Differences in the severity of respiratory syncytial virus (RSV)-induced lower respiratory disease in infants have been attributed to multiple environmental and genetic factors. To identify the genetic factor(s) influencing RSV susceptibility, we examined RSV infection in eight inbred mouse strains. Lung RSV titers differed significantly between mouse strains: the RSV titers were 15-fold higher in AKR/J (permissive) mice compared with C57BL/6J (resistant) mice at 4 days after inoculation. This strain-specific difference in RSV titers suggested that susceptibility to RSV infection was attributable to genetic differences between strains. To examine the mode of inheritance of RSV susceptibility, F1 and backcross (F1 x AKR/J) progeny were infected and RSV titers determined. RSV titers in the F1 progeny were similar to those found in the resistant (C57BL/6J) parent, suggesting resistance was inherited as a dominant trait. The distribution of RSV titers in backcross progeny were discordant with that predicted for a single gene effect, suggesting susceptibility was influenced by more than one gene. These data suggest that RSV susceptibility is a multigenic trait that should be amenable to resolution by genomic analysis.

No abstract

Patients on a statin regimen have a decreased risk of death due to bacterial sepsis. We have found that protection by simvastatin includes the inhibition of host cell invasion by Staphylococcus aureus, the most common etiologic agent of sepsis.

Acute lung injury, an often fatal condition, can result from a wide range of insults leading to a complex series of biologic responses. Despite extensive research, questions remain about the interplay of the factors involved and their role in acute lung injury. We proposed that assessing the temporal and functional relationships of differentially expressed genes after pulmonary insult would reveal novel interactions in the progression of acute lung injury. Specifically, 8,734 sequence-verified murine complementary DNAs were analyzed in mice throughout the initiation and progression of acute lung injury induced by particulate nickel sulfate. This study revealed the expression patterns of genes previously associated with acute lung injury in relationship to one another and also uncovered changes in expression of a number of genes not previously associated with acute lung injury. The overall pattern of gene expression was consistent with oxidative stress, hypoxia, cell proliferation, and extracellular matrix repair, followed by a marked decrease in pulmonary surfactant proteins. Also, expressed sequence tags (ESTs), with nominal homology to known genes, displayed similar expression patterns to those of known genes, suggesting possible roles for these ESTs in the pulmonary response to injury. Thus, this analysis of the progression and response to acute lung injury revealed novel gene expression patterns.

To delineate the functional protein domains necessary for the biological activity of hepatocyte growth factor-like protein (HGFL), we created various site-directed and deletion mutated cDNAs coding for this protein. Wild-type and mutated versions of HGFL were produced after transfection of the corresponding cDNAs into tissue culture cells. The biological importance of the domains within HGFL was then examined by addition of recombinant wild-type or mutant forms of HGFL to assays aimed at elucidating regions involved in the stimulation of DNA synthesis, the induction of shape changes in macrophages, and the ability to stimulate cell scattering. Mutant proteins lacking the serine protease-like domain (light chain) were not biologically active in any of the assays tested and could not compete with wild-type HGFL in cell scattering experiments. These data, in addition to direct enzyme-linked immunosorbent assay analyses, suggest that the light chain may play an important role in the interaction of HGFL with its receptor, Ron. Elimination of the proposed protease cleavage site between the heavy and light chains (by mutation of Arg-483 to Glu) produced a protein with activity comparable to wild-type HGFL. Further studies with this mutated protein uncovered an additional proteolytic cleavage site that produces biologically active protein. Deletion of the various kringle domains or the amino-terminal hairpin loop had various effects in the multiple assays. These data suggest that the heavy chain may play a pivotal role in determining the functional aspects of HGFL.Hepatocyte growth factor-like protein (HGFL) 1 was initially isolated by virtue of its sequence homology to domains in proteins involved in blood coagulation and fibrinolysis (1-3) and by virtue of its functional ability to induce macrophage activation (4, 5). HGFL has been shown to be synthesized as a single polypeptide chain that is proteolytically cleaved to an active disulfide-linked heterodimer. The large ␣ or heavy chain encodes an amino-terminal hairpin loop structure, homologous to the preactivation peptide in plasminogen, and four kringle domains. Kringle domains are triple disulfide-bonded loop structures composed of approximately 80 amino acids that are thought to be important in protein:protein interactions. The small ␤ or light chain contains a serine protease-like domain in which the three active site amino acids have been changed such that protease activity is unlikely. The cDNA for HGFL codes for a protein of approximately 80 kDa containing 711 amino acids and three potential N-linked carbohydrate addition sites (1). However, the exact size of the two chains of HGFL as determined by Western analysis has been placed at anywhere from 45 kDa to 62 kDa for the heavy chain and approximately 25-35 kDa for the light chain (6 -8).HGFL has been classified in the same growth factor family as hepatocyte growth factor (HGF), with both proteins having a strikingly similar domain structure composed of the NH 2 -terminal hairpin loop, four kringle domains, an...

Acute lung injury (ALI), a severe respiratory syndrome, develops in response to numerous insults and responds poorly to therapeutic intervention. Recently, cDNA microarray analyses were performed that indicated several pathogenic responses during nickel-induced ALI, including marked macrophage activation. Macrophage activation is mediated, in part, via the receptor tyrosine kinase Ron. To address the role of Ron in ALI, the response of mice deficient in the cytoplasmic domain of Ron (Ron tk-/-) were assessed in response to nickel exposure. Ron tk-/- mice succumb to nickel-induced ALI earlier, express larger, early increases in interleukin-6, monocyte chemoattractant protein-1, and macrophage inflammatory protein-2, display greater serum nitrite levels, and exhibit earlier onset of pulmonary pathology and augmented pulmonary tyrosine nitrosylation. Increases in cytokine expression and cellular nitration can lead to tissue damage and are consistent with the differences between genotypes in the early onset of pathology and mortality in Ron tk-/- mice. These analyses indicate a role for the tyrosine kinase receptor Ron in ALI.

Often fatal, acute lung injury has a complicated etiology. Previous studies from our laboratory in mice have demonstrated that survival during acute lung injury is a complex trait governed by multiple loci. We also found that the increase in metallothionein (MT) is one of the greatest noted in transcriptome-wide analyses of gene expression. To assess the role of MT in nickel-induced acute lung injury, the survival of Mt-transgenic, Mt1/2 (ϩ/ϩ) , and Mt1/2 (Ϫ/Ϫ) mice was compared. Pulmonary inflammation and global gene expression were compared in Mt1/2 (ϩ/ϩ) and Mt1/2 (Ϫ/Ϫ) mice. Genetargeted Mt1/2 (Ϫ/Ϫ) mice were more susceptible than Mt1/2 (ϩ/ϩ) mice to nickel-induced inflammation, surfactant-associated protein B transcript loss, and lethality. Similarly, Mt-transgenic mice exhibited increased survival. MAPPFinder analyses also noted significant decreases in genes involved in protein processing (e.g., ubiquitination, folding), which were greater in Mt1/2 (Ϫ/Ϫ) mice as compared with Mt1/2 (ϩ/ϩ) mice early in the progression of acute lung injury, possibly due to a zinc-mediated transcript destabilization. In contrast, transcript levels of genes associated with the inflammatory response, extracellular matrix regulation, and coagulation/fibrinolysis were increased more in Mt1/2 (Ϫ/Ϫ) mice as compared with Mt1/2 (ϩ/ϩ) mice late in the development of acute lung injury. Thus, MT ultimately improves survival in the progression of acute lung injury in mice. Transcriptome-wide analysis suggests that this survival may be mediated through changes in the destabilization of transcripts associated with protein processing, the subsequent augmentation of transcripts controlling inflammation, extracellular matrix regulation, coagulation/fibrinolysis, and disruption of surfactant homeostasis.

In an e ort to understand the mechanisms governing the regulation of the mouse Ron receptor gene, a mouse genomic library was screened and overlapping clones coding for the Ron gene and¯anking DNA were identi®ed. Continuous DNA sequence was obtained for approximately 16.4 kilobases. The gene, from the initiator methionine to the polyadenylation site, is contained within 13 244 basepairs and contains 19 exons. Primer extension analyses were performed to determine the transcription start site of the mouse Ron transcript. Multiple transcription start sites were found which also appear to be used in transfected reporter constructs containing Ron 5'¯anking DNA. To determine the location of sites which may be critical for the function of the Ron gene promoter, a series of chimeric genes containing serial deletions of the Ron gene promoter fused to the coding sequences for the chloramphenicol acetyltransferase gene were constructed. Transient transfection analyses of these hybrid genes into various cell lines demonstrated that two regions of the Ron gene promoter, encompassing nucleotides 7585 to 7465 and from 7465 to 7285, are important for expression of this transcript in CMT-93 cells. Further analysis of the Ron promoter utilizing gel mobility shift analyses suggests that regions encompassing nucleotides 7585 to 7508 and nucleotides 7375 to 7285 appear to bind speci®c proteins which may be involved in the negative and positive regulation, respectively, of the mouse Ron gene.