The role of arginine 310 in catalysis and substrate specificity in xanthine dehydrogenase from Rhodobacter capsulatus

The rapid reaction kinetics of wild-type xanthine dehydrogenase from Rhodobacter capsulatus and variants at Arg-310 in the active site have been characterized for a variety of purine substrates. With xanthine as substrate, k(red) (the limiting rate of enzyme reduction by substrate at high [S]) decreased approximately 20-fold in an R310K variant and 2 x 10(4)-fold in an R310M variant. Although Arg-310 lies on the opposite end of the substrate from the C-8 position that becomes hydroxylated, its interaction with substrate still contributed approximately 4.5 kcal/mol toward transition state stabilization. The other purines examined fell into two distinct groups: members of the first were effectively hydroxylated by the wild-type enzyme but were strongly affected by the exchange of Arg-310 to methionine (with a reduction in k(red) greater than 10(3)), whereas members of the second were much less effectively hydroxylated by wild-type enzyme but also much less significantly affected by the amino acid exchanges (with a reduction in k(red) less than 50-fold). The effect was such that the 4000-fold range in k(red) seen with wild-type enzyme was reduced to a mere 4-fold in the R310M variant. The data are consistent with a model in which "good" substrates are bound "correctly" in the active site in an orientation that allows Arg-310 to stabilize the transition state for the first step of the overall reaction via an electrostatic interaction at the C-6 position, thereby accelerating the reaction rate. On the other hand, "poor" substrates bound upside down relative to this "correct" orientation. In so doing, they are unable to avail themselves of the additional catalytic power provided by Arg-310 in wild-type enzyme but, for this reason, are significantly less affected by mutations at this position. The kinetic data thus provide a picture of the specific manner in which the physiological substrate xanthine is oriented in the active site relative to Arg-310 and how this residue is used catalytically to accelerate the reaction rate (rather than simply bind substrate) despite being remote from the position that is hydroxylated.     

Determinants of RNA quality from FFPE samples

The large archives of formalin-fixed paraffin-embedded (FFPE) tissue specimens that exist are a highly valuable source of sample material for molecular biological analysis, including gene expression profiling. However, current data on adverse effects of standard pathological practice on the usefulness of biomolecular analytes obtained from such archived specimens is largely anecdotal. Here, we present a systematic examination of the most relevant parameters for integrity and useability of RNA obtained from FFPE samples, including storage time and conditions, fixation time, and specimen size. The results are particularly relevant for any application relying on cDNA synthesis as an initial step of the procedure, such as RT-PCR, and microarray analysis.     

A novel technique to propagate primary human preadipocytes without loss of differentiation capacity

Objective: The study of human preadipocytes is hampered by the limited availability of adipose tissue and low yield of cell preparation. Proliferation of preadipocytes using common protocols, including fetal bovine serum (FBS), results in a markedly reduced differentiation capacity. Therefore, we were interested in developing an improved culture system that allows the proliferation of human preadipocytes without loss of differentiation capacity. Research Methods and Procedures: Adipose tissue samples were taken from subjects undergoing elective abdominal surgery. Cells were seeded at various densities and cultured using different formulations of proliferation media including factors such as fibroblast growth factor‐2 (basic fibroblast growth factor), epidermal growth factor, insulin, and FBS either alone or in combination. Cells were counted and induced to differentiate after confluence. After complete differentiation, cells were harvested, and glycerol‐3‐phosphate dehydrogenase activity was measured. Cells were subcultured for up to five passages. Results: The proliferation medium with 4 growth factors (PM4), consisting of 2.5% FBS, 10 ng/mL epidermal growth factor, 1 ng/mL basic fibroblast growth factor, and 8.7 µM insulin, resulted in lower doubling times at all seeding densities tested (0.05 × 10⁴ to 1.5 × 10⁴) compared with medium supplemented with 10% FBS. In contrast to cells in FBS medium, cells grown with PM4 medium retained full differentiation rate (glycerol‐3‐phosphate dehydrogenase activity, 493 ± 215 vs. 41 ± 17 mU/mg, p < 0.01). Differentiation capacity was fully retained at least for up to three passages in PM4 medium. Discussion: The use of PM4 medium results in substantial proliferation of human preadipocytes with preserved differentiation capacity. This novel technique represents a valuable tool for the study of human adipose tissue development and function starting from small samples.     

Site-directed mutagenesis of the active site loop of the rhodanese-like domain of the human molybdopterin synthase sulfurase MOCS3. Major differences in substrate specificity between eukaryotic and bacterial homologs

Sequence alignments of human molybdopterin synthase sulfurase, MOCS3, showed that the N-terminal domain is homologous to Escherichia coli MoeB, whereas the C-terminal domain is homologous to rhodanese-like proteins. Previous studies showed that the activity of the separately purified rhodanese-like domain of MOCS3 displayed 1000-fold lower activity in comparison to bovine rhodanese with thiosulfate as sulfur source. When the six amino acid active site loop of MOCS3 rhodanese-like domain was exchanged with the loop found in bovine rhodanese, thiosulfate:cyanide sulfurtransferase activity was increased 165-fold. Site-directed mutagenesis of each individual residue of the active site loop of the MOCS3 rhodanese-like domain showed that the charge of the last amino acid determines thiosulfate sulfurtransferase activity. Replacing Asp417 by threonine resulted in 90-fold increased activity, whereas replacing it by arginine increased the activity 470-fold. Using a fully defined in vitro system containing precursor Z, MOCS2A, E. coli MoaE, E. coli MoeB, Mg-ATP, MOCS3 rhodanese-like domain, and thiosulfate, it was shown that sulfur transfer to MOCS2A was also affected by the alterations, but not as drastically. Our studies revealed that in humans and most eukaryotes thiosulfate is not the physiologic sulfur donor for MOCS3, whereas in bacterial homologs, which have an arginine at the last position of the active site loop, thiosulfate can be used as a sulfur source for molybdenum cofactor biosynthesis. The phylogenetic analysis of MoeB homologs showed that eukaryotic homologs are of bacterial origin. Furthermore, it could be shown that an MoeB homolog named MoeZ, where the dual CXXC zinc-binding motif of the MoeB domain is not present, arose independently several times during evolution.     

Genetic engineering of brewing yeast to reduce the content of ethanol in beer

The GPD1 gene encoding the glycerol-3-phosphate dehydrogenase was overexpressed in an industrial lager brewing yeast (Saccharomyces cerevisiae ssp. carlsbergensis) to reduce the content of ethanol in beer. The amount of glycerol produced by the GPD1-overexpressing yeast in fermentation experiments simulating brewing conditions was increased 5.6 times and ethanol was decreased by 18% when compared to the wild-type. Overexpression of GPD1 does not affect the consumption of wort sugars. Only minor changes in the concentration of higher alcohols, esters and fatty acids could be observed in beer produced by the GPD1-overexpressing brewing yeast. However, the concentrations of several other by-products, particularly acetoin, diacetyl and acetaldehyde, were considerably increased.     

The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants

Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles.     

Cryptic niche conservatism among evolutionary lineages of an invasive lizard

Aim There is increasing evidence that the quality and breadth of ecological niches vary among individuals, populations, evolutionary lineages and therefore also across the range of a species. Sufficient knowledge about niche divergence among clades might thus be crucial for predicting the invasion potential of species. We tested for the first time whether evolutionary lineages of an invasive species vary in their climate niches and invasive potential. Furthermore, we tested whether lineage‐specific models show a better performance than combined models. Location Europe. Methods We used species distribution models (SDMs) based on climatic information at native and invasive ranges to test for intra‐specific niche divergence among mitochondrial DNA (mtDNA) clades of the invasive wall lizard Podarcis muralis. Using DNA barcoding, we assigned 77 invasive populations in Central Europe to eight geographically distinct evolutionary lineages. Niche similarity among lineages was assessed and the predictive power of a combination of clade‐specific SDMs was compared with a combined SDM using the pooled records of all lineages. Results We recorded eight different invasive mtDNA clades in Central Europe. The analysed clades had rather similar realized niches in their native and invasive ranges, whereas inter‐clade niche differentiation was comparatively strong. However, we found only a weak correlation between geographic origin (i.e. mtDNA clade) and invasive occurrences. Clades with narrow realized niches still became successful invaders far outside their native range, most probably due to broader fundamental niches. The combined model using data for all invasive lineages achieved a much better prediction of the invasive potential. Conclusions Our results indicate that the observed niche differentiation among evolutionary lineages is mainly driven by niche realization and not by differences in the fundamental niches. Such cryptic niche conservatism might hamper the success of clade‐specific niche modelling. Cryptic niche conservatism may in general explain the invasion success of species in areas with apparently unsuitable climate.     

Molecular cloning and characterization of the gene coding for the aerobic azoreductase from Xenophilus azovorans KF46F

The gene coding for an aerobic azoreductase was cloned from Xenophilus azovorans KF46F (formerly Pseudomonas sp. strain KF46F), which was previously shown to grow with the carboxylated azo compound 1-(4'-carboxyphenylazo)-2-naphthol (carboxy-Orange II) as the sole source of carbon and energy. The deduced amino acid sequence encoded a protein with a molecular weight of 30,278 and showed no significant homology to amino acid sequences currently deposited at the relevant data bases. A presumed NAD(P)H-binding site was identified in the amino-terminal region of the azoreductase. The enzyme was heterologously expressed in Escherichia coli and the azoreductase activities of resting cells and cell extracts were compared. The results suggested that whole cells of the recombinant E. coli strains were unable to take up sulfonated azo dyes and therefore did not show in vivo azoreductase activity. The turnover of several industrially relevant azo dyes by cell extracts from the recombinant E. coli strain was demonstrated.     

Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus

To identify Rhodobacter capsulatus nif genes necessary for the alternative nitrogenase, strains carrying defined mutations in 32 genes and open reading frames of nif region A, B or C were constructed. The ability of these mutants to grow on nitrogen-free medium with molybdenum (Nif phenotype) or in a nifHDK deletion background on medium without molybdenum (Anf phenotype) was tested. Nine nif genes and nif-associated coding regions are absolutely essential for the alternative nitrogenase. These genes comprise nifV and nifB, the nif-specific ntr system (nifR1, R2, R4) and four open reading frames, which exhibit no homology to known genes. In addition, a significantly reduced activity of both the alternative nitrogenase and the molybdenum-dependent nitrogenase was found for fdxN mutants. By random Tn5 mutagenesis of a nifHDK deletion strain 42 Anf^? mutants were isolated. Southern hybridization experiments demonstrated that 17 of these Tn5 mutants were localized in at least 13 different restriction fragments outside of known nif regions. Ten different Anf^? Tn5 mutations are clustered on a 6 kb DNA fragment of the chromosome designated anf region A. DNA sequence analysis revealed that this region contained the structural genes of the alternative nitrogenase (anfHDGK). The identification of several Tn5 insertions mapping outside of anf region A indicated that at least 10 genes specific for the alternative nitrogenase are present in R. capsulatus.     

3D nuclear organization of telomeres in the Hodgkin cell lines U-HO1 and U-HO1-PTPN1: PTPN1 expression prevents the formation of very short telomeres including "t-stumps"

Background  

In cancer cells the three-dimensional (3D) telomere organization of interphase nuclei into a telomeric disk is heavily distorted and aggregates are found. In Hodgkin's lymphoma quantitative FISH (3D Q-FISH) reveals a major impact of nuclear telomere dynamics during the transition form mononuclear Hodgkin (H) to diagnostic multinuclear Reed-Sternberg (RS) cells. In vitro and in vivo formation of RS-cells is associated with the increase of very short telomeres including "t-stumps", telomere loss, telomeric aggregate formation and the generation of "ghost nuclei".