Functional characterisation of mutations in the ligand-binding domain of the androgen receptor gene in patients with androgen insensitivity syndrome

Five mutations in the ligand-binding domain of the androgen receptor gene were identified in patients with complete (A765T, C784Y, R831X and M895T) or partial (R840G) androgen insensitivity. A765T and R831X have been reported previously whereas the other three mutations are novel. Receptors carrying these mutations were transiently expressed in COS-1 cells, and androgen binding and capacity to transactivate an androgen-responsive reporter gene were assayed. C784Y led to abolished androgen binding and transactivating capacity, R840G and M895T showed reduced specific binding and partial transactivation. The in vitro functions of the R840G and M895T mutants were improved with supraphysiological concentrations of steroid. Received: 10 June 1998 / Accepted: 10 September 1998     

The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4′,6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.     

Overcoming Microstructural Limitations in Water Processed Organic Solar Cells by Engineering Customized Nanoparticulate Inks

The application of conjugated polymer and fullerene water‐based nanoparticles (NP) as ecofriendly inks for organic photovoltaics (OPVs) is reported. A low bandgap polymer diketopyrrolopyrrole–quinquethiophene (PDPP5T‐2) and the methanofullerene PC₇₁BM are processed into three types of nanoparticles: pristine fullerene NPs, pristine polymer NPs, and mixed polymer:fullerene NPs, allowing the formation of bulk heterojunction (BHJ) composites with different domain sizes. Mild thermal annealing is required to melt the nanospheres and enable the formation of interconnected pathways within mixed phases. This BHJ is accompanied by a shrinkage of film, whereas the more compact layers show enhanced mobility. Consistently reduced recombination and better performance are found for mixed NP, containing both, the polymer and the fullerene within a single NP. The optimized solar cell processed by ultrasmall NPs delivers a power conversion efficiency of about 3.4%. This is among the highest values reported for aqueous processed OPVs but still lacks performance compared to those being processed from halogenated solvents. Incomplete crystallization is identified as the main root for reduced efficiency. It is nevertheless believed that postprocessing does not cut attraction from printing aqueous organic NP inks as a trendsetting strategy for the reliable and ecofriendly production of organic solar cells.     

Biomechanical characterization and clinical implications of artificially induced toe-walking: differences between pure soleus, pure gastrocnemius and combination of soleus and gastrocnemius contractures

The purpose of this study was to characterize biomechanically three different toe-walking gait patterns, artificially induced in six neurologically intact subjects and to compare them to selected cases of pathological toe-walking. The subjects, equipped with lightweight mechanical exoskeleton with elastic ropes attached to the left leg's heel on one end and on shank and thigh on the other end in a similar anatomical locations where soleus and gastrocnemius muscles attach to skeleton, walked at speed of approximately 1m/s along the walkway under four experimental conditions: normal walking (NW), soleus contracture emulation (SOL), gastrocnemius contracture emulation (GAS) and emulation of both soleus and gastrocnemius contractures (SOLGAS). Reflective markers and force platform data were collected and ankle, knee and hip joint angles, moments and powers were calculated using inverse dynamic model for both legs. Characteristic peaks of averaged kinematic and kinetic patterns were compared among all four experimental conditions in one-way ANOVA. In the left leg SOL contracture mainly influenced the ankle angle trajectory, while GAS and SOLGAS contractures influenced the ankle and knee angle trajectories. GAS and SOLGAS contractures significantly increased ankle moment during midstance as compared to SOL contracture and NW. All three toe-walking experimental conditions exhibited significant power absorption in the ankle during loading response, which was absent in the NW condition, while during preswing significant decrease in power absorption as compared to NW was seen. In the knee joint SOL contracture diminished, GAS contracture increased while SOLGAS contracture approximately halved knee extensor moment during midstance as compared to NW. All three toe-walking experimental conditions decreased hip range of motion, hip flexor moment and power requirements during stance phase. Main difference in the right leg kinematic and kinetic patterns was seen in the knee moment trajectory, where significant increase in the knee extensor moment took place in terminal stance for GAS and SOLGAS experimental conditions as compared to SOL and NW. The kinetic trajectories under SOL and GAS experimental conditions were qualitatively compared to two selected clinical cases showing considerable similarity. This implies that distinct differences in kinetics between SOL, GAS and SOLGAS experimental conditions, as described in this paper, may be clinically relevant in determining the relative contribution of soleus and gastrocnemius muscles contractures to toe-walking in particular pathological gait.     

Cloning of the Bacillus subtilis DNA fragment containing the genes for lysine and riboflavin biosynthesis

The SalI fragment of chromosomal DNA of Bacillus subtilis carrying the gene for lysine biosynthesis and the regulatory operator region (ribO) from the riboflavin gene was cloned into Escherichia coli cells. This fragment was shown to contain the gene coding for lysine synthesizing enzyme. Localization of this gene in Bac. subtili was determined. New plasmids pLRS33 and pLRB4 were constructed using pBR322; they carry a fragment homologous to pLP102 plasmid containing the operon for riboflavin biosynthesis.     

Effect of hydrophobic mutations in the H2-H3 subdomain of prion protein on stability and conversion in vitro and in vivo

Prion diseases are fatal neurodegenerative diseases, which can be acquired, sporadic or genetic, the latter being linked to mutations in the gene encoding prion protein. We have recently described the importance of subdomain separation in the conversion of prion protein (PrP). The goal of the present study was to investigate the effect of increasing the hydrophobic interactions within the H2-H3 subdomain on PrP conversion. Three hydrophobic mutations were introduced into PrP. The mutation V209I associated with human prion disease did not alter protein stability or in vitro fibrillization propensity of PrP. The designed mutations V175I and T187I on the other hand increased protein thermal stability. V175I mutant fibrillized faster than wild-type PrP. Conversion delay of T187I was slightly longer, but fluorescence intensity of amyloid specific dye thioflavin T was significantly higher. Surprisingly, cells expressing V209I variant exhibited inefficient proteinase K resistant PrP formation upon infection with 22L strain, which is in contrast to cell lines expressing wild-type, V175I and T187I mPrPs. In agreement with increased ThT fluorescence at the plateau T187I expressing cell lines accumulated an increased amount of the proteinase K-resistant prion protein. We showed that T187I induces formation of thin fibrils, which are absent from other samples. We propose that larger solvent accessibility of I187 in comparison to wild-type and other mutants may interfere with lateral annealing of filaments and may be the underlying reason for increased conversion efficiency.     

Role of immune cytolysis and its thymosin stimulation in experimental influenza

In experiments on C57BL and CC57W mice the acute or chronic course of experimental influenza infection has been shown to correlate with the activity of immune cytolysis. At a low level of the cytolytic activity of T-lymphocytes the prolonged persistence of influenza virus develops. The stimulation of cell-mediated immunity with thymosin prevents the development of chronic influenza infection.     

Sequential changes in the macrostructure and RNA-synthesizing activity of nucleolar and extranucleolar chromatin from rat liver cells during induction of DNA synthesis

The dynamics of structural changes and RNA-polymerase activity in rat liver cell chromatin caused by drastic changes in the rates of protein synthesis was investigated. Inhibition of protein synthesis after a single injection of animals with cycloheximide (0.3 mg/100 g of body weight) increased the total condensibility of chromatin. Under these conditions, the stepwise activation of RNA-polymerases I and II correlated with decondensation of chromatin. By the 6-12th hour following cycloheximide injection, a chromatin fraction enriched with RNA-polymerase I and a RNA-polymerase II-rich fraction could be isolated from liver cell nuclei.