Acylation of Bowman–Birk Soybean Proteinase Inhibitor by Unsaturated Fatty Acid Derivatives

A procedure was developed for acylation of Bowman–Birk soybean proteinase inhibitor (BBI) by N-hydroxysuc-cinimide esters of oleic, linoleic, and -linolenic acids in a dimethyl sulfoxide–dioxane–pyridine mixture. BBI derivatives containing two acylated amino groups were prepared with high yield. The use of the reversible modifier citraconic anhydride in the first stage of synthesis permitted the synthesis of hydrophobized BBI derivatives retaining high antitrypsin and antichymotrypsin activities. It was found that the insertion of two long chain moieties in the BBI molecule decreases its thermostability.     

2'-modified oligonucleotides from methoxyoxalamido and succinimido precursors: synthesis, properties, and applications

Synthesis of 2'-modified oligonucleotides from 2'-methoxyoxalamido (MOX) and 2'-succinimido (SUC) precursors is described. Their physical and biochemical properties were assessed. Synthesized oligonucleotides were used as primers in advanced DNA sequencing protocols. An example of sequencing directly off genomic DNA template without prior cloning or PCR amplification is presented.     

Distribution and localization of CMP-N-acetylneuraminic acid hydroxylase and N-glycolylneuraminic acid-containing glycoconjugates in porcine lymph node and peripheral blood lymphocytes

An immunohistochemical analysis was performed on paraplast-embedded sections of porcine lymph node with antibodies specific for CMP-N-acetylneuraminic acid hydroxylase (h-3 antibody) and glycoconjugate-bound N-glycolylneuraminic acid (Neu5Gc), which appears as a result of the hydroxylase reaction (a-Gc antibody). The observed localization of the enzyme in cells of the perifollicular zone, including lymphocytes, was reflected in a similar distribution of glycoconjugate-bound Neu5Gc. This result confirms previous biochemical investigations on the role of the hydroxylase in regulating Neu5Gc biosynthesis in vitro on a histological level. An analysis of lymphocytes isolated from porcine thymus, spleen, lymph node and peripheral blood revealed differences in the amount of Neu5Gc in the various lymphocytes that correlated well with the activity of the hydroxylase determined in these cells. The largest amount of Neu5Gc and highest activity of the enzyme were detected in the peripheral blood lymphocytes (PBL). Immunohistochemical studies with a-Gc and h-3 antibodies on sections of paraplast-embedded PBL showed that these antigens were located at the cell surface and in the cytosol, respectively. Ultrastructural immunocytochemistry with the h-3 antibody and immunogold labelling was used to investigate the subcellular localization of the hydroxylase. The enzyme was detected in the cytosol in the vicinity of the nuclear membrane and the outer membrane of mitochondria, in particular those close to the nucleus. The antigen was also detected on cytoplasmic tubular structures. In addition, a weak labelling of the Golgi apparatus was also observed occasionally. The possibility that this localization may be related to the availability of the substrate CMP-Neu5Ac and the redox partner cytochrome b5 is discussed.     

Analyzing proteome topology and function by automated multidimensional fluorescence microscopy

Temporal and spatial regulation of proteins contributes to function. We describe a multidimensional microscopic robot technology for high-throughput protein colocalization studies that runs cycles of fluorescence tagging, imaging and bleaching in situ. This technology combines three advances: a fluorescence technique capable of mapping hundreds of different proteins in one tissue section or cell sample; a method selecting the most prominent combinatorial molecular patterns by representing the data as binary vectors; and a system for imaging the distribution of these protein clusters in a so-called toponome map. By analyzing many cell and tissue types, we show that this approach reveals rules of hierarchical protein network organization, in which the frequency distribution of different protein clusters obeys Zipf's law, and state-specific lead proteins appear to control protein network topology and function. The technology may facilitate the development of diagnostics and targeted therapies.     

A pulse-periodic torch in a coaxial waveguide: Formation dynamics and spatial structure

Results are presented from experimental studies of the formation dynamics, spatial structure, and parameters of a pulse-periodic microwave discharge excited in a coaxial waveguide. The experimental setup allows the stable generation of a plasma jet in molecular and atomic gas flows at pressures close to atmospheric pressure without applying additional initiators. The complicated sequence of processes leading to torch formation cannot be adequately described with conventional models of a discharge sustained by a surface electromagnetic wave.     

Analysis of the structure-functional features of a highly-repeating centromere sequence from the Fringilla coelebs L. (Aves: Passeriformes) genome]

A highly repetitive centromeric Fringilla coelebs PstI (FCP) element was cloned and sequenced. The FCP tandem repeats with unit 505 or 506 nt accounted for about 0.9% of the entire genome and had 57% GC. Direct genomic sequencing with FCP-specific primers and ThermoFidelase 2A revealed the consensus sequence and the five most common single-nucleotide polymorphisms (SNPs) for the FCP unit. FCP may be transcribed and may play a role in spatial arrangement of the genome.     

Structural and Functional Characterization of a Centromeric Highly Repetitive DNA Sequence of Fringilla coelebsL. (Aves: Passeriformes)

A highly repetitive centromeric Fringilla coelebs PstI (FCP) element was cloned and sequenced. The FCP tandem repeats with a unit length 505 or 506 accounted for about 0.9% of the entire genome and had 57% GC. Direct genomic sequencing with FCP-specific primers and ThermoFidelase 2A revealed the consensus sequence and the five most common single-nucleotide polymorphisms (SNPs) for the FCP unit. FCP may be transcribed and may play a role in the spatial arrangement of the genome.     

Self-assembling systems based on amphiphilic poly-N-vinylpyrrolidone and their interaction with model proteins

Polymeric particles formed by stearoyl-poly-N-vinylpyrrolidone (PVP-stear) of M_n = 2600 were obtained in aqueous solution, and their shape and size distribution were characterized. The size of the particles was shown to decrease with an increase in the ionic strength of the solution. Interaction of PVP-stear and its aggregates with model proteins (Bowman–Birk soybean proteinase inhibitor (BBI) and its hydrophobized derivatives) was studied. The possibility of inclusion of both native BBI and oleoylic derivative of BBI in the PVP-stear polymeric aggregates was investigated. It was established that polymeric particles with a diameter of 30 nm formed under certain concentration ratios between PVP-stear and poorly soluble dioleoyl BBI are capable of solubilization of dioleoyl BBI as well as prevention of its inactivation at low pH values.     

Novel biotin phosphoramidites with super-long tethering arms

A number of novel biotin phosphoramidites, possessing exceptionally long and uncharged tethering arms, were synthesized from methoxyoxalamido (MOX) and succinimido (SUC) precursors. Included among these monomers is a uridine derivative with the biotin moiety attached through the 2'-position. Some of these phosphoramidites were used to make 5'-biotinylated primers, which were applied in direct sequencing of genomic DNA and capture of Sanger fragment pools.     

Biological properties of N-acyl and N-haloacetyl neuraminic acids: processing by enzymes of sialic acid metabolism,and interaction with influenza virus

Several unnatural N-acyl neuraminic acids (N-propionyl, N-hexanoyl, N-benzoyl, N-trifluoroacetyl, N-chloroacetyl, N-difluoroacetyl) were prepared enzymatically using immobilised sialic acid aldolase. N-Trifluoroacetyl-, N-chloroacetyl- and N-difluoroacetyl neuraminic acids were shown to enhance up to 10-fold the rate of association of influenza virus A to a sialoglycolipid neomembrane by surface plasmon resonance, and were found to act as weak inhibitors (K(iapp) 0.45-2.0 mM) of influenza virus neuraminidase. The N-propionyl, N-chloroacetyl- and N-difluoroacetyl neuraminic acids were found to be substrates for recombinant Escherichia coli CMP sialate synthase, to give the corresponding CMP-N-acyl-neuraminic acids. CMP-N-propionyl neuraminic acid was found not to be a substrate for CMP-N-acetyl neuraminic acid hydroxylase from pig submandibular gland.