Purification and primary structure of pituitary adenylate cyclase activating polypeptide (PACAP) from the brain of an elasmobranch, stingray, Dasyatis akajei.

Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalami and found to exist as two amidated forms with 38 (PACAP 38) and 27 (PACAP 27) residues. The amino acid sequences of PACAPs isolated from the vertebrates, such as a bird, a frog and teleost fish, appear to be well conserved. In the present study, we attempted to isolate PACAP from the brain of an elasmobranch fish, Dasyatis akajei (stingray), which belongs to the Chondrichthyes (cartilaginous fish), by extraction of the acetone-dried powder with acetic acid, followed by successive high-performance liquid chromatography (HPLC) on a gel-filtration, a cation-exchange and two reverse-phase columns. Purification was monitored by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting analysis using an anti-PACAP 27 serum. The PACAP thus obtained consisted of 44 residues. The amino acid sequence of the comparable portion of its N-terminal 38 residues showed 92%, 89%, 89%, and 82% identity with those of mammalian, chicken, frog and teleost PACAPs with 38 residues, respectively. The extra six C-terminal residues of the stingray resembled those of tetrapod and teleost PACAP precursors which were deduced from the respective cDNAs. These results indicate that PACAP, which has an amino acid sequence showing high similarity with those of tetrapod and teleost PACAPs, is present in the elasmobranch brain.

Rapid increase in plasma IL-6 after hemorrhage, and posthemorrhage reduction of the IL-6 response to LPS, in conscious rats: interrelation with plasma corticosterone levels.

To study whether hemorrhage stimulates interleukin-6 (IL-6) production in conscious rats, 30% of the total blood was withdrawn over 3 min through an indwelling venous catheter and the shedblood was reinfused 1 h later. Plasma adrenocorticotropic hormone (ACTH), corticosterone and IL-6 concentration rapidly increased. Plasma ACTH levels peaked at 10 min and corticosterone and IL-6 peaked at 60 min; all started to decrease after reinfusion. In adrenalectomized (ADX) rats with or without a corticosterone pellet implant, there was an inverse relationship between IL-6 and corticosterone concentrations, greatest in ADX rats and lowest in ADX rats in which plasma corticosterone was elevated by crushing the implanted pellet. However, the ADX rats in which plasma corticosterone was maintained at normal or slightly elevated levels showed greater IL-6 responses to hemorrhage and elevated basal plasma IL-6 levels compared to sham-operated control rats. Twenty-four hours after hemorrhage/reinfusion, ACTH, corticosterone, and IL-6 responses to i.v. injection of lipopolysaccharide (LPS) were all reduced compared to the non-hemorrhaged animals, indicating that hemorrhage impaired general host defense. Although very high plasma corticosterone concentrations markedly suppressed the IL-6 response to LPS, in ADX rats in which plasma corticosterone was maintained at slightly higher levels than normal, the reduced IL-6 response to LPS in the posthemorrhage period was not reversed, but enhanced. Thus corticosterone has biphasic effects on the IL-6 response to hemorrhage and the response to LPS during the posthemorrhage period, which has important clinical implications with regard to the optimal dose of glucocorticoid for maintaining the host defense response.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitronectin diversity in evolution but uniformity in ligand binding and size of the core polypeptide.

We isolated vitronectins from the plasma or sera of 14 animal species including mouse and rat by heparin affinity chromatography. They cross-reacted with anti-vitronectin antibody and their amino terminal sequences showed strong homology. They also promoted spreading of BHK cells and were bound to heparin and collagen in the same way. Therefore, these properties appear to be essential for vitronectin function. However, the apparent molecular weights of these vitronectins varied considerable from 59 to 78 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, the number of bands also varied from 1 to 3. To search for the uniformity of vitronectin polypeptide, vitronectins were deglycosylated and examined by Ferguson plot analysis. The size of the polypeptide portion of vitronectins was estimated to range from 40 to 57 kDa which was 19-26 kDa smaller than original values. Supposing a possible cleavage site at 5-13 kDa far from the carboxyl terminus, all vitronectin polypeptides were speculated to be synthesized de novo in the size range of 50-57 kDa. Proteins reacting with anti-vitronectin antibody were also detected on the immunoblot of 13 more species including Drosophila and Physarum. Almost all of these vitronectin-like proteins showed marked species-specific variations in their apparent molecular weights from 51 to 96 kDa in SDS-PAGE.

[Effect of vitronectin on the healing of rabbit corneal epithelial damage].

Vitronectin is one of the major cell-adhesive glycoproteins in mammalian plasma. We investigated the effect of topically applied vitronectin on the healing of rabbit corneal epithelial damage. Vitronectin was purified from rabbit serum by heparin-Sepharose affinity chromatography and autoclaved at 121 degrees C for 20 min after adjusting the concentration to 0.2 mg/ml with saline. Rabbit corneal epithelium was abraded with ethanol and a laser blade in the central portion of the cornea which had been demarcated with a 6.5 mm trephine. Vitronectin eye drops were instilled into the right eye, and sterile saline drops into the left eye as control. The dimensions of the circular epithelial defect were measured at various times after wounding. The area of epithelial damage of vitronectin-treated eyes was smaller than that of control eyes at 2hr-12hr after abrasion (p less than 0.01). These results suggested that topically applied vitronectin might accelerate the corneal epithelial wound healing at an early stage. Vitronectin can be considered as a candidate for treatment of corneal epithelial damage.

Diversities in animal vitronectins. Differences in molecular weight, immunoreactivity and carbohydrate chains.

Six animal plasma vitronectins, human, horse, porcine, bovine, rabbit and chicken vitronectins purified by a novel method using two successive heparin affinity columns, showed marked diversity in molecular weight, immunoreactivity and carbohydrate composition. Chicken vitronectin had a distinctly different amino acid composition from the mammalian vitronectins; and bovine vitronectin was the only one to contain N-glycolylneuraminic acid as well as N-acetylneuraminic acid. Binding studies with horseradish peroxidase-labelled lectins indicated that all the vitronectins contained complex-type, sialylated N-linked sugar chains and that only porcine vitronectin had a fucosylated sugar chain. D-Galactosamine determinations and binding studies with horseradish peroxidase-peanut lectin on native and asialovitronectins revealed that the mammalian vitronectins other than human vitronectin contained O-linked sugar chains with sialic acid, chicken vitronectin contained unsialylated chains, and human vitronectin contained neither. The results indicate that diversities in vitronectins are apparent in their molecular weights and glycosylations, especially in the number and structure of O-linked sugar chains.

Novel purification of vitronectin from human plasma by heparin affinity chromatography.

The glycoprotein vitronectin (also called S-protein, serum spreading factor, or epibolin) promotes spreading of a variety of cultured cells, inhibits the cytotoxicity of membrane attack complex C5b-9, and modulates thrombin-antithrombin III activity. We developed a strikingly simple method to purify vitronectin from human plasma by heparin affinity chromatography. Serum was obtained from plasma by adding calcium and then centrifuging. The heparin-binding activity of vitronectin in human serum was activated with 8 M urea. The activated vitronectin specifically bound to heparin-Sepharose in 8 M urea and was eluted with 0.5 M NaCl containing 8 M urea. This procedure resulted in an approximately 250-fold purification of vitronectin with a 15-30% recovery; 3-6 mg of pure vitronectin were obtained from 100 ml human plasma within 2 days. The purified vitronectin preparations promoted spreading of BHK fibroblastic cells on substrates with a half-maximal activity at only 0.1 microgram/ml. This new method is very simple, rapid, inexpensive, and flexible. It could probably be readily scaled up for commercial applications.