Role of oxidants in NF-kappa B activation and TNF-alpha gene transcription induced by hypoxia and endotoxin.

The transcription factor NF-kappa B stimulates the transcription of proinflammatory cytokines including TNF-alpha. LPS (endotoxin) and hypoxia both induce NF-kappa B activation and TNF-alpha gene transcription. Furthermore, hypoxia augments LPS induction of TNF-alpha mRNA. Previous reports have indicated that antioxidants abolish NF-kappa B activation in response to LPS or hypoxia, which suggests that reactive oxygen species (ROS) are involved in NF-kappa B activation. This study tested whether mitochondrial ROS are required for both NF-kappaB activation and the increase in TNF-alpha mRNA levels during hypoxia and LPS. Our results indicate that hypoxia (1.5% O2) stimulates NF-kappa B and TNF-alpha gene transcription and increases ROS generation as measured by the oxidant sensitive dye 2',7'-dichlorofluorescein diacetate in murine macrophage J774.1 cells. The antioxidants N-acetylcysteine and pyrrolidinedithiocarbamic acid abolished the hypoxic activation of NF-kappa B, TNF-alpha gene transcription, and increases in ROS levels. Rotenone, an inhibitor of mitochondrial complex I, abolished the increase in ROS signal, the activation of NF-kappa B, and TNF-alpha gene transcription during hypoxia. LPS stimulated NF-kappa B and TNF-alpha gene transcription but not ROS generation in J774.1 cells. Rotenone, pyrrolidinedithiocarbamic acid, and N-acetylcysteine had no effect on the LPS stimulation of NF-kappa B and TNF-alpha gene transcription, indicating that LPS activates NF-kappa B and TNF-alpha gene transcription through a ROS-independent mechanism. These results indicate that mitochondrial ROS are required for the hypoxic activation of NF-kappa B and TNF-alpha gene transcription, but not for the LPS activation of NF-kappa B.

Differential display identification of plunc, a novel gene expressed in embryonic palate, nasal epithelium, and adult lung.

We have identified a novel gene transcript of approximately 1.1 kilobases in length that is expressed in the presumptive nasal epithelium of the mouse embryo. In situ hybridization analysis shows discrete regions of expression associated with the palate, nasal septum, and nasal conchae. This transcript is also expressed strongly in the trachea and bronchi of the adult lung. Screening of a mouse heart cDNA library yielded several overlapping clones to give a continuous sequence of 1113 bases, containing an open reading frame of 278 codons comprising the complete mRNA. No significant homologies with known genes were observed at the nucleotide level; limited amino acid homology with two salivary gland-specific proteins was noted. A search for functionally significant protein motifs revealed consensus sequences for N-glycosylation, protein kinase C and casein kinase phosphorylation, and a leucine zipper. Additionally, we observed a unique amino acid sequence pattern, consisting of the residues Gly-(Leu/Pro/Gln)-(Pro/Leu)-Leu-Pro-Leu, repeated four times near the amino-terminal portion of the protein with two amino acid residues separating the repeats. Based on these observations, we propose that we have identified a new gene, which we call plunc (for palate, lung, and nasal epithelium clone; GenBankTM accession number U69172).

Identification of distinct molecular phenotypes in cultured gastrointestinal smooth muscle cells.

BACKGROUND & AIMS: Cultured gastrointestinal smooth muscle cells have been shown to dedifferentiate and reinitiate their myogenic program in vitro. The aim of this study was to determine whether the cellular phenotypes observed in vitro were similar to those previously characterized in vivo. METHODS: Differential isoactin expression was examined in primary cultures of intestinal smooth muscle cells (ISMCs) by Northern blot and immunohistochemical analysis. Cellular phenotype was determined for cultured ISMCs grown at high density, at low density, in the presence and absence of serum supplementation, and on several distinct substrates including collagen type IV, laminin, fibronectin, and plastic. RESULTS: The unique patterns of isoactin protein and gene expression observed in cultured ISMCs indicate that distinct cellular phenotypes were present in vitro. The production and maintenance of these distinct smooth muscle cell phenotypes was dependent on cell density, serum supplementation, and substrate used. CONCLUSIONS: Cultured ISMCs appear to recapitulate a portion of their in vivo myogenic program in vitro, providing a unique opportunity for the molecular mechanisms controlling gastrointestinal smooth muscle myogenesis and pathogenesis to begin to be identified.

Molecular determination of the malignant potential of smooth muscle neoplasms.

BACKGROUND: The determination of the malignant potential of smooth muscle neoplasms remains ambiguous, and yet has far reaching clinical, therapeutic, and social implications. METHODS: In this pilot study, the authors examined smooth muscle isoactin gene expression by polymerase chain reaction in a variety of smooth muscle tumors. RESULTS: A lack of gamma-smooth muscle isoactin gene expression correlated 100% with a pathologic diagnosis of sarcoma. These results suggest that gamma-smooth muscle isoactin gene expression represents a unique molecular marker of oncogenic transformation. CONCLUSIONS: gamma-Smooth muscle isoactin gene expression provides a valuable molecular adjunct to the diagnosis of smooth muscle neoplasms.

Differential antigen recognition by T cell populations from strains of mice developing polar forms of granulomatous inflammation in response to eggs of Schistosoma mansoni.

In humans, infection with schistosome helminths can lead to dissimilar forms of clinical disease. Likewise, in the experimental mouse system, identical infection protocols with Schistosoma mansoni cause a more severe granulomatous disease in the C3H strain than in the C57BL/6 strain. To address this difference, we developed panels of schistosomal egg antigen (SEA)-specific T cell hybridomas to compare the responses of C3H and C57BL/6 mice to the major egg antigen p40. All derived C3H T cell hybridomas, despite being clonally distinct and restricted by either I-Ak or I-Ek, responded to recombinant fragment 15-1 of the p40 antigen, while none of the C57BL/6 T cell hybridomas did. Consistent with the observed monoclonal T cell responses, polyclonal lymph node cells from schistosome-infected C3H mice reacted strongly to fragment 15-1, which contrasted sharply with the weak response displayed by the C57BL/6 strain. Moreover, studies with congenic mice demonstrated that the strong CD4+ T cell response to fragment 15-1 was under major histocompatibility complex control and segregated with the H-2k haplotype. These findings suggest that a dominant T cell response against a major egg antigen may represent a risk factor for the development of severe disease.

Isolation and characterization of a novel short chain alcohol dehydrogenase-like isozyme by differential display of distinct smooth muscle cell phenotypes.

Gastrointestinal smooth muscle development proceeds by the linear differentiation of distinct smooth muscle cell phenotypes. In an effort to identify specific gene products associated with distinct smooth muscle cell phenotypes, we performed differential display on smooth muscle myoblasts versus immature smooth muscle myocytes. This analysis identified a novel short-chain alcohol dehydrogenase-like isozyme which was preferentially expressed in smooth muscle myoblasts over immature and mature smooth muscle myocytes. We postulate that this novel short-chain alcohol dehydrogenase-like isozyme may play a role in potentiating the dedifferentiation of smooth muscle cells in vitro.

Schistosoma mansoni: genetic non-response to p40, the major protein antigen of the egg, reveals a novel mechanism enhancing IgM production during infection.

p40 is the major protein antigen in eggs and miracidia of Schistosoma mansoni. Immunization with recombinant p40 produced in bacteria and with p40 from miracidia reveals a conventional immune response gene effect in which H-2b mice fail to produce antibody against p40. This is true when either denatured recombinant p40 and non-denatured miracidial p40 are used as immunogens. In contrast, during infection all strains of mice produce antibodies to p40. However, non-responder H-2b mice produce only IgM to p40 and never any IgG. Thus, H-2b mice appear to be producing specific IgM to p40 in the absence of MHC-restricted T-cell help. The mechanism revealed in these non-responder mice might play an important role in stimulating the production of IgM 'blocking' antibodies to antigens from schistosomula which cross-react with egg antigens.