Susumu Funakoshi

Magainin 1-induced leakage of entrapped calcein out of negatively-charged lipid vesicles

Effects of magainin 1, a novel antimicrobial peptide, on the permeability of lipid vesicles were investigated by using calcein as a trapped fluorescent marker. Magainin 1 induces the leakage of calcein specifically out of negatively-charged vesicles. The peptide binds to bovine brain phosphatidylserine sonicated vesicles according to the Langmuir isotherm with a binding constant of 3.8.10(5) M-1 and a binding-site number of 0.10 per lipid molecule. The leakage seems to occur at a critical binding number of approx. 0.03 per lipid molecule. A circular dichroism study revealed that magainin 1 conforms mainly to an unordered structure both in an aqueous solution and in the presence of egg yolk phosphatidylcholine vesicles, whereas to an amphiphilic helix with the phosphatidylserine vesicles. In conclusion, magainin 1 interacts with acidic lipids through electrostatic interactions followed by hydrophobic interactions to form an amphiphilic helix, inducing the leakage.

A novel anti-HIV synthetic peptide, T-22 ([Tyr5,12,Lys7]-polyphemusin II)

Tachyplesin and polyphemusin are antimicrobial peptides recently isolated from the hemocytes of horseshoe crabs (Tachypleus tridentatus and Limulus polyphemus). We synthesized them and their analogs and examined their antiviral activity against human immunodeficiency virus (HIV) type 1 in vitro. The infection of human T cells with the virus was markedly inhibited by some of them at low concentrations. In this structure-activity study, we found that [Tyr5,12, Lys7]-polyphemusin II, which was designated as T22, had extremely high anti-HIV activity. Its 50% inhibitory concentration (EC50) was 0.008 micrograms/ml, while its 50% cytotoxic concentration (CC50) was 54 micrograms/ml and these values were comparable to those of AZT. This result indicates that T22 would be a potential candidate for the therapy of HIV infection.

New strategy for the chemical synthesis of proteins

Abstract For the chemical synthesis of proteins, an efficient deprotecting procedure by combination of a hard acid, trimethylsilyl trifluoromethanesulfonate, and a soft nucleophile, such as thioanisole, is described. For disulfide bond formation, two new procedures are presented; the one by oxidation with thallium(III) trifluoroacetate and the other by the acid-catalyzed reaction, involving S-substituted cysteine sulfoxides.

A comparative study of the solution structures of tachyplesin I and a novel anti-HIV synthetic peptide, T22 ([Tyr5,12, Lys7]-polyphemusin II), determined by nuclear magnetic resonance

The solution structure of tachyplesin I, which was isolated from membrane acid extracts of the hemocytes from the Japanese horseshoe crab (Tachypleus tridentatus), was determined by nuclear magnetic resonance (NMR) and distance geometry calculation. Tachyplesin I takes an antiparallel beta-sheet structure with a type-II beta-turn. Recently, among more than 20 synthetic peptides associated with tachyplesin and its isopeptide (polyphemusin), we found that a novel compound, which we designated as T22 ([Tyr5,12, Lys7]-polyphemusin II), strongly inhibited the human immunodeficiency virus (HIV)-1-induced cytopathic effect and viral antigen expression. The solution structure of T22 was investigated using NMR, and its secondary structure was confirmed to be similar to that of tachyplesin I. The anti-parallel beta-sheet structure and the several amino-acid side chains on the plane of the beta-sheet of T22 are thought to be associated with the expression of anti-HIV activity.

Bioactivity of synthetic human pancreastatin on exocrine pancreas

A biological activities of synthetic human pancreastatin (1-52) and its C-terminal fragment (24-52) were evaluated for the first time in the conscious rats. Both pancreastatins inhibited CCK-stimulated pancreatic secretion in a range of 20-200 pmol/kg/h with the same potency, indicating that the C-terminal portion of this peptide has a full biological activity. The relative molar potency of this substance compared to that of porcine pancreastatin was equivalent. This study suggests that human pancreastatin has the same biological activity as that of porcine, and plays a biological action in the exocrine pancreas.

Tissue-specific expression of the rat secretin precursor gene

Secretin is a 27-amino acid gastrointestinal hormone that stimulates the secretion of bicarbonate-rich pancreatic fluid. We previously demonstrated that the secretin precursor gene is expressed in the brain as well as in the small intestine. In this study, we demonstrated that the abundance of secretin precursor mRNA in the heart, lung, and kidney was comparable to that of the small intestine. The nucleotide sequences of the coding regions of the secretin precursor mRNAs in these tissues were identical to those of the small intestine, indicating that secretin precursor proteins produced in these tissues are identical to those in the small intestine. This is the first report that the secretin precursor gene is also expressed in the heart, lung, kidney, and testis as well as in the gastrointestinal tract and brain.

High Plasma Pancreastatinlike Immunoreactivity in a Patient With Malignant Insulinoma

High levels of pancreastatinlike immunoreactivity were detected in the plasma (2.9 pmol/ml, greater than 200-fold the normal level), pancreas (2.9 nmol/g wet wt, greater than 450-fold the normal level), and liver (1.6 nmol/g wet wt) of a patient with pancreatic insulinoma with metastasis to the liver by a sensitive and specific radioimmunoassay for human pancreastatin. Antiserum was produced against the C-terminal fragment of human pancreastatin-(24-52), which was synthesized according to the sequence of human chromogranin A corresponding to that of pancreastatin. With the antiserum, intense immunocytochemical staining was detected in the tumors. Sephadex G-50 gel filtration showed that the tumors and plasma contained two molecular forms of pancreastatinlike immunoreactivity--a molecular form coeluted with synthetic human pancreastatin-52 and a larger molecular form (Mr approximately 12,000-15,000). The smaller form eluted in the same position as synthetic human pancreastatin-52 on reverse-phase high-performance liquid chromatography.

Isolation and characterization of a tumor-derived human pancreastatin-related protein.

A protein with pancreastatin-like immunoreactivity has been isolated and purified from liver metastasis of a patient with insulinoma. NH2-terminal sequence analysis in conjunction with the use of antibodies specific for the C-terminal structure of pancreastatin identified this protein as a 186-amino acid residue protein corresponding to human chromogranin A-116-301. Using a sensitive radioimmunoassay it was found that serum from the patient with insulinoma contains two peptide species; one comigrates with the 186-amino acid residue pancreastatin and the other the 48-residue pancreastatin.

Inhibitory effect of pancreastatin on pancreatic exocrine secretions : Pancreastatin inhibits central vagal nerve stimulation

The effects of the C-terminal fragment of rat pancreastatin on exocrine pancreatic secretions induced by several neural stimulations [IV injection of 75 or 15 mg/kg of 2-deoxy-D-glucose (central vagal nerve stimulation), injection of 2 mg of cisapride (proposed to elicit acetylcholine release from cholinergic nerve ending), and infusion of 1 or 3 mg.kg-1.h-1 of bethanechol (direct stimulation of acinar cells)] were examined in conscious rats. Rats with external bile and pancreatic fistulae were used. All the stimulations caused significant increases in pancreatic exocrine secretions. Pancreastatin at 100 pmol.kg-1.h-1 inhibited pancreatic secretions stimulated by IV injection of 2-deoxy-D-glucose but not those induced by the infusion of bethanechol or the injection of cisapride. Because these findings showed that pancreastatin inhibited pancreatic secretions induced by central vagal nerve stimulation, the effect of pancreastatin on cholecystokinin-stimulated pancreatic secretions in vagotomized rats was examined. Pancreastatin at 100 pmol.kg-1.h-1 did not inhibit pancreatic secretions stimulated by cholecystokinin octapeptide at 100 pmol.kg-1.h-1 in conscious rats after bilateral truncal vagotomy. These results suggest that pancreastatin inhibits pancreatic exocrine secretions by inhibiting vagal efferent nerve activity.

Bioactivity of human pancreastatin and its localization in pancreas

Synthetic human pancreastatin and its C-terminal fragment were first evaluated with respect to the biological activity on the insulin secretion in the isolated rat islets. Both these pancreastatins inhibited glucose-stimulated insulin secretion at a concentration of 100 nM. The relative molar potency of human pancreastatin compared to that of porcine pancreastatin was equivalent. The pancreastatin-reactive cells were widely located in the islets of Langerhans, and not observed in exocrine acinar cells by immunocytochemistry using human pancreastatin C-terminal specific antibody. These results suggest that human pancreastatin may modulate endocrine function of the pancreas, especially insulin secretion.