Effect of lung-protective ventilation on severe Pseudomonas aeruginosa pneumonia and sepsis in rats

Pneumonia caused by Pseudomonas aeruginosa carries a high rate of morbidity and mortality. A lung-protective strategy using low tidal volume (V(T)) ventilation for acute lung injury improves patient outcomes. The goal of this study was to determine whether low V(T) ventilation has similar utility in severe P. aeruginosa infection. A cytotoxic P. aeruginosa strain, PA103, was instilled into the left lung of rats anesthetized with pentobarbital. The lung-protective effect of low V(T) (6 ml/kg) with or without high positive end-expiratory pressure (PEEP, 10 or 3 cmH(2)O) was then compared with high V(T) with low PEEP ventilation (V(T) 12 ml/kg, PEEP 3 cmH(2)O). Severe lung injury and septic shock was induced. Although ventilatory mode had little effect on the involved lung or septic physiology, injury to noninvolved regions was attenuated by low V(T) ventilation as indicated by the wet-to-dry weight ratio (W/D; 6.13 +/- 0.78 vs. 3.78 +/- 0.26, respectively) and confirmed by histopathological examinations. High PEEP did not yield a significant protective effect (W/D, 4.03 +/- 0.32) but, rather, caused overdistension of noninvolved lungs. Bronchoalveolar lavage revealed higher concentrations of TNF-alpha in the fluid of noninvolved lung undergoing high V(T) ventilation compared with those animals receiving low V(T). We conclude that low V(T) ventilation is protective in noninvolved regions and that the application of high PEEP attenuated the beneficial effects of low V(T) ventilation, at least short term. Furthermore, low V(T) ventilation cannot protect the involved lung, and high PEEP did not significantly alter lung injury over a short time course.     

NO reduction by nitric-oxide reductase from denitrifying bacterium Pseudomonas aeruginosa: characterization of reaction intermediates that appear in the single turnover cycle

Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N(2)O at the binuclear catalytic center consisting of high spin heme b(3) and non-heme Fe(B). The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device. In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous Fe(B)-NO and the penta-coordinated ferrous heme b(3)-NO species were observed. The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state. The time- and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N(2)O formation, in good agreement with the so-called "trans" mechanism. It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the mu-oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated. On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR.     

Influence of anesthesia on ocular effects and temperature in rabbit eyes exposed to microwaves

To investigate the effect of systemic anesthesia on ocular effects and temperature in rabbit eyes exposed to microwaves, one eye each of 43 male pigmented rabbits (Dutch, 1.8-2.2 kg) was exposed at 2.45 GHz for 60-20 min (300 mW/cm2; 108 W/kg), either under anesthesia (ketamine hydrochloride (5 mg/kg) + xylazine (0.23 mg/kg)) or without anesthesia. Changes in the anterior segment were evaluated by image analysis utilizing a Scheimpflug camera, specular microscopy, and a laser flare cell meter. Temperatures within the eye were measured during microwave exposure by a Fluoroptic thermometer. The exposed eyes showed miosis, conjunctival congestion, corneal edema, and an increase in the light scattering of the anterior shallow cortex in the pupillary area of the lens. The group under systemic anesthesia showed much stronger symptoms than those treated without anesthesia. All of the anterior ocular changes disappeared within a week. The highest temperature during exposure was in the vitreous, followed by the anterior chamber, and the retrobulbar cavity of the orbit. The ocular temperatures of the rabbits under systemic anesthesia were 2-9 degrees C higher than those without anesthesia. Body temperature showed an increase of 1 degrees C during the exposure. Acute high intensity microwave exposure temporarily induced anterior segments inflammation and lens changes. The more pronounced ocular effects in the anesthetized rabbits were associated with the significantly higher ocular temperatures in the anesthetized animals. The influence of systemic anesthesia on ocular changes should be considered.     

Stopped-flow spectrophotometric and resonance Raman analyses of aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis

On stopped-flow analysis of aliphatic aldoxime dehydratase (OxdA), a novel hemoprotein, a spectrum derived from a reaction intermediate was detected on mixing ferrous OxdA with butyraldoxime; it gradually changed into that of ferrous OxdA with an isosbestic point at 421 nm. The spectral change on the addition of butyraldoxime to the ferrous H320A mutant showed the formation of a substrate-coordinated mutant, the absorption spectrum of which closely resembled that of the above intermediate. These observations and the resonance Raman investigation revealed that the substrate actually binds to the heme in OxdA, forming a hexa-coordinate low-spin heme.     

Practical range of effective dose for Cre recombinase-expressing recombinant adenovirus without cell toxicity in mammalian cells

The site-specific recombinase Cre is valuable for regulation of gene expression not only in vitro but also in vivo. We previously reported that replication-deficient recombinant adenovirus (rAd) expressing Cre can mediate efficient and strict regulation in 100% of cultured cells. Recently, the constitutive-expression of Cre using retrovirus or lentivirus vector reportedly inhibited cell-growth, but the effect of transient Cre expression have not yet been examined. Here we showed that an excess amount of Cre produced from Cre-expressing rAd caused a deleterious effect in cells even when Cre was transiently expressed. We used three rAds carrying promoters with different activities: the SV40 early promoter (AxSVENCre), the SR alpha promoter (AxSRCre) and the CAG promoter (AxCANCre). Cell toxicity was clearly caused by Cre itself and was distinguishable from that caused by rAd virions when the cytopathic effects of these rAds were compared with that of a control virus lacking the Cre expression unit. Cre toxicity was strongly correlated with the expression level of Cre. Importantly, AxSRCre and AxCANCre gave a 60-fold range of effective MOIs ("effective range") sufficient for gene activation without causing cell toxicity from either the rAd particles or Cre itself, while AxSVENCre failed to give such a range because the expression level of Cre was too low. When Cre was tagged with a nuclear localization signal (NLS), not only its activity but also Cre toxicity was increased fourfold, and the effective range was unchanged. Therefore, AxSRNCre might be more useful to control cell toxicity from the rAd virions than AxSRCre. Cre-induced cell toxicity can be avoided by pre-examining the "effective range" using the purpose cell lines before starting experiments utilizing the experiment of Cre-expressing rAd.     

Supramolecular assemblies for the cytoplasmic delivery of antisense oligodeoxynucleotide: polyion complex (PIC) micelles based on poly(ethylene glycol)-SS-oligodeoxynucleotide conjugate

A novel cytoplasmic delivery system of antisense oligodeoxynucleotide (asODN) was developed by assembling a PEG-asODN conjugate with disulfide linkage (smart linkage) (PEG-SS-asODN) into polyion complex (PIC) micelles through the complexation with branched poly(ethylenimine) (B-PEI). The PIC micelle thus prepared showed a significant antisense effect against luciferase gene expression in HuH-7 cells, far more efficient than nonmicelle systems (asODN and PEG-SS-asODN in free form) and PIC micelle encapsulating the conjugate without the disulfide linkage. Use of poly(l-lysine) (PLL) instead of the B-PEI for PIC micellization led to a substantial decrease in the antisense effect. These results indicate that the PIC micelles formulated from PEG-SS-asODN conjugate and B-PEI is successfully transported from the endosomal compartment into the cytoplasm by the buffering effect of the B-PEI, releasing hundreds of active asODN molecules via cleavage of the disulfide linkage into the cellular interior, responding to a high glutathione concentration in the cytoplasmic compartment. Furthermore, the type of smart linkage (glutathione-sensitive SS linkage vs pH-sensitive linkage) in the conjugates substantially affected the antisense effect of the PIC micelles, depending on the nature of the counter polycation (B-PEI vs PLL).     

An outbreak of neonatal toxic shock syndrome-like exanthematous disease (NTED) caused by methicillin-resistant Staphylococcus aureus (MRSA) in a neonatal intensive care unit

Neonatal toxic shock syndrome-like exanthematous disease (NTED) is a new entity of methicillin-resistant Staphylococcus aureus (MRSA) infection. Most of NTED cases reported previously in the literature were sporadic ones. In the present report, we describe an outbreak of NTED that occurred in a neonatal intensive care unit (NICU) between April, 1999 and April, 2000 in Japan. All MRSA strains isolated from 14 patients (6 NTED, 2 infections and 6 colonizations) in this outbreak belonged to the group of coagulase II and produced toxic shock syndrome toxin 1 (TSST-1). Of these, 14 strains produced staphylococcal enterotoxin C (SEC). No other superantigenic toxins were produced by these strains. The pulsed field gel electrophoresis (PFGE) patterns of genomic DNA digested with SmaI were indistinguishable each other due to no band shifting in all of the 13 strains except for strain O-21 and M56. Strain M56 was different from the dominant type in the positions of only 2 bands, whereas the pattern of strain O-21 had no similarity with the other pattern, suggesting that this outbreak was associated with the spread of a unique MRSA strain in the NICU. Two-dimensional electrophoresis (2-DE) analysis of exoproteins revealed that the patterns of these 14 strains were very indistinguishable to each other, and that these strains produced very large amounts of TSST-1 and SEC3 subtype superantigens, as measured with computer-assisted image analysis of the intensity of 2-DE spots. The 2-DE gel of O-21 showed the different pattern from the others. These results as well as the profiles of toxin production also supported the conclusion drawn from PFGE analysis. Based on these results, the involvement of TSST-1 and SEC3 in the pathogenesis of NTED is discussed.     

Inhibition of gastric acid secretion and mucosal blood flow induced by intraventricularly applied neurotensin in rats

To investigate the central effect of neurotensin in gastric functions, changes in gastric acid secretion and mucosal blood flow (MBF) following administration were examined in rats anesthetized with urethane. Neurotensin in doses 1–10 μg/animal injected into the lateral ventricle decreased the basal value of both gastric acid output and MBF. This effect of neurotensin on these gastric parameters was completely blocked by pretreatment of animals with reserpine (2 mg/kg, i.p., 24 hr) or 6-OH-dopamine (250 μg/animal, intraventricularly, 10–14 days). These results indicate that exogenously applied neurotensin induces an inhibition of gastric functions by a central mechanism and suggest that an interaction exists between central catecholamines and the effect of neurotensin on gastric functions.