The proteomic profile of circulating pentraxin 3 (PTX3) complex in sepsis demonstrates the interaction with azurocidin 1 and other components of neutrophil extracellular traps

Pentraxin 3 (PTX3), a long pentraxin subfamily member in the pentraxin family, plays an important role in innate immunity as a soluble pattern recognition receptor. Plasma PTX3 is elevated in sepsis (~200 ng/ml) and correlates with mortality. The roles of PTX3 in sepsis, however, are not well understood. To investigate the ligands of PTX3 in sepsis, we performed a targeted proteomic study of circulating PTX3 complexes using magnetic bead-based immunopurification and shotgun proteomics for label-free relative quantitation via spectral counting. From septic patient fluids, we successfully identified 104 candidate proteins, including the known PTX3-interacting proteins involved in complement activation, pathogen opsonization, inflammation regulation, and extracellular matrix deposition. Notably, the proteomic profile additionally showed that PTX3 formed a complex with some of the components of neutrophil extracellular traps. Subsequent biochemical analyses revealed a direct interaction of bactericidal proteins azurocidin 1 (AZU1) and myeloperoxidase with PTX3. AZU1 exhibited high affinity binding (K(D) = 22 ± 7.6 nm) to full-length PTX3 in a calcium ion-dependent manner and bound specifically to an oligomer of the PTX3 N-terminal domain. Immunohistochemistry with a specific monoclonal antibody generated against AZU1 revealed a partial co-localization of AZU1 with PTX3 in neutrophil extracellular traps. The association of circulating PTX3 with components of the neutrophil extracellular traps in sepsis suggests a role for PTX3 in host defense and as a potential diagnostic target.     

Long-term results of cell-free biodegradable scaffolds for in situ tissue-engineering vasculature: in a canine inferior vena cava model

We have developed a new biodegradable scaffold that does not require any cell seeding to create an in-situ tissue-engineering vasculature (iTEV). Animal experiments were conducted to test its characteristics and long-term efficacy. An 8-mm tubular biodegradable scaffold, consisting of polyglycolide knitted fibers and an L-lactide and ε-caprolactone copolymer sponge with outer glycolide and ε-caprolactone copolymer monofilament reinforcement, was implanted into the inferior vena cava (IVC) of 13 canines. All the animals remained alive without any major complications until euthanasia. The utility of the iTEV was evaluated from 1 to 24 months postoperatively. The elastic modulus of the iTEV determined by an intravascular ultrasound imaging system was about 90% of the native IVC after 1 month. Angiography of the iTEV after 2 years showed a well-formed vasculature without marked stenosis or thrombosis with a mean pressure gradient of 0.51 ± 0.19 mmHg. The length of the iTEV at 2 years had increased by 0.48 ± 0.15 cm compared with the length of the original scaffold (2-3 cm). Histological examinations revealed a well-formed vessel-like vasculature without calcification. Biochemical analyses showed no significant differences in the hydroxyproline, elastin, and calcium contents compared with the native IVC. We concluded that the findings shown above provide direct evidence that the new scaffold can be useful for cell-free tissue-engineering of vasculature. The long-term results revealed that the iTEV was of good quality and had adapted its shape to the needs of the living body. Therefore, this scaffold would be applicable for pediatric cardiovascular surgery involving biocompatible materials.     

Cyclooxygenase-Independent Neuroprotective Effects of Aspirin Against Dopamine Quinone-Induced Neurotoxicity

Prostaglandin H synthase exerts not only cyclooxygenase activity but also peroxidase activity. The latter activity of the enzyme is thought to couple with oxidation of dopamine to dopamine quinone. Therefore, it has been proposed that cyclooxygenase inhibitors could suppress dopamine quinone formation. In the present study, we examined effects of various cyclooxygenase inhibitors against excess methyl L-3,4-dihydroxyphenylalanine (L-DOPA)-induced quinoprotein (protein-bound quinone) formation and neurotoxicity using dopaminergic CATH.a cells. The treatment with aspirin inhibited excess methyl L-DOPA-induced quinoprotein formation and cell death. However, acetaminophen did not show protective effects, and indomethacin and meloxicam rather aggravated these methyl L-DOPA-induced changes. Aspirin and indomethacin did not affect the level of glutathione that exerts quenching dopamine quinone in dopaminergic cells. In contrast with inhibiting effects of higher dose in the previous reports, relatively lower dose of aspirin that affected methyl L-DOPA-induced quinoprotein formation and cell death failed to prevent cyclooxygenase-induced dopamine chrome generation in cell-free system. Furthermore, aspirin but not acetaminophen or meloxicam showed direct dopamine quinone-scavenging effects in dopamine-semiquinone generating systems. The present results suggest that cyclooxygenase shows little contribution to dopamine oxidation in dopaminergic cells and that protective effects of aspirin against methyl L-DOPA-induced dopamine quinone neurotoxicity are based on its cyclooxygenase-independent property.     

Inhomogeneous response of expiratory muscle activity to cold block of the ventral medullary surface.

We assessed the effects of cooling the ventral medullary surface (VMS) on the activity of chest wall and abdominal expiratory muscles in eight anesthetized artificially ventilated dogs after vagotomy and denervation of the carotid sinus nerves. Electromyograms (EMGs) of the triangularis sterni, internal intercostal, abdominal external oblique, abdominal internal oblique, and transversus abdominis muscles were measured with EMG of the diaphragm as an index of inspiratory activity. Bilateral localized cooling (2 x 2 mm) in the thermosensitive intermediate part of the VMS produced temperature-dependent reduction in the EMG of diaphragm and abdominal muscles. The rib cage expiratory EMGs were little affected at 25 degrees C; their amplitudes decreased at lower VMS temperatures (less than 20 degrees C) but by significantly fewer degrees than the diaphragmatic and abdominal expiratory EMGs at a constant VMS temperature. With moderate to severe cooling (less than 20 degrees C) diaphragmatic EMG disappeared, but rib cage expiratory EMGs became tonic and resumed a phasic pattern shortly before the recovery of diaphragmatic EMG during rewarming of the VMS. These results indicate that the effects of cooling the VMS differ between the activity of rib cage and abdominal expiratory muscles. This variability may be due to inhomogeneous inputs from the VMS to expiratory motoneurons or to a different responsiveness of various expiratory motoneurons to the same input either from the VMS or the inspiratory neurons.     

An Escherichia coli Mutant Having Altered D-Xylose Uptake Activity and Cloning of a Gene for D-Xylose Uptake

An Escherichia coli C600 mutant having an altered D-xylose uptake activity was isolated. The growth rate and D-xylose uptake activity of the mutant grown on the minimal medium with D-xylose at 25°C were much lower than those of the parental strain grown under the same conditions, although the activities of D-xylose-binding proteins and the enzymes involved in D-xylose metabolism were almost the same for the two strains. An uptake study on sugars at the low temperature (25°C) indicated that the mutant was deficient in D-xylose uptake activity. A gene responsible for the D-xylose uptake activity at the low temperature was isolated and cloned onto vector plasmid pBR322. The gene specifically improved the D-xylose uptake activity of the mutant at the low temperature when it was introduced into the mutant cells. Based on these results, it was suggested that another D-xylose transport system other than the D-xylose-binding protein mediated system might be functioning in E. coli cells.     

Rapid determination of isoamyl nitrite in pharmaceutical preparations by flow injection analysis with on‐line UV irradiation and luminol chemiluminescence detection

Isoamyl nitrite is used as a therapeutic reagent for cardiac angina and as an antidote for cyanide poisoning, but it is abused because of its euphoric properties. Therefore, a method to determine isoamyl nitrite is required in many fields, including pharmaceutical and forensic studies. In this study, a simple, rapid and sensitive method for the determination of isoamyl nitrite was developed using a flow injection analysis system equipped with a chemiluminescence detector and on‐line photoreactor. This method is based on on‐line ultraviolet irradiation of isoamyl nitrite and subsequent luminol chemiluminescence detection without the addition of an oxidant. A linear standard curve was obtained up to 1.0 μM of isoamyl nitrite with a detection limit (blank + 3SD) of 0.03 μM. The method was successfully applied to determine isoamyl nitrite content in pharmaceutical preparations. Copyright © 2013 John Wiley & Sons, Ltd.     

Gonadal sex differentiation and effect of rearing temperature on sex ratio in black rockfish (Sebastes schlegeli)

Temperature-dependent sex determination has been demonstrated in some species of fish, and a high temperature during the period of sex differentiation typically produces a male-dominant population. This research investigated the gonadal sex differentiation and effect of rearing temperature on the sex ratio in larval black rockfish Sebastes schlegeli, which is a viviparous species. Two types of gonads were histologically distinguishable in fish 20 mm in total length (TL). The putative ovary started forming an ovarian cavity, while the putative testis was not clearly differentiated until 51 mm TL. In a temperature-controlled experiment, the proportions of females were 45% at 10°C, 46% at 14°C, 50% at 18°C, 63% at 22°C, and 83% (significantly different from 1:1 sex ratio) at 24°C. These results suggest that morphological sex differentiation in black rockfish occurs at approximately 20 mm in TL, and it is possible that high temperatures (24°C) induce not a male- but a female-dominant population in this species.