Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide‐dependent mechanism

Protein tyrosine (Tyr) nitration is a post‐translational modification yielding 3‐nitrotyrosine (NO₂–Tyr). Formation of NO₂–Tyr is generally considered as a marker of nitro‐oxidative stress and is involved in some human pathophysiological disorders, but has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as an O₂ transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO₂–Tyr in Lb. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO₂–Tyr25 and NO₂–Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving and were found to decrease during senescence. This demonstrates formation of nitric oxide (˙NO) and by alternative means to nitrate reductase, probably via a ˙NO synthase‐like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lb revealed that Tyr nitration requires  + H₂O₂ and that peroxynitrite is not an efficient inducer of nitration, probably because Lb isomerizes it to . Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO₂–Tyr in Lb is a consequence of active metabolism in functional nodules, where Lb may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.     

The Arrival of Siberian Ancestry Connecting the Eastern Baltic to Uralic Speakers further East

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Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering the molecular signals that cooperatively guide neuronal migration in the embryonic brain is therefore important to understand how the complex neural networks form which later support postnatal life. Facial branchiomotor (FBM) neurons in the mouse embryo hindbrain migrate from rhombomere (r) 4 caudally to form the paired facial nuclei in the r6-derived region of the hindbrain. Here we provide a detailed protocol for wholemount ex vivo culture of mouse embryo hindbrains suitable to investigate the signaling pathways that regulate FBM migration. In this method, hindbrains of E11.5 mouse embryos are dissected and cultured in an open book preparation on cell culture inserts for 24 hr. During this time, FBM neurons migrate caudally towards r6 and can be exposed to function-blocking antibodies and small molecules in the culture media or heparin beads loaded with recombinant proteins to examine roles for signaling pathways implicated in guiding neuronal migration.     

Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of α-ketobutyrate to l-isoleucine

Summary Corynebacterium glutamicum ATCC 13 032 produces 13 g/l l-isoleucine from 200 mM -ketobutyrate as a synthetic precursor. In fed batch cultures up to 19 g/l l-isoleucine is formed. For optimal conversion the addition of 0.3 mM l-valine plus 0.3 mM l-leucine to the fermentation medium is required. The affinity constants for the acetohydroxy acid synthase (AHAS) were determined. (This enzyme directs the flow of -ketobutyrate plus pyruvate towards l-isoleucine and that of two moles of pyruvate to l-valine and l-leucine, respectively.) For -ketobutyrate the K _m is 4.8×10^-3 M, and V _max 0.58 U/mg, for pyruvate the K _m is 8.4×10^-3 M, and V _max 0.37 U/mg. Due to these characteristics the presence of high -ketobutyrate concentrations apparently results in a l-valine, l-leucine deficiency. This in turn leads to a derepression of the AHAS synthesis from 0.03 U/mg to 0.29 U/mg and high l-isoleucine production is favoured. The derepression of the AHAS synthesis induced by the l-valine, l-leucine shortage was directly proven with a l-valine, l-leucine, l-isoleucine auxotrophic mutant where the starvation of each amino acid resulted in an increased AHAS level. This is in accordance with the fact that only one AHAS enzyme could be verified by chromatographic and electrophoretic separations as being responsible for the synthesis of all three branched-chain amino-acids.     

Growth and morphological responses to different UV wavebands in cucumber (Cucumis sativum) and other dicotyledonous seedlings

We examined the influence of short-term exposure of different UV wavebands on the fine-scale kinetics of hypocotyl growth of dim red light-grown cucumbers (Cucumis sativus L.) and other selected dicotyledonous seedlings to evaluate: (1) whether responses induced by UV-B radiation (280-320 nm) are qualitatively different from those induced by UV-A (320-400 nm) radiation, and (2) whether different wavebands within the UV-B elicit different responses. Responses to brief (30 min) irradiations with 3 different UV wavebands all included transient inhibition of elongation during irradiation followed by wavelength specific responses. Irradiations with proportionally greater short wavelength UV-B (37% of UV-B between 280 and 300 nm) induced inhibition of hypocotyl elongation within 20 min of onset of irradiation, while UV-B including only wavelengths longer than 290 nm (and only 8% of UV-B between 290 and 300 nm) induced inhibition of hypocotyl elongation with a lag of 1-2 h. The response to short wavelength UV-B was persistent for at least 24 h, while the response to long wavelength UV-B lasted only 2-3 h. The UV-A treatment induced reductions in elongation rates of approximately 6-9 h following exposure followed by a continued decline in rates for the following 15-18 h. Short wavelength UV-B also induced positive phototropic curvature in both cucumber and Arabidopsis seedlings, and this response was present in nph-1 mutant Arabidopsis seedlings defective in normal blue light phototropism. Reciprocity was not found for the response to short wavelength UV-B. The short wavelength and long wavelength UV-B responses differed in dose-response relationships and both short wavelength responses (phototropic curvature and elongation inhibition) increased sharply at wavelengths below 300 nm. These results indicate that different photosensory processes are involved in mediating growth and morphological responses to short wavelength UV-B (280-300 nm), long wavelength UV-B (essentially 300-320 nm) and UV-A. The existence of two separate types of hypocotyl inhibition responses to UV-B, with one that depends on the intensity of the light source, provides alternate interpretations to findings in other studies of UV-B induced photomorphogenesis and may explain inconsistencies between action spectra for inhibition of stem growth.     

Estrogen-induced contraction of coronary arteries is mediated by superoxide generated in vascular smooth muscle

Although previous studies demonstrated beneficial effects of estrogen on cardiovascular function, the Women's Health Initiative has reported an increased incidence of coronary heart disease and stroke in postmenopausal women taking hormone replacement therapy. The objective of the present study was to identify a molecular mechanism whereby estrogen, a vasodilatory hormone, could possibly increase the risk of cardiovascular disease. Isometric contractile force recordings were performed on endothelium-denuded porcine coronary arteries, whereas molecular and fluorescence studies identified estrogen signaling molecules in coronary smooth muscle. Estrogen (1-1,000 nM) relaxed arteries in an endothelium-independent fashion; however, when arteries were pretreated with agents to uncouple nitric oxide (NO) production from NO synthase (NOS), estrogen contracted coronary arteries with an EC(50) of 7.3 +/- 4 nM. Estrogen-induced contraction was attenuated by reducing superoxide (O(2)(-)). Estrogen-stimulated O(2)(-) production was detected in NOS-uncoupled coronary myocytes. Interestingly, only the type 1 neuronal NOS isoform (nNOS) was detected in myocytes, making this protein a likely target mediating both estrogen-induced relaxation and contraction of endothelium-denuded coronary arteries. Estrogen-induced contraction was completely inhibited by 1 muM nifedipine or 10 muM indomethacin, indicating involvement of dihydropyridine-sensitive calcium channels and contractile prostaglandins. We propose that a single molecular mechanism can mediate the dual and opposite effect of estrogen on coronary arteries: by stimulating type 1 nNOS in coronary arteries, estrogen produces either vasodilation via NO or vasoconstriction via O(2)(-).     

Lipopolysaccharide-induced Lung Injury Involves the Nitration-mediated Activation of RhoA

Acute lung injury (ALI) is characterized by increased endothelial hyperpermeability. Protein nitration is involved in the endothelial barrier dysfunction in LPS-exposed mice. However, the nitrated proteins involved in this process have not been identified. The activation of the small GTPase RhoA is a critical event in the barrier disruption associated with LPS. Thus, in this study we evaluated the possible role of RhoA nitration in this process. Mass spectroscopy identified a single nitration site, located at Tyr³⁴ in RhoA. Tyr³⁴ is located within the switch I region adjacent to the nucleotide-binding site. Utilizing this structure, we developed a peptide designated NipR1 (nitration inhibitory peptide for RhoA 1) to shield Tyr³⁴ against nitration. TAT-fused NipR1 attenuated RhoA nitration and barrier disruption in LPS-challenged human lung microvascular endothelial cells. Further, treatment of mice with NipR1 attenuated vessel leakage and inflammatory cell infiltration and preserved lung function in a mouse model of ALI. Molecular dynamics simulations suggested that the mechanism by which Tyr³⁴ nitration stimulates RhoA activity was through a decrease in GDP binding to the protein caused by a conformational change within a region of Switch I, mimicking the conformational shift observed when RhoA is bound to a guanine nucleotide exchange factor. Stopped flow kinetic analysis was used to confirm this prediction. Thus, we have identified a new mechanism of nitration-mediated RhoA activation involved in LPS-mediated endothelial barrier dysfunction and show the potential utility of “shielding” peptides to prevent RhoA nitration in the management of ALI.