An asymmetric ion channel derived from gramicidin A. Synthesis, function and NMR structure

The biological ion channel gramicidin A (gA) was modified by synthetic means to obtain the tail-to-tail linked asymmetric gA-derived dimer compound 3. Single-channel current measurements for 3 in planar lipid bilayers exhibit an Eisenman I ion selectivity for alkali cations. The structural asymmetry does not lead to an observable functional asymmetry. The structure of 3 in solution without and with Cs cations was investigated by 1H-NMR spectroscopy. In CDCl3/CD3OH (1 : 1, v/v), 3 forms a mixture of double-stranded beta-helices. Upon addition of excess CsCl, the double-stranded species are converted completely into one new conformer: the right-handed single-stranded beta-helix. A combination of DQF-COSY and TOCSY was used for the assignment of the 1H-NMR spectrum of the Cs-3 complex in CDCl3/CD3OH (1 : 1, v/v). A total of 69 backbone, 27 long-range, and 64 side-chain distance restraints were obtained from NOESY together with 25 phi and 14 chi1 torsion angles obtained from coupling constants. These data were used as input for structure calculation with dyana built in sybyl 6.8. A final set of 11 structures with an average rmsd for the backbone of 0.45 A was obtained (PDB: 1TKQ). The structure of the Cs-3 complex in solution is equivalent to the bioactive channel conformation in the membrane environment.     

Immediate early gene X1 (IEX-1) is organized in subnuclear structures and partially co-localizes with promyelocytic leukemia protein in HeLa cells

Immediate early gene X1 (IEX-1) represents a stress response gene involved in growth control and modulation of apoptosis. Here, we report a detailed analysis of IEX-1 with respect to its intracellular localization. By means of confocal laser scanning microscopy, a green fluorescent protein-IEX-1 fusion protein transfected into HeLa cells, as well as endogenous IEX-1, could be detected in distinct subnuclear structures. This particular subnuclear localization of IEX-1 was not observed with a green fluorescent protein-IEX-1 fusion protein lacking a putative nuclear localization sequence, along with a decreased effect on apoptosis. Double immunofluorescence staining revealed a partial co-localization of endogenous promyelocytic leukemia protein (PML) and IEX-1 in these subnuclear structures. Nuclear localization of IEX-1 is also enhanced upon treatment of cells with leptomycin B, an inhibitor of the nuclear exporter CRM1. These observations indicate that IEX-1 is specifically shuttled to and from the nucleus. Overexpression experiments using PML isoforms III and IV revealed distinct intranuclear interaction of IEX-1 and PML. Coprecipitation experiments showed physical interaction between IEX-1 and PML. The close structural relation of IEX-1-containing nuclear subdomains and PML nuclear bodies suggests a function of IEX-1 related to the multiple functions of these unique subnuclear regions, particularly during stress response and growth control.     

Effect of genetic variation on the thermal stability of human serum albumin

Reversible thermal denaturation of 33 genetic variants of human serum albumin (HSA) appeared to be a two-state process when studied by circular dichroism (CD). Fourteen single-residue variants have Tm values (midpoint of denaturation) higher than, and nine have Tm values lower than, their endogenous, wild-type counterpart. Nine single-residue variants have DeltaHv values (van't Hoff enthalpy) higher than, and 14 have DeltaHv values lower than, normal albumin. All types of combinations of positive and negative DeltaTm values and Delta(DeltaHv) values were found. Good linear correlations between mutation-induced changes of alpha-helical content and Delta(DeltaHv) values, but not DeltaTm values, were found especially for the variants mutated in domains I and III. The effect of altered chain length and glycosylation on Tm and DeltaHv was also studied. For all variants, no clear relationship was found between the changes in the thermodynamic parameters and the type of substitution, changes in protein charge or hydrophobicity. However, the protein changes taking place in domain I have a rather uniform effect (almost all of the nine variants have positive DeltaTm values and negative Delta(DeltaHv) values, i.e., they denature more easily than normal albumin but they do so at a higher temperature). The present results can be of both protein chemical relevance and of clinical interest, because they could be useful when designing stable, recombinant HSAs for clinical applications.     

Reactive alveolar epithelium in chondroid hamartoma of the lung

OBJECTIVE: To determine the morphologic characteristics of the nonciliated epithelium found in chondroid hamartoma of the lung. STUDY DESIGN: The morphologic characteristics and immunohistochemical reaction for surfactant protein A of the nonciliated epithelium in chondroid hamartoma of the lung was studied by immunohistochemistry. Alveolar epithelium in normal lung tissue and lung tissue surrounding primary lung cancer or metastatic lung lesions was used as a control. RESULTS: In all cases, the nonciliated epithelium in chondroid hamartoma showed the morphologic criteria of hyperplastic alveolar type II cells and a very strong positive surfactant protein A reaction in the cytoplasm when compared with alveolar epithelium of the normal lung. Similar hyperplastic type II cells were also found in the alveolar lung around metastatic or primary lung tumors. CONCLUSION: These findings may indicate that the nonciliated cells found in chondroid hamartoma of the lung are hyperplastic type II cells. This suggests that the alveolar epithelium found in chondroid hamartoma of the lung is a secondary reaction around the hamartoma and not a primary component of the lesion.     

A small molecule alpha 4 beta 1 antagonist prevents development of murine Lyme arthritis without affecting protective immunity

After infection with Borrelia burgdorferi, humans and mice under certain conditions develop arthritis. Initiation of inflammation is dependent on the migration of innate immune cells to the site of infection, controlled by interactions of a variety of adhesion molecules. In this study, we used the newly synthesized compound S18407, which is a prodrug of the active drug S16197, to analyze the functional importance of alpha4beta1-dependent cell adhesion for the development of arthritis and for the antibacterial immune response. S16197 is shown to interfere specifically with the binding of alpha4beta(1 integrin to its ligands VCAM-1 and fibronectin in vitro. Treatment of B. burgdorferi-infected C3H/HeJ mice with the alpha4beta1 antagonist significantly ameliorated the outcome of clinical arthritis and the influx of neutrophilic granulocytes into ankle joints. Furthermore, local mRNA up-regulation of the proinflammatory mediators IL-1, IL-6, and cyclooxygenase-2 was largely abolished. Neither the synthesis of spirochete-specific Igs nor the development of a Th1-dominated immune response was altered by the treatment. Importantly, the drug also did not interfere with Ab-mediated control of spirochete load in the tissues. These findings demonstrate that the pathogenesis, but not the protective immune response, in Lyme arthritis is dependent on the alpha4beta1-mediated influx of inflammatory cells. The onset of inflammation can be successfully targeted by treatment with S18407.     

Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents

Cytochrome p450 BM-3 (EC 1.14.14.1) catalyzes the hydroxylation and/or epoxidation of a broad range of substrates, including alkanes, alkenes, alcohols, fatty acids, amides, polyaromatic hydrocarbons, and heterocycles. For many of these notoriously water-insoluble compounds, p450 BM-3's K(m) values are in the millimolar range. Polar organic cosolvents are therefore added to increase substrate solubility and achieve high catalytic efficiency. Using p450 BM-3 as a catalyst for these important transformations requires that we improve its ability to tolerate the cosolvents. By directed evolution, we improved the activity of p450 BM-3 in the presence of dimethylsulfoxide (DMSO) and tetrahydrofuran (THF), achieving increases in specific activity up to 10-fold in 2% (v/v) THF and 6-fold in 25% (v/v) DMSO. The engineered p450 BM-3's are also significantly more resistant to acetone, acetonitrile, dimethylformamide, and ethanol as cosolvents in the reaction.     

Regulation of the DNA replication fork: a way to fight genomic instability

DNA replication is a complex mechanism that functions due to the coordinated interplay of many factors. In the last few years, numerous studies have suggested that DNA replication factors are closely implicated in several DNA transaction events that maintain the integrity of the genome. Therefore, DNA replication fork factors have to be considered as part of a general process that aims to protect and replicate the genome in order to allow correct functioning of a cell and its eventual daughter cells. This is illustrated by the numerous factors that have a well-defined function at the DNA replication fork, but also play crucial roles in different DNA repair pathways such as base excision repair, nucleotide excision repair, double-strand break repair, and mismatch repair. Moreover, several of the replisome proteins have also been shown to be essential in sensing and transducing DNA damages through the checkpoint cascade pathways, including the recently characterised alternative clamps and clamp-loaders. In this review we present DNA replication factors that are involved in different DNA transaction and checkpoint regulation pathways, with emphasis on the link between DNA replication and maintenance of genomic stability.     

Application of the yeast Yarrowia lipolytica as a model to analyse human pathogenic mutations in mitochondrial complex I (NADH:ubiquinone oxidoreductase)

While diagnosis and genetic analysis of mitochondrial disorders has made remarkable progress, we still do not understand how given molecular defects are correlated to specific patterns of symptoms and their severity. Towards resolving this dilemma for the largest and therefore most affected respiratory chain enzyme, we have established the yeast Yarrowia lipolytica as a eucaryotic model system to analyse respiratory chain complex I. For in vivo analysis, eYFP protein was attached to the 30-kDa subunit to visualize complex I and mitochondria. Deletions strains for nuclear coded subunits allow the reconstruction of patient alleles by site-directed mutagenesis and plasmid complementation. In most of the pathogenic mutations analysed so far, decreased catalytic activities, elevated K(M) values, and/or elevated I(50) values for quinone-analogous inhibitors were observed, providing plausible clues on the pathogenic process at the molecular level. Leigh mutations in the 49-kDa and PSST homologous subunits are found in regions that are at the boundaries of the ubiquinone-reducing catalytic core. This supports the proposed structural model and at the same time identifies novel domains critical for catalysis. Thus, Y. lipolytica is a useful lower eucaryotic model that will help to understand how pathogenic mutations in complex I interfere with enzyme function.