Polypyrimidine tract‐binding protein homologues from Arabidopsis underlie regulatory circuits based on alternative splicing and downstream control

Alternative splicing (AS) of precursor mRNAs is a widespread phenomenon in plants; however, many questions, especially regarding its regulation and functional implications, remain to be elucidated. In vertebrates, polypyrimidine tract‐binding proteins (PTBs) have been identified as key splicing factors influencing splice site selection and orchestrating coordinated splicing programmes during developmental processes. Here, we analysed three PTB homologues from Arabidopsis thaliana and provide evidence for their gene regulatory potential based on AS and a splicing‐independent mechanism. Our data reveal that Arabidopsis PTB homologues are subject to extensive auto‐ and cross‐regulation via AS‐coupled nonsense‐mediated decay, thereby establishing a basis for interlinking their expression. Furthermore, the multiple modes of action of Arabidopsis PTB homologues are reflected in their subcellular localization in the nucleus, cytosol and processing bodies. This work provides insight into the regulation of AS in plants and highlights the regulatory potential of the multifunctional plant PTB homologues, which might have important implications in diverse biological processes.     

Novel tetrahydrochinoline derived CETP inhibitors

In the course of our efforts to identify orally active cholesteryl ester transfer protein (CETP) inhibitors, we have continued to explore tetrahydrochinoline derivatives. Based on BAY 19-4789 structural modifications led to the discovery of novel cycloalkyl substituted compounds. Thus, example 11b is a highly potent CETP inhibitor both in vitro and in vivo in transgenic mice with favourable pharmacokinetic properties for clinical development.     

Fungal metabolites: structural diversity as incentive for anticancer drug development

Natural products play an important role in the development of anticancer drugs. To date, predominantly metabolites from plants and bacteria served as lead structures for anticancer agents. Fungal metabolites and derivatives thereof are much less investigated for their potential in cancer therapy. There are, however, some promising candidates derived from fungi in clinical phases I and II studies. This review gives an overview on the role of natural products in cancer therapy and summarises some of the latest results of our group in this area.     

Modelling of the provisional side-branch stenting approach for the treatment of atherosclerotic coronary bifurcations: effects of stent positioning

The most common approach to treat atherosclerosis in coronary bifurcations is the provisional side-branch (PSB) stenting, which consists sequentially of the insertion of a stent in the main branch (MB) of the bifurcation and a dilatation of the side branch (SB) passing through the struts of the stent at the bifurcation. This approach can be followed by a redilatation of the MB only or by a Final Kissing Balloon (FKB) inflation, both strategies leading to a minor stent distortion in the MB. The positioning of the stent struts in the bifurcation and the stresses generated in the stent and vessel wall are worthy of investigation for a better understanding of the mechanobiology of the system. For this purpose, a computer model of an atherosclerotic coronary bifurcation based on the finite element method was developed; the effects of performing the final redilatation with the two strategies utilising one or two balloons and those created by a different stent strut positioning around the SB were investigated. Results correlate well with previous experimental tests regarding the deformation following balloon expansion. Furthermore, results confirm firstly that the re-establishment of an optimal spatial configuration of the stent after the PSB approach is achieved with both strategies; secondly, results show that case of stent positioning with one cell placed centrally (with regard to the SB) should be preferred, avoiding the presence of struts inside the vessel lumen, which may reduce hemodynamic disturbances. The central positioning also resulted in a better solution in terms of lower stresses in the stent struts and, more importantly, in the vascular tissues.     

Continuous Turnover of Carotenes and Chlorophyll a in Mature Leaves of Arabidopsis Revealed by 14CO2 Pulse-Chase Labeling

Carotenoid turnover was investigated in mature leaves of Arabidopsis (Arabidopsis thaliana) by ¹⁴CO₂ pulse-chase labeling under control-light (CL; 130 μmol photons m⁻² s⁻¹) and high-light (HL; 1,000 μmol photons m⁻² s⁻¹) conditions. Following a 30-min ¹⁴CO₂ administration, photosynthetically fixed ¹⁴C was quickly incorporated in β-carotene (β-C) and chlorophyll a (Chl a) in all samples during a chase of up to 10 h. In contrast, ¹⁴C was not detected in Chl b and xanthophylls, even when steady-state amounts of the xanthophyll-cycle pigments and lutein increased markedly, presumably by de novo synthesis, in CL-grown plants under HL. Different light conditions during the chase did not affect the ¹⁴C fractions incorporated in β-C and Chl a, whereas long-term HL acclimation significantly enhanced ¹⁴C labeling of Chl a but not β-C. Consequently, the maximal ¹⁴C signal ratio between β-C and Chl a was much lower in HL-grown plants (1:10) than in CL-grown plants (1:4). In lut5 mutants, containing α-carotene (α-C) together with reduced amounts of β-C, remarkably high ¹⁴C labeling was found for α-C while the labeling efficiency of Chl a was similar to that of wild-type plants. The maximum ¹⁴C ratios between carotenes and Chl a were 1:2 for α-C:Chl a and 1:5 for β-C:Chl a in CL-grown lut5 plants, suggesting high turnover of α-C. The data demonstrate continuous synthesis and degradation of carotenes and Chl a in photosynthesizing leaves and indicate distinct acclimatory responses of their turnover to changing irradiance. In addition, the results are discussed in the context of photosystem II repair cycle and D1 protein turnover.Carotenoids are classified as accessory pigments in photosynthesis because they augment light harvesting in the blue spectral region by transferring the absorbed light energy to chlorophyll (Chl). However, the universal occurrence of carotenoids in photosynthetic cells, from bacteria to higher plants, indicates their essential roles, rather than mere accessory roles, in photosynthesis. Under excess light, carotenoids provide protection against photooxidative damage by facilitating dissipation of excitation energy from singlet- or triplet-state Chl and scavenging highly reactive singlet oxygen, which is generated through interaction between triplet excited Chl and oxygen (Demmig-Adams, 1990; Müller et al., 2001). These photoprotective functions make carotenoids indispensable for oxygenic photosynthesis, as demonstrated by lethal effects of inhibitors of carotenoid biosynthesis in plants (Bramley, 1993). Regulation of light harvesting and photoprotection by carotenoids requires their close proximity as well as the proper orientation to Chl molecules in pigment-protein complexes of PSI and PSII. Furthermore, a small fraction of non-protein-bound carotenoids serves as antioxidants in the lipid phase of photosynthetic membranes (Havaux and Niyogi, 1999; Havaux et al., 2004) and influences the structure and fluidity of the lipid bilayer (Gruszecki and Strzałka, 2005). Despite these and other lines of defense, the PSII reaction center polypeptide D1, and to a lesser extent also D2, undergo frequent photooxidative damage and repair in the light (Melis, 1999; Baena-González and Aro, 2002). When the repair process cannot keep up with the rate of photodamage, the overall quantum yield of PSII declines.Carotenoids are derived from isoprenoid precursors in plastids (for reviews on carotenoid biosynthesis in plants, see Lichtenthaler, 1999; Hirschberg, 2001; DellaPenna and Pogson, 2006; Giuliano et al., 2008; Tanaka et al., 2008; Cazzonelli and Pogson, 2010). Following the formation of linear C₄₀ lycopene, the pathway splits into two branches of major cyclic carotenoids: the β,β-branch gives rise to β-carotene (β-C) having two β-rings, whereas the β,ϵ-branch leads to formation of α-carotene (α-C) having one β-ring and one ϵ-ring. Hydroxylation of β-C and α-C produces the xanthophylls zeaxanthin (Z) and lutein (L), respectively. In the β,β-branch, epoxidation of the β-rings of Z results in successive synthesis of antheraxanthin (A) and violaxanthin (V); subsequently, V can be converted to neoxanthin (N), the last carotenoid product of the β,β-branch. Except for some species (García-Plazaola et al., 2007), L does not undergo β-ring epoxidation and the β,ϵ-branch thus stops with L, the most abundant carotenoid in leaves.Each of these carotenoids occupies specific binding sites in the photosynthetic apparatus to fulfill distinct roles. In both PSI and PSII, carotenes (α-C and β-C) are generally bound in core complexes, which also harbor Chl a molecules, while the majority of xanthophylls (L, Z, A, V, and N) are bound in light-harvesting antenna complexes together with Chl a and Chl b molecules (Bassi et al., 1993; Lee and Thornber, 1995). Accumulation of β-C in core complexes is a common feature of diverse photosynthetic organisms, whereas the occurrence of α-C in addition to β-C is restricted to certain taxa. For higher plants, α-C has been found in leaves of some, but not all, shade-tolerant species (Thayer and Björkman, 1990; Demmig-Adams and Adams, 1992; Demmig-Adams, 1998; Matsubara et al., 2009). Based on this photoacclimatory behavior, it has been proposed that α-C may function as a light-harvesting pigment while β-C may contribute to photoprotection (Krause et al., 2001), presumably by scavenging singlet oxygen and mediating a cyclic electron transfer around PSII (Tracewell et al., 2001; Telfer, 2005).Pronounced light-dependent changes are also observed for xanthophyll composition in light-harvesting antenna complexes. In a short term (minutes to hours), operation of the xanthophyll cycle, involving Z, A, and V, modulates levels of Z in a light-dependent manner. It is widely accepted that Z is able to enhance the dissipation of excess light energy from singlet excited Chl while V is not (Demmig-Adams, 1990; Müller et al., 2001). Long-term acclimation (days) to strong irradiance typically results in an increased pool size of the xanthophyll-cycle pigments (V + A + Z) and downsizing of PSII antenna, as indicated by a greater Chl a-to-Chl b ratio (Demmig-Adams and Adams, 1992; Demmig-Adams, 1998; Matsubara et al., 2009). Based on the observed changes in steady-state amounts of xanthophylls and carotenes following irradiance shifts, alterations in the balance between biosynthesis and degradation, or turnover, have been implicated as a mechanism for long-term adjustment of carotenoid levels in leaves (Förster et al., 2009). However, just how much biosynthesis and degradation of different carotenoids occurs in photosynthesizing green leaves is an open question to date.In order to gain insight into carotenoid turnover of mature leaves, we conducted ¹⁴CO₂ pulse-chase labeling experiments with Arabidopsis (Arabidopsis thaliana) plants. Carotenoid turnover has been studied in algae in the past by applying [¹⁴C]bicarbonate (Blass et al., 1959; Grumbach et al., 1978); for example, no more than 1% to 2% of the photosynthetically incorporated ¹⁴C was allocated to the lipophilic fraction containing Chl and carotenoid in Chlorella pyrenoidosa after a 2-h pulse application (Grumbach et al., 1978). Even lower labeling efficiency is expected for photosynthetic pigments in nongrowing green leaves, in which pigment turnover takes place almost exclusively as part of the maintenance and acclimation of photosynthetic membranes. To overcome this intrinsic but anticipated difficulty, a ¹⁴CO₂ application setup was established for efficient and reproducible ¹⁴CO₂ incorporation in detached leaves of Arabidopsis during a short (30-min) pulse period. Moreover, a method of pigment separation was developed for ¹⁴C detection in concentrated leaf pigment extracts using a radio-HPLC system. Because carotenoid composition exhibits marked sun-shade responses in leaves (Demmig-Adams and Adams, 1992; Demmig-Adams, 1998; Matsubara et al., 2009), ¹⁴CO₂ labeling patterns were studied in three different sets of Arabidopsis plants: (1) plants grown under 130 μmol photons m⁻² s⁻¹ (control light [CL]) and exposed to CL during a chase period of up to 10 h (CL plants); (2) plants acclimated to 1,000 μmol photons m⁻² s⁻¹ (high light [HL]) for 2 weeks and exposed to HL during the chase period (HL plants); and (3) plants grown under CL but exposed to HL during the chase period (CL→HL plants). These treatments simulated short-term (CL→HL) and long-term (CL or HL) responses to irradiance. Finally, as ¹⁴C was found to be rapidly incorporated in β-C and Chl a molecules in leaves of wild-type plants, in which β-C represents the only carotene species, ¹⁴C labeling experiments were also conducted with leaves of lut5 mutants containing both α-C and β-C (Fiore et al., 2006; Kim and DellaPenna, 2006) to compare turnover of the two carotenes.     

The SSR-based molecular profile of 1005 grapevine (Vitis vinifera L.) accessions uncovers new synonymy and parentages, and reveals a large admixture amongst varieties of different geographic origin

A collection of 1005 grapevine accessions was genotyped at 34 microsatellite loci (SSR) with the aim of analysing genetic diversity and exploring parentages. The comparison of molecular profiles revealed 200 groups of synonymy. The removal of perfect synonyms reduced the database to 745 unique genotypes, on which population genetic parameters were calculated. The analysis of kinship uncovered 74 complete pedigrees, with both parents identified. Many of these parentages were not previously known and are of considerable historical interest, e.g. Chenin blanc (Sauvignon × Traminer rot), Covè (Harslevelu selfed), Incrocio Manzoni 2–14 and 2–15 (Cabernet franc × Prosecco), Lagrein (Schiava gentile × Teroldego), Malvasia nera of Bolzano (Perera × Schiava gentile), Manzoni moscato (Raboso veronese × Moscato d’Amburgo), Moscato violetto (Moscato bianco × Duraguzza), Muscat of Alexandria (Muscat blanc à petit grain × Axina de tres bias) and others. Statistical robustness of unexpected pedigrees was reinforced with the analysis of an additional 7–30 SSRs. Grouping the accessions by profile resulted in a weak correlation with their geographical origin and/or current area of cultivation, revealing a large admixture of local varieties with those most widely cultivated, as a result of ancient commerce and population flow. The SSRs with tri- to penta-nucleotide repeats adopted for the present study showed a great capacity for discriminating amongst accessions, with probabilities of identity by chance as low as 1.45 × 10^?27 and 9.35 × 10^?12 for unrelated and full sib individuals, respectively. A database of allele frequencies and SSR profiles of 32 reference cultivars are provided.     

IncP-1-beta plasmid pGNB1 isolated from a bacterial community from a wastewater treatment plant mediates decolorization of triphenylmethane dyes

Plasmid pGNB1 was isolated from bacteria residing in the activated sludge compartment of a wastewater treatment plant by using a transformation-based approach. This 60-kb plasmid confers resistance to the triphenylmethane dye crystal violet and enables its host bacterium to decolorize crystal violet. Partial sequencing of pGNB1 revealed that its backbone is very similar to that of previously sequenced IncP-1beta plasmids. The two accessory regions of the plasmid, one located downstream of the replication initiation gene trfA and the other located between the conjugative transfer modules Tra and Trb, were completely sequenced. Accessory region L1 contains a transposon related to Tn5501 and a gene encoding a Cupin 2 conserved barrel protein with an unknown function. The triphenylmethane reductase gene tmr and a truncated dihydrolipoamide dehydrogenase gene that is flanked by IS1071 and another putative insertion element were identified in accessory region L2. Subcloning of the pGNB1 tmr gene demonstrated that this gene is responsible for the observed crystal violet resistance phenotype and mediates decolorization of the triphenylmethane dyes crystal violet, malachite green, and basic fuchsin. Plasmid pGNB1 and the associated phenotype are transferable to the alpha-proteobacterium Sinorhizobium meliloti and the gamma-proteobacterium Escherichia coli. This is the first report of a promiscuous IncP-1beta plasmid isolated from the bacterial community from a wastewater treatment plant that harbors a triphenylmethane reductase gene. The pGNB1-encoded enzyme activity is discussed with respect to bioremediation of sewage polluted with triphenylmethane dyes.     

A marine Mesorhizobium sp. produces structurally novel long-chain N-acyl-L-homoserine lactones

Our study focused on a Mesorhizobium sp. that is phylogenetically affiliated by 16S rRNA gene sequence to other marine and saline bacteria of this genus. Liquid chromatography-mass spectrometry investigations of the extract obtained from solid-phase extraction of cultures of this bacterium indicated the presence of several N-acyl homoserine lactones (AHLs), with chain lengths of C(10) to C(16). Chromatographic separation of the active bacterial extract yielded extraordinarily large amounts of two unprecedented acylated homoserine lactones, 5-cis-3-oxo-C(12)-homoserine lactone (5-cis-3-oxo-C(12)-HSL) (compound 1) and 5-cis-C(12)-HSL (compound 2). Quorum-sensing activity of compounds 1 and 2 was shown in two different biosensor systems [Escherichia coli MT102(pSB403) and Pseudomonas putida F117(pKR-C12)]. Furthermore, it was shown that both compounds can restore protease and pyoverdin production of an AHL-deficient Pseudomonas aeruginosa PAO1 lasI rhlI double mutant, suggesting that these signal molecules maybe used for intergenus signaling. In conclusion, these data indicate that the quorum-sensing activity of compounds 1 and 2 is modulated by the chain length and functional groups of the acyl moiety. Additionally, compound 1 showed antibacterial and cytotoxic activities.