Poly(L-lysine)-mediated immobilisation of oligonucleotides on carboxy-rich polymer surfaces

The immobilisation efficiency of the complexes of oligonucleotide/poly(L-lysine) on two polymeric carboxy-rich surfaces, i.e. poly(styrene/maleic acid) (PSMA) and poly(styrene/maleic anhydride) (PSMAA), has been investigated using X-ray photoelectron spectroscopy, atomic force microscopy (AFM) and fluorescence-based measurements of DNA attachment. A molecularly thin layer of either electrostatically or covalently (via amide bond) bound poly(L-lysine) allows the 'switching' from COOH-based to NH(2)-based surface functionality. The results indicate that approximately 54-57% and 55-62% of the applied oligonucleotides bind to polymeric surfaces via the route of electrostatic adsorption of poly(L-lysine) and covalent bonding of poly(L-lysine), respectively. This system can be applied conveniently for the detection of nucleic acids in both disposable and reusable biosensors.     

Neutral density liquid formulations for nematode-based biopesticides

Entomopathogenic nematodes are widely used as biological insecticides for agricultural and horticultural pests. In the vast majority of cases, commercial products contain nematodes partially dehydrated on to inert solid carriers. Nematode survival in these products is generally poor, they are difficult to handle and are not suitable for use with all nematode species. We have developed a non-viscous, non-adhesive and non-toxic liquid formulation for nematode storage and transport based on neutral density colloidal silica suspensions. Survival and virulence of nematodes stored in this formulation without aeration was superior to nematodes stored in aerated quarter strength Ringer's solution.     

Monomers for oligonucleotide synthesis with linkers carrying reactive residues: II. Synthesis of phosphoamidites based of uridine and cytosine and containing a linker with methoxyoxalamide groups in position 2'

A convenient preparative synthesis of 2'-amino-2'-deoxyuridine was developed. Starting from 2'-amino-2'-deoxyuridine and 2'-amino-2'-deoxycytidine, monomers for the phosphoamidite oligonucleotide synthesis were obtained that carry a linker with methoxyoxalamide groups in position 2'. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.     

Ribonuclease activity of the peptides with alternating arginine and leucine residues conjugated to tetrathymidilate

RNA cleaving conjugates have been prepared by attachment of oligodeoxyribonucleotide TTTT to peptides containing arginine, leucine, proline and serine residues. The highest activity was displayed by the conjugates containing peptides with alternating arginine and leucine residues (LR)4G-amide. Ribonuclease activity of the conjugates pep-T4 decreases in the order T4-(LR)4G > T4-(LR)2G > T4-(LLRR)2G > T4-(LR)2PRLRG > S2R3-Hmda-T4 > or = R5 double dagger (LR)3. According to CD spectra, the free peptide (LR)4G-amide in water solution at neutral pH and physiological ionic strength has no pronounced secondary structure whereas conjugated to oligonucleotide it acquires a folding similar to alpha-helix.     

Influence of Combined Microinjection of Gene Engineering Construct and Endonuclease on Preimplantation Development of Mouse Embryos in vitro

We studied the influence of combined microinjection of a gene engineering construct and site-specific endonuclease SalIin the pronucleus on preimplantation development of (CBA × C57BL)F₁ mouse embryos in vitro. The rate of survival of the embryos was estimated according to their capacity to develop until the blastocyst stage and hatch from zona pellucida. The results obtained suggest that the microinjection of exogenous DNA jointly with endonuclease SalI at concentrations from 0.1 to 0.01 U/l decreased reliably the rate of survival, as compared to the control (p < 0.05 and p < 0.01, respectively). However, a decrease of endonuclease SalI concentration in the injection mixture to 0.01 U/l enhanced the capacity of mouse embryos to develop until the blastocyst stage and hatch from zona pellucida, as compared to the embryos microinjected with exogenous DNA and endonuclease SalI at a higher concentration.     

Monomers for Oligonucleotide Synthesis with Linkers Carrying Reactive Residues: II. The Synthesis of Phosphoamidites on the Basis of Uridine and Cytosine and Containing a Linker with Methoxyoxalamide Groups in Position 2′

A convenient preparative synthesis of 2-amino-2-deoxyuridine was developed. Starting from 2-amino-2-deoxyuridine and 2-amino-2-deoxycytidine, monomers for the phosphoamidite oligonucleotide synthesis were obtained that carry a linker with methoxyoxalamide groups in position 2.     

Investigation of erythrocyte shape, plasma membrane fluidity and conformation of haemoglobin haemoporphyrin under the influence of long-term space flight

The investigation of long-term space flight (SF) effect on the blood cells function is of great importance for modern space biology and medicine. We established that the number of discocytes decreased in the period of early rehabilitation after long-term SF. After SF plasma membrane fluidity and phospholipid content decreased and cholesterol content increased. After SF the amount of haemoglobin decreased and the parameters characterizing haemoglobin haemoporyphyrin (HH) conformation changed. We suppose that erythrocyte shape, membrane fluidity and HH conformation are among factors affecting oxygen transfer during and after space flight.     

The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities

LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A. We have compared the substrate specificities of LmrA and MsbA in detail. Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E. coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A. LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity. Reciprocally, the expression of MsbA conferred multidrug resistance on E. coli. Similar to LmrA, MsbA interacted with photoactivatable substrate [3H]azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein. Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities. Our observations imply the presence of structural elements in the recently published crystal structures of MsbA in E. coli and Vibrio cholera (Chang, G., and Roth, C. B. (2001) Science 293, 1793-1800; Chang, G. (2003) J. Mol. Biol. 330, 419-430) that support drug-protein interactions and suggest a possible role for LmrA in lipid trafficking in L. lactis.