Crystal structures of TM0549 and NE1324--two orthologs of E. coli AHAS isozyme III small regulatory subunit

Crystal structures of two orthologs of the regulatory subunit of acetohydroxyacid synthase III (AHAS, EC 2.2.1.6) from Thermotoga maritima (TM0549) and Nitrosomonas europea (NE1324) were determined by single-wavelength anomalous diffraction methods with the use of selenomethionine derivatives at 2.3 A and 2.5 A, respectively. TM0549 and NE1324 share the same fold, and in both proteins the polypeptide chain contains two separate domains of a similar size. Each protein contains a C-terminal domain with ferredoxin-type fold and an N-terminal ACT domain, of which the latter is characteristic for several proteins involved in amino acid metabolism. The ferredoxin domain is stabilized by a calcium ion in the crystal structure of NE1324 and by a Mg(H2O)(6)2+ ion in TM0549. Both TM0549 and NE1324 form dimeric assemblies in the crystal lattice.     

Costs of resistance to fungal pathogens in genetically modified wheat

Aims Many resistance genes against fungal pathogens show costs of resistance. Genetically modified (GM) plants that differ in only one or a few resistance genes from control plants present ideal systems for measuring these costs in the absence of pathogens.Methods To assess the ecological relevance of costs of pathogen resistance, we grew individual plants of four transgenic spring wheat lines in a field trial with three pathogen levels and varied the genetic diversity of the crop.Important findings We found that two lines with a Pm3b transgene were more resistant to powdery mildew than their sister lines of the variety Bobwhite, whereas lines with chitinase (A9) or chitinase and glucanase (A13) transgenes were not more resistant than their mother variety Frisal. Nevertheless, in the absence of the pathogen, both the GM lines of Bobwhite as well as those of Frisal performed significantly worse than their controls, i.e. Pm3b #1 and Pm3b #2 had 39% or 53% and A9 and A13 had 14% or 23% lower yields. In the presence of the pathogen, all GM lines except Pm3b #2 could increase their yields and other fitness-related traits, reaching the performance levels of the control lines. Line Pm3b #2 seemed to have lost its phenotypic plasticity and had low performance in all environments. This may have been caused by very high transgene expression. No synergistic effects of mixing different GM lines with each other were detected. This might have been due to high transgene expression or the similarity between the lines regarding their resistance genes. We conclude that costs of resistance can be high for transgenic plants with constitutive transgene expression and that this can occur even in cases where the non-transgenic control lines are already relatively resistant, such as in our variety Frisal. Transgenic plants could only compete with conventional varieties in environments with high pathogen pressure. Furthermore, the large variability among the GM lines, which may be due to unpredictable transgene expression, suggests that case-by-case assessments are necessary to evaluate costs of resistance.     

Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs

In cultured pulmonary artery endothelial cells and other cell types, overexpression of mt-targeted DNA repair enzymes protects against oxidant-induced mitochondrial DNA (mtDNA) damage and cell death. Whether mtDNA integrity governs functional properties of the endothelium in the intact pulmonary circulation is unknown. Accordingly, the present study used isolated, buffer-perfused rat lungs to determine whether fusion proteins targeting 8-oxoguanine DNA glycosylase 1 (Ogg1) or endonuclease III (Endo III) to mitochondria attenuated mtDNA damage and vascular barrier dysfunction evoked by glucose oxidase (GOX)-generated hydrogen peroxide. We found that both Endo III and Ogg1 fusion proteins accumulated in lung cell mitochondria within 30 min of addition to the perfusion medium. Both constructs prevented GOX-induced increases in the vascular filtration coefficient. Although GOX-induced nuclear DNA damage could not be detected, quantitative Southern blot analysis revealed substantial GOX-induced oxidative mtDNA damage that was prevented by pretreatment with both fusion proteins. The Ogg1 construct also reversed preexisting GOX-induced vascular barrier dysfunction and oxidative mtDNA damage. Collectively, these findings support the ideas that mtDNA is a sentinel molecule governing lung vascular barrier responses to oxidant stress in the intact lung and that the mtDNA repair pathway could be a target for pharmacological intervention in oxidant lung injury.     

Macro- and Microelement Status in Animal and Human Hypertension: the Role of the ACE Gene I/D Polymorphism

Growth hormone (GH) and zinc (Zn) were evaluated for their potential to prevent radiation injury using a rat model of radiation-induced skin injury. Sprague–Dawley rats were divided into five groups: a control group not receiving Zn, GH, or irradiation: a radiation (RT) group receiving a single 30 Gy dose of gamma irradiation to the right hind legs; a radiation + GH group (RT + GH) receiving a single 30 Gy dose of gamma irradiation plus the subcutaneous administration of 0.01 IU kg d^?1 GH; a radiation + Zn group (RT + Zn) receiving a single 30 Gy dose plus 5 mg kg d^?1 Zn po; and a radiation + GH + Zn group (RT + GH + Zn) group receiving a single 30 Gy dose plus subcutaneous 0.01 IU kg d^?1 GH and 5 mg kg d^?1 Zn po. Acute skin reactions were assessed every 3 days by two radiation oncologists grouping. Light microscopic findings were assessed blindly by two pathologists. Groups receiving irradiation were associated with dermatitis as compared to the control group (P < 0.05). The severity of radiodermatitis in the RT + GH, RT + Zn, and RT + GH + Zn groups was significantly lower than that in the RT group (P < 0.05). Furthermore, radiodermatitis was observed earlier in the RT group than in the other treatment groups (P < 0.05). GH and Zn effectively prevented epidermal atrophy, dermal degeneration, and hair follicle atrophy. The highest level of protection against radiation dermatitis was observed in the combination group.     

Influence of Factors of Cryopreservation and Hypothermic Storage on Survival and Functional Parameters of Multipotent Stromal Cells of Placental Origin

Human placenta is a highly perspective source of multipotent stromal cells (MSCs) both for the purposes of patient specific auto-banking and allogeneic application in regenerative medicine. Implementation of new GMP standards into clinical practice enforces the search for relevant methods of cryopreservation and short-term hypothermic storage of placental MSCs. In this paper we analyze the effect of different temperature regimes and individual components of cryoprotective media on viability, metabolic and culture properties of placental MSCs. We demonstrate (I) the possibility of short-term hypothermic storage of these cells; (II) determine DMSO and propanediol as the most appropriate cryoprotective agents; (III) show the possibility of application of volume expanders (plasma substituting solutions based on dextran or polyvinylpyrrolidone); (IV) reveal the priority of ionic composition over the serum content in cryopreservation media; (V) determine a cooling rate of 1°C/min down to -40°C followed by immersion into liquid nitrogen as the optimal cryopreservation regime for this type of cells. This study demonstrates perspectives for creation of new defined cryopreservation methods towards GMP standards.     

Correction of autoradiographic grain count in respect to precisely calculated background

Summary It was endeavored to find a criterion for significantly labeled cells in quantitative autoradiography. Measurements of the autoradiographic background were performed and it was found that: 1. the value of the background over the non-proliferating epithelial cells from an animal injected with ³H-thymidine is higher than over the same cells from animals not injected with an isotope, 2. the value of the background in emulsion over the tissue specimen is higher than away from the specimen. Therefore, one should take into account the background over the tissue. Nomograms are shown for quick evaluation of the percentage of cells labeled with 1, 2, 3 or 4 grains, which should be disregarded as due to the background. To obtain this percentage for a given experiment its appropriate parameters: the labeling index, the mean grain count over the cell, the standard deviation of the grain count distribution and the background grain count distribution should be taken into account.     

Structure of the extremely slow GTPase Rab6A in the GTP bound form at 1.8 Å resolution

Rab/Ypt GTPases represent a>60 member large family of membrane traffic regulators in eukaryotic cells. Members of this group display intrinsic GTPase activity varying over two orders of magnitude. Here, we show that Rab6A represents the RabGTPase with the slowest spontaneous GTPase activity yet measured (5x10(-6)s(-1)). Due to the very low intrinsic hydrolysis rate we were able to crystallise and solve the structure of the Rab6A:GTP complex to 1.82A resolution. Analysis of the structure suggests that low catalytic activity of the Rab6A might be due to high flexibility of the Switch II region and a low degree of constraint of critically important for catalysis Gln 72.