Hemoglobinuria-related acute kidney injury is driven by intrarenal oxidative reactions triggering a heme toxicity response.

Intravascular hemolysis can result in hemoglobinuria with acute kidney injury. In this study we systematically explored two in vivo animal models and a related cell culture system to identify hemoglobinuria-triggered damage pathways. In models of stored blood transfusion and hemoglobin (Hb) exposure in guinea pigs and beagle dogs we found that hemoglobinuria led to intrarenal conversion of ferrous Hb(Fe(2+)) to ferric Hb(Fe(3+)), accumulation of free heme and Hb-cross-linking products, enhanced 4-hydroxynonenal reactivity in renal tissue, and acute tubule injury. These changes were associated in guinea pigs with activation of a renal cortex gene expression signature indicative of oxidative stress and activation of the unfolded protein response (UPR). Tubule cells of hemolytic animals demonstrated enhanced protein expression of heme oxygenase and heat shock protein and enhanced expression of acute kidney injury-related neutrophil gelatinase-associated lipocalin. These adverse changes were completely prevented by haptoglobin treatment. The in vivo findings were extrapolated to a MS-based proteome analysis of SILAC-labeled renal epithelial cells that were exposed to free heme within a concentration range estimate of renal tubule heme exposure. These experiments confirmed that free heme is a likely trigger of tubule barrier deregulation and oxidative cell damage and reinforced the hypothesis that uncontrolled free heme could trigger the UPR as an important pathway of renal injury during hemoglobinuria.

A multi-frequency pulse EPR and ENDOR approach to study strongly coupled nuclei in frozen solutions of high-spin ferric heme proteins.

In spite of the tremendous progress in the field of pulse electron paramagnetic resonance (EPR) in recent years, these techniques have been scarcely used to investigate high-spin (HS) ferric heme proteins. Several technical and spin-system-specific reasons can be identified for this. Additional problems arise when no single crystals of the heme protein are available. In this work, we use the example of a frozen solution of aquometmyoglobin (metMb) to show how a multi-frequency pulse EPR approach can overcome these problems. In particular, the performance of the following pulse EPR techniques are tested: Davies electron nuclear double resonance (ENDOR), hyperfine correlated ENDOR (HYEND), electron-electron double resonance (ELDOR)-detected NMR, and several variants of hyperfine sublevel correlation (HYSCORE) spectroscopy including matched and SMART HYSCORE. The pulse EPR experiments are performed at X-, Q- and W-band microwave frequencies. The advantages and drawbacks of the different methods are discussed in relation to the nuclear interaction that they intend to reveal. The analysis of the spectra is supported by several simulation procedures, which are discussed. This work focuses on the analysis of the hyperfine and nuclear-quadrupole tensors of the strongly coupled nuclei of the first coordination sphere, namely, the directly coordinating heme and histidine nitrogens and the 17O nucleus of the distal water ligand. For the latter, 17O-isotope labeling was used. The accuracy of our results and the spectral resolution are compared in detail to an earlier single-crystal continuous-wave ENDOR study on metMb, and it will be shown how additional information can be obtained from the multi-frequency approach. The current work is therefore prone to become a template for future EPR/ENDOR investigations of HS ferric heme proteins for which no single crystals are available.

[Anaphylaxis after carrying out prick tests]. / Anafilaxia tras realización de pruebas cutáneas en prick.

The skin prick test (SPT) is a simple and fast method used routinely in allergology practice. Systemic reactions have been described with this technique on few occasions. We are presenting a case of anaphylaxis with hemodynamic consequences after carrying out skin prick test with a cat dander extract. A 23 years old female who suffered rhinoconjunctivitis and asthma following contact with cats. We performed skin prick test with a battery of the usual inhalants. Twenty minutes after carrying out the prick test the patient showed intense ocular irritation and reddening followed by dysphonia and a feeling of pharyngeal occupation. Although skin prick test is a safe diagnostic approach, it should be performed only in places equipped to treat anaphylaxis and for trained specialists .

Anafilaxia tras realización de pruebas cutáneas en prick / Anaphylaxis after carrying out prick tests

Las pruebas cutáneas en prick (SPT) son un método sencillo y muy útil en la práctica alergológica diaria. Se han descrito reacciones sistémicas con este método diagnóstico en contadas ocasiones. Presentamos un caso de anafilaxia tras la realización de SPT con epitelio de gato. Se trataba de una mujer de 23 años que presentaba rinoconjuntivitis y asma bronquial al contacto con gato. Realizamos SPT con bateria de aeroalérgenos habituales, presentando cuadro de irritación ocular, disfonía y sensación de ocupación faríngea a los 20 minutos. Aunque el riesgo de reacción sistémica tras SPT es escaso, recomendamos la realización de esta prueba diagnóstica en lugares preparados para tratar reacciones anafilácticas y por personal experto

The skin prick test (SPT) is a simple and fast method used routinely in allergology practice. Systemic reactions have been described with this technique on few occasions. We are presenting a case of anaphylaxis with hemodynamic consequences after carrying out skin prick test with a cat dander extract. A 23 years old female who suffered rhinoconjunctivitis and asthma following contact with cats. We performed skin prick test with a battery of the usual inhalants. Twenty minutes after carrying out the prick test the patient showed intense ocular irritation and reddening followed by dysphonia and a feeling of pharyngeal occupation. Although skin prick test is a safe diagnostic approach, it should be performed only in places equipped to treat anaphylaxis and for trained specialists

Fixed exanthema from systemic tobramycin.

Eye drops contain several ophthalmic medications which can produce allergic reactions. We report the case of a patient with contact dermatitis from neomycin and a probable fixed exanthema after parenteral administration of tobramycin who tolerated topical tobramycin and other aminoglycosides.

Detergent effect on cytochrome b559 electron paramagnetic resonance signals in the photosystem II reaction centre.

The detergent effect on Cytochrome b559 from spinach photosystem II was studied by electron paramagnetic resonance (EPR) spectroscopy in D1-D2-Cyt b559 complex preparations. Various n-dodecyl-beta-D-maltoside concentrations from 0 to 0.2% (w/v) were used to stabilise the D1-D2-Cyt b559 complexes. Low spin heme EPR spectra were obtained but the g(z) feature positions changed depending on the detergent conditions Redox potentiometric titrations showed a unique redox potential cytochrome b559 form (E'm = + 123-150 mV) in all the D1-D2-Cyt b559 complex preparations indicating that detergent does not affect this property of the protein in those conditions. A similar effect on Cytochrome b559 EPR spectrum was observed in more intact photosystem II preparations independently of their aggregation state. This finding indicates that changes due to detergent could be a common phenomenon in photosystem II complexes. Results are discussed in terms of the environment each detergent provides to the protein.

Spin label electron paramagnetic resonance study in thylakoid membranes from a new herbicide-resistant D1 mutant from soybean cell cultures deficient in fatty acid desaturation.

The effect of fatty acid desaturation on lipid fluidity in thylakoid membranes isolated from the STR7 mutant was investigated by electron paramagnetic resonance (EPR) using spin label probes. The spectra of both 5- and 16-n-doxylstearic acid probes were measured as a function of the temperature between 10 and 305 K and compared to those of the wild type. This complete thermal evolution provides a wider picture of the dynamics. The spectra of the 5-n-doxylstearic acid probe as well as their temperature evolution were identical in both STR7 mutant and wild type thylakoids. However, differences were found with the 16-n-doxylstearic acid probe at temperatures between 230 and 305 K. The differences in the thermal evolution of the EPR spectra can be interpreted as a 5-10 K shift toward higher temperatures of the probe motional rates in the STR7 mutant as compared with that in the wild type. At temperatures below 230 K no differences were observed. The results indicated that the lipid motion in the outermost region of the thylakoids is the same in the STR7 mutant as in the wild type while the fluidity in the inner region of the STR7 mutant membrane decreases. Our data point out a picture of the STR7 thylakoid membrane in which the lipid motion is slower most probably as a consequence of fatty acid desaturation deficiency.