SPAK kinase is a substrate and target of PKCtheta in T-cell receptor-induced AP-1 activation pathway

Protein kinase C-theta (PKCtheta) plays an important role in T-cell activation via stimulation of AP-1 and NF-kappaB. Here we report the isolation of SPAK, a Ste20-related upstream mitogen-activated protein kinase (MAPK), as a PKCtheta-interacting kinase. SPAK interacted with PKCtheta (but not with PKCalpha) via its 99 COOH-terminal residues. TCR/CD28 costimulation enhanced this association and stimulated the catalytic activity of SPAK. Recombinant SPAK was phosphorylated on Ser-311 in its kinase domain by PKCtheta, but not by PKCalpha. The magnitude and duration of TCR/CD28-induced endogenous SPAK activation were markedly impaired in PKCtheta-deficient T cells. Transfected SPAK synergized with constitutively active PKCtheta to activate AP-1, but not NF-kappaB. This synergistic activity, as well as the receptor-induced SPAK activation, required the PKCtheta-interacting region of SPAK, and Ser-311 mutation greatly reduced these activities of SPAK. Conversely, a SPAK-specific RNAi or a dominant-negative SPAK mutant inhibited PKCtheta- and TCR/CD28-induced AP-1, but not NF-kappaB, activation. These results define SPAK as a substrate and target of PKCtheta in a TCR/CD28-induced signaling pathway leading selectively to AP-1 (but not NF-kappaB) activation.     

Modulation of epithelial tubule formation by Rho kinase

We have developed a model system for studying integrin regulation of mammalian epithelial tubule formation. Application of collagen gel overlays to Madin-Darby canine kidney (MDCK) cells induced coordinated disassembly of junctional complexes that was accompanied by lamellipodia formation and cell rearrangement (termed epithelial remodeling). In this study, we present evidence that the Rho signal transduction pathway regulates epithelial remodeling and tubule formation. Incubation of MDCK cells with collagen gel overlays facilitated formation of migrating lamellipodia with membrane-associated actin. Inhibitors of myosin II and actin prevented lamellipodia formation, which suggests that actomyosin function was involved in regulation of epithelial remodeling. To determine this, changes in myosin II distribution, function, and phosphorylation were studied during epithelial tubule biogenesis. Myosin II colocalized with actin at the leading edge of lamellipodia thereby providing evidence that myosin is important in epithelial remodeling. This possibility is supported by observations that inhibition of Rho kinase, a regulator of myosin II function, alters formation of lamellipodia and results in attenuated epithelial tubule development. These data and those demonstrating myosin regulatory light-chain phosphorylation at the leading edge of lamellipodia strongly suggest that Rho kinase and myosin II are important modulators of epithelial remodeling. They support a hypothesis that the Rho signal transduction pathway plays a significant role in regulation of epithelial tubule formation. signaling pathway; polarity     

Canonical labeling of proteome maps

We propose a canonical labeling of proteome maps, which enables one to sort and catalog the maps in a simple way. The canonical label of a proteome map is based on the canonical labeling of vertexes of Hasse diagram embedded in the map resulting in the adjacency matrix, the rows of which when viewed as binary numbers are the smallest possible such numbers. The use of the approach in documentation is illustrated with the proteome maps of liver cells of healthy male Fisher F344 rats and the rats treated with different peroxisome proliferators.     

Nd6p, a novel protein with RCC1-like domains involved in exocytosis in Paramecium tetraurelia

In Paramecium tetraurelia, the regulated secretory pathway of dense core granules called trichocysts can be altered by mutation and genetically studied. Seventeen nondischarge (ND) genes controlling exocytosis have already been identified by a genetic approach. The site of action of the studied mutations is one of the three compartments, the cytosol, trichocyst, or plasma membrane. The only ND genes cloned to date correspond to mutants affected in the cytosol or in the trichocyst compartment. In this work, we investigated a representative of the third compartment, the plasma membrane, by cloning the ND6 gene. This gene encodes a 1,925-amino-acid protein containing two domains homologous to the regulator of chromosome condensation 1 (RCC1). In parallel, 10 new alleles of the ND6 gene were isolated. Nine of the 12 available mutations mapped in the RCC1-like domains, showing their importance for the Nd6 protein (Nd6p) function. The RCC1 protein is well known for its guanine exchange factor activity towards the small GTPase Ran but also for its involvement in membrane fusion during nuclear envelope assembly. Other proteins with RCC1-like domains are also involved in intracellular membrane fusion, but none has been described yet as involved in exocytosis. The case of Nd6p is thus the first report of such a protein with a documented role in exocytosis.     

Landscape determinants of genetic differentiation,inbreeding and genetic drift in the hazel dormouse (<Emphasis Type="Italic">Muscardinus avellanarius</Emphasis>)

The dispersal process is crucial in determining the fate of populations over time, but habitat fragmentation limits or prevents it. Landscape genetic is an effective tool to assess the degree to which dispersal still occurs in fragmented landscapes. The purpose of this study was to investigate the landscape determinants of genetic differentiation in the hazel dormouse (Muscardinus avellanarius), a forest-dependent species of conservation concern. By comparing subpopulations in a continuous (SLR) and a fragmented (VTH) population, we (i) searched for the presence of Isolation-by-Resistance (IBR); (ii) estimated migration rates; (iii) evaluated the degree of inbreeding and genetic drift, and searched for their landscape determinants. We found an IBR effect in VTH, which heavily hindered the dispersal process. The overall number of migrants among VTH subpopulations was very low (1 per generation, compared to 15 in SLR), although a between-patch displacement of about 4 km along a well-structured hedgerow probably occurred. The inbreeding (F?>?0.2 in most subpopulations) and the genetic drift (four out five subpopulations showed private alleles on several loci, with relatively high frequencies) are of particular concern in VTH. However, they were found to be limited in large patches or in patches connected by hedgerows with a high number of neighbouring patches. As a conservation strategy in the VTH landscape, characterized by small patches, we suggest that the dispersal process among subpopulations is enhanced to sustain a functional metapopulation. For this purpose, an effective ecological network should be created by enhancing the continuity and the internal features of hedgerows.     

Effect of switch trimming on udder and teat hygiene of dairy cows

The study objective was to determine the effects of trimming the switch of dairy cows on teat-end bacterial counts and udder hygiene scores. Cows (n = 102) were blocked by days in milk, milk production, and parity and then assigned to (a) treatment (trimming of their tail switch using a commercially available trimmer), or (b) control (unaltered tails). Udder hygiene was recorded for cows on Days 0 (initiation of treatment), 32, and 64. A subset of cows (n = 21) was used to assess Streptococci and coliform bacterial populations on teat ends. Samples were collected by swabbing the left front teat end before milking on Days 0, 32, and 64 and were cultured within 24 hr of sampling. The GLIMMIX and PROC Frequency (SAS Version 9.3) were used to analyze data. There were no treatment effects of switch trimming on hygiene scores or bacterial counts. These findings suggest that udder hygiene may not be driven by tail status. Environmental and management factors, such as cleanliness, stall bedding, and stall design, may be more important contributing factors in maintaining udder health.     

Simultaneous Improvement in Efficiency and Stability of Low‐Temperature‐Processed Perovskite Solar Cells by Interfacial Control

In most current state‐of‐the‐art perovskite solar cells (PSCs), high‐temperature (≈500 °C)‐sintered metal oxides are employed as electron‐transporting layers (ETLs). To lower the device processing temperature, the development of low‐temperature‐processable ETL materials (such as solution‐processed ZnO) has received growing attention. However, thus far, the use of solution‐processed ZnO is limited because the reverse decomposition reaction that occurs at ZnO/perovskite interfaces significantly degrades the charge collection and stability of PSCs. In this work, the reverse decomposition reaction is successfully retarded by sulfur passivation of solution‐processed ZnO. The sulfur passivation of ZnO by a simple chemical means, efficiently reduces the oxygen‐deficient defects and surface oxygen‐containing groups, thus effectively preventing reverse decomposition reactions during and after formation of the perovskite active layers. Using the low‐temperature‐processed sulfur‐passivated ZnO (ZnO–S), perovskite layers with higher crystallinity and larger grain size are obtained, while the charge extraction at the ZnO/perovskite interface is significantly improved. As a result, the ZnO–S‐based PSCs achieve substantially improved power‐conversion‐efficiency (PCE) (19.65%) and long‐term air‐storage stability (90% retention after 40 d) compared with pristine ZnO‐based PSCs (16.51% and 1% retention after 40 d). Notably, the PCE achieved is the highest recorded (19.65%) for low‐temperature ZnO‐based PSCs.     

Cytosolic Ribosomal Mutations That Abolish Accumulation of Circular Intron in the Mitochondria without Preventing Senescence of Podospora Anserina

The filamentous fungus Podospora anserina presents a degeneration syndrome called Senescence associated with mitochondrial DNA modifications. We show that mutations affecting the two different and interacting cytosolic ribosomal proteins (S7 and S19) systematically and specifically prevent the accumulation of senDNAα (a circular double-stranded DNA plasmid derived from the first intron of the mitochondrial cox1 gene or intron α) without abolishing Senescence nor affecting the accumulation of other usually observed mitochondrial DNA rearrangements. One of the mutant proteins is homologous to the Escherichia coli S4 and Saccharomyces cerevisiae S13 ribosomal proteins, known to be involved in accuracy control of cytosolic translation. The lack of accumulation of senDNAα seems to result from a nontrivial ribosomal alteration unrelated to accuracy control, indicating that S7 and S19 proteins have an additional function. The results strongly suggest that modified expression of nucleus-encoded proteins contributes to Senescence in P. anserina. These data do not fit well with some current models, which propose that intron α plays the role of the cytoplasmic and infectious Determinant of Senescence that was defined in early studies.     

Purification and partial immunochemical characterization of proteins of fimbriæ F107 fromEscherichia coli isolated from edema disease of pigs

The paper describes the isolation, purification and characterization of F107-fimbrial proteins, obtained by thermoelution fromEscherichia coli 107/86. Isolation of the pure F107 protein was done by FPLC chromatography, employing Superose 12, Mono Q, and Phenyl-Superose columns. The highest purity of the F107 protein was achieved with Superose 12 HR 10/30. Purity checking by a HPLC system Waters 625 LC (Millipore) proved the absence of protein admixtures in a fraction from Superose 12. Analysis of the molar mass of F107 proteins by SDS PAGE revealed that F107 fimbriæ consist of two proteins, one ofM=43 kDa (minor), and other ofM=18.9 kDa (major). Western blot analysis with rabbit polyclonal antiserum confirmed that the 18.9 kDa protein was the major characteristic unit of F107 fimbriæ.