Evidence for an interferon-inducible gene, Ifi202, in the susceptibility to systemic lupus.

The Nba2 locus is a major genetic contribution to disease susceptibility in the (NZB x NZW)F(1) mouse model of systemic lupus. We generated C57BL/6 mice congenic for this NZB locus, and these mice produced antinuclear autoantibodies characteristic of lupus. F(1) offspring of congenic and NZW mice developed high autoantibody levels and severe lupus nephritis similar to (NZB x NZW)F(1) mice. Expression profiling with oligonucleotide microarrays revealed only two differentially expressed genes, interferon-inducible genes Ifi202 and Ifi203, in congenic versus control mice, and both were within the Nba2 interval. Quantitative PCR localized increased Ifi202 expression to splenic B cells and non-T/non-B cells. These results, together with analyses of promoter region polymorphisms, strain distribution of expression, and effects on cell proliferation and apoptosis, implicate Ifi202 as a candidate gene for lupus.

Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis.

The molecular mechanisms of pulmonary fibrosis are poorly understood. We have used oligonucleotide arrays to analyze the gene expression programs that underlie pulmonary fibrosis in response to bleomycin, a drug that causes lung inflammation and fibrosis, in two strains of susceptible mice (129 and C57BL/6). We then compared the gene expression patterns in these mice with 129 mice carrying a null mutation in the epithelial-restricted integrin beta6 subunit (beta6(-/-)), which develop inflammation but are protected from pulmonary fibrosis. Cluster analysis identified two distinct groups of genes involved in the inflammatory and fibrotic responses. Analysis of gene expression at multiple time points after bleomycin administration revealed sequential induction of subsets of genes that characterize each response. The availability of this comprehensive data set should accelerate the development of more effective strategies for intervention at the various stages in the development of fibrotic diseases of the lungs and other organs.

cGMP and cAMP cause pulmonary vasoconstriction in the presence of hemolysate.

We recently reported that addition of a small amount of hemolysate to the salt solution that perfused isolated rat lungs hypersensitized the vasculature to subsequent additions of ANG II or exposure to hypoxia, and addition of NO gas (. NO) to the perfusate that contained hemolysate caused a strong vasoconstrictor rather than a vasodilator response. In the present study, we demonstrate that CO and the secondary messengers cGMP and cAMP (usually associated with vasodilation) exert similar effects in hemolysate-perfused lungs. Analogs of the cyclic nucleotides cGMP or cAMP (8-bromo-cGMP and dibutyryl-cAMP, respectively) caused profound vasoconstriction in the isolated rat lung perfused with a salt solution that contained hemolysate. The cGMP- or cAMP-analog-induced vasoconstriction was inhibited by chemically dissimilar Ca2+ antagonists, by the protein phosphatase inhibitor okadaic acid, and, to a lesser degree, by protein kinase inhibitor H-7. Antiphosphothreonine immunoblotting demonstrated that lungs perfused with hemolysate exhibit increased phosphorylation of several proteins. These data indicate that, in the presence of hemolysate, pulmonary vasculature responds to nominally vasodilatory stimuli, including analogs of cGMP and cAMP, with vasoconstriction rather than vasodilation. The importance of our finding is the paradoxical nature of the response to (analogs of) cyclic nucleotides because, to our knowledge, cyclic nucleotide-induced vasoconstriction has not been previously reported.

Mutational analysis of the SRC homology 2 domain protein-tyrosine phosphatase Corkscrew.

The SRC homology 2 (SH2) domain protein-tyrosine phosphatase, Corkscrew (CSW) is required for signaling by receptor tyrosine kinases, including the Sevenless receptor tyrosine kinase (SEV), which directs Drosophila R7 photoreceptor cell development. To investigate the role of the different domains of CSW, we constructed domain-specific csw mutations and assayed their effects on CSW function. Our results indicate that CSW SH2 domain function is essential, but either CSW SH2 domain can fulfill this requirement. We also found that CSW and activated SEV are associated in vivo in a manner that does not require either CSW SH2 domain function or tyrosine phosphorylation of SEV. In contrast, the interaction between CSW and Daughter of Sevenless, a CSW substrate, is dependent on SH2 domain function. These results suggest that the role of the CSW SH2 domains during SEV signaling is to bind Daughter of Sevenless rather than activated SEV. We also found that although CSW protein-tyrosine phosphatase activity is required for full CSW function, a catalytically inactive CSW is capable of providing partial function. In addition, we found that deletion of either the CSW protein- tyrosine phosphatase insert or the entire CSW carboxyl terminus, which includes a conserved DRK/GRB2 SH2 domain binding sequence, does not abolish CSW function.

Plasma and lipids from stored packed red blood cells cause acute lung injury in an animal model.

Transfusion-related acute lung injury (TRALI) is a serious complication of hemotherapy. During blood storage, lipids are generated and released into the plasma. In this study, the role of these lipids in TRALI was investigated using an isolated, perfused rat lung model. Rats were pretreated with endotoxin (LPS) or saline in vivo and the lungs were isolated, ventilated, and perfused with saline, or (a) 5% (vol/ vol) fresh human plasma, (b) plasma from stored blood from the day of isolation (D.0) or from the day of outdate (D.42), (c) lipid extracts from D.42 plasma, or (d) purified lysophosphatidylcholines. Lungs from saline or LPS-pretreated rats perfused with fresh (D.0) plasma showed no pulmonary damage as compared with saline perfused controls. LPS pretreatment/D.42 plasma perfusion caused acute lung injury (ALI) manifested by dramatic changes in both pulmonary artery pressure and edema. Incubation of LPS pre-tx rats with mibefradil, a Ca2+ channel blocker, or WEB 2170, a platelet-activating factor (PAF) receptor antagonist, inhibited ALI caused by D.42 plasma. Lung histology showed neutrophil sequestration without ALI with LPS pretreatment/saline or D.0 plasma perfusion, but ALI with LPS pretreatment/D.42 plasma perfusion, and inhibition of D.42 plasma induced ALI with WEB 2170 or mibefradil. A significant increase in leukotriene E4 was present in LPS-pretreated/D.42 plasma-perfused lungs that was inhibited by WEB 2170. Lastly, significant pulmonary edema was produced when lipid extracts of D.42 plasma or lysophosphatidylcholines were perfused into LPS-pretreated lungs. Lipids caused ALI without vasoconstriction, except at the highest dose employed. In conclusion, both plasma and lipids from stored blood produced pulmonary damage in a model of acute lung injury. TRALI, like the adult respiratory distress syndrome, may be the result of two insults: one derived from stored blood and the other from the clinical condition of the patient.

Prostaglandins induce vascular endothelial growth factor in a human monocytic cell line and rat lungs via cAMP.

Prostaglandins have emerged as a therapeutic option for patients with peripheral vascular disease as well as pulmonary hypertension as a means to increase blood flow. We tested the hypothesis that prostaglandins regulate vascular endothelial growth factor (VEGF) expression in the human monocytic THP-1 cell line and in isolated perfused rat lungs. Our data show that the stable PGI2-analogue iloprost induces VEGF gene expression (predominantly VEGF121, but also VEGF165 isoforms) and VEGF protein synthesis in THP-1 cells. This effect is abolished by dexamethasone and by Rp-cAMP, a specific inhibitor of cAMP-dependent protein kinase (PKA) activation. The calcium channel blocker diltiazem has no effect on the iloprost-induced VEGF gene expression, and depletion of intracellular Ca2+ stores by long-term exposure (16 h) of THP-1 cells to thapsigargin does not inhibit iloprost-induced VEGF gene expression, suggesting that an increase in intracellular Ca2+ is not essential for VEGF gene induction by iloprost. However, an increase of intracellular Ca2+ by a short-term (2 h) exposure of THP-1 cells to thapsigargin or to the calcium-ionophore A23187 increases VEGF mRNA levels, indicating that a change in intracellular Ca2+ by itself can alter VEGF gene expression. The effects of thapsigargin or A23187 on VEGF gene expression are also mediated via cAMP-PKA since they are inhibited by Rp-cAMP. In isolated perfused rat lungs, PGI2 and PGE2 increases VEGF mRNA abundance whereas Rp-cAMP inhibits the prostaglandin-induced VEGF gene activation. Thus, our data suggest that prostaglandins stimulate VEGF gene expression in monocytic cells and in rat lungs via a cAMP-dependent mechanism.

Daughter of sevenless is a substrate of the phosphotyrosine phosphatase Corkscrew and functions during sevenless signaling.

The SH2 domain-containing phosphotyrosine phosphatase Corkscrew (CSW) is an essential component of the signaling pathway initiated by the activation of the sevenless receptor tyrosine kinase (SEV) during Drosophila eye development. We have used genetic and biochemical approaches to identify a substrate for CSW. Expression of a catalytically inactive CSW was used to trap CSW in a complex with a 115 kDa tyrosine-phosphorylated substrate. This substrate was purified and identified as the product of the daughter of sevenless (dos) gene. Mutations of dos were identified in a screen for dominant mutations which enhance the phenotype caused by overexpression of inactive CSW during photoreceptor development. Analysis of dos mutations indicates that DOS is a positive component of the SEV signaling pathway and suggests that DOS dephosphorylation by CSW may be a key event during signaling by SEV.

The SH2-containing tyrosine phosphatase corkscrew is required during signaling by sevenless, Ras1 and Raf.

The sevenless gene encodes a receptor tyrosine kinase which is required for the development of the R7 photoreceptor cell in each ommatidium of the Drosophila eye. We have previously used a sensitized genetic screen to identify mutations, designated Enhancers of sevenless (E(sev)), which affect genes that encode components of the sevenless signaling pathway. Here, we report that one of these mutations, E(sev)1Ae0P is a dominantly inhibiting allele of corkscrew, which encodes an SH2 domain-containing protein tyrosine phosphatase (Perkins et al., 1992). We show that corkscrew function is essential for sevenless signaling and that expression of a membrane-targeted form of corkscrew can drive R7 photoreceptor development in the absence of sevenless function. Furthermore, we have used the dominantly inhibiting corkscrew allele to examine the role of corkscrew during signaling by activated forms of Ras1 and Raf. Our analysis indicates that corkscrew function is still required during signaling by activated forms Ras1 and Raf proteins. These results define a function for corkscrew that is either downstream of Ras1 activation or in a parallel pathway that acts with activated Ras1/Raf to specify R7 photoreceptor development.

Sequence of a class E tetracycline resistance gene from Escherichia coli and comparison of related tetracycline efflux proteins.

We determined the nucleotide sequence of the class E tetA gene on plasmid pSL1456 from Escherichia coli SLH1456A. The deduced amino acid sequence of the class E TetA protein shows 50 to 56% identity with the sequences of five related TetA proteins (classes A through D and G). Hydrophobicity profiles identify 12 putative transmembrane segments with similar boundaries in all six TetA sequences. The N-terminal alpha domain of the six sequences is more highly conserved than the C-terminal beta domain; the central hydrophilic loop connecting the alpha and beta domains is the least conserved region. Amino acid residues that have been shown to be important for class B (Tn10) TetA function are conserved in all six TetA sequences. Unlike the class B tetA gene, the class D and E tetA genes do not exhibit a negative gene dosage effect when present on multicopy plasmids derived from pACYC177.

Nucleotide sequence of class D tetracycline resistance genes from Salmonella ordonez.

Plasmid pIP173, isolated from Salmonella ordonez strain BM2000, confers resistance to tetracycline and a number of other antibiotics. We determined the nucleotide sequence of the pIP173 tetR repressor and tetA resistance genes. The pIP173 tetR gene is essentially identical to the class D tetR gene from plasmid RA1. The pIP173 tet genes are flanked by directly repeated copies of the insertion sequence IS26. Interestingly, the 3' end of the tetR gene, encoding the C-terminal 16 amino acids of the TetR protein, extends into the flanking IS26 sequence. The relationships between the class A, B, C, and D TetA sequences parallel the relationships between the corresponding TetR sequences; class D is more closely related to class B than to either class A or C. Overall, the four TetA sequences show 38% identity and 57% similarity.

Membrane topology of the pBR322 tetracycline resistance protein. TetA-PhoA gene fusions and implications for the mechanism of TetA membrane insertion.

The tetracycline resistance gene of pBR322 encodes a 41-kDa inner membrane protein (TetA) that acts as a tetracycline/H+ antiporter. Based on hydrophobicity profiles, we identified 12 potential transmembrane segments in TetA. We used oligonucleotide deletion mutagenesis to fuse alkaline phosphatase (PhoA) to the C-terminal edge of each of the predicted periplasmic and cytoplasmic segments of TetA. In general, the PhoA activities of the TetA-PhoA fusions support a TetA topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, several TetA-PhoA fusions have unexpected properties. One PhoA fusion to a predicted cytoplasmic segment (C6) has high activity. However, previous protease accessibility studies on the related Tn10 TetA protein indicated that C6 is cytoplasmically localized as predicted (Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264, 11663-11670). PhoA fusions to three predicted periplasmic segments (P1, P2, and P5) have low to intermediate activity. In each case, the preceding transmembrane segment (TM1, TM3, and TM9) contains an aspartate (Asp17, Asp86, and Asp287). We show that these aspartates act like signal sequence mutations for PhoA export: (i) Asp----Ala mutations increase the PhoA activity of fusions to P1, P2, and P5. (ii) The signal sequence mutation suppressor prlA402 increases the PhoA activity of these same fusions. We also show that the aspartates in TM1, TM3, and TM9 are critical for wild-type TetA function; they are conserved in related TetA proteins and Asp----Ala mutations reduce or eliminate tetracycline resistance. The properties of the anomalous TetA-PhoA fusions suggest that TetA sequences C-terminal to some cytoplasmic and periplasmic segments are required for the proper localization of those segments, i.e. long range interactions may be more important in determining the membrane topology of TetA than suggested in some general models.

Analysis of Escherichia coli TonB membrane topology by use of PhoA fusions.

Alkaline phosphatase (PhoA) fusions to TonB amino acids 32, 60, 125, 207, and 239 (the carboxy terminus) all showed high PhoA activity; a PhoA fusion to TonB amino acid 12 was inactive. The full-length TonB-PhoA fusion protein was associated with the cytoplasmic membrane and retained partial TonB function. These results support a model in which TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder of the protein, including its hydrophobic carboxy terminus, extending into the periplasm.