Prothoracicotropic hormone (PTTH), a brain secretory polypeptide of insects, stimulates the prothoracic glands to produce and release ecdysone, the steroid essential to insect development. The complementary DNAs encoding PTTH of the silkmoth Bombyx mori were cloned and characterized, and the complete amino acid sequence was deduced. The data indicated that PTTH is first synthesized as a 224-amino acid polypeptide precursor containing three proteolytic cleavage signals. The carboxyl-terminal component (109 amino acids) that follows the last cleavage signal represents one PTTH subunit. Two PTTH subunits are linked together by disulfide bonds, before or after cleavage from prepro-PTTH, to form a homodimeric PTTH. When introduced into Escherichia coli cells, the complementary DNA directed the expression of an active substance that was functionally indistinguishable from natural PTTH. In situ hybridization showed the localization of the prepro-PTTH mRNA to two dorsolateral neurosecretory cells of the Bombyx brain.

Three molecular forms of prothoracicotropic hormone were isolated from the head of the adult silkworm, Bombyx mori, and the amino acid sequence of 19 amino acid residues in the amino terminus of these prothoracicotropic hormones was determined. These residues exhibit significant homology with insulin and insulin-like growth factors.

No abstract

We have determined the complete amino acid sequence of 4K-PTTH-H, one of three forms of the Mr 4400 prothoracicotropic hormone of the silkworm Bombyx mori, active to brainless pupae of Samia cynthia ricini. Like vertebrate insulin, it consists of two nonidentical peptide chains (A and B chains). The A chain consists of 20 amino acid residues. The B chain is a mixture of four microheterogeneous peptides, two of which consist of 28 residues, and the other two, of 26 residues. 4K-PTTH-II has considerable sequence homology (40%) with human insulin, and it resembles porcine relaxin both in the carboxyl-terminal cysteine residue of the A chain and in the amino-terminal pyroglutamic acid residue of the B chain. The identical distribution ofthe six cysteine residues also indicates that 4K-PTTH-ll belongs to the insulin family. Carboxypeptidase A Digestion. A suspension of carboxypeptidase A (treated with diisopropyl fluorophosphate, Sigma) in water was centrifuged at 1000 x g for 5 min and the supernatant was removed to eliminate free amino acids. The precipitated carboxypeptidase A was dissolved in 0.1 M ammonium acetate (pH 9.0) at a concentration of 1 ,g/,u and 1 ,ul of this solution was added to 19,ul of the same buffer containing 4K-PTTH-II (0.7 nmol), DA (1.1 nmol), DB1 (0.6 nmol), or DB2 (1.0 nmol). The digestions were performed at 37°C. Aliquots (5 ,ul each) were taken at 0.5, 1, 2, and 4 hr after the initiation of incubation, and the released amino acids were quantified by RP-HPLC (Senshu Pak SEQ-4; Senshu Kagaku, Tokyo) after derivatization to phenylthiocarbamoyl amino acids (7).Pyroglutamate Aminopeptidase Digestion. Tryptic peptides T2 (1.8 nmol) and T3 (5.0 nmol), and DB2 (0.5 nmol) were dissolved in 10 ,ul of 0.1 M phosphate buffer (pH 7.5) with 30 mM dithiothreitol. To these solutions, 4 ,ug of pyroglutamate aminopeptidase (Boehringer Mannheim) in 10 Al of the same buffer was added. After incubation at 35°C for 1 hr, the mixture was subjected to RP-HPLC on a TSK ODS-120T column developed with a gradient of 5-22.5% acetonitrile in 0.1% trifluoroacetic acid.Chymotrypsin Digestion. DB1 (3.0 nmol) and DB2 (7.0 nmol) were dissolved in 20 ,ul of 0.1 M Tris'HCl buffer (pH 7.8). To these solutions, 0.5 jig of a-chymotrypsin (Sigma) in 1 ,ul of 1 mM HCO was added. After incubation at 25°C for 3 hr, the resulting peptides were separated by RP-HPLC on a Develosil 5-ODS column with a linear gradient of 5-40% acetonitrile in 10 mM ammonium acetate.Amino Acid Analyses. Peptides (30-200 pmol) were hydrolyzed in constant-boiling HCO (Pierce) at 110°C for 20 hr.Abbreviations: PTTH, prothoracicotropic hormone; RP-HPLC, reversed-phase high-performance liquid chromatography. 5840The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

No abstract

We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm, Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for alpha-amino group. Three disulfide bonds were bridged step by step (A6-A11, A20-B22, and A7-B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides.

Prothoracicotropic hormone (PTTH) of the silkworm, Bombyx mori, was purified and its primary structure determined for the most part. From sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified PTTH under non-reducing and reducing conditions, and the amino acid sequence and composition analyses of the carboxamidomethylated PTTH, we concluded that Bombyx PTTH has a dimeric structure consisting of two identical, or nearly identical subunits, held together by disulfide bond(s). There exist subunit variants that differ by deletion of only a few amino acid residues at the N-terminus, and possibly at the C-terminus also, giving rise to a high heterogeneity in the PTTH molecule. The amino acid sequence up to the 104th residue from the N-terminus of the longest subunit, except for the 41st residue, was determined by sequence analysis of fragment peptides produced by lysyl endopeptidase, chymotrypsin and V8 protease digestions, leaving only a short C-terminal sequence undetermined. Bombyx PTTH is expected to contain a carbohydrate chain bound to an asparagine at position 41, deduced from the cDNA sequence.

The myc target gene Mina53 was reported to be overexpressed in esophageal cancer with a poor prognosis. The purpose of the present study was to examined Mina53 expression and its relationship to clinicopathological parameters in human renal cell carcinoma (RCC). Mina53 and Ki-67 expression was examined on immunohistochemistry for 64 surgically resected RCC and non-cancerous tissue. In addition, the relationship between Mina53 expression and clinicopathological prognostic factors of RCC such as age, stage, microvenous invasion (MVI), histological subtype, Ki-67 labeling index (LI), and prognosis, was examined. Mina53 was expressed in the nuclei of tumor cells and tubular nuclei of normal renal tissue. The expression level of Mina53 was significantly higher in patients with poor prognostic factors (stage IV, MVI-positive, and sarcomatoid RCC, and high Ki-67 LI). The prognosis of high Mina53-expressing tumors was significantly poorer than that of non-Mina53-high tumors (P < 0.0001). In conclusion, Mina53 is overexpressed in RCC tissue from patients with poor prognostic factors, suggesting that Mina53 overexpression is one of the factors for poor prognosis in RCC.