Biodistribution and gene expression of lipid/plasmid complexes after systemic administration

The objectives of this study were to investigate the influence of physicochemical properties of lipid/plasmid complexes on in vivo gene transfer and biodistribution characteristics. Formulations based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and novel biodegradable cationic lipids, such as ethyl dioleoyl phosphatidylcholine (EDOPC), ethyl palmitoyl myristyl phosphatidylcholine (EPMPC), myristyl myristoyl carnitine ester (MMCE), and oleyl oleoyl L-carnitine ester (DOLCE), were assessed for gene expression after tail vein injection of lipid/plasmid complexes in mice. Gene expression was influenced by cationic lipid structure, cationic lipid-to-colipid molar ratios, plasmid-to-lipid charge ratios, and precondensation liposome size. Detectable levels of human growth hormone (hGH) in serum, human factor IX (hFIX) in plasma, and chloramphenicol acetyltransferase (CAT) in the lung and liver were observed with positively charged lipid/plasmid complexes prepared from 400-nm extruded liposomes with a cationic lipid-to-colipid ratio of 4:1 (mol/mol). Intravenous administration of lipid/CAT plasmid complexes resulted in distribution of plasmid DNA mainly to the lung at 15 min after injection. Plasmid DNA accumulation in the liver increased with time up to 24 hr postinjection. There was a 10-fold decrease in the amount of plasmid DNA in the lung at 15 min after injection, when the lipid/plasmid complex charge ratio was decreased from 3:1 to 0.5:1 (+/-). Bright fluorescent aggregates were evident in in vivo-transfected lung with the positively charged pCMV-CAT/DOLCE:dioleyl phosphatidylethanolamine (DOPE) (1:1, mol/mol) complexes, while more discrete punctate fluorescence was observed with a 4:1 molar ratio of cationic lipid:colipid formulations. Preinjection of polyanions such as plasmid, dextran sulfate, polycytidic acid, and polyinosinic acid decreased hGH expression, whereas the preinjection of both positively charged and neutral liposomes had no effect on hGH serum levels. Of the cationic lipids tested, DOLCE was found to be the most effective potentially biodegradable cationic lipid. A correlation between gene expression and cationic lipid:colipid ratios and lipid-to-plasmid charge ratio was also observed for DOTMA- and DOLCE-based formulations.     

Regulation of CDK/cyclin complexes during the cell cycle

We have examined the effects of base sequence on nucleosome array formation using randomly selected chicken genomic DNA sequences. DNA clones were assembled into chromatin under identical conditions using a defined in vitro system capable of generating physiologically spaced nucleosomes on some sequences. The nucleosome arrangements in native chromatin on the selected sequences were also examined in liver nuclei. Variations in nucleosome ladders were found among the different sequences that were similar in vitro and in nuclei. Differences in both the degree of regularity of nucleosome arrays and in the value of the nucleosome repeat length were observed. Analysis of an approximately 100 kilobase-pair contiguous region using cosmid clones suggested that well-ordered regions of chromatin, generally less than two kilobase-pairs in extent, alternate with less-ordered regions. This mosaic arrangement for chromatin organization appears to be largely a consequence of information encoded in the DNA base sequence. Nucleosome ordering with a 210 base-pair periodicity in a highly ordered ten-nucleosome array appeared to result from linker histone-dependent alignment with respect to each of two positioned nucleosomes, approximately 1000 base-pairs apart.     

Differential modulation of G1-S-phase cyclin-dependent kinase 2/cyclin complexes occurs during the acquisition of a polyploid DNA content

Despite a growing understanding of the biochemical mechanisms controlling the cell cycle, information regarding the temporal ordering of S phase and M phase remains scarce. Polyploid cells represent a useful model for examining S- and M-phase control, because their cell cycle machinery must be modulated to retain high levels of DNA content (ploidy) within a single nucleus. To evaluate the mechanisms of S-phase control during the process of polyploidization, we investigated the modulations that occur in cyclin-dependent kinase (CDK) complexes during the induction of megakaryocyte differentiation in human erythroleukemia cells. We report that during polyploidization, megakaryocytic human erythroleukemia cells undergo a dramatic modulation in the subunit composition of G1-associated and S phase-associated CDK complexes and a marked increase in their specific activities. This, in turn, is facilitated by a differential loss of the p21 or p27 CDK-inhibitory protein/kinase-inhibitory proteins (CIP/KIP) bound to specific cyclin/CDK complexes. The data show that the loss of S- and M-phase control in polyploid cells occurs within the context of an up-regulated function in those CDK complexes associated with both G1-S-phase transit and S-phase progression. Additional studies regarding the regulation of these complex CDK interactions will be important to understand cell cycle control in such diverse processes as megakaryocyte differentiation or the types of genomic instability that occur in cancer cells.     

Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability

PURPOSE: Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery. With the aim of developing this system, various biophysical characteristics of chitosan-condensed DNA complexes were measured, and transfections were performed. METHODS: Transmission electronic microscopy (TEM) visualizations, sedimentation experiments, dynamic light scattering (DLS), and zeta potential measurements were realized. Transfections were made by using the luciferase reporter gene. RESULTS: In defined conditions, plasmid DNA formulated with chitosan produced homogenous populations of complexes which were stable and had a diameter of approximately 50-100 nm. Discrete particles of nicely condensed DNA had a donut, rod, or even pretzel shape. Chitosan/DNA complexes efficiently transfected HeLa cells, independently of the presence of 10% serum, and did not require an added endosomolytic agent. In addition, gene expression gradually increased over time. from 24 to 96 hours, whereas in the same conditions the efficacy of polyethylenimine-mediated transfection dropped by two orders of magnitude. At 96 hours, chitosan was found to be 10 times more efficient than PEI. However, chitosan-mediated transfection depended on the cell type. This dependency is discussed here. CONCLUSIONS: Chitosan presents some characteristics favorable for gene delivery, such as the ability to condense DNA and form small discrete particles in defined conditions.     

Stability of peptide-condensed plasmid DNA formulations

Low molecular weight homogeneous peptides were used to form peptide/DNA condensates. A peptide possessing 18 lysines was found to protect plasmid DNA from serum endonuclease and sonicative-induced degradation whereas a shorter peptide possessing 8 lysines dissociated in 0.1 M sodium chloride and failed to protect DNA from enzymatic degradation. Peptide-condensed DNA showed no change in the ratio of supercoiled to circular DNA following 100 W sonication for up to 60 s and was able to transfect HepG2 cells with equivalent efficiency as untreated condensed plasmid DNA. Alternatively, uncondensed plasmid DNA was rapidly fragmented by sonication and serum endonucleases and resulted in negligible gene expression following condensation with peptide. Cationic lipid/DNA complexes were only partially effective at stabilizing DNA in serum compared to the complete stabilization afforded by peptide/DNA condensation. These results indicate that the stabilization afforded by condensation with a peptide protects DNA during formulation and preserves its structure in serum. These functions are important to achieve optimal gene expression from a nonviral gene delivery system.     

Biodistribution of radiolabeled lipid-DNA complexes and DNA in mice

The tissue biodistribution and expression of [33P]DNA-1-[2-[9-(Z)-octadecenoyloxy]ethyl]]-2-[8](Z)-heptadece nyl]-3 -[hydroxyethyl]imidazolinium chloride (DOTIM):cholesterol complexes and 33P-radiolabeled DNA expressing chloramphenicol acetyl transferase (CAT; 4.7 kB) were studied after intravenous (iv) injection in ICR mice. Mice were injected with 200 microL of complex containing DNA at 3 mg/kg or DNA alone. One group received 8 microCi of radioactivity and were sacrificed at 5 and 20 min, and 1, 2, 4 and 24 h post-dose (n = 4/time point). A second group received the equivalent of 3.9 microCi of radioactivity and were sacrificed at 20 min, and 2 and 24 h for subsequent whole body autoradiographic analysis (WBA; n = 2/time point). The tissue distribution of intact DNA was assessed by Southern blot at 24 h post-dose, whereas the integrity of complexes and DNA incubated in heparinized whole blood was studied separately. In further studies, the time course of expression in lung tissue over a 48-h period was examined, and the relative lung-expression of purified open circular (OC) versus supercoiled (SC) DNA at 24 h was evaluated. Approximately 42% of the radioactivity was found in the lungs 5 min after injection and about half this percentage was found in the liver. By 2 h, only 5% remained in the lungs, but 48% was present in the liver. No other tissue accumulated >5% of the dose throughout the duration of the study. WBA radiograms confirmed the tissue distribution results and highlighted significant accumulation of radioactivity in bone over time. Southern Blot analysis demonstrated intact DNA in many tissues 24 h after dosing. In contrast, the majority of DNA incubated in blood was degraded within 2 h, although the complexes afforded some protection relative to DNA alone. The OC DNA expressed equivalently to SC DNA in lung tissue (OC = 1035 +/- 183 pg; SC = 856 +/- 257 pg/mg soluble protein, n = 6, mean +/- SEM) at 24 h, and detectable levels of CAT were present within 2 h of dosing (21.3 +/- 7.2 pg, n >/= 8, mean +/- SD). The results confirm that DNA-DOTIM:cholesterol complexes are initially deposited in the lungs after iv administration.     

Significance of lipid consumption during lactation

Human milk lipids are the main source of energy to support optimum growth of the breast-fed infant. The content and composition of milk lipids come from three main sources of fatty acids: the diet, mobilization of body fat stores and fatty acid synthesis de novo by the mammary gland. On account of these, the consumption and composition of the lipids from the diet and also the nutritional state, specifically the body fat percentage of the lactating woman, are elements that maintain a close relation with the content and composition of milk lipids which translates into the energy content given to the baby. The evidence suggests that the body fat stores significantly provide the demand imposed by lactation, and under suboptimal nutritional conditions where body fat stores are depleted, dietary lipid consumption is essential. It is necessary to elucidate the physiological regulatory mechanisms involved in the utilization of dietary lipids on milk synthesis. This information will be of great practical value, since it may allow the development of optimum diets for lactating women.     

Effect of serum components on the physico-chemical properties of cationic lipid/oligonucleotide complexes and on their interactions with cells

Two behavioral rhythm phenotypes, oviposition and locomotor activity, have been compared in the four period genotypes (per+, pers, per0, and per1) of Drosophila. Period, signal-to-noise ratio, and phase were all analyzed and the genetic penetrance of the two characters was estimated. Significant rhythmicity of both oviposition and locomotor activity was evident in all four genotypes. The entrained and free-running periods of the activity rhythms of per+, pers, and per1 were within the range reported for these flies by previous workers, and rhythmic behavior was also shown by the per0 flies. The free-running period of the oviposition rhythm varied similarly between the four genotypes and showed significant correlation with that of the locomotor activity rhythm. It is suggested that both rhythm phenotypes are determined by the period gene, and estimates of the genetic penetrance of rhythmicity in oviposition and locomotor activity, based on period and signal-to-noise ratios (SNRs) of the different strains, are consistent with this hypothesis. The phase of maximum oviposition and locomotor activity showed greater variability between the genotypes and was not significantly correlated with period, suggesting that this rhythm characteristic is independent of mutations at the period locus.     

Multilamellar structures of DNA complexes with cationic liposomes

Studies of DNA complexes with cationic liposomes are prompted by the search for nonviral DNA carriers for gene therapy. Recent experiments have identified a stable multilamellar phase in which ordered smectic layers of DNA alternate with cationic bilayers. In this paper we identify the forces governing DNA adsorption on cationic lamellae, including a membrane-induced attraction between the adsorbed DNA. Calculating the DNA interhelical spacing as a function of system composition, the model successfully explains recent surprising observations.     

Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system

Currently in vivo gene delivery by synthetic vectors is hindered by the limited diffusibility of complexes in extra-cellular fluids and matrices. Here we show that certain formulations of plasmid DNA with linear polyethylenimine (22 kDa PEI, ExGene 500) can produce complexes that are sufficiently small and stable in physiological fluids so as to provide high diffusibility. When plasmid DNA was formulated with 22 kDa PEI in 5% glucose, it produced a homogeneous population of complexes with mean diameters ranging from 30 to 100 nm according to the amount of PEI used. In contrast, formulation in physiological saline produced complexes an order of magnitude greater (> or = 1 micron). Intraventricular injection of complexes formulated in glu-cose showed the complexes to be highly diffusible in the cerebrospinal fluid of newborn and adult mice, diffusing from a single site of injection throughout the entire brain ventricular spaces. Transfection efficiency was followed by histochemistry of beta-galactosidase activity and double immunocytochemistry was used to identify the cells transfected. Transgene expression was found in both neurons and glia adjacent to ventricular spaces. Thus, this method of formulation is promising for in vivo work and may well be adaptable to other vectors and physiological models.     

The interaction of DNA-targeted platinum phenanthridinium complexes with DNA

Cisplatin analogues were synthesised that consisted of platinum(II) diamine complexes tethered via a polymethylene chain ( n = 3, 5, 8 and 10) to a phenanthridinium cation. Both chloro and iodo leaving groups were examined. DNA adduct formation was quantitatively analysed using a linear amplification system with the plasmid pGEM-3Zf(+). This system utilised Taq DNA polymerase to extend from an oligonucleotide primer to the damage site. This damage site inhibited the extension of the DNA polymerase. The products were electrophoresed on a DNA sequencing gel enabling adduct formation to be determined at base pair resolution. The damage intensity at each site was determined by densitometry. The platinum phenanthridinium complexes were shown to damage DNA at shorter incubation times than cisplatin. To produce similar levels of damage, an 18 h incubation was required for cisplatin compared to 30 min for the n = 3 platinum phenanthridinium complexes; this indicates that the intercalating chromophore causes a large increase in the rate of platination. A reaction mechanism involving direct displacement of the chloride by the N-7 of guanine may account for the rate increase. These results indicate that further development of these compounds could lead to more effective cancer chemotherapeutic agents.     

Smoking-associated mitochondrial DNA mutations and lipid peroxidation in human lung tissues

BACKGROUND/AIMS: The objective of the present study was to analyze the expression and regulation of intercellular adhesion molecule-1 (ICAM-1) in organotypic cultures of rat liver slices, which preserve the normal microenvironment of liver cells. METHODS: Rat liver slices were maintained in culture for 15 min to 24 h and examined for ICAM-1 expression by immunohistochemistry and Western blotting in basal conditions and after stimulation with 1000 IU/ml interferon-gamma (IFNgamma), 1000 IU/ml tumor necrosis factor-alpha (TNF alpha) and 50 microg/ml endotoxin. Immunohistochemical results were evaluated using a semiquantitative scoring system. RESULTS: In uncultured slices, ICAM-1 was not detected on hepatocytes. In unstimulated liver slices maintained in organotypic culture, ICAM-1 was induced at the surface of scattered hepatocytes (score at 15 min, 0.33+/-0.47 and at 24 h, 1.17+/-0.69). After 4 h of stimulation, a significant increase in ICAM-1 expression by hepatocytes and adjacent sinusoidal cells, but not by intra-hepatic biliary epithelial cells, was observed for IFNgamma (score: 2.35+/-0.47) and endotoxin (score: 2.67+/-0.47), but not with TNF alpha (score: 0.66+/-0.47). After 24 h of stimulation, a further increase in the extent of ICAM-1 expression by hepatocytes was observed for IFNgamma (score: 3.67+/-0.47) and endotoxin (score: 4.0+/-0.0), and a significant overexpression of ICAM-1 by hepatocytes was detectable after treatment with TNF alpha (score: 3.67+/-0.47). CONCLUSIONS: In rat liver organotypic cultures, TNF alpha, IFNgamma and endotoxin induce the expression of ICAM-1 in hepatocytes and adjacent sinusoidal endothelial cells, but not in portal tracts.     

Stability and change in temperament during adolescence.

This study assessed genetic and environmental contributions to temperament during adolescence within the Nonshared Environment and Adolescent Development project (NEAD; D. Reiss, J. M. Neiderhiser, E. M. Hetherington, & R. Plomin, 2000). NEAD is a national study that includes twins and other sibling types who vary in regard to genetic relatedness. Seven hundred twenty sibling pairs (aged 12.1-13.5 years) participated at Time 1, and 395 sibling pairs (aged 14.7-16.2 years) participated again at Time 2. At both Times, mothers and fathers rated their children's temperament (emotionality, activity, sociability, and shyness). At Times 1 and 2, genetic and nonshared environmental factors accounted for variance in temperament, whereas shared environmental contributions were negligible. However, at Time 1, genetic contributions were inflated, and shared environmental contributions were masked if sibling contrast effects were not taken into account. At Time 2, sibling interaction effects had little impact on estimates of genetic and environmental contributions to temperament. Last, temperament stability was primarily explained by genetic factors, whereas both genetic and nonshared environmental factors accounted for change. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

岩体边坡开挖卸荷稳定性分析

笔者应用弹塑性力学及断裂力学的相关理论，分析讨论了裂纹的分形特性及裂隙面的压剪起裂破坏机理；分析了静载、动载和不同类型裂隙的相互作用对边坡稳定性的影响规律。结合工程实际问题，研究了边坡开挖前后其应力应变的分布规律，提出了由于开挖卸荷而引起裂纹扩展的作用机理。     

Theory of cooperative transition of DNA complexes with multimodal ligands

The theory of the cooperative transition of DNA.ligand complexes have been developed, in which a model was implied where the multimodal ligands simultaneously interacted with DNA. Obtained formula express the dependence of the experimentally estimated values of the changes of transition point and width of transition on the concentration of the ligands. These expressions make possible to obtain the thermodynamic parameters of the interaction of the multimodal ligands with DNA (binding constants, number of base pairs corresponding to one binding site of DNA etc.) by comparing the theoretical and the experimental data.