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.

Enhanced nitrogen removal using C/N load adjustment and real-time control strategy in sequencing batch reactors for swine wastewater treatment.

The laboratory-scale sequencing batch reactor (SBR) was used to study the effectiveness of an integrated strategy of real time control with C/N ratio adjustment for practical swine wastewater treatment. Swine waste was used as the external carbon source for continuous treatment in the SBR reactors. Oxidation-reduction potential and pH were used as parameters to control the continuous denitrification and nitrification process, respectively. A constant effluent quality could be obtained, despite drastic variations in the characteristics of influent wastewater. Also, a relatively complete removal of nutrients was always ensured, since the optimum quantity of the external carbon source could be provided for complete denitrification, and a flexible hydraulic retention time was achieved by the successful real-time control strategy. The average removal efficiencies of total organic carbon and nitrogen were over 94% and 95%, respectively.

Effect of C/N ratio on nitrous oxide emission from swine wastewater treatment process.

To evaluate control parameters for nitrous oxide (N2O) emissions in the swine wastewater treatment process, the N2O emission was compared in the activated sludge from SBRs acclimated in different carbon/nitrogen (C/N) ratios. N2O emission from a denitrification phase was very strongly dependent on C/N ratio of swine wastewater, and the total N2O emission in the operating condition of BOD5/TN ratio of 2.6 was approximately 270 times greater than that in BOD5/TN ratio of 4.5. However, the effect of C/N ratio on N2O emission amount from nitrification was not significant in an oxic phase study. It is considered that stabilization of the C/N ratio through optimal solid-liquid separation of slurry or use of an external carbon source is indispensable for effective N2O emission control from nitrogen removal process of swine wastewater.

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.

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.

Cloning of a novel G protein-coupled receptor, SLT, a subtype of the melanin-concentrating hormone receptor.

A DNA fragment encoding an amino acid sequence possessing common features to the G protein-coupled receptor (GPCR) superfamily was found in the human genomic sequence, and from this information, the full-length cDNA of a novel GPCR, designated SLT, was cloned from the human hippocampus cDNA library. SLT showed the highest homology to the melanin-concentrating hormone (MCH) receptor, SLC-1 (31.5% identity), and to a lesser extent, to the somatostatin (SST) receptor subtypes. MCH exhibited agonistic behavior when applied to the SLT-expressing CHO cells at subnanomolar doses whereas more than 200 known peptides, including SST and cortistatin, did not. These results indicated that MCH is the cognate ligand of the SLT receptor and that this newly cloned GPCR is the second subtype of the MCH receptor. Quantitative polymerase chain reaction analysis of the SLT gene expression in human tissues showed that the SLT receptor is expressed mainly in brain areas including the cerebral cortex, amygdala, hippocampus, and corpus callosum, as well as in a limited number of peripheral tissues. The distribution of the SLT nearly overlapped that of SLC-1, suggesting that some of the neural functions of MCH may be mediated by both of these receptor subtypes.

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.

Prenatal MR findings in a case of aneurysm of the vein of Galen.

We describe a case of aneurysm of the vein of Galen (AVG), which was diagnosed by intrauterine US, MRI and MRA. The baby girl was born at 35 weeks' gestation. She had severe clinical symptoms at birth and died at 29 h of age from intractable congestive heart failure. Intrauterine US detected an intracranial aneurysm and cardiomegaly due to excessive arteriovenous shunting. Intrauterine MRI (SSFSE) confirmed the diagnosis of AVG, and intrauterine MRA (2D-TOF) successfully demonstrated the anatomical structure of the AVG. MRA may be a useful additional sequence to evaluate AVG, and 2D-TOF is thought to be an appropriate technique for scanning fetal AVG.

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.

Improved preparation and crystallization of 25 kDa human fibroblast growth factor-9.

We prepared 25 kDa human fibroblast growth factor-9 (hFGF-9 N33) on a large scale after overproduction in Escherichia coli MM294 (DE3)/pTG931. The purification was performed by a combination of hydrophobic chromatography and HPLC with an ion exchange column, a heparin affinity column and a gel filtration column. This improved procedure was rapid and simple, and the purified hFGF-9 N33 was found to be homogeneous as judged by various criteria, such as amino acid analysis, N-terminal amino acid sequence, C-terminal amino acid analysis and biological activity. Furthermore, as determined by low endotoxin and DNA content, the protein was of high purity. In addition, the hFGF-9 N33 prepared in the present study was easily crystallized by the vapour diffusion method.

Sexual and aggressive interactions in a visible burrow system with provisioned burrows.

The visible burrow system (VBS) is a habitat providing burrows and an open area for mixed-set rat colonies. Provisioning of food and water in the burrows makes it unnecessary for potentially defensive animals to leave the burrows to eat/drink on the surface, and enables evaluation of new types of agonistic interactions that may emerge when this necessity is removed. In such colonies, subordinate males showed high magnitude tunnel guarding behavior, occupying a tunnel opening onto the surface and confronting the dominant. Dominants, in response, made lunges into the tunnels, but quickly retreated without gaining entry, apparently stopped by contact with the defender's vibrissae. Dominants also made and continued to make lateral attacks to the wall adjacent to the tunnels guarded by subordinates, although these were useless in terms of affording contact with the subordinate. Dominant-female agonistic interactions were more frequent than those of dominants and subordinates. These were largely initiated by the male, and involved female defensive behavior. Nonetheless, females, unlike subordinates, failed to show tunnel guarding and continued to utilize the surface freely. They also spent more time in the vicinity of the dominant over days of colony formation. This apparent paradox may reflect that females were seldom wounded, and that the initial site of male contact with females was the female's anogenital area, findings suggesting that interactions of males and females often reflect male sexual advances, countered by female defenses that effectively protect nonestrus females from mounting and copulation.

Role of leukocytes in uterine hypoperfusion and fetal growth retardation induced by ischemia-reperfusion.

We investigated leukocyte involvement in uterine hypoperfusion and intrauterine fetal growth retardation (IUGR) induced by ischemia-reperfusion (I/R) in Sprague-Dawley rats. On day 17 of gestation, leukocyte accumulation in the uterus and placenta subjected to 30 min of ischemia, followed by reperfusion, was assessed by measuring myeloperoxidase (MPO) activity. Uterine MPO activity was significantly higher after 1 h of reperfusion than it was before ischemia (P < 0.05), without any increase in placental MPO activity. Immunohistochemical staining showed leukocyte accumulation in the uterus subjected to I/R. The effects of treatment with monoclonal antibodies against CD11a (WT1) and CD18 (WT3) at a dose of 0.8 mg/kg on uterine blood flow and IUGR were investigated. Laser-Doppler flowmetry demonstrated that uterine hypoperfusion at 2 h after ischemia (blood flow, -51.7 +/- 1.2%; P < 0.01) was inhibited by WT1 and WT3 treatment. I/R-induced IUGR at full term (P < 0.05 vs. nonischemic horn) was prevented by WT1 and WT3 treatment on day 17. These results indicate that leukocyte accumulation may play an important role in the pathogenesis of uterine hypoperfusion and IUGR induced by I/R in pregnant rats.