Metastin suppresses the motility and growth of CHO cells transfected with its receptor.

We recently reported having identified of the ligand for an orphan G-protein-coupled receptor, hOT7T175, as the gene product (68-121)-amide of the metastasis suppressor gene KiSS-1. We further showed that the ligand, which we named "metastin," inhibits chemotaxis and invasion of Chinese hamster ovary (CHO) cells transfected with hOT7T175 cDNA (CHO/h175) in vitro, and pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. In the present study, we investigated the activity of metastin in CHO/h175 cells in greater detail. Metastin significantly suppressed motility in a chemotaxis assay and wound healing assay at 10-100 nM order concentrations. Two N-terminally truncated peptides, metastin(40-54) and metastin(45-54) inhibited the migration of CHO/h175 cells as potently as metastin itself. Metastin also inhibited the spreading, monolayer growth and colony formation in agar (0.8%) of CHO/h175 cells at 10-100 nM concentrations. These results indicate that metastin is a potent inhibitor of cell motility, leading to suppression of cell growth and antimetastatic activity, and suggest that low molecular chemical compounds could replace its activity as a novel antimetastatic agent.

Novel betacellulin derivatives. Separation of the differentiation activity from the mitogenic activity.

Betacellulin (BTC) is a member of the epidermal growth factor family. It has two biological activities: mitogenic activity in fibroblasts and vascular smooth muscle cells, and differentiation activity for the differentiation of pancreatic acinar AR42J cells into insulin-secreting cells. The previous finding that recombinant BTC promotes the neogenesis of beta-cells in a mouse model supports the possibility that BTC is a therapeutic protein. However, the mitogenic activity of BTC may not be needed for differentiation into beta-cells and may cause a side effect in clinical use. We prepared several derivatives of BTC to segregate the two activities, to decrease the mitogenic activity, and to maintain the differentiation activity. We succeeded in obtaining BTC derivatives segregated by the two biological activities by preparing truncated-type derivatives. A derivative of BTC, BTC24-76, with a truncated N-terminal 23 amino acids and C-terminal 4 amino acids, was 2.5-fold more active in differentiation and had one-tenth of the mitogenic activity. The derivatives described in the present study should be helpful in future applications as therapeutic proteins and in basic research for discovery of a BTC-specific receptor.

A novel secreted tumor antigen with a glycosylphosphatidylinositol-anchored structure ubiquitously expressed in human cancers.

In a search for novel genes expressed in human cancers, we identified one gene from an assembled expressed sequence tag database. Northern blot analysis revealed that the gene, termed alcan, was expressed in various types of human cancer cell lines and in the fetus, but not in normal tissues. The alcan gene is located on chromosome 6 and is encoded on a 246-amino-acid protein with weak homology to classical major histocompatibility complex class I. Its gene product, ALCAN, had hydrophobic amino acid clusters at both the N- and C-terminal regions and was predicted to be a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Flow cytometric analysis revealed that ALCAN was detected on the surface of human cancer cells and on alcan-transfected CHO-K1 cells. ALCAN was also secreted from these cells, suggesting that some portion of the molecules was secreted by enzymatic cleavage by, for example, phospholipases. Mutational analysis of ALCAN suggested that the GPI-anchored position was the Ser(216) residue. These findings indicate that ALCAN may be a potential target for cancer diagnosis or therapy.

Stimulation effect of galanin-like peptide (GALP) on luteinizing hormone-releasing hormone-mediated luteinizing hormone (LH) secretion in male rats.

Galanin-like peptide (GALP) is a recently isolated hypothalamic peptide which has sequence homology to galanin and binds to galanin receptors with high affinity. It has been shown that GALP neurons are localized in the arcuate nucleus and that GALP-immunoreactive fibers are in close apposition with LHRH neurons in the medial preoptic area (MPA). In the present study, we found that intracerebroventricular (icv) administration of GALP increased the plasma LH level but did not change the levels of other hormones. Concomitantly, accumulation of c-Fos protein was dramatically increased in the nuclei of LHRH-positive cells in the MPA by icv GALP administration. Furthermore, the GALP-induced plasma LH response was completely abolished by pretreatment with Cetrorelix, a LHRH receptor antagonist. On the other hand, GALP did not affect the release of LH, FSH, TSH, ACTH, GH or PRL directly from dispersed rat pituitary cells in vitro. These results strongly suggest a role for GALP in the control of gonadotropin secretion through a hypothalamic mechanism involving the release of LHRH.

Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor.

Metastasis is a major cause of death in cancer patients and involves a multistep process including detachment of cancer cells from a primary cancer, invasion of surrounding tissue, spread through circulation, re-invasion and proliferation in distant organs. KiSS-1 is a human metastasis suppressor gene, that suppresses metastases of human melanomas and breast carcinomas without affecting tumorigenicity. However, its gene product and functional mechanisms have not been elucidated. Here we show that KiSS-1 (refs 1, 4) encodes a carboxy-terminally amidated peptide with 54 amino-acid residues, which we have isolated from human placenta as the endogenous ligand of an orphan G-protein-coupled receptor (hOT7T175) and have named 'metastin'. Metastin inhibits chemotaxis and invasion of hOT7T175-transfected CHO cells in vitro and attenuates pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. The results suggest possible mechanisms of action for KiSS-1 and a potential new therapeutic approach.

Molecular properties of apelin: tissue distribution and receptor binding.

We analyzed the tissue distribution of apelin mRNA in rats by a quantitative reverse transcription-polymerase chain reaction and that of immunoreactive apelin (ir-apelin) by an enzyme immunoassay (EIA) using a monoclonal antibody. The expression levels of apelin mRNA and ir-apelin seemed to be consistent among tissues: they were highly expressed in the lung and mammary gland. By the combination of gel filtration and EIA, we found that the molecular forms of apelin differ among respective tissues: apelin molecules with sizes close to apelin-36 (long forms) were major components in the lung, testis, and uterus, but both long and short (whose sizes were close to [

Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma.

Airway hyperresponsiveness (AHR), goblet cell metaplasia, and mucus overproduction are important features of bronchial asthma. To elucidate the molecular mechanisms behind these pulmonary pathologies, we examined for genes preferentially expressed in the lungs of a murine model of allergic asthma by using suppression subtractive hybridization (SSH). We identified a gene called gob-5 that had a selective expression pattern in the airway epithelium with AHR. Here, we show that gob-5, a member of the calcium-activated chloride channel family, is a key molecule in the induction of murine asthma. Intratracheal administration of adenovirus-expressing antisense gob-5 RNA into AHR-model mice efficiently suppressed the asthma phenotype, including AHR and mucus overproduction. In contrast, overexpression of gob-5 in airway epithelia by using an adenoviral vector exacerbated the asthma phenotype. Introduction of either gob-5 or hCLCA1, the human counterpart of gob-5, into the human mucoepidermoid cell line NCI-H292 induced mucus production as well as MUC5AC expression. Our results indicated that gob-5 may play a critical role in murine asthma, and its human counterpart hCLCA1 is therefore a potential target for asthma therapy.

Pyloricidins, novel anti-helicobacterpylori antibiotics produced by Bacillus sp. I. Taxonomy, fermentation and biological activity.

Novel anti-Helicobacter pylori antibiotics, pyloricidins A, A1, A2, B, C and D, were discovered in the culture broth of two bacilli strains. Pyloricidins selectively inhibited the growth of H. pylori. Pyloricidin B was efficacious in the treatment of gastric infection caused by H. pylori in Mongolian gerbils and may be promising for cure of H. pylori infection as a single agent.

Pyloricidins, novel anti-Helicobacter pylori antibiotics produced by bacillus sp. II. Isolation and structure elucidation.

Novel anti-Helicobacter pylori antibiotics, pyloricidins A, A1, A2, B, C and D were isolated from Bacillus sp. HC-70 and Bacillus sp. HC-72 by column chromatographies using adsorption and ion exchange resins. Their structures have been elucidated based on spectroscopic and degradation studies and shown to be peptide-like compounds. These compounds contained two unusual amino acids, viz., 5-amino-2,3,4,6-tetrahydroxyhexanoic acid and 3-amino-3-phenylpropionic acid (beta-phenylalanine). The structure-activity relationship studies suggested that 3-(5-amino-2,3,4,6-tetrahydroxyhexanoyl)amino-3-phenylpropionic acid moiety was essential for anti-H. pylori activity.

Analyses for susceptibility of rat anterior pituitary cells to prolactin-releasing peptide.

We validated the effect of prolactin-releasing peptide (PrRP) on prolactin (PRL) secretion from rat anterior pituitary cells in in vitro culture. We found that culture conditions considerably influenced the response of the anterior pituitary cells to PrRP. Longer culture term (4 d) was required to obtain better responses of the anterior pituitary cells to PrRP in comparison to thyrotropin-releasing hormone (TRH). Under the culture conditions employed here, PrRP was comparable to TRH in the potency promoting PRL secretion, and the action of PrRP was very specific for PRL secretion. The susceptibility of the anterior pituitary cells to PrRP varied in female rats depending on the process of reproduction: the cells prepared from lactating rats were the most sensitive to PrRP compared with those from random-cycle and pregnant rats. Because the expression levels of PrRP receptor mRNA in the pituitary varied during the reproductive process, we speculated that the susceptibility of the anterior pituitary cells would reflect cellular changes including the expression level of PrRP receptors. In addition, treatment with estrogen in vivo enhanced the susceptibility of the cultured anterior pituitary cells in male rats. Our results indicate that the susceptibility of the rat anterior pituitary cells to PrRP is regulated by physiological mechanisms.

Discovery of novel, potent, and selective small-molecule CCR5 antagonists as anti-HIV-1 agents: synthesis and biological evaluation of anilide derivatives with a quaternary ammonium moiety.

The search for new small-molecule CCR5 antagonists by high-throughput screening (HTS) of the Takeda chemical library using [(125)I]RANTES and CHO/CCR5 cells led to the discovery of lead compounds (A, B) with a quaternary ammonium or phosphonium moiety, which were synthesized to investigate new MCP-1 receptor antagonists. A series of novel anilide derivatives 1 with a quaternary ammonium moiety were designed, synthesized, and tested for their CCR5 antagonistic activity. Through the optimization of lead compounds, we have found N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6, 7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydr o-2 H-pyran-4-aminium chloride (1r, TAK-779) as a highly potent and selective nonpeptide CCR5 antagonist with a IC(50) value of 1.4 nM in the binding assay. Compound 1r also inhibited the replication of macrophage (M)-tropic HIV-1 (Ba-L strain) in both MAGI-CCR5 cells and PBMCs with EC(50) values of 1.2 and 3.7 nM, respectively. The synthesis and structure-activity relationships of 1r and its related compounds are detailed.

Isolation and identification of melanin-concentrating hormone as the endogenous ligand of the SLC-1 receptor.

Melanin-concentrating hormone (MCH), which is an orexigenic peptide, was isolated and identified as the endogenous ligand of the SLC-1 receptor. We established a CHO cell line expressing the rat SLC-1 receptor to search for its endogenous ligand. The extract of rat whole brain showed inhibition of intracellular forskolin-induced cAMP accumulation in rat SLC-1-expressing CHO cells and was purified. Using HPLC purification, we isolated and identified MCH as the endogenous ligand of the SLC-1 receptor. The authentic MCH demonstrated a dose-dependent inhibitory effect on cAMP accumulation in forskolin-stimulated rat and human SLC-1-expressing CHO cells with an EC(50) value of 0.2 nM for both the rat and human SLC-1 receptors. This is the first description of the functional receptor for MCH.

Synthesis of new peptides with prolactin-releasing activity by a combination of recombinant DNA technology and a cysteine-specific cyanylation reaction.

A newly isolated peptide from bovine hypothalamus with prolactin-releasing activity (prolactin-releasing peptide; PrRP) was synthesized by a combination of recombinant DNA technology and a cysteine-specific cyanylation reaction, together with rat and human homologs. The peptides were expressed in the form of fusion proteins with basic fibroblast growth factor mutein, which were purified by heparin-affinity chromatography. The fusion proteins were cleaved at the cysteine residues of the junction site by cyanylation, followed by treatment with ammonia for C-terminal amidation. Purification of the resulting crude peptides was performed using chromatography on a gel-filtration column, a cation-exchange column, and a reversed-phase column. As an example, about 90 mg of bovine PrRP (bPrRP) was obtained from 201 of culture bloth. The purified b PrRP showed full biological activities in binding to its receptor expressed on CHO cells and releasing arachidonic acid metabolite from the same cells, while the C-terminal acid form of bPrRP had little of these activities. These results indicate that the C-terminal amide structure is very important for expressing biological activity. The peptides obtained here might be very useful for studies on their biological significance and roles in vivo.

Renaturation of recombinant human neurotrophin-3 from inclusion bodies using a suppressor agent of aggregation.

Escherichia coli has been widely used in the production of recombinant proteins. One of the drawbacks inherent in this method is that the proteins produced in the cells often form inactive inclusion bodies. Usually, the inclusion bodies can be separated from other cell components, solubilized by denaturants such as guanidine hydrochloride or urea, and then renatured through a refolding process such as dilution or dialysis. However, it has been shown that biologically active recombinant human neurotrophin-3 cannot be obtained at high yield by this procedure due to aggregation and precipitation of the protein. We applied the refolding process using the aggregation suppressor L-arginine in the renaturation of neurotrophin-3, and obtained biologically active neurotrophin-3 at high yield from the inclusion bodies. Consequently, about 10 mg of purified neurotrophin-3 was prepared from 1 litre of culture broth.

A prolactin-releasing peptide in the brain.

Hypothalamic peptide hormones regulate the secretion of most of the 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.

Studies of polymorphic DNA fingerprinting and lipid pattern of Mycobacterium tuberculosis patient isolates in Japan.

Strain differentiation by DNA restriction fragment length polymorphism (RFLP) has been used mainly for the epidemiological purpose of Mycobacterium tuberculosis infection. In this study, we tried to connect the molecular and phenotypic characteristics of M. tuberculosis patient isolates by comparing the DNA fingerprints obtained by RFLP using IS6110 and lipid patterns using two-dimensional thin-layer chromatography (2-D TLC) with silica gel, since M. tuberculosis has a lipid-rich cell envelope which contributes to the virulence and immunomodulatory properties. We found that 66 isolates of M. tuberculosis from tuberculosis patients showed that the occurrence of IS6110 varied from 1 to 24 copies. The IS6110 patterns were highly variable among isolates. Fifty different RFLP patterns were observed, and 12 RFLP patterns were shared by two or more strains. By computerized analysis of the RFLP patterns of M. tuberculosis patient isolates, we found that 95% of the isolates fell into seven clusters, from A to G, with at least two isolates in each (> 30% similarity). Among the cellular lipids, the phospholipid composition did not differ by strain, whereas the glycolipid pattern differed markedly. Especially, the relative concentration of cord factor and sulfolipid, both of which were known as virulent factors, varied by strain. The fingerprints of some strains showed an association between the DNA and glycolipid patterns, even though some of the same DNA fingerprint strains showed differences in lipid patterns. Among the patient isolates, M. tuberculosis strain 249 possessed a specific glycolipid with 2-O-methyl-L-rhamnose and L-rhamnose, which is rarely found in other strains. This glycolipid showed serological activity against the sera of tuberculosis patients, even if the reactivity was not as strong as trehalose dimycolate. It also showed the inhibition of phagosome-lysosome fusion in macrophages, suggesting involvement with virulence. These results suggest that RFLP analysis using IS6110 is useful for clustering the human isolates of M. tuberculosis, however, for further strain differentiation on virulence, a lipid analysis provides more information.

[A case of Pryce's type I pulmonary sequestration with hemoptysis and bruit].

A 20-year-old man was admitted to our hospital with the complaint of hemoptysis. A bruit was best heard at the lower portion of the left posterior chest. CT scan and MRI revealed clearly the existence of a large aberrant vessel from the descending thoracic aorta. Aorto- and pulmonary arteriography demonstrated that a large systemic artery from the descending thoracic aorta supplied the basilar segments of the left lower lobe, and to which there was no pulmonary artery supply. The pulmonary veins from these segments drained into the left atrium. The properative diagnosis was Pryce's type I pulmonary sequestration. A left lower lobectomy was performed. The aberrant vessel was 15 mm in diameter and was elastic walled. The systemic artery of the basilar segments revealed proliferation of media and obstructive changes. Some discussions were made about hemoptysis and bruit in 23 cases of Pryce's type I pulmonary sequestration in Japan.

Minute hepatocellular carcinoma originating in the caudate lobe with a long-term surviving of 9 years 6 months after surgical resection: a case report.

An extremely rare case of minute hepatocellular carcinoma originating in the caudate lobe who was resected and surviving for 9 years 6 months is presented. A 52 year-old male patient was diagnosed as having a minute hepatocellular carcinoma originating in the caudate lobe by lipiodol computed tomography and celiac angiography. Although the caudate lobectomy was successfully performed, refractory ascites developed for 4 months after surgical procedure. After remission of ascites, the patient is well and surviving for 9 years 6 months without recurrence of hepatocellular carcinoma. This case suggests that early detection and treatment of hepatocellular carcinoma can lead to a long-term survival even when the caudate lobe of the liver is involved.

[Chest wall reconstruction after the resection of primary chest wall tumors].

Between April 1983 and June 1994, we performed chest wall reconstructions on 9 patients (7 males and 2 females) out of 27 all of whom underwent the radical resection for the primary chest wall tumors. Histological diagnosis of tumors included 6 chondrosarcomas, 1 Ewing's sarcoma, 1 plasmacytoma and 1 giant cell tumor. Six lesions were located on the rib and 3 on the sternum. For the materials for reconstruction of the chest wall use a polyethylene plate in 2 patients, a Marlex mesh in 5 patients and a Marlex mesh with a methyl methacrylate utilizing the sandwich method in 2 patients. The polyethylene plate proved to be satisfactory for maintaining stability of the chest wall. However, our experience using this plate showed intensive foreign body reactions, and discovering these we have not use the polyethylene plate. Marlex mesh had an affinity with human tissue and was very easy to use and produced no complications. We use Marlex mesh for flat surfaces on the anterior or posterior chest wall. Marlex mesh and methyl methacrylate utilizing the sandwich method, combined the solidity and shaping of methyl methacrylate with the benefit such as easy fixation and excellent incorporation of the mesh.

Production of human PTH(1-34) via a recombinant DNA technique.

The production of hPTH(1-34) by site specific chemical cleavage of [Cys35]hPTH(1-84) obtained by a biotechnology technique is described. The amino-peptide bond of S-cyanylated cysteine residues in peptides or proteins is specifically cleaved by exposure to an alkaline solution (Stark, G. R. (1977) Methods Enzymol, 47, 129-132). However, when applying this method to [Cys35]hPTH(1-84), we observed many by-products. Formation of these by-products was suppressed using a short reaction in 0.03N NaOH containing 6M urea at low temperature, and hPTH(1-34) was specifically produced. The product was indistinguishable from standard hPTH(1-34) with respect to chemical and biological characterization.