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Our aims were to examine whether oxidative DNA damage was elevated in brain cells of male C57BL/6 mice after oxidative stress, and to determine whether neuronal nitric oxide synthase (nNOS) was involved in such damage. Oxidative stress was induced by occluding both common carotid arteries for 90 min, followed by reperfusion. Escherichia coli exonuclease III (Exo III) removes apyrimidinic or apurinic (AP) sites and 3'-phosphate termini in single-strand breaks, and converts these lesions to 3'OH termini. These ExoIII-sensitive sites (EXOSS) can then be postlabeled using digoxigenin-11-dUTP and Klenow DNA polymerase-I, and detected using fluorescein isothiocyanate-IgG against digoxigenin. Compared with the non-ischemia controls, the density of EXOSS-positive cells was elevated at least 20-fold (P < 0.01) at 15 min of reperfusion, and remained elevated for another 30 min. EXOSS mainly occurred in the cell nuclei of the astrocytes and neurons. Signs of cell death were detected at 24 h of reperfusion and occurred mostly in the neurons. Both DNA damage and cell death in the cerebral cortical neurons were abolished by treatment with 3-bromo-7-nitroindazole (30 mg/kg, intraperitoneal), which specifically inhibited nNOS. Our results suggest that nNOS, its activator (calcium), and peroxynitrite exacerbate oxidative DNA damage after brain ischemia.-Huang, D., Shenoy, A., Cui, J., Huang, W., Liu, P. In situ detection of AP sites and DNA strand breaks bearing 3'-phosphate termini in ischemic mouse brain.

An octacosapeptide that we named pro-somatostatin has been isolated from acid extracts of porcine hypothalami and found to have the amino acid sequence Ser-AlaAsn-Ser-Asn-Pro-:Ala-Met-Ala-Pro-Arg-Glu-Arg>Lys-Ala-GlyCys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-C's. This octacosapeptide possesses high somatotropin (growth hormone) and prolactin release-inhibiting activity in vitro. It also crossreacts strongly with antisera generated against the somatostatin tetradecapeptide. This octacosapeptide is most likely a precursor (pro-hormone) of somatostatin in the hypothalamus. The existence of still larger molecular size precursors of somatostatin was also observed.More than 5 years ago, our laboratory reported larger, more basic forms of somatostatin in fractions of pig hypothalamic extract (1, 2). We also recognized two types of immunoreactive somatostatin in extracts of rat pancreas, stomach, and duodenum (3). We suggested, on the basis of physicochemical, biological, and immunological data, that these substances represented precursors of somatostatin (1-3). Later, we postulated that the hypothalamic pro-somatostatin has an extension at the NH2 terminus containing one or more arginine residues (4-6). Manual degradation by the Edman-dansyl method showed the NH2 terminal sequence to be Ser-Ala-Asn-Ser. However, because of the small amounts available, we were unable to'complete the entire sequence. Recent developments in automated amino acid sequence analysis (7), which led to an increase in sensitivity of several thousandfold, permitted the successful elucidation of the whole sequence. In this paper we describe the isolation, biological and immunological characterization, and structural elucidation of putative pro-somatostatin from porcine hypothalami. The sequence of hypothalamic pro-somatostatin is identical with that of a putative precursor of somatostatin from pig intestine recently reported by Pradayrol et al. (8). We conclude that in the hypothalamus, as in the gut, somatostatin also exists in the form of the prohormone.MATERIALS AND METHODS Purification. A total of 630,000 pig hypothalami was dissected, defatted, and extracted as described (9, 10). The major part of this extract, corresponding to 470,000 hypothalami, was the same as that used for the isolation of porcine somatostatin (2) and pro-[Met]enkephalin (11). This extract was purified first by gel filtration on Sephadex G-25 (9, 10). Fractions containing immunoreactive somatostatin from the gel filtration were extracted with the upper phase of 0.1% acetic acid/1-butanol/ pyridine, 11:5:3 (vol/vol), (system I) and 1yophilized. This material was then further purified by preparative countercurrent distribution (CCD) in the same system by the C-2 model apparatus (H. 0. Post, New York), followed by chromatography on carboxymethyl (CM)-cellulose, another CCD in system I for 1800 transfers in the A-4 apparatus, chromatography on SESephadex, analytical gel filtration on Sephadex G-25, and rechromatography on SE-Sephadex. A smaller part of this extract, ...