Tsuneo Asano

A prolactin-releasing peptide in the brain

Hypothalamic peptide hormones regulate the secretion of most ofthe anterior pituitary hormones, that is, growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and adrenocorticotropin,. These peptides do not regulate the secretion of prolactin,, at least in a specific manner, however. The peptides act through specific receptors, which are referred to as seven-transmembrane-domain receptors or G-protein-coupled receptors. Although prolactin is important in pregnancy and lactation in mammals, and is involved in the development of the mammary glands and the promotion of milk synthesis,, a specific prolactin-releasing hormone has remained unknown. Here we identify a potent candidate for such a hormone. We first proposed that there may still be unknown peptide hormone factors that control pituitary function through seven-transmembrane-domain receptors. We isolated the complementary DNA encoding an ‘orphan’ receptor (that is, one for which the ligand is unknown). This receptor, hGR3, is specifically expressed in the human pituitary. We then searched for the hGR3 ligand in the hypothalamus and identified a new peptide, which shares no sequence similarity with known peptides and proteins, as an endogenous ligand. We show that this ligand is a potent prolactin-releasing factor for rat anterior pituitary cells; we have therefore named this peptide prolactin-releasing peptide.

Purification and characterization of recombinant human interleukin-2 produced in Escherichia coli

Recombinant human interleukin-2 (rIL-2) produced in Escherichia coli was purified to apparent homogeneity by cation exchange chromatography and reverse phase high performance liquid chromatography. The amino acid composition, amino terminal amino acid sequence, and carboxyl terminal amino acid were consistent with those deduced from the cDNA sequence. Besides the molecular species with the amino terminal Ala, the purified preparation contained another species having an additional Met residue at the amino terminus corresponding to the initiation codon AUG. The molar absorption coefficient of rIL-2 was determined to be 9.58 X 10(3) M-1 cm-1 at 280nm in water. Ultracentrifugal analyses revealed that it existed as a monomeric form in 0.1 M NaCl. The apparent sedimentation coefficient (S20,w) was calculated to be 1.8 S.

Gelation of limulus amoebocyte lysate by an antitumor (1→3)-β-D-glucan

Abstract An antitumor carboxymethylated (1→3)-β-D-glucan (CMPS) was found to have a potent ability to cause gelation of the amoebocyte lysate of horseshoe crab at concentrations as low as 10−6 mg/ml. The gelation of the lysate and the activation of the proclotting enzyme in the lysate caused by CMPS were unique in that these reactions occurred at CMPS concentrations ranging from 10−6 to 10−3 mg/ml. The optimum concentration was 10−5 – 10−4 mg/ml and at concentrations above 10−2 mg/ml no gelation occurred. This gelation pattern was also observed in common with other antitumor polysaccharides. The mechanism of the gelation caused by CMPS was revealed to be distinctly different from that working in the gelation caused by endotoxins.

Recombinant hepatitis B virus surface antigen carrying the pre-S2 region derived from yeast: purification and characterization

Abstract Modified hepatitis B virus surface antigen (HBsAg) carrying the pre-S2 region (HBsAg M-P31c) has been highly purified from the recombinant yeast Saccharomyces cerevisiae . The purified HBsAg M-P31c formed spherical particles with a diameter of about 20 nm, which were similar in both shape and size to human plasma-derived HBsAg small particles (h-HBsAg). The M-P31c particles were composed of two major glycoproteins with molecular masses of 37 kDa (GP37) and 34 kDa (GP34). GP37 and GP34 had identical polypeptide backbones (P31) of 31 kDa and had N-linked sugar chains with a molecular mass of about 3 kDa at the Asn 4 residue. GP37 has additional sugar chain(s) in the pre-S2 region. The sugar chains were composed of mannose and N -acetylglucosamine. Like h-HBsAg, the particles also contained phospholipids, triglycerides, and free fatty acids. On the other hand, little cholesterol was contained in the particles. M-P31c vaccine adsorbed on Al(OH) 3 gel elicited anti-pre-S2 antibodies in addition to anti-S antibodies. These results demonstrate that M-P31c particles are promising as an improved immunogen for the hepatitis B vaccine.

Production of recombinant human relaxin 3 in AtT20 cells

Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 microM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.

Purification and characterization of human T-cell leukemia virus type I protease produced in Escherichia coli

Human T‐cell leukemia virus type I (HTLV‐I) protease has been purified to homogeneity from a strain of recombinant Escherichia coli. The protease was expressed as a larger precursor, which was autoprocessed to form a mature protease. Protein chemical analyses revealed the coding sequence of mature protease, which agreed with the putative sequence predicted from the sequence of bovine leukemia virus protease, The purified protease processed the natural substrate gag precursor (p53) to form gag p19 and gag p24. The protease activity was inhibited by pepstatin A. These results provide direct evidence that this protease belongs to the aspartic protease family and has an activity consistent with the protease in HTLV‐I virion.

Escherichia coli-derived human interferon-γ with CysTyrCys at the N-terminus is partially Nα-acylated

Abstract Purified preparations of recombinant human interferon-γ (rIFN-γ) with CysTyrCys at the N-terminus ([CysTyrCys]IFN-γ) derived from Escherichia coli gave two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two peaks on reversed-phase high-performance liquid chromatography (rpHPLC). In contrast, rIFN-γ without CysTyrCys and rIFN-γ in which both Cys-1 and Cys-3 were substituted with serine behaved as a single species on both SDS-PAGE and rpHPLC. These results suggest that the N-terminal portion of rIFN-γ is heterogeneous. To elucidate the structure of the N-terminal portion, the N-terminal peptide preparation was obtained by binding rIFN-γ to thiopropyl-Sepharose 6B gel with disulfide linkage followed by trypsin digestion and elution with 2-mercaptoethanol. The preparation gave four peaks (NT-1, NT-2, NT-3, and NT-4, in order of elution) on rpHPLC; all four were found to be Cys-1-Lys-9 by amino acid analysis after acid hydrolysis. Various analyses indicate that NT-1 is the intact nonapeptide, that NT-3 and NT-4 are N α -formyl and N α -acetyl forms of NT-1, respectively, and that NT-2 may be S-blocked at Cys-1. It is concluded that E. coli -derived [CysTyrCys]IFN-γ is partially N α -acylated. The data also suggest that N α -acylation does not affect the biological activity of [CysTyrCys]-IFN-γ.

Activation of the alternative pathway of complement by an antitumor (1----3)-beta-D-glucan from Alcaligenes faecalis var. myxogenes IFO 13140, and its lower molecular weight and carboxymethylated derivatives.

An antitumor (1----3)-beta-D-glucan with a number-average degree of polymerization (DP) of 540 from Alcaligenes faecalis var. myxogenes IFO 13140, and its lower molecular weight derivatives were found to activate the alternative pathway of complement (APC), as judged by hemolytic and immunoelectrophoretic analyses. Of the native and derivative (1----3)-beta-D-glucans measured, the smallest one that showed APC-activating ability was that with a DP of about 20. The effect of carboxymethylation of the (1----3)-beta-D-glucans with DPs of 49, 131 and 540 on their APC-activating ability was investigated. In any (1----3)-beta-D-glucan the ability was decreased with the increase of carboxymethyl substitution and was completely lost when about one carboxymethyl group per glucose residue was incorporated. In contrast, strong inhibitory ability against C1 hemolytic activity appeared on carboxymethylation.

Production of human protein disulfide isomerase by Bacillus brevis

Human protein disulfide isomerase (PDI; EC 5.3.4.1) was expressed and secreted into the culture medium using Bacillus brevis as host and pNU200 which codes the promoter and signal sequence of major cell wall protein of B. brevis as vector. The accumulation of recombinant human PDI (rhPDI) reached about 5 mg l-1 in the late exponential phase of the bacterial growth. The purified rhPDI was found to be exactly processed at the carboxyl terminus of the signal sequence. It was as active as natural PDI derived from human placenta as determined by its ability to reactivate scrambled ribonuclease that was a fully oxidized mixture containing randomly formed disulfide bonds. The activity was significantly accelerated in the presence of dithiothreitol or a mixture of reduced and oxidized glutathione. These indicate that the characteristics of rhPDI are similar to those reported for mammalian PDI and that it can be used for refolding inactive proteins having incorrect disulfide bonds.

Cell distributions in and sound absorption characteristics of flexible polyurethane foams

In this article, the two-dimensional distributions of cells from the cross section of some flexible polyurethane foams were cleared, and the three-dimensional distributions of cells based on Saltykovs theory were estimated further. As a result, it was found that a mean of the two-dimensional distributions of cells was a good linear relation with a mean of the three-dimensional distributions of cells, and it was confirmed that cell structure of the foams which should have been analyzed in the three-dimensional distributions was evaluated by analysis of the two-dimensional distributions fully. It was also found that not only cell number but also cell distribution was necessary in the evaluation of flexible polyurethane foams, and cell diameter was closely related to the sound absorption coefficient in polyester-based flexible polyurethane foams.