Molecular mechanisms of allosteric probe dependence in μ opioid receptor

Allostery is one of the most important features of proteins. It greatly contributes to the complexity of life, since it enables possibility of precise tuning of protein function, as well as performing more than one function per protein. Probe dependence is one of the unique features of allostery. It allows a protein to respond differently to the same allosteric modulator when different drugs or transmitters are bound. Unfortunately, allosteric mechanisms are difficult to investigate experimentally. Instead, they can be reproduced artificially in simulations. We simulated in silico a native-like cell membrane fragment with an active-state human μ opioid receptor (MOR) in order to investigate diverse effects of a receptor’s positive allosteric modulator on various agonists. Particular emphasis on native-likeness of the environment was put. We managed to reproduce the experimentally observed effects, which allowed us to take deeper insight into their underlying mechanisms. We found an allosteric pathway in the receptor, leading from the ligand binding site to the intracellular, effector site. We observed that the modulator affected the pathway, inducing different resultant responses for full and partial agonists.     

Night shift work and osteoporosis: evidence and hypothesis

Osteoporosis is an important public health problem worldwide. Among the countries with a very high population risk of fractures, there are those with the highest level of economic development. Osteoporotic fractures are the main cause of disability among elderly people, and the resultant disabilities require particularly large financial support associated not only with the direct treatment of the fracture but also with the necessity for long-term rehabilitation and care for the disabled person. Many well-established factors can have impact on bone mass and fracture risk. Recently, it has been hypothesized that working during nighttime which leads to endocrine disorders may have an indirect impact on bone physiology among night shift workers. Therefore, it can be presumed that the night shift work may contribute to the etiology of osteoporosis. The aim of our work was to make a review of the epidemiological evidence on the association between night shift work and bone mineral density or fracture risk as well as to discuss the potential biological mechanisms linking the work under this system with the development of osteoporosis. We have identified only four studies investigating the association between system of work and bone mineral density or fracture risk among workers. The findings of three out of four studies support the hypothesis. None of the studies has investigated a potential relationship between night shift work and bone turnover markers. Given that there have been no epidemiological studies in European countries that would concern working populations and the noticeable difference in the risk of osteoporosis between communities, further studies are warranted to elucidate the problem. It is presumed that further in-depth studies will not only identify the underlying factors of the disease but also contribute to developing guidelines for policy makers and employers for primary prevention of osteoporosis in workplace.     

Ergosterol and Fusarium mycotoxins content in two maize cultivars under different forms of nitrogen fertilizers

Mycotoxins are secondary metabolites produced by many filamentous fungi, such as Fusarium, and are widespread in nature and can adversely affect almost all organisms. Mycotoxins can be formed in very different conditions such as when plants are growing or in crops that are stored in improper conditions. One of the methods to prevent the formation of mycotoxins in powdered feed is using varieties of appropriate quality and proper mineral fertilization, primarily nitrogen. In this study, the concentration of ergosterol and mycotoxins in the kernels of two maize cultivars from different maturity groups in Poland during the harvest of 2011 and 2012 was investigated using HPLC methods. We aimed to analyse the concentration of ergosterol as a fungal biomass indicator and mycotoxins from two maize cultivars under six different forms of nitrogen fertilizers. The “stay‐green” hybrid ES Paroli was characterized by a significantly lower ergosterol and the ability to accumulate the mycotoxins, compared to the classic cultivar ES Palazzo. The varieties of maize that remain green for a longer time period, the so‐called stay‐green type, are characterized by a different coefficient of nitrogen remobilization compared to the classic hybrids. Their dynamics of growth and accumulation of nutrients indicates a nitrogen fertilization system, indicating that slow‐release fertilizers are potentially more adapted to the cycles of maize vegetation. Hence, compared to the classic variant, the “stay‐green” variety uses nitrogen more effectively from mineral fertilizers. Such hybrids are healthier and remain in good condition for a longer time; therefore, they are characterized by a lower pathogen pressure. Thus, the cultivation of such varieties can be considered as one of the elements of integrated maize production. We observed identical reactions of the studied experimental factors for both vegetation seasons, which indicates the lack of environmental impact on the functioning and interaction of experimental factors.     

Experimental Models of Protein–RNA Interaction: Isolation and Analyses of tRNAPhe and U1 snRNA-Binding Peptides from Bacteriophage Display Libraries

Peptides that bind either U1 small nuclear RNA (U1 snRNA) or the anticodon stem and loop of yeast tRNA^Phe (tRNA _AC ^Phe ) were selected from a random-sequence, 15-amino acid bacteriophage display library. An experimental system, including an affinity selection method, was designed to identify primary RNA-binding peptide sequences without bias to known amino acid sequences and without incorporating nonspecific binding of the anionic RNA backbone. Nitrocellulose binding assays were used to evaluate the binding of RNA by peptide-displaying bacteriophage. Amino acid sequences of RNA-binding bacteriophage were determined from the foreign insert DNA sequences, and peptides corresponding to the RNA-binding bacteriophage inserts were chemically synthesized. Peptide affinities for the RNAs (K _d ? 0.1–5.0 μM) were analyzed successfully using fluorescence and circular dichroism spectroscopies. These methodologies demonstrate the feasibility of rapidly identifying, isolating, and initiating the analyses of small peptides that bind to RNAs in an effort to define better the chemistry, structure, and function of protein–RNA complexes.     

Effects of anesthetics on systemic hemodynamics in mice

The aim of this study was to compare the systemic hemodynamic effects of four commonly used anesthetic regimens in mice that were chronically instrumented for direct and continuous measurements of cardiac output (CO). Mice (CD-1, Swiss, and C57BL6 strains) were instrumented with a transit-time flow probe placed around the ascending aorta for CO measurement. An arterial catheter was inserted into the aorta 4 or 5 days later for blood pressure measurements. After full recovery, hemodynamic parameters including stroke volume, heart rate, CO, mean arterial pressure (MAP), and total peripheral resistance were measured with animals in the conscious state. General anesthesia was then induced in these mice using isoflurane (Iso), urethane, pentobarbital sodium, or ketamine-xylazine (K-X). The doses and routes of administration of these agents were given as required for general surgical procedures in these animals. Compared with the values obtained for animals in the conscious resting state, MAP and CO decreased during all anesthetic interventions, and hemodynamic effects were smallest for Iso (MAP, -24 +/- 3%; CO, -5 +/- 7%; n = 15 mice) and greatest for K-X (MAP, -51 +/- 6%; CO, -37 +/- 9%; n = 8 mice), respectively. The hemodynamic effects of K-X were fully antagonized by administration of the alpha(2)-receptor antagonist atipamezole (n = 8 mice). These results indicate that the anesthetic Iso has fewer systemic hemodynamic effects in mice than the nonvolatile anesthetics.     

Mouse development and cell proliferation in the absence of D-cyclins

D-type cyclins (cyclins D1, D2, and D3) are regarded as essential links between cell environment and the core cell cycle machinery. We tested the requirement for D-cyclins in mouse development and in proliferation by generating mice lacking all D-cyclins. We found that these cyclin D1(-/-)D2(-/-)D3(-/-) mice develop until mid/late gestation and die due to heart abnormalities combined with a severe anemia. Our analyses revealed that the D-cyclins are critically required for the expansion of hematopoietic stem cells. In contrast, cyclin D-deficient fibroblasts proliferate nearly normally but show increased requirement for mitogenic stimulation in cell cycle re-entry. We found that the proliferation of cyclin D1(-/-)D2(-/-)D3(-/-) cells is resistant to the inhibition by p16(INK4a), but it critically depends on CDK2. Lastly, we found that cells lacking D-cyclins display reduced susceptibility to the oncogenic transformation. Our results reveal the presence of alternative mechanisms that allow cell cycle progression in a cyclin D-independent fashion.     

Infiltration with <Emphasis Type="Italic">Agrobacterium</Emphasis> — the method for stable transformation avoiding tissue culture

A number of in planta transformation protocols that avoid long culture under sterile conditions were developed for Arabidopsis thaliana. The most widely used methods are based on vacuum infiltration and floral dip. These methods were adapted for transformation of other species as well. Successful in planta transformations of alfalfa, radish, pakchoi and petunia were reported recently. In this short review we present several modified procedures originally developed for Arabidopsis thaliana and in some cases adapted to other species. We emphasize the crucial parameters involved in in planta transformation. We also describe here the studies attempting to shed light on the mechanisms and estimating the cellular target of transformation, which may help in transforming new plant species.     

Validation of semiempirical methods for modeling of corrinoid systems

Several semiempirical methods (MNDO-d, PM3tm, PM3-d, PM5, PM6, and AM1-d) have been tested against experimental data and density functional theory (DFT) results in search for the best methods that can be used for quantum-mechanical-molecular mechanics (QM/MM) modeling of corrinoid systems of vitamin B(12) co-factor. It has been found that the PM6 parametrization in its present form gives results closest to hybrid DFT calculations that are most widely used thus far. In comparison with pure DFT and experimental data the best agreement is obtained for PM3tm parametrization, while PM6 yields slightly worse results. AM1-d yields bad geometry of the corrin moiety. The worst performance was observed for MNDO-d, which has severe problem with position and orientation of the alpha-ligands.     

Effect of restricted feed intake and addition of vitamins B2 and B6 and folic acid on the liver concentration of zinc and copper in rats

The aim of the present study was to investigate the influence of nutritional deficiency and dietary addition of vitamins (B₂, B₆, and folate) on hepatic concentration of zinc and copper in rats. The experiment was performed on 260 growing male Wistar rats divided into 13 groups. Animals of 11 groups were fed isocaloric diets (14.7 MJ/kg) in which the 20% of energy was derived from protein. Another two groups of rats were offered diets with 9% or 4.5% of energy originating from protein. Animals of both mentioned groups and of the control group (20% of energy from protein) were offered diets ad libitum. The other 10 groups were offered 50% and 30% of the amount consumed in the control group. Eight groups, from those 10 restricted ones, were differentiated by dietary addition of vitamins B₂ and B₆ and folate (300% addition). Restricted feed intake did not affect the liver zinc concentration but significantly increased the copper concentration. The addition of vitamin B₆ decreased the liver Zn concentration. The highest liver Cu concentration was noted in rats offered restricted diets to only 30% of intake in the control group and high in vitamin B₂ and in rats supplemented with all of studied vitamins together. It suggests that vitamin B₂ had the strongest impact on liver Cu concentration in rats fed restricted diets.