Figure 2.

(A) Representative differential gene expression in murine lungs after reperfusion: mRNA differential display. Total RNAs were isolated from the lungs of control, clamping alone, or clamping–reperfusion mice. RNA was reverse transcribed, amplified, and displayed in a sequencing gel. The oligonucleotides 5′-ACGACTCACTATAGGGCTTTTTTTTTTTTGG-3′ and 5′-ACAATTTCACACAGGAGCTAGCATGG-3′ were used as anchored and arbitrary primers, respectively. Autoradiogram illustrates the prominent differential display of RT-PCR products designated L5B1, which is expressed in lungs of mice that underwent 3 h of hind-limb clamping and 3 h of reperfusion. (B) Northern analysis of L5B1 mRNA expression in lung tissues of control mice and mice that underwent lower-limb ischemia–reperfusion. Total RNAs were isolated and samples (15 μg per lane) were loaded and run on a formaldehyde/agarose gel, then transferred onto a Hybond-N+ membrane (Amersham Life Science) and hybridized with [α-³²P]dCTP-labeled L5B1 cDNA fragment (specific activity ≈ 1.2 × 10⁹ cpm/μg; 30 × 10⁶ cpm/10 ml of hybridization buffer). A unique ≈4-kb mRNA of L5B1 was detected (arrow). RNA loading was monitored by ethidium bromide-stained 28S and 18S rRNA on the same gel, displayed beside the Northern hybridization blot. (C) DNA sequence comparison between L5B1 cDNA fragment and mouse AMP deaminase 3 (heart-type) gene exon 15. The 476-nt sequence of the L5B1 cDNA fragment is aligned with the 3′ untranslated sequence of exon 15 (GenBank accession no. D88984S11) of the mouse AMPD3 (heart-type) gene. Identical nucleotides found for L5B1 cDNA are indicated by vertical lines. AATAAA, the putative polyadenylation signal sequence, is double underlined, and the sequence of the poly(A) tail is underlined with dashes. (D) Distribution of the up-regulated AMPD3 gene transcripts in remote lung tissue after ischemia–reperfusion. Left and Center represent lung biopsy sections from the same reperfused mouse hybridized with sense and antisense oligonucleotides for AMPD3 mRNA, respectively. In Right, lung tissue section from control mouse (without ischemia–reperfusion) was hybridized with the same antisense oligonucleotides used in Center. The dark blue-stained spots are selectively distributed in bronchial and alveolar epithelium. (×200.)