Biodynamic parameters of micellar diminazene in sheep erythrocytes and blood plasma

In this work, we used a preparation of diminazene, which belongs to the group of aromatic diamidines. This compound acts on the causative agents of blood protozoan diseases produced by both flagellated protozoa (Trypanosoma) and members of the class Piroplasmida (Babesia, Theileria, and Cytauxzoon) in various domestic and wild animals, and it is widely used in veterinary medicine. We examined the behavior of water-disperse diminazene (immobilized in Tween 80 micelles) at the cellular and organismal levels. We assessed the interaction of an aqueous and a water-disperse preparation with cells of the reticuloendothelial system. We compared the kinetic parameters of aqueous and water-disperse diminazene in sheep erythrocytes and plasma. The therapeutic properties of these two preparations were also compared. We found that the surface-active substances improved intracellular penetration of the active substance through interaction with the cell membrane. In sheep blood erythrocytes, micellar diminazene accumulated more than its aqueous analog. This form was also more effective therapeutically than the aqueous analog. Our findings demonstrate that use of micellar diminazene allows the injection dose to be reduced by 30%

Synthesis of silymarin−selenium nanoparticle conjugate and examination of its biological activity in vitro

Silymarin (Sil) was conjugated to selenium nanoparticles (SeNPs) to increase Sil bioavailability. The conjugates were monodisperse; the average diameter of the native SeNPs was ~ 20-50 ± 1.5 nm, whereas that of the conjugates was 30-50 ± 0.5 nm. The use of SeNPs to increase the bioavailability of Sil was examined with the MH-22a, EPNT-5, HeLa, Hep-2, and SPEV-2 cell lines. The EPNT-5 (glioblastoma) cells were the most sensitive to the conjugates compared to the conjugate-free control. The conjugates increased the activity of cellular dehydrogenases and promoted the penetration of Sil into the intracellular space. Possibly, SeNPs play the main part in Sil penetration of cells and Sil penetration is not associated with phagocytosis. Thus, SeNPs are promising for use as a Sil carrier and as protective antigens

Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab

<p>Abstract</p> <p>Background</p> <p>Nucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods. Thus, the search for novel nucleases with potentially exploitable functions remains an important scientific undertaking.</p> <p>Results</p> <p>Using degenerative primers and the rapid amplification of cDNA ends (RACE) procedure, we cloned the Duplex-Specific Nuclease (DSN) gene from the hepatopancreas of the Kamchatka crab and determined its full primary structure. We also developed an effective method for purifying functional DSN from the crab hepatopancreas. The isolated enzyme was highly thermostable, exhibited a broad pH optimum (5.5 – 7.5) and required divalent cations for activity, with manganese and cobalt being especially effective. The enzyme was highly specific, cleaving double-stranded DNA or DNA in DNA-RNA hybrids, but not single-stranded DNA or single- or double-stranded RNA. Moreover, only DNA duplexes containing at least 9 base pairs were effectively cleaved by DSN; shorter DNA duplexes were left intact.</p> <p>Conclusion</p> <p>We describe a new DSN from Kamchatka crab hepatopancreas, determining its primary structure and developing a preparative method for its purification. We found that DSN had unique substrate specificity, cleaving only DNA duplexes longer than 8 base pairs, or DNA in DNA-RNA hybrids. Interestingly, the DSN primary structure is homologous to well-known Serratia-like non-specific nucleases structures, but the properties of DSN are distinct. The unique substrate specificity of DSN should prove valuable in certain molecular biology applications.</p

Bovine serum albumin adsorbed on eremomycin and grafted on silica as new mixed-binary chiral sorbent for improved enantioseparation of drugs

A new silica-based, mixed-binary chiral sorbent grafted with the macrocyclic antibiotic eremomycin and bovine serum albumin (BSA) was obtained. The sorbent-filled high-performance liquid chromatography column was capable of enantioseparation of racemic drugs, such as profens, in reversed-phase-chromatography mode. The mixed-binary eremomycin-BSA-sorbent showed better capability for profens enantioseparation as compared with a sorbent containing eremomycin only. BSA grafted onto the sorbent surface significantly reduced retention times of other proteins from the analyte solution, and free proteins (including BSA) injected as analytes were not retained on the column, and subsequently eluted with a dead volume. The drastic difference observed in the binding of profens and other proteins using the sorbent was tested for determination and enantioseparation of profens in artificial-urine solutions

Enantioseparation of β-Blockers Using Silica-Immobilised Eremomycin Derivatives as Chiral Stationary Phases in HPLC

The regularities of chromatographic retention and separation enantioselectivity of the selected β-blockers (propranolol, pindolol, alprenolol, atenolol, oxprenalol, metoprolol, clenbuterol, sotalol, pronethalol, salbutamol, and labetalol) were studied with eight chiral stationary phases (CSPs) in polar ionic mode (PIM) elution system. A range of novel CSPs was prepared by immobilisation of macrocyclic glycopeptide antibiotic eremomycin (E-CSP); structurally related antibiotics chloreremomycin (Chloro-E-CSP) and semi-synthetic oritavancin (O-CSP); and five eremomycin derivatives including amide- (Amide-E-CSP), adamantyl-2-amide- (Adamantylamide-E-CSP), eremomycin aglycon (EAg-CSP), eremosaminyl eremomycin aglycon (EEA-CSP), and des-eremosamynyl eremomycin (DEE-CSP) onto microspherical silica (Kromasil, particle size 5 micron, pore size 11 nm). The effect of different functional groups in eremomycin structure on chiral recognition of β-blockers was studied. The original E-CSP revealed moderate enantioseparation for all studied β-blockers. The presence of a free carboxylic group in a chiral selector molecule is found to be critical for the general retention of enantiomers as no separation enantioselectivity was recorded for Amide-E-CSP and Adamantyl-E-CSP. Modification of the aromatic system of eremomycin by the introduction of a chloro- substituent in the aromatic ring (Chloro-E-CSP) or a hydrophobic 4’-chlorobiphenylmethyl substituent to the disaccharide sugar residue (O-CSP) resulted in decreased enantioselectivity. The best enantioseparation of β-blockers was obtained for CSPs with eremosaminyl eremomycin aglycon and des-eremosamynyl eremomycin as chiral selectors

A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants.

We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu(222)-->Gly (E222G) substitution, which removed the evolutionarily invariant Glu(222). Re-introduction of Glu(222) into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of 'humanized' aceGFP and beta-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells