Large scale application of French reed beds: municipal wastewater treatment for a 20,000 inhabitant's town in Moldova.

A two-stage vertical flow treatment wetlands system (French reed beds) was realized in 2012-2013 for the Orhei's town in Moldova. The treatment system occupies a total area of about 5 ha and operates in cold climate conditions during winter, with air temperatures below -20 °C. The first 2 years (2013-2015) of treatment performances for this system are presented here, with a particular highlight on the analysis of the commissioning phase and the operative choices taken along this period basing on the observed results. The specific classification of this application of constructed wetlands (CWs) for the primary and secondary treatment of municipal wastewater as a medium-large size system makes this technical report a relevant reference for demonstrating the possible extension to the highest numbers of inhabitants for the common application range of this family of technologies (CWs) for municipal wastewater. The observed performances for organic carbon (both as chemical oxygen demand (COD) and biochemical oxygen demand (BOD5)), suspended solids and ammonia removals in the whole first operational period consistently satisfied the national limits for discharge in rivers, respectively, with average values of 86%, 96% and 66%. The treated daily flow was measured in the range of 1,000-2,000 m3/d.

Upflow anaerobic sludge blanket and aerated constructed wetlands for swine wastewater treatment: a pilot study.

Swine wastewater management is often affected by two main issues: a too high volume for optimal reuse as a fertilizer and a too high strength for an economically sustainable treatment by classical solutions. Hence, an innovative scheme has been tested to treat swine wastewater, combining a low cost anaerobic reactor, upflow anaerobic sludge blanket (UASB), with intensified constructed wetlands (aerated CWs) in a pilot scale experimental study. The swine wastewater described in this paper is produced by a swine production facility situated in North Italy. The scheme of the pilot plant consisted of: (i) canvas-based thickener; (ii) UASB; (iii) two intensified aerated vertical subsurface flow CWs in series; (iv) a horizontal flow subsurface CW. The influent wastewater quality has been defined for total suspended solids (TSS 25,025 ± 9,323 mg/l), organic carbon (chemical oxygen demand (COD) 29,350 ± 16,983 mg/l), total reduced nitrogen and ammonium (total Kjeldahl nitrogen (TKN) 1,783 ± 498 mg/l and N-NH4+ 735 ± 251 mg/l) and total phosphorus (1,285 ± 270 mg/l), with nitrates almost absent. The overall system has shown excellent performances in terms of TSS, COD, N-NH4+ and TKN removal efficiencies (99.9%, 99.6%, 99.5%, and 99.0%, respectively). Denitrification (N-NO3- effluent concentration equal to 614 ± 268 mg/l) did not meet the Italian quality standards for discharging in water bodies, mainly because the organic carbon was almost completely removed in the intensified CW beds.

Beneficial effects of melatonin on nicotine-induced vasculopathy.

Tobacco smoking is responsible for death of many people each year and increases the risk of developing numerous disorders, particularly cardiovascular disease and cancer. Among the components of cigarette smoke, nicotine is known to excert proatherosclerotic, prothrombotic and proangiogenic effects on vascular endothelial cells. The current study was designed to investigate the mechanisms by which nicotine induces endothelial dysfunction and further to examine whether melatonin protects against nicotine-induced vasculopathy. Four groups of male rats (controls, melatonin-treated, nicotine treated [100 microg/mL in drinking water], and nicotine plus melatonin [5 mg/kg/day] treated) were used in this study. After 28 days all the animals were killed by decapitation and the aorta was removed. We evaluated the hydroxyproline content, and the different expression of proteins involved in several types of stress (ERK1/2), in fibrosis (TGF-beta1, NF-kappaB) and in recruitment of circulating leukocytes onto the vessel wall, including intercellular adhesion molecule-1 (ICAM-1) and vascular cellular adhesion molecule-1 (VCAM-1). These metabolic pathways are important in the development of nicotine-induced atherosclerosis and hypertension. Our results show that nicotine induces marked structural and functional alterations in the aorta. Nicotine receptor binding results in activation and phosphorylation of ERK 1/2. This enzyme, in turn, activates both TGF-beta1 and NF-kappaB; they stimulate respectively the synthesis of type I collagen, responsible of fibrosis, and moreover ICAM-1, VCAM-1 and reactive oxygen species. Based on these findings, melatonin is able to minimize the negative effects of nicotine by blocking the activation of ERK and the other signalling pathways in which this enzyme is involved.

Lessons learnt from a pilot study on residual dye removal by an aerated treatment wetland.

Treatment wetlands (TWs) have shown good capacity in dye removal from textile wastewater. However, the high hydraulic retention times (HRTs) required by these solutions and the connected high area requirements, remain a big drawback towards the application of TWs for dye treatment at full scale. Aerated TWs are interesting intensified solutions that attempt to reduce the TW required area. Therefore, an aerated CW pilot plant, composed of a 20 m2 horizontal subsurface flow TW (HF) and a 21 m2 Free Water System (FWS), equipped with aeration pipelines, was built and monitored to investigate the potential reduction of required area for dye removal from the effluent wastewater of a centralized wastewater treatment plant (WWTP). During a 8 months long study, experimenting with different hydraulic retention times (HRTs - 1.2, 2.6 and 3.5 days) and aeration modes (intermittent and continuous), the pilot plant has shown a normal biological degradation for organic matter and nutrients, while the residual dye removal has been very low, as demonstrated by the absorbance measure at three wavelengths: at 426 nm (blue) the removal varies from -55% at influent absorbance of 0.010 to 41% at 0.060; at 558 nm (yellow) the removal is negative at 0.005 (-58%) and high at higher influent concentrations (72% at 0.035 of absorbance for the inlet); at 660 nm (red) -82% of removal efficiency was obtained at influent absorbance of 0.002 and 74% at 0.010. These results are a consequence of the biological oxidation processes taking place in the WWTP, so that the residual dye seems to be resistant to further aerobic degradation. Therefore, TWs enhanced by aeration can provide only a buffer effect on peak dye concentrations.

Tolerance to hydraulic and organic load fluctuations in constructed wetlands.

This paper describes a two-year performance evaluation of four different constructed wetland (CW) treatment systems designed by IRIDRA Srl, located in central Italy. All four CW systems were established to treat wastewater effluent from different tourist activities: (1) one single-stage CW for secondary treatment of domestic wastewater (30 p.e.) at a holiday farm site; (2) a hybrid compact system consisting of two stages, a horizontal flow (HF) system followed by a vertical flow (VF) system for the secondary treatment of effluent from a 140 p.e. tourist resort; (3) a single-stage vertical flow (VF) CW for a 100 p.e. mountain shelter; and (4) a pair of single-stage, HF CWs for the secondary treatment of segregated grey and black water produced by an 80 p.e. camping site. These tourism facilities are located in remote areas and share some common characteristics concerning their water management: they have high variability of water consumption and wastewater flow, depending on the season, weather and weekly regularities; they have no connection to a public sewer and most sites are located in a sensitive environment. Total suspended solids (TSS), chemical oxygen demand (COD), biochemical oxygen demand (BOD5), ammonium (N-NH4+), nitrate (N-NOx), total nitrogen (Ntot), total phosphorus (Ptot), total coliform (TC), faecal coliform (FC), E. coli removal efficiencies for all four CW systems are presented. The results from this study demonstrate the potential of CWs as a suitable technology for treating wastewater from tourism facilities in remote areas. A very efficient COD reduction (83-95%) and pathogen elimination (3-5 logs) have been achieved. Furthermore, the CWs are easily maintained, robust (not sensitive to peak flows), constructed with local materials, and operate with relatively low cost.

Lysosomal acid phosphatase is not involved in the dephosphorylation of mannose 6-phosphate containing lysosomal proteins.

The mannose 6-phosphate (Man6P) residues that are necessary for the targeting of newly synthesized lysosomal proteins are dephosphorylated after delivery of lysosomal proteins to lysosomes. To examine the role of lysosomal acid phosphatase (LAP) for the dephosphorylation of Man6P residues in lysosomal proteins, the phosphorylation of endogenous lysosomal proteins and of internalized arylsulfatase A was analyzed in mouse L-cells that overexpress human LAP. Non-transfected L-cells dephosphorylate endogenous lysosomal proteins slowly (half time approximately 13 h) as well as internalized arylsulfatase A. A more than 100-fold overexpression of LAP in these cells did not affect the dephosphorylation rate. Control experiments showed that the internalized arylsulfatase A and overexpressed LAP partially colocalize and that under in vitro conditions purified LAP does not dephosphorylate arylsulfatase A. Taken together, these results indicate that LAP is not the mannose 6-phosphatase that dephosphorylates lysosomal proteins after their delivery to lysosomes.

Ganglioside GM2 is substrate for a sialidase in MDCK cells.

GM1 ganglioside carrying a fluorescent fatty acid in substitution of the natural one, has been administered to cultured Madin-Darby canine kidney (MDCK) cells for different pulse times (0.5-24 h), and its metabolic fate was followed. The fluorescent GM2, asialo-GM2, asialo-GM1 and ceramide were the only detectable metabolites. The complete absence of fluorescent GM3 is consistent with the presence in these cells of a sialidase working on GM1 and GM2 gangliosides. After treatment of the cells with chloroquine the fluorescent GM1 remained essentially undegraded, indicating a catabolic processing at lysosomal level.

Overexpression of wild-type and mutant mucolipin proteins in mammalian cells: effects on the late endocytic compartment organization.

Mucolipin-1 is a 65-kDa membrane protein encoded by the MCOLN1 gene, which is mutated in patients with mucolipidosis type IV (MLIV), a rare neurodegenerative lysosomal storage disorder. We studied the subcellular localization of wild-type and three different mutant forms (T232P, F408del and F465L) of mucolipin by expressing Myc-tagged proteins in HeLa cells. The overexpressed wild-type mucolipin colocalizes to late endocytic structures and induces an aberrant distribution of these compartments. F408del and F465L MLIV mutant proteins show a distribution similar to the wild-type protein, whereas T232P is retained in the endoplasmic reticulum. Among the mutants, only F408del induces a redistribution of the late endocytic compartment. These findings suggest that the overexpression of the mucolipin cation channel influences the dynamic equilibrium of late endocytic compartments.

Nerve growth factor induces sphingomyelin accumulation in pheochromocytoma cells.

The pheochromocytoma cells are a well-known model for studying the nerve growth factor (NGF)-induced molecular changes during the differentiation process. The involvement of sphingomyelin (SM) was studied using the fluorescent analogue of ceramide, i.e. N-lissamine rhodaminyl-(12-aminododecanoyl) D-erythro-sphingosine (C12-LRh-Cer). This fluorescent analogue is metabolically active and can be used to follow the biosynthesis of SM in intact cells. NGF induces a 4-fold increase of fluorescent SM content in PC12 cells, when loaded with C12-LRh-Cer. Treatment of PC12 cells with actinomycin D or cycloheximide completely abolishes the NGF-induced elevation of SM. Inhibition of p140(trkA) receptor by AG-879 prevents extracellular signal-regulated kinase 1/2 phosphorylation and suppresses the increase of SM. Inhibition of protein kinase C (PKC), protein kinase A (PKA) and phosphatidylinositol 3-kinase does not have any effect on NGF-induced C12-LRh-SM accumulation. On the other hand, activation of PKA or PKC with simultaneous treatment with NGF has a synergistic effect on increase of SM content. The NGF-induced SM increase in PC12 cells is an effect promoted by other differentiating agents like dibutyryl cyclic AMP or fibroblast growth factor-2 but not by a mitogenic agent like epidermal growth factor.

Silencing of membrane-associated sialidase Neu3 diminishes apoptosis resistance and triggers megakaryocytic differentiation of chronic myeloid leukemic cells K562 through the increase of ganglioside GM3.

In chronic myeloid leukemia K562 cells, differentiation is also blocked because of low levels of ganglioside GM3, derived by the high expression of sialidase Neu3 active on GM3. In this article, we studied the effects of Neu3 silencing (40-70% and 63-93% decrease in protein content and activity, respectively) in these cells. The effects were as follows: (a) gangliosides GM3, GM1, and sialosylnorhexaosylceramide increased markedly; (b) cell growth and [(3)H]thymidine incorporation diminished relevantly; (c) as mRNA, cyclin D2, and Myc were much less expressed, whereas cyclin D1 was expressed more like its inhibitor p21; (d) as mRNA, pro-apoptotic proteins Bax and Bad increased with concurrent decrease and increase in the anti-apoptotic proteins Bcl-2 and Bcl-XL, respectively; (e) the apoptosis inducers etoposide and staurosporine were active on Neu3 silencing cells but not on mock cells; (f) as mRNA, the megakaryocytic markers CD10, CD44, CD41, and CD61 increased similar to the case of mock cells stimulated with PMA; (g) the signaling cascades mediated by PLC-beta2, PKC, RAF, ERK1/2, RSK90, and JNK were largely activated. The induction of a GM3-rich ganglioside pattern in K562 cells by treatment with brefeldin A elicited a phenotype similar to that of Neu3 silencing cells. In conclusion, upon Neu3 silencing, K562 cells show a decrease in proliferation, propensity to undergo apoptosis, and megakaryocytic differentiation.

Dephosphorylation of the mannose-6-phosphate recognition marker is localized in later compartments of the endocytic route. Identification of purple acid phosphatase (uteroferrin) as the candidate phosphatase.

The mannose-6-phosphate (Man6P) recognition marker in lysosomal proteins is known to be dephosphorylated after the delivery of lysosomal proteins to the endosome/lysosome compartment. The rate of Man6P recognition marker inactivation depends on the cell type and lysosomal protein. In the present study we show that in BHK 21 cells, which rapidly dephosphorylate lysosomal proteins, the recognition marker is stable in the endosomal compartment, to which lysosomal enzymes such as arylsulfatase A are delivered during endocytosis at 20 degrees C. Dephosphorylation depends on the transfer of internalized lysosomal enzymes from the 20 degrees C compartment to later compartments, most likely lysosomes. This transfer is sensitive to NH4C1 and nocodazole. In vitro experiments identified purple acid phosphatase (uteroferrin) as a candidate for the lysosomal phosphatase catalyzing in vivo the dephosphorylation of Man6P recognition marker.

Effect of insulin-like growth factor II on uptake of arylsulfatase A by cultured rat hepatocytes and Kupffer cells.

Mannose 6-phosphate/insulin-like growth factor II receptors have been characterized in hepatocytes and Kupffer cells isolated from adult rat liver. Affinity labeling with [125I]insulin-like growth factor II revealed a protein of Mr 250,000 in both cell types. Labeling was inhibited by an antiserum against the mannose 6-phosphate/insulin-like growth factor II receptor. In Kupffer cells, [125I]insulin-like growth factor II was also cross-linked to a second protein of Mr 130,000. In both cell types, insulin-like growth factor II was 10 times more potent than insulin-like growth factor I in displacing [125I]insulin-like growth factor II from its receptor. The mannose 6-phosphate-specific uptake of [125I]arylsulfatase A via the mannose 6-phosphate/insulin-like growth factor II receptor was inhibited by insulin-like growth factor II and antibodies against the receptor, but was not affected by insulin-like growth factor I, insulin or transforming growth factor beta 1. Cell surface iodination followed by immunoprecipitation of the mannose 6-phosphate/insulin-like growth factor II receptor showed that expression of the mannose 6-phosphate/insulin-like growth factor II receptors at the plasma membrane was increased two-fold by insulin-like growth factor II. These results suggest that binding of insulin-like growth factor II to the mannose 6-phosphate/insulin-like growth factor II receptor blocks the binding and uptake of mannose 6-phosphate-containing lysosomal enzymes and may be directly involved in a co-ordinate regulation of ligand uptake from plasma into hepatocytes and Kupffer cells.

The 46 kDa mannose-6-phosphate receptor contains a signal for basolateral sorting within the 19 juxtamembrane cytosolic residues.

The cytosolic domain of the 46 kDa mannose-6-phosphate receptor (MPR 46) contains a signal that mediates sorting of the receptor and of a reporter protein to the basolateral surface domain of Madin-Darby canine kidney cells. Progressive truncation of the 67 cytosolic residues indicated that the 19 juxtamembrane residues are sufficient for basolateral sorting. Alanine/glycine-scanning mutagenesis identified Glu-11 and Ala-17 as the critical residues between residues 7 and 19. Glu-11 is also of critical importance for the one of the three internalization signals in the cytosolic tail of the receptor [Denzer, Weber, Hille-Rehfeld, von Figura and Pohlmann (1997) Biochem. J. 326, 497-505]. Although overlapping, the signals for basolateral sorting and internalization depend on different residues. The basolateral sorting signal of MPR 46 is distinct from tyrosine- or dileucine-based basolateral sorting signals and also lacks similarity to the few other basolateral signals that do not fall into these two classes.

Insulin-like growth factor II overexpression does not affect sorting of lysosomal enzymes in NIH-3T3 cells.

The sorting of newly synthesized mannose 6-phosphate (M6P)-containing proteins and of the major excreted protein (MEP), a lysosomal thiol proteinase, was studied in NIH-3T3 cells transfected with the cDNA of human insulin-like growth factor II (IGF II) or with the vector alone. Extracts from media and cells labelled with [35S] methionine were used for chromatography on a M6P/IGF II receptor affinity matrix or for immunoprecipitation to assess the distribution of newly synthesized M6P-containing proteins and MEP, respectively. The results indicate that the overexpression of IGF II did not affect the synthesis and the sorting of M6P-containing proteins and of MEP. The binding and uptake of the lysosomal enzyme arylsulfatase A were not affected in IGF II overexpressing cells.

Rapid preparation of a distinct lysosomal population from myelinating mouse brain using Percoll gradients.

To study the vesicular lysosome-associated transport and the metabolism of some brain macromolecules (in particular, sialoglycoconjugates), we developed a rapid procedure to obtain a distinct lysosomal population starting from myelinating mouse brain. This procedure is based on an initial differential centrifugation step producing a 1,000-17,500-g fraction (P2), followed by isopycnic centrifugation of fraction P2 on a self-generated colloidal silica gel (Percoll) gradient. The heaviest subfraction thus obtained is very rich in acid hydrolase activities like beta-galactosidase, arylsulfatase A, and acid phosphatase. The enrichment of these enzymes is approximately 100-fold as compared with the starting homogenate, whereas the markers of other subcellular organelles, such as mitochondria, plasma membranes, or the Golgi apparatus, are virtually absent. The lysosomal preparation contains approximately 12-14% of the total acid hydrolase activities, with a protein yield of approximately 0.12%. Electron microscopy shows that the lysosomal fraction is composed of an approximately 90% pure population of lysosomes. Therefore, the procedure described here is suitable for obtaining a highly purified lysosome preparation from myelinating mouse brain.

Occurrence in brain lysosomes of a sialidase active on ganglioside.

A lysosomal preparation, obtained from brain homogenate of 17-day-old C57BL mice by centrifugation on a self-generating Percoll linear density gradient, showed relative specific activity (RSA) values for typical lysosomal enzymes of 40-120 and for mitochondria, plasma membrane, and cytosol markers of much lower than 1, a result indicating a high degree of homogeneity. The lysosomal preparation contained a sialidase activity that was assayed radiometrically with ganglioside [3H]GD1a and fluorimetrically with 4-methylumbelliferyl-1-alpha-D-N-acetylneuraminic acid (MUB-NeuAc). The properties of the lysosomal enzyme were compared with those of the plasma membrane-bound sialidase contained in a purified synaptosomal plasma membrane fraction that was prepared from the same homogenate and assayed with the same substrates. The optimal pH was 4.2 for the lysosomal and 5.1 for the plasma membrane-bound enzyme. The apparent Km values for GD1a and MUB-NeuAc were 1.5 X 10(-5) and 4.2 X 10(-5) M, respectively, for the lysosomal enzyme and 2.7 X 10(-4) and 6.3 X 10(-5) M for the plasma membrane-bound one. Triton X-100 had a predominantly inhibitory effect on the lysosomal enzyme, whereas it strongly activated the plasma membrane-bound one. The lysosomal enzyme was highly unstable on storage and freezing and thawing cycles, whereas the plasma membrane-bound one was substantially stable. The RSA value of the lysosomal sialidase in the lysosomal fraction closely resembled that of authentic lysosomal enzymes, whereas the RSA value of plasma membrane-bound sialidase in the plasma membrane fraction was very similar to that of typical plasma membrane markers.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and characterization of NEU4, the fourth member of the human sialidase gene family.

Several mammalian sialidases have been cloned so far and here we describe the identification and expression of a new member of the human sialidase gene family. The NEU4 gene, identified by searching sequence databases for entries showing homologies to the human cytosolic sialidase NEU2, maps in 2q37 and encodes a 484-residue protein. The polypeptide contains all the typical sialidase amino acid motifs and, apart from an amino acid stretch that appears unique among mammalian sialidases, shows a high degree of homology for NEU2 and the plasma membrane-associated (NEU3) sialidases. RNA dot-blot analysis showed a low but wide expression pattern, with the highest level in liver. Transient transfection in COS7 cells allowed the detection of a sialidase activity toward the artificial substrate 4MU-NeuAc in the acidic range of pH. Immunofluorescence staining and Western blot analysis demonstrated the association of NEU4 with the inner cell membranes.