C. Samuel Bradford

Physiological Effects of Collecting and Transporting Emigrating Juvenile Chinook Salmon past Dams on the Columbia River

Abstract Emigrating juvenile salmonids are collected at McNary Dam on the Columbia River and transported past the three downstream dams to avoid mortalities caused by passage through power-generating turbines. During the 1982–1984 seaward migrations of juvenile fall and spring chinook salmon Oncorhynchus tshawytscha, we used an array of physiological measurements (plasma cortisol and glucose, white blood cell counts) and challenge tests (saltwater challenge, secondary stress, and swimming endurance) to identify the stressful elements in these activities. Sequential increases in plasma cortisol titers offish sampled at the physically separable points in the collection system led us to conclude that the elements of the system stressed fish cumulatively. Furthermore, there were decreases in numbers of white blood cells, in osmoregulatory ability, and in swimming endurance during the first 24 h after fish were collected. Increasing the water flow rate in the system after the 1982 season seemed to reduce total...

Evidence for ultra-short-loop feedback in ACTH-induced interrenal steroidogenesis in coho salmon: acute self-suppression of cortisol secretion in vitro.

Interrenal tissues from coho salmon (Oncorhynchus kisutch) were incubated in a defined medium under blood-gas atmosphere at 17 degrees. Rates of cortisol secretion by tissues incubated in media containing 50 mU/ml porcine-ACTH were initially much greater than those of resting tissues in hormone-free media, but after 3 to 6 hr returned to resting rates. The time course of cortisol accumulation in ACTH-containing media was the same when tissues were incubated in different volumes; the final concentrations of cortisol in these incubations were similar to each other and resembled peak in vivo concentrations in juvenile coho subjected to acute stress. Cortisol secretion rates of tissues sequentially transferred to fresh ACTH-containing media every 6 hr did not return to resting levels but remained elevated for at least 24 hr. Cortisol secretion in response to ACTH was attenuated or completely abolished in tissues incubated in media containing exogenous cortisol; this effect was reversible and dose-dependent. Our results suggest that in coho salmon, cortisol may exert ultra-short-loop negative feedback directly at the level of the interrenal gland to effect self-suppression.

Cell cultures from zebrafish embryos and adult tissues

The zebrafish is increasingly popular as a nonmammalian model for studies of vertebrate developmental biology, genetics, and toxicology. The availability of cell culture systems makes it possible to address many basic questions using in vitro approaches. Here we describe materials and procedures for initiating cell cultures from zebrafish early (blastula- and gastrula-stage) diploid and haploid embryos and adult tissues (gills, fins, liver, viscera). Zebrafish cells are grown in a complex basal nutrient medium supplemented with insulin, selenite, fetal bovine serum, trout serum, and an extract prepared from rainbow trout embryos. The procedure for preparing trout embryo extract (demonstrated to be mitogenic for a variety of piscine cell lines), is also described.

Molecular cloning, sequence analysis and expression distribution of rainbow trout (Oncorhynchus mykiss) cystatin C

Cystatin C is one of a family of proteinase inhibitors of cathepsins and other cysteine proteinases. Among warm-blooded vertebrates, small functional regions of cystatin amino acid sequences are well conserved among species, but major portions of cystatin amino acid sequences vary evolutionarily. Although considerable attention has been given to mammalian and avian cystatins, little data exist on cystatins from other vertebrates. A cDNA clone for trout cystatin C was isolated from a lambda gt11 cDNA library of rainbow trout (Oncorhynchus mykiss) liver. An apparently full-length cDNA clone of 674 bp encoding 132 amino acid residues was obtained. Sequence analysis indicated that trout cystatin C contains an N-terminal signal sequence extension of 21 amino acids and a mature sequence of 111 amino acid residues, with amino acid residues conserved in functional regions relative to mammalian and avian cystatin C. Using cloned cDNA as a probe, we investigated expression of the cystatin C gene in trout tissues, several cell lines of trout liver or liver tumor, and cell cultures of liver tumor origin. Cystatin C mRNA was in high abundance in trout embryo tissue, a tumor-derived liver cell line and some normal adult tissues. Southern hybridization analysis indicated one copy of the trout cystatin C gene per haploid genome, and sequence comparisons indicated considerable divergence in large portions of the coding region of the trout cystatin C gene relative to a variety of species.

Identification and characterization of mesotocin and V1a-like vasotocin receptors in a urodele amphibian, Taricha granulosa.

The cDNA sequences encoding the mesotocin receptor (MTR) and vasotocin 1a receptor (VTR-1a) were identified in a urodele amphibian, the rough-skinned newt, Taricha granulosa. Saturation binding of [(3)H]oxytocin (OT) to the Taricha MTR (tMTR) was best fit by a two-state model; a high affinity-low abundance site and a lower affinity-high abundance site. Competition-binding studies found the following rank-order affinities for the tMTR: mesotocin (MT)>OT≈vasotocin (VT)>vasopressin (VP)>isotocin (IT). Inositol phosphate (IP) accumulation studies demonstrated functional activity of both the tMTR and Taricha VTR-1a (tVTR-1a) in a heterologous cell culture system. The rank-order potencies for the tMTR were MT>OT>VT≈VP>IT. The combined binding and IP results indicate that VT may act as a partial agonist of the tMTR. Rank-order potencies for the tVTR-1a were VT>VP>MT≈OT>IT. For both receptors, stimulation of IP accumulation was blocked by d(CH(2))(5)[Tyr(Me)(2)]AVP (Manning compound) and d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2)]OVT (OTA). OTA was a more potent antagonist for the transiently expressed tMTR while Manning compound was relatively more potent at inhibiting IP accumulation in tVTR-1a expressing cells. In contradiction to earlier assumptions, the absolute IC(50) of Manning compound was lower for the tMTR (27nM±13) than the tVTR-1a (586nM±166) indicating its potential higher affinity for the tMTR, a finding with special relevance to interpretation of comparative studies investigating the behavioral and physiological actions of neurohypophysial peptides in non-mammalian species.

Reciprocal regulation of BMF and BIRC5 (Survivin) linked to Eomes overexpression in colorectal cancer

Eomesodermin (Eomes) is a T-box transcription factor that has been implicated in the etiology of colorectal cancer and other human malignancies. We screened a panel of human primary colon cancers and patient-matched controls (n = 30) and detected Eomes overexpression at the mRNA and protein level. Similar results were obtained in a panel of rat colon tumors and adjacent normal-looking colonic mucosa (n = 24). In human colon cancer cells, forced overexpression of Eomes enhanced cell viability and protected against staurosporine-induced apoptosis. On the other hand, knocking down Eomes resulted in reduced cell viability, G2/M cell cycle arrest, and apoptosis induction. The apoptotic mechanism centered on the reciprocal downregulation of anti-apoptotic BIRC5 (Survivin) and upregulation of proapoptotic Bcl-2 modifying factor (BMF). In patients with colorectal cancer, high EOMES expression (n = 95) was associated with poor overall survival compared with individuals exhibiting low EOMES levels (n = 80). We conclude from the current investigation, and prior literature, that Eomes has a divergent role in cancer development, with evidence for tumor suppressor and oncogenic functions, depending on stage and tissue context. Further studies are warranted on the apoptotic mechanisms linked to the reciprocal regulation of BMF and BIRC5 in human colorectal cancers characterized by Eomes overexpression.