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.

Evaluation of extremely shallow vertical subsurface flow constructed wetland for nutrient removal.

Mesocosm-scale vertical subsurface flow constructed wetlands (SSF, 0.5 m length, 0.3 m width) with different reed-bed thickness, including standard SSF (SD, 0.6 m deep), shallow SSF (S, 0.3 m deep) and extremely shallow SSF (ES, 0.075 m deep) were set up at sewage treatment plant and their nutrient removal efficiencies from the sewage plant effluent were compared under three hydraulic loading rate (HLR) conditions of 0.15, 0.45 and 0.75 m(3) m(-2) d(-1). A very interesting characteristics was found for the extremely shallow SSF, in which a high nitrogen removal efficiency was obtained despite the effective hydraulic retention time was only 1/8 times as long as the standard SSF. The results of kinetic analysis confirmed that the high volumetric nitrogen removal efficiency observed in the extremely shallow SSF did not depend on high response against the water temperature but on much higher basic nitrogen removal activity compared with other SSF. The phosphorus removal depending on the adsorption to sand in the reed-bed filter was, however, the lowest in the extremely shallow SSF although the volumetric removal efficiency was much higher compared with other SSF. Results of morphological analysis of rhizosphere collected from respective reed-bed suggested that the extremely shallow SSF lead to a very high-density rhizosphere, resulting in a high basic nitrogen removal activity and volumetric phosphorus removal efficiency.

Differences in adsorption mechanisms of heavy metal by two different plant biomasses: reed and brown seaweed.

The adsorption of Pb(II) by two different biomaterials, reed (Phragmites australis) and brown seaweed (Sargassum horneri) biomass pretreated with CaCl(2), were compared in an attempt to explain the differences in adsorption performance between the two biosorbents. A very interesting characteristic was found in their individual adsorption performances; the Pb(II) adsorption capacity of brown seaweed (Q(max)=0.45 mmol/g) was much higher than that of reed (Q(max)=0.05 mmol/g), but its adsorption affinity (b=112 L/mmol) was much lower compared with that of reed (b=471 L/mmol). To elucidate the mechanism, the elemental components, ion exchange phenomenon and roles of functional groups of these two biosorbents were compared. The higher Pb(II) adsorption by brown seaweed could be due to its richness in total functional groups and calcium contents on its surface. In contrast, the functional complexity, higher zeta potential and pK(a) value (deprotonation state) of reed are believed to lead to its high adsorption affinity.

Marine macroalga Sargassum horneri as biosorbent for heavy metal removal: roles of calcium in ion exchange mechanism.

Brown seaweed Sargassum horneri, a troublesome biomass scattered along the seashore, was utilized as a biosorbent for Pb(II) removal from aqueous solutions. The Pb(II) adsorption by brown seaweed was enhanced by pretreatment with CaCl(2), and the Langmuir adsorption isotherm equation showed a maximum capacity of a Q(max) of 0.696 mmol/g and a b value of 94.33 L/mmol. Results obtained from the mass-balance equation derived from the simulation model of the Langmuir adsorption isotherm suggested that the adsorption performance of brown seaweed biosorbent was sufficient to reduce the concentration of Pb(II) to meet the range of WHO guideline. The mechanism, as elucidated using pH monitoring, adsorption rate and ion exchange model, involved the rapid pH change of metal solutions that led to high reaction rate and Pb(II) uptake in the first 30 min of the biosorption process. The energy X-ray analysis's result confirmed the sharp reduction of calcium content in the biosorbent after Pb(II) adsorption. The amount of calcium ions released from the biosorbent was about 1.5 times the amount of Pb(II) adsorbed and proved the role of calcium in the ion exchange mechanism. These adsorption equilibrium and mechanistic studies provide useful information for system design and performance prediction of biosorption processes.

Pb(II) biosorption on reed biosorbent derived from wetland: effect of pretreatment on functional groups.

Reed biomass harvested from wetland constructed for water purification was modified into a biosorbent for Pb(ll) removal from aqueous solution. The enhancement of Pb(ll) adsorption by reed biosorbent depended not only on the types of reagent used for pretreatment, but also on the pH during the pretreatment process. The mechanisms, as elucidated using relational data obtained from Boehm titration, Fisher esterification and FTIR, involved the conversion of carboxylic groups into carboxylate groups, and proved the role of the carboxylate group, which occupied more than 80% in binding Pb(ll). The Langmuir sorption isotherm of Pb(ll) by R-NaOH-12 showed QO, and b values of 0.082 mmol/g and 312.5 g/mmol, suggesting enough adsorption performance to reduce the concentration of Pb(ll) to meet the range of WHO guidelines. The salinity of aqueous solution containing NaH2PO4 and NaN03 promoted the adsorption of Pb(ll), while NaCl and Na2SO4 suppressed the adsorption capacity of Pb(ll). The adsorption mechanism of reed biosorbent provides valuable insight on the pretreatment effects and the advantages of utilizing this plant as biosorbent for Pb(ll) and other heavy metals.

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 [

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.

Acute cocaine effects on stereotype and defense: an ethoexperimental approach.

Cocaine administration to laboratory animals may produce locomotor hyperactivity and stereotypies that include sniffing and rearing, in addition to anxiety-like effects. A time-sampling study of the effects of 3, 10 or 30 mg/kg cocaine (i.p.) over time following injection indicated early enhancement of locomotion and crouching, with the latter most increased in low- and intermediate-dose cocaine groups, with increased rearing and standing during the second hour of the test period. Additional analyses at 30-60 min post-injection suggested qualitative changes in rearing, with high dose animals showing more, but shorter, rears, and a higher frequency of sniffing. The high dose cocaine enhancement of sniffing was strongly associated with rear and stand behaviors, but also occurred while the animal was crouching. This pattern of changes, with initial crouching/freezing and locomotion (flight?), followed by rearing, standing, and sniffing behaviors similar to those seen in risk assessment suggests that cocaine, particularly at high doses, may elicit defense. An additional study using only saline or the high (30 mg/kg) dose indicated that cocaine produced more sniffing regardless of the direction from which the air stream entered the test cage (i.e. top or bottom). However, cocaine animals oriented their sniffing behaviors toward the incoming air, with reliably more sniffs up in cages with the air stream entering from the top, and more sniffs down, when the air stream entered through a wire mesh cage bottom. Controls showed the same pattern, but their sniff orientation differences were not reliable. These results indicate that the sniffing that follows acute high dose cocaine administration is appropriately oriented toward relevant environmental stimuli, a factor disconsonant with the interpretation of sniffing as a stereotypical behavior, but one that is in agreement with the view that it may reflect a risk assessment component of the defense pattern.

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.

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.

Human interferon-gamma lacking 23 COOH-terminal amino acids is biologically active.

We constructed five mutated cDNAs encoding human interferon-gamma (IFN-gamma) derivatives lacking 19-23 COOH-terminal residues and expressed them in Escherichia coli. All the derivatives were purified to homogeneity. They showed substantially the same order of antiviral activity in vitro as the intact molecule, and behaved as a dimer. The far- and near-UV circular dichroism spectra of the derivatives were quite similar to those of the intact one. These results indicate that the 23 COOH-terminal amino acids at least are not essential for achieving the full antiviral activity and constructing the higher structure of human IFN-gamma.

Amino acid sequence of heat-stable enterotoxin produced by Vibrio cholerae non-01.

The amino acid sequence of heat-stable enterotoxin, produced by Vibrio cholerae non-01 and isolated from its culture supernatant, was determined by both Edman degradation of native and reductively carboxymethylated enterotoxin and also a combination of fast atom bombardment mass spectrometry and carboxypeptidase Y digestion of native enterotoxin to be as follows: Ile-Asp-Cys-Cys-Glu-Ile-Cys-Cys-Asn-Pro-Ala-Cys-Phe-Gly-Cys-Leu-Asn. This sequence is very similar, but not identical, to those of heat-stable enterotoxins produced by enterotoxigenic Escherichia coli and Yersinia enterocolitica.

Cloning of rat brain protein kinase C complementary DNA.

Four peptides derived from rat brain protein kinase C were partially sequenced. Using synthetic oligonucleotides deduced from the amino acid sequences as probes, a clone of complementary DNA (cDNA) was isolated from a cDNA library prepared from the same tissue. The nucleotide sequence of this cDNA clone revealed the primary structure of the carboxyl-terminal region as having 224 amino acids, with significant sequence homology with cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

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.

Tissue distribution of prolactin-releasing peptide (PrRP) and its receptor.

Prolactin-releasing peptide (PrRP) is a novel bioactive peptide, originally isolated from bovine hypothalamus by utilizing an orphan seven-transmembrane-domain receptor expressed in the human pituitary gland. In this paper, we analyzed the tissue distribution of rat and human PrRP and their receptor mRNAs by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Northern blotting. In RT-PCR analysis, rat PrRP receptor mRNA was detected in the central nervous system, and the highest expression was detected in the pituitary gland. In addition, in situ hybridization revealed that rat PrRP receptor mRNA was highly expressed in the anterior lobe of the pituitary. On the other hand, rat PrRP mRNA was most abundantly expressed in the medulla oblongata, while significant levels of expression were widely detected in other tissues. In Northern blot analyses, human PrRP receptor mRNA was detected only in the pituitary gland among tissues examined. Human PrRP mRNA was detected in the medulla oblongata and in the pancreas. In contrast to the pattern of mRNA expression, the highest content of bioactive PrRP was found in the hypothalamus rather than the medulla oblongata in the rat brain, indicating that PrRP mRNA does not always parallel with mature PrRP in tissue distribution. The wide distribution of PrRP and its receptor suggests that they have various functions not only in the pituitary gland but also in the other tissues.

Topical application of neurotrophin-3 attenuates ischemic brain injury after transient middle cerebral artery occlusion in rats.

In order to examine the effect of neurotrophin-3 (NT-3) on ischemic brain injury, NT-3 was topically applied to brain surface just after 90 min of middle cerebral artery occlusion (MCAO) in rats. NT-3 significantly reduced the infarct size at 24 h of reperfusion. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick labeling (TUNEL) staining and immunohistochemical study for caspase-3 and heat shock protein 72 (HSP72) showed that NT-3 treatment decreased the number of cells with DNA fragmentation and caspase-3 and HSP72 expressions. These data suggest that NT-3 protects neuronal cells from ischemic injury, and it is possibly associated with inhibition of DNA fragmentation.

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.

Central administration of prolactin-releasing peptide stimulates oxytocin release in rats.

The prolactin-releasing peptide (PrRP) is a novel hypothalamic peptide that has been purified as a ligand of an orphan receptor which is expressed in pituitary cells, and is known to stimulate prolactin release both in vitro and in vivo. We previously determined the immunocytochemical localization of PrRP neurons in the rat brain and our results suggest that PrRP takes part in a variety of brain functions. Additionally, in rats we have demonstrated the synaptic contact of PrRP neurons with oxytocin cell bodies in the paraventricular hypothalamic nucleus (PVH) and supraoptic nucleus (SON). This observation indicates that PrRP may regulate oxytocin secretion. In the present study, we performed intra-cerebroventricular administration of PrRP to conscious rats, and examined the effect of PrRP on the plasma levels of oxytocin and vasopressin. Our results show that central administration of PrRP increased the plasma oxytocin and vasopressin levels in female rats, but in male rats only oxytocin was increased. These results suggest that the PrRP acts as a neuro-modulator of the function of magnocellular neurons, especially oxytocin neurons, in the brain.

Reduction of tyrosine kinase B and tyrosine kinase C inductions by treatment with neurotrophin-3 after transient middle cerebral artery occlusion in rat.

Infarct volume and immunoreactivities for trkB and trkC in rat brain were compared at 24 h after 90 min of transient middle cerebral artery occlusion (MCAO) between animal groups with or without neurotrophin-3 (NT-3, 10 microg/250 g animal). Treatment of rat brain with topical application of NT-3 significantly reduced infarct volume (P = 0.02) and trkB and trkC inductions. These data suggest that NT-3 reduced the ischemic injury along with the reduction of trkB and trkC inductions.

A novel procedure for the preparation of biologically active recombinant peptides using a cyanylation reaction.

A cysteine specific cleavage reaction was used for the preparation of biologically active peptides from recombinant fusion proteins. The fusion protein through cysteine was prepared by a recombinant DNA technology and then treated with cyanylating reagents such as 2-nitro-5-thiocyanatobenzoic acid (NTCB) and 1-cyano-4-(dimethylamino) pyridinium tetrafluoroborate (DMAP-CN) to release the desired product. As an example, we have selected a glucagon-like peptide 1 (7-37) (termed insulinotropin). We constructed an expression vector for a fusion protein in which insulinotropin and human basic fibroblast growth factor (hbFGF) mutein (abbreviated as CS 23) are connected by cysteine and then expressed it in Escherichia coli cells. The fusion protein, after refolding, was purified by heparin affinity chromatography, since CS23 has a strong affinity for heparin. The affinity-purified fusion protein was treated with NTCB or DMAP-CN to give crude insulinotropin, which was then purified by reversed phase (rp) high-performance liquid chromatography (HPLC). From various criteria such as amino acid analysis, amino acid sequence and the biological activity, the purified material obtained was found to be methionylated insulinotropin (Met-insulinotropin) with full activity. The specificity and simplicity of the present method make it versatile and convenient for the preparation of biologically active peptides.