Characterization and functional analysis of the murine Frat2 gene

The Frat1 proto-oncogene was first identified as a gene contributing to tumor progression in T-cell lymphomas induced by retroviral insertional mutagenesis with the Moloney murine leukemia virus. The biological function of Frat remained elusive until its Xenopus homologue GBP was isolated as a glycogen synthase kinase 3 (GSK3)-binding protein and was shown to be an essential component of the maternal Wnt-signaling pathway. To date two Frat homologues have been described in the mouse, Frat1 and Frat3. The proteins encoded by these two genes are 84% identical. Here we describe the cloning and characterization of a third murine Frat homologue, Frat2, which is the mouse ortholog of human FRAT2. Frat1 and Frat2 are juxtaposed on chromosome 19 in a chromosomal organization conserved between man and mouse. We show that Frat1 and Frat2 are phosphorylated, which is the first evidence that these proteins are subject to posttranslational modification. Like Frat1, Frat2 is able to bind to GSK3beta. However, a side-by-side comparison of the murine Frat proteins for their capacity to induce signaling through beta-catenin/T-cell factor reveals that Frat2 is a less potent activator of the canonical Wnt pathway. Frat2 protein accumulates to higher levels upon transfection into 293T cells than either Frat1 or Frat3. Thus, whereas Frat1 may be a core component of canonical Wnt-signaling, Frat2 might very well be part of a divergent intracellular GSK3beta pathway.     

Detection of bacterial DNA in blood samples from febrile patients: underestimated infection or emerging contamination?

We applied real-time broad-range polymerase chain reaction (PCR) to detect bacteraemia in blood from febrile patients. Interpretation of amplification results in relation to clinical data and blood culture outcome was complex, although the reproducibility of the PCR results was good. Sequencing analysis of the PCR products revealed the presence of Burkholderia species DNA while no Burkholderia species grew in culture. The source of this contamination was shown to be the commercial DNA isolation kit used in the automated MagNA Pure Isolation Robot. A high degree of suspicion is required when uncommon or unexpected pathogens are diagnosed by molecular methods as clinical consequences can be serious.     

Molecular systematics ofCardamine and allied genera (Brassicaceae): Its and non-coding chloroplast DNA

Representatives of the generaCardamine, Dentaria, Nasturtium, Rorippa andArmoracia (Brassicaceae) were analyzed to elucidate their phylogenetic relationships based on nuclear (ITS) and non-coding chloroplast (cp) DNA sequences.Dentaria seems to be polyphyletic. The two studiedDentaria species group with differentCardamine clades, and it is argued thatD. bulbifera is an allopolyploid originating from a hybridization between aCardamine and aDentaria species. In the ITS tree,Nasturtium andRorippa form well supported clades but their relationship toCardamine andArmoracia remains unresolved. In the cpDNA tree,Nasturtium groups together withCardamine. Hybridization events apparently played a role in the evolution ofNasturtium. TheCardamine/Nasturtium clade is separated from a clade placingRorippa andArmoracia together.Armoracia is closely related toRorippa. Analyses of the 19Cardamine species studied revealed three main groupings, a northern hemispheric and two southern hemispheric groups. Within the northern hemisphere taxa theC. pratensis complex forms a well supported clade which seems to be closely related toC. amara, C. raphanifolia andC. flexuosa. The positions ofC. hirsuta andC. impatiens are uncertain. The two southern hemisphere clades consist of New Guinean species and south-eastern Australian/Tasmanian and subantarctic species, respectively. They may reflect migration routes from the northern to the southern hemisphere, but further studies are necessary to fully understand the evolution of the bihemispheric distribution pattern ofCardamine.     

Repair and replication of plasmids with site-specific 8-oxodG and 8-AAFdG residues in normal and repair-deficient human cells.

The in vivo mutagenicity of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and N-(guanin-8-yl)-N-acetyl-2-aminofluorene (8-AAFdG) in human cells was determined by transfecting various cell lines with plasmids that carried a single adduct at a defined site. 8-OxodG is one of the many DNA modifications formed by oxygen radicals, and was found to be highly miscoding during replication with purified DNA polymerases in vitro. Here we show that the frequency of mutations induced by 8-oxodG during replication in vivo is at most only 2% above background. The most predominant mutation found was a single G----T transversion. The frequency of this transversion was found to be 3 to 5-fold increased in excision repair deficient XP-A cells. Interestingly, also the replication of 8-oxodG containing plasmids was significantly impaired (approximately 4-fold) in the XP-A cells, but not in HeLa cells, normal fibroblasts or XP-A revertant cells. When 8-AAFdG containing plasmids were used, the mutation frequencies did not exceed background levels (less than 2%) with any of the cell lines tested. The presence of 8-AAFdG almost completely inhibited plasmid replication (more than 50-fold) in XP-A cells. Apparently, both 8-AAFdG and 8-oxodG are not or poorly repaired in these cells, causing a block of DNA replication. This suggests that both lesions are substrates for excision repair, although to a varying extent.     

Vascular adaptation to deconditioning and the effect of an exercise countermeasure: results of the Berlin Bed Rest study.

Deconditioning is a risk factor for cardiovascular disease. The physiology of vascular adaptation to deconditioning has not been elucidated. The purpose of the present study was to assess the effects of bed rest deconditioning on vascular dimension and function of leg conduit arteries. In addition, the effectiveness of resistive vibration exercise as a countermeasure for vascular deconditioning during bed rest was evaluated. Sixteen healthy men were randomly assigned to bed rest (BR-Ctrl) or to bed rest with resistive vibration exercise (BR-RVE). Before and after 25 and 52 days of strict horizontal bed rest, arterial diameter, blood flow, flow-mediated dilatation (FMD), and nitroglycerin-mediated dilatation were measured by echo Doppler ultrasound. In the BR-Ctrl group, the diameter of the common femoral artery decreased by 13 +/- 3% after 25 and 17 +/- 1% after 52 days of bed rest (P < 0.001). In the BR-RVE group this decrease in diameter was significantly attenuated (5 +/- 2% after 25 days and 6 +/- 2% after 52 days, P < 0.01 vs. BR-Ctrl). Baseline blood flow did not change after bed rest in either group. After 52 days of bed rest, FMD and nitroglycerin-mediated dilatation of the superficial femoral artery were increased in both groups, possibly by increased nitric oxide sensitivity. In conclusion, bed rest deconditioning is accompanied by a reduction in the diameter of the conduit arteries and by an increased reactivity to nitric oxide. Resistive vibration exercise effectively attenuates the diameter decrease of leg conduit arteries after bed rest.     

Genetic and clinical mosaicism in a patient with neurofibromatosis type 1

Patients with typical features of neurofibromatosis type 1 (NF1) limited to a specific body segment are usually referred to as having segmental NF1, which is generally assumed to be the result of somatic mosaicism for a NF1 mutation. Mosaicism has also been demonstrated at the molecular level in some sporadic cases with phenotypically classic NF1. In the present report, we describe a patient with NF1 disease manifestations throughout the whole body, but leaving a few sharply delineated segments of the skin unaffected, suggestive of revertant mosaicism. A large intragenic deletion was found by mutation analysis using long-range RT-PCR. The intra-exonic breakpoints were characterized in exon 13 and exon 28, resulting in a deletion of 99,571 bp at the genomic level. The presence of two genetically distinct cell populations, confirming mosaicism for this NF1 mutation, was shown by analysis of several tissues. Revertant mosaicism was excluded by demonstrating heterozygosity for markers residing in the deletion region. The findings in this patient demonstrate two things: (1) although the entire body is affected, mosaicism can still be suspected at clinical examination and proven by DNA analysis and skin biopsies; (2) long-range RT-PCR is a feasible method for demonstrating large intragenic deletions in NF1.     

CP-arene oxides: the ultimate, active mutagenic forms of cyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs)

The bacterial mutagenic response (Ames-assay, Salmonella typhimurium strain TA98+/-S9-mix) of a series of monocyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs) identified in combustion exhausts, viz. cyclopenta[cd]pyrene (1), acephenanthrylene (2), aceanthrylene (3) and cyclopenta[hi]chrysene (4), is re-evaluated. The mutagenic effects are compared with those exerted by the corresponding partially hydrogenated derivatives, 3,4-dihydrocyclopenta[cd]pyrene (5), 4,5-dihydroacephenanthrylene (6), 1,2-dihydroaceanthrylene (7) and 4,5-dihydrocyclopenta[hi]chrysene (8). It is shown that the olefinic bond of the externally fused five-membered ring of 1, 3 and 4 is of importance for a positive mutagenic response. In contrast, whilst CP-PAH 2 is found inactive, its dihydro analogue (6) shows a weak metabolism-dependent response. The importance of epoxide formation at the external olefinic bond in the five-membered ring is substantiated by the bacterial mutagenic response of independently synthesized cyclopenta[cd]pyrene-3,4-epoxide (9), acephenanthrylene-4,5-epoxide (10), aceanthrylene-1,2-epoxide (11) and cyclopenta[hi]chrysene-4,5-epoxide (12). Their role as ultimate, active mutagenic forms, when CP-PAHs 1, 3 and 4 exhibit a positive mutagenic response, is confirmed. Semi-empirical Austin Model 1 (AM1) calculations on the formation of the CP-arene oxides (9-12) and their conversion into the monohydroxy-carbocations (9a-12a and 9b-12b) via epoxide-ring opening support our results. For 2 and 4, which also possess a bay-region besides an annelated cyclopenta moiety, the calculations rationalize that epoxidation at the olefinic bond of the cyclopenta moiety is favoured.     

FtsH-mediated repair of the photosystem II complex in response to light stress

A common feature of light stress in plants, algae, and cyanobacteria is the light-induced damage to the photosystem II complex (PSII), which catalyses the photosynthetic oxidation of water to molecular oxygen. A repair cycle operates to replace damaged subunits within PSII, in particular, the D1 reaction centre polypeptide, by newly synthesized copies. As yet the molecular details of this physiologically important process remain obscure. A key aspect of the process that has attracted much attention is the identity of the protease or proteases involved in D1 degradation. The results are summarized here of recent mutagenesis experiments that were designed to assess the functional importance of the DegP/HtrA and FtsH protease families in the cyanobacterium Synechocystis sp. PCC 6803. Based on these results and the analysis of Arabidopsis mutants, a general model for PSII repair is suggested in which FtsH complexes alone are able to degrade damaged D1.     

Automated, on-line two-dimensional nano liquid chromatography tandem mass spectrometry for rapid analysis of complex protein digests

Evaluation of cellular processes and their changes at the level of protein expression and post-translational modifications may allow identification of novel proteins and the mechanisms involved in pathogenic processes. However, the number of proteins and, after tryptic digestion, of peptides from a single cell can be tremendously high. Separation and analysis of such complex peptide mixtures can be performed using multidimensional separation techniques such as two-dimensional gel electrophoresis or two-dimensional-high-performance liquid chromatography (2-D-HPLC). The aim of this work was to establish a fully automated on-line 2-D-HPLC separation method with column switching for the separation of complex tryptic digests. A model mixture of five proteins as well as a nuclear matrix protein sample were digested with trypsin and separated using a strong cation exchange (SCX) column in the first dimension and nano reversed phase in the second dimension. Separated peptides were detected using an ion trap mass spectrometer. The advantages of this new fully automated method are rapid sample loading, the possibility of injecting large volumes and no introduction of salt into the mass spectrometer. Furthermore, column switching allows the independent control and optimization of the two dimensions independently.