Response to ADP-ribose by activation of TRPM2 in the CRI-G1 insulinoma cell line.

The response to intracellular ADP-ribose in the rat CRI-G1 insulinoma cell line was studied using a patch-clamp method. Dialysis of ADP-ribose into cells induced a response in a dose-dependent manner. The reversal potentials in various solutions showed that the ADP-ribose-gated channel was a Ca2+-permeable nonselective cation channel. In inside-out recordings, ADP-ribose and b-NAD induced responses in the same patch. The single-channel current-voltage relationships for ADP-ribose- and b-NAD-induced responses were almost identical, indicating that ADP-ribose and b-NAD activated the same channel. The physiological properties of the ADP-ribose-gated channel are similar to those we reported previously for the cloned transient receptor potential channel TRPM2. Moreover, RT-PCR analysis showed that TRPM2 was abundantly expressed in CRI-G1 cells, suggesting that the ADP-ribose-gated channel represents the native TRPM2 channel in CRI-G1 cells. These results suggest that ADP-ribose can be an endogenous modulator of Ca2+ influx through the TRPM2 channel into CRI-G1 cells.

Fixation of osteochondral lesions of the talus using cortical bone pegs.

We have treated osteochondral lesions of the talus using cortical bone pegs. We examined 27 ankles (27 patients) after a mean follow-up of 7.0 years (2 to 18.8). The mean age of the patients was 27.8 years (12 to 62). An unstable osteochondral fragment or osteosclerotic changes in the bed of the talus were regarded as indications for the procedure. The clinical results were good in 24 ankles (89%) and fair in three (11%); none had a poor result. There was also radiological improvement in 24 ankles. Repair of the articular surface and stability of the lesion can be achieved even in unstable chronic lesions.

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.

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 [

Prolactin-releasing peptide as a novel stress mediator in the central nervous system.

A1/A2 noradrenergic neurons in the medulla oblongata are well known to mediate stress signals in the central nervous system. Stress activates A1/A2 noradrenergic neurons, and then noradrenaline (NA) stimulates ACTH secretion through hypothalamic CRH. On the other hand, PRL-releasing peptide (PrRP) was recently isolated and was found to be produced by some A1/A2 neurons and the dorsomedial hypothalamic nucleus. We previously demonstrated that PrRP neurons make synapse-like contact with hypothalamic CRH neurons. In fact, we demonstrated that the central administration of PrRP stimulates CRH-mediated ACTH secretion. Furthermore, it has been reported that PrRP neurons in A1/A2 cell groups are colocalized with tyrosine hydroxylase (TH), which is known as the marker enzyme of catecholaminergic neurons. These data strongly suggest that PrRP is related to stress-responsive signal transduction, and PrRP and NA cooperatively modulate the hypothalamo-pituitary-adrenal axis. We therefore examined the effect of water immersion-restraint stress on c-Fos protein accumulation in PrRP- and TH-immunoreactive neurons. The synergistic effects of PrRP and NA on plasma ACTH elevation were also examined. The results clearly showed that c-Fos protein accumulation dramatically increased in the nuclei of A1/A2 and dorsomedial hypothalamic nucleus PrRP neurons. In addition, it was revealed that c-Fos protein was specifically expressed in the PrRP/TH double positive cells in the A1/A2 cell groups. We also demonstrated that the central administration of PrRP and NA in combination at subactive (noneffective) doses clearly induced plasma ACTH elevation. Here we report that PrRP is a novel and important mediator of the hypothalamo-pituitary-adrenal axis for the stress response.

Conformation of a peptide ligand bound to its G-protein coupled receptor.

Many peptide hormones elicit a wide array of physiological effects by binding to G-protein coupled receptors. We have determined the conformation of pituitary adenylate cyclase activating polypeptide, PACAP(1--21)NH(2), bound to a PACAP-specific receptor by NMR spectroscopy. Residues 3--7 form a unique beta-coil structure that is preceded by an N-terminal extended tail. This beta-coil creates a patch of hydrophobic residues that is important for receptor binding. In contrast, the C-terminal region (residues 8--21) forms an alpha-helix, similar to that in the micelle-bound PACAP. Thus, the conformational difference between PACAP in the receptor-bound and the micelle-bound states is limited to the N-terminal seven residues. This observation is consistent with the two-step ligand transportation model in which PACAP first binds to the membrane nonspecifically and then diffuses two-dimensionally in search of its receptor; a conformational change at the N-terminal region then allows specific interactions between the ligand and the receptor.

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.

Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum.

By using a strategy that we have developed to search for the ligands of orphan seven-transmembrane-domain receptors [S. Hinuma et al., Nature 393 (1998) 272-276], we have recently identified a natural ligand, apelin, for the orphan 7TMR, APJ [K. Tatemoto et al., Biochem. Biophys. Res. Commun. 251 (1998) 471-476]. In this paper, we isolated rat and mouse apelin cDNAs, and analyzed the tissue distribution of apelin mRNA in rats. Although apelin mRNA was widely detected in a variety of tissues, the highest expression of apelin mRNA was detected in the mammary gland of pregnant rats. In the mammary gland, biologically active apelin and its mRNA considerably increased during pregnancy and lactation, and reached a maximal level around parturition. Moreover, a large amount of apelin (14-93 pmol/ml) was found to be secreted in the bovine colostrum, and it was still detectable even in commercial bovine milk. Since apelin partially suppressed cytokine production by mouse spleen cells in response to T cell receptor/CD3 cross-linking, the oral intake of apelin in the colostrum and milk might modulate immune responses in neonates.

Long-term results of Watson-Jones tenodesis of the ankle. Clinical and radiographic findings after ten to eighteen years of follow-up.

Thirty-seven chronically unstable ankles in thirty-six patients were operated on with use of a Watson-Jones tenodesis. Thirty-four ankles (thirty-three patients) were followed for a mean duration of thirteen years and eight months (range, ten to eighteen years) after the operation. There were nine male and twenty-four female patients. The mean age of the patients was thirty-one years (range, fourteen to fifty-seven years) at the time of the operation and forty-four years (range, twenty-eight to seventy years) at the time of the latest follow-up. At the time of the most recent follow-up evaluation, twenty-seven patients (twenty-eight ankles) were examined directly by one of us and twenty-five patients (twenty-six ankles) also were evaluated radiographically. The other six patients were interviewed, with use of a questionnaire, by telephone. Of the thirty-four ankles, nineteen had an excellent result (grade 1), eleven had a good result (grade 2), three had a fair result (grade 3), and one had a poor result (grade 4) according to the rating system of Good et al. The mean score (and standard deviation) on the ankle-hindfoot scale of the American Orthopaedic Foot and Ankle Society for the twenty-eight ankles that were examined directly by one of us was 90 +/- 9.3 points (range, 68 to 100 points). Progression of an exostosis at the edge of the joint was detected in eighteen (69 percent) of the twenty-six ankles that were examined radiographically, but narrowing of the joint space was not seen in any ankle. No relationship was detected between the clinical results and radiographic osteoarthrotic changes or the duration of follow-up. The results did not deteriorate over the long term.

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.

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.

Expression, purification, and reconstitution of receptor for pituitary adenylate cyclase-activating polypeptide. large-scale purification of a functionally active G protein-coupled receptor produced in Sf9 insect cells.

Human pituitary adenylate cyclase-activating polypeptide (PACAP) receptor was expressed in Sf9 insect cells and Chinese hamster ovary (CHO) cells. The recombinant receptor in Sf9 cell membranes had low affinity for 125I-PACAP27 (Kd = 155.3 pM) and was insensitive to guanosine 5'-O-3-thiotriphosphate (GTPgammaS), whereas the receptor in CHO membranes had a high affinity (Kd = 44.4 pM) and was GTPgammaS sensitive. The receptor in Sf9 membranes was converted to a high affinity state (Kd = 20-40 pM) following solubilization with digitonin. A large quantity (2 mg from 8 liters of insect cells) of the purified PACAP receptors (Bmax = 23.9 nmol/mg of protein) were obtained in a digitonin-induced high affinity state (Kd = 17.3 pM) using biotinylated ligand affinity chromatography. The apparent molecular weight of the purified receptor (Mr = 48,000) was smaller than that of the receptor from CHO cells (Mr = 58,000) due to differences in asparagine-linked sugar chains. The purified receptor reverted to a low affinity state (Kd = 182.6 pM) upon reconstitution into lipid vesicles, however, the receptor reconstituted with Gs protein had a high affinity (Kd = 40.2 pM) and was GTPgammaS sensitive. [35S]GTPgammaS binding to the reconstituted Gs protein was enhanced by PACAP27 and PACAP38 (EC50 = 42.5 and 9.4 pM, respectively) but not by antagonist PACAP(6-38), indicating that the purified receptor was functionally active.

Identification and characterization of a novel human cortistatin-like peptide.

Expressed sequence tags (ESTs) that showed significant homology to rat cortistatin (CST) were found in a human fetal brain cDNA library. A protein coded by the cDNA showed 55% identity to rat preprocortistatin in amino acid. Similarly in the generation of mature peptides from rat preprocortistatin, it was expected that cleavage at dibasic amino acids in the C-terminal portion of the coded protein might produce at least two different sizes of mature peptides with 29 and 17 amino acid residues, respectively. We chemically synthesized the predicted mature peptide with 17 amino acid residues (hCS-17) and examined its biological activities. It bound to all human somatostatin receptor (SSTR) subtypes in almost the same manner as rat CST-14. It also inhibited cAMP production induced by forskolin through SSTRs. Administration of hCS-17 to the cerebral ventricle showed flattening of cortical and hippocampal electroencephalograms in rats. These results indicate that a bioactive peptide encoded by the cDNA is a human counterpart corresponding to rat CST.

Application of a unique automated synthesis system for solution-phase peptide synthesis.

An automated synthesis system, which is suitable for repetitive syntheses using similar reaction procedures, was used to synthesize systematically a library of all possible dipeptides (25) and tripeptides (125) from 5 protected amino acids. The apparatus has also been applied to the automated synthesis of 10 fragment tripeptide derivatives that are constituents of the hormone PACAP-27. The measured molecular optical rotation values of the library of 125 tripeptides were found to correlate well with calculated values obtained by summation of the molecular optical rotation values for the constituent amino acids.

Long amyloid beta-protein secreted from wild-type human neuroblastoma IMR-32 cells.

The 39- to 43-amino acid amyloid beta-protein (A beta) is deposited as amyloid in Alzheimer's disease. Recent studies have suggested that short A beta (A beta 39 or A beta 40) and long A beta (A beta 42 or A beta 43) play different roles in Alzheimer-type pathology. However, little attempt has been made to investigate the cellular mechanisms underlying the generation of short and long A beta individually. In the present report, we first measured the amount of short and long A beta that are secreted from wild-type human and rodent cells with neuron- or glia-like properties using highly sensitive sandwich-ELISAs that discriminate long A beta from short A beta. The results showed that long A beta secreted by all cells constitutes approximately 10% of the total A beta. To identify the molecular species of long A beta, we next isolated the A beta species secreted from human neuroblastoma IMR-32 cells by affinity chromatography, gel-filtration HPLC, and reverse-phase HPLC. Mass spectrometric analysis demonstrated unequivocally that IMR-32 cells produce A beta 1-42 together with A beta 1-37, A beta 1-38, A beta 1-39, and most predominantly, A beta 1-40. Finally, to investigate the cellular mechanisms that generate A beta 1-42, we studied the effects of brefeldin A and monensin on the production of A beta 1-40 and A beta 1-42 in IMR-32 cells. These reagents reduced the production of both A beta 1-40 and A beta 1-42 simultaneously in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis of human endothelins in transformants expressing cDNAs for human prepro-endothelins.

To understand the biosynthesis of the human ET family, three kinds of cloned cDNA for human prepro-endothelin-1 (prepro-ET-1), prepro-ET-2, and prepro-ET-3 were stably expressed in Chinese hamster ovary cells (CHO-K1). Immunoreactive (ir-) ET polypeptides in the culture media of the transformants were purified by reverse-phase high-performance liquid chromatography (HPLC) coupled with sandwich-type enzyme immunoassay (EIA). Amino acid sequencing and FAB mass spectrometry of the purified ir-ET polypeptides revealed the presence of human big ET-1 (1-38), big ET-2 (1-38), and big ET-3 (1-41)NH2 as intermediate forms. These results directly revealed the biosynthetic pathways of three human ETs at the peptide level.