Fe-Mn binary oxides improve the methanogenic performance and reduce the environmental health risks associated with antibiotic resistance genes during anaerobic digestion.

Increasing evidence indicates that metal oxides can improve the methanogenic performance during anaerobic digestion (AD) of piggery wastewater. However, the impacts of composite metal oxides on the methanogenic performance and risk of antibiotic resistance gene (ARG) transmission during AD are not fully understood. In this study, different concentrations of Fe-Mn binary oxides (FMBO at 0, 250, 500, and 1000 mg/L) were added to AD to explore the effects of FMBO on the process. The methane yield was 7825.1 mL under FMBO at 250 mg/L, 35.2% higher than that with FMBO at 0 mg/L. PICRUSt2 functional predictions showed that FMBO promoted the oxidation of acetate and propionate, and the production of methane from the substrate, as well as increasing the abundances of most methanogens and genes encoding related enzymes. Furthermore, under FMBO at 250 mg/L, the relative abundances of 14 ARGs (excluding tetC and sul2) and four mobile gene elements (MGEs) decreased by 24.7% and 55.8%, respectively. Most of the changes in the abundances of ARGs were explained by microorganisms, especially Bacteroidetes (51.20%), followed by MGEs (11.98%). Thus, the methanogenic performance of AD improved and the risk of horizontal ARG transfer decreased with FMBO, especially at 250 mg/L.

Functional Tuning of Intrinsic Endothelial Ca2+ Dynamics in Swine Coronary Arteries.

RATIONALE: Recent data from mesenteric and cerebral beds have revealed spatially restricted Ca(2+) transients occurring along the vascular intima that control effector recruitment and vasodilation. Although Ca(2+) is pivotal for coronary artery endothelial function, spatial and temporal regulation of functional Ca(2+) signals in the coronary endothelium is poorly understood. OBJECTIVE: We aimed to determine whether a discrete spatial and temporal profile of Ca(2+) dynamics underlies endothelium-dependent relaxation of swine coronary arteries. METHODS AND RESULTS: Using confocal imaging, custom automated image analysis, and myography, we show that the swine coronary artery endothelium generates discrete basal Ca(2+) dynamics, including isolated transients and whole-cell propagating waves. These events are suppressed by depletion of internal stores or inhibition of inositol 1,4,5-trisphosphate receptors but not by inhibition of ryanodine receptors or removal of extracellular Ca(2+). In vessel rings, inhibition of specific Ca(2+)-dependent endothelial effectors, namely, small and intermediate conductance K(+) channels (K(Ca)3.1 and K(Ca)2.3) and endothelial nitric oxide synthase, produces additive tone, which is blunted by internal store depletion or inositol 1,4,5-trisphosphate receptor blockade. Stimulation of endothelial inositol 1,4,5-trisphosphate-dependent signaling with substance P causes idiosyncratic changes in dynamic Ca(2+) signal parameters (active sites, event frequency, amplitude, duration, and spatial spread). Overall, substance P-induced vasorelaxation corresponded poorly with whole-field endothelial Ca(2+) measurements but corresponded precisely with the concentration-dependent change in Ca(2+) dynamics (linearly translated composite of dynamic parameters). CONCLUSIONS: Our findings show that endothelium-dependent control of swine coronary artery tone is determined by spatial and temporal titration of inherent endothelial Ca(2+) dynamics that are not represented by tissue-level averaged Ca(2+) changes.

TRPC4 inactivation confers a survival benefit in severe pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary arterial pressure with lumen-occluding neointimal and plexiform lesions. Activation of store-operated calcium entry channels promotes contraction and proliferation of lung vascular cells. TRPC4 is a ubiquitously expressed store-operated calcium entry channel, but its role in PAH is unknown. We tested the hypothesis that TRPC4 promotes pulmonary arterial constriction and occlusive remodeling, leading to right ventricular failure in severe PAH. Severe PAH was induced in Sprague-Dawley rats and in wild-type and TRPC4-knockout Fischer 344 rats by a single subcutaneous injection of SU5416 [SU (semaxanib)], followed by hypoxia exposure (Hx; 10% O2) for 3 weeks and then a return to normoxia (Nx; 21% O2) for 3 to 10 additional weeks (SU/Hx/Nx). Although rats of both backgrounds exhibited indistinguishable pulmonary hypertensive responses to SU/Hx/Nx, Fischer 344 rats died within 6 to 8 weeks. Normoxic and hypertensive TRPC4-knockout rats recorded hemodynamic parameters similar to those of their wild-type littermates. However, TRPC4 inactivation conferred a striking survival benefit, due in part to preservation of cardiac output. Histological grading of vascular lesions revealed a reduction in the density of severely occluded small pulmonary arteries and in the number of plexiform lesions in TRPC4-knockout rats. TRPC4 inactivation therefore provides a survival benefit in severe PAH, associated with a decrease in the magnitude of occlusive remodeling.

The rat kidney contains high levels of prouroguanylin (the uroguanylin precursor) but does not express GC-C (the enteric uroguanylin receptor).

The peptide uroguanylin (Ugn) regulates enteric and renal electrolyte transport. Previous studies have shown that Ugn and its receptor GC-C (a ligand-activated guanylate cyclase) are abundant in the intestine. Less is known about Ugn and GC-C expression in the kidney. Here, we identify a 9.4-kDa polypeptide in rat kidney extracts that appears, based on its biochemical and immunological properties, to be authentic prouroguanylin (proUgn). This propeptide is relatively plentiful in the kidney (~16% of intestinal levels), whereas its mRNA is marginally present (<1% of intestinal levels), and free Ugn peptide levels are below detection limits (<0.4% of renal proUgn levels). The paucity of preproUgn-encoding mRNA and free Ugn peptide raises the possibility that the kidney might absorb intact proUgn from plasma, where the concentration of propeptide greatly exceeds that of Ugn. However, immunocytochemical analysis reveals that renal proUgn is found exclusively in distal tubular segments, sites previously shown not to accumulate radiolabeled proUgn after intravascular infusions. Thus proUgn appears to be synthesized within the kidney, but the factors that determine its abundance (rates of transcription, translation, processing, and secretion) must be balanced quite differently than in the gut. Surprisingly, we also find negligible expression of GC-C in the rat kidney, a result confirmed both by RT-PCR and by functional assays that measure Ugn-activated cGMP synthesis. Taken together, these data provide evidence for an intrarenal Ugn system that differs from the well-described intestinal system in its regulatory mechanisms and in the receptor targeted by the peptide.

Circulating prouroguanylin is processed to its active natriuretic form exclusively within the renal tubules.

The intestine and kidney are linked by a mechanism that increases salt excretion in response to salt intake. The peptide uroguanylin (UGn) is thought to mediate this signaling axis. Therefore, it was surprising to find (as reported in a companion publication) that UGn is stored in the intestine and circulates in the plasma almost exclusively in the form of its biologically inactive propeptide precursor, prouroguanylin (proUGn), and, furthermore, that infused proUGn leads to natriuretic activity. Here, we investigate the fate of circulating proUGn. Kinetic studies show rapid renal clearance of radiolabeled propeptide. Radiolabel accumulates at high specific activity in kidney (relative to other organs) and urine (relative to plasma). The principal metabolites found in kidney homogenates are free cysteine and methionine. In contrast, urine contains cysteine, methionine, and three other radioactive peaks, one comigrating with authentic rat UGn15. Interestingly, proUGn is not converted to these or other metabolites in plasma, indicating that circulating proUGn is not processed before entering the kidney. Therefore, our findings suggest that proUGn is the true endocrine agent released in response to salt intake and that the response of the kidney is dependent on conversion of the propeptide to an active form after it reaches the renal tubules. Furthermore, proUGn metabolites (other than small amounts of cysteine and methionine) are not returned to the circulation from the kidney or any other organ. Thus, to respond to proUGn released from the gut, any target organ must use a local mechanism for production of active peptide.

Dephosphorylation of 2,3-bisphosphoglycerate by MIPP expands the regulatory capacity of the Rapoport-Luebering glycolytic shunt.

The Rapoport-Luebering glycolytic bypass comprises evolutionarily conserved reactions that generate and dephosphorylate 2,3-bisphosphoglycerate (2,3-BPG). For >30 years, these reactions have been considered the responsibility of a single enzyme, the 2,3-BPG synthase/2-phosphatase (BPGM). Here, we show that Dictyostelium, birds, and mammals contain an additional 2,3-BPG phosphatase that, unlike BPGM, removes the 3-phosphate. This discovery reveals that the glycolytic pathway can bypass the formation of 3-phosphoglycerate, which is a precursor for serine biosynthesis and an activator of AMP-activated protein kinase. Our 2,3-BPG phosphatase activity is encoded by the previously identified gene for multiple inositol polyphosphate phosphatase (MIPP1), which we now show to have dual substrate specificity. By genetically manipulating Mipp1 expression in Dictyostelium, we demonstrated that this enzyme provides physiologically relevant regulation of cellular 2,3-BPG content. Mammalian erythrocytes possess the highest content of 2,3-BPG, which controls oxygen binding to hemoglobin. We determined that total MIPP1 activity in erythrocytes at 37 degrees C is 0.6 mmol 2,3-BPG hydrolyzed per liter of cells per h, matching previously published estimates of the phosphatase activity of BPGM. MIPP1 is active at 4 degrees C, revealing a clinically significant contribution to 2,3-BPG loss during the storage of erythrocytes for transfusion. Hydrolysis of 2,3-BPG by human MIPP1 is sensitive to physiologic alkalosis; activity decreases 50% when pH rises from 7.0 to 7.4. This phenomenon provides a homeostatic mechanism for elevating 2,3-BPG levels, thereby enhancing oxygen release to tissues. Our data indicate greater biological significance of the Rapoport-Luebering shunt than previously considered.

Integration of inositol phosphate signaling pathways via human ITPK1.

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a reversible, poly-specific inositol phosphate kinase that has been implicated as a modifier gene in cystic fibrosis. Upon activation of phospholipase C at the plasma membrane, inositol 1,4,5-trisphosphate enters the cytosol and is inter-converted by an array of kinases and phosphatases into other inositol phosphates with diverse and critical cellular activities. In mammals it has been established that inositol 1,3,4-trisphosphate, produced from inositol 1,4,5-trisphosphate, lies in a branch of the metabolic pathway that is separate from inositol 3,4,5,6-tetrakisphosphate, which inhibits plasma membrane chloride channels. We have determined the molecular mechanism for communication between these two pathways, showing that phosphate is transferred between inositol phosphates via ITPK1-bound nucleotide. Intersubstrate phosphate transfer explains how competing substrates are able to stimulate each others' catalysis by ITPK1. We further show that these features occur in the human protein, but not in plant or protozoan homologues. The high resolution structure of human ITPK1 identifies novel secondary structural features able to impart substrate selectivity and enhance nucleotide binding, thereby promoting intersubstrate phosphate transfer. Our work describes a novel mode of substrate regulation and provides insight into the enzyme evolution of a signaling mechanism from a metabolic role.

Further studies on aberrant gene expression associated with arsenic-induced malignant transformation in rat liver TRL1215 cells.

Chronic arsenic exposure of rat liver epithelial TRL1215 cells induced malignant transformation in a concentration-dependent manner. To further define the molecular events of these arsenic-transformed cells (termed CAsE cells), gene expressions associated with arsenic carcinogenesis or influenced by methylation were examined. Real-time RT-PCR showed that at carcinogenic concentrations (500 nM, and to a less extent 250 nM of arsenite), the expressions of alpha-fetoprotein (AFP), Wilm's tumor protein-1 (WT-1), c-jun, c-myc, H-ras, c-met and hepatocyte growth factor, heme oxygenase-1, superoxide dismutase-1, glutathione-S-transferase-pi and metallothionein-1 (MT) were increased between 3 to 12-fold, while expressions of insulin-like growth factor II (IGF-II) and fibroblast growth factor receptor (FGFR1) were essentially abolished. These changes were not significant at the non-carcinogenic concentration (125 nM), except for IGF-II. The positive cell-cycle regulators cyclin D1 and PCNA were overexpressed in CAsE cells, while the negative regulators p21 and p16 were suppressed. Western-blot confirmed increases in AFP, WT-1, cyclin D1 and decreases in p16 and p21 protein in CAsE cells. The CAsE cells over-expressed MT but the demethylating agent 5-aza-deoxycytidine (5-aza-dC, 2.5 microM, 72 h) stimulated further MT expression. 5-Aza-deoxycytidine restored the loss of expression of p21 in CAsE cells to control levels, but did not restore the expression of p16, IGF-II, or FGFR1, indicating the loss of expression of these genes is due to factors other than DNA methylation changes. Overall, an intricate variety of gene expression changes occur in arsenic-induced malignant transformation of liver cells including oncogene activation and alterations in expression of genes critical to growth regulation.

scyllo-inositol pentakisphosphate as an analogue of myo-inositol 1,3,4,5,6-pentakisphosphate: chemical synthesis, physicochemistry and biological applications.

myo-Inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P(5)), an inositol polyphosphate of emerging significance in cellular signalling, and its C-2 epimer scyllo-inositol pentakisphosphate (scyllo-InsP(5)) were synthesised from the same myo-inositol-based precursor. Potentiometric and NMR titrations show that both pentakisphosphates undergo a conformational ring-flip at higher pH, beginning at pH 8 for scyllo-InsP(5) and pH 9 for Ins(1,3,4,5,6)P(5). Over the physiological pH range, however, the conformation of the inositol rings and the microprotonation patterns of the phosphate groups in Ins(1,3,4,5,6)P(5) and scyllo-InsP(5) are similar. Thus, scyllo-InsP(5) should be a useful tool for identifying biologically relevant actions of Ins(1,3,4,5,6)P(5), mediated by specific binding sites, and distinguishing them from nonspecific electrostatic effects. We also demonstrate that, although scyllo-InsP(5) and Ins(1,3,4,5,6)P(5) are both hydrolysed by multiple inositol polyphosphate phosphatase (MINPP), scyllo-InsP(5) is not dephosphorylated by PTEN or phosphorylated by Ins(1,3,4,5,6)P(5) 2-kinases. This finding both reinforces the value of scyllo-InsP(5) as a biological control and shows that the axial 2-OH group of Ins(1,3,4,5,6)P(5) plays a part in substrate recognition by PTEN and the Ins(1,3,4,5,6)P(5) 2-kinases.

On the contribution of stereochemistry to human ITPK1 specificity: Ins(1,4,5,6)P4 is not a physiologic substrate.

Ins(1,4,5,6)P4, a biologically active cell constituent, was recently advocated as a substrate of human Ins(3,4,5,6)P4 1-kinase (hITPK1), because stereochemical factors were believed relatively unimportant to specificity [Miller, G.J., Wilson, M.P., Majerus, P.W. and Hurley, J.H. (2005) Specificity determinants in inositol polyphosphate synthesis: crystal structure of inositol 1,3,4-triphosphate 5/6-kinase. Mol. Cell. 18, 201-212]. Contrarily, we provide three examples of hITPK1 stereospecificity. hITPK1 phosphorylates only the 1-hydroxyl of both Ins(3,5,6)P3 and the meso-compound, Ins(4,5,6)P3. Moreover, hITPK1 has >13,000-fold preference for Ins(3,4,5,6)P4 over its enantiomer, Ins(1,4,5,6)P4. The biological significance of hITPK1 being stereospecific, and not physiologically phosphorylating Ins(1,4,5,6)P4, is reinforced by our demonstrating that Ins(1,4,5,6)P4 is phosphorylated (K(m) = 0.18 microM) by inositolphosphate-multikinase.

Microglial NADPH oxidase mediates leucine enkephalin dopaminergic neuroprotection.

Here, we report that leucine enkephalin (LE) is neuroprotective to dopaminergic (DA) neurons at femtomolar concentrations through anti-inflammatory properties. Mesencephalic neuron-glia cultures pretreated with femtomolar concentrations of LE (10(-15)-10(-13) M) protected DA neurons from lipopolysaccharide (LPS)-induced DA neurotoxicity, as determined by DA uptake assay and tyrosine hydroxylase (TH) immunocytochemistry (ICC). However, des-tyrosine leucine enkephalin (DTLE), an LE analogue that is missing the tyrosine residue required for binding to the kappa opioid receptor, was also neuroprotective (10(-15)-10(-13) M), as determined by DA uptake assay and TH ICC. Both LE and DTLE (10(-15)-10(-13) M) reduced LPS-induced superoxide production from microglia-enriched cultures. Further, both LE and DTLE (10(-14), 10(-13) M) reduced the LPS-induced tumor necrosis factor-alpha (TNFalpha) mRNA and TNFalpha protein from PHOX+/+ microglia, as determined by quantitative real-time RT-PCR and ELISA analysis in mesencephalic neuron-glia cultures, respectively. However, both peptides failed to inhibit TNFalpha expression in PHOX-/- cultures, which are unable to produce extracellular superoxide in response to LPS. Additionally, LE and DTLE (10(-14), 10(-13) M) failed to show any neuroprotection against LPS in PHOX-/- cultures. Together, these data indicate that LE and DTLE are neuroprotective at femtomolar concentrations through the inhibition of oxidative insult associated with microglial NADPH oxidase and the attenuation of the ROS-mediated amplification of TNFalpha gene expression in microglia.

Interactive role of the toll-like receptor 4 and reactive oxygen species in LPS-induced microglia activation.

Microglia are activated by lipopolysaccharide (LPS) to produce neurotoxic pro-inflammatory factors and reactive oxygen species (ROS). While a multitude of LPS receptors and corresponding pathways have been identified, the detailed mechanisms mediating the microglial response to LPS are unclear. Using mice lacking a functional toll-like receptor 4 (TLR4), we demonstrate that TLR4 and ROS work in concert to mediate microglia activation, where the contribution from each pathway is dependent on the concentration of LPS. Immunocytochemical staining of microglia in neuron-glia cultures with antibodies against F4/80 revealed that while TLR4(+/+) microglia were activated the low concentration of 1 ng/ml of LPS, TLR4(-/-) microglia exhibit activated morphology in response to LPS only at higher concentrations (100-1,000 ng/ml). Additionally, tumor necrosis factor-alpha (TNF-alpha) was only produced from higher concentrations (100-1,000 ng/ml) of LPS in TLR4(-/-) enriched microglia cultures. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, reduced TNF-alpha production from TLR4(-/-) microglia. The influence of TLR4 on LPS-induced superoxide production was tested in rat enriched microglia cultures, where the presence or absence of serum failed to show any effect on the superoxide production. Further, both TLR4(-/-) and TLR4(+/+) microglia showed a similar increase in extracellular superoxide production when exposed to LPS (1-1,000 ng/ml). These data indicate that LPS-induced superoxide production in microglia is independent of TLR4 and that ROS derived from the production of extracellular superoxide in microglia mediates the LPS-induced TNF-alpha response of both the TLR4-dependent and independent pathway.

The Ins(1,3,4)P3 5/6-kinase/Ins(3,4,5,6)P4 1-kinase is not a protein kinase.

Among inositol phosphate kinases, Ins(3,4,5,6)P4 1-kinase has been considered to be an outsider with disparate sequence, a proclaimed capacity to also phosphorylate proteins and apparent 1-phosphatase activity. Such multifunctionality, coupled with ignorance of its operational domains, complicates any mechanistic rationale behind literature reports that Ins(3,4,5,6)P4 1-kinase regulates apoptosis, salt and fluid secretion, and transcription. We have expressed poly(His)-tagged human Ins(3,4,5,6)P4 1-kinase in Sf9 insect cells and purified the enzyme using Ni-agarose chromatography. Protein kinase activity was eluted from the Ni-agarose column, but this did not co-elute with the Ins(3,4,5,6)P4 1-kinase, indicating that the protein kinase and inositol kinase activities belong to separate proteins. To pursue this conclusion, we prepared catalytically inactive mutants of the Ins(3,4,5,6)P4 1-kinase by identifying and targeting the ATP-binding site. Our strategy was based on sequence alignments suggesting homology of the Ins(3,4,5,6)P4 1-kinase with ATP-grasp metabolic enzymes. Individual mutation of four candidate MgATP-binding participants, Lys157, Asp281, Asp295 and Asn297, severely compromised Ins(3,4,5,6)P4 1-kinase activity. Yet, these mutations did not affect the protein kinase activity. We conclude that the Ins(3,4,5,6)P4 1-kinase is not a protein kinase, contrary to earlier reports [e.g. Wilson, Sun, Cao and Majerus (2001) J. Biol. Chem. 276, 40998-41004]. Elimination of protein kinase activity from the enzyme's repertoire and recognition of its ATP-grasp homology together indicate that structural, functional and catalytic relationships between Ins(3,4,5,6)P4 1-kinase and other inositol phosphate kinases are closer than previously thought [Gonzalez, Schell, Letcher, Veprintsev, Irvine and Williams (2004) Mol. Cell 15, 689-701].