Naoki Sakura

Toxic effect of a β-amyloid peptide (β22–35) on the hippocampal neuron and its prevention

A synthetic truncated beta-amyloid peptide, beta 22-35, was shown to have a cytotoxic effect on cultured neurons from the rat hippocampus in serum-free medium. The peptide formed aggregates and typical amyloid fibrils resembling those of the beta-amyloid protein (AP) in neutral buffer solution and showed characteristic staining with Congo red and thioflavin-S. The neurotoxicity of beta 22-35 was suppressed by addition of calf serum, dibutyryl cAMP or insulin to culture medium, but not by addition of NGF or substance P. beta 22-35 had no effect on the glial cells. These results suggest that the AP can induce neurotoxicity in the hippocampal cells in vitro and the toxicity may involve a disorder in the intracellular signal transduction.

Novel drug delivery system to bone using acidic oligopeptide: pharmacokinetic characteristics and pharmacological potential.

We synthesized fifteen oligopeptides consisting of Asp or Glu conjugated with a fluorescent probe, 9- fluorenylmethylchloroformate (Fmoc). In the in vitro binding assay to putative hydroxy apatite (HA), the affinities of these conjugates depended only on the number of amino acid residues, not on their optical characters (L or D) or their species (Asp or Glu). In an in vivo experiment involving a single i.v. injection of Fmoc-D-Asp oligopeptides into mice, peptides consisting of over six Asp residues were selectively distributed to the bone. Then, we synthesized estradiol-17β-succinate-(L-Asp)6 [E2-(L-Asp)6] and studied its pharmacokinetic characteristics and its antiosteoporotic effects on ovariectomized (OVX) mice. Although the distribution volume of E2-(L-Asp)6 was significantly smaller than that of E2, E2-(L-Asp)6 was selectively distributed in the bone after i.v. injection and gradually decreased during 7 days. E2-(L-Asp)6 effectively prevented OVX-induced bone loss, without altering the uterine weight, in the dosage range of 0.11 to 1.1 μmol/kg once a week, while E2 increased both the bone mineral density and uterine weight at 0.37 μmol/kg every third day. The results suggest that acidic oligopeptide may be useful for drug delivery to bone and E2-(L-Asp)6is a good candidate as an anti-osteoporosis drug without the adverse side effects of E2.

Expression of N-acetyllactosamine and β1,4-Galactosyltransferase (β4GalT-I) During Adenoma-Carcinoma Sequence in the Human Colorectum

We set out to determine the expression profiles of glycoproteins possessing N-acetyllactosamine, a precursor carbohydrate of sialyl Lex, during colorectal cancer development. We immunohistochemically analyzed the distribution of N-acetyllactosamine as well as of β4GalT-I, a member of the β1,4-galactosyltransferase family responsible for N-acetyllactosamine biosynthesis, in normal mucosa and in adenoma and carcinoma of the human colorectum. Using monoclonal antibody H11, N-acetyllactosamine was barely detectable in the normal mucosa. In low-grade adenoma, however, N-acetyllactosamine was weakly but definitely expressed on the cell surface, and its expression level was moderately increased in high-grade adenoma and markedly increased in carcinoma in situ as well as in advanced carcinoma. To detect β4GalT-I, we used a newly developed polyclonal antibody (designated A18G), which is specific for the stem region of human β4GalT-I. Faint expression of β4GalT-I was detectable in normal mucosa, and the expression level was moderately increased in low-grade adenoma and in high-grade adenoma and markedly increased in carcinoma in situ and advanced carcinoma. The expression of N-acetyllactosamine was highly correlated with the expression of β4GalT-I in these tumor cells. These results indicate that the expression level of β4GalT-I is apparently enhanced during tumorigenesis in the colorectum and that β4GalT-I mostly directs the carcinoma-associated expression of N-acetyllactosamine on the colorectal tumor cell surface.

Syntheses of C-peptides and Human Proinsulin

Syntheses of human, dog, rat, and duck C-peptides and their analogues and preliminary results on the total synthesis of human proinsulin are described. In the syntheses of the C-peptides, chain elongation was performed exclusively by the azide-fragment condensation method in solution. The synthetic human, dog, rat, and duck C-peptides and their analogues were proved to be homogeneous by several analytic means. With these synthetic peptides, radioimmunoassay systems for dog, rat, and duck C-peptides were developed. For the total synthesis of human proinsulin, 10 protected peptide hydrazides were prepared, and the linearly protected hexaoctacontapeptide having the proposed sequence of human proinsulin was constructed by the azide-fragment condensation method in solution starting from the C-terminal undecapeptide (HP 75–86). After deblocking of the α-amino protection, the partially protected hexaoctacontapeptide was treated with sodium in liquid ammonia. The ensuing sulfhydryl form was converted to the S-sutfonate form, which was reduced and then air-oxidized. The oxidized material was purified by gel filtration on Sephadex G-50 (fine) followed by ion-exchange chromatography on DEAE-cellulose. The crossreactivity in the insulin radioimmunoassay of the ensuing product was 62.5 per cent of porcine proinsulin on a weight basis at B/B0 = 60 per cent. Acid hydrolysis and amino acid analysis of this product gave the theoretically expected ratios. In addition, this peptide, as well as the S-sutfonate form of the hexaoctacontapeptide, showed displacement curves superimposable on that of synthetic human C-peptide on an equimolar basis in the human C-peptide radioimmunoassay (antiserum 527). These results confirm the synthesis of human proinsulin.

Synthesis of Polypeptides Related to Porcine Proinsulin

Syntheses are described of eleven protected peptide fragments which cover the entire sequence of porcine proinsulin and of two partially protected polypeptides which correspond to the 17–84 and 15–84 amino acid sequences, respectively, of porcine proinsulin. The amino acid sequences of the two partially protected polypeptides are illustrated in the text. In these peptides, the sulfhydryl group of cysteine is protected with the ethylcarbamyl function, and the s-amino group of lysine is blocked with the formyl function. The partially protected octahexacontapeptide I cross-reacted with anti-synthetic porcine connecting peptide antisera to the same extent as did the porcine proinsulin or the synthetic connecting peptide. Heptacontapeptide II cross-reacted with antisera to porcine proinsulin and slightly with antisera to porcine insulin.

Hydrolytic removal of acetyl-amino acid from acetyl- peptide in methanesulfonic acid

Acylation of the N-terminus of a protein with an acetyl (Ac) group is a common modification. Proteins and peptides with such blocking groups cannot be sequenced by Edman degradation. In the present study, we investigated the stability and susceptibility of the peptide bonds of Ac-X-Ala-Phe-OH (X = Gly, Ala, Pro, Tyr) towards aqueous methanesulfonic acid (MSA). This paper describes selective hydrolytic removal of Ac-amino acid from Ac-peptide in 70% MSA.

Selective removal of pyroglutamic acid residue from biologically active pyroglutamyl-peptides by aqueous methanesulfonic acid

A number of native proteins and peptides have been shown to possess a pGlu residue at the N-terminal, which is thought to afford protection against aminopeptidase digestion. Our recent studies [1–4] indicated that pGlu-peptide is highly sensitive to acid under mild conditions, generating not only the ringopened product of the pyrrolidone moiety of the pGlu residue, but also the cleavage product of the pGlu-peptide linkage, and pGlu-OH is selectively cleaved from pGlu-peptide in aqueous methanesulfonic acid (MSA). In this study, the selective removal of pGlu residue was examined from biologically active pGlu-peptides by aqueous MSA at 25°C.