Characterization of the transcriptionally active form of dephosphorylated DctD complexed with dephospho-IIAGlc.

Bacterial enhancer-binding proteins (bEBPs) acquire a transcriptionally active state via phosphorylation. However, transcriptional activation by the dephosphorylated form of bEBP has been observed in DctD, which belongs to Group I bEBP. The formation of a complex between dephosphorylated DctD (d-DctD) and dephosphorylated IIAGlc (d-IIAGlc) is a prerequisite for the transcriptional activity of d-DctD. In the present study, characteristics of the transcriptionally active complex composed of d-IIAGlc and phosphorylation-deficient DctD (DctDD57Q) of Vibrio vulnificus were investigated in its multimeric conformation and DNA-binding ability. DctDD57Q formed a homodimer that could not bind to the DNA. In contrast, when DctDD57Q formed a complex with d-IIAGlc in a 1:1 molar ratio, it produced two conformations: dimer and dodecamer of the complex. Only the dodecameric complex exhibited ATP-hydrolyzing activity and DNA-binding affinity. For successful DNA-binding and transcriptional activation by the dodecameric d-IIAGlc/DctDD57Q complex, extended upstream activator sequences were required, which encompass the nucleotide sequences homologous to the known DctD-binding site and additional nucleotides downstream. This is the first report to demonstrate the molecular characteristics of a dephosphorylated bEBP complexed with another protein to form a transcriptionally active dodecameric complex, which has an affinity for a specific DNA-binding sequence.IMPORTANCEResponse regulators belonging to the bacterial two-component regulatory system activate the transcription initiation of their regulons when they are phosphorylated by cognate sensor kinases and oligomerized to the appropriate multimeric states. Recently, it has been shown that a dephosphorylated response regulator, DctD, could activate transcription in a phosphorylation-independent manner in Vibrio vulnificus. The dephosphorylated DctD activated transcription as efficiently as phosphorylated DctD when it formed a complex with dephosphorylated form of IIAGlc, a component of the glucose-phosphotransferase system. Functional mimicry of this complex with the typical form of transcriptionally active phosphorylated DctD led us to study the molecular characteristics of this heterodimeric complex. Through systematic analyses, it was surprisingly determined that a multimer constituted with 12 complexes gained the ability to hydrolyze ATP and recognize specific upstream activator sequences containing a typical inverted-repeat sequence flanked by distinct nucleotides.

Transition of Dephospho-DctD to the Transcriptionally Active State via Interaction with Dephospho-IIAGlc.

Exopolysaccharides (EPSs), biofilm-maturing components of Vibrio vulnificus, are abundantly produced when the expression of two major EPS gene clusters is activated by an enhancer-binding transcription factor, DctD2, whose expression and phosphorylation are induced by dicarboxylic acids. Surprisingly, when glucose was supplied to V. vulnificus, similar levels of expression of these clusters occurred, even in the absence of dicarboxylic acids. This glucose-dependent activation was also mediated by DctD2, whose expression was sequentially activated by the transcription regulator NtrC. Most DctD2 in cells grown without dicarboxylic acids was present in a dephosphorylated state, known as the transcriptionally inactive form. However, in the presence of glucose, a dephosphorylated component of the glucose-specific phosphotransferase system, d-IIAGlc, interacted with dephosphorylated DctD2 (d-DctD2). While d-DctD2 did not show any affinity to a DNA fragment containing the DctD-binding sequences, the complex of d-DctD2 and d-IIAGlc exhibited specific and efficient DNA binding, similar to the phosphorylated DctD2. The d-DctD2-mediated activation of the EPS gene clusters' expression was not fully achieved in cells grown with mannose. Furthermore, the degrees of expression of the clusters under glycerol were less than those under mannose. This was caused by an antagonistic and competitive effect of GlpK, whose expression was increased by glycerol, in forming a complex with d-DctD2 by d-IIAGlc. The data demonstrate a novel regulatory pathway for V. vulnificus EPS biosynthesis and biofilm maturation in the presence of glucose, which is mediated by d-DctD2 through its transition to the transcriptionally active state by interacting with available d-IIAGlc. IMPORTANCE Transcription regulation by bacterial two-component systems is achieved by a response regulator upon its transition to the transcriptionally active form via kinase activity of its cognate sensor under specific conditions. A well-known response regulator, DctD, is converted to its phosphorylated form when DctB senses ambient dicarboxylic acids. Phospho-DctD induces expression of its regulon, including the gene clusters for biosynthesis of exopolysaccharides (EPSs), the essential constituents of biofilm matrix. In the absence of dicarboxylic acids, however, DctD-mediated induction of these EPS gene clusters and biofilm maturation was observed if glucose was supplied. This suggests that dephospho-DctD could play a role in activating the transcription of target genes. A component of glucose-phosphotransferase system, IIAGlc, was present in a dephosphorylated state in the presence of glucose. Dephospho-DctD formed a complex with dephospho-IIAGlc and was converted to a transcriptionally active state. These findings suggest the other response regulators could also have alternative pathways of activation independent of phosphorylation.

Transcription activation of two clusters for exopolysaccharide biosynthesis by phosphorylated DctD in Vibrio vulnificus.

NtrC-mediated production of exopolysaccharides (EPS), essential components for Vibrio vulnificus biofilms, is highly increased in the presence of dicarboxylic or tricarboxylic acids. Gel-shift assays showed that regulation of the EPS-gene cluster I (EPS-I cluster) by NtrC was direct via binding of phosphorylated NtrC (p-NtrC) to the regulatory region of the EPS-I cluster. In contrast, p-NtrC did not bind to the EPS-II and EPS-III clusters, suggesting that NtrC regulation was not direct and another transcription factor belonging to an NtrC-regulon might play a role in activating their transcription. A candidate transcription factor, DctD, of which expression was induced by NtrC, activated the expression of the EPS-II and EPS-III clusters via direct binding to their upstream regions. Under growth conditions with either dicarboxylic or tricarboxylic acids, the expression of NtrC was induced and the transcription of dctD was activated. Furthermore, DctD exhibited higher transcriptional activity under the conditions with dicarboxylic acids than with tricarboxylic acids. Therefore, this study demonstrates that under dicarboxylate-rich conditions, both the abundance and activity of DctD were markedly induced, which activates the expression of two EPS clusters to maximize biosynthesis of EPS facilitating biofilm maturation in V. vulnificus.

Visual loss due to optic nerve infarction and central retinal artery occlusion after spine surgery in the prone position: A case report.

RATIONALE: Visual loss after spine surgery in the prone position is a serious complication. Several cases of central retinal artery occlusion with ophthalmoplegia after spine surgery have been reported in patients with ophthalmic arteries fed by the internal carotid artery (ICA) in a normal manner. PATIENT CONCERNS: A 74-year-old man developed visual loss after undergoing a spinal decompression and fusion operation in the prone position that lasted approximately 5 hours. DIAGNOSES: We detected an extremely rare case of visual loss due to optic nerve infarction and central retinal artery occlusion through fundoscopic examination, fluorescein angiogram, brain magnetic resonance imaging, and magnetic resonance angiography. The patient's visual loss may have been caused by compromised retrograde collateral circulation of the ophthalmic artery from branches of the external carotid artery in the presence of proximal ICA occlusion after a spinal operation in the prone position. INTERVENTIONS: To recover movement of the left extraocular muscles, the patient received intravenous injections of methylprednisolone for 3 days and then oral prednisolone for 6 days. OUTCOMES: Twenty days after the treatment, the motion of the left extraocular muscles was significantly improved. However, recovery from the left visual loss did not occur until 4 months after the operation. LESSONS: In high-risk patients with retrograde collateral circulation of the ophthalmic artery from the external carotid artery due to proximal ICA occlusion, various measures, including the use of a head fixator to provide a position completely free of direct compression of the head and face, should be considered to decrease the risk of postoperative visual loss.

Dexmedetomidine Inhibits Phenylephrine-induced Contractions via Alpha-1 Adrenoceptor Blockade and Nitric Oxide Release in Isolated Rat Aortae.

The goal of this in vitro study was to examine the effect of the alpha-2 adrenoceptor agonist dexmedetomidine on phenylephrine (alpha-1 adrenoceptor agonist)-induced contraction in isolated rat aortae and to elucidate the associated cellular mechanisms, with a particular focus on alpha-1 adrenoceptor antagonism. Dexmedetomidine dose-response curves were generated in isolated endothelium-intact and endothelium-denuded rat aortae precontracted with phenylephrine or 5-hydroxytryptamine. Endothelium-denuded aortic rings were pretreated with either dexmedetomidine or the reversible alpha-1 adrenoceptor antagonist phentolamine, followed by post-treatment with the irreversible alpha-1 adrenoceptor blocker phenoxybenzamine. Control rings were treated with phenoxybenzamine alone. All rings were repeatedly washed with Krebs solution to remove all pretreatment drugs, including phenoxybenzamine, phentolamine and dexmedetomidine. Phenylephrine dose-response curves were then generated. The effect of rauwolscine on the dexmedetomidine-mediated change in phenylephrine-induced endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein endothelial cells was examined using western blotting. The magnitude of the dexmedetomidine-mediated inhibition of phenylephrine-induced contraction was higher in endothelium-intact aortae than in endothelium-denuded aortae or endothelium-intact aortae treated with Nω-nitro-L-arginine methyl ester. However, dexmedetomidine did not significantly alter 5-hydroxytryptamine-induced contraction. In further experiments, prazosin attenuated dexmedetomidine-induced contraction. Additionally, pretreatment with either dexmedetomidine plus phenoxybenzamine or phentolamine plus phenoxybenzamine produced greater phenylephrine-induced contraction than phenoxybenzamine alone, suggesting that dexmedetomidine protects aortae from the alpha-1 adrenoceptor blockade induced by phenoxybenzamine. Rauwolscine attenuated the dexmedetomidine-mediated enhancement of phenylephrine-induced eNOS phosphorylation. Taken together, these results suggest that dexmedetomidine attenuates phenylephrine-induced contractions via alpha-1 adrenoceptor blockade and endothelial nitric oxide release in the isolated rat aorta.

A Lipid Emulsion Reverses Toxic-Dose Bupivacaine-Induced Vasodilation during Tyrosine Phosphorylation-Evoked Contraction in Isolated Rat Aortae.

The goal of this in vitro study was to examine the effect of a lipid emulsion on toxic-dose bupivacaine-induced vasodilation in a model of tyrosine phosphatase inhibitor sodium orthovanadate-induced contraction in endothelium-denuded rat aortae and to elucidate the associated cellular mechanism. The effect of a lipid emulsion on vasodilation induced by a toxic dose of a local anesthetic during sodium orthovanadate-induced contraction was examined. In addition, the effects of various inhibitors, either bupivacaine alone or a lipid emulsion plus bupivacaine, on protein kinase phosphorylation induced by sodium orthovanadate in rat aortic vascular smooth muscle cells was examined. A lipid emulsion reversed the vasodilation induced by bupivacaine during sodium orthovanadate-induced contraction. The lipid emulsion attenuated the bupivacaine-mediated inhibition of the sodium orthovanadate-induced phosphorylation of protein tyrosine, c-Jun NH2-terminal kinase (JNK), myosin phosphatase target subunit 1 (MYPT1), phospholipase C (PLC) γ-1 and extracellular signal-regulated kinase (ERK). These results suggest that a lipid emulsion reverses toxic-dose bupivacaine-induced vasodilation during sodium orthovanadate-induced contraction via the activation of a pathway involving either tyrosine kinase, JNK, Rho-kinase and MYPT1 or tyrosine kinase, PLC γ-1 and ERK, and this reversal is associated with the lipid solubility of the local anesthetic and the induction of calcium sensitization.

Lipid Emulsion Inhibits Apoptosis Induced by a Toxic Dose of Verapamil via the Delta-Opioid Receptor in H9c2 Rat Cardiomyoblasts.

The goals of this study were to investigate the effects of lipid emulsion (LE) on apoptosis induced by a toxic dose of verapamil in H9c2 cells and to elucidate the associated cellular mechanism. The effects of LE alone and combined with an inhibitor on the decreases in cell counts and viability induced by verapamil and diltiazem were examined using the MTT assay. The effects of verapamil alone, combined LE and verapamil treatment, and combined inhibitor, LE and verapamil treatment on cleaved caspase-3, caspase-8 and Bax expression, were examined using Western blotting. The effects of verapamil alone and combined with LE on the number of TUNEL-positive H9c2 cells were also examined. LE attenuated the decreases in cell counts and viability induced by verapamil and diltiazem. However, the magnitude of the LE-mediated attenuation of decreased cell viability was enhanced by verapamil compared with diltiazem treatment. Naloxone, naltrindole hydrochloride, LY294002 and MK-2206 inhibited the LE-mediated attenuation of increased cleaved caspase-3 and caspase-8 expression induced by verapamil. LE attenuated the increase in the number of TUNEL-positive cell induced by verapamil. These results suggest that LE attenuates apoptosis induced by verapamil via activation of the delta-opioid receptor, phosphoinositide 3-kinase and Akt.

Lipid emulsion inhibits vasodilation induced by a toxic dose of bupivacaine by suppressing bupivacaine-induced PKC and CPI-17 dephosphorylation but has no effect on vasodilation induced by a toxic dose of mepivacaine.

BACKGROUND: The goal of this in vitro study was to investigate the effect of lipid emulsion on vasodilation caused by toxic doses of bupivacaine and mepivacaine during contraction induced by a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), in an isolated endothelium-denuded rat aorta. METHODS: The effects of lipid emulsion on the dose-response curves induced by bupivacaine or mepivacaine in an isolated aorta precontracted with PDBu were assessed. In addition, the effects of bupivacaine on the increased intracellular calcium concentration ([Ca2+]i) and contraction induced by PDBu were investigated using fura-2 loaded aortic strips. Further, the effects of bupivacaine, the PKC inhibitor GF109203X and lipid emulsion, alone or in combination, on PDBu-induced PKC and phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) was examined by western blotting. RESULTS: Lipid emulsion attenuated the vasodilation induced by bupivacaine, whereas it had no effect on that induced by mepivacaine. Lipid emulsion had no effect on PDBu-induced contraction. The magnitude of bupivacaine-induced vasodilation was higher than that of the bupivacaine-induced decrease in [Ca2+]i. PDBu promoted PKC and CPI-17 phosphorylation in aortic VSMCs. Bupivacaine and GF109203X attenuated PDBu-induced PKC and CPI-17 phosphorylation, whereas lipid emulsion attenuated bupivacaine-mediated inhibition of PDBu-induced PKC and CPI-17 phosphorylation. CONCLUSIONS: These results suggest that lipid emulsion attenuates the vasodilation induced by a toxic dose of bupivacaine via inhibition of bupivacaine-induced PKC and CPI-17 dephosphorylation. This lipid emulsion-mediated inhibition of vasodilation may be partly associated with the lipid solubility of local anesthetics.

Mepivacaine attenuates vasodilation induced by ATP-sensitive potassium channels in rat aorta.

The goal of this in vitro study was to investigate the effect of mepivacaine on vasodilation induced by the ATP-sensitive potassium (KATP) channel opener levcromakalim in isolated endothelium-denuded rat aortas. The effects of mepivacaine and the KATP channel inhibitor glibenclamide, alone or in combination, on levcromakalim-induced vasodilation were assessed in the isolated aortas. The effects of mepivacaine or combined treatment with a protein kinase C (PKC) inhibitor, GF109203X, and mepivacaine on this vasodilation were also investigated. Levcromakalim concentration-response curves were generated for isolated aortas precontracted with phenylephrine or a PKC activator, phorbol 12,13-dibutyrate (PDBu). Further, the effects of mepivacaine and glibenclamide on levcromakalim-induced hyperpolarization were assessed in rat aortic vascular smooth muscle cells. Mepivacaine attenuated levcromakalim-induced vasodilation, whereas it had no effect on this vasodilation in isolated aortas pretreated with glibenclamide. Combined treatment with GF109203X and mepivacaine enhanced levcromakalim-induced vasodilation compared with pretreatment with mepivacaine alone. This vasodilation was attenuated in aortas precontracted with PDBu compared with those precontracted with phenylephrine. Mepivacaine and glibenclamide, alone or in combination, attenuated levcromakalim-induced membrane hyperpolarization. Taken together, these results suggest that mepivacaine attenuates vasodilation induced by KATP channels, which appears to be partly mediated by PKC.

Mepivacaine-induced intracellular calcium increase appears to be mediated primarily by calcium influx in rat aorta without endothelium.

BACKGROUND: Mepivacaine induces contraction or decreased blood flow both in vivo and in vitro. Vasoconstriction is associated with an increase in the intracellular calcium concentration ([Ca(2+)]i). However, the mechanism responsible for the mepivacaine-evoked [Ca(2+)]i increase remains to be determined. Therefore, the objective of this in vitro study was to examine the mechanism responsible for the mepivacaine-evoked [Ca(2+)]i increment in isolated rat aorta. METHODS: Isometric tension was measured in isolated rat aorta without endothelium. In addition, fura-2 loaded aortic muscle strips were illuminated alternately (48 Hz) at two excitation wavelengths (340 and 380 nm). The ratio of F340 to F380 (F340/F380) was regarded as an amount of [Ca(2+)]i. We investigated the effects of nifedipine, 2-aminoethoxydiphenylborate (2-APB), gadolinium chloride hexahydrate (Gd(3+)), low calcium level and Krebs solution without calcium on the mepivacaine-evoked contraction in isolated rat aorta and on the mepivacaine-evoked [Ca(2+)]i increment in fura-2 loaded aortic strips. We assessed the effect of verapamil on the mepivacaine-evoked [Ca(2+)]i increment. RESULTS: Mepivacaine produced vasoconstriction and increased [Ca(2+)]i. Nifedipine, 2-APB and low calcium attenuated vasoconstriction and the [Ca(2+)]i increase evoked by mepivacaine. Verapamil attenuated the mepivacaine-induced [Ca(2+)]i increment. Calcium-free solution almost abolished mepivacaine-induced contraction and strongly attenuated the mepivacaineinduced [Ca(2+)]i increase. Gd(3+) had no effect on either vasoconstriction or the [Ca(2+)]i increment evoked by mepivacaine. CONCLUSIONS: The mepivacaine-evoked [Ca(2+)]i increment, which contributes to mepivacaine-evoked contraction, appears to be mediated mainly by calcium influx and partially by calcium released from the sarcoplasmic reticulum.

Mepivacaine-induced contraction involves phosphorylation of extracellular signal-regulated kinase through activation of the lipoxygenase pathway in isolated rat aortic smooth muscle.

Mepivacaine is an aminoamide local anesthetic with an intermediate duration that intrinsically produces vasoconstriction both in vivo and in vitro. This study investigated the arachidonic acid metabolic pathways involved in mepivacaine-induced contraction, and elucidated the associated cellular mechanism with a particular focus on extracellular signal-regulated kinase (ERK) in endothelium-denuded rat aorta. Isolated rat thoracic aortic rings were suspended for isometric tension recording. Cumulative mepivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: quinacrine dihydrochloride, nordihydroguaiaretic acid, phenidone, AA-861, indomethacin, NS-398, SC-560, fluconazole, PD 98059, and verapamil. Mepivacaine-induced ERK phosphorylation, 5-lipoxygenase (5-LOX) expression, and cyclooxygenase (COX)-2 expression in rat aortic smooth muscle cells were detected by Western blot analysis in the presence or absence of inhibitors. Mepivacaine produced tonic contraction in isolated endothelium-denuded rat aorta. Quinacrine dihydrochloride, nordihydroguaiaretic acid, phenidone, AA-861, NS-398, PD 98059, and verapamil attenuated mepivacaine-induced contraction in a concentration-dependent manner. However, fluconazole had no effect on mepivacaine-induced contraction. PD 98059, quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone, and indomethacin attenuated mepivacaine-induced ERK phosphorylation. Mepivacaine upregulated 5-LOX and COX-2 expression. These results suggest that mepivacaine-induced contraction involves ERK activation, which is primarily mediated by the 5-LOX pathway and in part by the COX-2 pathway.

A Patient with Kikuchi's Disease: What Should Pain Clinicians Do?

Kikuchi's disease (KD) is an idiopathic and self-limiting necrotizing lymphadenitis that predominantly occurs in young females. It is common in Asia, and the cervical lymph nodes are commonly involved. Generally, KD has symptoms and signs of lymph node tenderness, fever, and leukocytopenia, but there are no reports on treatment for the associated myofacial pain. We herein report a young female patient who visited a pain clinic and received a trigger point injection 2 weeks before the diagnosis of KD. When young female patients with myofascial pain visit a pain clinic, doctors should be concerned about the possibility of KD, which is rare but can cause severe complications.