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Gaye F. White Sonya M. Schermann Justin Bradley Andrew Roberts Nicholas P. Greene Ben C. Berks Andrew J. Thomson 《The Journal of biological chemistry》2010,285(4):2294-2301
The Tat system is used to transport folded proteins across the cytoplasmic membrane in bacteria and archaea and across the thylakoid membrane of plant chloroplasts. Multimers of the integral membrane TatA protein are thought to form the protein-conducting element of the Tat pathway. Nitroxide radicals were introduced at selected positions within the transmembrane helix of Escherichia coli TatA and used to probe the structure of detergent-solubilized TatA complexes by EPR spectroscopy. A comparison of spin label mobilities allowed classification of individual residues as buried within the TatA complex or exposed at the surface and suggested that residues Ile12 and Val14 are involved in interactions between helices. Analysis of inter-spin distances suggested that the transmembrane helices of TatA subunits are arranged as a single-walled ring containing a contact interface between Ile12 on one subunit and Val14 on an adjacent subunit. Experiments in which labeled and unlabeled TatA samples were mixed demonstrate that TatA subunits are exchanged between TatA complexes. This observation is consistent with the TatA dynamic polymerization model for the mechanism of Tat transport. 相似文献
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Sonya C. Bardswell Friederike Cuello Alexandra J. Rowland Sakthivel Sadayappan Jeffrey Robbins Mathias Gautel Jeffery W. Walker Jonathan C. Kentish Metin Avkiran 《The Journal of biological chemistry》2010,285(8):5674-5682
Protein kinase D (PKD), a serine/threonine kinase with emerging cardiovascular functions, phosphorylates cardiac troponin I (cTnI) at Ser22/Ser23, reduces myofilament Ca2+ sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser22/Ser23 remains to be established. To determine the role of cTnI phosphorylation at Ser22/Ser23 in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser22/Ser23 are substituted by nonphosphorylatable Ala (cTnI-Ala2). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser22/Ser23 and decreased the Ca2+ sensitivity of force. In contrast, PKD had no effect on the Ca2+ sensitivity of force in myocardium from cTnI-Ala2 mice, in which Ser22/Ser23 were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala2 mice. Because cardiac myosin-binding protein C (cMyBP-C) phosphorylation underlies cAMP-dependent protein kinase (PKA)-mediated acceleration of cross-bridge cycle kinetics, we explored whether PKD phosphorylates cMyBP-C at its PKA sites, using recombinant C1C2 fragments with or without site-specific Ser/Ala substitutions. Kinase assays confirmed that PKA phosphorylates Ser273, Ser282, and Ser302, and revealed that PKD phosphorylates only Ser302. Furthermore, PKD phosphorylated Ser302 selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala2 mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca2+ sensitivity through cTnI phosphorylation at Ser22/Ser23 but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser302, which may mediate the latter effect. 相似文献
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Lu S Yao Y Cheng X Mitchell S Leng S Meng S Gallagher JW Shelness GS Morris GS Mahan J Frase S Mansbach CM Weinberg RB Black DD 《The Journal of biological chemistry》2006,281(6):3473-3483
Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans. 相似文献
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Artificial reef systems play an important role in the increase of natural production of biological marine resources and they have been deployed worldwide. In Portugal, seven artificial systems have been deployed along the southern coast of the Algarve. Research to date has focussed mainly on fish populations, particularly those of economical importance. The present work aims to study the macrobenthic communities of the artificial reef structures, as these will enhance the food resources and shelter, making the reefs more attractive to fish. In particular, we experimentally analysed the sequence of colonisation of macrobenthic communities of the Ancão artificial reef system, which was deployed in the summer of 2002. The study of the colonisation of benthic communities was done using samples of concrete cubic units (15 × 15 cm) that were suspended at the reef modules at a depth of 20 m, at the time of the reef immersion. Four replicate samples were collected by SCUBA diving from two groups of the Ancão reef every three months from the starting date. Sampling was done using essentially non-destructive methods to assess the percentage cover of macrobenthic organisms in both vertical and horizontal surfaces. The percentage cover of the taxonomic groups was compared within the different surfaces of the samples and between the two reef groups. The bottom surface of cubic samples had a significantly higher colonisation related to the dominance of barnacle cover, probably due to lower sedimentation levels. Samples from both reef groups showed a similar pattern of colonisation. Barnacles, bryozoans and serpulids dominated the samples three months immediately after the beginning of the experiment. Other invertebrates groups, such as Porifera, Hydrozoa, Anthozoa, other sessile Polychaeta, Decapoda, Gastropoda and Bivalvia, were more abundant after six months of colonisation. 相似文献
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Bader S Kortholt A Snippe H Van Haastert PJ 《The Journal of biological chemistry》2006,281(29):20018-20026
Dictyostelium discoideum cells possess multiple cyclic nucleotide phosphodiesterases that belong either to class I enzymes that are present in all eukaryotes or to the rare beta-lactamase class II. We describe here the identification and characterization of DdPDE4, the third class I enzyme of Dictyostelium. The deduced amino acid sequence predicts that DdPDE4 has a leader sequence, two transmembrane segments, and an extracellular catalytic domain that exhibits a high degree of homology with human cAMP-specific PDE8. Expression of the catalytic domain of DdPDE4 shows that the enzyme is a cAMP-specific phosphodiesterase with a K(m) of 10 microm; cGMP is hydrolyzed at least 100-fold more slowly. The full-length protein is shown to be membrane-bound with catalytic activity exposed to the extracellular medium. Northern blots and activity measurements reveal that expression of DdPDE4 is low during single cell stages and increases at 9 h of starvation, corresponding with mound stage. A function during multicellular development is confirmed by the phenotype of ddpde4(-) knock-out strains, showing normal aggregation but impaired development from the mound stage on. These results demonstrate that DdPDE4 is a unique membrane-bound phosphodiesterase with an extracellular catalytic domain regulating intercellular cAMP during multicellular development. 相似文献
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Bertozzi CC Chang CY Jairaj S Shan X Huang J Weber BL Chu CS Carroll RG 《In vitro cellular & developmental biology. Animal》2006,42(3-4):58-62
Summary To increase the efficiency of stable cell line establishment from primary ovarian cancer specimens, we simultaneously initiated
cultures under, multiple conditions, varying extracellular matrices and the inclusion of supplements (e.g., serum or serum
albumin), while minimizing exposure to xenogeneic antigens (e.g., fetal calf serum). Primary cultures were initiated from
30 specimens; cell lines were established from 10 of these for a success rate of 33%. In some instances, multiple cell lines
were established from the same specimen. Five lines were characterized extensively with respect to growth properties, antigen
expression, and genomic alterations. Although these lines are all low-passage, marked heterogeneity was observed, even between
lines derived from the same specimen. The culture approach outlined herein will facilitate generation of reagents useful for
many aspects of ovarian cancer biology.
Equal contribution. 相似文献