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Recent Deceleration of the Ice Elevation Change of Ecology Glacier (King George Island, Antarctica)
Get PDFPhosphorylation of thymidylate synthase affects slow-binding inhibition by 5-fluoro-dUMP and N4-hydroxy-dCMP
Get PDFEndogenous thymidylate synthases, isolated from tissues or cultured cells of the same specific origin, have been reported to show differing slow-binding inhibition patterns. These were reflected by biphasic or linear dependence of the inactivation rate on time and accompanied by differing inhibition parameters. Considering its importance for chemotherapeutic drug resistance, the possible effect of thymidylate synthase inhibition by post-translational modification was tested, e.g. phosphorylation, by comparing sensitivities to inhibition by two slow-binding inhibitors, 5-fluoro-dUMP and N4-hydroxy-dCMP, of two fractions of purified recombinant mouse enzyme preparations, phosphorylated and non-phosphorylated, separated by metal oxide/hydroxide affinity chromatography on Al(OH)3 beads. The modification, found to concern histidine residues and influence kinetic properties by lowering Vmax, altered both the pattern of dependence of the inactivation rate on time from linear to biphasic, as well as slow-binding inhibition parameters, with each inhibitor studied. Being present on only one subunit of at least a great majority of phosphorylated enzyme molecules, it probably introduced dimer asymmetry, causing the altered time dependence of the inactivation rate pattern (biphasic with the phosphorylated enzyme) and resulting in asymmetric binding of each inhibitor studied. The latter is reflected by the ternary complexes, stable under denaturing conditions, formed by only the non-phosphorylated subunit of the phosphorylated enzyme with each of the two inhibitors and N5,10-methylenetetrahydrofolate. Inhibition of the phosphorylated enzyme by N4-hydroxy-dCMP was found to be strongly dependent on [Mg2+], cations demonstrated previously to also influence the activity of endogenous mouse TS isolated from tumour cells
PCNA SUMOylation protects against PCNA polyubiquitination-mediated, Rad59-dependent, spontaneous, intrachromosomal gene conversion.
Get PDFPhage display-selected peptides for binding and synthesis of nanoparticles: ZnO as an example
Get PDFNanoparticles of metal oxides are widely used in bionanotechnology, particularly in bio-medical applications; e.g., construction of biosensors, separation of biological materials, molecular imaging, and anticancer and antimicrobial therapies. However,
synthesis of these nanoparticles using physico-chemical methods is problematic, because such procedures require high-temperature processes and harsh chemical treatments. The use of peptides specifically binding particular nanoparticles or nanostructures and facilitating their synthesis appears to be an encouraging alternative. Specific peptides capable of such reactions may be identified with the use of the phage display method. In this mini-review, zinc oxide is discussed as an exemple material whose nanoparticles can be bound and synthesized by such peptides exposed on the surface of bacteriophage capsids. An analysis of reports on studies into methods of peptide-aided synthesis of ZnO nanoparticles has indicated that, despite the encouraging results obtained so far, further studies are necessary to optimize such procedures. This may also be true for nanoparticles of other materials, particularly metal oxides
Isoprenoid generating systems in plants—A handy toolbox how to assess contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthetic process
Get PDFIsoprenoids comprise an astonishingly diverse group of metabolites with numerous potential and actual applications
in medicine, agriculture and the chemical industry. Generation of efficient platforms producing isoprenoids is a target of numerous laboratories. Such efforts are generally enhanced if the native biosynthetic routes can be
identified, and if the regulatory mechanisms responsible for the biosynthesis of the compound(s) of interest can be determined. In this review a critical summary of the techniques applied to establish the contribution of the two alternative routes of isoprenoid production operating in plant cells, the mevalonate and methylerythritol pathways, with a focus on their co-operation (cross-talk) is presented. Special attention has been paid to methodological aspects of the referred studies, in order to give the reader a deeper understanding for the nuances of these powerful techniques. This review has been designed as an organized toolbox, which might offer the researchers comments useful both for project design and for interpretation of results obtained
Structural Dynamics of the Vimentin Coiled-Coil Contact Regions involved in Filament Assembly as revealed by Hydrogen-Deuterium Exchange
Get PDFIntermediate filaments (IF) are major constituents of the cytoskeleton of metazoan cells. They not only are responsible for the mechanical properties but also for various physiological activities in different cells and tissues. The building blocks of IFs are extended coiled-coil−forming proteins exhibiting a characteristic central α-helical domain (″rod″). The fundamental principles of the filament assembly mechanism and the network formation have been widely elucidated for the cytoplasmic IF protein vimentin. Also, a comprehensive structural model for the tetrameric complex of vimentin has been obtained by X-ray crystallography in combination with various biochemical and biophysical techniques. To extend these static data and investigate the dynamic properties of the full-length proteins in solution during the various assembly steps, we analyzed the patterns of hydrogen-deuterium exchange (HDex) in vimentin and in four variants carrying point mutations in the IF consensus motifs present at either end of theα-helical rod that cause an assembly arrest at the unit-length filament (ULF) stage. The results yielded unique insights into the structural properties of subdomains within full-length vimentin, in particular in regions of contact in α-helical and linker segments that stabilize different oligomeric forms such as tetramers, ULFs, and mature filaments. Moreover, HDex analysis of the point-mutated variants directly demonstrated the active role of the IF-consensus motifs in the oligomerization mechanism of tetramers during ULF formation. Ultimately, using molecular dynamics simulation procedures, we provide a structural model for the subdomain-mediated tetramer−tetramer interaction via ″cross-coiling″ as the first step of the assembly process
Network of protein interactions within the Drosophilainner kinetochore
Get PDFThe kinetochore provides a physical connection between microtubules and the centromeric regions of chromosomes that is critical for their equitable segregation. The trimeric Mis12 sub-complex of the Drosophila kinetochore binds to the mitotic centromere using CENP-C as a platform. However, knowledge of the precise connections between Mis12 complex components and CENP-C has remained elusive despite the fundamental importance of this part of the cell division machinery. Here, we employ hydrogen–deuterium exchange coupled with mass spectrometry to reveal that Mis12 and Nnf1 form a dimer maintained by interacting coiled-coil (CC) domains within the carboxy-terminal parts of both proteins. Adjacent to these interacting CCs is a carboxy-terminal domain that also interacts with Nsl1. The amino-terminal parts of Mis12 and Nnf1 form a CENP-C-binding surface, which docks the complex and thus the entire kinetochore to mitotic centromeres. Mutational analysis confirms these precise interactions are critical for both structure and function of the complex. Thus, we conclude the organization of the Mis12–Nnf1 dimer confers upon the Mis12 complex a bipolar, elongated structure that is critical for kinetochore function
Codon optimization of antigen coding sequences improves the immune potential of DNA vaccines against avian influenza virus H5N1 in mice and chickens
Get PDFBACKGROUND:
Highly pathogenic avian influenza viruses are a serious threat to domestic poultry and can be a source of new human pandemic and annual influenza strains. Vaccination is the main strategy of protection against influenza, thus new generation vaccines, including DNA vaccines, are needed. One promising approach for enhancing the immunogenicity of a DNA vaccine is to maximize its expression in the immunized host.
METHODS:
The immunogenicity of three variants of a DNA vaccine encoding hemagglutinin (HA) from the avian influenza virus A/swan/Poland/305-135V08/2006 (H5N1) was compared in two animal models, mice (BALB/c) and chickens (broilers and layers). One variant encoded the wild type HA while the other two encoded HA without proteolytic site between HA1 and HA2 subunits and differed in usage of synonymous codons. One of them was enriched for codons preferentially used in chicken genes, while in the other modified variant the third position of codons was occupied in almost 100 % by G or C nucleotides.
RESULTS:
The variant of the DNA vaccine containing almost 100 % of the GC content in the third position of codons stimulated strongest immune response in two animal models, mice and chickens. These results indicate that such modification can improve not only gene expression but also immunogenicity of DNA vaccine.
CONCLUSION:
Enhancement of the GC content in the third position of the codon might be a good strategy for development of a variant of a DNA vaccine against influenza that could be highly effective in distant hosts, such as birds and mammals, including humans
Protein phosphatase type 2C PP2CA together with ABI1 inhibits SnRK2.4 activity and regulates plant responses to salinity
Get PDFProtein phosphatases 2C (PP2Cs) are important regulators of plant responses to abiotic stress. It is established that clade A PP2Cs inhibit ABA-activated SNF1-related protein kinases 2 (SnRK2s). Our recently published results show that ABI1, a member of clade A of PP2C is also a negative regulator of SnRK2.4, a kinase not activated in response to ABA. Here, we show that another member of this clade - PP2CA, interacts with and inhibits SnRK2.4. The salt-induced SnRK2.4/SnRK2.10 activity is higher in abi1-2 pp2ca-1 mutant than in wild type or single abi1 or pp2ca mutants, indicating that both phosphatases are inhibitors of SnRK2.4 and are at least partially redundant. Moreover, PP2CA together with ABI1 and SnRK2.4 regulates root growth in response to salinity