Iron as electron donor for denitrification: The efficiency, toxicity and mechanism.

For the treatment of low C/N wastewaters, methanol or acetate is usually dosed as electron donor for denitrification but such organics makes the process costly. To decrease the cost, iron which is the fourth most abundant element in lithosphere is suggested as the substitution of methanol and acetate. The peak volumetric removal rate (VRR) of nitrate nitrogen in the ferrous iron-dependent nitrate removal (FeNiR) reactor was 0.70 ± 0.04 kg-N/(m3·d), and the corresponding removal efficiency was 98%. Iron showed toxicity to cells by decreasing the live cell amount (dropped 56%) and the live cell activity (dropped 70%). The toxicity of iron was mainly expressed by the formation of iron encrustation. From microbial community data analysis, heterotrophs (Paracocccus, Thauera and Azoarcus) faded away while the facultative chemolithotrophs (Hyphomicrobium and Anaerolineaceae_uncultured) dominated in the reactor after replacing acetate with ferrous iron in the influent. Through scanning electron microscope (SEM) and transmission electron microscope (TEM), two iron oxidation sites in FeNiR cells were observed and accordingly two FeNiR mechanisms were proposed: 1) extracellular FeNiR in which ferrous iron was bio-oxidized extracellularly; and 2) intracellular FeNiR in which ferrous iron was chemically oxidized in periplasm. Bio-oxidation (extracellular FeNiR) and chemical oxidation (intracellular FeNiR) of ferrous iron coexisted in FeNiR reactor, but the former one predominated. Comparing with the control group without electron donor in the influent, FeNiR reactor showed 2 times higher and stable nitrate removal rate, suggesting iron could be used as electron donor for denitrification. However, further research works are still needed for the practical application of FeNiR in wastewater treatment.

Parathyroid hormone-related protein regulates osteoclast inhibitory lectin expression via multiple signaling pathways in osteoblast-like cells.

Osteoclast inhibitory lectin (OCIL) is a recently identified inhibitor of osteoclast formation. A variety of osteotropic factors regulate OCIL expression in osteoblastic cells, however, little information is available to date concerning how this gene is controlled. Using real-time RT-PCR, we examined the regulation of OCIL expression by PTHrp and the signaling pathways used. We demonstrated in rat osteoblast-like UMR-106 cells, rat calvarial primary osteoblastic cells, and murine MC3T3-E1 cells, PTHrp(1-34) increased OCIL expression. In UMR-106 cells, the increase began and reached maximum later than RANKL induction and OPG suppression. cAMP/PKA signaling activators PTH(1-31), forskolin and dibutyryl cAMP (db-cAMP), and calcium ionophore A23187 all increased OCIL levels. In contrast, PKC activator phorbol-12-myristate-13-acetate reduced OCIL expression in short term but induced OCIL mRNA in long term. PKA inhibitor KT5720, mitogen-activated protein kinase (MAPK) cascade inhibitor PD98059, calmodulin antagonist W-7, and Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) inhibitor KN-62 all significantly blunted PTHrp-stimulated OCIL expression. Moreover, PD98059 blocked the stimulation of OCIL by FSK or db-cAMP but not that by A23187. In primarily cultured osteoblasts, the PTHrp induction of OCIL was blocked by KT5720, W-7, and PD98059 as well. The data established that PTHrp(1-34) regulates OCIL expression in vitro through cAMP/PKA, Ca(2+)/CaMK II, and MAPK signaling pathways.

Recombinant soluble receptor activator of nuclear factor-kappaB inhibits parathyroid hormone-induced osteoclastogenesis in vitro.

The recent identification of receptor activator of nuclear factor-kappaB ligand (RANKL)/RANK/osteoprotegerin (OPG) cytokine system has led to a new molecular perspective on osteoclast biology and bone homeostasis. Specifically, the interaction between RANKL and RANK is responsible for osteoclast differentiation. In the present study, we evaluated whether soluble RANK (sRANK) could act as an antagonist of RANKL and down-regulate osteoclastogenesis and bone resorption in vitro. The prokaryotic expression vector coding for sRANK was constructed. Then the construct was introduced into E. coli Origami B (DE3) competent cells and recombinant sRANK was successfully produced and purified through affinity chromatography. sRANK reduced osteoclast-like cell (OLC) formation and resorption pit formation induced by parathyroid hormone (PTH) in a dose-dependent manner. In addition, sRANK significantly inhibited PTH-induced mRNA expression of carbonic anhydrase II and tartrate-resistant acid phosphatase in murine bone marrow cells as confirmed by using semi-quantitative RT-PCR. The down-regulation was highly correlated with the effect of sRANK on OLC formation from marrow cells. These data demonstrate the anti-resorptive effects of sRANK in vitro and highlight the potential of sRANK as a novel therapeutic approach to bone disorders characterized by enhanced bone resorption.

[Impacts of osteoclast-like cells cultured on bone wafer and their sub-cellular structures on osteoblast].

OBJECTIVE: To investigate the effects of osteoclast-like cells (OLC) and its sub-cellular structures on the osteoblast (OB) differentiation and function. METHODS: Spleen cells from C57 mice administrated with 5-fluorouracil were induced by IL-3, 6 and granulocyte-macrophage colony stimulating factor (GM-CSF), and 1alpha, 25-(OH)(2)D(3) to obtain massive OLCs. These OLC cells were cultured in culture fluid and on bone wafers (called bolcs). Osteoblasts were cultured and added with NaF, OLCs of two kinds, culture fluid free of OLC, and sub-unit structures such as nucleus, mitochondria, and cytoplasma from OLCs for 5 days. The proliferation rate of OBs was measured by MTT method and the alkaline phophatase (ALP) activity was measured by PNPP method. Immunochemistry was used to detect the core-binding factor alpha1 (Cbfalpha1) in the OBs, Enzyme linked immunosorbent assay was used to measure the osteocalcin. RESULTS: The OB number was lower in the OLC (1.288 +/- 0.039), OLC cytoplasm (1.138 +/- 0.024), 50% OLC culture fluid (1.203 +/- 0.033), 50% OLC culture medium of OLCs cultured on bone wafer (1.128 +/- 0.028) in comparison with the pure OB group (1.393 +/- 0.016, all P < 0.05). The increase functions of OBs by OLC cultured on bone wafer and their nucleus and mitochondria were all more significant than those of the OLCs not cultured on bone wafer. The ALP activity was increased in the NaF (1.027 +/- 0.024), OCL cytoplasm (1.850 +/- 0.033), 50% OLC medium (2.074 +/- 0.065), 50% OLC medium of OLCs cultured on bone wafer (1.718 +/- 0.048), and mitochondria and cytoplasm of the OLC cultured on bone wafer groups (1.246 +/- 0.037, all P < 0.05). NaF (0.0825 +/- 0.0025), OLCs (0.0775 +/- 0.0025), nucleus (0.0775 +/- 0.0025), mitochondria (0.0875 +/- 0.0025), and cytoplasm of OLCs (0.1100 +/- 0.0007), 50% OLC medium (0.0900 +/- 0.0000), 50% OLC medium of OLCs cultured on bone wafer (0.1200 +/- 0.0041), OLCs cultured on bone wafer and nucleus, mitochondria, and cytoplasm of OLCs cultured on bone wafer all significantly increase the oeteocalcin activity of OBs (0.525 +/- 0.0063, all P < 0.05). NaF (57.6% +/- 2.6%), OLC cytoplasm (45.3% +/- 4.7%), 50% OLC medium (46.6% +/- 3.3%), 50% medium of OLCs cultured on bone wafer (54.0% +/- 2.1%), OLCs cultured on bone wafer (44.8% +/- 3.0%), and cytoplasm of OLCs cultured on bone wafer (48.7% +/- 3.5%) all significantly increased the Cbfalpha1 protein in the OBs (32.8% +/- 4.5%, all P < 0.05). CONCLUSION: The sub-cellular elements of OLC and the supernatant of OLC culture media free of OLC promote the functions of OB, especially the OLCs cultured on bone wafer.

[Distribution of tetracycline-arginine-glycine-aspartate-tyrosine in mice and its effect on bone].

OBJECTIVE: To investigate the distribution of tetracycline-arginine-glycine-aspartate-tyrosine (T-RGDY) in mice and its effect on bone. METHODS: 125-labeled T-RGDY was studied for its distribution in mice and for its effects on bone by histomorphometry in ovariectomized rats. RESULTS: The 125I-labeled T-RGDY was more concentrated in the osteoporotic bone than in the normal bone. Compared with ovariectomy group, the morphologic index such as trabecular bone volume/total tissue volume (TBV/TTV), TBV/sponge bone volume (SBV), and mean trabecular plate thickness (MTPT) in T-RGDY group significantly increased (P < 0.05). As compared with sham operation group, MTPT significantly increased in T-RGDY group (P < 0.05), while TBV/SBV and mean trabecular plate density significantly decreased (P < 0.05), and TBV/TYV and mean trabecular plate spacing were almost the same as those in sham operation group (P > 0.05). CONCLUSION: T-RGDY may concentrate in bone tissue to a certain degree, which is closely related with the status of bone remodeling. T-RGDY may inhibit the bone loss caused by ovariectomy.