Diversity and vertical distribution of magnetotactic bacteria along chemical gradients in freshwater microcosms

The vertical distribution of magnetotactic bacteria along various physico-chemical gradients in freshwater microcosms was analyzed by a combined approach of viable cell counts, 16S rRNA gene analysis, microsensor profiling and biogeochemical methods. The occurrence of magnetotactic bacteria was restricted to a narrow sediment layer overlapping or closely below the maximum oxygen and nitrate penetration depth. Different species showed different preferences within vertical gradients, but the largest proportion (63-98%) of magnetotactic bacteria was detected within the suboxic zone. In one microcosm the community of magnetotactic bacteria was dominated by one species of a coccoid "Alphaproteobacterium", as detected by denaturing gradient gel electrophoresis in sediment horizons from 1 to 10 mm depth. Maximum numbers of magnetotactic bacteria were up to 1.5 x 10(7) cells/cm3, which corresponded to 1% of the total cell number in the upper sediment layer. The occurrence of magnetotactic bacteria coincided with the availability of significant amounts (6-60 microM) of soluble Fe(II), and in one sample with hydrogen sulfide (up to 40 microM). Although various trends were clearly observed, a strict correlation between the distribution of magnetotactic bacteria and individual geochemical parameters was absent. This is discussed in terms of metabolic adaptation of various strains of magnetotactic bacteria to stratified sediments and diversity of the magnetotactic bacterial communities.     

Unc5B Interacts with FLRT3 and Rnd1 to Modulate Cell Adhesion in Xenopus Embryos

The FLRT family of transmembrane proteins has been implicated in the regulation of FGF signalling, neurite outgrowth, homotypic cell sorting and cadherin-mediated adhesion. In an expression screen we identified the Netrin receptors Unc5B and Unc5D as high-affinity FLRT3 interactors. Upon overexpression, Unc5B phenocopies FLRT3 and both proteins synergize in inducing cell deadhesion in Xenopus embryos. Morpholino knock-downs of Unc5B and FLRT3 synergistically affect Xenopus development and induce morphogenetic defects. The small GTPase Rnd1, which transmits FLRT3 deadhesion activity, physically and functionally interacts with Unc5B, and mediates its effect on cell adhesion. The results suggest that FLRT3, Unc5B and Rnd1 proteins interact to modulate cell adhesion in early Xenopus development.     

Checkpoint kinase 1 negatively regulates somatic hypermutation

Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis.     

Preparative free-solution isotachophoresis for separation of human plasma lipoproteins: apolipoprotein and lipid composition of HDL subfractions

We have previously shown that plasma lipoproteins can be separated by analytical capillary isotachophoresis (ITP) according to their electrophoretic mobility in a defined buffer system. As in lipoprotein electrophoresis, HDL show the highest mobility followed by VLDL, IDL, and LDL. Chylomicrons migrate according to their net-charge between HDL and VLDL, because ITP has negligible molecular sieve effects. Three HDL subfractions were obtained which were designated fast-, intermediate-, and slow-migrating HDL. To further characterize these HDL subfractions, a newly developed free-solution ITP (FS-ITP)-system was used, that allows micro-preparative separation of human lipoproteins directly from whole plasma (B?ttcher, A. et al. 1998. Electrophoresis. 19: 1110-1116). The fractions obtained by FS-ITP were analyzed for their lipid and apolipoprotein composition and by two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-GGE) with subsequent immunoblotting. fHDL are characterized by the highest proportion of esterified cholesterol of all three subfractions and are relatively enriched in LpA-I. Together with iHDL they contain the majority of plasma apoA-I, while sHDL contain the majority of plasma apoA-IV, apoD, apoE, and apoJ. Pre-beta-HDL were found in separate fractions together with triglyceride-rich fractions between sHDL and LDL. In summary, ITP can separate the bulk of HDL into lipoprotein subfractions, which differ in apolipoprotein composition and electrophoretic mobility. While analytical ITP permits rapid separation and quantitation for diagnostic purposes, FS-ITP can be used to obtain these lipoprotein subfractions on a preparative scale for functional analysis. As FS-ITP is much better suited for preparative purposes than gel electrophoresis, it represents an important novel tool for the functional analysis of lipoprotein subclasses.     

Occurrence, genes and expression of the W/Se-containing class II benzoyl-coenzyme A reductases in anaerobic bacteria

Benzoyl-coenzyme A (CoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds and catalyse the reductive dearomatization of benzoyl-CoA to cyclohexa-1,5-dienoyl-1-carboxyl-CoA. Class I BCRs are ATP-dependent FeS enzymes, whereas class II BCRs are supposed to be ATP-independent and contain W, FeS clusters, and most probably selenocysteine. The active site components of a putative eight subunit class II BCR, BamBCDEFGHI, were recently characterized in Geobacter metallireducens. In this organism bamB was identified as structural gene for the W-containing active site subunit; bamF was predicted to code for a selenocysteine containing electron transfer subunit. In this work the occurrence and expression of BCRs in a number of anaerobic, aromatic compound degrading model microorganisms was investigated with a focus on the BamB and BamF components. Benzoate-induced class II BCR in vitro activities were determined in the soluble protein fraction in all obligately anaerobic bacteria tested. Where applicable, the results were in agreement with Western blot analysis using BamB targeting antibodies. By establishing a specific bamB targeting PCR assay, bamB homologues were identified in all tested obligately anaerobic bacteria with the capacity to degrade aromatic compounds; a number of bamB sequences from Gram-negative/positive sulfate-reducing bacteria were newly sequenced. In several organisms at least two bamB paralogues per genome were identified; however, in nearly all cases only one of them was transcribed during growth on an aromatic substrate. These benzoate-induced bamB genes are proposed to code for the active site subunit of class II BCRs; the major part of them group into a phylogenetic subcluster within the bamB homologues. Results from in silico analysis suggested that all class II BCRs contain selenocysteine in the BamF, and in many cases also in the BamE subunit. The results obtained indicate that the distribution of the two classes of BCRs in anaerobic bacteria appears to be strictly ruled by the available free energy from the oxidation of the aromatic carbon source rather than by phylogenetic relationships.     

Role of Vaspin in Human Eating Behaviour

Objective

The adipokine vaspin (visceral adipose tissue derived serine protease inhibitor, serpinA12) follows a meal-related diurnal variation in humans and intracerebroventricular vaspin administration leads to acutely reduced food intake in db/db mice. We therefore hypothesized that vaspin may play a role in human eating behaviour.

Materials and Methods

We measured serum vaspin concentrations in 548 subjects from a self-contained population of Sorbs (Germany) who underwent detailed metabolic testing including eating behaviour assessments using the three-factor eating questionnaire. In addition, genetic variation within vaspin was assessed by genotyping 28 single nucleotide polymorphisms (SNPs) in all study subjects.

Results

Serum vaspin concentrations correlated positively with restraint, disinhibition and hunger (all P<0.05), although the correlations did not withstand further adjustments for age, gender and BMI (all P>0.05). Independent of observed correlations, genetic variants in vaspin were associated with serum vaspin levels but showed no significant association with any of the eating behaviour phenotypes after accounting for multiple testing (P≥0.05 after adjusting for age, gender and BMI).

Conclusion

Our data suggest that serum vaspin concentrations might modulate human eating behaviour, which does not seem to be affected by common genetic variation in vaspin.     

Synthesis, characterisation and natural abundance Os NMR spectroscopy of hydride bridged triosmium clusters with chiral diphosphine ligands

Treatment of [Os₃(μ-H)₂(CO)₁₀] with the chiral diphosphines BINAP, tolBINAP [(R)-2,2′-bis(di-4-tolylphosphino)-1,1′-binaphthyl], DIOP [(4R,5R)-(−)-O-isopropenylidene-2,3-dihydroxy-1,4-bis(diphenylphosphino)butane] affords [Os₃(μ-H)₂(CO)₈(μ-L)] (L = BINAP (1), tolBINAP (2), DIOP (4)) in high yield. The X-ray structures for 1, 2 and 4 are reported, and structural and spectroscopic comparisons are made between these clusters and [Os₃(μ-H)₂(CO)₈(μ-L)] (L = dppm (5), dppe (6), dppp (7)) which were synthesised similarly. Compounds 5 to 7 were previously synthesised by hydrogenation of 1,2-[Os₃(CO)₁₀(μ-L)] but the route from [Os₃(μ-H)₂(CO)₁₀] is preferable. The H-bridged Os?Os distances are similar in 1, 2 and 4 indicating that these species are formally unsaturated 46-electron clusters. The P?P distances vary from 4.24 to 4.30 Å in 1 and 2, respectively, to 4.53 Å in 4 and there are related changes in the angles associated with the ligand set around the H-bridged osmium atoms. Introduction of the diphosphine ligands completely suppresses the ability to add CO, to insert acetylene to form a μ-η¹,η²-vinyl compound, and to exchange hydride ligands with styrene-d₈, which are reactions characteristic of [Os₃(μ-H)₂(CO)₁₀]. Clusters 2 and 5-7 were also used to examine the potential of natural abundance ¹⁸⁷Os NMR spectroscopy through techniques based on inverse detection by HMQC, HSQC and HMBC spectroscopy.     

A highly sensitive HPLC method for determination of nanomolar concentrations of dipicolinic acid, a characteristic constituent of bacterial endospores

A high-performance liquid chromatographic method with indirect fluorescence detection has been developed for quantification of dipicolinic acid, a major constituent of bacterial endospores. After separation on a reversed-phase column, a post-column reagent of sodium acetate at 1 mol l(-1) with 50 micromol l(-1) terbium chloride was added for complexation of dipicolinic acid. Terbium monodipicolinate complexes formed were quantified by measuring the fluorescence emission maximum at 548 nm after excitation with UV light at 270 nm wavelength. Parameters of post-column complexation were optimized to achieve a detection limit of 0.5 nmol DPA l(-1), corresponding to about 10(3) Desulfosporosinus orientis endospores per ml. The method was applied to the analysis of spore contamination in tuna and for estimating the endospore numbers in marine sediments.     

Dealing with particles in different conformational states by electron microscopy and image processing

Electron microscopy and image processing are powerful tools for investigating different conformational states of enzymes. It is not always possible to isolate these often unstable intermediates as single species. As a result electron micrographs show a snapshot of enzymes in various conformational states. We describe here how to recognize that the imaged particles have different conformations and how to obtain for each species a three-dimensional model using single-particle image processing. We investigated the ATP synthase from chloroplasts, which has a molecular mass of about 550 kDa. It is a membrane-bound enzyme and consists of two segments, a membrane-embedded hydrophobic F(0) part and a hydrophilic F(1) part. Analysis of the particle images indicated that the molecules were in two different conformations. For both conformations three-dimensional models were calculated, which showed that the structures differed mainly in the tilt of the F(0) part with respect to the F(1) part.