About 10% of the nalidixic acid-resistant (Nal r ) mutants in a transposition-induced library exhibited a growth factor requirement as the result of cysH, icdA, metE, or purB mutation. Resistance in all of these mutants required a functional AcrAB-TolC efflux pump, but the EmrAB-TolC pump played no obvious role. Transcription of acrAB was increased in each type of Nal r mutant. In the icdA and purB mutants, each of the known signaling pathways appeared to be used in activating the AcrAB-TolC pump. The metabolites that accumulate upstream of the blocks caused by the mutations are hypothesized to increase the levels of the AcrAB-TolC pump, thereby removing nalidixic acid from the organism.Quinolone antibiotics constitute one of the most widely used classes of antibacterial agent. Although the original quinolone, nalidixic acid (Nal) (Fig. 1), was effective primarily against gram-negative bacteria, fluoroquinolone derivatives are potent against a wide variety of gram-negative and gram-positive bacteria. The primary targets of the quinolones are DNA topoisomerase II (DNA gyrase) and topoisomerase IV (10,19,37).Because of the therapeutic importance of these antibiotics, the appearance of bacterial mutants resistant to quinolones is of considerable concern. Many such mutants in a variety of bacteria have been characterized. These fall into two broad classes: those altered in the target topoisomerases and those failing to accumulate the drug to levels seen in the wild type. No mutant resistance stemming from the breakdown or modification of quinolones has been described.Resistant mutants of Escherichia coli and of a wide variety of other gram-negative and gram-positive bacteria selected in the laboratory or isolated from clinical settings have been shown to be altered in one or more of the genes encoding components of the two topoisomerases (gyrA, gyrB, parC, and parE in E. coli) (34). These mutations produce resistance of various levels, and further mutations often increase the resistance (18).Quinolone-resistant mutants containing mutations in genes other than those for the topoisomerases are also obtained frequently when only a low level of drug is used in selection or when a secondary mutation increases resistance beyond that of the primary mutation. Where the basis for such mutation has been determined, it has usually resulted from the activation of an efflux pump (31, 32) that removes the quinolone during or after entry so it does not reach a toxic level. In E. coli, activation of either the AcrAB-TolC pump (4, 9, 28) or the EmrABTolC pump (26) provides resistance. AcrAB-TolC appears to be the main pump providing intrinsic resistance to low levels of many toxic compounds in nature (38). Three signaling pathways controlling the expression of the acrAB genes and the formation of the AcrAB-TolC pump are known; these involve the MarAB, SoxRS, and RobA proteins (5,11,15,33,39).Thus, there are several potential ways of generating low-level resistance to quinolones through the activation of efflux pumps.Screening mutant...

Urokinase plasminogen activator receptor-associated protein (uPARAP, or Endo180) is a transmembrane endocytic receptor that mediates collagen internalization and degradation. uPARAP may be a novel pathway for collagen turnover and matrix remodeling in the lung. The function of uPARAP in lung injury has not been described. We analyzed the pulmonary mechanics of uPARAP 2/2 and wild-type mice at baseline and examined their response after bleomycin instillation. We compared collagen internalization in primary mouse lung fibroblasts (MLFs) from wild-type and uPARAP 2/2 mice using flow cytometry and fluorescent microscopy, and we examined the role of cytokines in regulating uPARAP expression and collagen internalization. We show that uPARAP is highly expressed in the lung, and that uPARAP 2/2 mice have increased lung elastance at baseline and after injury. uPARAP 2/2 mice are protected from changes in lung permeability after acute lung injury and have increased collagen content after bleomycin injury. uPARAP is the primary pathway for internalization of collagens in MLFs. Furthermore, collagen internalization through uPARAP does not require matrix metalloproteinase digestion and is independent of integrins. Mediators of lung injury, including transforming growth factor-b, TNF-a, and IL-1, down-regulate both uPARAP expression and collagen internalization. uPARAP is highly expressed in the murine lung, and loss of uPARAP leads to differences in lung mechanics, lung permeability, and collagen content after injury. uPARAP is required for collagen internalization by MLFs. Thus, uPARAP is a novel pathway that regulates matrix remodeling in the lung after injury.Keywords: urokinase plasminogen activator receptor-associated protein; Endo180; collagen internalization; lung fibroblasts; matrix remodeling Urokinase plasminogen activator receptor-associated protein (uPARAP, Endo180, or mannose receptor, C type 2) is a 180-kD transmembrane receptor that can bind and internalize both fibrillar and nonfibrillar collagens (1, 2). After internalization, uPARAP targets collagen to the lysosome for degradation and then uPARAP recycles to the plasma membrane (3). uPARAP is expressed in mesenchymal cells, predominantly fibroblasts. In addition, dermal macrophage and human placental endothelial cells express low levels of uPARAP (1,4,5).We previously demonstrated high expression of uPARAP in the mesenchyme throughout lung development (6). Despite the high expression of uPARAP in the developing lung, development proceeds normally in uPARAP 2/2 mice, and is not associated with any differences in matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinases, or collagen expression in the lung (6). uPARAP 2/2 mice appear phenotypically normal in the unchallenged state, and have a normal lifespan. One possible explanation for the lack of lung phenotype is use of an alternative pathway for collagen internalization. Although previous work demonstrated that uPARAP was the primary receptor for collagen internalization in dermal fibroblast...

Fibroblast growth factor-10 (FGF-10), an alveolar epithelial cell (AEC) mitogen that is critical for lung development, may promote AEC repair. We determined whether FGF-10 attenuates H2O2-induced, A549 and rat alveolar type II cell DNA damage. We show that FGF-10 prevents H2O2-induced DNA damage assessed by an alkaline elution, ethidium bromide fluorescence as well as by a comet assay. Mitogen-activated protein kinase inhibitors abolished the protective effect of FGF-10 against H2O2-induced DNA damage yet had no effect on H2O2-induced DNA damage. A Grb2-SOS inhibitor (SH3 binding peptide), an Ras inhibitor (farnesyl transferase inhibitor 277), and an Raf-1 inhibitor (forskolin) each prevented FGF-10- and H2O2-induced A549 cell ERK1/2 phosphorylation. Also, FGF-10 and H2O2 each induced negligible ERK1/2 phosphorylation in Ras dominant-negative (N17) cells. Inhibitors of Ras and Raf-1 blocked the protective effect of FGF-10 against H2O2-induced DNA damage but had no effect on H2O2-induced DNA damage. Furthermore, cold conditions and aphidicolin, an inhibitor of DNA polymerase-alpha, -delta, and -epsilon, each blocked the protective effects of FGF-10, suggesting a role for DNA repair. We conclude that FGF-10 attenuates H2O2-induced AEC DNA damage by mechanisms that involve activation of Grb2-SOS/Ras/RAF-1/ERK1/2 pathway and DNA repair.

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