Diquaternary ammonium compounds as transfection agents

Diquaternary ammonium salts constitute a new class of reagent for mediating transfection of DNA in mammalian cell lines. N,N'-dioleyl-N,N,N',N'-tetramethyl-1,2-ethanediamine (TmedEce), N,N'-dioleyl-N,N,N',N'-tetramethyl-1,3-propanediamine (PropEce), N,N'-dioleyl-N,N,N',N'-tetramethyl-1,6-hexanediamine (HexEce), and their corresponding N,N'-dicetyl saturated analogues (TmedAce, PropAce and HexAce) have all been synthesized and characterized. They were prepared via a bis-Menshutkin reaction of the corresponding tetramethyldiamine with 2.2 M equiv of a long-chain alkyl halide (saturated or unsaturated). The reaction was run in anhydrous acetonitrile for ca. 3 days at 60 degrees C, which produced the diquaternary ammonium halides in good to nearly quantitative yields for most derivatives. DNA transfection comparable to commercially available reagents such as Lipofectin, Lipofectace, Lipofectamine, and O-ethyldioleoylphosphatidylcholinium triflate has been achieved in vitro with these new reagents. There was no need to use a colipid for effective transfection, but serum did significantly inhibit transfection. The saturated and the unsaturated derivatives differed with respect to hydration behavior. The saturated derivatives appeared to retain a lamellar-type crystalline array structure upon hydration, whereas the unsaturated versions formed micelles and/or liposomes, depending on the ionic strength: HexEce was micellar in both water and saline; PropEce was micellar in water but lamellar in saline; and TmedEce was lamellar in both. Despite these different hydration patterns, all of these unsaturated derivatives formed productive transfection complexes with DNA. Varying the distance between the quaternary sites affected transfection efficacy in the order HexAce > TmedAce = PropAce for the saturated derivatives and in the order PropEce = HexEce > TmedEce, with a smaller spread, for the unsaturated derivatives.     

The clinical course of HIV infection in children who were parenterally infected

The data on the study of the clinical course of HIV infection in 127 children, 124 from these infected in nosocomial foci. The overwhelming majority of the children were infected at the period of their stay in hospitals of Elista, Volgograd, Rostov-on-Don, Shakhty (Rostov Province) and Stavropol'. At the end of 9-9.5 years elapsed since the appearance of the first documented cases of parenteral infection 33 children (26%) died. The study revealed that at the age under 1 year the course of HIV infection could rapidly progress into the stage of AIDS in the presence of aggravated premorbid background linked with the unfavorable course of pregnancy in the mother and a severe disease in the child prior to contacting HIV infection.     

O-Alkyl dioleoylphosphatidylcholinium compounds: the effect of varying alkyl chain length on their physical properties and in vitro DNA transfection activity

1,2-Dioleoyl-sn-3-ethylphosphocholine (EDOPC) has been previously shown be a highly effective DNA transfection reagent in vitro. To assess the effect of alkyl chain length on transfection efficiency, the O-methyl, O-propyl, O-hexyl, O-decyl, and O-octadecyl derivatives have been prepared from dioleoylphosphatidylcholine using the corresponding alkyl trifluoromethylsulfonate. The methyl, ethyl, and propyl derivatives formed liposomes which were very large and unilamellar. The ethyl and propyl derivatives were equally efficient at mediating transfection (even in the presence of serum) of BHK cells, but the chemically labile methyl derivative was a much weaker transfection agent. The O-decyl and O-octadecyl compounds, which assume the inverted hexagonal phase in excess water (as determined by X-ray diffraction), were almost inactive after manual agitation in both water and in saline; however, after sonication, these compounds exhibited good transfection activity. The O-hexyl derivative displayed novel behavior, assuming the lamellar phase at low and a cubic phase at high ionic strength. All compounds, whether lamellar or not, formed lamellar structures when complexed with DNA. In water, where the hexyl compound dispersed well, sonication diminished transfection activity, whereas at physiological ionic strength, which led to poor manual dispersion, sonication was essential for good transfection. These results emphasize the importance of optimal dispersion of a cationic lipid: too little, and interaction with DNA is handicapped, too much, and the resultant particle transfects poorly. Lipid dispersibility is thus an important variable in assessing lipid transfection agents, and caution is advised in attributing too much significance to chemical structure until interaction with DNA has been optimized.     

Factors governing the assembly of cationic phospholipid-DNA complexes

The interaction of DNA with a novel cationic phospholipid transfection reagent, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), was investigated by monitoring thermal effects, particle size, vesicle rupture, and lipid mixing. By isothermal titration calorimetry, the heat of interaction between large unilamellar EDOPC vesicles and plasmid DNA was endothermic at both physiological and low ionic strength, although the heat absorbed was slightly larger at the higher ionic strength. The energetic driving force for DNA-EDOPC association is thus an increase in entropy, presumably due to release of counterions and water. The estimated minimum entropy gain per released counterion was 1.4 cal/mole- degrees K (about 0.7 kT), consistent with previous theoretical predictions. All experimental approaches revealed significant differences in the DNA-lipid particle, depending upon whether complexes were formed by the addition of DNA to lipid or vice versa. When EDOPC vesicles were titrated with DNA at physiological ionic strength, particle size increased, vesicles ruptured, and membrane lipids became mixed as the amount of DNA was added up to a 1.6:1 (+:-) charge ratio. This charge ratio also corresponded to the calorimetric end point. In contrast, when lipid was added to DNA, vesicles remained separate and intact until a charge ratio of 1:1 (+:-) was exceeded. Under such conditions, the calorimetric end point was 3:1 (+:-). Thus it is clear that fundamental differences in DNA-cationic lipid complexes exist, depending upon their mode of formation. A model is proposed to explain the major differences between these two situations. Significant effects of ionic strength were observed; these are rationalized in terms of the model. The implications of the analysis are that considerable control can be exerted over the structure of the complex by exploiting vectorial preparation methods and manipulating ionic strength.     

Cytotoxic properties of a recombinant hybrid of the A protein of Staphylococcus aureus with a fragment of exotoxin A of Pseudomonas aeruginosa]

By gene-engineering technique a chimeric protein made up of fragments of Staphylococcus aureus protein A and .Pseudomonas aeruginosa exotoxin A has been constructed. The chimeric protein was shown to preserve features characteristic of its both constituents--it ADP-ribosylates elongation factor 2 and binds to Ig. Cytotoxic properties of the chimeric protein were studied in two model systems. Treatment of target cells in both systems was performed successively with antibodies against corresponding antigens and after washing--with recombinant chimeric toxin which bound to antibodies on the surface of target cells. In the first model system human B-lymphoma cells (Daudi line) carrying Ig molecules on their surface were treated with polyclonal antibodies against human Ig L-chains. In the other system, human T-lymphoma cells (Jurkat line) were treated successively with monoclonal antibodies against cell surface CD5 antigen and further on--with polyclonal antibodies against mouse Ig. In both systems, only a slight inhibition of the target cells' growth was registered. The probable reasons of low cytotoxic activity of the chimeric protein and prospects of increasing it are discussed.     

New observations and reinterpretation on the enigmatic taxon Colombitherium (?Pyrotheria,Mammalia) from Colombia

Abstract: The controversial taxon Colombitherium tolimense (Mammalia) (probably Late Eocene in age) from Colombia, although known for nearly 40 years, still bears much mystery. Aside from the problematic ordinal attribution of the holotype and only specimen, its determination as an upper or lower jaw remains a highly debated issue. New observations include the presence of a contact facet on the distal face of the most posterior tooth, which indicates that the fragmentary jaw preserves three premolars and two molars; the M3, unpreserved but present, being most probably reduced. This new interpretation completely fits the morphology of the teeth. Furthermore, the shape of these latter and the deeper wear encompassed by their lingual part relative to the labial one is typical of upper dentition. This is in agreement with the internal curving of the roots of the anterior premolars and with several other arguments that lead interpreting the holotype of C. tolimense as a maxillary bearing P2‐M2. This new interpretation deepens the morphological gap between Colombitherium and other pyrotherians (except Proticia) and challenges further its referral to Pyrotheria. The peculiar morphology of Colombitherium relative to other pyrotherians is indeed striking. In fact, Colombitherium has nothing in common with pyrotherians but bilophodont cheek teeth, a feature largely widespread in placental mammals. It is here referred to ?Pyrotheria until additional evidence of its relationships is known. Associated with the putative removal of Proticia from Pyrotheria as argued by some authors, the hypothetical removal of Colombitherium from the order would adjust the widely accepted assumption that the pyrotherian bilophodont cheek teeth originated from bunodont cheek teeth. It would also make an origin from lophodont forms plausible. This in turn would have critical relevance, especially to the hypothesis that pyrotherians are notoungulates.     

Azygos Vein Lead Implantation For High Defibrillation Thresholds In Implantable Cardioverter Defibrillator Placement

Evaluation of defibrillation threshold is a standard of care during implantation of implantable cardioverter defibrillator. High defibrillation thresholds are often encountered and pose a challenge to electrophysiologists to improve the defibrillation threshold. We describe a case series where defibrillation thresholds were improved after implanting a defibrillation lead in the azygos vein.     

Feather ornaments are dynamic traits in the Great Tit Parus major

In behavioural studies it has been common to quantify plumage colours or ornaments over a range of dates and link them to fitness characteristics without accounting for seasonal changes in these traits. Such changes are likely to be widespread among birds, yet we lack assessments of this variability within individuals. We studied both within‐ and between‐individual temporal changes in Great Tit Parus major ornaments, specifically the melanin‐based black breast stripe and the pigment‐free white cheek patch. During the non‐breeding season both ornaments varied. In juveniles and adult females, the area of the breast stripe first rose and then, from near the end of December, decreased. In adult males there was a linear decrease. In the cheek patch, the irregularity of the cheek borders showed either a linear (adults) or a non‐linear (juveniles) increase as the season progressed. In individuals repeatedly sampled during the same winter, the decrease in the size of the breast stripe was larger for males than females and there was an overall decrease in the regularity of the cheek borders. There was no relationship between the size of the breast stripe and the white cheek patch irregularities or the cheek patch area. These results imply that more attention should be paid to quantification, within individuals, of the components of expression of phenotypic traits. In addition, we suggest that further research should focus on explaining the causes and functions of ornament change.