Photodegradation Pathways of Protein Disulfides: Human Growth Hormone.

PURPOSE: Comprehensive product characterization was performed for the photodegradation of protein disulfides, representatively of human growth hormone (somatotropin; hGH), in order to provide a product database, which will be useful for the general analysis of protein stability. METHODS: HGH was photo-irradiated at λ = 254 and λ > 295 nm and tryptic digests were analyzed by HPLC-MS to investigate light-induced disulfide degradation pathways. RESULTS: A total of 60 products were detected, and structures/tentative structures were assigned to the products by MS2 and MS3 analysis. The main products were reduced Cys residues, dithiohemiacetal, thioether and disulfide scrambling products. In addition, we detected Cys degradation products such as Cys thioaldehyde, dehydroalanine (Dha), Ala, Ser semialdehyde, Ser, S-sulfocysteine, and Gly. Frequently, the tryptic fragments contained more than one modification, i.e. a Cys degradation product in close proximity to a dehydrated amino acid. Several novel cross-links were detected between Cys and Tyr, Cys, Ser and Phe, Cys and Trp, and Trp and Tyr. Photo-induced protein fragmentation was detected specifically at or in close proximity to the disulfide bond between T6 and T16. An in-house packed 75 cm nano-column enabled us to resolve various isomers/diastereomers of the photo-degradation products. CONCLUSION: A comprehensive analysis of photodegradation products revealed a variety of novel photo-products, including cross-links, originating from disulfide degradation. The mechanisms of product formation are discussed.

Why selenocysteine replaces cysteine in thioredoxin reductase: a radical hypothesis.

Thioredoxin reductases, important biological redox mediators for two-electron transfers, contain either 2 cysteines or a cysteine (Cys) and a selenocysteine (Sec) at the active site. The incorporation of Sec is metabolically costly, and therefore surprising. We provide here a rationale: in the case of an accidental one-electron transfer to a S-S or a S-Se bond during catalysis, a thiyl or a selanyl radical, respectively would be formed. The thiyl radical can abstract a hydrogen from the protein backbone, which subsequently leads to the inactivation of the protein. In contrast, a selanyl radical will not abstract a hydrogen. Therefore, formation of Sec radicals in a GlyCysSecGly active site will less likely result in the destruction of a protein compared to a GlyCysCysGly active site.

Prospective model-based comparison of different laryngoscopes for difficult intubation in infants.

BACKGROUND: Difficult intubation in infants is uncommon but may be a challenge for the anesthesiologist. Many optical-assisted techniques are available to ease endotracheal placement of tube but have not been systemically evaluated for pediatric practice. AIM: The study was performed to compare conventional pediatric Macintosh - with different optical laryngoscopes in difficult endotracheal intubation in infants. We hypothesized that inexperienced anesthetists would perform more successful with optical devices and that differences between the devices would be found. METHODS/MATERIALS: In this randomized controlled study, 30 anesthesia residents performed endotracheal intubation in an infant model of difficult airway presenting with airway obstruction and neck immobilization. Primary endpoints were intubation success rate and intubation time. Beyond that glottis view, dental trauma and difficulty of technique were evaluated and measured by a study observer. Macintosh, Airtraq(®), Storz DCI(®) -, and Gyrus Infant Bullard(®) laryngoscopes were used in random order. After standardized briefing every resident had three attempts of at most 120 s with every device to place a 3-mm tube into the trachea. Glottis view and difficulty of technique were rated by the residents using classification of Cormack/Lehane and Visual Analogue Scale (VAS; 0 = easy to 10 = very difficult). RESULTS: Success rate was 41% with conventional Macintosh, 43% with Airtraq(®), 62% with Storz DCI(®), and 100% with Bullard(®) laryngoscopes. Median time from passing the lips to first ventilation was 67 s (Storz DCI(®) laryngoscope), 54 s (Macintosh laryngoscope), 45 s (Airtraq(®) laryngoscope), and 21 s (Bullard(®) laryngoscope), respectively. Dental trauma did not occur with Bullard(®) laryngoscope and was frequent with Storz DCI(®) laryngoscope (39%) and Macintosh laryngoscope (42%). Glottis view was best with Bullard(®) laryngoscope (Grade 1 in 100%) and worst with Macintosh laryngoscope (Grade 1 in 2%). Difficulty of technique was rated with a VAS score of 2 (Bullard(®) laryngoscope), 4.5 (Storz DCI(®) laryngoscope) and 6 (Airtraq(®) - and Macintosh laryngoscopes). CONCLUSIONS: Inexperienced anesthetists have higher success rates and shorter intubation times with optical-assisted laryngoscopes compared with conventional Macintosh laryngoscope. Gyrus Infant Bullard(®) laryngoscope significantly undertakes best success rate and shortest intubation time with mildest impact to maxillary dents and easiest technique. Our findings support the hypothesis that optical laryngoscopes can be used successfully by inexperienced anesthetists in simulated difficult pediatric airway conditions.

Comparison of Bullard and Airtraq laryngoscopes with conventional laryngoscopy in a manikin study of simulated difficult intubation.

BACKGROUND: When airway management is difficult, various measures can be taken to facilitate tracheal intubation. The Bullard and Airtraq laryngoscopes were developed for this purpose. We hypothesised that the Bullard and Airtraq laryngoscopes would perform better than a conventional laryngoscope in the management of a simulated difficult airway. We also hypothesised that the indirect laryngoscopes would perform comparably. METHODS: In a randomised controlled study, 60 anaesthetists (30 with no or little experience and 30 with broader experience in the use of the Bullard laryngoscope, referred to as beginners and experts, respectively) performed three successive intubation attempts using conventional, Bullard and Airtraq laryngoscopes in two simulated difficult airway scenarios: neck immobilisation (scenario A) and neck immobilisation with additional tongue oedema (scenario B). The primary endpoint was overall intubation success rate. Secondary endpoints were time required for successful intubation, the amount of dental stress exerted during laryngoscopy and satisfaction with each airway device. RESULTS: In scenario A, intubation success rates were 97-100% with all devices. In scenario B, all participants failed to intubate the trachea using the conventional laryngoscope. When using the Bullard laryngoscope, intubation success rates of 87-97% did not differ significantly (P > 0.05) from those during scenario A and between groups (beginners vs. experts). In contrast, when using the Airtraq laryngoscope, the overall intubation success rate was significantly lower (P < 0.05) compared with scenario A and compared with use of the Bullard laryngoscope, and differed between beginners and experts (20 and 50%, respectively). In cases of successful intubation, intubation times were comparable between devices and groups. Intubation times were longer during scenario B. Dental stress was always lower (P < 0.05) during use of the Bullard and Airtraq laryngoscopes compared with the conventional laryngoscope, lowest (P < 0.05) during use of the Bullard laryngoscope and (with the exception of use of the conventional laryngoscope by the experts) higher during scenario B than during scenario A. In scenario A, participants preferred both video laryngoscopes to the conventional laryngoscope. CONCLUSION: In a moderately difficult airway scenario, all laryngoscopes performed equally well. However, in a more difficult airway scenario, the Bullard and Airtraq laryngoscopes performed better than the conventional laryngoscope, with the Bullard device performing better than the Airtraq. This may be in part related to differing prior experiences of operators with the respective airway devices.

Why do proteins use selenocysteine instead of cysteine?

Selenocysteine is present in a variety of proteins and catalyzes the oxidation of thiols to disulfides and the reduction of disulfides to thiols. Here, we compare the kinetic and thermodynamic properties of cysteine with its selenium-containing analogon, selenocysteine. Reactions of simple selenols at pH 7 are up to four orders of magnitude faster than their sulfur analogs, depending on reaction type. In redox-related proteins, the use of selenium instead of sulfur can be used to tune electrode, or redox, potentials. Selenocysteine could also have a protective effect in proteins because its one-electron oxidized product, the selanyl radical, is not oxidizing enough to modify or destroy proteins, whereas the cysteine-thiyl radical can do this very rapidly.

Distance-dependent diffusion-controlled reaction of •NO and O2•- at chemical equilibrium with ONOO-.

The fast reaction of (•)NO and O(2)(•-) to give ONOO(-) has been extensively studied at irreversible conditions, but the reasons for the wide variations in observed forward rate constants (3.8 ≤ k(f) ≤ 20 × 10(9) M(-1) s(-1)) remain unexplained. We characterized the diffusion-dependent aqueous (pH > 12) chemical equilibrium of the form (•)NO + O(2)(•-) = ONOO(-) with respect to its dependence on temperature, viscosity, and [ONOO(-)](eq) by determining [ONOO(-)](eq) and [(•)NO](eq). The equilibrium forward reaction rate constant (k(f)(eq)) has negative activation energy, in contrast to that found under irreversible conditions. In contradiction to the law of mass action, we demonstrate that the equilibrium constant depends on ONOO(-) concentration. Therefore, a wide range of k(f)(eq) values could be derived (7.5-21 × 10(9) M(-1) s(-1)). Of general interest, the variations in k(f) can thus be explained by its dependence on the distance between ONOO(-) particles (sites of generation of (•)NO and O(2)(•-)).

[Anaesthesia for children with epidermolysis bullosa]. / Epidermolysis bullosa - Anästhesieführung bei Kindern.

Epidermolysis bullosa (EB) is a heterogeneous group of inherited rare diseases, which are characterized by trauma-induced blister formation of the skin and mucosa. The underlying cause is a functional deficiency of structural proteins of the epidermis or the dermis. Depending on the level of the blister formation, EB is divided into EB simplex (intra-epidermal), junctional EB (within the lamina lucida), dystrophic EB (below the lamina lucida) and Kindler syndrome (variable level of split formation). Besides different distinct blister formation and pain symptoms secondary problems like anaemia, oesophageal stenosis, cardiomyopathy or squamous cell carcinoma may occur. Since causal therapies are not available strict prevention of friction and trauma is essential to avoid blister formation. Anaesthesia challenges exist in the field of bedding procedures, care of the skin, monitoring, airway management und analgesia. This article gives a review over the EB and highlights in detail the corresponding anaesthesia characteristics.

Selenium and sulfur in exchange reactions: a comparative study.

Cysteamine reduces selenocystamine to form hemiselenocystamine and then cystamine. The rate constants are k(1) = 1.3 × 10(5) M(-1) s(-1); k(-1) = 2.6 × 10(7) M(-1) s(-1); k(2) = 11 M(-1) s(-1); and k(-2) = 1.4 × 10(3) M(-1) s(-1), respectively. Rate constants for reactions of cysteine/selenocystine are similar. Reaction rates of selenium as a nucleophile and as an electrophile are 2-3 and 4 orders of magnitude higher, respectively, than those of sulfur. Sulfides and selenides are comparable as leaving groups.

Acute tension pneumothorax following cardiac herniation after pneumonectomy.

A tension pneumothorax is one of the main causes of cardiac arrest in the initial postoperative period after thoracic surgery. Tension pneumothorax and cardiac herniation must be taken into account in hemodynamically unstable patients after pneumonectomy. We report an unusual case of successful treatment of acute tension pneumothorax following cardiac herniation and intrathoracic bleeding after pneumonectomy.

Reduction of protein radicals by GSH and ascorbate: potential biological significance.

The oxidation of proteins and other macromolecules by radical species under conditions of oxidative stress can be modulated by antioxidant compounds. Decreased levels of the antioxidants glutathione and ascorbate have been documented in oxidative stress-related diseases. A radical generated on the surface of a protein can: (1) be immediately and fully repaired by direct reaction with an antioxidant; (2) react with dioxygen to form the corresponding peroxyl radical; or (3) undergo intramolecular long range electron transfer to relocate the free electron to another amino acid residue. In pulse radiolysis studies, in vitro production of the initial radical on a protein is conveniently made at a tryptophan residue, and electron transfer often leads ultimately to residence of the unpaired electron on a tyrosine residue. We review here the kinetics data for reactions of the antioxidants glutathione, selenocysteine, and ascorbate with tryptophanyl and tyrosyl radicals as free amino acids in model compounds and proteins. Glutathione repairs a tryptophanyl radical in lysozyme with a rate constant of (1.05±0.05)×10(5) M(-1) s(-1), while ascorbate repairs tryptophanyl and tyrosyl radicals ca. 3 orders of magnitude faster. The in vitro reaction of glutathione with these radicals is too slow to prevent formation of peroxyl radicals, which become reduced by glutathione to hydroperoxides; the resulting glutathione thiyl radical is capable of further radical generation by hydrogen abstraction. Although physiologically not significant, selenoglutathione reduces tyrosyl radicals as fast as ascorbate. The reaction of protein radicals formed on insulin, ß-lactoglobulin, pepsin, chymotrypsin and bovine serum albumin with ascorbate is relatively rapid, competes with the reaction with dioxygen, and the relatively innocuous ascorbyl radical is formed. On the basis of these kinetics data, we suggest that reductive repair of protein radicals may contribute to the well-documented depletion of ascorbate in living organisms subjected to oxidative stress.

A lung area estimation method for analysis of ventilation inhomogeneity based on electrical impedance tomography.

PURPOSE: To evaluate a novel method for lung area estimation (LAE method) in electrical impedance tomography (EIT) images as a prerequisite of quantitative analysis of ventilation inhomogeneity. METHODS: The LAE method mirrors the lung regions in the functional EIT (fEIT) image and subsequently subtracts the cardiac related areas. In this preliminary study, 51 mechanically ventilated patients were investigated, including 39~patients scheduled for thoracic surgery (test group); 10 patients scheduled for orthopedic surgery without pulmonary disease (control group) and 2 ICU patients undergoing chest computed tomography (CT) examination. EIT data was recorded in all groups. The results of the LAE method were compared to those obtained with the fEIT method and to CT images. RESULTS: The lung area size determined with fEIT in control group is S(C,fEIT) = 361 +/- 35 (mean +/- SD) and in test group S(T,fEIT) = 299 +/- 61 (p< 0.01). The sizes estimated with the LAE method in control group S(C,LAE) = 353 +/- 27 and in test group S(T,LAE) = 353 +/- 61 (p=0.41). The result demonstrates that the novel LAE method improves the identification of lung region in EIT images, from which the analysis of ventilation distribution will benefit. The preliminary comparison with CT images exemplary indicates a closer match of the lung area shapes after the LAE than after the fEIT-based analysis. CONCLUSION: The LAE method is a robust lung area determination method, suitable for patients with healthy or seriously injured lungs.

Kinetics of tyrosyl radical reduction by selenocysteine.

The rate constant for the reduction of the tyrosyl radical with selenocysteine has been measured to investigate whether selenocysteine is capable of repair of protein radicals. Tyrosyl radicals, both free in solution and in insulin, were generated by means of pulse radiolysis and laser flash photolysis in aqueous solution. The rate constant for the reaction of free N-acetyl-tyrosyl-amine radicals with selenocysteine is (8 +/- 2) x 10 (8) M (-1) s (-1), and that for tyrosyl radicals in insulin is (1.6 +/- 0.4) x 10 (8) M (-1) s (-1). The rate constant for the reaction of selenoglutathione with the N-acetyl-tyrosyl-amine radical is (5 +/- 2) x 10 (8) M (-1) s (-1). In contrast, cysteine and glutathione react more slowly than their selenium analogues with the tyrosyl radical: the reactions of N-acetyl-tyrosyl-amine radicals with cysteine and glutathione are 3 and 5 orders of magnitude slower, respectively, than those with selenocysteine and selenoglutathione, while those of tyrosyl radicals in insulin are 3 and 2 orders of magnitude slower, respectively.

Inhibition of hepatitis C virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis C.

Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis worldwide. The study of antibody-mediated virus neutralization has been hampered by the lack of an efficient and high-throughput cell culture system for the study of virus neutralization. The HCV structural proteins have been shown to assemble into noninfectious HCV-like particles (HCV-LPs). Similar to serum-derived virions, HCV-LPs bind and enter human hepatocytes and hepatoma cell lines. In this study, we developed an HCV-LP-based model system for a systematic functional analysis of antiviral antibodies from patients with acute or chronic hepatitis C. We demonstrate that cellular HCV-LP binding was specifically inhibited by antiviral antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, we identified an epitope in the N-terminal E2 region (SQKIQLVNTNGSWHI; amino acid positions 408 to 422) as one target of human antiviral antibodies inhibiting cellular particle binding. Using a large panel of serum samples from patients with acute and chronic hepatitis C, we demonstrated that the presence of antibodies with inhibition of binding activity was not associated with viral clearance. In conclusion, antibody-mediated inhibition of cellular HCV-LP binding represents a convenient system for the functional characterization of human anti-HCV antibodies, allowing the mapping of envelope neutralization epitopes targeted by naturally occurring antiviral antibodies.