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71.
Schmitz-Esser S Haferkamp I Knab S Penz T Ast M Kohl C Wagner M Horn M 《Journal of bacteriology》2008,190(17):5746-5752
ATP/ADP translocases are a hallmark of obligate intracellular pathogens related to chlamydiae and rickettsiae. These proteins catalyze the highly specific exchange of bacterial ADP against host ATP and thus allow bacteria to exploit their hosts' energy pool, a process also referred to as energy parasitism. The genome sequence of the obligate intracellular pathogen Lawsonia intracellularis (Deltaproteobacteria), responsible for one of the most economically important diseases in the swine industry worldwide, revealed the presence of a putative ATP/ADP translocase most similar to known ATP/ADP translocases of chlamydiae and rickettsiae (around 47% amino acid sequence identity). The gene coding for the putative ATP/ADP translocase of L. intracellularis (L. intracellularis nucleotide transporter 1 [NTT1(Li)]) was cloned and expressed in the heterologous host Escherichia coli. The transport properties of NTT1(Li) were determined by measuring the uptake of radioactively labeled substrates by E. coli. NTT1(Li) transported ATP in a counterexchange mode with ADP in a highly specific manner; the substrate affinities determined were 236.3 (+/- 36.5) microM for ATP and 275.2 (+/- 28.1) microM for ADP, identifying this protein as a functional ATP/ADP translocase. NTT1(Li) is the first ATP/ADP translocase from a bacterium not related to Chlamydiae or Rickettsiales, showing that energy parasitism by ATP/ADP translocases is more widespread than previously recognized. The occurrence of an ATP/ADP translocase in L. intracellularis is explained by a relatively recent horizontal gene transfer event with rickettsiae as donors. 相似文献
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73.
Cheng-Chung Wu Hong-Jin Wu Chia-Hui Wang Chia-Hua Lin Shu-Ching Hsu Yi-Rong Chen Michael Hsiao Scott C Schuyler Frank Leigh Lu Nianhan Ma Jean Lu 《Cell cycle (Georgetown, Tex.)》2015,14(8):1207-1217
Mouse embryonic stem cells (ES cells) can proliferate indefinitely. To identify potential signals involved in suppression of self-renewal, we previously screened a kinase/phosphatase expression library in ES cells, and observed that inhibition of Dual Leucine zipper-bearing Kinase (DLK) increased relative cell numbers. DLK protein was detected in both the pluripotent and differentiated states of mouse ES cells while DLK kinase activity increased upon differentiation. Overexpression of DLK in mouse ES cells displayed reductions in relative cell/colony numbers and Nanog expression, suggesting a suppressive role of DLK in self-renewal. By examining protein sequences of DLK, we identified 2 putative Akt phosphorylation sites at S584 and T659. Blocking PI3K/Akt signaling with LY-294002 enhanced DLK kinase activity dramatically. We found that Akt interacts with and phosphorylates DLK. Mutations of DLK amino acid residues at putative Akt phosphorylation sites (S584A, T659A, or S584A and T659A) diminished the level of DLK phosphorylation. While the mutated DLKs (S584A, T659A, or S584A and T659A) were expressed, a further reduction in cell/colony numbers and Nanog expression appeared in mouse ES cells. In addition, these mutant DLKs (S584A, T659A, or S584A and T659A) exhibited more robust kinase activity and cell death compared to wild type DLK or green fluorescence (GFP) controls. In summary, our results show that DLK functions to suppress self-renewal of mouse ES cells and is restrained by Akt phosphorylation. 相似文献
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76.
Simulation models of nutrient uptake of root systems starting with one-dimensional single root approaches up to complex three-dimensional
models are increasingly used for examining the interacting of root distribution and nutrient uptake. However, their accuracy
was seldom systematically tested. The objective of the study is to compare one-dimensional and two-dimensional modelling approaches
and to test their applicability for simulation of nutrient uptake of heterogeneously distributed root systems giving particular
attention to the impact of spatial resolution. Therefore, a field experiment was carried out with spring barley (Hordeum vulgare L. cv. Barke) in order to obtain data of in situ root distribution patterns as model input. Results indicate that a comparable
coarse spatial resolution can be used with sufficient modelling results when a steady state approximation is applied to the
sink cells of the two-dimensional model. Furthermore, the accuracy of the model was clearly improved compared to a simple
zero sink approach assuming both near zero concentrations within the sink cell and a linear gradient between the sink cell
and its adjacent neighbours. However, for modelling nitrate uptake of a heterogeneous root system a minimum number of grid
cells is still necessary. The tested single root approach provided a computational efficient opportunity to simulate nitrate
uptake of an irregular distributed root system. Nevertheless, two-dimensional models are better suited for a number of applications
(e.g. surveys made on the impact of soil heterogeneity on plant nutrient uptake). Different settings for the suggested modelling
techniques are discussed. 相似文献
77.
78.
Role of the cytoplasmic tail domains of Bunyamwera orthobunyavirus glycoproteins Gn and Gc in virus assembly and morphogenesis
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The M RNA genome segment of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, encodes a precursor polyprotein that is proteolytically cleaved to yield two structural proteins, Gn and Gc, and a nonstructural protein called NSm. Gn and Gc are type I integral transmembrane glycoproteins. The Gn protein contains a predicted cytoplasmic tail (CT) of 78 residues, and Gc has a shorter CT of 25 residues. Little is known about the role of the Gn and Gc CT domains in the virus replication cycle. We generated a series of mutant glycoprotein precursor constructs containing either deletions or alanine substitutions in the CT domains of Gn and Gc. We examined the effects of these mutations on glycoprotein maturation, cell surface expression, and low pH-induced syncytium formation. In addition, the effects of these mutations were also assessed using a reverse genetics-based virus assembly assay and a virus rescue system. Our results show that the CT domains of both Gn and Gc play crucial roles in BUNV-mediated membrane fusion, virus assembly, and morphogenesis. 相似文献
79.
Elevated levels of the pro-inflammatory cytokine, interleukin-18 (IL-18) have recently been demonstrated in osteoarthritic cartilage. However, the effects of IL-18 on chondrocyte signalling and matrix biosynthesis are poorly understood. Therefore, the present study was undertaken to further characterize the impact of IL-18 on human articular chondrocyte in vitro. Human articular chondrocytes were stimulated with various concentrations of recombinant human IL-18 (1, 10, 100 ng/ml) for 0, 4, 8, 12, 24, 48, 72 h in vitro. The effects of IL-18 on the cartilage-specific matrix protein collagen type II, the cytoskeletal protein vinculin, the cell matrix signal transduction receptor beta-integrin, key signalling proteins of the MAPKinase pathway (such as SHC (Sarc Homology Collagen) and activated MAPKinase [ERK-1/-2]), the pro-inflammatory enzyme cyclo-oxygenase-2 (COX-2) and the apoptosis marker activated caspase-3 were evaluated by Western blot analysis and immunofluorescence labelling. Morphological features of IL-18 stimulated chondrocytes were estimated by transmission electron microscopy. IL-18 lead to inhibition of collagen type II-deposition, decreased beta-integrin receptor and vinculin synthesis, SHC and MAPKinase activation, increased COX-2 synthesis and activation of caspase-3 in chondrocytes in a time- and dose-dependent manner. Furthermore, chondrocytes treated with IL-18 exhibited typical morphological features of apoptosis as revealed by transmission electron microscopy. Taken together, the results of the present study underline key catabolic events mediated by IL-18 signalling in chondrocytes such as loss of cartilage-specific matrix and apoptosis. Inhibition of MAPKinase signalling is hypothesized to contribute to these features. Future therapeutics targeting IL-18 signalling pathways may be beneficial in rheumatoid arthritis and osteoarthritis therapy. 相似文献
80.
Oberholzer A John T Kohl B Gust T Müller RD La Face D Hutchins B Zreiqat H Ertel W Schulze-Tanzil G 《Cell and tissue research》2007,328(2):383-390
Gene transfer into cultured chondrocytes by using adenoviral vectors has potential applications in treating cartilage disorders.
The present study was undertaken to compare and optimize two chondrocyte culture conditions for adenoviral transduction efficacy
by using primary human articular chondrocytes cultivated either directly in a monolayer condition or as outgrowths from alginate-stored
chondrocyte cultures. Isolated primary chondrocytes from human articular cartilage were either immediately transduced with
an EGFP (enhanced green fluorescent protein)-gene-bearing adenoviral vector (1,000 and 3,000 virus particles/cell) or cultured
in alginate before transduction. Immunohistochemistry and flow cytometric analysis were employed to determine the expression
of extracellular matrix proteins and of the αvβ5 integrin receptor involved in adenoviral cell entry. Monolayer chondrocytes
exhibited moderate transduction rates (mean 22.2% and 46.9% EGFP-positive cells at 1,000 and 3,000 virus particles/cell by
72 h post-transduction), whereas alginate-derived chondrocytes revealed significantly higher transduction efficacies (95.7%
and 99%). Both monolayer and alginate-derived chondrocytes expressed αvβ5 integrin, type II collagen and cartilage proteoglycans.
The mean fluorescence intensity of type II collagen was significantly higher in the alginate-derived chondrocytes, whereas
that of αvβ5 integrin was higher in the monolayer chondrocytes. Our results indicate that transduction efficacy is independent
of αvβ5 integrin expression levels in chondrocytes. Moreover, adenoviral transduction of alginate-derived chondrocytes is
more efficient than that for monolayer chondrocytes and may be a suitable tool to achieve sufficient numbers of transduced
and differentiated chondrocytes for experimental applications and cartilage repair.
Dr. Gundula Schulze-Tanzil is supported by a grant awarded by the Rahel Hirsh Foundation from the Charité Medical Schools
Berlin. The study was supported by a grant from the Deutsche Arthrosehilfe e.V. 相似文献