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Several extremely halophilic archaea produce proteinaceous gas vesicles consisting of a gas-permeable protein wall constituted mainly by the gas vesicle proteins GvpA and GvpC. Eight additional accessory Gvp are involved in gas vesicle formation and might assist the assembly of this structure. Investigating interactions of halophilic proteins in vivo requires a method functioning at 2.5-5 M salt, and the split-GFP method was tested for this application. The two fragments NGFP and CGFP do not assemble a fluorescent GFP protein when produced in trans, but they assemble a fluorescent GFP when fused to interacting proteins. To adapt the method to high salt, we used the genes encoding two fragments of the salt-stable mGFP2 to construct four vector plasmids that allow an N- or C-terminal fusion to the two proteins of interest. To avoid a hindrance in the assembly of mGFP2, the fusion included a linker of 15 or 19 amino acids. The small gas vesicle accessory protein GvpM and its interaction partners GvpH, GvpJ, and GvpL were investigated by split-GFP. Eight different combinations were studied in each case, and fluorescent transformants indicative of an interaction were observed. We also determined that GvpF interacts with GvpM and uncovered the location of the interaction site of each of these proteins in GvpM. GvpL mainly interacted with the N-terminal 25-amino acid fragment of GvpM, whereas the other three proteins bound predominately to the C-terminal portion. Overall, the split-GFP method is suitable to investigate the interaction of two proteins in haloarchaeal cells. In future experiments, we will study the interactions of the remaining Gvps and determine whether some or all of these accessory Gvp proteins form (a) protein complex(es) during early stages of the assembly of the gas vesicle wall.

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This study focused on the unique properties of both the Ldlr knockout defect (closely mimicking the human situation) and the BALB/c (C) inbred mouse strain (Th-2 slanted immune response). We generated two immunodeficient strains with severe combined B- and T-cell immunodeficiency with or without a complete lack of natural killer cells to revisit the role of adaptive immune responses on atherogenesis. C-Ldlr-/- Rag1-/- mice, which show severe combined B- and T-cell immunodeficiency and C-Ldlr-/- Rag1-/- Il2rg-/- mice, which combine the T- and B-cell defect with a complete lack of natural killer cells and inactivation of multiple cytokine signalling pathways were fed an atherogenic Western type diet (WTD). Both B6-Ldlr-/- and C-Ldlr-/- immunocompetent mice were used as controls. Body weights and serum cholesterol levels of both immunodeficient strains were significantly increased compared to C-Ldlr-/- controls, except for cholesterol levels of C-Ldlr-/- Rag1-/- double mutants after 12 weeks on the WTD. Quantification of the aortic sinus plaque area revealed that both strains of immunodeficient mice developed significantly more atherosclerosis compared to C-Ldlr-/- controls after 24 weeks on the WTD. Increased atherosclerotic lesion development in C-Ldlr-/- Rag1-/- Il2rg-/- triple mutants was associated with significantly increased numbers of macrophages and significantly decreased numbers of smooth muscle cells compared to both C-Ldlr-/- wild type and C-Ldlr-/- Rag1-/- double mutants pointing to a plaque destabilizing effect of NK cell loss. Collectively, the present study reveals a previously unappreciated complexity with regard to the impact of lymphocytes on lipoprotein metabolism and the role of lymphocyte subsets in plaque composition.

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Gas vesicles consist predominantly of the hydrophobic GvpA and GvpC, and the accessory proteins GvpF through GvpM are required in minor amounts during formation. GvpM and its putative interaction partners were investigated. GvpM interacted with GvpH, GvpJ and GvpL, but not with GvpG. Interactions were also observed in vivo in Haloferax volcanii transformants using Gvp fusions to the green fluorescent protein smGFP. Cells producing the hydrophobic M(GF)P contained a single fluorescent aggregate per cell, whereas cells containing L(GFP) or H(GFP) were fully fluorescent. The soluble L(GFP) formed stable co-aggregates with GvpM in L(GFP)M transformants, but the presence of GvpH resulted in the absence of M(GF)P foci in HM(GFP) transformants. Substitution- and deletion mutants of GvpM determined functionally important amino acids (aa). Substitution of a polar by a non-polar aa in the N-terminal region of GvpM had no effect, whereas a substitution of a non-polar by a polar aa in this region inhibited gas vesicle formation in transformants. Substitutions in region 44-48 of GvpM strongly reduced the number of gas vesicles, and deletions at the N-terminus resulted in Vac(-) transformants. Gas vesicle morphology was not affected by any mutation, implying that GvpM is required during initial stages of gas vesicle assembly.

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Despite its very narrow tropism for erythroid progenitor cells, human parvovirus B19 (B19V) has recently been shown to replicate and form infectious progeny virus in 293 cells in the presence of early adenoviral functions provided either by infection with adenovirus type 5 or by addition of the pHelper plasmid encoding the E2a, E4orf6, and VA RNA functions. In the present study we dissected the individual influence of these functions on B19V genome replication and expression of structural proteins VP1 and VP2. We show that, in the presence of the constitutively expressed E1A and E1B, E4orf6 alone is able to promote B19V DNA replication, resulting in a concomitant increase in VP expression levels. The stimulatory effects of E4orf6 require the integrity of the BC box motifs, which target cellular proteins such as p53 and the Mre11 DNA repair complex for proteosomal degradation through formation of an E3 ubiquitin ligase complex with E1B. VA RNA also strongly induces VP expression but, in contrast to E4orf6, in a replication-independent manner. This stimulation could be attributed exclusively to the VA I RNA transcript and does not involve major activating effects at the level of the B19V p6 promoter, but the nucleotide residues required for the well-defined pathway of VA I RNA mediated stimulation of translation through functional inactivation of protein kinase R. These data show that the cellular pathways regulating B19V replication may be very similar to those governing the productive cycle of the helper-dependent parvoviruses, the adeno-associated viruses.

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Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome.

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Platelet aggregation and secretion play a crucial role in acute coronary syndromes. In Galpha(q) knock-out mice (Galpha(q)(-/-)) platelet function is eliminated in terms of aggregation and secretion of cytokines. We investigated whether restricted platelet aggregation and secretion reduces myocardial infarct size in vivo. Thirty minute regional myocardial ischemia was followed by 24 h reperfusion (I/R) in vivo. Infarct size was determined by counterstaining. Left ventricular function was measured by ultrasound. Infarct size to area at risk ratio was significantly smaller in Galpha(q)(-/-) mice (5.6+/-1.6%) compared to wild-type (WT) mice (27.2+/-3.0%, p<0.01). Fractional shortening was improved in Galpha(q)(-/-) mice compared to WT (42.2+/-1.4% versus 30.5+/-1.4%, respectively, p<0.01). WT mice, transplanted with Galpha(q)(-/-) bone marrow showed a significant reduction in infarct size compared to control (7.8+/-2.2% versus 18.4+/-2.7%, respectively, p<0.01). Platelets of Galpha(q)(-/-) mice had an impaired aggregation and secretion phenotype. In the in vivo model of ischemia and reperfusion, beyond impaired platelet aggregation, platelet secretion plays an additional role in myocardial infarct extension. Blocking platelet aggregation in combination with secretion might be a promising supplementary therapeutic strategy in acute myocardial infarction.

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