A high-resolution radiation hybrid map of the human genome draft sequence.

We have constructed a physical map of the human genome by using a panel of 90 whole-genome radiation hybrids (the TNG panel) in conjunction with 40,322 sequence-tagged sites (STSs) derived from random genomic sequences as well as expressed sequences. Of 36,678 STSs on the TNG radiation hybrid map, only 3604 (9.8%) were absent from the unassembled draft sequence of the human genome. Of 20,030 STSs ordered on the TNG map as well as the assembled human genome draft sequence and the Celera assembled human genome sequence, 36% of the STSs had a discrepant order between the working draft sequence and the Celera sequence. The TNG map order was identical to one of the two sequence orders in 60% of these discrepant cases.

Hyperoxic regulation of lung heme oxygenase in neonatal rats.

In the neonatal lung, hyperoxic exposure is associated with induction of various genes and critical antioxidants. Heme oxygenase, specifically the HO-1 isoenzyme, is regulated in oxidant stress and may also serve to limit oxidative damage. However, it is not known whether neonatal lung HO-1 is regulated in hyperoxia specifically and, if so, what type of regulation occurs. Therefore, we attempted to answer these questions using newly born (< 12 h) Wistar rats exposed to hyperoxia for 3 d. Neonatal rat lungs were evaluated daily for total HO activity, immunoreactive HO-1 protein, and steady state levels of HO-1 mRNA and compared with air-exposed controls. In neonatal rats, we noted an increased lung HO activity after 3 d of hyperoxic exposure. Additionally, evaluation of HO activity after immunoprecipitation of HO-1 protein suggested that HO-1 contributed most of the increase in lung total HO activity observed in hyperoxia. Nonetheless, we did not see a significant difference in immunoreactive HO-1 protein in neonatal lungs after 3 d of hyperoxic exposure, although we did so on d 2. Also, in contrast with previous reports, we did not detect any significant differences in steady state levels of HO-1 mRNA on any day of hyperoxic exposure compared with air. We therefore conclude that neonatal rat lung HO-1 is regulated in hyperoxia and speculate that the regulation of neonatal lung HO-1 occurs by posttranscriptional mechanisms, at least within the first days of hyperoxic exposure.

Hepatic heme oxygenase is inducible in neonatal rats during the early postnatal period.

Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme to bilirubin. Cobalt chloride (CoCl2) and many other agents that generate oxidant stresses induce the HO-1 isoform. Furthermore, HO-1 has been shown to protect against oxidant stress in vitro and in vivo by mechanisms involving increased ferritin synthesis. However, little is known about the inducibility of hepatic HO-1 during the very early postnatal period, and whether HO-1 induction is associated with increased ferritin synthesis in neonates. Therefore, we studied hepatic HO-1 mRNA, HO-1 protein concentration, total HO activity, and ferritin protein levels in neonatal rats. Neonatal rats 0-5 d of age were injected with 250 mumol/kg body weight of CoCl2. 6H2O in saline or with an equal volume of saline in age-matched controls. Liver samples were collected 4 h after injection for HO-1 mRNA analysis and 20 h after injection for analysis of HO-1 protein concentration, total HO activity, and ferritin protein levels. In CoCl2-treated rats, hepatic HO-1 mRNA was 3-10 times the levels in control rats (p < 0.05), HO-1 protein concentration was 2-5 times the levels in control rats (p < 0.05), and total HO activity was higher by 20-80% than in control rats (p < 0.05). There were no differences in hepatic ferritin protein levels between CoCl2-treated neonatal rats and controls; however, in CoCl2-treated adult rats, hepatic ferritin protein levels were 1.6 times the levels in controls (p < 0.05). Thus, neonatal rats can up-regulate hepatic HO-1 mRNA, HO-1 protein concentration, and total HO activity in response to CoCl2; however, no upregulation of hepatic ferritin protein levels was observed in neonatal rats after CoCl2 administration or subsequent HO-1 induction. We speculate that neonatal rats induce hepatic HO-1 and up-regulate ferritin by different mechanisms than do adult rats.

Heme oxygenase-mediated resistance to oxygen toxicity in hamster fibroblasts.

The role of heme oxygenase (HO)-1 was evaluated in the oxygen-resistant hamster fibroblast cell line, O2R95, which moderately overexpress HO when compared with the parental cell line, HA-1. To suppress HO-1 expression, O2R95 were transfected with HO-1 antisense oligonucleotide or treated with tin-mesoporphyrin (SnMP). To increase HO-1 expression, cells were transfected with HO-1 cDNA in a pRC/cytomegalovirus (CMV) vector. All cells were challenged with a 48-h exposure to 95% O2 (hyperoxia). When HO activity was suppressed, O2R95 cells had significantly decreased cell viability, increased susceptibility to lipid peroxidation, and increased protein oxidation in hyperoxia. In contrast, further overexpression of HO-1 did not improve resistance to oxygen toxicity. Antisense-transfected cells and SnMP-treated cells with lowered HO activity showed increased levels of cellular heme compared with controls. In the HO-1 cDNA-transfected O2R95 cells, cellular heme was lowered compared with controls; however, cellular redox active iron levels were increased. We conclude that HO mediates cytoprotection to oxygen toxicity within a narrow range of expression. We speculate that this protective effect may be mediated in part through increased metabolism of the pro-oxidant heme but that higher levels of HO activity obviate protection by increased redox active iron release.