Lipoplex size determines lipofection efficiency with or without serum

In order to identify factors affecting cationic Iiposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining Iipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.     

Surface charge density determines the efficiency of cationic gemini surfactant based lipofection

The efficiencies of the binary liposomes composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and cationic gemini surfactant, (2S,3R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide as transfection vectors, were measured using the enhanced green fluorescent protein coding plasmid and COS-1 cells. Strong correlation between the transfection efficiency and lipid stoichiometry was observed. Accordingly, liposomes with X(SR-1) > or = 0.50 conveyed the enhanced green fluorescent protein coding plasmid effectively into cells. The condensation of DNA by liposomes with X(SR-1) > 0.50 was indicated by static light scattering and ethidium bromide intercalation assay, whereas differential scanning calorimetry and fluorescence anisotropy of diphenylhexatriene revealed stoichiometry dependent reorganization in the headgroup region of the liposome bilayer, in alignment with our previous Langmuir-balance study. Surface charge density and the organization of positive charges appear to determine the mode of interaction of DNA with (2S,3R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide/1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes, only resulting in DNA condensation when X(SR-1) > 0.50. Condensation of DNA in turn seems to be required for efficient transfection.     

A polymorphism affecting apolipoprotein A-II translational efficiency determines high density lipoprotein size and composition

High density lipoproteins (HDL) are heterogeneous particles consisting of about equal amounts of lipid and protein that are thought to mediate the transport of cholesterol from peripheral tissues to liver. We show that a previously identified polymorphism affecting HDL electrophoretic mobility in mice is due to a monogenic variation controlling HDL size and apolipoprotein composition. Thus, the HDL particles of various inbred strains of mice exhibit a striking difference in the ratio fo the two major apolipoproteins of HDL, apoA-I and apoA-II. HDL particles in all strains examined contain an average of about five apoA-I molecules; however, whereas the strains with small HDL contain two to three apoA-II molecules per particle, the strains with large HDL contain about five apoA-II molecules per particle. This increase in the protein content of the large HDL is also accompanied by increased lipid content. The HDL size polymorphism and apoA-II levels cosegregate with the apoA-II structural gene on mouse chromosome 1, indicating that a mutation of the apoA-II gene locus is responsible. The rates of synthesis of apoA-II are increased in the strains with large HDL and high apoA-II levels as compared to the strains with small HDL and low apoA-II levels. On the other hand, the fractional catabolic rates of both apoA-I and apoA-II among the strains are very similar, confirming that apoA-II concentrations are controlled at the level of synthesis. Despite the difference in rates of apoA-II synthesis between strains, the apoA-II mRNA levels in the strains are not discernibly different, suggesting that a mutation of the apoA-II structural gene controls apoA-II translational efficiency. This was confirmed by translating apoA-II mRNA in vitro using a rabbit reticulocyte lysate system. Sequencing of apoA-II cDNA from the strains revealed a number of nucleotide substitutions, which may affect translational efficiency. We conclude that the assembly of apoA-II into HDL does not have a set stoichiometry but, rather, is controlled by the production of apoA-II. As apoA-II levels increase, the HDL particles become larger and acquire more lipid, but apoA-I content per particle remains unchanged. These studies with mice provide a model for the metabolic relationships between apoA-I, apoA-II, and HDL lipid in humans.     

Lectin enhancement of the lipofection efficiency in human lung carcinoma cells

Poor transfection efficiency of human lung carcinoma cells by lipofection begs further development of more efficient gene delivery strategies. The purpose of this study was to determine whether lectins can improve the lipofection efficiency in lung carcinoma cells. A549, Calu3, and H292 cells grown to 90% confluence were transfected for 18 h with a plasmid DNA containing a beta-galactosidase reporter gene (pCMVlacZ) using lipofectin plus a lectin as the vector. Ten different lectins, which exhibit a wide range of carbohydrate-binding specificities, were examined for their abilities to enhance the efficiency of lipofection. The transfected cells were assessed for transfection efficiency by beta-galactosidase activity (units/microg protein) and % blue cells following X-Gal stain. Lipofectin supplemented with Griffonia simplicifolia-I (GS-I) yields largest enhancement of the lipofection efficiency in A549 and Calu3 cells (5.3- and 28-fold, respectively). Maackia amurensis gives the largest enhancement (6.5-fold) of lipofection efficiency in H292 cells. The transfection efficiency correlates with the amounts of DNA delivered to the nucleus. Binding of FITC-labeled GS-I and the enhancement of the lipofection efficiency by GS-I were inhibited by alpha-methyl-D-galactopyranoside, indicating an alpha-galactoside-mediated gene transfer to lung carcinoma cells. We conclude that lectin-facilitated lipofection is an efficient gene delivery strategy. Employment of cell type-specific lectins may allow for efficient cell type-specific gene targeting.     

On the possible involvement of bovine serum albumin precursor in lipofection pathway

Protein factors involved in lipofection pathways remain elusive. Using avidin-biotin affinity chromatography and mass finger printing analysis technique, herein we report the identification of a 70 kDa size protein (bovine serum albumin precursor, BSAP) which binds strongly with lipoplexes and may play role in lipofection pathway. Using multiple cultured animal cells and three structurally different cationic transfection lipids, we show that the efficiencies of liposomal transfection vectors get significantly enhanced (by ~2.5- to 5.0-fold) in cells pre-transfected with lipoplexes of reporter plasmid construct encoding BSAP. Findings in the cellular uptake experiments in A549 cells cultured in DMEM supplemented with 10% (w/w) BODIPY-labelled BSAP are consistent with the supposition that BSAP enters cell cytoplasm from the cell culture medium (DMEM supplemented with 10% FBS) used in lipofection. Cellular uptake studies by confocal microscopy using BODIPY-labelled BSAP and FITC-labelled plasmid DNA revealed co-localization of plasmid DNA and BSAP within the cell cytoplasm and nucleus. In summary, the present findings hint at the possible involvement of BSAP in lipofection pathway.     

A method for high efficiency YAC lipofection into murine embryonic stem cells.

We describe a modified protocol for introducing yeast artificial chromosomes (YACs) into murine embryonic stem (ES) cells by lipofection. With a decreased DNA:cell ratio, increased concentration of condensing agents and altered culture conditions, this protocol reduces the requirement for YAC DNA to a few micrograms, improves the recovery of neomycin-resistant ES colonies and increases the yield of clones containing both flanking vector markers and insert. These modifications enable generation of sufficient 'intact' transgenic clones for biological analysis with a single experiment.     

Coupled pacing improves cardiac efficiency during acute atrial fibrillation with or without cardiac dysfunction

Coupled pacing (CP), a method for controlling ventricular rate during atrial fibrillation (AF), consists of a single electrical stimulation applied to the ventricles after each spontaneous activation. CP results in a mechanical contraction rate approximately one-half the rate during AF. Paired stimulation in which two electrical stimuli are delivered to the ventricles has also been proposed as a therapy for heart failure. Although paired stimulation enhances contractility, it greatly increases energy consumption. The primary hypothesis of the present study is that CP improves cardiac function during acute AF without a similar increase in energy consumption because of the reduced rate of ventricular contractions. In a canine model, CP was applied during four stages: sinus rhythm (SR), acute AF, cardiac dysfunction (CD), and AF in the presence of cardiac dysfunction. The rate of ventricular contraction decreased in all four stages as the result of CP. In addition, we determined the changes in external cardiac work, myocardial oxygen consumption, and myocardial efficiency in the each of four stages. CP partially reversed the effects of AF and CD on external cardiac work, whereas myocardial oxygen consumption increased only moderately. In all stages but SR, CP increased myocardial efficiency because of the marked increases in cardiac work compared with the moderate increases in total energy consumed. Thus this pacing therapy may be a viable therapy for patients with concurrent atrial fibrillation and heart failure.     

Radioimmunoassay of 17-hydroxyprogesterone in serum with or without thin-layer chromatographic purification.

A radioimmunoassay for the measurement of 17-hydroxyprogesterone (17-OHP) is described. The antigen 11-desoxycortisol-21-hemisuccinate-bovine serum albumin has been used to produce two antisera of different antibody populations in the same animal. The thin-layer chromatographic system described can be used to separate all the cross-reacting steroids investigated from 17-OHP. A simplified method is also presented using preliminary solvent extraction only. The mean 17-OHP levels measured, using this method, were, for normal men, 0.86 +/- 0.19 ng/ml (SD), for normal females 0.30 +/- 0.19 ng/ml in the follicular phase and 1.72 +/- 0.18 ng/ml in the luteal phase of the menstrual cycle, and 0.45 +/- 0.17 ng/ml in normal postmenopausal women.     

Cortical hypercolumn size determines stereo fusion limits

The size of a pair of cortical ocular dominance columns determines a basic anatomical module of V-1 which Hubel and Wiesel have termed the hypercolumn. Does this correspond to a basic functional, or psychophysically measurable, module as well? This is the basic question addressed in the present paper. Since the ocular dominance column architecture is presumed to be related to stereo vision, it is natural to assume that hypercolumn size should provide a modular basis for basic phenomena of stereopsis. In previous work, we have suggested that local nonlinear filtering via the cepstral transform, operating on a local window of cortical tissue scaled by hypercolumn size, provides such a modular model of stereopsis. In the present paper, we review this model and then discuss a number of issues related to the biological plausibility and implementation of this algorithm. Then, we present the main result of this paper: we have analyzed a number of experiments related to stereo fusion limits (Panum's area) and to disparity gradient and disparity scaling, and demonstrate that there is a simple unifying explanation for these phenomena in terms of a constant cortical module whose size is determined by a pair of ocular dominance columns. As a corollary, Panum's area must increase according to (inverse) cortical magnification factor. We show that this is supported by all existing experimental data.     

Level of HgCl2-mediated phosphorylation of intracellular proteins determines death of thymic T-lymphocytes with or without DNA fragmentation

Exposure to Hg²⁺ at a wide range of concentrations (approximately 1–100 μM) more or less caused the death of murine thymic T-lymphocytes, and exposure to 1 μM but not 10 μM (or more) of Hg²⁺ induced DNA fragmentation. Exposure of cells to Hg²⁺ caused phosphorylation of multiple cellular proteins at the tyrosine residue in a concentration-dependent manner. We found that not only the DNA fragmentation induced by 1 μM Hg²⁺ but also the cell death bypassing DNA fragmentation caused by 10 μM or more Hg²⁺ was partly inhibited by protein kinase inhibitors such as staurosporine and herbimycin A. This result suggested the involvement of a protein phosphorylation-linked signal in the mechanism of the Hg²⁺-mediated cell death with or without DNA fragmentation. Analysis of proteins by both one- and two-dimensional electrophoresis and immunoblot showed that a 52-kDa Shc protein was heavily phosphorylated by an early signal delivered by a high concentration of Hg²⁺, which also phosphorylated extracellular signal-regulated kinase 1 (ERK1; p44) and ERK2 (p42) of the mitogen-activated protein kinase (MAPK) family in a concentration- and time-dependent manner. The c-Jun amino terminal kinase (p54), which is a distant relative of the MAPK family, was also phosphorylated by the treatment with Hg²⁺. This eventually formed the signaling cascade that ended with a nuclear target by phosphorylating c-jun at the serine 73. This phosphorylation of c-jun was inhibited by staurosporine. These results suggest that a high level of Hg²⁺-mediated protein phosphorylation-linked signal induces rapid cell death bypassing DNA fragmentation, whereas a lower level induces cell death accompanying DNA fragmentation. This conclusion in turn implies that DNA fragmentation is not always a prerequisite for the signal transduction-dependent cell death of T-lymphocytes. J. Cell. Biochem. 71:243–253, 1998. © 1998 Wiley-Liss, Inc.     

Development of bovine oocytes reconstructed with different donor somatic cells with or without serum starvation

We conducted this study to examine whether serum starvation in culture contributes to better development of bovine reconstructed oocytes and to evaluate which serum-starved somatic cell is the most effective for cloned calf production. In Experiment 1, donor cells of four different types (cumulus cells, ear fibroblasts, oviduct cells and uterine cells) were either serum-starved or not before fusion with enucleated oocytes, and reconstructed oocytes were further cultured for 168 h. Regardless of serum starvation, cumulus cells or ear fibroblasts yielded higher (P < 0.05) rates of fusion than other cells (62.6-69.3 versus 33.3-38.7%). In the serum-starved group, the first cleavage after reconstruction was significantly increased in cumulus cells and ear fibroblasts, compared with oviduct cells (93.4-94.3 versus 78.8-86.0%), and oocytes reconstructed with either of these yielded more blastocysts than oocytes reconstructed with oviduct or uterine cells (40.6-43.8 versus 20.3-19.0%). We observed a similar pattern in the non-starved group, but we found a significant increase in blastocyst formation was found only in cumulus cells compared with other donor cells (42.6 versus 15.4-27.7%). Overall comparison showed that serum starvation increased the rates of cleavage and development to the blastocyst stage, but we found a statistical significance only in the cleavage rate (80.0 versus 89.5%). In Experiment 2, we transferred randomly selected 59 blastocysts that were developed from oocytes reconstructed with serum-starved cells to 44 synchronised recipients. Of those recipients, 23 became pregnant on Day 60 after transfer (52.3%) and 12 (27.3%) delivered cloned calves. The mean gestation length and birth weight was 275 +/- 8 days and 39.6 +/- 15.6 kg, respectively. Although there was no significant difference among donor cells, blastocysts that were derived from oocytes reconstructed with ear fibroblasts yielded the highest rates of pregnancy (50.0%) and delivery (27.3%). In conclusion, serum starvation is effective for improving preimplantation development of oocytes reconstructed with cumulus or ear fibroblast cells and it may positively influence on obtaining better pregnancy outcome.     

Body size determines eyespot size and presence in coral reef fishes

Numerous organisms display conspicuous eyespots. These eye‐like patterns have been shown to effectively reduce predation by either deflecting strikes away from nonvital organs or by intimidating potential predators. While investigated extensively in terrestrial systems, determining what factors shape eyespot form in colorful coral reef fishes remains less well known. Using a broadscale approach we ask: How does the size of the eyespot relate to the actual eye, and at what size during ontogeny are eyespots acquired or lost? We utilized publicly available images to generate a dataset of 167 eyespot‐bearing reef fish species. We measured multiple features relating to the size of the fish, its eye, and the size of its eyespot. In reef fishes, the area of the eyespot closely matches that of the real eye; however, the eyespots “pupil” is nearly four times larger than the real pupil. Eyespots appear at about 20 mm standard length. However, there is a marked decrease in the presence of eyespots in fishes above 48 mm standard length; a size which is tightly correlated with significant decreases in documented mortality rates. Above 75–85 mm, the cost of eyespots appears to outweigh their benefit. Our results identify a “size window” for eyespots in coral reef fishes, which suggests that eyespot use is strictly body size‐dependent within this group.     

Giant males or dwarf females: what determines the extreme sexual size dimorphism in Lamprologus callipterus?

In the Lake Tanganyika cichlid Lamprologus callipterus , males were >12 times heavier than females, the most extreme sexual size dimorphism in this direction among animals. L. callipterus males construct nests of empty snail shells in which the females breed. If the ancestors of L. callipterus were small cichlids, both sexes may have used shells for shelter. If the ancestors were larger, snail shells could not be used as shelters and would be important only for reproduction. In the field and the laboratory, females bred only in shells and the largest spawned in the largest shells. In the field, females chose larger shells than the average available in males' territories and did not copy the mate choice of other females. They never hid from predators in snail shells and they occurred commonly in areas without any potential shell shelters. In laboratory experiments females used shells only for reproduction and hardly for shelter. Therefore, it seems unlikely that L. callipterus descended from small shell-brooding cichlids which used shells for shelter, but more likely that the ancestors were of large or intermediate size, and that female size is constrained by the sizes of snail shells, which appear to be optimal breeding substrata.     

Hyperoxia decreases lung size of amphibian tadpoles without changing GSH-peroxidases or tissue peroxidation

1. During the development of D. pictus larvae (Amphibia) in normoxia, selenium (Se) GSH-Px increased whereas non-Se GSH-Px did not change. 2. Acclimation to 60 or 100% O2 did not change Se GSH-Px or non-Se GSH-Px. 3. Hyperoxia did not change tissue peroxidation (TBA-RS) confirming the good capacity of D. pictus tadpoles for O2-adaptation. 4. Since hyperoxic induction of catalase (CAT) has been previously described in D. pictus tadpoles, it is concluded that CAT is more important than both GSH-Px for the establishment of O2-adaptation. 5. Increases of Se GSH-Px, SOD and CAT, are probably important for adaptation to the change from aquatic to aerial environment during metamorphosis in normoxia. 6. Chronic exposure to 100% O2 enormously reduced the lung size of D. pictus larvae.     

Evidence for suppression of serum LH without elevation in serum estradiol or prolactin with a brain-enhanced redox delivery system for estradiol

We developed a redox system for brain-enhanced delivery of estradiol based on an interconvertible dihydropyridine in equilibrium pyridinium salt carrier. Estradiol (E2), when combined with the lipoidal carrier, readily crosses the blood-brain barrier. The carrier, when oxidized, reduces the rate of exit of the estradiol-carrier complex from the brain. Subsequent hydrolysis of the carrier provides sustained production of estradiol in the brain. The aim of the study was to evaluate the effects of single vs. multiple injections of the estradiol-chemical delivery system (E2-CDS) on both central and peripheral estrogen-responsive tissues. Ovariectomized Sprague-Dawley rats received an intravenous injection of E2-CDS at 10, 33, 100 or 333 micrograms/kg BW or the drug vehicle, dimethyl sulfoxide (DMSO; 0.5 ml/kg) every 2 days for 7 injections (2 weeks) or a single injection only at 2 days before sacrifice. With a single injection, E2-CDS did not affect serum luteinizing hormone (LH) levels at the 10 micrograms/kg dose but caused a dose-dependent reduction in serum LH of 39-52% at the dose range of 33 to 333 micrograms/kg. By contrast, multiple injections of E2-CDS caused a 32 to 76% reduction in serum LH levels at doses ranging from 10 micrograms/kg to 333 micrograms/kg. Additionally, multiple doses of E2-CDs caused a dose-dependent reduction in body weight at the 10 and 33 micrograms/kg doses with the higher doses causing no further weight reduction. For both single and multiple dosage groups, serum E2 levels remained unchanged after doses of E2-CDS of 10 and 33 micrograms/kg, then increased to 21 pg/ml for the single dosage group and to 23 pg/ml for the multiple dosage group at the 100 micrograms/kg dose, and to 59 pg/ml for singly-injected rats and 60 pg/ml for multiply-injected rats at the 333 micrograms/kg dose. Serum prolactin concentrations were closely correlated with serum E2 levels for both the single and multiple dose groups. These data reveal that a single or multiple doses of E2-CDS can reduce serum LH levels without elevating serum E2 or prolactin concentrations, supporting the concept of brain-enhanced delivery of estradiol with an estradiol chemical delivery system.