Gene structure and functional characterization of growth hormone in dogfish, Squalus acanthias.

Dogfish (Squalus acanthias) growth hormone (GH) was identified by cDNA cloning and protein purification from the pituitary gland. Dogfish GH cDNA encoded a prehormone of 210 amino acids (aa). Sequence analysis of purified GH revealed that the prehormone is composed of a signal peptide of 27 aa and a mature protein of 183 aa. Dogfish GH showed 94% sequence identity with blue shark GH, and also showed 37-66%, 26%, and 48-67% sequence identity with GH from osteichtyes, an agnathan, and tetrapods. The site of production was identified through immunocytochemistry to be cells of the proximal pars distalis of the pituitary gland. Dogfish GH stimulates both insulin-like growth factor-I and II mRNA levels in dogfish liver in vitro. The dogfish GH gene consisted of five exons and four introns, the same as in lamprey, teleosts such as cypriniforms and siluriforms, and tetrapods. The 5'-flanking region within 1082 bp of the transcription start site contained consensus sequences for the TATA box, Pit-1/GHF-1, CRE, TRE, and ERE. These results show that the endocrine mechanism for growth stimulation by the GH-IGF axis was established at an early stage of vertebrate evolution, and that the 5-exon-type gene organization might reflect the structure of the ancestral gene for the GH gene family.

Identification of two insulin-like growth factor IIs in the Japanese eel, Anguilla japonica: cloning, tissue distribution, and expression after growth hormone treatment and seawater acclimation.

To better understand the role of IGFs in Japanese eel, Anguilla japonica, we cloned insulin-like growth factor-II (IGF-II) cDNAs and examined their mRNA expression in several tissues. Two eel IGF-II cDNAs, eIGF-II-1 and eIGF-II-2, were cloned from the liver. A signal peptide and a mature peptide of both preproIGF-IIs were composed of 47 amino acids (aa) and 69 aa, but they differed at 17 aa and 13 aa, respectively. The E domain of eIGF-II-1 was 49 aa longer than that of eIGF-II-2, and differed at 22 aa within 52 aa. The highest eIGF-II-1 and II-2 mRNA levels were observed in the liver, with detectable levels also found in all tissues examined. The eIGF-II-1 mRNA levels in the liver, heart, and muscle were higher in females than in males, whereas those in the stomach and intestine were lower in the females. The eIGF-II-2 mRNA levels in the liver and swim-bladder were also higher in females than in males whereas those in the stomach, spleen, and intestine were lower in the females. The eIGF-II-1 mRNA levels in the liver were higher in large compared to small glass eels, while the eIGF-II-2 mRNA levels did not correlate with body weight. Both eIGF-II mRNA levels in the liver increased after eel GH treatments in vivo and in vitro. No differences in both eIGF-II mRNA levels were observed in the gills, liver, stomach and whole kidney between seawater- and freshwater-reared eels.

Impairments in recognition memory for object and for location after transient brain ischemia in monkeys.

Using an object recognition or location memory test employing either small or large sets of training stimuli, we examined the effects of selective damage to the hippocampus after ischemia in Japanese monkeys. Ischemic (ISC) monkeys were significantly impaired in learning a delayed matching-to-sample (DMS) task (10 sec) when the sample and test objects were drawn from a set of 45 possible stimuli, but not in learning a DMS when a set of 300 possible stimuli was used ISC monkeys were also impaired in a delayed matching-to-location (DML) task that employed a 3-well tray but not in one that employed a 10-well tray. After criterion learning was attained, ISC monkeys were impaired significantly only in the 300-stimuli version and at the longest delay tested (10 min). These results suggest that the hippocampus might be involved in processing comparisons and forming relationships between current and recent stimuli, but not in the remembering of familiar objects, and in long-term maintenance (more than 10 min) of stimulus memory.

Identification of growth hormone in the sea lamprey, an extant representative of a group of the most ancient vertebrates.

GH was identified in the sea lamprey, an extant representative of a group of the most ancient vertebrates, the Agnatha. A putative GH-cDNA was cloned from the pituitary by RT-PCR. The entire coding region comprised an open-reading frame of 203 amino acids (aa). The mature protein was also isolated from pituitaries, and fractionated by gel filtration and reverse-phase HPLC. A putative GH was monitored by Western blotting with a rabbit antiserum against a synthetic peptide corresponding to pre-GH sequence (aa 29-45). Sequence analysis of the purified protein demonstrated that the prehormone consists of a signal peptide of 22 aa and the mature protein of 181 aa, which shows 25% sequence identity with sturgeon GH. The site of production was identified through immunohistochemistry to be cells of the dorsal half of the proximal pars distalis of the pituitary. Following cDNA cloning of lamprey IGF cDNA, it was shown using RT-PCR that lamprey GH stimulates IGF expression in lamprey liver. This is the first study in which a member of the GH/prolactin/somatolactin family has been identified in an agnathan. In addition, GH appears to be the only member of this hormone family in the sea lamprey. Evidence suggests that GH is the ancestral hormone in the molecular evolution of the GH family and that the endocrine mechanism for growth stimulation was established at an early stage of vertebrate evolution.