Nucleic acids in mummified plant seeds : biochemistry and molecular genetics,of pre-Columbian maize

SummaryNucleic acids fractions were isolated from pre-Columbian maize seeds and characterized using different approaches such as polyacrylamide gel electrophoresis, anti-DNA antibody binding, HPLC fractionation, molecular hybridization with cloned genes, and DNA amplification by the polymerase chain reaction. The nucleic acids were found to be very depolymerized (≤140 base pairs in length) and composed mainly of ribosomal RNA. Despite the very low amount and degree of polymerization of seed DNA, specific maize nuclear Mul, Mu4, Mu8 and, possibly, Mu5 element components could be detected, thanks to the use of amplification systems as short as 90 bp. The results suggest that evaluation of the relative proportions of Mu-type element components and, possibly, other maize genomic components in single mummified kernels, may offer a new key to the study of ancient maize populations

Universality of DNA Adsorption Behavior on the Cationic Membranes of Nanolipoplexes.

Nanolipoplexes have emerged worldwide as the most pRev.alent synthetic gene delivery system. Nowadays, it is accepted that complete DNA protection and a precise control of the physical attributes of emerging complexes are major steps toward rational design of efficient nanocarriers. Here we Rev.ise the mechanism of DNA adsorption to the cationic membranes of lipid nanovectors. Here we show that both the DNA-binding ability of cationic membranes and the one-dimensional DNA packing density inside the complex depen on the cationic lipid/anionic DNA charge ratio. Remarkably, both these distributions are rescaled on universal curves when plotted against γ, a dimensionless quantity expressing the ratio between the area of cationic membranes and that occupied by DNA molecules. As a result, the DNA condensation on the surface of lipid nanocarriers can be regarded as a two-step process. Our findings indicate a successful way to the rational design of next-generation drug delivery nanocarriers

Surface area of lipid membranes regulates the DNA-binding capacity of cationic liposomes.

We have applied electrophoresis on agarose gels to investigate the DNA-binding capacity of cationic liposomes made of cationic DC-cholesterol and neutral dioleoylphosphatidylethanolamine as a function of membrane charge density and cationic lipid/DNA charge ratio. While each cationic liposome formulation exhibits a distinctive DNA-protection ability, here we show that such a capacity is universally regulated by surface area of lipid membranes available for binding in an aspecific manner. The relevance of DNA protection for gene transfection is also discussed

An Immunoenzyme Linked Assay (ELISA) for the Detection of Antibodies to Truncated Glycoprotein D (tgD) of Bovine Herpesvirus-1

Bovine herpesvirus-1 (BHV-1) is responsible for a variety of clinical signs. It is widespread in cattle and causes severe economic losses (Castrucci et al., 2002a, b). To prevent the infection several live and inactivated vaccines are commonly used. However, due to their short-term immunity and incomplete protection, new vaccine strategies have been proposed such as genetic vaccination (Babiuk et al., 1999). With this aim a DNA vaccine, with a plasmid expressing the tgD glycoprotein, known to be responsible for the virus antigenicity and consequent immunogenicity (Castrucci et al., 2004; Gupta et al., 1998), has been investigated. In the present study, the ELISA reaction was performed in order to detect specific antibodies in calves vaccinated with a DNA vaccine using the pcDNA3.1-tgD plasmid

Enhanced transfection efficiency of multicomponent lipoplexes in the regime of optimal membrane charge density

Recently, membrane charge density of lipid membranes, am, has been recognized as a universal parameter that controls the transfection efficiency of complexes made of binary cationic liposomes and DNA (binary lipoplexes). Three distinct regimes, most likely related to interactions between complexes and cells, have also been identified. The purpose of this work was to investigate the transfection efficiency behavior of multicomponent lipoplexes in the regime of optimal membrane charge density (1 < sigma(M) < 2 x 10(-2) e/angstrom(2)) and compare their performance with that of binary lipoplexes usually employed for gene delivery purposes. We found remarkable differences in transfection efficiency due to lipid composition, with maximum in efficiency being obtained when multicomponent lipoplexes were used to transfect NIH 3T3 cells. while binary lipoplexes were definitely less efficient. These findings suggested that multicomponent systems are especially promising lipoplex candidates. With the aim of providing new insights into the mechanism of transfection, we investigated the structural evolution of lipoplexes when interacting with anionic (cellular) lipids by means of synchrotron small-angle X-ray diffraction (SAXD), while the extent of DNA release upon interaction with anionic lipids was measured by electrophoresis on agarose gels. Interestingly, a clear trend was found that the transfection activity increased with the number of lipid components. These results highlight the compositional properties of carrier lipid/cellular lipid mixtures as decisive factors for transfection and suggest a strategy for the rational design of superior cationic lipid carriers

Structural stability against disintegration by anionic lipids rationalizes the efficency of cationic liposom/DNA complexes

Reported here is the correlation between the transfection efficiency of cationic liposome/DNA complexes (lipoplexes) and the structural evolution that they undergo when interacting with anionic membrane lipids. Multicomponent lipoplexes, incorporating from three to six lipid species simultaneously, presented a much higher transfection efficiency than binary lipoplexes, which are more commonly used for gene-delivery purposes. The discovery that a high transfection efficiency can be achieved by employing multicomponent complexes at a lower-than-ever-before membrane charge density of lipoplexes was of primary significance. Synchrotron small-angle X-ray diffraction (SAXD) experiments showed that anionic liposomes made of dioleoylphosphatidylglycerol (DOPG) disintegrated the lamellar phase of lipoplexes. DNA unbinding was measured by electrophoresis on agarose gels. Most importantly, structural changes induced by anionic lipids strictly depended on the lipid composition of lipoplexes. We found evidence of the existence of three different regimes of stability related to the interaction between complexes and anionic membranes. Both unstable (with low membrane charge density, !M) andhighly stable lipoplexes (withhigh !M) exhibited lowtransfection efficiency whereas highly efficient multicomponent lipoplexes exhibited an “optimal stability”. This intermediate regime reflects a compromise between two opposing constraints: protection of DNA in the cytosol and endosomal escape. Here we advance the concept that structural stability, upon interaction with cellular anionic lipids, is a key factor governing the transfection efficiency of lipoplexes. Possible molecular mechanisms underlying experimental observations are also discussed