The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 degrees C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184-193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.     

Romanowsky dyes and Romanowsky-Giemsa effect

Summary A reproducible Romanowsky-Giemsa staining (RGS) can be carried out with standardized staining solutions containing the two dyes azure B (AB) and eosin Y (EY). After staining, cell nuclei have a purple coloration generated by DNA-AB-EY complexes. The microspectra of cell nuclei have a sharp and intense absorption band at 18 100 cm^–1 (552 nm), the so called Romanowsky band (RB), which is due to the EY chromophore of the dye complexes. Other absorption bands can be assigned to the DNA-bound AB cations.Artificial DNA-AB-EY complexes can be prepared outside the cell by subsequent staining of DNA with AB and EY. In the first step of our staining experiments we prepared thin films of blue DNA-AB complexes on microslides with 1:1 composition: each anionic phosphodiester residue of the nucleic acid was occupied by one AB cation. Microspectrophotometric investigations of the dye preparations demonstrated that, besides monomers and dimers, mainly higher AB aggregates are bound to DNA by electrostatic and hydrophobic interactions. These DNA-AB complexes are insoluble in water. Therefore it was possible to stain the DNA-AB films with aqueous EY solutions and also to prepare insoluble DNA-AB-EY films in the second step of the staining experiments. After the reaction with EY, thin sites within the dye preparations were purple. The microspectra of the purple spots show a strong Romanowsky band at 18 100 cm^–1. Using a special technique it was possible to estimate the composition of the purple dye complexes. The ratio of the two dyes was approximately EY:AB1:3. The EY anions are mainly bound by hydrophobic interaction to the AB framework of the electrical neutral DNA-AB complexes. The EY absorption is red shifted by the interaction of EY with the AB framework of DNA-AB-EY. We suppose that this red shift is caused by a dielectric polarization of the bound EY dianions.The DNA chains in the DNA-AB complexes can mechanically be aligned in a preferred direction k. Highly orientated dye complexes prepared on microslides were birefringent and dichroic. The orientation is maintained during subsequent staining with aqueous EY solutions. In this way we also prepared highly orientated purple DNA-AB-EY complexes on microslides. The light absorption of both types of dye complexes was studied by means of a microspectrophotometer equipped with a polarizer and an analyser. The sites of best orientation within the dye preparations were selected under crossed nicols according to the quality of birefringence. Subsequently, the absorption spectra of the highly orientated dye complexes were measured with plane polarized light. We found that the transition moments, m _AB, of the bound AB cations in DNA-AB and DNA-AB-EY are orientated almost perpendicular to k, i.e. m _ABk. On the contrary, the transition moments, m _EY, of the bound EY anions in DNA-AB-EY are polarized parallel to k, i.e. m _EY k. The transition moments m _AB and m _EY lay in the direction of the long axes of the AB and EY chromophores. For that reason, in both DNA-AB and DNA-AB-EY the long molecular axes of the AB cations are orientated approximately perpendicular to the DNA chains, while the long molecular axes of the EY chromophores are polarized in the direction of the DNA chains. Therefore, in DNA-AB-EY the long axes of AB and EY are perpendicular to each other, m _ABm _EY. This molecular arrangement fully agrees with our quantitative measurements and with the theory of the absorption of plane polarized light by orientated dye complexes, which has been developed and discussed in detail.     

Derived amino acid sequences of the nosZ gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copper-binding residues. Implications for the CuA site of N2O reductase and cytochrome-c oxidase.

The nosZ genes encoding the multicopper enzyme nitrous oxide reductase of Alcaligenes eutrophus H16 and the type strain of Pseudomonas aeruginosa were cloned and sequenced for structural comparison of their gene products with the homologous product of the nosZ gene from Pseudomonas stutzeri [Viebrock, A. & Zumft, W. G. (1988) J. Bacteriol. 170, 4658-4668] and the subunit II of cytochrome-c oxidase (COII). Both types of enzymes possess the CuA binding site. The nosZ genes were identified in cosmid libraries by hybridization with an internal 1.22-kb PstI fragment (NS220) of nosZ from P. stutzeri. The derived amino acid sequences indicate unprocessed gene products of 70084 Da (A. eutrophus) and 70695 Da (P. aeruginosa). The N-terminal sequences of the NosZ proteins have the characteristics of signal peptides for transport. A homologous domain, extending over at least 50 residues, is shared among the three derived NosZ sequences and the CuA binding region of 32 COII sequences. Only three out of nine cysteine residues of the NosZ protein (P. stutzeri) are invariant. Cys618 and Cys622 are assigned to a binuclear center, A, which is thought to represent the CuA site of NosZ and is located close to the C terminus. Two conserved histidines, one methionine, one aspartate, one valine and two aromatic residues are also part of the CuA consensus sequence, which is the domain homologous between the two enzymes. The CuA consensus sequence, however, lacks four strictly conserved residues present in all COII sequences. Cys165 is likely to be a ligand of a second binuclear center, Z, for which we assume mainly histidine coordination. Of 23 histidine residues in NosZ (P. stutzeri), 14 are invariant, 7 of which are in regions with a degree of conservation well above the 50% positional identity between the Alcaligenes and Pseudomonas sequences. Conserved tryptophan residues are located close to several potential copper ligands. Trp615 may contribute to the observed quenching of fluorescence when the CuA site is occupied.     

Binding of the Heterogeneous Ribonucleoprotein K (hnRNP K) to the Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) Enhances Viral LMP2A Expression

The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.     

In vitro cell growth of marine archaeal-bacterial consortia during anaerobic oxidation of methane with sulfate

Anoxic sediment from a methane hydrate area (Hydrate Ridge, north-east Pacific; water depth 780 m) was incubated in a long-term laboratory experiment with semi-continuous supply of pressurized [1.4 MPa (14 atm)] methane and sulfate to attempt in vitro propagation of the indigenous consortia of archaea (ANME-2) and bacteria (DSS, Desulfosarcina/Desulfococcus cluster) to which anaerobic oxidation of methane (AOM) with sulfate has been attributed. During 24 months of incubation, the rate of AOM (measured as methane-dependent sulfide formation) increased from 20 to 230 micromol day(-1) (g sediment dry weight)(-1) and the number of aggregates (determined by microscopic counts) from 0.5 x 10(8) to 5.7 x 10(8) (g sediment dry weight)(-1). Fluorescence in situ hybridization targeting 16S rRNA of both partners showed that the newly grown consortia contained central archaeal clusters and peripheral bacterial layers, both with the same morphology and phylogenetic affiliation as in the original sediment. The development of the AOM rate and the total consortia biovolume over time indicated that the consortia grew with a doubling time of approximately 7 months (growth rate 0.003 day(-1)) under the given conditions. The molar growth yield of AOM was approximately 0.6 g cell dry weight (mol CH(4) oxidized)(-1); according to this, only 1% of the consumed methane is channelled into synthesis of consortia biomass. Concentrations of biomarker lipids previously attributed to ANME-2 archaea (e.g. sn-2-hydroxyarchaeol, archaeol, crocetane, pentamethylicosatriene) and Desulfosarcina-like bacteria [e.g. hexadecenoic-11 acid (16:1omega5c), 11,12-methylene-hexadecanoic acid (cy17:0omega5,6)] strongly increased over time (some of them over-proportionally to consortia biovolume), suggesting that they are useful biomarkers to detect active anaerobic methanotrophic consortia in sediments.     

ProfDist: a tool for the construction of large phylogenetic trees based on profile distances

SUMMARY: ProfDist is a user-friendly software package using the profile-neighbor-joining method (PNJ) in inferring phylogenies based on profile distances on DNA or RNA sequences. It is a tool for reconstructing and visualizing large phylogenetic trees providing new and standard features with a special focus on time efficency, robustness and accuracy. AVAILABILITY: A Windows version of ProfDist comes with a graphical user interface and is freely available at http://profdist.bioapps.biozentrum.uni-wuerzburg.de     

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.     

Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts

In MDCK cells, presenilin-1 (PS1) accumulates at intercellular contacts where it colocalizes with components of the cadherin-based adherens junctions. PS1 fragments form complexes with E-cadherin, beta-catenin, and alpha-catenin, all components of adherens junctions. In confluent MDCK cells, PS1 forms complexes with cell surface E-cadherin; disruption of Ca(2+)-dependent cell-cell contacts reduces surface PS1 and the levels of PS1-E-cadherin complexes. PS1 overexpression in human kidney cells enhances cell-cell adhesion. Together, these data show that PS1 incorporates into the cadherin/catenin adhesion system and regulates cell-cell adhesion. PS1 concentrates at intercellular contacts in epithelial tissue; in brain, it forms complexes with both E- and N-cadherin and concentrates at synaptic adhesions. That PS1 is a constituent of the cadherin/catenin complex makes that complex a potential target for PS1 FAD mutations.     

DNA ploidy and chromosomal imbalances in invasive ductal breast cancer. A comparative study of DNA image cytometry and comparative genomic hybridization (CGH).

Chromosomal imbalances were analyzed in 62 breast cancers with different DNA ploidy by CGH. The results of DNA image cytometry and CGH are consistent with peridiploid and aneuploid cases. The peritetraploid tumors harbored a high number of chromosomal imbalances, as a hint for an unfavorable prognosis. The quantitative analysis of imbalances highlighted the role of different physical constituents of the chromosome, and of chromosomal losses in different DNA ploidy groups. The peritetraploid and aneuploid tumors differed from the peridiploid tumors in losses at 8p and 18q. The peritetraploid cancers exhibited more gains at 8q, the aneuploid tumors more losses at 17p than their peridiploid counterparts. The aneuploid cases differed from the peritetraploid tumors in a higher number of losses at 11q and 14q. Combinations of imbalances provide further insights into the genetic background of DNA ploidy. Hypotheses for the progression from peridiploid to nondiploid breast cancers are given.     

Impact of selective genotyping in the training population on accuracy and bias of genomic selection

Estimating marker effects based on routinely generated phenotypic data of breeding programs is a cost-effective strategy to implement genomic selection. Truncation selection in breeding populations, however, could have a strong impact on the accuracy to predict genomic breeding values. The main objective of our study was to investigate the influence of phenotypic selection on the accuracy and bias of genomic selection. We used experimental data of 788 testcross progenies from an elite maize breeding program. The testcross progenies were evaluated in unreplicated field trials in ten environments and fingerprinted with 857 SNP markers. Random regression best linear unbiased prediction method was used in combination with fivefold cross-validation based on genotypic sampling. We observed a substantial loss in the accuracy to predict genomic breeding values in unidirectional selected populations. In contrast, estimating marker effects based on bidirectional selected populations led to only a marginal decrease in the prediction accuracy of genomic breeding values. We concluded that bidirectional selection is a valuable approach to efficiently implement genomic selection in applied plant breeding programs.