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Chemokines play a key role in the cellular infiltration of inflamed tissue. They are released by a wide variety of cell types during the initial phase of host response to injury, allergens, antigens, or invading microorganisms, and selectively attract leukocytes to inflammatory foci, inducing both migration and activation. Monocyte chemoattractant protein-1 (MCP-1), a member of the CC chemokine superfamily, functions in attracting monocytes, T lymphocytes, and basophils to sites of inflammation. MCP-1 is produced by monocytes, fibroblasts, vascular endothelial cells and smooth muscle cells in response to various stimuli such as tumour necrosis factor-a (TNF-a), interferon-g (IFN-g), and interleukin-1b (IL-1b). It also plays an important role in the pathogenesis of chronic inflammation, and overexpression of MCP-1 has been implicated in diseases including glomerulonephritis and rheumatoid arthritis. Oligonucleotide-directed triple helix formation offers a means to target specific sequences in DNA and interfere with gene expression at the transcriptional level. Triple helix-forming oligonucleotides (TFOs) bind to homopurine/homopyrimidine sequences, forming a stable, sequence-specific complex with the duplex DNA. Purine-rich sequences are frequent in gene regulatory regions and TFOs directed to promoter sequences have been shown to prevent binding of transcription factors and inhibit transcription initiation and elongation. Exogenous TFOs that bind homopurine/ homopyrimidine DNA sequences and form triple-helices can be rationally designed, while the intracellular delivery of single-stranded RNA TFOs has not been studied in detail before. In this study, expression vectors were constructed which directed transcription of either a 19 nt triplex-forming pyrimidine CU-TFO sequence targeting the human MCP-1 or two different 19 nt GU- or CA-control sequences, respectively, together with the vector encoded hygromycin resistance mRNA as one fusion transcript. HEK 293 cells were stable transfected with these vectors and several TFO and control cell lines were generated. Functional relevant triplex formation of a TFO with a corresponding 19 bp GC-rich AP-1/SP-1 site of the human MCP-1 promoter was shown. Binding of synthetic 19 nt CUTFO to the MCP-1 promoter duplex was verified by triplex blotting at pH 6.7. Underlining binding specificity, control sequences, including the GU- and CA-sequence, a TFO containing one single mismatch and a MCP-1 promoter duplex containing two mismatches, did not participate in triplex formation. Establishing a magnetic capture technique with streptavidin microbeads it was verified that at pH 7.0 the 19 nt TFO embedded in a 1.1 kb fusion transcript binds to a plasmid encoded MCP-1 promoter target duplex three times stronger than the controls. Finally, cell culture experiments revealed 76 ± 10.2% inhibition of MCP-1 protein secretion in TNF-a stimulated CU-TFO harboring cell lines and up to 88% after TNF-a and IFN-g costimulation in comparison to controls. Expression of interleukin-8 (IL-8) as one TNF-a inducible control gene was not affected by CU-TFO, demonstrating both highly specific and effective chemokine gene repression. Furthermore, another chemokine target, regulated upon activation normal T cell expressed and secreted (RANTES), which plays an essential role in inflammation by recruiting T lymphocytes, macrophages and eosinophils to inflammatory sites, was analysed using the triplex approach. A 28 nt TFO was designed targeting the murine RANTES gene promoter, and gel mobility shift assays demonstrated that the phosphodiester TFO formed a sequencespecific triplex with the double-stranded target DNA with a Kd of 2.5 x 10-7 M. It was analysed whether RANTES expression could be inhibited at the transcriptional level testing the TFO in two different cell lines, T helper-1 lymphocytes and brain microvascular endothelial cells (bend3 cells). Although there was a sequence-specific binding of the TFO detectable in the gel shift assays, there was no inhibitory effect of the exogenously added and phosphorothioate stabilised TFO on endogenous RANTES gene expression visible. Additionally, the small interfering RNA (siRNA) approach was tested as another strategy to inhibit expression of the pro-inflammatory chemokines MCP-1 and RANTES. Two different methods were pursuit, describing transient transfection with vector derived and synthetic siRNA. The vector pSUPER containing the siRNA coding sequence was used to suppress endogenous MCP-1 in HEK 293 cells. An empty vector without RNA sequence served as a control. Inhibition due to the siRNA was measured in stimulated and unstimulated cells. In TNF-a stimulated cells MCP-1 protein synthesis was decreased by 35 ± 11% after siRNA transfection. Using a synthetic double-stranded siRNA, the TNF-a induced MCP-1 protein secretion could be successfully inhibited about 62.3 ± 10.3% in HEK 293 cells, indicating that the siRNA is functional in these cells to suppress chemokine expression. The siRNA approach targeting murine RANTES in Th1 cells and b-end3 cells revealed no inhibition of endogenous gene expression. Gene therapy approaches rely on efficient transfer of genes to the desired target cells. A wide variety of viral and nonviral vectors have been developed and evaluated for their efficiency of transduction, sustained expression of the transgene, and safety. Among them, lentiviruses have been widely used for gene therapy applications. In order to improve the delivery of TFOs or siRNAs into the target cells, cloning of the lentiviral transfer vector SEW, the production of lentiviral particles by transient transfection were performed with the aim to generate lentiviral vector-derived TFOs in further experiments. Here, Th1 cells were transduced with infectious lentiviral particles and transduction efficacy was measured. Transduction efficacy higher than 82% could be achieved using the lentiviral vector SEW, opening optimal possibilities for the TFO or siRNA approach.
(Non)retroflexivity of slavic affricates and its motivation : Evidence from polish and czech <č>
(2005)
The goal of this paper is two-fold. First, it revises the common assumption that the affricate <č> denotes /t͡ʃ/ for all Slavic languages. On the basis of experimental results it is shown that Slavic <č> stands for two sounds: /t͡ʃ/ as e.g. in Czech and /ʈʂ/ as in Polish.
The second goal of the paper is to show that this difference is not accidental but it is motivated by perceptual relations among sibilants. In Polish, /t͡ʃ/ changed to /ʈʂ/ thus lowering its sibilant tonality and creating a better perceptual distance to /tɕ/, whereas in Czech /t͡ʃ/ did not turn to /ʈʂ/, as the former displayed sufficient perceptual distance to the only affricate present in the inventory, namely, the alveolar /t͡s/. Finally, an analysis of Czech and Polish affricate inventories is offered.
5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.
An account of the bryological activities on the Azores is given. Three phases can be distinguished: one at the end of the 19. century, when first collections of non-bryologists were made, which resulted in the discovery of less than one third of the presently known species and the description of many so called endemics. A second phase in the thirties of the 20. century is characterized by an intensive collecting activity of some bryologists, which enormously raised the species numbers. In a third phase at the end of the 20. century, tourism as well as activities of local bryologists raised again the species numbers. Revisions of genera reduced the number of endemic species and revisions of herbarium specimens of doubtful records even reduced the species numbers of hepatics.
A bryophyte checklist of the Ecological Reserve of Gurjaú (08º10’00'’-08º15’00"S; 35º02’30"- 35º05’00"W), a remnant of the Atlantic Forest in the State of Pernambuco, has been compiled. The Reserve covers an area of approximately 1362 ha, including several forest fragments of diverse sizes and stages of conservation. Specimens collected during the years 2000 and 2001, and voucher specimens from the UFP Herbarium at the Federal University of Pernambuco (UFPE) were analyzed. Bryophytes were collected on live and dead trunks, rocks, leaves and soil. The studied bryoflora is composed of 53 species of liverworts, 37 species of mosses and one hornwort. Among the twenty-three registered families, Lejeuneaceae (41 spp.) was the most representative one with the highest generic and specific richness, which confirms its status as predominant in tropical rain forests. Fissidens (7 spp.), Lejeunea and Cheilolejeunea (6 spp.), and Calymperes (4 spp.) were the most representative genera. The studied bryoflora is predominantly composed of species of neotropical distribution, widely spread throughout Brazil. The presence of an endemic species of the Atlantic Forest, Vitalianthus bichlerianus (Pôrto & Grolle) Schust. & Giancotti, and some other exclusively Brazilian species, e.g., Riccardia regnellii (Ängstr.) Hell have been recorded from the site.
The Distrito Federal, one of the most important centers of plant endemism in central Brazil, is located in the center of Brazil, with an area of 5,814 km2 at coordinates 15o30’ S, 16o03’W. Cerrado vegetation covers the region. The main city in the Distrito Federal is Brasília, the capital of Brazil with a population of 2.5 million. In the last two decades the anthropogenic action has reduced the original plant cover drastically. In this paper a checklist of the bryophytes (Marchantiophyta, Bryophyta and Anthocerotophyta) is presented. A total of 176 species are listed, 114 among Bryophyta, 61 among Marchantiophyta and 1 in the Anthocerotophyta. The families best represented are the Bryaceae and Sematophyllaceae (Bryophyta), and the Jubulaceae and Lejeuneaceae (Marchantiophya).
This paper proves correctness of Nöcker's method of strictness analysis, implemented in the Clean compiler, which is an effective way for strictness analysis in lazy functional languages based on their operational semantics. We improve upon the work of Clark, Hankin and Hunt did on the correctness of the abstract reduction rules. Our method fully considers the cycle detection rules, which are the main strength of Nöcker's strictness analysis. Our algorithm SAL is a reformulation of Nöcker's strictness analysis algorithm in a higher-order call-by-need lambda-calculus with case, constructors, letrec, and seq, extended by set constants like Top or Inf, denoting sets of expressions. It is also possible to define new set constants by recursive equations with a greatest fixpoint semantics. The operational semantics is a small-step semantics. Equality of expressions is defined by a contextual semantics that observes termination of expressions. Basically, SAL is a non-termination checker. The proof of its correctness and hence of Nöcker's strictness analysis is based mainly on an exact analysis of the lengths of normal order reduction sequences. The main measure being the number of 'essential' reductions in a normal order reduction sequence. Our tools and results provide new insights into call-by-need lambda-calculi, the role of sharing in functional programming languages, and into strictness analysis in general. The correctness result provides a foundation for Nöcker's strictness analysis in Clean, and also for its use in Haskell.
Multicomponent Tree Adjoining Grammars (MCTAG) is a formalism that has been shown to be useful for many natural language applications. The definition of MCTAG however is problematic since it refers to the process of the derivation itself: a simultaneity constraint must be respected concerning the way the members of the elementary tree sets are added. Looking only at the result of a derivation (i.e., the derived tree and the derivation tree), this simultaneity is no longer visible and therefore cannot be checked. I.e., this way of characterizing MCTAG does not allow to abstract away from the concrete order of derivation. Therefore, in this paper, we propose an alternative definition of MCTAG that characterizes the trees in the tree language of an MCTAG via the properties of the derivation trees the MCTAG licences.
Artificial drainage of agricultural land, for example with ditches or drainage tubes, is used to avoid water logging and to manage high groundwater tables. Among other impacts it influences the nutrient balances by increasing leaching losses and by decreasing denitrification. To simulate terrestrial transport of nitrogen on the global scale, a digital global map of artificially drained agricultural areas was developed. The map depicts the percentage of each 5’ by 5’ grid cell that is equipped for artificial drainage. Information on artificial drainage in countries or sub-national units was mainly derived from international inventories. Distribution to grid cells was based, for most countries, on the "Global Croplands Dataset" of Ramankutty et al. (1998) and the "Digital Global Map of Irrigation Areas" of Siebert et al. (2005). For some European countries the CORINE land cover dataset was used instead of the both datasets mentioned above. Maps with outlines of artificially drained areas were available for 6 countries. The global drainage area on the map is 167 Mio hectares. For only 11 out of the 116 countries with information on artificial drainage areas, sub-national information could be taken into account. Due to this coarse spatial resolution of the data sources, we recommended to use the map of artificially drained areas only for continental to global scale assessments. This documentation describes the dataset, the data sources and the map generation, and it discusses the data uncertainty.
The Land and Water Development Division of the Food and Agriculture Organization of the United Nations and the Johann Wolfgang Goethe University, Frankfurt am Main, Germany, are cooperating in the development of a global irrigation-mapping facility. This report describes an update of the Digital Global Map of Irrigated Areas for the continent of Asia. For this update, an inventory of subnational irrigation statistics for the continent was compiled. The reference year for the statistics is 2000. Adding up the irrigated areas per country as documented in the report gives a total of 188.5 million ha for the entire continent. The total number of subnational units used in the inventory is 4 428. In order to distribute the irrigation statistics per subnational unit, digital spatial data layers and printed maps were used. Irrigation maps were derived from project reports, irrigation subsector studies, and books related to irrigation and drainage. These maps were digitized and compared with satellite images of many regions. In areas without spatial information on irrigated areas, additional information was used to locate areas where irrigation is likely, such as land-cover and land-use maps that indicate agricultural areas or areas with crops that are usually grown under irrigation. Contents 1. Working Report I: Generation of a map of administrative units compatible with statistics used to update the Digital Global Map of Irrigated Areas in Asia 2. Working Report II: The inventory of subnational irrigation statistics for the Asian part of the Digital Global Map of Irrigated Areas 3. Working Report III: Geospatial information used to locate irrigated areas within the subnational units in the Asian part of the Digital Global Map of Irrigated Areas 4. Working Report IV: Update of the Digital Global Map of Irrigated Areas in Asia, Results Maps
The study offers a discourse-based account of the Spanish copula forms ser and estar, which are generally considered to be lexical exponents of the stage-level/individual-level contrast. It argues against the popular view that the distinction between SLPs and ILPs rests on a fundamental cognitive division of the world that is reflected in the grammar. As it happens, conceptual oppositions like “temporary vs. permanent” or “arbitrary vs. essential“ provide only a preference for the interpretation of estar and ser. In addition, the evidence for an SLP/ILP impact on the grammar turns out to be far less conclusive than is currently assumed. The study argues against event-based accounts of the ser/estar contrast in particular, showing that ser and estar pattern alike in failing all of the standard eventuality tests. The discourse-based account proposed instead assumes that ser and estar both display the same lexical semantics (which is identical to the semantics of English be, German sein, etc.); estar differs from ser only in presupposing a relation to a specific discourse situation. By using estar a speaker restricts his or her claim to a specific discourse situation, whereas by using ser, the speaker makes no such restriction. The preference for interpreting estar predications as denoting temporary properties and ser predications as denoting permanent properties follows from economy principles driving the pragmatic legitimation of estars discourse dependence. The analysis proposed in this paper can also account for the observation that ser predications do not give rise to thetic judgements. The proposal is couched in terms of the framework of DRT.