Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve interferon hyperresponsiveness without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depends on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acts on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to initiate their degradation via the immunoproteasome. Together, PARP9-DTX3L acts on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.
We demonstrate that the Bacillus subtilis fosB(yndN) gene encodes a fosfomycin resistance protein. Expression of fosB requires W , and both fosB and sigW mutants are fosfomycin sensitive. FosB is a metallothiol transferase related to the FosA class of Mn 2؉ -dependent glutathione transferases but with a preference for Mg 2؉ and L-cysteine as cofactors.Sequencing of the Bacillus subtilis genome revealed the presence of seven new factors, all members of the extracytoplasmic function subfamily (12, 13). We have begun to investigate their functions by mutation of each gene and the identification of target operons (8)(9)(10)(11). In this work, we demonstrate that yndN encodes a fosfomycin resistance (Fos r ) protein that depends on W for expression. We have renamed yndN as fosB, based on its similarity to the fosB gene identified on a Staphylococcus epidermidis plasmid (Fig. 1B).Transcription of fosB requires W . Previously, 15 W -dependent operons were identified by searching the genome for sequences matching the W autoregulatory site, P w : TGAAAC N 16 CGTA (10). Additional candidate promoters, including one for fosB (Fig. 1A), were identified with 17-bp spacer regions (10).To confirm the role of this predicted W -dependent promoter, we generated a P fosB -cat-lacZ operon fusion inserted ectopically in the SP prophage (HB8083; Table 1) and transduced the reporter fusion into wild-type, sigW, and rsiW mutant backgrounds. Promoter activity as determined in early-stationary-phase cells yielded 18.4 Miller units of -galactosidase in the wild-type strain (HB0052), and this was reduced to background levels (ϳ1 unit) in the sigW mutant (HB0023). In the rsiW (anti-W ) mutant (HB0012), expression was elevated approximately twofold (30.5 units). This pattern is precisely that expected for a W -dependent promoter. We used reverse transcriptase primer extension mapping to identify the transcriptional start site for fosB as a G residue 10 bases downstream from the Ϫ10 region CGTA motif (Fig. 2). There were no other start sites visible in the primer extension experiment, which is consistent with the idea that W is largely, if not exclusively, responsible for fosB transcription. The fosB gene is apparently monocistronic, as it is flanked on either side by genes transcribed from the complementary strand of the genome (Fig. 1A).fosB and sigW mutants are sensitive to fosfomycin. Both fosB (HB0008) and sigW (HB0020) mutants are fosfomycin sensitive: an MIC of 50 g/ml for the mutants compared to 800 g/ml for the wild type in liquid culture. Similarly, the sigW and fosB mutants have a much greater zone of growth inhibition in disk diffusion assays (ϳ25-mm zone for wild type versus Ͼ50 mm for the mutants). The fosB and sigW mutant strains did not display altered sensitivity to several other antibiotics, including vancomycin, cephalosporin C, penicillin G, D-cycloserine, tunicamycin, nisin, and bacitracin. Induction of fosB from a xylose-inducible promoter completely restores Fos r to either the sigW mutant (HB0081) or, as expected, t...
The genetic determination of eggshell coloration has not been determined in birds. Here we report that the blue eggshell is caused by an EAV-HP insertion that promotes the expression of SLCO1B3 gene in the uterus (shell gland) of the oviduct in chicken. In this study, the genetic map location of the blue eggshell gene was refined by linkage analysis in an F2 chicken population, and four candidate genes within the refined interval were subsequently tested for their expression levels in the shell gland of the uterus from blue-shelled and non-blue-shelled hens. SLCO1B3 gene was found to be the only one expressed in the uterus of blue-shelled hens but not in that of non-blue-shelled hens. Results from a pyrosequencing analysis showed that only the allele of SLCO1B3 from blue-shelled chickens was expressed in the uterus of heterozygous hens (O*LC/O*N). SLCO1B3 gene belongs to the organic anion transporting polypeptide (OATP) family; and the OATPs, functioning as membrane transporters, have been reported for the transportation of amphipathic organic compounds, including bile salt in mammals. We subsequently resequenced the whole genomic region of SLCO1B3 and discovered an EAV-HP insertion in the 5′ flanking region of SLCO1B3. The EAV-HP insertion was found closely associated with blue eggshell phenotype following complete Mendelian segregation. In situ hybridization also demonstrated that the blue eggshell is associated with ectopic expression of SLCO1B3 in shell glands of uterus. Our finding strongly suggests that the EAV-HP insertion is the causative mutation for the blue eggshell phenotype. The insertion was also found in another Chinese blue-shelled breed and an American blue-shelled breed. In addition, we found that the insertion site in the blue-shelled chickens from Araucana is different from that in Chinese breeds, which implied independent integration events in the blue-shelled chickens from the two continents, providing a parallel evolutionary example at the molecular level.
Insulin-secreting pancreatic islet -cells express a Group VIA Ca 2؉ -independent phospholipase A 2 (iPLA 2 ) that contains a calmodulin binding site and protein interaction domains. We identified Ca 2؉ /calmodulindependent protein kinase II (CaMKII) as a potential iPLA 2 -interacting protein by yeast two-hybrid screening of a cDNA library using iPLA 2  cDNA as bait. Cloning CaMKII cDNA from a rat islet library revealed that one dominant CaMKII isoform mRNA is expressed by adult islets and is not observed in brain or neonatal islets and that there is high conservation of the isoform expressed by rat and human -cells. Binary two-hybrid assays using DNA encoding this isoform as bait and iPLA 2  DNA as prey confirmed interaction of the enzymes, as did assays with CaMKII as prey and iPLA 2  bait. His-tagged CaMKII immobilized on metal affinity matrices bound iPLA 2 , and this did not require exogenous calmodulin and was not prevented by a calmodulin antagonist or the Ca 2؉ chelator EGTA. Activities of both enzymes increased upon their association, and iPLA 2  reaction products reduced CaMKII activity. Both the iPLA 2  inhibitor bromoenol lactone and the CaMKII inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both inhibit insulin secretion. CaMKII and iPLA 2  can be coimmunoprecipitated from INS-1 cells, and forskolin, which amplifies glucose-induced insulin secretion, increases the abundance of the immunoprecipitatable complex. These findings suggest that iPLA 2  and CaMKII form a signaling complex in -cells, consistent with reports that both enzymes participate in insulin secretion and that their expression is coinduced upon differentiation of pancreatic progenitor to endocrine progenitor cells.Phospholipases A 2 (PLA 2 ) 1 are a diverse group of enzymes that catalyze hydrolysis of sn-2 fatty acid substituents from glycerophospholipid substrates to yield a free fatty acid and a 2-lysophospholipid (1). The Group VIA PLA 2 designated iPLA 2  has a molecular mass of 84 -88 kDa and does not require Ca 2ϩ for catalysis (2). Various splice variants of iPLA 2  are expressed at high levels in testis (3), brain (4), and pancreatic islet -cells (5), among other tissues.Certain nutrients, hormones, neurotransmitters, and pharmacologic agents stimulate insulin secretion from -cells, and the dominant physiologic insulin secretagogue is D-glucose. A series of signals that result from glucose-induced ATP production and alterations of intracellular redox potentials trigger insulin secretion via a rise in cytosolic [Ca 2ϩ ], and Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) is an important -cell [Ca 2ϩ ] sensor and mediator of Ca 2ϩ -dependent events in insulin secretion (6 -11). Much evidence (12-22) suggests that iPLA 2  also participates in insulin secretion, including the facts that the mechanism-based bromoenol lactone (BEL) inhibitor of iPLA 2  suppresses glucose-induced hydrolysis of arachidonate from islet membrane phospholipids, the rise in -cell cytosolic...
There remain unmet clinical needs for safe and effective bone anabolic therapies to treat aging-related osteoporosis and to improve fracture healing in cases of nonunion or delayed union. Wnt signaling has emerged as a promising target pathway for developing novel bone anabolic drugs. Although neutralizing antibodies against the Wnt antagonist sclerostin have been tested, Wnt ligands themselves have not been fully explored as a potential therapy. Previous work has demonstrated Wnt7b as an endogenous ligand upregulated during osteoblast differentiation, and that Wnt7b overexpression potently stimulates bone accrual in the mouse. The earlier studies however did not address whether Wnt7b could promote bone formation when specifically applied to aged or fractured bones. Here we have developed a doxycycline-inducible strategy where Wnt7b is temporally induced in the bones of aged mice or during fracture healing. We report that forced expression of Wnt7b for 1 month starting at 15 months of age greatly stimulated trabecular and endosteal bone formation, resulting in a marked increase in bone mass. We further tested the effect of Wnt7b on bone healing in a murine closed femur fracture model. Induced expression of Wnt7b at the onset of fracture did not affect the initial cartilage formation but promoted mineralization of the subsequent bone callus. Thus, targeted delivery of Wnt7b to aged bones or fracture sites may be explored as a potential therapy.
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