gamma-Tocopherol nebulization by a lipid aerosolization device improves pulmonary function in sheep with burn and smoke inhalation injury

Fire accident victims who sustain both thermal injury to skin and smoke inhalation have gross evidence of systemic and pulmonary oxidant damage and acute lung injury. We hypothesized that gamma-tocopherol (gT), a reactive O(2) and N(2) scavenger, when delivered into the airway, would attenuate lung injury induced by burn and smoke inhalation. Acute lung injury was induced in chronically prepared, anesthetized sheep by 40% total burn surface area, third-degree skin burn and smoke insufflation (48 breaths of cotton smoke, <40 degrees C). The study groups were: (1) Sham (not injured, flaxseed oil (FO)-nebulized, n=6); (2) SA-neb (injured, saline-nebulized, n=6); (3) FO-neb (injured, FO-nebulized, n=6); and (4) gT+FO-neb (injured, gT and FO-nebulized, n=6). Nebulization was started 1 h postinjury, and 24 ml of FO with or without gT (51 mg/ml) was delivered into airways over 47 h using our newly developed lipid aerosolization device (droplet size: 2.5-5 microm). The burn- and smoke inhalation-induced pathological changes seen in the saline group were attenuated by FO nebulization; gT addition further improved pulmonary function. Pulmonary gT delivery along with a FO source may be a novel effective treatment strategy in management of patients with acute lung injury.     

Prasinophyte green algae <Emphasis Type="Italic">Tasmanites</Emphasis> and problematic fossils in the Upper Vendian Biota of the Zavkhan Basin,western Mongolia

A new species of spheromorphic microfossils of Tasmanites with a characteristically thick cover is established in the Zavkhan association of algae, microfossils, and problematic Upper Vendian organisms from the upper part of the siliciclastic–carbonate section of the Tsagaanolom Formation (632 ± 14 Ma) of the Zavkhan Basin in western Mongolia. Representatives of this genus are widespread in the Phanerozoic beds and their accumulations are recorded in the Domanik facies and near hydrocarbon deposits.     

Identification and characterization of a xyloglucan-specific family 74 glycosyl hydrolase from Streptomyces coelicolor A3(2)

The sco6545 gene of Streptomyces coelicolor A3(2) was nominated as a putative cellulase with 863 mature-form amino acids (90.58 kDa). We overexpressed and purified Sco6545 and demonstrated that the protein is not a cellulase but a xyloglucan-specific glycosyl hydrolase which cleaves xyloglucan at unbranched glucose residues.     

Carboxyhemoglobin formation following smoke inhalation injury in sheep is interrelated with pulmonary shunt fraction

Carboxyhemoglobin (COHb) formation is triggered by the inducible isoform of heme oxygenase (HO-1) catalyzing carbon monoxide (CO) production through breakdown of heme molecules, exposure to CO or both. In the setting of CO poisoning, COHb is regarded as a reliable marker characterizing both severity of injury and efficacy of treatment strategies. This study was designed as a prospective laboratory experiment to elucidate potential interdependencies between COHb generation, oxygenation, and pulmonary shunt fraction (Qs/Qt) in an ovine model of smoke inhalation injury. Chronically instrumented ewes (n=15) were repeatedly subjected to cotton smoke (4 x 12 breaths) according to an established protocol. This approach resulted in a progressive increase in COHb formation that was interrelated with the degree of Qs/Qt (P<0.001) and inversely correlated with both arterial and mixed venous HbO(2) saturation (r=-0.96 and -0.93). Although the arteriovenous COHb gradient successively decreased over time, COHb determined in venous blood underestimated the arterial content.     

Inhibition of mitochondrial membrane bound-glutathione transferase by mitochondrial permeability transition inhibitors including cyclosporin A

AimsEffect of mitochondrial permeability transition (MPT) inhibitors on mitochondrial membrane-bound glutathione transferase (mtMGST1) activity in rat liver was investigated in vitro.Main methodsWhen mitochondria were incubated with MPT inhibitors, mtMGST1 activity was decreased dose dependently and their 50% inhibition concentration (IC₅₀) were 1.2 μM (cyclosporin A; CsA), 31 μM (bongkrekic acid; BKA), 1.8 mM (ADP), and 3.2 mM (ATP). The decrease of mtMGST1 activity by the MPT inhibitors was not observed in the presence of detergent Triton X-100. On the contrary, mtMGST1 inhibition by GST inhibitors such as cibacron blue (IC₅₀, 4.2 μM) and S-hexylglutathione (IC₅₀, 480 μM) was not affected in the presence of detergent. Although mtMGST1 resides in both the inner (IMM) and outer mitochondrial membranes (OMM), only mtMGST1 in the IMM was inhibited by the MPT inhibitors in the absence of detergent. GST inhibitors decreased mtMGST1 activity both in the IMM and OMM regardless of the presence or absence of detergent. Cytosolic GSTs and microsomal MGST1 were not inhibited by the MPT inhibitors.Key findingsThese results indicate that mtMGST1 is inhibited by MPT inhibitors through membrane components, not directly by the inhibitors.SignificanceSince CsA binds to cyclophilin D (Cyp-D) in the mitochondrial matrix whereas BKA or ADP binds to adenine nucleotide translocator (ANT) in the IMM, it was suggested that mtMGST1 in the IMM interacts with Cyp-D/ANT and the binding of MPT inhibitors to Cyp-D or ANT causes their conformational change followed by an alteration of mtMGST1 conformation, resulting in decreasing mtMGST1 activity.     

Specific inhibition of nitric oxide synthases at different time points in a murine model of pulmonary sepsis

Excessive production of nitric oxide (NO) by NO synthase (NOS) and a subsequent oxidative stress reaction are thought to be critically involved in the pathophysiology of sepsis. Previous studies suggested that NO production by neuronal NOS (nNOS) and inducible NOS (iNOS) is implemented in the disease process at different time points after the injury. Here we tested the roles of selective pharmacological inhibition of nNOS and iNOS at different time points in a murine model of pulmonary sepsis. The injury was induced by intranasal administration of live Pseudomonas aeruginosa (3.2 × 10⁷ colony-forming units) in C57BL/6 wild-type mice. The animals received no treatment (control) or treatment with a specific nNOS inhibitor (4 or 8 h), iNOS inhibitor (4 or 8 h), or non-specific NOS inhibitor (4 or 8 h). In controls, the injury was associated with excessive releases of pro-inflammatory cytokines in the plasma, enhanced tissue lipid peroxidation, and decreased survival. Non-specific NOS inhibition at either time point did not influence survival and was not further investigated. While nNOS inhibition at 4 h was associated with a trend toward improved survival and significantly reduced contents of lung nitrite/nitrate (NO_x) and liver malondialdehyde, the blockade of nNOS at 8 h had no effect on these parameters. In contrast, early iNOS inhibition was associated with a trend toward decreased survival and no effects on lung NO_x and liver malondialdehyde contents, whereas later iNOS blockade was associated with decreased malondialdehyde content in liver homogenates. In conclusion, pulmonary sepsis in mice may be beneficially influenced by specific pharmacological nNOS inhibition at an earlier time point and iNOS inhibition at a later time points post-injury. Future investigations should identify the time changes of the expression and activation of NOS isoforms.     

PLU1 histone demethylase decreases the expression of KAT5 and enhances the invasive activity of the cells

Proteins SP-B and SP-C are essential to promote formation of surface-active films at the respiratory interface, but their mechanism of action is still under investigation. In the present study we have analysed the effect of the proteins on the accessibility of native, quasi-native and model surfactant membranes to incorporation of the fluorescent probes Nile Red (permeable) and FM 1-43 (impermeable) into membranes. We have also analysed the effect of single or combined proteins on membrane permeation using the soluble fluorescent dye calcein. The fluorescence of FM 1-43 was always higher in membranes containing SP-B and/or SP-C than in protein-depleted membranes, in contrast with Nile Red which was very similar in all of the materials tested. SP-B and SP-C promoted probe partition with markedly different kinetics. On the other hand, physiological proportions of SP-B and SP-C caused giant oligolamellar vesicles to incorporate FM 1-43 from the external medium into apparently most of the membranes instantaneously. In contrast, oligolamellar pure lipid vesicles appeared to be mainly labelled in the outermost membrane layer. Pure lipidic vesicles were impermeable to calcein, whereas it permeated through membranes containing SP-B and/or SP-C. Vesicles containing only SP-B were stable, but prone to vesicle-vesicle interactions, whereas those containing only SP-C were extremely dynamic, undergoing frequent fluctuations and ruptures. Differential structural effects of proteins on vesicles were confirmed by electron microscopy. These results suggest that SP-B and SP-C have different contributions to inter- and intra-membrane lipid dynamics, and that their combined action could provide unique effects to modulate structure and dynamics of pulmonary surfactant membranes and films.     

A murine model of sepsis following smoke inhalation injury

Acute lung injury (ALI) by smoke inhalation with subsequent pneumonia and sepsis represents a major cause of morbidity and mortality in burn patients. The aim of the present study was to develop a murine model of ALI and sepsis to enhance the knowledge of mechanistic aspects and pathophysiological changes in patients with these injuries. In deeply anesthetized female C57BL/6 mice, injury was induced by four sets of cotton smoke using an inhalation chamber. Afterward, live Pseudomonas aeruginosa (3.2 × 10⁷ colony-forming units) was administered intranasally. The indicated dose of bacteria was determined based on the results of a dose-response study (n = 47). The following study groups were monitored for survival over 96 h: (1) sham injury group, (2) only smoke inhalation group, (3) only bacteria group, and (4) smoke inhalation plus bacteria group. Each group included 10 mice. The survival rates were 100%, 90%, 30%, and 10%, respectively. The double hit injury was associated with excessive releases of pro-inflammatory cytokines in the plasma, and enhanced neutrophil accumulation, increased lipid peroxidation, and excessive formation of reactive nitrogen species in the lung. In mice receiving only smoke inhalation injury, no systemic cytokine release and increased lung tissue lipid peroxidation were observed. However, smoke alone significantly increased neutrophil accumulation and formation of reactive nitrogen species in lung tissue. In conclusion, bacterial pneumonia is predominantly responsible for mortality and morbidity in this novel murine model of smoke inhalation and pulmonary sepsis. Reactive oxygen and nitrogen species mediate the severity of lung injury.