Flocculation has been recognized for hundreds of years as an important phenomenon in brewing and wastewater treatment. However, the underlying molecular mechanisms remain elusive. The lack of a distinct phenotype to differentiate between slow-growing mutants and floc-forming mutants prevents the isolation of floc-related gene by conventional mutant screening. To overcome this, we performed a two-step Escherichia coli mutant screen. The initial screen of E. coli for mutants conferring floc production during high salt treatment yielded a mutant containing point mutations in 61 genes. The following screen of the corresponding single-gene mutants identified two genes, mrcB , encoding a peptidoglycan-synthesizing enzyme and cpxA , encoding a histidine kinase of a two-component signal transduction system that contributed to salt tolerance and flocculation prevention. Both single mutants formed flocs during high salt shock, these flocs contained cytosolic proteins. Δ cpxA exhibited decreased growth with increasing floc production and addition of magnesium to Δ cpxA suppressed floc production effectively. In contrast, the growth of Δ mrcB was inconsistent under high salt conditions. In both strains, flocculation was accompanied by the release of membrane vesicles containing inner and outer membrane proteins. Of 25 histidine kinase mutants tested, Δ cpxA produced the highest amount of proteins in floc. Expression of cpxP was up-regulated by high salt in Δ cpxA , suggesting that high salinity and activation of CpxR might promote floc formation. The finding that Δ mrcB or Δ cpxA conferred floc production indicates that cell envelope stress triggered by unfavorable environmental conditions cause the initiation of flocculation in E. coli .

Reactive sulfur species (RSS) modulate protein functions via S - polysulfidation of reactive Cys residues. Here, we report that Ca 2+ /calmodulin (CaM)-dependent protein kinase IV (CaMKIV) was reversibly inactivated by RSS via polysulfidation of the active-site Cys residue. CaMKIV is phosphorylated at Thr 196 by its upstream CaMK kinase (CaMKK), resulting in the induction of its full activity. In vitro incubation of CaMKIV with the exogenous RSS donors Na 2 S n ( n = 2–4) resulted in dose-dependent inhibition of the CaMKK-induced phospho-Thr 196 and consequent inactivation of the enzyme activity. Conversely, mutated CaMKIV (C198V) was refractory to the Na 2 S n -induced enzyme inhibition. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that Cys 198 in CaMKIV represents a target for S-polysulfidation. Furthermore, phosho-Thr 196 and CaMKIV activity were inhibited by incubation with cysteine hydropersulfide, a newly identified RSS that is generated from cystine by cystathionine-γ-lyase. In transfected cells expressing CaMKIV, ionomycin-induced CaMKIV phosphorylation at Thr 196 was decreased upon treatment with either Na 2 S 4 or the endoplasmic reticulum (ER) stress inducer thapsigargin, whereas cells expressing mutant CaMKIV (C198V) were resistant to this treatment. In addition, the ionomycin-induced phospho-Thr 196 of endogenous CaMKIV was also inhibited by treatment either with Na 2 S 4 or thapsigargin in Jurkat T lymphocytes. Taken together, these data define a novel signaling function for intracellular RSS in inhibiting CaMKIV activity via S - polysulfidation of its Cys 198 during the response to ER stress.

We previously demonstrated different spacial expression profiles of the neuronal nitric oxide (NO) synthase (nNOS) splice variants nNOS-µ and nNOS-α in the brain; however, their exact functions are not fully understood. Here, we used electron paramagnetic resonance to compare the electron-uncoupling reactions of recombinant nNOS-µ and nNOS-α that generate reactive oxygen species (ROS), in this case superoxide. nNOS-µ generated 44% of the amount of superoxide that nNOS-α generated. We also evaluated the ROS production in HEK293 cells stably expressing nNOS-α and nNOS-µ by investigating these electron-uncoupling reactions as induced by calcium ionophore A23187. A23187 treatment induced greater ROS production in HEK293 cells expressing nNOS-α than those expressing nNOS-µ. Also, immunocytochemical analysis revealed that A23187-treated cells expressing nNOS-α produced more 8-nitroguanosine 3′,5′-cyclic monophosphate, a second messenger in NO/ROS redox signaling, than did the cells expressing nNOS-µ. Molecular evolutionary analysis revealed that the ratio of nonsynonymous sites to synonymous sites for the nNOS-µ-specific region was higher than that for the complete gene, indicating that this region has fewer functional constraints than does the complete gene. These observations shed light on the physiological relevance of the nNOS-µ variant and may improve understanding of nNOS-dependent NO/ROS redox signaling and its pathophysiological consequences in neuronal systems.

Phosphorylation is considered a main mechanism modulating nNOS (neuronal nitric oxide synthase) function to reduce NO production. In the present study, the effects of nNOS phosphorylation on redox signalling, including that of NO, ROS (reactive oxygen species), and 8-nitro-cGMP (8-nitroguanosine 3′,5′-cyclic monophosphate), a downstream messenger of redox signalling, were investigated. In vitro experiments revealed that a phosphorylation-mimic mutant of nNOS (Ser 847 replaced with aspartic acid, 847D) increased uncoupling to produce a superoxide. In addition, nicotine, which triggers an influx of Ca 2+ , induced more ROS and 8-nitro-cGMP production in 847D-expressing PC12 cells than WT (wild-type)-expressing cells. Additionally, nicotine-induced phosphorylation of nNOS at Ser 847 and increased ROS and 8-nitro-cGMP production in rat CGNs (cerebellar granule neurons). In CGNs, the NOS (nitric oxide synthase) inhibitor L- NAME ( N G -nitro- L -arginine methyl ester) and superoxide dismutase completely inhibited ROS and 8-nitro-cGMP production, whereas the CaMK (Ca 2+ /calmodulin-dependent protein kinase) inhibitor KN93 mildly reduced this effect. Nicotine induced HO-1 (haem oxygenase 1) expression in CGNs and showed cytoprotective effects against apoptosis. Moreover, 8-nitro-cGMP treatment showed identical effects that were attenuated by KN93 pre-treatment. The present paper provides the first substantial corroboration for the biological effects of nNOS phosphorylation at Ser 847 on redox signalling, including ROS and intracellular 8-nitro-cGMP generation in neurons, which possibly play roles in neuroprotection.

8-Nitro-cGMP (8-nitroguanosine 3′,5′-cyclic monophosphate) is a nitrated derivative of cGMP, which can function as a unique electrophilic second messenger involved in regulation of an antioxidant adaptive response in cells. In the present study, we investigated chemical and biochemical regulatory mechanisms involved in 8-nitro-cGMP formation, with particular focus on the roles of ROS (reactive oxygen species). Chemical analyses demonstrated that peroxynitrite-dependent oxidation and myeloperoxidase-dependent oxidation of nitrite in the presence of H 2 O 2 were two major pathways for guanine nucleotide nitration. Among the guanine nucleotides examined, GTP was the most sensitive to peroxynitrite-mediated nitration. Immunocytochemical and tandem mass spectrometric analyses revealed that formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with lipopolysaccharide plus pro-inflammatory cytokines depended on production of both superoxide and H 2 O 2 . Using the mitochondria-targeted chemical probe MitoSOX™ Red, we found that mitochondria-derived superoxide can act as a direct determinant of 8-nitro-cGMP formation. Furthermore, we demonstrated that Nox2 (NADPH oxidase 2)-generated H 2 O 2 regulated mitochondria-derived superoxide production, which suggests the importance of cross-talk between Nox2-dependent H 2 O 2 production and mitochondrial superoxide production. The results of the present study suggest that 8-nitro-cGMP can serve as a unique second messenger that may be implicated in regulating ROS signalling in the presence of NO.

IL-1 (interleukin-1) acts as a key mediator of the degeneration of articular cartilage in RA (rheumatoid arthritis) and OA (osteoarthritis), where chondrocyte death is observed. It is still controversial, however, whether IL-1 induces chondrocyte death. In the present study, the viability of mouse chondrocyte-like ATDC5 cells was reduced by the treatment with IL-1β for 48 h or longer. IL-1β augmented the expression of the catalytic gp91 subunit of NADPH oxidase, gp91 phox , as well as inducible NO synthase in ATDC5 cells. Generation of nitrated guanosine and tyrosine suggested the formation of reactive nitrogen species including ONOO − (peroxynitrite), a reaction product of NO and O 2 − , in ATDC5 cells and rat primary chondrocytes treated with IL-1β. Death of ATDC5 cells after IL-1β treatment was prevented by an NADPH-oxidase inhibitor, AEBSF [4-(2-aminoethyl)benzenesulphonyl fluoride], an NO synthase inhibitor, L -NAME ( N G -nitro- L -arginine methyl ester), and a ONOO − scavenger, uric acid. The viability of ATDC5 cells was reduced by the ONOO − -generator 3-(4-morpholinyl)sydnonimine hydrochloride, but not by either the NO-donor 1-hydroxy-2-oxo-3-( N -methyl-2-aminopropyl)-3-methyl-1-triazene or S -nitrosoglutathione. Disruption of mitochondrial membrane potential and ATP deprivation were observed in IL-1β-treated ATDC5 cells, both of which were restored by L -NAME, AEBSF or uric acid. On the other hand, no morphological or biochemical signs indicating apoptosis were observed in these cells. These results suggest that the death of chondrocyte-like ATDC5 cells was mediated at least in part by mitochondrial dysfunction and energy depletion through ONOO − formation after IL-1β treatment.