A murine model of subglottic granulation.

OBJECTIVE: This study aimed to develop a functional model of subglottic stenosis by inducing direct airway irritation in transplanted mouse laryngotracheal complexes. METHODS: Laryngotracheal complexes from C57BL/6 mice were harvested and divided into three groups: uninjured, mechanically injured and chemically injured. Donor laryngotracheal complexes from each group were placed in dorsal subcutaneous pockets of recipient mice. Each week, the transplanted laryngotracheal complexes were harvested, and tissues were fixed, sectioned, and stained with haematoxylin and eosin. Representative slides were reviewed by a blinded pathologist, to determine the formation of granulation tissue, and graded as to the degree of granulation formation. RESULTS: Direct airway irritation induced granulation tissue formation under the disrupted epithelium of airway mucosa; this was seen as early as two weeks after chemical injury. CONCLUSION: Results indicate that granulation tissue formation in a murine model may be an efficient tool for investigating the development and treatment of subglottic stenosis.

adapt78 protects cells against stress damage and suppresses cell growth.

We have previously identified several genes whose RNA products are induced in HA-1 hamster cells under conditions where a cytoprotective adaptive response is observed. One of these genes, designated adapt78, was found to have a human homolog with some homology to glucose-regulated protein 78 (Grp78). We subsequently determined that adapt78 and grp78 mRNAs are induced by the same stress agents and conclude that adapt78 is a stress-response gene and putative new member of the grp stress gene family. Here we extend these studies to assess the effect of overexpressing adapt78 on stress protection and growth arrest. HA-1 cells stably transfected with adapt78 cDNA were found to exhibit significantly reduced calcium- and hydrogen peroxide-mediated cytotoxicity as compared with control transfectants. In addition, adapt78 stable overexpressors exhibited significantly reduced cell growth. Both cytoprotection and growth arrest accompanied only modest overexpression of adapt78. Flow cytometry revealed that the growth arrest occurred in G(1)-phase. Immunoflourescent analysis revealed that Adapt78 protein exhibits significant perinuclear staining suggestive of endoplasmic reticulum localization in addition to cytoplasmic localization. These data indicate that adapt78 is both cytoprotective and growth suppressive and that these effects may be mediated by Adapt78 protein at the endoplasmic reticulum.

adapt78, a stress-inducible mRNA, is related to the glucose-regulated protein family of genes.

We have recently reported a new oxidant- and calcium-inducible mRNA, adapt78, from hamster HA-1 cells. The adapt78 mRNA is induced in HA-1 cells under conditions where a protective adaptive response is observed and contains a translatable open reading frame whose protein product shows strong homology to a human sequence. Computer analysis of the predicted Adapt78 protein sequence also revealed a stretch of amino acids homologous to a portion of the glucose-regulated protein78 (Grp78). Based on this homology, we tested the hypothesis that adapt78 may be a new member of the grp gene family. Toward this, we assessed the modulation of adapt78 mRNA by stress agents known to induce grp78. In HA-1 cells, adapt78 mRNA was induced by the calcium ionophore A23187, 2-deoxyglucose, brefeldin A, tunicamycin, thapsigargin, and cyclopiazonic acid, with thapsigargin being the most potent inducer (7.3-fold). As expected, grp78 mRNA was also induced by these agents in our model system. In contrast, heat shock treatment produced little if any modulation of either grp78 or adapt78. Differences were also observed, as adapt78 mRNA but not grp78 mRNA was induced by 160 microM hydrogen peroxide, and adapt78 demonstrated earlier induction kinetics for certain agents compared with grp78. adapt78 mRNA was also found to be induced in several different human cell lines. A23187 had the strongest effect on adapt78 mRNA levels in human cells, inducing greater than 20-fold in all human cell cultures tested. Furthermore, in vitro transcription translation of human adapt78 cDNA produced an Adapt78 protein product. We conclude that adapt78 may be a new member of the grp family of genes and may represent an early response grp that complements the actions of grp78 and grp94.

Hamster adapt78 mRNA is a Down syndrome critical region homologue that is inducible by oxidative stress.

We are using the technique of mRNA differential display to identify RNAs that may be important in protecting cells against the damaging effects of oxidative stress. For these studies, we utilize a so-called "adaptive response" model system in which hamster HA-1 cells respond to a minimally toxic "pretreatment" dose of hydrogen peroxide by synthesizing RNAs and proteins that protect them against subsequent exposure to a highly cytotoxic concentration of hydrogen peroxide. Using this approach, we have recently reported several novel RNAs whose levels are increased under conditions of adaptive response. Here we report a new RNA, designated adapt78, whose steady-state level is significantly induced by a pretreatment dose of hydrogen peroxide. adapt78 mRNA was calculated to be 2.35 kb in size and inducible by the standard pretreatment dose of 4 micromol H2O2/10(7) cells. It was induced as early as 90 min after peroxide exposure and maximally at 5 h. Induction was strongly dependent upon calcium. Cloning and sequencing revealed a large predicted open reading frame of 197 amino acids. In vitro transcription and translation generated a protein of 25,000 Da. GenBank homology analysis revealed that much of adapt78 is strongly homologous to a sequence that has been mapped to the Down syndrome critical region (Fuentes et al., Hum. Mol. Genet. 4, 1935-1944, 1995). However, both the 5' and the 3' ends of adapt78 show no homology to any previously reported complete sequence. adapt78 represents a new oxidant-inducible RNA and marker of cellular oxidative stress and may provide new insight into our understanding of oxidant-related disorders and neural degeneration.

Oxidative stress induces the levels of a MafG homolog in hamster HA-1 cells.

The Maf family encodes nuclear proteins that recognize AP-1-like response elements. MafB, MafK, MafF, and MafG, are all able to heterodimerize with each other, c-fos, and erythroid cell-specific transcription factor NF-E2, to affect the transcription of target genes. Using the technique of differential display, we recently identified a new oxidant-inducible mRNA, designated adapt66, in HA-1 hamster fibroblasts. Cloning, partial sequencing, and GenBank analysis of adapt66 revealed strong homology to chicken mafG, a newly identified member of the maf oncogene family. The mafG homolog/adapt66 mRNA induction appeared to be dependent upon calcium; occurred as early as 90 minutes following exposure of HA-1 cells to hydrogen peroxide; and peaked between 5 and 10 hours after peroxide treatment. It has previously been demonstrated that several cellular transcription factors, including Fos, can be induced by oxidative stress. The induction of the DNA binding sequence mafG homolog/adapt66 by hydrogen peroxide, and it's known interaction with c-fos, may represent important mechanisms by which oxidative stress can modulate gene expression.

The antioxidant, U74389, ameliorates the depression of vascular reactivity caused by lipopolysaccharide.

There is growing evidence that an oxidant stress contributes to the deleterious effects of bacterial lipopolysaccharide (LPS). The present study evaluated the ability of the antioxidant, U74389, to prevent the depression of vascular reactivity caused by LPS. Aortic rings taken from rats given LPS showed a depression of maximum force in response to phenylephrine that was reversed by an inhibitor of nitric oxide synthase. Pretreatment of animals with U74389 attenuated this depression of vascular reactivity. U74389 did not limit the increase in serum tumor necrosis factor levels caused by LPS. These results show that U74389 can ameliorate the depression of vascular reactivity caused by LPS possibly by interfering with the induction of nitric oxide synthase.