Trityl-cyanoethylidene condensation of azidosaccharide derivatives as a route to hexosaminoglycans. Synthesis of the O-antigenic polysaccharide of Pseudomonas aeruginosa X (Meitert)]

Derivatives of azidosugars were shown to be stable under conditions of trityl-cyanoethylidene condensation. Tritylated 1,2-O-(1-cyano)ethylidene derivative of 2-azido-2-deoxy-beta-D-mannopyranosyl-(1----4)-L-rhamnopyranose was used as a starting material for the synthesis of [----3)-beta-D-ManNAc-(1----4)-alpha-L-Rha-(1----]n, the O-specific polysaccharide of Pseudomonas aeruginosa X (Meitert).     

Self-assembling and membrane modifying properties of a lipopeptaibol studied by CW-ESR and PELDOR spectroscopies.

Trichogin GA IV is a short lipopeptaibol antibiotic that is capable of enhancing the transport of small cations through the phospholipid double layer of the membrane. The antibiotic activity of the undecapeptide is thought to be based on either its self-assembling or membrane-modifying property. The chemical equilibrium between self-aggregated and non-aggregated molecular states was studied by CW-ESR spectroscopy using solutions of TOAC nitroxide spin-labelled trichogin analogues in an apolar solvent to mimic the membrane bound state. At room temperature the two different sets of signals observed in the spectrum were attributed to the presence of both monomers and aggregates in the sample. The ESR spectra of the monomeric and aggregated forms were separated and the dependence of the fraction of monomeric peptide molecules on concentration was obtained over the range 5 x 10(-6) to 7 x 10(-4) M. A two-step aggregation mechanism is proposed: dimerization of peptide molecules followed by aggregation of dimers to assemblies of four peptide molecules per aggregate. The equilibrium constants were estimated for both steps. In addition, the lower lifetime limit was determined for dimers and tetramers. It is shown that when the peptide concentration exceeds 10(-5) M. the major part of the peptide molecules in solution has the form of tetrameric aggregates. Independently, the PELDOR technique was used to investigate the concentration dependence of the parameters of the dipole-dipole interaction between spin labels in frozen (77 K] glassy solutions of aggregates of mono-labelled TOAC analogues. The number of molecules in aggregates as well as the frequency and amplitude of PELDOR signal oscillations were found to be concentration independent in the range 5 x 10(-4) to 8 x 10(-3) M. In the frozen glassy solution state, the number of peptide molecules per aggregate was determined to be close to four, which is in agreement with the value obtained for spin-labelled trichogin at room temperature. The present data provide experimental evidence in favour of a self-assembling rather than a membrane-modifying ion conduction mechanism.     

Interaction of chromatin-associated Plk1 and Mcm7

Plk1 is a multifunctional protein kinase involved in regulation of mitotic entry, chromosome segregation, centrosome maturation, and mitotic exit. Plk1 is a target of DNA damage checkpoints and aids resumption of the cell cycle during recovery from G2 arrest. The polo-box domain (PBD) of Plk1 interacts with phosphoproteins and localizes Plk1 to some mitotic structures. In a search for proteins that interact with the PBD of Plk1, we identified two of the minichromosome maintenance (MCM) proteins, Mcm2 and Mcm7. Co-immunoprecipitation and immunoblot analysis showed an interaction between full-length Plk1 and all other members of the MCM2-7 protein complex. Endogenous Plk1 co-immunoprecipitates with basal forms of Mcm7 as well as with slower migrating forms of Mcm7, induced in response to DNA damage. The strongest interaction between endogenous Plk1 and Mcm7 was detected in a soluble chromatin fraction. These findings suggest a new function for Plk1 in coordination of DNA replication and mitotic events.     

Assessment of genetic diversity of wild soybean (Glycine soja Siebold et Zucc.) in the far eastern region of Russia

Polymorphism of RAPD markers was analyzed in the wild soybean populations from the Far East region of Russia. The level of RAPD marker polymorphism was significantly higher in the wild than in the cultivated soybean. The results obtained suggest active development of genetically different groups of wild soybean. Geographically isolated subpopulations showed maximum distance from the main population of wild soybean.     

Synthesis, NMR and conformational studies of fucoidan fragments. VI. Fragments, content of alpha-(1--->2)-bound fucobioside unit

A series of selectively sulfated di- and trisaccharide derivatives corresponding to the potential fragments of fucoidans with a (1-->2)-alpha-bound fucobioside unit were synthesized and studied by 1H and 13C NMR spectroscopy. NOE experiments and molecular modeling were used for a conformational analysis of the compounds synthesized. In the case of disaccharides, the experimental NOE values were found to agree with those obtained using modeling with the use of density functional theory (DFT) and differ from those resulting from modeling by the molecular mechanics MM3 force field. Trisaccharide fragments partially or completely sulfated in position 4 turned out to be correctly described by both MM3 force field and DFT computation. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

Study of the Carbohydrate Specificity of Antibodies Against <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> Using the Library of Synthetic Mycoantigens

The carbohydrate specificity of antibodies obtained by immunizing laboratory animals with immunogens prepared from the Aspergillus fumigatus cell wall was analyzed using the library of biotinylated synthetic oligosaccharides. It has been shown that the main part of antipolysaccharide antibodies recognizes galactomannan in the studied immunogens with preference to its fragments containing more than two β-(1→5)-linked galactofuranosyl units. The data obtained can form the basis for the development of enzyme immunoassay for the detection of this dangerous fungal pathogen.     

Acquisition of the sequence of a new subclass of human alpha- satellite DNA, localized in the centromere regions of two pairs of acrocentric chromosomes

The results are presented in the paper on obtaining of a clone of sub-group of alpha-satellite human DNA localized in heterochromatin regions of two pairs of acrocentric chromosomes. The obtained repeated DNA sequence is represented in a genome by some 20,000 copies. Using of the blot-hybridization method made it possible to show the polymorphic restriction variant distribution being preserved in different individuals genomes. Intergenomic structural heterogeneity in this alpha-satellite DNA sub-group has been found only by using restriction endonuclease BamHI.     

The pool of histones in the nucleosol and cytosol of proliferating Friend cells is small, uneven and chasable.

This study examines the histones pools in the nucleosol and cytosol of proliferating Friend cells. By using the conventional approach, detectable amounts of these molecules were found in both compartments; however, only H3 and H2B were identified in nucleosol, and H3, H2B and H4 in cytosol. The authenticity of each of these histones was verified by two independent methods, migration in SDS/polyacrylamide gels and peptide mapping. When the sensitivity of the approach was increased by radiolabelling with 125I, two additional proteins, migrating as H2A and H4, were observed in nucleosol. Even by this approach, however, H1 was not detected. Direct quantitative measurements of the histones in both compartments indicated that these pools are uneven and small. This was found also in experiments involving inhibition of protein synthesis by cycloheximide. Considered together, our data do not support the idea of the existence of preformed histone heterocomplexes or octamers. Instead the assembly of nucleosomes during replication occurs by a successive deposition of individual core histones.     

Mode of deposition of the histone subtypes during replication

By using various approaches, we have observed in cycling mouse erythroleukemia cells a characteristic sequential deposition of newly synthesized histones. Control experiments confirmed that this deposition is replicative and is not associated with a process of histone replacement. Interestingly, each of the newly synthesized variants of histones H3, H2B, and H2A was found to deposit simultaneously. This observation indicates that the mechanisms governing the nucleosome assembly do not discriminate among the variants and thus supports the idea that they have equal biological significance. In contrast, a different mode of deposition was found for the regular subtypes H1A and H1B, suggesting a structural and functional difference between these two histones.