we show that the proteasome activity, but not its abundance, is significantly increased in Arabidopsis upon treatment with benzothiadiazole (BTH). This upregulation of proteasome activity depends on NPR1, and occurs mostly in the cytoplasm. The simplicity, robustness and versatility of this method will make this method widely applicable in plant science.
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Targeted metabolite and transcript profiling for elucidating enzyme function: isolation of novel N-methyltransferases from three benzylisoquinoline alkaloid-producing species.
Plant J.
PUBLISHED: 07-16-2009
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An integrated approach using targeted metabolite profiles and modest EST libraries each containing approximately 3500 unigenes was developed in order to discover and functionally characterize novel genes involved in plant-specialized metabolism. EST databases have been established for benzylisoquinoline alkaloid-producing cell cultures of Eschscholzia californica, Papaver bracteatum and Thalictrum flavum, and are a rich repository of alkaloid biosynthetic genes. ESI-FTICR-MS and ESI-MS/MS analyses facilitated unambiguous identification and relative quantification of the alkaloids in each system. Manual integration of known and candidate biosynthetic genes in each EST library with benzylisoquinoline alkaloid biosynthetic networks assembled from empirical metabolite profiles allowed identification and functional characterization of four N-methyltransferases (NMTs). One cDNA from T. flavum encoded pavine N-methyltransferase (TfPavNMT), which showed a unique preference for (+/-)-pavine and represents the first isolated enzyme involved in the pavine alkaloid branch pathway. Correlation of the occurrence of specific alkaloids, the complement of ESTs encoding known benzylisoquinoline alkaloid biosynthetic genes and the differential substrate range of characterized NMTs demonstrated the feasibility of bilaterally predicting enzyme function and species-dependent specialized metabolite profiles.
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Analysis of phenolic choline esters from seeds of Arabidopsis thaliana and Brassica napus by capillary liquid chromatography/electrospray- tandem mass spectrometry.
J Mass Spectrom
PUBLISHED: 07-16-2009
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Total phenolic choline ester fractions prepared from seeds of Arabidopsis thaliana and Brassica napus were analyzed by capillary LC/ESI-QTOF-MS and direct infusion ESI-FTICR-MS. In addition to the dominating sinapoylcholine, 30 phenolic choline esters could be identified based on accurate mass measurements, interpretation of collision-induced dissociation (CID) mass spectra, and synthesis of selected representatives. The compounds identified so far include substituted hydroxycinnamoyl- and hydroxybenzoylcholines, respective monohexosides as well as oxidative coupling products of phenolic choline esters and monolignols. Phenolic choline esters are well separable by reversed-phase liquid chromatography and sensitively detectable using electrospray ionization mass spectrometry in positive ion mode. CID mass spectra obtained from molecular ions facilitate the characterization of both the type and substitution pattern of such compounds. Therefore, LC/ESI-MS/MS represents a valuable tool for comprehensive qualitative and quantitative analysis of this compound class.
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New pyridine alkaloids from rove beetles of the genus Stenus (Coleoptera: Staphylinidae).
Z. Naturforsch., C, J. Biosci.
PUBLISHED: 06-17-2009
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Three new pyridine alkaloids were detected in the pygidial glands of some Stenus species. The chemotaxonomic significance of the occurrence of these alkaloids and stenusine in different Stenus species is discussed. The antimicrobial properties of (Z)- and (E)-3-(2-methyl-1-butenyl)-pyridine and the deterrent activities of stenusine and norstenusine were investigated.
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Functional characterization of a novel benzylisoquinoline O-methyltransferase suggests its involvement in papaverine biosynthesis in opium poppy (Papaver somniferum L).
Plant J.
PUBLISHED: 05-28-2009
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The benzylisoquinoline alkaloids are a highly diverse group of about 2500 compounds which accumulate in a species-specific manner. Despite the numerous compounds which could be identified, the biosynthetic pathways and the participating enzymes or cDNAs could be characterized only for a few selected members, whereas the biosynthesis of the majority of the compounds is still largely unknown. In an attempt to characterize additional biosynthetic steps at the molecular level, integration of alkaloid and transcript profiling across Papaver species was performed. This analysis showed high expression of an expressed sequence tag (EST) of unknown function only in Papaver somniferum varieties. After full-length cloning of the open reading frame and sequence analysis, this EST could be classified as a member of the class II type O-methyltransferase protein family. It was related to O-methyltransferases from benzylisoquinoline biosynthesis, and the amino acid sequence showed 68% identical residues to norcoclaurine 6-O-methyltransferase. However, rather than methylating norcoclaurine, the recombinant protein methylated norreticuline at position seven with a K(m) of 44 mum using S-adenosyl-l-methionine as a cofactor. Of all substrates tested, only norreticuline was converted. Even minor changes in the benzylisoquinoline backbone were not tolerated by the enzyme. Accordingly, the enzyme was named norreticuline 7-O-methyltransferase (N7OMT). This enzyme represents a novel O-methyltransferase in benzylisoquinoline metabolism. Expression analysis showed slightly increased expression of N7OMT in P. somniferum varieties containing papaverine, suggesting its involvement in the partially unknown biosynthesis of this pharmaceutically important compound.
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Evolution of morphine biosynthesis in opium poppy.
Phytochemistry
PUBLISHED: 05-07-2009
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Benzylisoquinoline alkaloids (BIAs) are a group of nitrogen-containing plant secondary metabolites comprised of an estimated 2500 identified structures. In BIA metabolism, (S)-reticuline is a key branch-point intermediate that can be directed into several alkaloid subtypes with different structural skeleton configurations. The morphinan alkaloids are one subclass of BIAs produced in only a few plant species, most notably and abundantly in the opium poppy (Papaver somniferum). Comparative transcriptome analysis of opium poppy and several other Papaver species that do not accumulate morphinan alkaloids showed that known genes encoding BIA biosynthetic enzymes are expressed at higher levels in P. somniferum. Three unknown cDNAs that are co-ordinately expressed with several BIA biosynthetic genes were identified as enzymes in the pathway. One of these enzymes, salutaridine reductase (SalR), which is specific for the production of morphinan alkaloids, was isolated and heterologously overexpressed in its active form not only from P. somniferum, but also from Papaver species that do not produce morphinan alkaloids. SalR is a member of a class of short chain dehydrogenase/reductases (SDRs) that are active as monomers and possess an extended amino acid sequence compared with classical SDRs. Homology modelling and substrate docking revealed the substrate binding site for SalR. The amino acids residues conferring salutaridine binding were compared to several members of the SDR family from different plant species, which non-specifically reduce (-)-menthone to (+)-neomenthol. Previously, it was shown that some of these proteins are involved in plant defence. The recruitment of specific monomeric SDRs from monomeric SDRs involved in plant defence is discussed.
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Proteomic analysis of the phytopathogenic fungus Botrytis cinerea during cellulose degradation.
Proteomics
PUBLISHED: 05-06-2009
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The ascomycete Botrytis cinerea is a phytopathogenic fungus infecting and causing significant yield losses in a number of crops. Moreover, in the last few years, B. cinerea has been adopted as an important model system in molecular phytopathology. In spite of these contributions, the molecular basis of the infection cycle remains unclear. Proteomic approaches have revealed significant information about the infective cycle of several pathogens, including B. cinerea. The main aim of this study is to make available a proteomic database containing a significant number of identified proteins from B. cinerea. In brief, three independent B. cinerea cultures supplemented with carboxymethylcellulose were used, and the extracted proteins were independently separated by 2-D PAGE to obtain the proteome map from B. cinerea. Two hundred and sixty-seven spots were selected for MALDI TOF/TOF MS analysis, resulting in 303 positive identifications, mostly representing unannotated proteins. Identified proteins were then classified into categories using the PANTHER classification system (www.pantherdb.org), showing the relevance of protein metabolism and modification process and oxidoreductase activity. Since cellulose is one of the major components of the plant cell wall, many of the identified proteins may have a crucial role in the pathogenicity process. In brief, this proteomic map of B. cinerea will be a useful basis for exploring the proteins involved in the infection cycle, which will in turn provide new targets for crop diagnosis and focused fungicide design.
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Effects of glutamate and alpha2-noradrenergic receptor antagonists on the development of neurotoxicity produced by chronic rotenone in rats.
Toxicol. Appl. Pharmacol.
PUBLISHED: 04-28-2009
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Systemic inhibition of complex I by rotenone in rats represents a model of Parkinsons disease (PD). The aim of this study was to elucidate whether neramexane (NMDA, nicotinic alpha9/alpha10 and 5-HT3 receptor antagonist), idazoxan (alpha2-adrenoceptor antagonist) or 2-methyl-6-(phenyl-ethyl)-pyrimidine (MPEP, metabotropic glutamate receptor 5 antagonist) prevents rotenone-induced parkinsonian-like behaviours and neurochemical changes in rats. Rotenone (2.5 mg/kg i.p. daily) was administered over 60 days together with saline, neramexane (5 mg/kg i.p., b.i.d.), idazoxan (2.5 mg/kg i.p., b.i.d.) or MPEP (2.5 mg/kg i.p., b.i.d.). The same doses of neramexane, idazoxan and MPEP were administered to rats treated with vehicle instead of rotenone. Treatment-related effects on parkinsonian-like behaviours, such as hypokinesia/rigidity and locomotor activity, were evaluated. Moreover, concentrations of dopamine, serotonin and their metabolites were measured in rats from each experimental group. Over the 60-day treatment period, the rotenone+saline treated animals developed hypokinesia, expressed as an increase in the bar and grid descent latencies in the catalepsy test, and a decrease in locomotor activity. Neramexane and idazoxan partially prevented the development of catalepsy in rotenone-treated rats. Co-administration of MPEP with rotenone resulted only in a decrease in descent latency in the grid test on day 60. Chronic rotenone treatment reduced concentrations of dopamine and serotonin in the anterior striatum, which was blocked by co-treatment with neramexane or idazoxan but not with MPEP. Only neramexane treatment blocked the rotenone-induced decrease in dopamine levels in the substantia nigra pars compacta. In conclusion, neramexane and idazoxan counteracted to some extent the development of parkinsonian symptoms and neurochemical alterations in the rotenone model of Parkinsons disease.
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Solution structure and dynamics of ERp18, a small endoplasmic reticulum resident oxidoreductase .
Biochemistry
PUBLISHED: 04-14-2009
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Here we report the solution structure of oxidized ERp18 as determined using NMR spectroscopy. ERp18 is the smallest member of the protein disulfide isomerase (PDI) family of proteins to contain a Cys-Xxx-Xxx-Cys active site motif. It is an 18 kDa endoplasmic reticulum resident protein with unknown function although sequence similarity to individual domains of the thiol-disulfide oxidoreductase PDI suggests ERp18 may have a similar structure and function. Like the catalytic domains of PDI, ERp18 adopts a thioredoxin fold with a thioredoxin-like active site located at the N-terminus of a long kinked helix that spans the length of the protein. Comparison of backbone chemical shifts for oxidized and reduced ERp18 shows the majority of residues possess the same backbone conformation in both states, with differences limited to the active site and regions in close proximity. S(2) order parameters from NMR backbone dynamics were found to be 0.81 for oxidized and 0.91 for reduced ERp18, and these observations, in combination with amide hydrogen exchange rates, imply a more rigid and compact backbone for the reduced structure. These observations support a putative role for ERp18 within the cell as an oxidase, introducing disulfide bonds to substrate proteins, providing structural confirmation of ERp18s role as a thiol-disulfide oxidoreductase.
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A proteomic analysis of powdery mildew (Blumeria graminis f.sp. hordei) conidiospores.
Mol. Plant Pathol.
PUBLISHED: 02-25-2009
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Conidiospores are the asexual propagation units of many plant-pathogenic fungi. In this article, we report an annotated proteome map of ungerminated conidiospores of the ascomycete barley powdery mildew pathogen, Blumeria graminis f.sp. hordei. Using a combination of two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, we have identified the proteins in 180 spots, which probably represent at least 123 distinct fungal gene products. Most of the identified proteins have a predicted function in carbohydrate, lipid or protein metabolism, indicating that the spore is equipped for the catabolism of storage compounds as well as for protein biosynthesis and folding on germination.
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Variation in protein C(alpha)-related one-bond J couplings.
Magn Reson Chem
PUBLISHED: 02-10-2009
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Four types of polypeptide (1)J(C alpha X) couplings are examined, involving the main-chain carbon C(alpha) and either of four possible substituents. A total 3105 values of (1)J(C alpha H alpha), (1)J(C alpha C beta), (1)J(C alpha C), and (1)J(C alpha N) were collected from six proteins, averaging 143.4 +/- 3.3, 34.9 +/- 2.5, 52.6 +/- 0.9, and 10.7 +/- 1.2 Hz, respectively. Analysis of variances (ANOVA) reveals a variety of factors impacting on (1)J and ranks their relative statistical significance and importance to biomolecular NMR structure refinement. Accordingly, the spread in the (1)J values is attributed, in equal proportions, to amino-acid specific substituent patterns and to polypeptide-chain geometry, specifically torsions phi, psi, and chi(1) circumjacent to C(alpha). The (1)J coupling constants correlate with protein secondary structure. For alpha-helical phi, psi combinations, (1)J(C alpha H alpha) is elevated by more than one standard deviation (147.8 Hz), while both (1)J(C alpha N) and (1)J(C alpha C beta) fall short of their grand means (9.5 and 33.7 Hz). Rare positive phi torsion angles in proteins exhibit concomitant small (1)J(C alpha H alpha) and (1)J(C alpha N) (138.4 and 9.6 Hz) and large (1)J(C alpha C beta) (39.9 Hz) values. The (1)J(C alpha N) coupling varies monotonously over the phi torsion range typical of beta-sheet secondary structure and is largest (13.3 Hz) for phi around -160 degrees. All four coupling types depend on psi and thus help determine a torsion that is notoriously difficult to assess by traditional approaches using (3)J. Influences on (1)J stemming from protein secondary structure and other factors, such as amino-acid composition, are largely independent.
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Substrates related to chromatin and to RNA-dependent processes are modified by Arabidopsis SUMO isoforms that differ in a conserved residue with influence on desumoylation.
Plant Physiol.
PUBLISHED: 01-16-2009
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The higher plant Arabidopsis (Arabidopsis thaliana) has eight genes potentially coding for small ubiquitin-related modifier (SUMO) proteins. However, two well-expressed isoforms differ from fungal and animal consensus in a conserved glutamine (Gln) residue situated four residues from the carboxyl terminus. We tested deviations in this position in the background of SUMO1, the isoform with the highest expression level, and found that changes do not prevent conjugation to substrate proteins in vivo. Replacement of this conserved Gln by alanine resulted in a protein that was less readily removed from a substrate by SUMO protease EARLY IN SHORT DAYS4 in an in vitro reaction and apparently led to higher levels of SUMO conjugates when expressed in vivo. We used the SUMO1 variant with the Gln-to-alanine substitution, as well as SUMO3 and SUMO5 (which carry methionine and leucine, respectively, at this position), to enrich in vivo substrates. Identification of the most abundant proteins contained in these fractions indicated that they are involved in DNA-related, or in RNA-dependent, processes, such as regulation of chromatin structure, splicing, or translation. The majority of the identified bona fide substrates contain predicted sumoylation sites. A subset of the proteins was expressed in Escherichia coli and could be sumoylated in vitro.
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Negative ion tandem mass spectrometry of prenylated fungal metabolites and their derivatives.
Anal Bioanal Chem
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Liquid chromatography negative ion electrospray ionisation tandem mass spectrometry has been used for characterisation of naturally occurring prenylated fungal metabolites and synthetic derivatives. The fragmentation studies allow an elucidation of the decomposition pathways for these compounds. It could be shown, that the prenyl side chain is degraded by successive radical losses of C(5) units. Both the benzoquinones and the phenolic derivatives display significant key ions comprising the aromatic ring. In some cases, the formation of significant oxygen-free key ions could be evidenced by high-resolution MS/MS measurements. Furthermore, the different types of basic skeletons, benzoquinones and phenol type as well as cyclic prenylated compounds, can be differentiated by their MS/MS behaviour. Figure Fruiting bodies of Suillus bovinus, the structure of boviquinone-4 and its negative ion MS2 spectrum.
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New cardiolipin analogs synthesized by phospholipase D-catalyzed transphosphatidylation.
Chem. Phys. Lipids
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Cardiolipin (CL) and related diphosphatidyl lipids are hardly accessible because of the complexity of their chemical synthesis. In the present paper, the transphosphatidylation reaction catalyzed by phospholipase D (PLD) from Streptomyces sp. has been proven as an alternative enzyme-assisted strategy for the synthesis of new CL analogs. The formation of this type of compounds from phosphatidylcholine was compared for a series of N- and C2-substituted ethanolamine derivatives as well as non-charged alcohols such as glycerol and ethylene glycol. The rapid exchange of the choline head group by ethanolamine derivatives having a low molecular volume (diethanolamine and serinol) gave rise to an efficient production of the corresponding CL analogs. In contrast, the yields were comparably low in the reaction with bulky nitrogenous acceptor alcohols (triethanolamine, tris(hydroxymethyl)aminomethane, tetrakis(hydroxyethyl)ammonium) or the non-charged alcohols. Therefore, a strong dependence of the conversion of the monophosphatidyl to the diphosphatidyl compound on steric parameters and the head group charge was concluded. The enzyme-assisted strategy was used for the preparation of purified diphosphatidyldiethanolamine and diphosphatidylserinol.
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A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxy-copalyl diphosphate.
Phytochemistry
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The bicyclic diterpene (-)-sclareol is accumulated in glandular trichomes in Salvia sclarea (Schmiderer et al., 2008), and is a major terpenoid component of this plant species. It is used as the starting material for Ambrox synthesis, a synthetic ambergris analog used in the flavor and fragrance industry. In order to investigate the formation of sclareol, cDNA prepared from secretory cells of glandular trichomes from S. sclarea inflorescence were randomly sequenced. A putative copalyl diphosphate synthase encoding EST, SscTPS1, was functionally expressed in Escherichia coli. Whereas reaction of geranylgeranyl diphosphate with the putative copalyl diphosphate synthase followed by hydrolysis with alkaline phosphatase yielded a diastereomeric mixture of (13R)- and (13S)-manoyl oxide, HCl hydrolysis yielded (-)-sclareol (1) and 13-epi-sclareol as products. The product of the reaction of SscTPS1 with geranylgeranyl diphosphate was subjected to analysis by LC-negative ion ESI-MS/MS without prior hydrolysis. EPI scans were consistent with copalyl diphosphate to which 18 mass units had added (m/z 467 [M+H](-)). The enzymatic reaction was also carried out in the presence of 60% H2(18)O. LC-negative ion ESI-MS/MS analysis established an additional reaction product consistent with the incorporation of (18)O. Incubation in the presence of 60% (2)H2O resulted in the incorporation of one deuterium atom. These results suggest water capture of the carbocation intermediate, which is known to occur in reactions catalyzed by monoterpene synthases, but has been described only several times for diterpene synthases.
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Volume reduction of the jugular foramina in Cavalier King Charles Spaniels with syringomyelia.
BMC Vet. Res.
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Understanding the pathogenesis of the chiari-like malformation in the Cavalier King Charles Spaniel (CKCS) is incomplete, and current hypotheses do not fully explain the development of syringomyelia (SM) in the spinal cords of affected dogs. This study investigates an unconventional pathogenetic theory for the development of cerebrospinal fluid (CSF) pressure waves in the subarachnoid space in CKCS with SM, by analogy with human diseases. In children with achondroplasia the shortening of the skull base can lead to a narrowing of the jugular foramina (JF) between the cranial base synchondroses. This in turn has been reported to cause a congestion of the major venous outflow tracts of the skull and consequently to an increase in the intracranial pressure (ICP). Amongst brachycephalic dog breeds the CKCS has been identified as having an extremely short and wide braincase. A stenosis of the JF and a consequential vascular compromise in this opening could contribute to venous hypertension, raising ICP and causing CSF jets in the spinal subarachnoid space of the CKCS. In this study, JF volumes in CKCSs with and without SM were compared to assess a possible role of this pathologic mechanism in the development of SM in this breed.
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The effect of polyelectrolyte multilayer coated titanium alloy surfaces on implant anchorage in rats.
Acta Biomater
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Advances have been achieved in the design and biomechanical performance of orthopedic implants in the last decades. These include anatomically shaped and angle-stable implants for fracture fixation or improved biomaterials (e.g. ultra-high-molecular-weight polyethylene) in total joint arthroplasty. Future modifications need to address the biological function of implant surfaces. Functionalized surfaces can promote or reduce osseointegration, avoid implant-related infections or reduce osteoporotic bone loss. To this end, polyelectrolyte multilayer structures have been developed as functional coatings and intensively tested in vitro previously. Nevertheless, only a few studies address the effect of polyelectrolyte multilayer coatings of biomaterials in vivo. The aim of the present work is to evaluate the effect of polyelectrolyte coatings of titanium alloy implants on implant anchorage in an animal model. We test the hypotheses that (1) polyelectrolyte multilayers have an effect on osseointegration in vivo; (2) multilayers of chitosan/hyaluronic acid decrease osteoblast proliferation compared to native titanium alloy, and hence reduce osseointegration; (3) multilayers of chitosan/gelatine increase osteoblast proliferation compared to native titanium alloy, hence enhance osseointegration. Polyelectrolyte multilayers on titanium alloy implants were fabricated by a layer-by-layer self-assembly process. Titanium alloy (Ti) implants were alternately dipped into gelatine (Gel), hyaluronic acid (HA) and chitosan (Chi) solutions, thus assembling a Chi/Gel and a Chi/HA coating with a terminating layer of Gel or HA, respectively. A rat tibial model with bilateral placement of titanium alloy implants was employed to analyze the bones response to polyelectrolyte surfaces in vivo. 48 rats were randomly assigned to three groups of implants: (1) native titanium alloy (control), (2) Chi/Gel and (3) Chi/HA coating. Mechanical fixation, peri-implant bone area and bone contact were evaluated by pull-out tests and histology at 3 and 8 weeks. Shear strength at 8 weeks was statistically significantly increased (p<0.05) in both Chi/Gel and Chi/HA groups compared to the titanium alloy control. No statistically significant difference (p>0.05) in bone contact or bone area was found between all groups. No decrease of osseointegration of Chi/HA-coated implants compared to non-coated implants was found. The results of polyelectrolyte coatings in a rat model showed that the Chi/Gel and Chi/HA coatings have a positive effect on mechanical implant anchorage in normal bone.
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Identification of proteins associated with malting quality in a subset of wild barley introgression lines.
Proteomics
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Malted barley is an important ingredient used in the brewing and distilling industry worldwide. In this study, we used a proteomics approach to investigate the biochemical function of previously identified quantitative trait loci (QTLs) on barley chromosomes 1H and 4H that influence malting quality. Using a subset of barley introgression lines containing wild barley (Hordeum vulgare ssp. spontaneum) alleles at these QTLs, we validated that wild barley alleles at the chromosome 1H QTL reduced overall malting quality, whereas wild barley alleles at the chromosome 4H QTL improved the malting quality parameters ?-amylase activity, VZ45, and Kolbach index compared to the control genotype Scarlett. 2DE was used to detect changes in protein expression during the first 72 h of micromalting associated with these QTLs. In total, 16 protein spots showed a significant change in expression between the introgression lines and Scarlett, of which 14 were successfully identified with MS. Notably, the wild barley alleles in the line containing the chromosome 4H QTL showed a sixfold increased expression of a limit dextrinase inhibitor. The possible role of the identified proteins in malting quality is discussed. The knowledge gained will assist ongoing research toward cloning the genes underlying these important QTL.
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Transforming between discrete and continuous angle distribution models: application to protein ?? torsions.
J. Biomol. NMR
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Two commonly employed angular-mobility models for describing amino-acid side-chain ?(1) torsion conformation, the staggered-rotamer jump and the normal probability density, are discussed and performance differences in applications to scalar-coupling data interpretation highlighted. Both models differ in their distinct statistical concepts, representing discrete and continuous angle distributions, respectively. Circular statistics, introduced for describing torsion-angle distributions by using a universal circular order parameter central to all models, suggest another distribution of the continuous class, here referred to as the elliptic model. Characteristic of the elliptic model is that order parameter and circular variance form complementary moduli. Transformations between the parameter sets that describe the probability density functions underlying the different models are provided. Numerical aspects of parameter optimization are considered. The issues are typified by using a set of ?(1) related (3) J coupling constants available for FK506-binding protein. The discrete staggered-rotamer model is found generally to produce lower order parameters, implying elevated rotatory variability in the amino-acid side chains, whereas continuous models tend to give higher order parameters that suggest comparatively less variation in angle conformations. The differences perceived regarding angular mobility are attributed to conceptually different features inherent to the models.
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Biosynthesis of the defensive alkaloid cicindeloine in Stenus solutus beetles.
Naturwissenschaften
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To protect themselves from predation and microorganismic infestation, rove beetles of the genus Stenus produce and store bioactive alkaloids like stenusine, 3-(2-methyl-1-butenyl)pyridine, and cicindeloine in their pygidial glands. The biosynthesis of stenusine and 3-(2-methyl-1-butenyl)pyridine was previously investigated in Stenus bimaculatus and Stenus similis, respectively. Both molecules follow the same biosynthetic pathway, where the N-heterocyclic ring is derived from L-lysine and the side chain from L-isoleucine. The different alkaloids are finally obtained by slight modifications of shared precursor molecules. The piperideine alkaloid cicindeloine occurs as a main compound additionally to (E)-3-(2-methyl-1-butenyl)pyridine and traces of stenusine in the pygidial gland secretion of Stenus cicindeloides and Stenus solutus. Feeding of S. solutus beetles with [D,(15)N]-labeled amino acids followed by GC/MS analysis techniques showed that cicindeloine is synthesized via the identical pathway and precursor molecules as the other two defensive alkaloids.
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Proteomic analysis of latex from the rubber-producing plant Taraxacum brevicorniculatum.
Proteomics
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Many plants produce latex, a specialized, metabolically active cytoplasm. This is generally regarded as a defensive trait but latex may also possess additional functions. We investigated the role of latex in the dandelion species Taraxacum brevicorniculatum that contains considerable amounts of high-quality natural rubber by carrying out a comprehensive analysis of the latex proteome. We developed reliable protocols for the preparation of protein samples for one-dimensional gel electrophoresis, two-dimensional gel electrophoresis, and subsequent mass spectrometry analysis, which led to 278 unique identifications. A gene ontology classification system based on comparisons with known Arabidopsis thaliana root proteins showed that dandelion proteins involved in lipid metabolism and transport were enriched in the latex proteome, whereas those involved in stress responses were not. We also found that proteins involved in rubber biosynthesis were distributed among different fractions of the latex proteome.
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Chemical composition and biological activity of essential oil from Pulicaria undulata from Yemen.
Nat Prod Commun
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The chemical composition of the essential oil obtained from the leaves of Pulicaria undulata Gamal Ed Din (syn P. orientalis sensu Schwartz and P. jaubertii Gamal Ed Din) was analyzed by GC-MS. Major compounds of P. undulata oil were the oxygenated monoterpenenes, carvotanacetone (91.4%) and 2,5-dimethoxy-p-cymene (2.6.%). The antimicrobial activity of the essential oil was evaluated against six microorganisms, Escherichia coli Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis, and Candida albicans, using disc diffusion and broth microdilution methods. The oil showed the strongest bactericidal activity against Staphylococcus aureus and methicillin-resistant S. aureus, as well as Candida albicans. The essential oil showed moderate cytotoxic activity against MCF-7 breast tumor cells, with an IC50 of 64.6 +/- 13.7 microg/mL. Bioautographic assays were used to evaluate the acetylcholinesterase inhibitory effect as well as antifungal activity of the oil against Cladosporium cucumerinum.
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Analysis of furanocoumarins from Yemenite Dorstenia species by liquid chromatography/electrospray tandem mass spectrometry.
J Mass Spectrom
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A series of prevailing prenylated furanocoumarins from leaves of Dorstenia gigas and Dorstenia foetida (Moraceae) were investigated by liquid chromatography/electrospray tandem mass spectrometry. The mass spectral behavior of the furanocoumarins under positive ion electrospray conditions is discussed using both an ion trap and a triple quadrupole system. It is demonstrated that both methods represent valuable tools not only for the rapid classification of this type of compounds, but also with respect to their substitution pattern.
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