A novel, high-affinity, fluorescent progesterone receptor antagonist. Synthesis and in vitro studies

The present paper describes the chemical synthesis and in vitro characterization of a novel, high-affinity, fluorescent progesterone receptor (PR) antagonist. The three-step synthesis was carried out starting from mifepristone. After demethylation with calcium oxide, the methylamino group was alkylated with 6-bromohexanol, and the resulting compound was reacted with fluorescein 5-isothiocyanate, yielding the fluorescein-mifepristone conjugate. Interaction of the conjugate as well as of its precursors with PR was determined in cell culture (alkaline phosphatase assay and transactivation assay). Antiprogestagenic activity of the intermediates were comparable to that of the parent compound. Even after attachment of the bulky fluorescein moiety, considerable antiprogestagenic activity was maintained. Microscopic studies revealed that fluorescence of the conjugate was almost confined to the nuclei of steroid hormone receptor-positive cells, whereas the nuclei of steroid hormone receptor-negative cells remained unstained. To our knowledge, this is the first report on a fluorescent ligand for PR suitable for studies in living cells. It is proposed that the present fluorescent PR antagonist might serve as a lead compound for the development of contrast agents for PR imaging, e.g., by near-infrared optical imaging.     

Epidermolysis bullosa simplex-type mutations alter the dynamics of the keratin cytoskeleton and reveal a contribution of actin to the transport of keratin subunits

Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs.     

Helifulvanolsäkure—ein neues diterpen mit anomalem kohlenstoffgerüst aus Helichrysum fulvum

The aerial parts of Helichrysum fulvum afforded, in addition to beyerenic acid and ent-kaurenic acid, two new diterpenic acids with the hitherto unknown carbon skeleton of an isotrachylobane type. The structures of these acids, isolated as their methyl esters, were elucidated by extensive NMR studies, some chemical transformations and by X-ray structural analysis of the corresponding acetate. The related alcohol on reaction with pyridinochlorochromate afforded a homoconjugated diene probably formed by fragmentation of a cyclopropyl carbinol. The possible biogenesis of the new carbon skeleton is discussed briefly.     

Deletion of mdmB impairs mitochondrial distribution and morphology in Aspergillus nidulans

Mitochondria form a dynamic network of interconnected tubes in the cells of Saccharomyces cerevisiae or filamentous fungi such as Aspergillus nidulans, Neurospora crassa, or Podospora anserina. The dynamics depends on the separation of mitochondrial fragments, their movement throughout the cell, and their subsequent fusion with the other parts of the organelle. Interestingly, the microtubule network is required for the distribution in N. crassa and S. pombe, while S. cerevisiae and A. nidulans appear to use the actin cytoskeleton. We studied a homologue of S. cerevisiae Mdm10 in A. nidulans, and named it MdmB. The open reading frame is disrupted by two introns, one of which is conserved in mdm10 of P. anserina. The MdmB protein consists of 428 amino acids with a predicted molecular mass of 46.5 kDa. MdmB shares 26% identical amino acids to Mdm10 from S. cerevisiae, 35% to N. crassa, and 32% to the P. anserina homologue. A MdmB-GFP fusion protein co-localized evenly distributed along mitochondria. Extraction of the protein was only possible after treatment with a non-ionic and an ionic detergent (1% Triton X-100; 0.5% SDS) suggesting that MdmB was tightly bound to the mitochondrial membrane fraction. Deletion of the gene in A. nidulans affected mitochondrial morphology and distribution at 20 degrees C but not at 37 degrees C. mdmB deletion cells contained two populations of mitochondria at lower temperature, the normal tubular network plus some giant, non-motile mitochondria.     

Backbone dynamics of the human MIA protein studied by (15)N NMR relaxation: implications for extended interactions of SH3 domains

The melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma as enhanced values diagnose metastatic melanoma stages III and IV. Here, we report the backbone dynamics of human MIA studied by (15)N NMR relaxation experiments. The folded core of human MIA is found to be rigid, but several loops connecting beta-sheets, such as the RT-loop for example, display increased mobility on picosecond to nanosecond time scales. One of the most important dynamic features is the pronounced flexibility of the distal loop, comprising residues Asp 68 to Ala 75, where motions on time scales up to milliseconds occur. Further, significant exchange contributions are observed for residues of the canonical binding site of SH3 domains including the RT-loop, the n-Src loop, for the loop comprising residues 13 to 19, which we refer to as the"disulfide loop", in part for the distal loop, and the carboxyl terminus of human MIA. The functional importance of this dynamic behavior is discussed with respect to the biological activity of several point mutations of human MIA. The results of this study suggest that the MIA protein and the recently identified highly homologous fibrocyte-derived protein (FDP)/MIA-like (MIAL) constitute a new family of secreted proteins that adopt an SH3 domain-like fold in solution with expanded ligand interactions.     

Three dimensional modeling of the cerebrospinal fluid dynamics and brain interactions in the aqueduct of sylvius

A computational fluid dynamics (CFD) method is presented to investigate the flow of cerebro-spinal fluid (CSF) in the cerebral aqueduct. In addition to former approaches exhibiting a rigid geometry, we propose a model which includes a deformable membrane as the wall of this flow channel. An anatomical shape of the aqueduct was computed from magnetic resonance images (MRI) and the resulting meshing was immersed in a marker-and-cell (MAC) staggered grid for to take into account fluid-structure interactions. The time derivatives were digitized using the Crank-Nicolson scheme. The equation of continuity was modified by introducing an artificial compressibility and digitized by a finite difference scheme. Calculations were validated with the simulation of laminar flow in a rigid tube. Then, comparisons were made between simulations of a rigid aqueduct and a deformable one. We found that the deformability of the walls has a strong influence on the pressure drop for a given flow.     

A single intratracheal dose of the growth factor Fms-like tyrosine kinase receptor-3 ligand induces a rapid differential increase of dendritic cells and lymphocyte subsets in lung tissue and bronchoalveolar lavage,resulting in an increased local antibody production

Repetitive doses of the growth factor Fms-like tyrosine kinase receptor-3 ligand (Flt3L) have resulted in increased numbers of dendritic cells (DC) in various organs, and the effect on protective or tolerogeneic responses in the gut wall has been documented in the literature. In this study, for the first time, Flt3L was locally applied in the trachea of rats using a single dose only. A dose-dependent increase not only of DC, but also of T lymphocytes (CD4(+) and CD8(+)), was seen with a maximum on day 3. The effects on the cells in the lung interstitium and the bronchoalveolar space showed some differences. The use of tetanus toxoid as a model Ag applied intratracheally after the local Flt3L stimulation resulted in increased levels of specific IgA and IgG in the lung. Thus, this novel approach of locally stimulating APCs by topical application of a DC growth factor before applying the Ag offers a new vaccination strategy.     

Structural instability and fibrillar aggregation of non-expanded human ataxin-3 revealed under high pressure and temperature

Protein misfolding and formation of structured aggregates are considered to be the earliest events in the development of neurodegenerative diseases, but the mechanism of these biological phenomena remains to be elucidated. Here, we report a study of heat- and pressure-induced unfolding of human Q26 and murine Q6 ataxin-3 using spectroscopic methods. UV absorbance and fluorescence revealed that heat and pressure induced a structural transition of both proteins to a molten globule conformation. The unfolding pathway was partly irreversible and led to a protein conformation where tryptophans were more exposed to water. Furthermore, the use of fluorescent probes (8-anilino-1-naphthalenesulfonate and thioflavin T) allowed the identification of different intermediates during the process of pressure-induced unfolding. At high temperature and pressure, human Q26, but not murine Q6, underwent concentration-dependent aggregation. Fourier transform infrared and circular dichroism spectroscopy revealed that these aggregates are characterized by an increased beta-sheet content. As revealed by electron microscopy, heat- and pressure-induced aggregates were different; high temperature treatment led to fibrillar microaggregates (8-10-nm length), whereas high pressure induced oligomeric structures of globular shape (100 nm in diameter), which sometimes aligned to higher order suprastructures. Several intermediate structures were detected in this process. Two factors appear to govern ataxin unfolding and aggregation, the length of the polyglutamine tract and its protein context.     

Crystal structure of an archaeal class I aldolase and the evolution of (betaalpha)8 barrel proteins

Fructose-1,6-bisphosphate aldolase (FBPA) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate in the glycolytic pathway. FBPAs from archaeal organisms have recently been identified and characterized as a divergent family of proteins. Here, we report the first crystal structure of an archaeal FBPA at 1.9-A resolution. The structure of this 280-kDa protein complex was determined using single wavelength anomalous dispersion followed by 10-fold non-crystallographic symmetry averaging and refined to an R-factor of 14.9% (Rfree 17.9%). The protein forms a dimer of pentamers, consisting of subunits adopting the ubiquitous (betaalpha)8 barrel fold. Additionally, a crystal structure of the archaeal FBPA covalently bound to dihydroxyacetone phosphate was solved at 2.1-A resolution. Comparison of the active site residues with those of classical FBPAs, which share no significant sequence identity but display the same overall fold, reveals a common ancestry between these two families of FBPAs. Structural comparisons, furthermore, establish an evolutionary link to the triosephosphate isomerases, a superfamily hitherto considered independent from the superfamily of aldolases.