Airway eosinophil migration into lymph nodes in mice depends on leukotriene C4.

BACKGROUND: We previously demonstrated in mice that airway eosinophils traffic from the airway lumen into lung-draining paratracheal lymph nodes. However, mechanisms whereby eosinophils traverse from the lungs and home to paratracheal lymph nodes remain unclear. We investigated roles of cysteinyl leukotrienes in mediating eosinophil trafficking from lungs to paratracheal lymph nodes. METHODS: The expression of CCR7 was determined by flow cytometry. Transwell assays were used to test chemotactic responses of leukotriene C4 synthase-deficient and control airway eosinophils to the chemokine CCL19 ex vivo. Eosinophils from the spleens of IL-5 transgenic mice, fluorescently labeled ex vivo, were intratracheally injected into ovalbumin-sensitized and ovalbumin aerosol-challenged leukotriene C4 synthase-deficient and control mice. Eosinophils were identified by microscopy and flow cytometry in the lungs and paratracheal lymph nodes. RESULTS: Mouse eosinophils expressed CCR7, the receptor for CCL19, and responded chemotactically to CCL19. Leukotriene C4 synthase-deficient eosinophils exhibited impaired chemotaxis to CCL19 that was restored by exogenous leukotriene C4 . The migration of intratracheally injected eosinophils into paratracheal lymph nodes from distal alveolar lung was diminished in leukotriene C4 synthase-deficient mice compared with wild-type mice, with increased retention of eosinophils in the lungs of leukotriene C4 synthase-deficient mice. Exogenous administration of leukotriene C4 restored trafficking of eosinophils to paratracheal lymph nodes in leukotriene C4 synthase-deficient mice. CONCLUSIONS: Our findings that cysteinyl leukotrienes are involved in regulating airway and lung eosinophil migration into paratracheal lymph nodes identify previously unrecognized roles for the cysteinyl leukotrienes in regulating the pulmonary trafficking of eosinophils in experimental allergic asthma.

Editor's Choice - Subsequent Results for Arch Aneurysm Repair with Inner Branched Endografts.

OBJECTIVES: The aim was to evaluate the current results of aortic arch aneurysm repair using inner branched endografts performed in three high volume aortic endovascular centers and to compare them to the pioneering global experience with this technology. METHODS: Included patients underwent repair of aortic arch aneurysms >55 mm in diameter using inner branched endograft technology between April 2013 and November 2014. All patients were deemed unfit for open surgery. Inner branches were designed to perfuse the brachiocephalic trunk and the left common carotid artery in all cases. A left subclavian artery (LSA) revascularization was performed prior to the arch endovascular repair. Data were collected retrospectively in an electronic database. Parameters included length of procedure, fluoroscopy time, contrast volume, technical success, presence of endoleaks, early and late complications, and mortality. RESULTS: Twenty-seven patients were included in the study. Technical success was achieved in all cases. No patients died during the 30 day post-operative period. Early neurologic events included two major strokes (7.4%) and one minor stroke (3.7%). Transient spinal cord ischemia with full recovery was observed in two patients (7.4%). Four patients (14.8%) underwent early (<30 day) re-interventions; these were for an access complication, an ischemic limb and exploration of the left ventricle through a sternotomy in two patients. During follow up (median 12 months), one patient (3.7%) died from a remote thoraco-abdominal aneurysm rupture. There were three Type 2 endoleaks (11.1%). Two re-interventions (7.4%) were performed, one to treat a Type 2 endoleak and one to treat a septic false aneurysm. A significant decrease in overall mortality was observed when comparing patients from the early experience with patients from the current report. CONCLUSIONS: The early outcomes associated with this technology are favorable. Branched endografting of aortic arch aneurysms should be considered in patients unfit for open surgery.

Microwave endometrial ablation for hypermenorrhea treatment: a new era in Japan.

An estimated six million women in Japan suffer from excessive menstruation and the treatment of this disorder has been undergoing dramatic changes recently. In April 2012, microwave endometrial ablation (MEA) was approved for insurance coverage as a K863-3: a hysteroscopic endometrial ablation (17,810 points). Since the introduction of MEA to Shimane University Hospital in August 2007, authors (KN, KM) have performed the procedure in 96 patients with excessive menstruation. They have also evaluated its safety and its efficacy, not only by comparing it to the existing surgical treatment but by quantifying patients' satisfaction levels and symptom improvement. The authors conclude that MEA is a safe, effective, a low-cost treatment, and they recommend that it be considered as a standard treatment for conservative therapy-resistant excessive menstruation.

Two-dimensional crystallization conditions of human leukotriene C4 synthase requiring adjustment of a particularly large combination of specific parameters.

Human leukotriene C(4) synthase (LTC(4)S) forms highly ordered two-dimensional (2D) crystals under specific reconstitution conditions. It was found that control of a larger number of parameters than is usually observed for 2D crystallization of membrane proteins was necessary to induce crystal formation of LTC(4)S. Here, we describe the parameters that were optimized to yield large and well-ordered 2D crystals of LTC(4)S. Careful fractioning of eluates during the protein purification was essential for obtaining crystals. While the lipid-to-protein ratio was critical in obtaining order, four parameters were decisive in inducing growth of crystals that were up to several microns in size. To obtain a favorable diameter, salt, temperature, glycerol, and initial detergent concentration had to be controlled with great care. Interestingly, several crystal forms could be grown, namely the plane group symmetries of p2, p3, p312, and two different unit cell sizes of plane group symmetry p321.

Vacuum-assisted closure for bilateral severe ischemic foot after revascularization: a case report.

Vacuum-assisted closure (VAC) therapy is a unique system that helps promote wound healing. We report a case of severe ischemic foot in which VAC therapy markedly improved wound healing. A 73-year-old man underwent left axillopopliteal bypass and left 3rd, 4th , and 5th digital amputations for gangrene. Although his amputation stumps were complicated with methicillin-resistant Staphylococcus aureus (MRSA) infection, the stumps were successfully healed by VAC. He also had gangrene in his right 1st toe, which could not healed by VAC alone, and we performed right femoropopliteal bypass and right 1st digital amputation. The stump with MRSA infection was also successfully healed by VAC. Histopathologic examination revealed a lot of microvessels in the increased granulation tissue.

[Vacuum-assisted closure therapy for the treatment of sternal wound infection after cardiac surgery].

Sternal wound infection is still one of the critical and challenging complications after cardiac surgery. Vacuum-assisted closure (VAC) therapy is a unique and simple system that helps promote wound healing. We report 3 cases with the sternal wound infection after cardiac surgery, in which VAC therapy was applied between January, 2005 and April, 2007. Two of them had good response to VAC therapy and had their wound healed after 3 and 5 weeks, respectively. However, the remaining case, in which bilateral internal thoracic artery had been taken down for coronary artery bypass grafting (CABG) and osteomyelitis of the sternum was not well controlled, did not respond to VAC therapy. Our results suggested that VAC might facilitate wound healing of the patients with sternal wound infection only after abscess was drained and opened, while it might not be useful for the patents with osteomyelitis.

Cysteinyl leukotrienes mediate the enhancing effects of indomethacin and aspirin on eosinophil production in murine bone marrow cultures.

BACKGROUND: Prostaglandin E(2) (PGE(2)) suppresses, while indomethacin and aspirin enhance, eosinophil production in murine liquid bone-marrow cultures. Because cysteinyl leukotrienes (cys-LTs) enhance human eosinophil colony formation, we investigated whether the effects of indomethacin and aspirin on murine bone-marrow were due to blockade of PGE(2) production alone, or involved further promotion of cys-LTs production/signalling. EXPERIMENTAL APPROACH: BALB/c liquid bone-marrow cultures were established with IL-5, alone or associated with indomethacin, aspirin, or cys-LTs. The effects of preventing cys-LT production or signalling were assessed. KEY RESULTS: Indomethacin and aspirin counteracted the suppression of eosinophil production by exogenous PGE(2). LTD(4), LTC(4) and LTE(4) enhanced IL-5-dependent eosinophil production and further counteracted the effect of exogenous PGE(2). The 5-lipoxygenase activating protein (FLAP) inhibitor, MK886, a leukotriene synthesis inhibitor, zileuton, the CysLT(1) receptor antagonists, MK571 and montelukast, or inactivation of the LTC(4) synthase gene, abolished effects of indomethacin and aspirin. MK886 and zileuton were ineffective but MK571 and montelukast were effective, against LTD(4). Indomethacin, aspirin and LTD(4) failed to enhance eosinophil production in bone-marrow from CysLT1 receptor-deficient mice. Indomethacin, aspirin and LTD(4) no longer counteracted the effects of exogenous PGE(2) in the presence of MK571 and montelukast. MK886, MK571 and montelukast had no effect by themselves, or in association with PGE(2). CONCLUSIONS AND IMPLICATIONS: Dependence on the FLAP/5-lipoxygenase/LTC(4) synthase pathway and receptor signalling shows that cyclo-oxygenase inhibitors act here through endogenous cys-LTs. While PGE(2) does not act by suppressing cys-LT production, cys-LTs override PGE(2) signalling. Eosinophil production is therefore coordinately regulated by both pathways.

The six-year results of a combined surgical and endovascular repair for thoracoabdominal aortic aneurysm involving the visceral arteries.

The authors report a case of a combined endovascular and open repair (hybrid procedure) for a mycotic thoracoabdominal aneurysm (TAAA) including its 6-year result. A 72-year-old man with diabetes mellitus, old brain infarction and moderate aortic stenosis was transferred to the hospital because of obstinate fever and back pain, The initial computed tomography (CT) scan revealed giant (TAAA), and from the laboratory findings, the white blood cell and C-reactive protein (CRP) were significantly elevated 12,400/mm3 and 23.9 mg/dL respectively. Based on the CT and laboratory findings, a mycotic TAAA was highly suspected. After the remission of inflammation, graft replacement with reconstruction of celiac trunk (CA) and superior mesenteric artery (SMA) was performed via spiral incision under extracorporeal circulation. two months after the first operation, the patient complained about his back pain again. CT showed a pseudoaneurysm which formed at the distal anastomotic site. A hybrid procedure was deemed to be the most appropriate for such patient who needs a second operation. First bilateral renal artery bypass (ilio-renal artery bypass) were done using the saphenous vein grafts (SVGs). Following bypass grafting to renal arteries, endovascular aneurysm repair was performed with handmade stent-graft which was fabricated using a self-expanding "Z" stent and woven Dacron graft. The postoperative course was uneventful, and follow-up CT showed the aneurysm to have shrunk with no endoleaks. At six months after hybrid procedure, the shrinkage of the aneurysm sac and the patency of the graft to renal arteries were confirmed by a CT scan. A hybrid procedure is considered to be useful and feasible for the poor surgical candidate with severe comorbidities, hostile abdomen and a complex anatomy. The long-term results of this hybrid procedure is considered to be promising.

Isolation of a mammalian homologue of a fission yeast differentiation regulator.

In the fission yeast Schizosaccharomyces pombe the nrd1(+) gene encoding an RNA binding protein negatively regulates the onset of differentiation. Its biological role is to block differentiation by repressing a subset of the Ste11-regulated genes essential for conjugation and meiosis until the cells reach a critical level of nutrient starvation. By using the phenotypic suppression of the S. pombe temperature-sensitive pat1 mutant that commits lethal haploid meiosis at the restrictive temperature, we have cloned ROD1, a functional homologue of nrd1(+), from rat and human cDNA libraries. Like nrd1(+), ROD1 encodes a protein with four repeats of typical RNA binding domains, though its amino acid homology to Nrd1 is limited. When expressed in the fission yeast, ROD1 behaves in a way that is functionally similar to nrd1(+), being able to repress Ste11-regulated genes and to inhibit conjugation upon overexpression. ROD1 is predominantly expressed in hematopoietic cells or organs of adult and embryonic rat. Like nrd1(+) for fission yeast differentiation, overexpressed ROD1 effectively blocks both 12-O-tetradecanoyl phorbol-13-acetate-induced megakaryocytic and sodium butyrate-induced erythroid differentiation of the K562 human leukemia cells without affecting their proliferative ability. These results suggest a role for ROD1 in differentiation control in mammalian cells. We discuss the possibility that a differentiation control system found in the fission yeast might well be conserved in more complex organisms, including mammals.

Cyclin G: a new mammalian cyclin with homology to fission yeast Cig1.

A new gene encoding a cyclin-like protein has been isolated from a rat fibroblast cDNA library by cross-hybridization with a mixture of c-src family proto-oncogene kinase domains as a probe. This putative cyclin, called cyclin G, contains a typical cyclin box at the N-terminus but no apparent 'destruction box' or 'PEST' sequence. Interestingly, in its C-terminus region, it has a sequence homologous with a tyrosine phosphorylation site of the epidermal growth factor receptor. Although this cyclin is phylogenetically related to HCS26 of Saccharomyces cerevisiae, it most resembles Cig1, a B-type cyclin, of Schizosaccharomyces pombe, which has been suggested to act at the G1/S phase of the cell cycle. Cyclin G mRNA is induced within 3 h after growth stimulation and remains elevated with no apparent cell cycle dependency, indicating its close association with growth stimuli but not with the cell cycle.

Enantiomeric specificity at the deacylation process of tryptic catalysis.

Trypsin-specific substrate analogs, 'inverse substrates', carrying chiral acyl group were synthesized. Kinetic analysis of the trypsin-catalyzed hydrolysis of these substrates revealed that the deacylation process is also appropriate for discrimination between enantiomers. The enantiomeric preference during the deacylation process was analyzed by comparing the hydrolytic rates corresponding to eight enantiomeric pairs of the substrates. The spatial requirement of the enzyme active site for catalytic efficiency is discussed, based on the steric characteristics of the optically active acyl residues.

A site-directed antibody that inhibits phosphorylation of the rat-brain sodium channel by cyclic-AMP-dependent protein kinase.

Antibodies were raised against three peptides corresponding to the potential protein phosphorylation sites of rat-brain sodium channels by the cAMP-dependent protein kinase (PKA). One of the antibody against sequence (C561-575) reacted to the channel molecule. This immunoreaction occurred in a sequence-specific manner, as it was inhibited by the antigen peptide itself but not inhibited by two other peptides. Although PKA phosphorylates two synthetic peptides, C561-575 and C681-689, of the three, anti-(C561-575) antibody can only inhibit the phosphorylation of peptide (C561-575). PKA catalyzed the incorporation of 3.1-3.5 mol of phosphates into the alpha subunit of the purified sodium channel. The anti-(C561-575) antibody inhibited the channel phosphorylation by 40%. Digestion of the phosphorylated sodium channel with lysyl endoproteinase yielded four major phosphorylated fragments of 3.5, 5.0, 7.0, and 10 kDa. However, similar digestion of the channel that was phosphorylated in the presence of anti-(C561-575) antibody did not yield the phosphorylated fragment of 3.5 kDa and gave the 7.0 kDa fragment in reducing yield. Inspection of these phosphorylated fragments by the predicted sizes of the peptide fragments containing the five potential phosphorylation sites gives a conclusion that anti-(C561-575) antibody inhibits the phosphorylation on Ser-573 completely, and on either Ser-610 or Ser-623 partially, probably due to their proximity orientation in the tertiary structure.

Comparison of Streptomyces griseus and bovine trypsin by active site analysis using fluorescent acyl groups.

The preparation of fluorescence labeled acyl enzymes (Streptomyces griseus trypsin) was successfully carried out using specific trypsin substrates, 'inverse substrates'. The topographical analysis of the structures of the area around the active site was carried out by measuring the fluorescence spectra of the acyl enzyme preparations and these results were compared with those of bovine trypsin. It was found that the polarity of the active site vicinity at pH 5 was similar to that of bovine trypsin, whereas considerable differences were noticed at lower pH as a result of pH-induced transformation. Conformational changes of the active site induced by the interaction with the specific ligand were analyzed from the fluorescence spectra. In these responses the two enzymes were quite distinguishable. The two enzymes active sites were also different in the energy transfer experiments. The spatial arrangements of the catalytic residues relative to the intrinsic tryptophan residues were suggested to be substantially different for the two enzymes.

Specific spin-labeling at trypsin active site. Application of 'inverse substrate' to the structural analysis of the active site.

Specific and reversible spin-labeling of trypsin (EC 3.4.21.4) active site was carried out by 'inverse substrate', 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl and 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolinyloxyl-p-amidinophenyl esters. The paramagnetic resonance spectra of the labeled trypsin in the form of acyl enzyme intermediate were investigated. The 2T value, separation of the outer hyperfine extrema, was observed to be sensitive to pH of the medium. These results are discussed in terms of pH-dependent conformational change at the vicinity of active site.

'Inverse' substrates for butyrylcholinesterase.

1. 'Inverse'-type substrates for butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8), i.e., p- and o-nitrophenyl esters of (3-carboxypropyl)-trimethylammonium iodide, (4-carboxybutyl)trimethylammonium iodide and (5-carboxypentyl)trimethylammonium iodide were prepared, and their kinetic parameters for butyrylcholinesterase-catalyzed hydrolysis were determined. 2. The hydrolysis of these 'inverse'-type substrates were found to proceed through specific binding with the enzyme and efficient production of acyl enzyme intermediates, a pathway essentially identical with that followed by choline esters, normal type substrates.

Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry. III. Changes in the conformation of tropomyosin associated with functional states.

The local conformational changes in the tropomyosin molecule under various conditions were studied by means of fluorimetry using SH-directed fluorescent dyes, N-(1-anilinonaphthyl-4)maleimide (ANM) and N-(3-pyrene)maleimide (PRM). 1. The fluorescence intensity, polarization and the emmission maximum of ANM-tropomyosin were found to be susceptible to ionic strength, but in different ways. The changes in these parameters suggest that the fluorescence-labeled sulfhydryl group or groups become more buried in a hydrophobic internal region by salt-induced depolymerization of aggregate and by adding F-actin to tropomyosin. 2. Titration of the labeled tropomyosin with F-actin revealed a cooperative nature in ANM labeling and a simple saturation kinetics in PRM labeling. The dissociation constant of F-actin to PRM-tropomyosin was calculated to be 5.8-10(-6) M. 3. Temperature dependence of the fluorescence polarization showed a thermal transition in the conformation of ANM- or PRM-tropomyosin at around 30 degrees C. Flexibility or segmental motion of the region containing the fluorophore was suppressed significantly on adding troponin and markedly on adding F-actin. 4. Measurements of the quantum yield and polarization of the ANM-tropomyosin-F-actin complex suggested that troponin strengthened the binding between the two proteins and that Ca2+ reversed this effect.

Fluorescent thiol reagents. VI. N-(1-Anilinonaphthyl-4)maleimide; a fluorescent hydrophobic probe directed to thiol groups in protein.

N-(1-Anilinonaphthyl-4)maleimide (ANM) (m.p. 207-208.5 degrees C), nonfluorescent, reacts selectively with thiols to give addition products which are strongly fluorescent (excitation maximum 355 nm, emission maximum 448 nm, in ethanol). 0.7 mole of ANM was introduced into thiol groups of egg albumin without modifying any other amino acid residues. In the fluorescence spectra of the reaction products of ANM with thiols, the quantum yields increase and the emission maxima shift towards the blue as the solvent polarity decreases. This remarkable solvent dependence was described in terms ofZ value of the solvents. ANM is thus expected to be a useful fluorescent hydrophobic probe directed to thiol groups in protein.

A topographical study of the electroplax sodium channel with site-directed antibodies.

Three peptides corresponding to residues (13-23C), (Y1419-1431), and (1809-1820) of the electric eel sodium channel have been synthesized and used to raise antisera in rabbits. All the antibodies produced specifically recognized the corresponding peptides in an ELISA assay. However, their avidities to the channel protein were different. Two antibodies, against the sequence (1809-1820) and (13-23C) which are directed to the C-terminus region and to the adjacent portion of N-terminus, respectively, recognized the 250 kDa channel protein in the immunoblot and an ELISA assay. Binding of the two antibodies to the sodium channel in oriented electroplax membrane vesicles was increased 4-5-fold after permeabilizing the vesicles with 0.01% saponin, implying cytoplasmic orientation for the two peptides. The cytoplasmic orientation of the N- and C-terminal regions were further confirmed by immunogold electron microscopy. By contrast, antibody raised against the sequence (Y1419-1431) which has been proposed to be the transmembrane segment S4 of internal repeat IV, did not react with the channel protein not only after the saponin treatment but also even under the immunoblot conditions following SDS-PAGE. The antibody could recognize fragments of the channel protein after digestion with lysyl endoproteinase, suggesting that the region may apparently form a strictly well-ordered conformation in the transmembrane part of the channel molecule.

Anhydrosubtilisin-catalyzed peptide synthesis.

Anhydrosubtilisin was found to be a good catalyst for the peptide synthesis though its hydrolytic activity was fully inhibited. Amino acid (peptide) p-chlorophenyl ester (acyl donor) was coupled with amino acid (peptide) amide in the presence of the modified enzyme. The method did not afford by-products resulting from the hydrolysis of the acyl donor. This is the most advantageous characteristic of the method, since the hydrolysis of the acyl donor is unavoidable for the methods using catalytically active proteases. Anhydrosubtilisin was further shown to be useful for the fragment condensation of peptides.

Differentiation of tryptic enzymes based on enantiomeric specificity at the deacylation step.

The chiral specificity of tryptic enzymes at their deacylation step has been determined for the first time by virtue of 'inverse substrates' carrying optically active acyl groups. Differentiation of tryptic enzymes was also successful with these substrates. The stability of acyl-thrombin is substantially higher than those of trypsin and plasmin when the (S)-dihydrocoumarilyl group is applied. This is in contrast to the result with its (R)-antipode in which all three enzymes are not differentiated. The use of chiral p-amidinophenyl esters is proposed as a versatile methodology for the design of specific inhibitors capable of discriminating among tryptic enzymes.