Ferrocenyl-substituted allenylidene complexes of chromium, molybdenum and tungsten: Synthesis, structure and reactivity

Bis(ferrocenyl)-substituted allenylidene complexes, [(CO)₅MCCCFc₂] (1a-c, Fc = (C₅H₄)Fe(C₅H₅), M = Cr (a), Mo (b), W (c)) were obtained by sequential reaction of Fc₂CO with Me₃Si-CCH, KF/MeOH, n-BuLi, and [(CO)₅M(THF)]. For the synthesis of related mono(ferrocenyl)allenylidene chromium complexes, [(CO)₅CrCCC(Fc)R] (R = Ph, NMe₂), three different routes were developed: (a) reaction of the deprotonated propargylic alcohol HCCC(Fc)(Ph)OH with [(CO)₅Cr(THF)] followed by desoxygenation with Cl₂CO, (b) Lewis acid induced alcohol elimination from alkenyl(alkoxy)carbene complexes, [(CO)₅CrC(OR)CHC(NMe₂)Fc], and (c) replacement of OMe in [(CO)₅CrCCC(OMe)NMe₂] by Fc. Complex 1a was also formed when the mono(ferrocenyl)allenylidene complex [(CO)₅CrCCC(Fc)NMe₂] was treated first with Li[Fc] and the resulting adduct then with SiO₂. The replacement route (c) was also applied to the synthesis of an allenylidene complex (7a) with a CC spacer in between the ferrocenyl unit and C_γ of the allenylidene ligand, [(CO)₅CrCCC(NMe₂)-CCFc]. The related complex containing a CHCH spacer (9a) was prepared by condensation of [(CO)₅CrCCC(Me)NMe₂] with formylferrocene in the presence of NEt₃. The bis(ferrocenyl)-substituted allenylidene complexes 1a-c added HNMe₂ across the C_α-C_β bond to give alkenyl(dimethylamino)carbene complexes and reacted with diethylaminopropyne by regioselective insertion of the CC bond into the C_β-C_γ bond to afford alkenyl(diethylamino)allenylidene complexes, [(CO)₅MCCC(NEt₂)CMeCFc₂]. The structures of 5a, 7a, and 9a were established by X-ray diffraction studies.     

Solving the structure of PTB in complex with pyrimidine tracts: an NMR study of protein-RNA complexes of weak affinities

NMR spectroscopy has proven to be a powerful tool for the structure determination of protein/RNA complexes. However, the quality of these structures depends critically on the number of unambiguous intermolecular and intra-RNA nuclear Overhauser effect (NOE) constraints that can be derived. This number is often limited due to exchange phenomena that can cause signal line broadening and the fact that unambiguous NOE assignments are challenging in systems that exchange between different conformations in the intermediate to fast exchange limit. These exchange processes can include exchange between free and bound form, as well as exchange of the ligand between different binding sites on the protein. Furthermore, for the large class of RNA metabolizing proteins that bind repetitive low-complexity RNA sequences in multiple register, exchange of the protein between these overlapping binding sites introduces additional exchange pathways. Here, we describe the strategy we used to overcome these exchange processes and to reduce significantly the line width of the RNA resonances in complexes of the RNA recognition motifs (RRMs) of the polypyrimidine tract-binding protein (PTB) in complex with pyrimidine tracts and hence allowed a highly precise structure determination. This method could be employed to derive structures of other protein/single-stranded nucleic acid complexes by NMR spectroscopy. Furthermore, we have determined the affinities of the individual RRMs of PTB for pyrimidine tracts of different length and sequence. These measurements show that PTB binds preferentially to long pyrimidine tracts that contain cytosine and hence confirm the structure of PTB in complex with RNA. Furthermore, they provide quantitative insight into the question of which pyrimidine sequences within alternatively spliced pre-mRNAs will be preferentially bound by PTB.     

Effect of alternative NAD<Superscript>+</Superscript>-regenerating pathways on the formation of primary and secondary aroma compounds in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> glycerol-defective mutant

Saccharomyces cerevisiae maintains a redox balance under fermentative growth conditions by re-oxidizing NADH formed during glycolysis through ethanol formation. Excess NADH stimulates the synthesis of mainly glycerol, but also of other compounds. Here, we investigated the production of primary and secondary metabolites in S. cerevisiae strains where the glycerol production pathway was inactivated through deletion of the two glycerol-3-phosphate dehydrogenases genes (GPD1/GPD2) and replaced with alternative NAD⁺-generating pathways. While these modifications decreased fermentative ability compared to the wild-type strain, all improved growth and/or fermentative ability of the gpd1Δgpd2Δ strain in self-generated anaerobic high sugar medium. The partial NAD⁺ regeneration ability of the mutants resulted in significant amounts of alternative products, but at lower yields than glycerol. Compared to the wild-type strain, pyruvate production increased in most genetically manipulated strains, whereas acetate and succinate production decreased in all strains. Malate production was similar in all strains. Isobutanol production increased substantially in all genetically manipulated strains compared to the wild-type strain, whereas only mutant strains expressing the sorbitol producing SOR1 and srlD genes showed increases in isoamyl alcohol and 2-phenyl alcohol. A marked reduction in ethyl acetate concentration was observed in the genetically manipulated strains, while isobutyric acid increased. The synthesis of some primary and secondary metabolites appears more readily influenced by the NAD⁺/NADH availability. The data provide an initial assessment of the impact of redox balance on the production of primary and secondary metabolites which play an essential role in the flavour and aroma character of beverages.     

Preparative enzymatic solid phase synthesis of cis(+)-12-oxo-phytodienoic acid - physical interaction of AOS and AOC is not necessary

The pathway of jasmonic acid (JA) biosynthesis was established in the 1980s by Vick and Zimmerman but, until now, the preparative biosynthesis of the jasmonic acid precursors 12-oxo-phytodienoic acid (OPDA) and 3-oxo-2-[2'-pentenyl]-cyclopentan-1-octanoic acid (OPC-8:0) in their endogenous and biologically relevant cis(+)-configuration was only possible in small amounts and had to put up with high costs. This was mainly due to the lack of high amounts of pure and enzymatically active allene oxide cyclase (AOC), which is a key enzyme in the biosynthesis of jasmonates in that it releases, in a coupled reaction with allene oxide synthase (AOS), the first cyclic and biological active metabolite - OPDA. We describe here the expression and purification of AOS and AOC and their subsequent coupling to solid matrices to produce an enantioselective, reusable bioreactor for octadecanoid production. With the method described here it is possible to produce optically pure enantiomers of octadecanoids in high amounts in a cost- and time-efficient manner. Furthermore, it could be demonstrated that a physical interaction of AOS and AOC, hitherto postulated to be required for substrate channeling from AOS to AOC, is not necessary for the in vitro cyclization of the unstable epoxide generated by the AOS reaction.     

The NOD2 single nucleotide polymorphisms rs2066843 and rs2076756 are novel and common Crohn's disease susceptibility gene variants

Background

The aims were to analyze two novel NOD2 variants (rs2066843 and rs2076756) in a large cohort of patients with inflammatory bowel disease and to elucidate phenotypic consequences.

Methodology/Principal Findings

Genomic DNA from 2700 Caucasians including 812 patients with Crohn''s disease (CD), 442 patients with ulcerative colitis (UC), and 1446 healthy controls was analyzed for the NOD2 SNPs rs2066843 and rs2076756 and the three main CD-associated NOD2 variants p.Arg702Trp (rs2066844), p.Gly908Arg (rs2066847), and p.Leu1007fsX1008 (rs2066847). Haplotype and genotype-phenotype analyses were performed. The SNPs rs2066843 (p = 3.01×10⁻⁵, OR 1.48, [95% CI 1.23-1.78]) and rs2076756 (p = 4.01×10⁻⁶; OR 1.54, [95% CI 1.28-1.86]) were significantly associated with CD but not with UC susceptibility. Haplotype analysis revealed a number of significant associations with CD susceptibility with omnibus p values <10⁻¹⁰. The SNPs rs2066843 and rs2076756 were in linkage disequilibrium with each other and with the three main CD-associated NOD2 mutations (D''>0.9). However, in CD, SNPs rs2066843 and rs2076756 were more frequently observed than the other three common NOD2 mutations (minor allele frequencies for rs2066843 and rs2076756: 0.390 and 0.380, respectively). In CD patients homozygous for these novel NOD2 variants, genotype-phenotype analysis revealed higher rates of a penetrating phenotype (rs2076756: p = 0.015) and fistulas (rs2076756: p = 0.015) and significant associations with CD-related surgery (rs2076756: p = 0.003; rs2066843: p = 0.015). However, in multivariate analysis only disease localization (p<2×10⁻¹⁶) and behaviour (p = 0.02) were significantly associated with the need for surgery.

Conclusion/Significance

The NOD2 variants rs2066843 and rs2076756 are novel and common CD susceptibility gene variants.     

Efficient analysis and extraction of MS/MS result data from Mascot™ result files

Background  

Mascot™ is a commonly used protein identification program for MS as well as for tandem MS data. When analyzing huge shotgun proteomics datasets with Mascot™'s native tools, limits of computing resources are easily reached. Up to now no application has been available as open source that is capable of converting the full content of Mascot™ result files from the original MIME format into a database-compatible tabular format, allowing direct import into database management systems and efficient handling of huge datasets analyzed by Mascot™.     

PIT-tagging Italian spined loach (Cobitis bilineata): Methodology,survival and behavioural effects

The Italian spined loach (Cobitis bilineata) is an elongated, small-sized (<12 cm) spined loach native to northern Italy, Slovenia and Croatia. As for loaches in general, little is known about the individual movements of this loach in nature. Passive integrated transponders (PIT-tags) are small (typically 7–32 mm), relatively cheap and allow tracking of individual fish movements and behaviour. A fundamental assumption in animal telemetry is that the performance of a tagged animal does not deviate substantially from its natural performance. Although PIT-tagged fish often display high survival and tag retention, the effect varies between species and contexts, and few studies have looked at behavioural effects of PIT-tagging. Here we demonstrate a PIT-tagging methodology for spined loaches, and compare survival, activity and provoked escape response (maximum swimming speed) between tagged and control fish. We also track tag retention in the tagged fish. Italian spined loaches tagged with 12 mm PIT-tags displayed high tag retention and no extra mortality, and no effects of tagging on activity or maximum swimming speed were observed. The tag-to-fish weight and length ratios in our study ranged from 2% to 5% and from 10% to 16%, respectively, and we conclude that PIT-tagging, within these ratios, appears suitable for Italian spined loach.     

Plasma transport and tissue distribution of β-carotene, vitamin A and retinol-binding protein in domestic cats

The objective of this study was to determine retinol, retinyl esters and retinol-binding protein (RBP) as well as carotenoids in plasma, urine, liver and kidneys of randomly selected domestic cats. Retinol (240±64 ng/ml, mean±S.D.) represented one-third of total retinyl esters (736±460 ng/ml) in plasma. Retinyl esters were stearate, palmitate and oleate representing 61±6, 36±13 and 5±3% of total retinyl esters, respectively. In half of the cats, retinyl esters (22±21 ng/ml) were found in the urine. Vitamin A in the livers (4317±1956 μg/g) was significantly higher than in the kidney cortex and medulla (14.16±8.92 and 7.59±4.52 μg/g, respectively, both P<0.001). RBP was detected in the plasma but not in the urine. Immunoreactive RBP was observed in hepatocytes and in the cells of the proximal tubules. β-Carotene was present in plasma but never in tissues. The results show that similar to canines differences in vitamin A metabolism in cats are related to the occurrence of retinyl esters in plasma. They differ, however, with regard to the tissue distribution of β-carotene and the excretion of vitamin A in the urine.     

Sapling communities in Amazonian white-water forests

Aim Structure and floristic composition of forest regeneration (trees between 1 and 10 cm diameter at breast height (d.b.h), ≥ 1 m growth height) was described in three forest types of Amazonian white‐water forests (várzea), in order to analyse whether floristic composition of saplings is related to the successional stage of the forests, whether it differs in comparision with the mature flora, and if there exists a zonation of sapling species along the gradients of flooding and irradiation. Location Mamirauá Sustainable Development Reserve, Western Brazilian Amazon. Methods The investigated forests were of the low and the high várzea type, on an annual average flooded around 4 and 1 months, respectively. The two low‐várzea forests belonged to the secondary and to the late‐successional stage, the high‐várzea forest also to the late‐successional stage. A total of 24 circular sample plots covering 1885 m² were installed. They were nested within three rectangular 1 ha permanent sample plots where individuals ≥ 10 cm d.b.h. were formerly inventoried. Average inundation and radiation [relative photosynthetically active radiation (rPAR)] at the forest floor was recorded in all inventoried plots. The Mean spatial Distribution Center (MDC; Ebdon, 1998 ) for all sapling species was calculated. Sapling species were grouped into associations with respect to their light‐demand and their location along the flood‐level gradient. Results Average flood height and average rPAR at the forest floor was highest in the secondary stage of the low várzea and lowest in the high‐várzea forest. Overall average density and basal area of the saplings averaged 2250 individuals and 2.3 m² ha^?1 in the secondary stage, 2330 individuals and 2.6 m² ha^?1 in the late‐successional stage of the low várzea and 5000 individuals and 4.8 m² ha^?1 in the high várzea. In all forest types, species richness of saplings was lower than species richness of trees ≥ 10 cm d.b.h., and amounted to 25 species in the secondary stage, to 35 species in the late‐successional stage of the low várzea and to 88 species in the high‐várzea forest. The amount of ‘immigrants’ ( Bazzaz, 1991 ) at the sapling level increased with proceeding forest succession and amounted to 24 and 29% in the low várzea and to 31% in the high várzea. Sapling species distribution, species richness and individual density was linked to both, the gradient of flooding and the gradient of irradiation. Species richness and individual density was highest in the high várzea and decreased with increasing influence of flooding. The high várzea was predominated by pronounced shade‐tolerant sapling species, whereas in the low várzea occured both, light demanding and shade‐tolerant species groups. Main conclusions Influence of flooding seems to be the main factor triggering species composition and structure of sapling communities in várzea forests. Proceeding forest succession reduces the impact of flooding because of the biogenical induced silting up of the forested sites. However, forest succession also alters forest architecture of the overstory and such the light conditions at the sapling level. Therefore, radiation is an important factor influencing species composition of sapling communities in várzea forests.