Rabbit muscle aldolase (RAMA) as a catalyst in a new approach for the synthesis of 3-deoxy-D-manno-2-octulosonic acid and analogues

A new approach leading to 3-deoxy-D-manno-2-octulosonic acid (KDO) and 4-deoxy-KDO is described. The key step is the formation of the C₅---C₆ bond catalysed by fructose-1,6-bisphosphate aldolase, which controls the stereochemistry of these two centres. The important step of the aldehyde substrates (1a, 1b, 1c) synthesis is the Barbier reaction of ethyl-bromomethylacrylate or bromomethylacrylonitrile with the monoacetal of glyoxal in the presence of indium.     

Distinct lytic vacuolar compartments are embedded inside the protein storage vacuole of dry and germinating Arabidopsis thaliana seeds

Plant cell vacuoles are diverse and dynamic structures. In particular, during seed germination, the protein storage vacuoles are rapidly replaced by a central lytic vacuole enabling rapid elongation of embryo cells. In this study, we investigate the dynamic remodeling of vacuolar compartments during Arabidopsis seed germination using immunocytochemistry with antibodies against tonoplast intrinsic protein (TIP) isoforms as well as proteins involved in nutrient mobilization and vacuolar acidification. Our results confirm the existence of a lytic compartment embedded in the protein storage vacuole of dry seeds, decorated by γ-TIP, the vacuolar proton pumping pyrophosphatase (V-PPase) and the metal transporter NRAMP4. They further indicate that this compartment disappears after stratification. It is then replaced by a newly formed lytic compartment, labeled by γ-TIP and V-PPase but not AtNRAMP4, which occupies a larger volume as germination progresses. Altogether, our results indicate the successive occurrence of two different lytic compartments in the protein storage vacuoles of germinating Arabidopsis cells. We propose that the first one corresponds to globoids specialized in mineral storage and the second one is at the origin of the central lytic vacuole in these cells.     

Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.      

Molecular models of NS3 protease variants of the Hepatitis C virus

Background  

Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed.     

Relationships between tree dimension and coarse root biomass in mixed stands of European beech (Fagus sylvatica L.) and Norway spruce (Picea abies[L.] Karst.)

Relationships between tree parameters above ground and the biomass of the coarse root system were examined in six mixed spruce-beech stands in the Solling Mountain region in northwest Germany. The selected stands were located on comparable sites and covered an age range of 44 to 114 years. Coarse roots (d?\ge?2 mm) of 42 spruce and 27 beech trees were sampled by excavating the entire root system. A linear model with logarithmic transformation of the variables was developed to describe the relationship between the coarse root biomass (CRB, dry weight) and the corresponding tree diameter at breast height (DBH). The coefficients of determination (R ²) attained values between 0.92 for spruce and 0.94 for beech; the logarithmic standard deviation values were between 0.29 and 0.43. A significantly different effect of tree species on the model estimates could not be detected by an analysis of co-variance (ANCOVA). For spruce, the derived relationships were similar to those reported in previous studies, but not for beech. Biomass partitioning in the tree compartments above and below ground differs significantly between spruce (coarse root/shoot ratio 0.16±0.06) and beech (coarse root/shoot ratio 0.10±0.03) in the mixed stands. These results are similar to those given in other studies involving pure spruce and beech stands on comparable sites in the region, although the ratios of pure stands in other regions growing under different site conditions are somewhat higher. Comparing trees of the same DBH classes, root/shoot ratios of spruce are 1.2 to 3 times higher than those of beech. Dominant spruce trees (DBH>60 cm) attained the highest ratios, suppressed beech trees (DBH<10 cm) the lowest. Site conditions of varying climate and soils and interspecific tree competition are likely to affect root/shoot ratio and DBH-coarse root biomass relationships. The greater variability in beech compared with spruce indicates a high 'plasticity' and adaptability of beech carbon allocation. Thus, the derived equations are useful for biomass estimates of coarse roots involving trees of different ages in mixed stands of spruce and beech in the Solling Mountains. However, application of these relationships to stands in other regions would need further testing.     

Reset of a critically disturbed microbial ecosystem: faecal transplant in recurrent Clostridium difficile infection

Recurrent Clostridium difficile infection (CDI) can be effectively treated by infusion of a healthy donor faeces suspension. However, it is unclear what factors determine treatment efficacy. By using a phylogenetic microarray platform, we assessed composition, diversity and dynamics of faecal microbiota before, after and during follow-up of the transplantation from a healthy donor to different patients, to elucidate the mechanism of action of faecal infusion. Global composition and network analysis of the microbiota was performed in faecal samples from nine patients with recurrent CDI. Analyses were performed before and after duodenal donor faeces infusion, and during a follow-up of 10 weeks. The microbiota data were compared with that of the healthy donors. All patients successfully recovered. Their intestinal microbiota changed from a low-diversity diseased state, dominated by Proteobacteria and Bacilli, to a more diverse ecosystem resembling that of healthy donors, dominated by Bacteroidetes and Clostridium groups, including butyrate-producing bacteria. We identified specific multi-species networks and signature microbial groups that were either depleted or restored as a result of the treatment. The changes persisted over time. Comprehensive and deep analyses of the microbiota of patients before and after treatment exposed a therapeutic reset from a diseased state towards a healthy profile. The identification of microbial groups that constitute a niche for C. difficile overgrowth, as well as those driving the reinstallation of a healthy intestinal microbiota, could contribute to the development of biomarkers predicting recurrence and treatment outcome, identifying an optimal microbiota composition that could lead to targeted treatment strategies.     

Evidence for para Dechlorination of Polychlorobiphenyls by Methanogenic Bacteria

When microorganisms eluted from upper Hudson River sediment were cultured without any substrate except polychlorobiphenyl (PCB)-free Hudson River sediment, methane formation was the terminal step of the anaerobic food chain. In sediments containing Aroclor 1242, addition of eubacterium-inhibiting antibiotics, which should have directly inhibited fermentative bacteria and thereby should have indirectly inhibited methanogens, resulted in no dechlorination activity or methane production. However, when substrates for methanogenic bacteria were provided along with the antibiotics (to free the methanogens from dependence on eubacteria), concomitant methane production and dechlorination of PCBs were observed. The dechlorination of Aroclor 1242 was from the para positions, a pattern distinctly different from, and more limited than, the pattern observed with untreated or pasteurized inocula. Both methane production and dechlorination in cultures amended with antibiotics plus methanogenic substrates were inhibited by 2-bromoethanesulfonic acid. These results suggest that the methanogenic bacteria are among the physiological groups capable of anaerobic dechlorination of PCBs, but that the dechlorination observed with methanogenic bacteria is less extensive than the dechlorination observed with more complex anaerobic consortia.     

Endocrine Profiles, Haematology and Pregnancy Outcomes of Late Pregnant Holstein Dairy Heifers Sired by Bulls Giving a High or Low Incidence of Stillbirth

The high incidence of stillbirth in Swedish Holstein heifers has increased continuously during the last 15 years to an average of 11% today. The pathological reasons behind the increased incidence of stillbirth are unknown. The present experiment was undertaken to investigate possible causes of stillbirth and to study possible physiological markers for predicting stillbirth. Twenty Swedish Holstein dairy heifers sired by bulls with breeding values for a high risk of stillbirth (n = 12) (experimental group) and a low risk of stillbirth (n = 8) (control group, group B) were selected based on information in the Swedish AI-data base. The experimental group consisted of 2 subgroups of heifers (groups A1 and A2) inseminated with 2 different bulls with 3.5% and 9% higher stillbirth rates than the average, and the control group consisted of heifers pregnant with 5 different bulls with 0%–6% lower stillbirth rates than the average. The bull used for group A1 had also calving difficulties due to large calves as compared to the bull in group A2 showing no calving difficulties. The heifers were supervised from 6–7 months of pregnancy up to birth, and the pregnancies and parturitions were compared between groups regarding hormonal levels, haematology, placental characteristics and calf viability. In group A1, 1 stillborn, 1 weak and 4 normal calves were recorded. In group A2, 2 stillborn and 4 normal calves were registered. All animals in the control group gave birth to a normal living calf without any assistance. The weak calf showed deviating profiles of body temperature, saturated oxygen and heart rates, compared with the normal living calves. No differences of the placentome thickness, measured in vivo by ultrasonography were seen between the groups. The number of leukocytes and differential cell counts in groups A1 and A2 followed the profiles found in the control group. In group A1, a slight decrease of oestrone sulphate (E1SO4) levels was found in the animal delivering a stillborn calf from the first 24-h blood sampling at 6 weeks to the second at 3 weeks prior to delivery, while the levels of E1SO4 at both periods in the animal delivering a weak calf followed the profile in animals delivering a normal living calf. During late pregnancy and at the time of parturition, the levels of E1SO4 and PAGs in animals delivering a stillborn or weak calf (from group A1) followed the normal profiles found in animals delivering a normal living calf. In group A2, low levels of E1SO4 and pregnancy associated glycoproteins (PAGs) over 24 h at both 3 and 6 weeks prior to parturition (<1.5 nmol/L) were recorded in animals delivering a stillborn calf. During late pregnancy and parturition, the levels of E1SO4 and PAGs were slightly lower during 30–50 days prior to delivery and increased with a lower magnitude at the time of parturition. In conclusion, our results indicate that the aetiology behind stillbirth varies depending on the AI-bulls used and is associated with dystocia or low viability of the calves. Deviating profiles of oestrone sulphate (E1SO4) and pregnancy associated glycoproteins (PAGs) in animals delivering a stillborn calf not caused by dystocia were observed, suggesting placental dysfunction as a possible factor. The finding suggests that the analyses of E1SO4 and PAGs could be used for monitoring foetal well-being in animals with a high risk of stillbirth at term.     

Identification and characterization of NAGNAG alternative splicing in the moss <Emphasis Type=Italic>Physcomitrella patens</Emphasis>

Background  

Alternative splicing (AS) involving tandem acceptors that are separated by three nucleotides (NAGNAG) is an evolutionarily widespread class of AS, which is well studied in Homo sapiens (human) and Mus musculus (mouse). It has also been shown to be common in the model seed plants Arabidopsis thaliana and Oryza sativa (rice). In one of the first studies involving sequence-based prediction of AS in plants, we performed a genome-wide identification and characterization of NAGNAG AS in the model plant Physcomitrella patens, a moss.     

Changes in genomic methylation patterns during the formation of triploid asexual dandelion lineages

DNA methylation is an epigenetic mechanism that has the potential to affect plant phenotypes and that is responsive to environmental and genomic stresses such as hybridization and polyploidization. We explored de novo methylation variation that arises during the formation of triploid asexual dandelions from diploid sexual mother plants using methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. In dandelions, triploid apomictic asexuals are produced from diploid sexual mothers that are fertilized by polyploid pollen donors. We asked whether the ploidy level change that accompanies the formation of new asexual lineages triggers methylation changes that contribute to heritable epigenetic variation within novel asexual lineages. Comparison of MS‐AFLP and AFLP fragment inheritance in a diploid × triploid cross revealed de novo methylation variation between triploid F₁ individuals. Genetically identical offspring of asexual F₁ plants showed modest levels of methylation variation, comparable to background levels as observed among sibs in a long‐established asexual lineage. Thus, the cross between ploidy levels triggered de novo methylation variation between asexual lineages, whereas it did not seem to contribute directly to variation within new asexual lineages. The observed background level of methylation variation suggests that considerable autonomous methylation variation could build up within asexual lineages under natural conditions.