Antiviral Activity and Resistance Profile of the Novel HIV-1 Non-Catalytic Site Integrase Inhibitor JTP-0157602.

HIV-1 integrase (IN) is an essential enzyme for viral replication. Non-catalytic site integrase inhibitors (NCINIs) are allosteric HIV-1 IN inhibitors and a potential new class of antiretrovirals. In this report, we identified a novel NCINI, JTP-0157602, with an original scaffold. JTP-0157602 exhibited potent antiviral activity against HIV-1 and showed a serum-shifted 90% effective concentration (EC90) of 138 nM, which is comparable to those of the FDA-approved IN strand transfer inhibitors (INSTIs). This compound was fully potent against a wide range of recombinant viruses with IN polymorphisms, including amino acids 124/125, a hot spot of IN polymorphisms. In addition, JTP-0157602 retained potent antiviral activity against a broad panel of recombinant viruses with INSTI-related resistance mutations, including multiple substitutions that emerged in clinical studies of INSTIs. Resistance selection experiments of JTP-0157602 led to the emergence of A128T and T174I mutations, which are located at the lens epithelium-derived growth factor/p75 binding pocket of IN. JTP-0157602 inhibited HIV-1 replication mainly during the late phase of the replication cycle, and HIV-1 virions produced by reactivation from HIV-1 latently infected Jurkat cells in the presence of JTP-0157602 were noninfectious. These results suggest that JTP-0157602 and analog compounds can be used to treat HIV-1 infectious diseases. IMPORTANCE Non-catalytic site integrase inhibitors (NCINIs) are allosteric HIV-1 integrase (IN) inhibitors that bind to the lens epithelium-derived growth factor (LEDGF)/p75 binding pocket of IN. NCINIs are expected to be a new class of anti-HIV-1 agents. In this study, we present a novel NCINI, JTP-0157602, which has potent activity against a broad range of HIV-1 strains with IN polymorphisms. Furthermore, JTP-0157602 shows strong antiviral activity against IN strand transfer inhibitor-resistant mutations, suggesting that JTP-0157602 and its analogs are potential agents for treating HIV-1 infections. Structural modeling indicated that JTP-0157602 binds to the LEDGF/p75 binding pocket of IN, and the results of in vitro resistance induction revealed the JTP-0157602 resistance mechanism of HIV-1. These data shed light on developing novel NCINIs that exhibit potent activity against HIV-1 with broad IN polymorphisms and multidrug-resistant HIV-1 variants.

Cystathionine γ-Lyase Self-Inactivates by Polysulfidation during Cystine Metabolism.

Cystathionine γ-lyase (CSE) is an enzyme responsible for the biosynthesis of cysteine from cystathionine in the final step of the transsulfuration pathway. It also has ß-lyase activity toward cystine, generating cysteine persulfide (Cys-SSH). The chemical reactivity of Cys-SSH is thought to be involved in the catalytic activity of particular proteins via protein polysulfidation, the formation of -S-(S)n-H on their reactive cysteine residues. The Cys136/171 residues of CSE have been proposed to be redox-sensitive residues. Herein, we investigated whether CSE polysulfidation occurs at Cys136/171 during cystine metabolism. Transfection of wild-type CSE into COS-7 cells resulted in increased intracellular Cys-SSH production, which was significantly increased when Cys136Val or Cys136/171Val CSE mutants were transfected, instead of the wild-type enzyme. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CSE polysulfidation occurs at Cys136 during cystine metabolism. In vitro incubation of CSE with CSE-enzymatically synthesized Cys-SSH resulted in the inhibition of Cys-SSH production. In contrast, the mutant CSEs (Cys136Val and Cys136/171Val) proved resistant to inhibition. The Cys-SSH-producing CSE activity of Cys136/171Val CSE was higher than that of the wild-type enzyme. Meanwhile, the cysteine-producing CSE activity of this mutant was equivalent to that of the wild-type enzyme. It is assumed that Cys-SSH-producing CSE activity could be auto-inactivated via the polysulfidation of the enzyme during cystine metabolism. Thus, the polysulfidation of CSE at the Cys136 residue may be an integral feature of cystine metabolism, which functions to down-regulate Cys-SSH synthesis by the enzyme.

Identification of the corticotropin-releasing factor receptor 1 antagonists as inhibitors of Chikungunya virus replication using a Gaussia luciferase-expressing subgenomic replicon.

The Chikungunya virus (CHIKV), an enveloped RNA virus that has been identified in over 40 countries and is considered a growing threat to public health worldwide. However, there is no preventive vaccine or specific therapeutic drug for CHIKV infection. To identify a new inhibitor against CHIKV infection, this study constructed a subgenomic RNA replicon expressing the secretory Gaussia luciferase (Gluc) based on the CHIKV SL11131 strain. Transfection of in vitro-transcribed replicon RNA to BHK-21 cells revealed that Gluc activity in culture supernatants was correlated with the intracellular replication of the replicon genome. Through a chemical compound library screen using the Gluc reporter CHIKV replicon, we identified several compounds that suppressed CHIKV infection in Vero cells. Among the hits identified, CP-154,526, a non-peptide antagonist of the corticotropin-releasing factor receptor type-1 (CRF-R1), showed the strongest anti-CHIKV activity and inhibited CHIKV infection in Huh-7 cells. Interestingly, other CRF-R1 antagonists, R121919 and NGD 98-2, also exhibited inhibitory effects on CHIKV infection. Time-of-drug addition and virus entry assays indicated that CP-154,526 suppressed a post-entry step of infection, suggesting that CRF-R1 antagonists acted on a target in the intracellular replication process of CHIKV. Therefore, the Gluc reporter replicon system established in this study would greatly facilitate the development of antiviral drugs against CHIKV infection.

Regulation of nitric oxide/reactive oxygen species redox signaling by nNOS splicing variants.

We previously demonstrated different expression patterns of the neuronal nitric oxide synthase (nNOS) splicing variants, nNOS-µ and nNOS-α, in the rat brain; however, their exact functions have not been fully elucidated. In this study, we compared the enzymatic activities of nNOS-µ and nNOS-α and investigated intracellular redox signaling in nNOS-expressing PC12 cells, stimulated with a neurotoxicant, 1-methyl-4-phenylpyridinium ion (MPP+), to enhance the nNOS uncoupling reaction. Using in vitro studies, we show that nNOS-µ produced nitric oxide (NO), as did nNOS-α, in the presence of tetrahydrobiopterin (BH4), an important cofactor for the enzymatic activity. However, nNOS-µ generated more NO and less superoxide than nNOS-α in the absence of BH4. MPP + treatment induced more reactive oxygen species (ROS) production in nNOS-α-expressing PC12 cells than in those expressing nNOS-µ, which correlated with the intracellular production of 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a downstream messenger of nNOS redox signaling, and apoptosis in these cells. Furthermore, post-treatment with 8-nitro-cGMP aggravated MPP+-induced cytotoxicity via activation of the H-Ras/extracellular signal-regulated kinase signaling pathway. In conclusion, our results provide strong evidence that nNOS-µ exhibits distinctive enzymatic properties of NO/ROS production, contributing to the regulation of intracellular redox signaling, including the downstream production of 8-nitro-cGMP.

miR103a-3p in extracellular vesicles from FcεRI-aggregated human mast cells enhances IL-5 production by group 2 innate lymphoid cells.

BACKGROUND: Mast cells (MCs) are key regulators of IgE-mediated allergic inflammation. Cell-derived extracellular vesicles (EVs) contain bioactive compounds such as microRNAs. EVs can transfer signals to recipient cells, thus using a novel mechanism of cell-to-cell communication. However, whether MC-derived EVs are involved in FcεRI-mediated allergic inflammation is unclear. OBJECTIVE: We sought to investigate the effect of EVs derived from FcεRI-aggregated human MCs on the function of human group 2 innate lymphoid cells (ILC2s). METHODS: Human cultured MCs were sensitized with and without IgE for 1 hour and then incubated with anti-IgE antibody, IL-33, or medium alone for 24 hours. EVs in the MC supernatant were isolated by using ExoQuick-TC. RESULTS: Coculture of ILC2s with EVs derived from the FcεRI-aggregated MCs significantly enhanced IL-5 production and sustained upregulation of IL-5 mRNA expression in IL-33-stimulated ILC2s, but IL-13 production and IL-13 mRNA expression were unchanged. miR103a-3p expression was upregulated in IL-33-stimulated ILC2s that had been cocultured with EVs derived from anti-IgE antibody-stimulated MCs. Transduction of an miR103a-3p mimic to ILC2s significantly enhanced IL-5 production by IL-33-stimulated ILC2s. miR103a-3p promoted demethylation of an arginine residue of GATA3 by downregulating protein arginine methyltransferase 5 (PRMT5) mRNA. Reduction of protein arginine methyltransferase 5 expression in ILC2s by using a small interfering RNA technique resulted in upregulation of IL-5 production by IL-33-stimulated ILC2s. Furthermore, the level of miR103a-3p expression was significantly higher in EVs from sera of patients with atopic dermatitis than in EVs from nonatopic healthy control subjects. CONCLUSION: Eosinophilic allergic inflammation may be exacerbated owing to ILC2 activation by MC-derived miR103a-3p.

Prophylactic innominate artery transection to prevent tracheoinnominate artery fistula: a retrospective review of single institution experiences.

PURPOSE: This study aimed to investigate the optimal indication and availability of prophylactic innominate artery transection (PIAT). METHODS: We retrospectively analyzed the medical records of the patients with neurological or neuromuscular disorders (NMDs) who underwent PIAT. Meanwhile, we originally defined the tracheal flatting ratio (TFR) and mediastinum-thoracic anteroposterior ratio (MTR) from preoperative chest computed tomography imaging and compared these parameters between non-PIAT and PIAT group. RESULTS: There were 13 patients who underwent PIAT. The median age was 22 years. PIAT was planned before in one, simultaneously in five, and after tracheostomy or laryngotracheal separation in seven patients. Image evaluations of the brain to assess circle of Willis were performed in all patients. Appropriate skin incisions with sternotomy to expose the innominate artery were made in four patients. All patients are still alive except one late death without any association with PIAT. No neurological complications occurred in any patients. As significant differences (p < 0.01) between two groups were observed for TFR and MTR, objective validity of the indication of PIAT was found. CONCLUSIONS: PIAT is safe and tolerable in case of innominate artery compression of the trachea with NMDs. TFR and MTR are useful objective indexes to judge the indication of PIAT.

Substrate and metabolic promiscuities of d-altronate dehydratase family proteins involved in non-phosphorylative d-arabinose, sugar acid, l-galactose and l-fucose pathways from bacteria.

The gene context in microorganism genomes is of considerable help for identifying potential substrates. The C785_RS13685 gene in Herbaspirillum huttiense IAM 15032 is a member of the d-altronate dehydratase protein family, and which functions as a d-arabinonate dehydratase in vitro, is clustered with genes related to putative pentose metabolism. In the present study, further biochemical characterization and gene expression analyses revealed that l-xylonate is a physiological substrate that is ultimately converted to α-ketoglutarate via so-called Route II of a non-phosphorylative pathway. Several hexonates, including d-altronate, d-idonate and l-gluconate, which are also substrates of C785_RS13685, also significantly up-regulated the gene cluster containing C785_RS13685, suggesting a possibility that pyruvate and d- or l-glycerate were ultimately produced (novel Route III). On the contrary, ACAV_RS08155 of Acidovorax avenae ATCC 19860, a homologous gene to C785_RS13685, functioned as a d-altronate dehydratase in a novel l-galactose pathway, through which l-galactonate was epimerized at the C5 position by the sequential activity of two dehydrogenases, resulting in d-altronate. Furthermore, this pathway completely overlapped with Route III of the non-phosphorylative l-fucose pathway. The 'substrate promiscuity' of d-altronate dehydratase protein(s) is significantly expanded to 'metabolic promiscuity' in the d-arabinose, sugar acid, l-fucose and l-galactose pathways.

Coordination between Calcium/Calmodulin-Dependent Protein Kinase II and Neuronal Nitric Oxide Synthase in Neurons.

Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is highly abundant in the brain and exhibits broad substrate specificity, thereby it is thought to participate in the regulation of neuronal death and survival. Nitric oxide (NO), produced by neuronal NO synthase (nNOS), is an important neurotransmitter and plays a role in neuronal activity including learning and memory processes. However, high levels of NO can contribute to excitotoxicity following a stroke and neurodegenerative disease. Aside from NO, nNOS also generates superoxide which is involved in both cell injury and signaling. CaMKII is known to activate and translocate from the cytoplasm to the post-synaptic density in response to neuronal activation where nNOS is predominantly located. Phosphorylation of nNOS at Ser847 by CaMKII decreases NO generation and increases superoxide generation. Conversely, NO-induced S-nitrosylation of CaMKII at Cys6 is a prominent determinant of the CaMKII inhibition in ATP competitive fashion. Thus, the "cross-talk" between CaMKII and NO/superoxide may represent important signal transduction pathways in brain. In this review, we introduce the molecular mechanism of and pathophysiological role of mutual regulation between CaMKII and nNOS in neurons.

Oxidative Stress Orchestrates MAPK and Nitric-Oxide Synthase Signal.

Reactive oxygen species (ROS) are not only harmful to cell survival but also essential to cell signaling through cysteine-based redox switches. In fact, ROS triggers the potential activation of mitogen-activated protein kinases (MAPKs). The 90 kDa ribosomal S6 kinase 1 (RSK1), one of the downstream mediators of the MAPK pathway, is implicated in various cellular processes through phosphorylating different substrates. As such, RSK1 associates with and phosphorylates neuronal nitric oxide (NO) synthase (nNOS) at Ser847, leading to a decrease in NO generation. In addition, the RSK1 activity is sensitive to inhibition by reversible cysteine-based redox modification of its Cys223 during oxidative stress. Aside from oxidative stress, nitrosative stress also contributes to cysteine-based redox modification. Thus, the protein kinases such as Ca2+/calmodulin (CaM)-dependent protein kinase I (CaMKI) and II (CaMKII) that phosphorylate nNOS could be potentially regulated by cysteine-based redox modification. In this review, we focus on the role of post-translational modifications in regulating nNOS and nNOS-phosphorylating protein kinases and communication among themselves.

Crystal structure of substrate-bound bifunctional proline racemase/hydroxyproline epimerase from a hyperthermophilic archaeon.

The hypothetical OCC_00372 protein from Thermococcus litoralis is a member of the ProR superfamily from hyperthermophilic archaea and exhibits unique bifunctional proline racemase/hydroxyproline 2-epimerase activity. However, the molecular mechanism of the broad substrate specificity and extreme thermostability of this enzyme (TlProR) remains unclear. Here we determined the crystal structure of TlProR at 2.7 Šresolution. Of note, a substrate proline molecule, derived from expression host Escherichia coli cells, was tightly bound in the active site of TlProR. The substrate bound structure and mutational analyses suggested that Trp241 is involved in hydroxyproline recognition by making a hydrogen bond between the indole group of Trp241 and the hydroxyl group of hydroxyproline. Additionally, Tyr171 may contribute to the thermostability by making hydrogen bonds between the hydroxyl group of Tyr171 and catalytic residues. Our structural and functional analyses provide a structural basis for understanding the molecular mechanism of substrate specificity and thermostability of ProR superfamily proteins.

Reactive sulfur species impair Ca2+/calmodulin-dependent protein kinase II via polysulfidation.

We previously reported that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is inhibited by S-nitrosylation of Cys6 in cells. Herein, we show that polysulfidation of CaMKII at Cys6 limits its enzyme activity following reactive sulfur species (RSS) stimulus. In vitro incubation of CaMKII with the RSS donor, Na2S4, induced the inhibition of the enzyme via its polysulfidation. Treatment with dithiothreitol reversed the polysulfidation and the subsequent inhibition. The inhibition of CaMKII by Na2S4 is competitive with ATP but not with the peptide substrate Syntide-2. In transfected cells expressing CaMKII, the enzyme activity decreased upon treatment with Na2S4, whereas cells expressing mutant CaMKII (C6A) were resistant to this treatment. In addition, the endogenous CaMKII was inhibited by treatment with Na2S4 in RAW264.7 murine macrophage cells. These results suggest a novel regulation of CaMKII by RSS via its Cys6 polysulfidation in cells.

The active-site cysteine residue of Ca2+/calmodulin-dependent protein kinase I is protected from irreversible modification via generation of polysulfidation.

Ca2+/calmodulin (CaM)-dependent protein kinase (CaMK) I is activated by the phosphorylation of a crucial activation loop Thr177 by upstream kinases, CaMK kinase (CaMKK), and regulates axonal or dendritic extension and branching. Reactive sulfur species (RSS) modulate protein functions via polysulfidation of the reactive Cys residues. Here, we report that the activity of CaMKI was reversibly inhibited via its polysulfidation at Cys179 by RSS. In vitro incubation of CaMKI with the exogenous RSS donor Na2S3 resulted in a dose-dependent inhibition of the phosphorylation at Thr177 by CaMKK and inactivation of the enzymatic activity. Dithiothreitol (DTT), a small molecule reducing reagent, rescued these inhibitions. Conversely, mutated CaMKI (C179V) was resistant to the Na2S3-induced inactivation. In transfected cells expressing CaMKI, ionomycin-induced CaMKI activity was decreased upon treatment with Na2S4, whereas cells expressing mutant CaMKI (C179V) proved resistant to this treatment. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CaMKI was a target for polysulfidation in cells. Furthermore, the polysulfidation of CaMKI protected Cys179 from its irreversible modification, known as protein succination. Thus, we propose that CaMKI was reversibly inhibited via polysulfidation of Cys179 by RSS, thereby protecting it from irreversible modification.

Involvement of nitric oxide/reactive oxygen species signaling via 8-nitro-cGMP formation in 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells and rat cerebellar granule neurons.

To investigate the role of nitric oxide (NO)/reactive oxygen species (ROS) redox signaling in Parkinson's disease-like neurotoxicity, we used 1-methyl-4-phenylpyridinium (MPP+) treatment (a model of Parkinson's disease). We show that MPP+-induced neurotoxicity was dependent on ROS from neuronal NO synthase (nNOS) in nNOS-expressing PC12 cells (NPC12 cells) and rat cerebellar granule neurons (CGNs). Following MPP+ treatment, we found production of 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a second messenger in the NO/ROS redox signaling pathway, in NPC12 cells and rat CGNs, that subsequently induced S-guanylation and activation of H-Ras. Additionally, following MPP+ treatment, extracellular signal-related kinase (ERK) phosphorylation was enhanced. Treatment with a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor attenuated MPP+-induced ERK phosphorylation and neurotoxicity. In conclusion, we demonstrate for the first time that NO/ROS redox signaling via 8-nitro-cGMP is involved in MPP+-induced neurotoxicity and that 8-nitro-cGMP activates H-Ras/ERK signaling. Our results indicate a novel mechanism underlying MPP+-induced neurotoxicity, and therefore contribute novel insights to the mechanisms underlying Parkinson's disease.

Subarachnoid hemorrhage induces neuronal nitric oxide synthase phosphorylation at Ser1412 in the dentate gyrus of the rat brain.

INTRODUCTION: We previously demonstrated that cyclic AMP-dependent protein kinase (PKA) phosphorylates neuronal nitric oxide synthase (nNOS) at Ser1412 in the hippocampal dentate gyrus after forebrain ischemia; this phosphorylation event activates NOS activity and might contribute to depression after cerebral ischemia. In this study, we revealed chronological and topographical changes in the phosphorylation of nNOS at Ser1412 immediately after subarachnoid hemorrhage (SAH). METHODS: In a rat single-hemorrhage model of SAH, the hippocampus and adjacent cortex were collected up to 24 h after SAH. Samples from rats that were not injected with autologous blood were used as controls. NOS was partially purified from crude samples via an ADP-agarose gel. Levels of nNOS, nNOS phosphorylated at Ser1412 (p-nNOS), PKA, and p-PKA at Thr197 were studied in the rat hippocampus and cortex using Western blot analyses and immunohistochemistry. RESULTS: According to the Western blot analysis, levels of p-nNOS at Ser1412 were significantly increased in the hippocampus, but not in the cortex, between 1 and 3 h after SAH. Immunohistochemistry revealed the phosphorylation of nNOS at Ser1412 and PKA at Thr197 in the dentate gyrus, but not in the CA1 area, 1 h after SAH. An injection of saline instead of blood also significantly increased levels of p-nNOS at Ser1412 in the hippocampus 1 h after the injection. CONCLUSIONS: An immediate increase in intracranial pressure (ICP) might induce transient cerebral ischemia and promote the PKA-mediated phosphorylation of nNOS at Ser1412 in the dentate gyrus. This signal transduction pathway induces the excessive production of nitric oxide (NO) and might be involved in cognitive dysfunction after SAH.

Characterization of cis-4-hydroxy-D-proline dehydrogenase from Sinorhizobium meliloti.

The hypO gene from Sinorhizobium meliloti, located within the trans-4-hydroxy-L-proline metabolic gene cluster, was first successfully expressed in the host Pseudomonas putida. Purified HypO protein functioned as a FAD-containing cis-4-hydroxy-D-proline dehydrogenase with a homomeric structure. In contrast to other known enzymes, significant activity for D-proline was found, confirming a previously proposed potential involvement in D-proline metabolism.

Reactive sulfur species inactivate Ca2+/calmodulin-dependent protein kinase IV via S-polysulfidation of its active-site cysteine residue.

Reactive sulfur species (RSS) modulate protein functions via S-polysulfidation of reactive Cys residues. Here, we report that Ca2+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) was reversibly inactivated by RSS via polysulfidation of the active-site Cys residue. CaMKIV is phosphorylated at Thr196 by its upstream CaMK kinase (CaMKK), resulting in the induction of its full activity. In vitro incubation of CaMKIV with the exogenous RSS donors Na2S n (n = 2-4) resulted in dose-dependent inhibition of the CaMKK-induced phospho-Thr196 and consequent inactivation of the enzyme activity. Conversely, mutated CaMKIV (C198V) was refractory to the Na2S n -induced enzyme inhibition. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that Cys198 in CaMKIV represents a target for S-polysulfidation. Furthermore, phosho-Thr196 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 Thr196 was decreased upon treatment with either Na2S4 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-Thr196 of endogenous CaMKIV was also inhibited by treatment either with Na2S4 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 Cys198 during the response to ER stress.

Superoxide generation from nNOS splice variants and its potential involvement in redox signal regulation.

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.

Characterization of human decidual mast cells and establishment of a culture system.

BACKGROUND: Although rodent decidual mast cells (MCs) reportedly play an important role in implantation and placenta formation, the characterization of human decidual MCs has been not well clarified. The aims of this study were to investigate the distribution and characteristics of MCs in human decidua and to establish a culture system for decidua-derived MCs. METHODS: Decidual tissues were obtained from patients who underwent a legal elective abortion (6th week to 9th week of pregnancy), and decidual MCs were enzymatically dispersed. Cultured decidua-derived MCs were generated by culturing decidual cells with stem cell factor. An ultrastructural analysis of primary decidual MCs and cultured decidua-derived MCs was performed using a transmission electron microscope. Receptor and protease expression was analyzed using FACS. Histamine released from MCs was measured using enzyme immune assays. RESULTS: A larger proportion of tryptase positive(+) MCs in decidua was present on the maternal side. Both enzymatically dispersed decidual MCs and cultured decidua-derived MCs showed an FcεRIα+Kit+tryptase+chymase+ phenotype. Their granules contenting particles exhibited variable amounts of electron-lucent space separating electron-dense particles. Both enzymatically dispersed decidual MCs and cultured decidua-derived MCs released comparable amounts of histamine following FcεRI aggregation. CONCLUSIONS: The isolation method for MCs from decidua during early pregnancy and the culture system for decidua-derived MCs may enable the roles of decidual MC during pregnancy to be explored.

Characterization of a Novel cis-3-Hydroxy-l-Proline Dehydratase and a trans-3-Hydroxy-l-Proline Dehydratase from Bacteria.

Hydroxyprolines, such as trans-4-hydroxy-l-proline (T4LHyp), trans-3-hydroxy-l-proline (T3LHyp), and cis-3-hydroxy-l-proline (C3LHyp), are present in some proteins including collagen, plant cell wall, and several peptide antibiotics. In bacteria, genes involved in the degradation of hydroxyproline are often clustered on the genome (l-Hyp gene cluster). We recently reported that an aconitase X (AcnX)-like hypI gene from an l-Hyp gene cluster functions as a monomeric C3LHyp dehydratase (AcnXType I). However, the physiological role of C3LHyp dehydratase remained unclear. We here demonstrate that Azospirillum brasilense NBRC 102289, an aerobic nitrogen-fixing bacterium, robustly grows using not only T4LHyp and T3LHyp but also C3LHyp as the sole carbon source. The small and large subunits of the hypI gene (hypIS and hypIL, respectively) from A. brasilense NBRC 102289 are located separately from the l-Hyp gene cluster and encode a C3LHyp dehydratase with a novel heterodimeric structure (AcnXType IIa). A strain disrupted in the hypIS gene did not grow on C3LHyp, suggesting its involvement in C3LHyp metabolism. Furthermore, C3LHyp induced transcription of not only the hypI genes but also the hypK gene encoding Δ1-pyrroline-2-carboxylate reductase, which is involved in T3LHyp, d-proline, and d-lysine metabolism. On the other hand, the l-Hyp gene cluster of some other bacteria contained not only the AcnXType IIa gene but also two putative proline racemase-like genes (hypA1 and hypA2). Despite having the same active sites (a pair of Cys/Cys) as hydroxyproline 2-epimerase, which is involved in the metabolism of T4LHyp, the dominant reaction by HypA2 was clearly the dehydration of T3LHyp, a novel type of T3LHyp dehydratase that differed from the known enzyme (Cys/Thr).IMPORTANCE More than 50 years after the discovery of trans-4-hydroxy-l-proline (generally called l-hydroxyproline) degradation in aerobic bacteria, its genetic and molecular information has only recently been elucidated. l-Hydroxyproline metabolic genes are often clustered on bacterial genomes. These loci frequently contain a hypothetical gene(s), whose novel enzyme functions are related to the metabolism of trans-3-hydroxyl-proline and/or cis-3-hydroxyl-proline, a relatively rare l-hydroxyproline in nature. Several l-hydroxyproline metabolic enzymes show no sequential similarities, suggesting their emergence by convergent evolution. Furthermore, transcriptional regulation by trans-4-hydroxy-l-proline, trans-3-hydroxy-l-proline, and/or cis-3-hydroxy-l-proline significantly differs between bacteria. The results of the present study show that several l-hydroxyprolines are available for bacteria as carbon and energy sources and may contribute to the discovery of potential metabolic pathways of another hydroxyproline(s).

Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling.

Using methodology developed herein, it is found that reactive persulfides and polysulfides are formed endogenously from both small molecule species and proteins in high amounts in mammalian cells and tissues. These reactive sulfur species were biosynthesized by two major sulfurtransferases: cystathionine ß-synthase and cystathionine γ-lyase. Quantitation of these species indicates that high concentrations of glutathione persulfide (perhydropersulfide >100 µM) and other cysteine persulfide and polysulfide derivatives in peptides/proteins were endogenously produced and maintained in the plasma, cells, and tissues of mammals (rodent and human). It is expected that persulfides are especially nucleophilic and reducing. This view was found to be the case, because they quickly react with H2O2 and a recently described biologically generated electrophile 8-nitroguanosine 3',5'-cyclic monophosphate. These results indicate that persulfides are potentially important signaling/effector species, and because H2S can be generated from persulfide degradation, much of the reported biological activity associated with H2S may actually be that of persulfides. That is, H2S may act primarily as a marker for the biologically active of persulfide species.