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.

Effect of obesity and insulin on immunity in non-insulin-dependent diabetes mellitus.

OBJECTIVE: To understand the effect of obesity and insulin on immune functions in non-insulin-dependent diabetes mellitus (NIDDM). SUBJECT: Fourteen obese NIDDM (body mass index (BMI)=30.6+/-1.1), seven non-obese NIDDM (BMI=24.2+/-0.5) and five obese non-NIDDM (BMI=28.3+/-0.67). INTERVENTIONS: We first examined the influence of insulin on the proliferation of several human cell lines. Second, we compared several immune functions between obese and non-obese NIDDM, and obese non-NIDDM patients using peripheral blood mononuclear cells. RESULT: Insulin decreased proliferation of T-cell lines but not that of other types of cell lines. Furthermore, obesity augmented the production of IL-1beta which could have cytotoxity against islet beta cells in NIDDM. CONCLUSION: Our data suggested that the pathophysiology of NIDDM could be affected by the change of immunity due to obesity, and the treatment of obesity in NIDDM may be important from an immunological aspect.

Characteristics and distribution of endogenous RFamide-related peptide-1.

We have recently identified RFamide-related peptide (RFRP) gene that would encode three peptides (i.e., RFRP-1, -2, and -3) in human and bovine, and demonstrated that synthetic RFRP-1 and -3 act as specific agonists for a G protein-coupled receptor OT7T022. However, molecular characteristics and tissue distribution of endogenous RFRPs have not been determined yet. In this study, we prepared a monoclonal antibody for the C-terminal portion of rat RFRP-1. As this antibody could recognize a consensus sequence among the C-terminal portions of rat, human, and bovine RFRP-1, we purified endogenous RFRP-1 from bovine hypothalamus on the basis of immunoreactivity to the antibody. The purified bovine endogenous RFRP-1 was found to have 35-amino-acid length that corresponds to 37-amino-acid length in human and rat. We subsequently constructed a sandwich enzyme immunoassay using the monoclonal antibody and a polyclonal antibody for the N-terminal portion of rat RFRP-1, and analyzed the tissue distribution of endogenous RFRP-1 in rats. Significant levels of RFRP-1 were detected only in the central nervous system, and the highest concentration of RFRP-1 was detected in the hypothalamus. RFRP-1-positive nerve cells were detected in the rat hypothalamus by immunohistochemical analyses using the monoclonal antibody. In culture, RFRP-1 lowered cAMP production in Chinese hamster ovary cells expressing OT7T022 and it was abolished by pre-treatment with pertussis toxin, suggesting that OT7T022 couples G(i)/G(o) in the signal transduction pathway.

Solution-phase automated synthesis of tripeptide derivatives.

An improved general method for automated synthesis of tripeptides was developed, in which methanesulfonic acid (MSA) was used in place of trifluoroacetic acid (TFA), thus making it possible to avoid, 1) corrosion of the apparatus by strong acid vapor, 2) formation of emulsions, and 3) use of the restricted solvent, dichloromethane. As an application of the automated synthesis apparatus, 216 fragment tripeptide derivatives were synthesized systematically using the MSA method, in excellent yield and with increased efficiency.

Application of an automated synthesis suite to parallel solution-phase peptide synthesis.

An in-house developed automated synthesis suite was used to prepare a library of 72 tetrapeptide derivatives, the starting materials for pharmaceutically attractive pentapeptides, employing a convergent strategy. An initial set of 18 dipeptides were synthesized on a large-scale (100-1000 g) using automated synthesis workstations, and then 72 tetrapeptides were synthesized on a medium scale (5-10 g) using an automated system. Each di- or tetrapeptide was prepared in a single operating cycle using a modified methanesulfonic acid method, then a sub-library of 56 pentapeptides were synthesized in parallel, on a small-scale (100 mg-1 g) using a robotic workstation.

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.

Distribution of galanin-like peptide in the rat brain.

Galanin-like peptide (GALP) is a novel galanin-like peptide isolated from the porcine hypothalamus. To determine the distribution of GALP in the rat brain, we performed immunohistochemical studies using a monoclonal antibody toward the N-terminal sequence of GALP. GALP-immunoreactive neuronal cell bodies were observed only in the arcuate nucleus (Arc), which was further confirmed by in situ hybridization studies using digoxigenin-labeled antisense GALP riboprobe. Additional immunostained cells were found in the median eminence and infundibular stalk. The GALP neurons found in the Arc were further characterized by double label immunohistochemistry. More than 85% of the GALP neurons were immunostained with leptin receptor antibody. However, the GALP neurons and fibers found in the Arc were not labeled with alpha-MSH, somatostatin, neuropeptide Y, agouti-related protein, or galanin antibodies, indicating that GALP is found in neurons other than these known Arc neurons. Dense staining of GALP-containing fibers was found in the anterior parvicellular part of the paraventricular hypothalamic nucleus, in the ventral part of the lateral septal nucleus, and in the bed nucleus of the stria terminalis. Relatively dense staining was noted in the medial preoptic area (MPA), and weak staining was noted in the periventricular hypothalamic nucleus. Detailed double labeling studies in the paraventricular hypothalamic nucleus demonstrated that GALP-containing fibers converged in a more rostral direction than did agouti-related protein-containing fibers. Furthermore, GALP-immunoreactive fibers were in close apposition with GnRH-immunoreactive fibers in the MPA and bed nucleus of the stria terminalis, and about 6% of GnRH-positive neurons in the MPA showed close contact with the GALP-immunoreactive fibers. Our findings indicate that GALP neurons, as leptin-responsive neurons, may participate in the regulation of feeding behavior and/or reproductive functions.

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.

Differential expression of two cytochrome P450s involved in the biosynthesis of flavones and anthocyanins in chemo-varietal forms of Perilla frutescens.

In Perilla frutescens, there are two varietal forms of anthocyanin accumulation, i.e. red and green forms. The cDNA clones encoding flavone synthase II (FSII) and flavonoid 3'-hydroxylase (F3'H), two cytochrome P450s that are involved in the biosynthesis of flavones and anthocyanins, were isolated from P. frutescens. The FSII cDNA encoded a 57.1 kDa protein designated as CYP93B6, and the F3'H cDNA encoded 57.5 kDa protein designated as CYP75B4. Recombinant CYP93B6 expressed in yeast converted flavanones to flavones with K(m) values of 8.8-11.9 microM. Recombinant CYP75B4 catalyzed 3'-hydroxylation of flavanones to the corresponding compounds with K(m) values of 18-20 microM. The CYP93B6 transcript accumulated to an equal level in leaves of both red and green forms of P. frutescens, in agreement with the accumulation pattern of flavones in the leaves. However, the CYP75B4 transcript was predominantly expressed in the red form of P. frutescens, and its expression was induced by light in conjunction with other transcripts of biosynthetic enzymes of anthocyanin. These results indicate that gene expression of a set of anthocyanin biosynthetic enzymes including F3'H is regulated coordinately only in the red form of P. frutescens but not in the green form, whilst FSII gene expression is controlled in a similar manner in red and green forms of P. frutescens.

Apelin, the natural ligand of the orphan seven-transmembrane receptor APJ, inhibits human immunodeficiency virus type 1 entry.

In addition to the CCR5 and CXCR4 chemokine receptors, a subset of primary human immunodeficiency virus type 1 (HIV-1) isolates can also use the seven-transmembrane-domain receptor APJ as a coreceptor. A previously identified ligand of APJ, apelin, specifically inhibited the entry of primary T-tropic and dualtropic HIV-1 isolates from different clades into cells expressing CD4 and APJ. Analysis of apelin analogues demonstrated that potent and specific antiviral activity was retained by a 13-residue, arginine-rich peptide. Antiviral potency was influenced by the integrity of methionine 75, which contributes to APJ-binding affinity, and by the retention of apelin residues 63 to 65. These studies demonstrate the ability of a small peptide ligand to block the function of APJ as an HIV-1 coreceptor, identify apelin sequences important for the inhibition, and provide new reagents for the investigation of the significance of APJ to HIV-1 infection and pathogenesis.

New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals.

Only a few RFamide peptides have been identified in mammals, although they have been abundantly found in invertebrates. Here we report the identification of a human gene that encodes at least three RFamide-related peptides, hRFRP-1-3. Cells transfected with a seven-transmembrane-domain receptor, OT7T022, specifically respond to synthetic hRFRP-1 and hRFRP-3 but not to hRFRP-2. RFRP and OT7T022 mRNAs are expressed in particular regions of the rat hypothalamus, and intracerebroventricular administration of hRFRP-1 increases prolactin secretion in rats. Our results indicate that a variety of RFamide-related peptides may exist and function in mammals.

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.

Stimulation of corticotropin-releasing hormone-mediated adrenocorticotropin secretion by central administration of prolactin-releasing peptide in rats.

Prolactin-releasing peptide (PrRP) is a recently isolated hypothalamic peptide which is an endogenous ligand to an orphan receptor. We previously demonstrated that PrRP neurons are widely distributed throughout the rat brain and suggested that PrRP may have important functions in the central nervous system. To analyze the function of PrRP, we studied the effect of intracerebroventricular (i.c.v.) PrRP administration on c-Fos protein accumulation in the rat brain. The results clearly indicated that c-Fos protein accumulation was dramatically increased in the nuclei of corticotropin-releasing hormone (CRH)-positive parvocellular neurosecretory cells in the paraventricular nucleus (PVN). We also demonstrated synapse-like contact between PrRP neurons and CRH cell bodies in the PVN, which suggests that PrRP31 has some effect on CRH secretion. We therefore investigated the effect of i.c.v. administration of PrRP31 on the CRH-mediated increase in adrenocorticotropin (ACTH) levels, and found that plasma ACTH levels were indeed increased by i.c.v. PrRP31. In addition, animals pre-treated with intravenous alpha-helical CRH, a potent CRH antagonist, showed attenuated plasma ACTH responses after i.c.v. PrRP31 administration. These results strongly suggest that PrRP affects the hypothalamic-pituitary-adrenal axis.

Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin.

We have recently identified apelin as the endogenous ligand for human APJ. In rats, the highest expression of APJ mRNA was detected in the lung, suggesting that APJ and its ligand play an important role in the pulmonary system. When apelin-36 and its pyroglutamylated C-terminal peptide, [