Single-chain Tet transregulators

We demonstrate here that the Tet repressor (TetR), a dimeric allosterical regulatory protein, can be converted to a fully functional monomer when connected by a 29 amino acid linker. TetR-based transregulators are widely used to regulate gene expression in eukaryotes. They can be fused to form single-chain (sc) Tet transregulators with two TetR moieties and one eukaryotic regulatory domain. Sc variants of transactivator and transsilencer exhibit the same regulatory properties as their respective dimeric counterparts in human cell lines. In particular, the reverse ‘tet-on’ phenotype of rtTA variants is also present in the sc variants. Coexpression of a reverse transactivator and sc transsilencer leads to reduced background expression and shows full activation upon induction. The data demonstrate that sc Tet transregulators exhibit the phenotype of their respective dimers and lack functional interference when coexpressed in the same cell.     

Impact of DNA ligase IV on the fidelity of end joining in human cells

A DNA ligase IV (LIG4)-null human pre-B cell line and human cell lines with hypomorphic mutations in LIG4 are significantly impaired in the frequency and fidelity of end joining using an in vivo plasmid assay. Analysis of the null line demonstrates the existence of an error-prone DNA ligase IV-independent rejoining mechanism in mammalian cells. Analysis of lines with hypomorphic mutations demonstrates that residual DNA ligase IV activity, which is sufficient to promote efficient end joining, nevertheless can result in decreased fidelity of rejoining. Thus, DNA ligase IV is an important factor influencing the fidelity of end joining in vivo. The LIG4-defective cell lines also showed impaired end joining in an in vitro assay using cell-free extracts. Elevated degradation of the terminal nucleotide was observed in a LIG4-defective line, and addition of the DNA ligase IV–XRCC4 complex restored end protection. End protection by DNA ligase IV was not dependent upon ligation. Finally, using purified proteins, we demonstrate that DNA ligase IV–XRCC4 is able to protect DNA ends from degradation by T7 exonuclease. Thus, the ability of DNA ligase IV–XRCC4 to protect DNA ends may contribute to the ability of DNA ligase IV to promote accurate rejoining in vivo.     

A graphical method for the analysis of anisotropic rotational diffusion in proteins

A graphical method for the analysis of relaxation data is presented. It allows a fast estimation of the range of values of the components of the axially symmetric rotational diffusion tensor that are compatible with the experimental relaxation data. The graphical method clearly shows the contribution of different experimental relaxation parameters to the measured anisotropy. In particular, for proteins with moderate anisotropy, data from at least two N-H bonds forming angles close to 0° and 90° with respect to the principal axis of the rotational diffusional tensor are needed. For very anisotropic systems, combination of different relaxation parameters from a single residue is enough to characterize the local anisotropy.     

Oxidant-induced DNA damage by quartz in alveolar epithelial cells

Respirable quartz has recently been classified as a human carcinogen. Although, studies with quartz using naked DNA as a target suggest that formation of oxyradicals by particles may play a role in the DNA-damaging properties of quartz, it is not known whether this pathway is important for DNA damage in the target cells for quartz carcinogenesis, i.e. alveolar epithelial cells. Therefore, we determined in vitro DNA damage by DQ12 quartz particles in rat and human and alveolar epithelial cells (RLE, A549) using the single cell gel electrophoresis/comet assay. The radical generation capacity of quartz was analysed by electron spin resonance (ESR) and by immunocytochemical analysis of the hydroxyl radical-specific DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in the epithelial cells. Quartz particles as well as the positive control hydrogen peroxide, caused a dose-dependent increase in DNA strand breaks in both cell lines. DNA damage by quartz was significantly reduced in the presence of the hydroxyl-radical scavengers mannitol or DMSO. The involvement of hydroxyl radicals was further established by ESR measurements and was also demonstrated by the ability of the quartz to induce formation of 8-OHdG. In conclusion, our data show that quartz elicits DNA damage in rat and human alveolar epithelial cells and indicate that these effects are driven by hydroxyl radical-generating properties of the particles.     

Molecular basis of CIB binding to the integrin alpha IIb cytoplasmic domain

Integrin adhesion receptors appear to be regulated by molecules that bind to their cytoplasmic domains. We previously identified a 22-kDa, EF-hand-containing protein, CIB, which binds to the alpha(IIb) cytoplasmic tail of the platelet integrin, alpha(IIb)beta(3). Here we describe regions within CIB and alpha(IIb) that interact with one another. CIB binding to alpha(IIb) cytoplasmic tail peptides, as measured by intrinsic tryptophan fluorescence, indicates a CIB-binding site within a hydrophobic, 15-amino acid, membrane-proximal region of alpha(IIb). This region is analogous to the alpha-helical targets of other EF-hand-containing proteins, such as calcineurin B or calmodulin. A homology model of CIB based upon calcineurin B and recoverin indicated a conserved hydrophobic pocket within the C-terminal EF-hand motifs of CIB as a potential integrin-binding site. CIB engineered to contain alanine substitutions in the implicated regions retained wild type secondary structure as determined by circular dichroism, yet failed to bind alpha(IIb) in 11 of 12 cases, whereas CIB mutated within the N terminus retained binding activity. Thus, specific hydrophobic residues in the C terminus of CIB appear necessary for CIB binding to alpha(IIb). The identification of essential interacting regions within alpha(IIb) and CIB provides tools for further probing potential interrelated functions of these proteins.     

Binding of transmissible gastroenteritis coronavirus to cell surface sialoglycoproteins

The surface glycoprotein S of transmissible gastroenteritis virus (TGEV) has two binding activities. (i) Binding to porcine aminopeptidase N (pAPN) is essential for the initiation of infection. (ii) Binding to sialic acid residues on glycoproteins is dispensable for the infection of cultured cells but is required for enteropathogenicity. By comparing parental TGEV with mutant viruses deficient in the sialic acid binding activity, we determined the contributions of both binding activities to the attachment of TGEV to cultured cells. In the presence of a functional sialic acid binding activity, the amount of virus bound to two different porcine cell lines was increased sixfold compared to the binding of the mutant viruses. The attachment of parental virus was reduced to levels observed with the mutants when sialic acid containing inhibitors was present or when the cells were pretreated with neuraminidase. In virus overlay binding assays with immobilized cell surface proteins, the mutant virus only recognized pAPN. In addition, the parental virus bound to a high-molecular-mass sialoglycoprotein. The recognition of pAPN was sensitive to reducing conditions and was not dependent on sialic acid residues. On the other hand, binding to the sialic acid residues of the high-molecular-mass glycoprotein was observed regardless of whether the cellular proteins had been separated under reducing or nonreducing conditions. We propose that binding to a surface sialoglycoprotein is required for TGEV as a primary attachment site to initiate infection of intestinal cells. This concept is discussed in the context of other viruses that use two different receptors to infect cells.     

Solute complexation degree with human serum albumin: biochromatographic approach

A mathematical model was developed for the study of the D,L-dansylamino acid retention mechanism in reversed-phase liquid chromatography using a C18 column as a stationary phase and human serum albumin (HSA) as an eluent modifier. The solute retention factor is dependent on the HSA concentration in the eluent as well as the binding constant of the guest-HSA complex. A determination of the degree of complexation n(c) (the percent of the complexed guest) could be carried out. Different Van 't Hoff plot shapes of the degree of complexation were observed with different eluent pH, confirming a change in the solute complexation mechanism for physiological pH (between 7-7.5). Enthalpy-entropy compensation was also analysed in relation to this mathematical model to confirm the solute complexation behavior with HSA. These results finally confirmed that at physiological pH and temperature (approximately 35 degrees C) values the HSA was in a favorable structural conformation for its binding with a great majority of drugs.     

Flanking sequence tags in Arabidopsis thaliana T-DNA insertion lines: a pilot study

Eight hundred and fifty Arabidopsis thaliana T-DNA insertion lines have been selected on a phenotypic basis. The T-DNA flanking sequences (FST) have been isolated using a PCR amplification procedure and sequenced. Seven hundred plant DNA sequences have been obtained revealing a T-DNA insertion in, or in the immediate vicinity of 482 annotated genes. Limited deletions of plant DNA have been observed at the site of insertion of T-DNA as well as in its left (LB) and right (RB) T-DNA signal sequences. The distribution of the T-DNA insertions along the chromosomes shows that they are essentially absent from the centrometric and pericentrometric regions.     

Pseudomonas aeruginosa displays an epidemic population structure

Bacteria can have population structures ranging from the fully sexual to the highly clonal. Despite numerous studies, the population structure of Pseudomonas aeruginosa is still somewhat contentious. We used a polyphasic approach in order to shed new light on this issue. A data set consisting of three outer membrane (lipo)protein gene sequences (oprI, oprL and oprD), a DNA-based fingerprint (amplified fragment length polymorphism), serotype and pyoverdine type of 73 P. aeruginosa clinical and environmental isolates, collected across the world, was analysed using biological data analysis software. We observed a clear mosaicism in the results, non-congruence between results of different typing methods and a microscale mosaic structure in the oprD gene. Hence, in this network, we also observed some clonal complexes characterized by an almost identical data set. The most recent clones exhibited serotypes O1, 6, 11 and 12. No obvious correlation was observed between these dominant clones and habitat or, with the exception of some recent clones, geographical origin. Our results are consistent with, and even clarify, some seemingly contradictory results in earlier epidemiological studies. Therefore, we suggest an epidemic population structure for P. aeruginosa, comparable with that of Neisseria meningitidis, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise.