ABSTRACT. To assess oxidant stress responses in newborn infants treated with elevated concentrations of oxygen, we measured plasma concentrations of glutathione (GSH) and glutathione disulfide (GSSG) in newborn infants ranging from 23 to 42 wk gestational age. All infants recruited into the study were mechanically ventilated and had catheters placed in their umbilical arteries a s part of their normal clinical management. Blood samples were obtained on d 1,3, and 5 and weekly thereafter or until the catheters were removed. W e observed plasma concentrations of GSSG in these infants that were frequently an order of magnitude higher than the 0.1 to 0.3 pM we find in adults. Interestingly, plasma GSSG concentrations were inversely correlated to the inspired oxygen tensions. This effect appeared to arise from the patient selection criteria whereby, of the infants studied, those breathing the lowest partial pressures of oxygen were the smallest and gestationally youngest. A second observation was that plasma concentrations of G S H in the premature infants were substantially, indeed often dramatically, lower than we have observed in adult humans (6 to 10 pM). Finally, we found that in patients with both umbilical arterial and umbilical venous catheters arterial GSSG concentrations were consistently higher than venous concentrations; conversely, arterial GSI4 concentrations were lower than venous concentrations. The elevated GSSG concentrations we observed in these infants indicate marked oxidant stress responses in prematurely born infants, even in those infants exposed only to room air. The positive arteriovenous gradients of GSSG concentrations across the lungs of these infants suggest that a t least some of the increased plasma GSSG originates in the lung. The low plasma G S H concentrations we observed in these same infants suggest deficiencies in an antioxidant that has been shown in numerous animal studies to be critical for prevention of hyperoxia-induced lung injury. Finally, the negative arteriovenous gradients of G S H concentrations across the lung provide the first evidence in humans for pulmonary uptake of GSH. (Pediatr Res 34: 360-365,1993) Abbreviations Fi02, fraction inspired oxygen GGT, y-glutamyltranspeptidase GSH, glutathione GSSG, glutathione disulfide PAO~, alveolar oxygen tension Pao2, arterial oxygen tension

LPS inducibility of ICAM-1 mRNA in A549 cells is independent of TNF- and IL-1 in A549 cells, and the similar time course of mRNA induction and NF-kappaB activation suggest the induction of ICAM-1 is mediated, in part, by NF-kappaB.

Purpose: Bcl-2 is an apoptotic protein that is highly expressed in advanced melanoma. Several strategies have been employed to target the expression of this protein, including G3139, an 18-mer phosphorothioate oligodeoxyribonucleotide targeted to the initiation region of the Bcl-2 mRNA. This compound has recently completed phase III global clinical evaluation, but the function of Bcl-2 as a target in melanoma has not been completely clarified. To help resolve this question, we have permanently and stably down-regulated Bcl-2 protein and mRNA expression in 518A2 cells by two different technologies and evaluated the resulting clones both in vitro and in vivo. Experimental Design: 518A2 melanoma cells were transfected with plasmids engineered to produce either a single-stranded antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA or a short hairpin RNA also targeted to the Bcl-2 mRNA. In vitro growth, the apoptotic response to G3139, and the G3139-induced release of cytochrome c from isolated mitochondria were evaluated. Cells were then xenografted into severe combined immunodeficient mice and tumor growth was measured. Results: In vitro, down-regulation of Bcl-2 expression by either method produced no change either in the rate of growth or in sensitivity to standard cytotoxic chemotherapeutic agents. Likewise, the induction of apoptosis by G3139 was entirely Bcl-2 independent. In addition, the G3139-induced release from isolated mitochondria was also relatively independent of Bcl-2 expression. However, when xenografted into severe combined immunodeficient mice, cells with silenced Bcl-2, using either technology, either failed to grow at all or grew to tumors of low volume and then completely regressed. In contrast, control cells with “normal” levels of Bcl-2 protein expression expanded to be large, necrotic tumors. Conclusions: The presence of Bcl-2 protein profoundly affects the ability of 518A2 melanoma cells to grow as human tumor xenografts in severe combined immunodeficient mice. The in vivo role of Bcl-2 in melanoma cells thus differs significantly from its in vitro role, and these experiments further suggest that Bcl-2 may be an important therapeutic target even in tumors that do not contain the t14:18 translocation.

A set of single-stranded DNA (ssDNA) expression vectors, which can generate intracellularly any ssDNA or oligodeoxynucleotide (ODN) molecules, have been developed in our laboratory. Studies from our laboratory as well as our collaborators demonstrated that these ssDNA expression vectors are capable of producing: (1) 10-23 DNA enzyme for downregulating c-raf kinase gene expression and (2) triplexforming oligodeoxynucleotide (TFO) for inducing genomic recombination. We report here the construction of a new version of ssDNA expression vector. A b-galactosidase (bgal) reporter gene was used as a test target so that the alteration of gene expression can be easily measured using b-gal activity assay. We designed a 10-23 DNA enzyme molecule that specifically cleaves b-gal mRNA at protein translation starting site (ATG). Using a cell-free RNA cleavage assay, we confirmed that this DNA enzyme molecule could effectively cleave b-gal RNA. However, a single substitution from T to G in the catalytic domain of this DNA enzyme molecule abolished its RNA cleavage activity. We also constructed an expression vector that can generate DNA enzyme molecules in cells. A549 lung carcinoma cells were cotransfected with both DNA enzyme expression vector and the b-gal reporter gene. Compared to the cells that were transfected with the mutated DNA enzyme expression vector, significant reduction of b-gal gene expression (up to 76%) was observed in the cells transfected with DNA enzyme expression vector as indicated by the protein expression level as well as its enzyme activity. These results further suggest that the ssDNA expression vector has potential applications in the study of gene function and target validation.

No abstract