Determination and characterization of nitric oxide generation in mice by in vivo l-band EPR spectroscopy

The authors have shown direct, real-time, in vivo measurement of nitric oxide (NO) in mice by using the water soluble metal chelator complex, N-methyl-D-glucamine dithiocarbamate (MGD), and Fe(II) as monitored by EPR at L-band. The three-line EPR spectrum from the product [(MGD)₂-Fe(II)-NO] was observed noninvasively in lipopolysaccharide (LPS)-treated mice. The spectrum was markedly suppressed by the administration, before LPS injection, of phenyl N-tert-butyl nitrone (PBN), an inhibitor of the expression of induced nitric oxide synthase (iNOS). When ¹⁵N-arginine was administered to LPS-treated mice, a diagnostic EPR spectrum was observed, consisting of both three- and two-line EPR signals, due to (MGD)₂-Fe(II)-¹⁴NO and (MGD)₂-Fe(II)-¹⁶NO, respectively. The results strongly suggested that the NO detected in these experiments was synthesized by iNOS. In vivo EPR measurements of [(MGD)₂-Fe(II)-NO] at several regions in the body (from the head to the tail) indicated that the NO was generated mostly in the upper abdomen near the liver. These observations were confirmed by ex vivo EPR measurements on isolated organs where higher NO levels were detected in vivo in the liver and kidney. The spectroscopic results, combined with the pharmacokinetic data, support the model that NO detected in LPS-treated mice was produced mainly in the liver, and that it did not reflect NO-adduct complex accumulated in the liver via the blood circulation.     

Complementation of phytochrome chromophore-deficient Arabidopsis by expression of phycocyanobilin:ferredoxin oxidoreductase

The covalently bound phytochromobilin (PphiB) prosthetic group is required for the diverse photoregulatory activities of all members of the phytochrome family in vascular plants, whereas by contrast, green algal and cyanobacterial phytochromes use the more reduced linear tetrapyrrole pigment phycocyanobilin (PCB). To assess the functional consequence of the substitution of PphiB with PCB in plants, the phytochrome chromophore-deficient hy2 mutant of Arabidopsis was transformed with a constitutively expressed pcyA gene that encodes the cyanobacterial enzyme, PCB:ferredoxin oxidoreductase. Spectroscopic analyses of extracts from etiolated seedlings revealed that PcyA expression restored photoactive phytochrome to WT levels, albeit with blue-shifted absorption maxima, while also restoring light lability to phytochrome A. Photobiological measurements indicated that PcyA expression rescued phytochrome-mediated red high-irradiance responses, low-fluence red/far-red (FR) photoreversible responses, and very-low-fluence responses, thus confirming that PCB can functionally substitute for PphiB for these photoregulatory activities. Although PcyA expression failed to rescue phytochrome A-mediated FR high-irradiance responsivity to that of WT, our studies indicate that the FR high-irradiance response is fully functional in pcyA-expressing plants but shifted to shorter wavelengths, indicating that PCB can functionally complement this phytochrome-mediated response in vascular plants.     

Evaluation of radical scavenging properties of shikonin

With the aim of developing effective anti-inflammatory drugs, we have been investigating the biochemical effects of shikonin of “Shikon” roots, which is a naphthoquinone with anti-inflammatory and antioxidative properties. Shikonin scavenged reactive oxygen species like hydroxyl radical, superoxide anion (O₂^•−) and singlet oxygen in previous studies, but its reactivity with reactive oxygen species is not completely understood, and comparison with standard antioxidants is lacking. This study aimed elucidation of the reactivity of shikonin with nitric oxide radical and reactive oxygen species such as alkyl-oxy radical and O₂^•−. By using electron paramagnetic resonance spectrometry, shikonin was found unable of reacting with nitric oxide radical in a competition assay with oxyhemoglobin. However, shikonin scavenged alkyl-oxy radical from 2,2''-azobis(2-aminopropane) dihydrochloride with oxygen radical absorbance capacity, ORAC of 0.25 relative to Trolox, and showed a strong O₂^•−-scavenging ability (42-fold of Trolox; estimated reaction rate constant: 1.7 × 10⁵ M⁻¹s⁻¹) in electron paramagnetic resonance assays with CYPMPO as spin trap. Concerning another source of O₂^•−, the phagocyte NADPH oxidase (Nox2), shikonin inhibited the Nox2 activity by impairing catalysis when added before enzyme activation (IC₅₀: 1.1 µM; NADPH oxidation assay). However, shikonin did not affect the preactivated Nox2 activity, although having potential to scavenge produced O₂^•−. In conclusion, shikonin scavenged O₂^•− and alkyl-oxy radical, but not nitric oxide radical.     

Estimation of the following cardiac output using sympathetic tone and hemodynamics for the control of a total artificial heart.

A sympathetic neurogram is potentially useful for the development of a real time total artificial heart (TAH) control system. We used sympathetic tone and hemodynamic derivatives to estimate the following cardiac output in acute animal experiments using adult mongrel dogs. Moving averages of the mean left atrial pressure and mean aortic pressure were used as parameters of the preload and afterload, respectively. Renal sympathetic nerve activity (RSNA) was employed as a parameter of sympathetic tone. Equations for the following cardiac output were calculated using multiple linear regression analysis of the time series data. A significant correlation was observed between the estimated and following measured cardiac output. These results suggest the potential usefulness of the sympathetic neurogram for the real time TAH automatic control system.     

Cardiac-related sympathetic nerve activity during circulation with only the left ventricular assist device.

Circulatory maintenance with a left ventricular assist device (LVAD) alone during cardiac arrest until heart transplantation has been evaluated. To assess the effect on the autonomic nervous system, the sympathetic neurogram was analyzed by power spectrum and coherence function. LVAD were inserted between the left atrium and the descending aorta in seven adult mongrel dogs and ventricular fibrillation was induced electrically. Renal sympathetic nerve activity (RSNA) was detected by bipolar electrodes attached to the left renal sympathetic nerve. Values of squared coherence between the arterial pulse wave and RSNA were calculated. Under the condition of circulatory maintenance with only LVAD, coherence at the cardiac rhythm frequency was decreased, and coherence at the LVAD pumping rhythm frequency was increased. These results indicate that the arterial pulse wave observed during maintenance of the circulation with only LVAD contributed to the sympathetic neurogram.     

Effect of Propofol on Norepinephrine-induced Increases in [Ca2+] (i) and Force in Smooth Muscle of the Rabbit Mesenteric Resistance Artery

Background: Propofol (2,6-diisopropylphenol) possesses vasodilating activity in vivo and in vitro. The propofol-induced relaxation of agonist-induced contractions in small resistance arteries has not been clarified.

Methods: The effect of propofol was examined on the contractions induced by norepinephrine and high K+ in endothelium-denuded rabbit mesenteric resistance artery in vitro. The effects of propofol on the [Ca2+]i mobilization induced by norepinephrine and high K+ were studied by simultaneous measurement of [Ca2+]i using Fura 2 and isometric force in ryanodine-treated strips.

Results: Propofol attenuated the contractions induced by high K+ and norepinephrine, the effect being greater on the high K+-induced contraction than on the norepinephrine-induced contraction. In Ca2-free solution, norepinephrine produced a transient contraction resulting from the release of Ca2+ from storage sites that propofol attenuated. In ryanodine-treated strips, propofol increased the resting [Ca2+]i but attenuated the increases in [Ca2+]i and force induced by both high K+ and norepinephrine. In the presence of nicardipine, propofol had no inhibitory action on the residual norepinephrine-induced [Ca2+]i increase, whereas it still modestly increased resting [Ca2+]i, as in the absence of nicardipine.     

Postextubation airway management with nasal continuous positive airway pressure in a child with Down syndrome

We describe our experience with use of variable-flow nasal continuous positive airway pressure (NCPAP) to manage postextubation stridor in a 31-month-old child with Down syndrome (DS). Although it has been recognized that children with DS tend to develop obstruction of the upper airway postoperatively, little is known concerning appropriate management of this situation. Although there are surprisingly few reports of use of variable-flow NCPAP for children older than preterm infants, we successfully treated postextubation ventilatory complications by providing variable-flow NCPAP without complications such as pneumothorax.     

Inadvertent intrathecal cannulation in an infant, demonstrated by three-dimensional computed tomography: a rare complication of internal jugular vein catheterization

We describe a case of inadvertent intrathecal cannulation with a central venous catheter in an infant, confirmed by three-dimensional computed tomography, which clearly demonstrated the track of the catheter. We believe that this complication could have been related to two major factors: depth of needle insertion and penetration of the vein by a straight-tip guidewire. To avoid this complication, the depth of needle insertion must be carefully checked, a “J”-tipped rather than a straight-tipped guidewire should be used, and puncture should be guided by ultrasound.