Structures of the Substrate-free and Product-bound Forms of HmuO,a Heme Oxygenase from Corynebacterium diphtheriae: X-RAY CRYSTALLOGRAPHY AND MOLECULAR DYNAMICS INVESTIGATION*?

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe³⁺-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe³⁺-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe³⁺-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe³⁺-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.     

Individual closed chamber: an alternative method for quantifying 14C in both labeled organic and inorganic carbon substrates

Incubation of ¹⁴C-labeled substrates continues to be a widely used procedure in soil organic matter (OM) research due to its sensitiveness. When the labeling is found in liquid fractions (soil extracts, hydrolysates), ¹⁴C can be easily quantified by using an aliquot for scintillation counting. For this reason, converting a solid carbon sample into liquid form is a typical step for accurate ¹⁴C analysis. We have developed an alternative method to carry out this step, which uses standard glass hardware and does not require complex laboratory facilities. Carbon (both in organic or inorganic forms) is converted into CO₂ within a reaction vessel connected to a Twisselmann’s extractor with an alkali trap inside. This forms an individual closed chamber (ICC) for each sample, thus eliminating the risk of cross-contaminations. The alkali solution adsorbs the evolved CO₂ within the closed system, and the excess of pressure is easily overcome by the use of a balloon. We tested the procedure on a set of substrates and two contrasting soils, checking also the effect of different sample loads (from 20 to 160 mg C) on the CO₂ recovery of the process. The percentage of carbon recovered into the alkali (i.e. the efficiency of the process) ranged from 92% for the inorganic C to 93–95% for the organic C method, the latter being sensitive to the amount of sample used for analysis. The ICC method can be successfully applied to analyze ¹⁴C-labeling in both carbonates and OM from solid samples, thus representing an alternative method to some established protocols, and it is suitable for substrates with low or very low ¹⁴C contents, in which high volumes of sample must be analyzed in order to guarantee representative results.     

Differential sensitivity to detergents of actin cytoskeleton from nerve endings

Detergent-resistant membranes (DRM), an experimental model used to study lipid rafts, are typically extracted from cells by means of detergent treatment and subsequent ultracentrifugation in density gradients, Triton X-100 being the detergent of choice in most of the works. Since lipid rafts are membrane microdomains rich in cholesterol, depletion of this component causes solubilization of DRM with detergent. In previous works from our group, the lack of effect of cholesterol depletion on DRM solubilization with Triton X-100 was detected in isolated rat brain synaptosomes. In consequence, the aim of the present work is to explore reasons for this observation, analyzing the possible role of the actin cytoskeleton, as well as the use of an alternative detergent, Brij 98, to overcome the insensitivity to Triton X-100 of cholesterol-depleted DRM. Brij 98 yields Brij-DRM that are highly dependent on cholesterol, since marker proteins (Flotillin-1 and Thy-1), as well as actin, appear solubilized after MCD treatment. Pretreatment with Latrunculin A results in a significant increase in Flotillin-1, Thy-1 and actin solubilization by Triton X-100 after cholesterol depletion. Studies with transmission electron microscopy show that combined treatment with MCD and Latrunculin A leads to a significant increase in solubilization of DRM with Triton X-100. Thus, Triton-DRM resistance to cholesterol depletion can be explained, at least partially, thanks to the scaffolding action of the actin cytoskeleton, without discarding differential effects of Brij 98 and Triton X-100 on specific membrane components. In conclusion, the detergent of choice is important when events that depend on the actin cytoskeleton are going to be studied.     

Self-incompatibility genotypes in almond re-evaluated by PCR,stylar ribonucleases,sequencing analysis and controlled pollinations

As part of the almond breeding programme at IRTA, we investigated the S genotypes of several cultivars using a combination of RNase zymograms, testcrosses, pollen-tube growth analysis and molecular identification by PCR analysis. For some of the cultivars examined, discrepancies appeared between their S alleles as reported in the literature and those found in this investigation, leading to a re-evaluation of their S genotypes. Analysis of the stylar ribonucleases (RNases), which are known to correlate with S alleles, of cvs. Achaak, Ardechoise, Desmayo Largueta, Ferrastar, Gabaix, Garbí, Glorieta, Languedoc, Primorskiy and Texas revealed inconsistencies with respect to the S₅ and S₁₀ alleles. However, PCR with the conserved primer pair AS1II/AmyC5R failed to detect any of these inconsistencies. When the S alleles from Desmayo Largueta, Gabaix, Primorskiy and Texas were sequenced, Texas and Primorskiy were found to carry the reported S₅ allele, while Desmayo Largueta and Gabaix carried a new allele, which has been tentatively denoted as S₂₅ This new S allele, previously reported to be S₁₀, was also identified in Achaak, Ardechoise and Ferrastar. The proposed new S genotypes are Achaak (S₂S₂₅), Ardechoise (S₁S₂₅), Desmayo Largueta (S₁S₂₅), Ferrastar (S₂S₂₅) and Gabaix (S₁₀S₂₅). The S alleles of Garbí, Glorieta, Languedoc, Texas and Primorskiy remain as reported in the literature. Testcrosses in the field and laboratory confirmed the new S genotypes. One cultivar (Gabaix) could be assigned to the existing cross-incompatibility group O of unique genotypes, and two new groups were established (XVI and XVII) consisting of two cultivars each. The clarification of these S alleles will be useful in almond breeding programmes and for planning new commercial orchards in the future.     

Aspergillus galactomannan detection in allogenic hematopoietic cell transplantation

Invasive aspergillosis has become the leading cause of death after allogeneic hematopoietic stem cell transplantation. This is partially due to the lack of a prompt diagnosis. Recently the detection of Aspergillus galactomannan antigen by means an ELISA technique in serum has been described. The objective of this study was to validate its usefulness in the allogeneic hematopoietic stem cell transplantation setting.     

Optical measurement of isolated canine lung filtration coefficients after alloxan infusion

In this study, lung filtration coefficient(K_fc) wasmeasured in eight isolated canine lung preparations by using threemethods: standard gravimetric (Std), blood-corrected gravimetric (BC), and optical. The lungs were held in zone III conditions and were subjected to an average venous pressure increase of 8.79 ± 0.93 (mean ± SD) cmH₂O. Thepermeability of the lungs was increased with an infusion of alloxan (75 mg/kg). The resultingK_fc values (inmilliliters · min¹ · cmH₂O¹ · 100 g dry lung weight¹)measured by using Std and BC gravimetric techniques before vs. afteralloxan infusion were statistically different: Std, 0.527 ± 0.290 vs. 1.966 ± 0.283; BC, 0.313 ± 0.290 vs. 1.384 ± 0.290. However, the optical technique did not show any statisticaldifference between pre- and postinjury with alloxan, 0.280 ± 0.305 vs. 0.483 ± 0.297, respectively. The alloxan injury, quantified byusing multiple-indicator techniques, showed an increase in permeability and a corresponding decrease in reflection coefficient for albumin (_f). Because the opticalmethod measures the product ofK_fc and _f, this study shows thatalbumin should not be used as an intravascular optical filtrationmarker when permeability is elevated. However, the optical technique,along with another means of measuringK_fc (such as BC),can be used to calculate the _fof a tracer (in this study, _fof 0.894 at baseline and 0.348 after injury). Another important findingof this study was that the ratio of baseline-to-injury K_fc values wasnot statistically different for Std and BC techniques, indicating thatthe percent contribution of slow blood-volume increases does not changebecause of injury.

     

ZmMYB31 directly represses maize lignin genes and redirects the phenylpropanoid metabolic flux

Few regulators of phenylpropanoids have been identified in monocots having potential as biofuel crops. Here we demonstrate the role of the maize (Zea mays) R2R3-MYB factor ZmMYB31 in the control of the phenylpropanoid pathway. We determined its in vitro consensus DNA-binding sequence as ACC(T)/(A) ACC, and chromatin immunoprecipitation (ChIP) established that it interacts with two lignin gene promoters in vivo. To explore the potential of ZmMYB31 as a regulator of phenylpropanoids in other plants, its role in the regulation of the phenylpropanoid pathway was further investigated in Arabidopsis thaliana. ZmMYB31 downregulates several genes involved in the synthesis of monolignols and transgenic plants are dwarf and show a significantly reduced lignin content with unaltered polymer composition. We demonstrate that these changes increase cell wall degradability of the transgenic plants. In addition, ZmMYB31 represses the synthesis of sinapoylmalate, resulting in plants that are more sensitive to UV irradiation, and induces several stress-related proteins. Our results suggest that, as an indirect effect of repression of lignin biosynthesis, transgenic plants redirect carbon flux towards the biosynthesis of anthocyanins. Thus, ZmMYB31 can be considered a good candidate for the manipulation of lignin biosynthesis in biotechnological applications.