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861.
Aline Lara Denis D. Damasceno Rita Pires Robert Gros Enéas R. Gomes Mariana Gavioli Ricardo F. Lima Diogo Guimar?es Patricia Lima Carlos Roberto Bueno Jr. Anilton Vasconcelos Danilo Roman-Campos Cristiane A. S. Menezes Raquel A. Sirvente Vera M. Salemi Charles Mady Marc G. Caron Anderson J. Ferreira Patricia C. Brum Rodrigo R. Resende Jader S. Cruz Marcus Vinicius Gomez Vania F. Prado Alvair P. de Almeida Marco A. M. Prado Silvia Guatimosim 《Molecular and cellular biology》2010,30(7):1746-1756
862.
Nadine Katzke Solmaz Arvani René Bergmann Franco Circolone Annette Markert Vera Svensson Karl-Erich Jaeger Achim Heck Thomas Drepper 《Protein expression and purification》2010,69(2):137-146
The functional expression of heterologous genes using standard bacterial expression hosts such as Escherichia coli is often limited, e.g. by incorrect folding, assembly or targeting of recombinant proteins. Consequently, alternative bacterial expression systems have to be developed to provide novel strategies for protein synthesis exceeding the repertoire of the standard expression host E. coli.Here, we report on the construction of a novel expression system that combines the high processivity of T7 RNA polymerase with the unique physiological properties of the facultative photosynthetic bacterium Rhodobacter capsulatus. This system basically consists of a recombinant R. capsulatus T7 expression strain (R. capsulatus B10S-T7) harboring the respective polymerase gene under control of a fructose inducible promoter. In addition, a set of different broad-host-range vectors (pRho) was constructed allowing T7 RNA polymerase dependent and independent target gene expression in R. capsulatus and other Gram-negative bacteria. The expression efficiency of the novel system was studied in R. capsulatus and E. coli using the yellow fluorescent protein (YFP) as model protein. Expression levels were comparable in both expression hosts and yielded up to 80 mg/l YFP in phototrophically grown R. capsulatus cultures. This result clearly indicates that the novel R. capsulatus-based expression system is well suited for the high-level expression of soluble proteins. 相似文献
863.
Kararigas G Becher E Mahmoodzadeh S Knosalla C Hetzer R Regitz-Zagrosek V 《Biology of sex differences》2010,1(1):2-9
Background
Although circulating levels of sexual hormones in elderly men and women are low and quite similar, the adaptation of the elderly heart to stress differs between the sexes. We have hypothesized that the effects of sexual hormones in the heart may differ in men and women. Here, we assessed whether 17β-oestradiol regulates gene expression in the human heart in a sex-dependent manner. We selected the progesterone receptor as a well studied 17β-oestradiol target that may be pathologically linked to cardiac remodelling.Methods
In order to assess the ex vivo effects of 17β-oestradiol in intact human cardiac tissues, we developed a 24-h model for the culture of human atrial myocardium. We verified tissue viability after 24 h in culture with two standard assays to determine the degree of apoptosis and metabolic activity of cardiac tissues. Progesterone receptor mRNA and protein level were measured after 24-h treatment of tissues with 17β-oestradiol. Statistical analysis was performed by the Mann-Whitney U test and two-way ANOVA.Results
We established a tissue culture model that allows for the study of viable human cardiac tissue over a 24-h period. After 24 h, cultured cardiac tissues revealed low apoptosis, retained their metabolic activity and, therefore, remained viable. Treatment with 17β-oestradiol led to an induction of the progesterone receptor mRNA level in female (P = 0.001) but not in male tissues. Similarly, there was an increase in the level of progesterone receptor protein in female tissues (P = 0.03), while a decreasing trend was observed in male tissues (P = 0.079) exposed to 17β-oestradiol.Conclusions
Our novel finding may offer a molecular explanation for the sex-specific differences observed in cardiac remodelling. The culture model we established for human cardiac tissue will facilitate the study of cellular processes in health and disease and will be of use for pharmacological testing. 相似文献864.
Airich LG Tsyrenzhapova IS Vorontsova OV Feofanov AV Doroshenko VG Mashko SV 《Journal of molecular microbiology and biotechnology》2010,19(4):189-197
YddG is an inner membrane protein (IMP) that exports aromatic amino acids in Escherichia coli. Topology models of YddG produced by sequence-based analysis in silico have predicted the presence of 9 or 10 potential transmembrane segments. To experimentally analyze the membrane topology of YddG, we used randomly created fusions to β-lactamase (BlaM) as a reporter. The selection of such fusions under 50 μg/ml of ampicillin had to fit with the periplasmic location of the BlaM domain. Five periplasmic loops of YddG predicted by the 10-transmembrane (TM) helices model were identified via the characterization of 12 unique in-frame fusions distributed along the yddG coding region. To confirm the 10-TM helices model further, cytoplasmic regions of YddG were identified with the help of ZsGreen fluorescent protein as a reporter. The presence of four cytoplasmic regions and the cytoplasmic localization of the C-terminus were revealed. Therefore, a 10-TM helices topology with cytoplasmic locations of the N- and C-termini is supported. The present data confirm the 'positive-inside rule' for IMPs and the early results of other workers regarding the cytoplasmic location of the C-terminus of YddG. The pole-specific localization of YddG-ZsGreen in E. coli cells was detected by fluorescence microscopy. 相似文献
865.
866.
Olga Khorosjutina Paulina H. Wanrooij Julian Walfridsson Zsolt Szilagyi Xuefeng Zhu Vera Baraznenok Karl Ekwall Claes M. Gustafsson 《The Journal of biological chemistry》2010,285(39):29729-29737
The multiprotein Mediator complex is an important regulator of RNA polymerase II-dependent genes in eukaryotic cells. In contrast to the situation in many other eukaryotes, the conserved Med15 protein is not a stable component of Mediator isolated from fission yeast. We here demonstrate that Med15 exists in a protein complex together with Hrp1, a CHD1 ATP-dependent chromatin-remodeling protein. The Med15-Hrp1 subcomplex is not a component of the core Mediator complex but can interact with the L-Mediator conformation. Deletion of med15+ and hrp1+ causes very similar effects on global steady-state levels of mRNA, and genome-wide analyses demonstrate that Med15 associates with a distinct subset of Hrp1-bound gene promoters. Our findings therefore indicate that Mediator may directly influence histone density at regulated promoters. 相似文献
867.
Alexander Swidsinski Yvonne Dörffel Vera Loening-Baucke Werner Mendling Johannes Schilling Jennifer L. Patterson Hans Verstraelen 《Anaerobe》2010,16(5):478-482
Recent data point at the similarity between the perianal and vaginal microflora in terms of Lactobacillus species involved. Bacterial vaginosis, the most common perturbation of the vaginal microflora involving primarily overgrowth of Gardnerella vaginalis, has also been suggested to involve a recto-vaginal pathway. We addressed this issue with regard to bacteria of the Bifidobacteriaceae family. In particular, we investigated the putative concordance of the presence of G. vaginalis and a series of Bifidobacteria between the perianal and vaginal microflora in 10 patients with bacterial vaginosis through multicolor fluorescence in situ hybridization analysis of desquamated epithelial cells.G. vaginalis was found in a biofilm mode of growth at the perianal and vaginal sites. In most women at least one of the following species was detected perianally: Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium breves, Bifidobacterium bifidum and Bifidobacterium catenulatum. At the vaginal site, none of these Bifidobacteria was found.We conclude that bacterial vaginosis does not occur as a result of simple growth per continuum of perianal bacteria. Only some species originating from the intestinal tract do display pronounced vaginotropism, like G. vaginalis, whereas many other species do not. 相似文献
868.
Sven C. Neulinger Rüdiger Stöhr Vera Thiel Rolf Schmaljohann Johannes F. Imhoff 《Journal of microbiology (Seoul, Korea)》2010,48(4):411-418
Though spirochetes have been repeatedly found in marine sponges and other invertebrates, little attention has been paid to
the specificity of this association. This study demonstrates that different genoand morphotypes of spirochetes can reside
within the same sponge individual and develop in considerable numbers. Specimens of the calcareous sponge Clathrina clathrus collected from the Adriatic Sea off Rovinj (Croatia) were found to harbor spirochete-like bacteria, which were characterized
by scanning electron microscopy (SEM), 16S rRNA gene analysis, and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Two novel spirochete sequence types related to the Brachyspiraceae could be retrieved. By use of specifically designed CARD-FISH probes, the C. clathrus-associated sequences could be assigned to a linear and a helical spirochete morphotype. Both were located within the sponge
mesohyl and resembled the spirochete-like cells identified by SEM. In addition, from a Clathrina sp., most likely C. coriacea, that originated from Indonesian coastal waters, four different spirochete type sequences were recovered. Two of these also
affiliated with the Brachyspiraceae, the other two were found associated with the Spirochaetaceae, one with the genera Borrelia and Cristispira. 相似文献
869.
870.
Goncharova EA Lim P Goncharov DA Eszterhas A Panettieri RA Krymskaya VP 《Nature protocols》2006,1(6):2905-2908
Vascular and airway remodeling, which are characterized by airway smooth muscle (ASM) and pulmonary arterial vascular smooth muscle (VSM) proliferation, contribute to the pathology of asthma, pulmonary hypertension, restenosis and atherosclerosis. To evaluate the proliferation of VSM and ASM cells in response to mitogens, we perform a [3H]thymidine incorporation assay. The proliferation protocol takes approximately 48 h and includes stimulating cells synchronized in G0/G1 phase of the cell cycle with agonists, labeling cells with [3H]thymidine and examining levels of [3H]thymidine incorporation by scintillation counting. Although using radiolabeled [3H]thymidine incorporation is a limitation, the greatest benefit of the assay is providing reliable and statistically significant data. 相似文献