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PubMedDate not supplied

Glycofection in the presence of anionic fusogenic peptides: a study of the parameters affecting the peptide-mediated enhancement of the transfection efficiency.

Gene delivery mediated by polyplexes such as DNA complexed with polylysine conjugates is limited by the low efficiency of escape of DNA from the endosomes. One of the strategies which favors the transmembrane passage of polyplexes consists of adding anionic amphipathic peptides capable of destabilizing membranes in an acidic medium. Although less efficient than replication-defective adenoviruses, fusogenic peptides increase the expression of the reporter gene by a factor between 100 and 1000 depending on the cell line. However, the activity of a given peptide depends on the composition of the lipid bilayer. We were interested in developing a polyplex (glycoplex) formulation comprising a glycosylated polylysine, a fusogenic peptide and a plasmid which would be useful for efficient transfection (glycofection) of a large panel of cells, even in the presence of serum. We synthesized several peptides and tested their efficiency in combination with different glycoplex formulations. We found that glycofection with a quaternary complex (called one pot formulation) made of lactosylated-polylysine, polylysine, DNA, and the dimeric peptide (E5-WYGG)2-KA was less cell-type dependent than other peptide-based formulations. In addition, its efficiency was not affected by the presence of serum (up to 20%).

Amino Acid Sequence↗

PubMed2000

Separation efficiency in protein zone electrophoresis performed in capillaries of different diameters.

R-phycoerythrin (PHYCO, Mr 240 000), glucose-6-phosphate dehydrogenase (GPD, Mr 104 000) and two charge isomers of recombinant green fluorescent protein (GFP-1 and GFP-2, Mr 27 000) were subjected to capillary zone electrophoresis (CZE) in capillaries of 50, 100 and 150 microm inner diameter at various sample concentrations, electric field strengths, and lengths of the initial zone with the purpose of testing the hypothesis that protein - capillary wall interactions rather than thermal effects are predominantly responsible for the peak spreading of proteins in CZE. The efficiency of CZE was expressed in terms of the number of theoretical plates, N, or the plate height corrected by subtracting the contribution from initial zone length, H'. The latter has the advantage of solely reflecting contributions to the separation efficiency arising from intracolumn peak spreading in capillaries of different diameters. The separation efficiency measured varied widely, by two orders of magnitude, for these proteins under identical conditions, with GPD exhibiting the highest and PHYCO the lowest values of N. H' was found to be independent of sample concentrations within the concentration ranges studied, 1-100 microg/mL for PHYCO and 100-1000 microg/mL for GPD, while exhibiting a decrease with sample concentration for GFP, especially in 150 microm diameter capillaries, within the concentration range 1-100 microg/mL. H'was also found to be independent of electric field strength up to 300-400 V/cm for PHYCO and GFP. In all experiments, the CZE of proteins in 100 microm diameter capillaries provided a higher or, at least, equal efficiency, compared to that in 50 or 150 microm diameter capillaries. It may be concluded that the protein - capillary wall interactions and protein microheterogeneity are the dominant sources of peak spreading and their specific combinations are thought to be responsible for the wide variation in separation efficiency between proteins in CZE observed under identical conditions.

Electricity↗

PubMed1983

Cloning efficiency. A possible prognostic indicator in squamous cell carcinoma of the head and neck.

Seventy-three squamous cell carcinomas arising in the head and neck region were cultured using the soft agar clonogenic system. Thirty-six (49%) demonstrated clonal growth. The cloning efficiency was variable, ranging from 0.001% to 1.67% (median, 0.004%). Statistically significant correlations of high cloning efficiency with advanced local class, increased stage, and decreased survival were observed. There was no correlation between cloning efficiency and T-class or modified Jakobsson's histologic grade. A cloning efficiency of greater than or equal to 0.005% was associated with a high likelihood of death from disease or the development of recurrent disease. For head and neck squamous cell carcinomas, the cloning efficiency in soft agar appears to be a potentially useful prognostic indicator.

Carcinoma, Squamous Cell↗

PubMed2002

Nonaqueous capillary electrophoresis with alcoholic background electrolytes: separation efficiency under high electrical field strengths.

The effect of high voltage on capillary electrophoresis (CE) separations of anionic analytes in nonaqueous separation media was investigated. Methanol, ethanol, 1-propanol, and 1-butanol were tested as background electrolyte (BGE) solvents. Experiments were carried out with a laboratory-built CE instrument suitable for high-voltage separations. Potentials up to 60 kV were applied with reversed polarity to generate unusually high field strengths (e.g. 2000 Vcm-1) and so achieve fast and efficient separations. Highest separation efficiencies were obtained with propanol as BGE solvent, and the dependency of the efficiency on the separation voltage was more or less linear. With the other alcohols, separation efficiency decreased or remained roughly constant with increasing absolute voltage. The separation efficiencies are discussed in terms of longitudinal diffusion, Joule heating, and analyte interaction with the capillary wall. Capillary preconditioning had a varied effect on the separations in the different BGEs as the BGE and the conditioning process affected the electroosmotic flow (EOF) velocity and direction.

1-Butanol↗

PubMedDate not supplied

Effect of conformation on the chemiluminescence efficiency of light-producing 2-methyl-6-(4-methoxyphenyl)imidazo[1,2-alpha]pyrazin-3(7H)-ones.

Analogues of the light-producing substrate 2-methyl-6-(4-methoxyphenyl)imidazo[1,2-alpha]pyrazin-3(7H)-one, with restrictions on the dihedral angles between the pyrazine and the 4-methoxyphenyl rings, were synthesized. These compounds were used to investigate the effects of the conformation between the pyrazine and 4-methoxyphenyl rings on their chemiluminescence efficiencies in aqueous solutions. The effects of the conformation on the chemiluminescence efficiencies were clearly observed; the decrease of the dihedral angle caused a light-enhancing effect and, conversely, the increase of the angle dramatically decreased the chemiluminescence efficiency. These effects on the chemiluminescence efficiencies are attributed to the influence of the conformation on the fluorescence efficiency of the emitter.

Amides↗

PubMed2004

Highly efficient baculovirus-mediated gene transfer into rat chondrocytes.

To explore the potential of baculovirus serving as a gene delivery vector in tissue engineering of articular cartilage, the efficiencies of baculovirus-mediated gene delivery into primary rat chondrocytes were evaluated and the transduction protocol commonly employed by others (using concentrated virus at multiplicity of infection [MOI] 200 for 1 h) was found to be ineffective (<1%). Therefore, a modified protocol was adopted, which markedly enhanced the efficiency (68%). Optimization of the transduction parameters, such as incubation time (8 h), temperature (25 degrees C), and surrounding solutions (PBS), further increased the efficiency to 88% and prolonged the duration of expression to 21 days, suggesting that the cells previously considered nonpermissive to baculovirus transduction may be reexamined for their permissiveness using alternative transduction protocols. The elevated efficiency correlated well with increased virus uptake upon extended incubation time, as demonstrated by quantitative real-time polymerase chain reaction (Q-PCR). The Q-PCR also revealed the degradation of viral DNA over culture time. Although the virus transduction somewhat hindered the cell proliferation, growth rate could be restored in the long-term culture. More importantly, transduced cells could secrete articular cartilage-specific type II collagen and glycosaminoglycan as well as mock-transduced cells, confirming that normal differentiation state of rat chondrocytes is retained upon baculovirus transduction. Taken together, these data indicate that baculovirus is a safe and highly efficient gene delivery vehicle into rat chondrocytes.

Animals↗

PubMed1981

A flow system for partial automation of plating efficiency tests.

A flow system that sterilely deposits a known number of cells into a collection vessel has been constructed and evaluated. In this device, the cells from a single-cell suspension flow through a nozzle that constrains them to the center of the emerging jet. The cells in the jet are illuminated by a 0.5-mW helium-neon laser; they are detected, sized and counted on the basis of the light scattered as they pass through the laser beam. The device has been used for plating efficiency tests in a tissue culture laboratory. We found that it saved considerable labor by eliminating the dilution step from the standard plating efficiency procedure. The biologic effect of machine plating was determined by comparing plating efficiencies of KHT, CHO-T5, and CHO-AA8 cells established by hand plating with those established by machine plating. Results indicate that machine plating reduces the plating efficiency of the CHO-T5 cells while the plating efficiencies of the CHO-AA8 and KHT cells are unaffected.

Animals↗

PubMed2002

Visual search for high-level configural differences as well as low-level critical features is highly efficient early in childhood.

Visual search, or the ability to locate a single target from a field of distractors, is used as a means of assessing the capacity of the visual system with regard to the detection of low-level stimulus dimensions such as color, line orientation, and other specific perceptual features and higher level characteristics such as three-dimensional (3-D) orientation implied by pictorial cues to depth or occlusion. Target-distractor differences of importance to the visual system result in rapid (and possibly preattentive) target location while differences of lesser importance (or differences that are less salient) tend to elicit slower, less efficient search performance (search in this latter case may require attentional resources.) Adult abilities in this type of task have been tested extensively, but comparatively little is known about the development of these abilities. The present investigation was conducted to test, across a range of ages in childhood, the ability to detect visual search targets defined by a low-level critical feature (a texton; B. Julesz, 1981) and by static (pictorial) cues to depth. Children 2 to 9 years of age and adults were tested with each type of target. Previous data from infant studies predicted that the texton-defined differences would elicit highly efficient search performance, and this proved to be the case. When no texton difference was present, however, children's search was highly inefficient. Children between 4 to 9 years of age also proved to be quite efficient at locating a target, differing only by a pictorially rendered 3-D orientation from the distractors, but were much less efficient when the static cues to 3-D orientation were removed. These findings confirm previous reports that the basic processes underlying visual search are functional early in development, but suggest that children's search is less efficient than adult search when attention is required for object localization.

Child↗

PubMed2002

Relative efficiency of ambiguous vs. directly measured haplotype frequencies.

Haplotypes are useful for both fine-mapping of susceptibility loci and evaluation of sequence variation at multiple sites along a chromosome. However, they are difficult to directly measure over long stretches of DNA in diploid organisms. Consequently, multiple genetic markers are typically measured, without linkage phase information, giving rise to a subject's diplotype. From diplotype data, haplotypes are often inferred by pedigree information, or treated as partially missing data when haplotype frequencies are estimated among unrelated subjects. This latter ambiguity can increase the variance of the estimated haplotype frequencies. Douglas et al. ([2001] Nat. Genet. 28:361-364) recently quantified the relative efficiency of estimating haplotype frequencies from the diplotypes of unrelated subjects, relative to directly measured haplotypes via somatic cell hybrids (conversion technology), and demonstrated that unknown linkage phase can lead to a large loss of efficiency. However, their results were based on linkage equilibrium among marker loci, which may not be realistic for closely linked markers. We extend their relative efficiency calculations by several aspects: 1) allowance for linkage disequilbrium (LD) among marker loci; 2) evaluation of different patterns of LD; and 3) evaluation of nuclear families with and without parents. We show that although the loss in efficiency of haplotype frequencies among unrelated subjects decreases as LD increases to its maximum value, the general conclusions of Douglas et al. ([2001] Nat. Genet. 28:361-364) hold true for a variety of LD patterns and magnitudes. However, our results also demonstrate that trios of parents+one child are highly efficient for haplotype frequency estimation, that additional children offer little information, and that siblings without parents can be grossly inefficient. Genet. Epidemiol. 23:426-443, 2002.

Algorithms↗

PubMed1991

Poly(A) site efficiency reflects the stability of complex formation involving the downstream element.

A critical step in mRNA biogenesis is the generation of the mRNA 3' end through an endonucleolytic cleavage of the primary transcript followed by the addition of a approximately 200 nucleotide (nt) poly(A) tail. The efficiency of poly(A) site function can vary widely and for those genes with multiple poly(A) sites, the choice can be a regulated event. A functional poly(A) site is characterized by cis-acting RNA sequences including the well-conserved AAUAAA hexamer, located 10-30 nt upstream of the cleavage site, and a highly variable downstream GU- or U-rich element. The gene specific nature of the downstream sequence suggests that it may be a primary determinant of poly(A) site efficiency. Several recent studies have detailed the purification of factors that mediate the cleavage and polyadenylation reaction and that recognize the cis-acting signals. Two of these factors are responsible for the formation of a stable, committed ternary complex with the pre-RNA. In order to define the role of this stable complex in poly(A) site function, we have compared the processing efficiency of several pre-mRNAs with the stability of the complex that forms on these RNAs. We show that ternary complex stability reflects both the in vivo and the in vitro efficiency of the poly(A) site and that the stability of this complex is dependent on the nature of the downstream sequence element. We conclude that the stability of these protein--RNA interactions, dictated by the downstream element, plays a major role in determining the processing efficiency of a particular poly(A) site.

Base Sequence↗

PubMedDate not supplied

Jet nebulization of PEI/DNA polyplexes: physical stability and in vitro gene delivery efficiency.

BACKGROUND: Aerosol drug delivery currently represents the most acceptable and convenient delivery system for repeated drug application to the lungs. Although polyethyleneimine (PEI) has recently been demonstrated to mediate gene transfer successfully to mouse lungs via aerosol delivery, the effect of the jet nebulization process on the properties of PEI/DNA polyplexes has not yet been examined. METHODS: PEI/DNA polyplexes were generated in several commonly used solvents, such as distilled water, HEPES buffered saline (HBS), and 5% glucose. The complex parameters, such as particle size, zeta potential, DNA integrity, and transfection efficiency, were examined before and after jet nebulization. RESULTS: The complex parameters and the transfection efficiency were influenced by the solvent that was used for complex formulation and by the nebulization process itself. When polyplexes were formulated in HBS, the particle size, zeta potential, and DNA concentration were affected by the nebulization process and the transfection efficiency decreased dramatically. Polyplexes formulated in 5% glucose were less susceptible to the nebulization process, as indicated by only minor changes of the zeta potential and particle size when compared with HBS. The resulting transfecion efficiency was very low both before and after nebulization. Polyplexes formulated in distilled water had the most resistant behavior with the nebulization process. Zeta potential, particle size, and DNA integrity were influenced least of all by nebulization. CONCLUSION: As a result, the transfection efficiency of PEI/DNA complexes remained constant throughout the nebulization process only when formulated in distilled water. These data suggest that distilled water represents the most appropriate solvent for polyplex formulation when delivered by jet nebulization.

Animals↗

PubMedDate not supplied

Cationic lipids derived from glycine betaine promote efficient and non-toxic gene transfection in cultured hepatocytes.

BACKGROUND: The low efficiency and toxicity of transfection in a primary culture of hepatocytes using cationic lipids remains a limiting step to the study of gene function and the setting up of non-viral gene therapy. METHODS: A novel class of cationic lipids (GBs) derived from natural glycine betaine compounds covalently linked to acyl chains by enzymatically hydrolysable peptide and ester bonds, a structure designed to reduce cytotoxicity, was used to improve transfection efficiency in a primary culture of rat hepatocytes. The relationship between lipid structure, lipoplex formulation and transfection efficiency was studied using six GBs (12-14-16, 22-24-26) varying in their spacer and acyl chains. RESULTS: GB12, characterized by short [(CH(2))(10)] acyl chains and spacer, allowed plasmid uptake in all cells and reporter gene expression in up to 40% of hepatocytes with a low cytotoxicity, a much higher efficiency compared with transfections using other reagents including Fugene6 and Lipofectin. We also showed that numerous cells accumulated high amounts of plasmids demonstrating that GB12 promoted a very efficient DNA transfer through plasma membrane leading to an increase in nuclear plasmid translocation, allowing a much higher gene expression. Moreover, GB12-transfected hepatocytes survived to injection in normal livers and were found to express the LacZ reporter gene. CONCLUSIONS: The non-toxic GB12 formulation is a powerful vehicle for plasmid delivery in cultured hepatocytes with relevance in liver gene therapy.

Animals↗

PubMed2004

Short-term culture of myeloid leukemic cells allows efficient transduction by adenoviral vectors.

BACKGROUND: Ex vivo gene therapy of acute myeloid leukemia (AML) requires efficient transduction of leukemic cells. Recombinant adenovirus has been reported to be a poorly efficient vector in leukemic cells. We investigated leukemic cell culture as a possible method of improving the efficacy of this vector. METHODS: Leukemic cell lines and primary cultured AML cells were incubated with adenoviral vectors carrying GFP, LacZ, or IL-12 cDNA. Transduction efficiency was evaluated by measuring adenoviral genome copy number and transgene expression in leukemic cells. The expression of the coxsackie/adenovirus receptor (CAR), CD29, CD49e, and CD51/61 was measured, as was the effect of blocking integrin on adenoviral transduction. RESULTS: Increasing the multiplicity of infection (MOI) to 300 plaque-forming units per cell enhanced transduction of leukemic cell lines and to a lesser degree of AML cells. Analysis of adenoviral genome copy per cell showed only a partial correlation between gene transfer efficiency and transgene expression. Culture of AML cells for 3 days prior to adenoviral transduction increased both adenoviral copy number per cell and the percentage of transgene-expressing cells. CD29, CD49e, and CD51/61 but not CAR expression increased in cultured AML cells between days 0 and 3 and integrin-blocking experiments showed inhibition of transduction in two of four AML samples tested. CONCLUSIONS: Efficient ex vivo gene transfer in primary cultured AML cells can be achieved by short-term culture of leukemic cells prior to gene transfer with adenoviral vectors at a high MOI. This effect appears to be at least partially mediated by enhanced integrin expression.

Acute Disease↗

PubMed2007

Specific adeno-associated virus serotypes facilitate efficient gene transfer into human and non-human primate mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) show great promise for ex vivo gene and cell-mediated therapies. The immunophenotype and in vitro differentiation capacity of primary baboon MSCs was demonstrated to be near-identical to that observed in human MSCs. To optimize gene transfer efficiency, we compared the efficiency of serotypes 1, 2, 3, 4, 5, 6, and 8 of adeno-associated virus (AAV) vectors for their ability to mediate transduction of human and baboon MSCs. AAV serotype 2 vectors were the most efficient in transducing MSCs from humans and baboons. As a reference, human Ad293 cells were transduced with these seven AAV serotypes, and were found to have the highest transduction levels followed by baboon MSCs, and then human MSCs. The order of increasing transduction efficiency for the serotypes tested was similar for human and baboon MSCs, but was different for human Ad293 cells. The transduction efficiency of MSCs isolated from different individuals was comparable within the same species. We also demonstrated that baboon MSCs transduced with AAV serotype 2 vectors retain their potential to differentiate into adipocytes in vitro, and can incorporate into injured muscle tissue of NODSCID mice in vivo. We detected beta-galactosidase reporter gene expression in host muscle tissue for up to 9 weeks in this study, indicating engraftment of transduced baboon MSCs and sustained transgene expression in vivo.

Adipocytes↗

PubMed2001

Adaptive vessel tracking: automated computation of vessel trajectories for improved efficiency in 2D coronary MR angiography.

A new method was investigated for improving the efficiency of ECG-gated coronary magnetic resonance angiography (CMRA) by accurate, automated tracking of the vessel motion over the cardiac cycle. Vessel tracking was implemented on a spiral gradient-echo pulse sequence with sub-millimeter in-plane spatial resolution as well as high image signal to noise ratio. Breath hold 2D CMRA was performed in 18 healthy adult subjects (mean age 46 +/- 14 years). Imaging efficiency, defined as the percentage of the slices where more than 30 mm of the vessel is visualized, was computed in multi-slice spiral scans with and without vessel tracking. There was a significant improvement in the efficiency of the vessel tracking sequence compared to the multi-slice sequence (56% vs. 32%, P < 0.001). The imaging efficiency increased further when the true motion of the coronary arteries (determined using a cross correlation algorithm) was used for vessel tracking as opposed to a linear model for motion (71% vs. 57%, P < 0.05). The motion of the coronary arteries was generally found to be linear during the systolic phase and nonlinear during the diastolic phase. The use of subject-tailored, automated tracking of vessel positions resulted in improved efficiency of coronary artery illustration on breath held 2D CMRA.

Coronary Angiography↗

PubMed1987

The relative efficiency of the sets and the cusum techniques in monitoring the occurrence of a rare event.

The efficiency of each of two methods for monitoring the occurrence of a rare event, the cusum, carried out monthly, and the sets techniques, was assessed by use of simulated datasets. The monthly cusum parameters used in this study were those recommended for monitoring of birth defects in a monthly analysis. The sets technique parameters, recommended for monitoring of low-level epidemics, were adjusted to match the average interval between false alarms to the corresponding values observed for the monthly cusum technique. The results indicate that the relative efficiency of the two techniques depends on the baseline frequency of diagnoses. The sets technique is shown to be more efficient than monthly cusum when the number of cases expected in a year is no greater than five, but less efficient otherwise. In the former case, lengthening the interval between cusum analyses can make the cusum technique more efficient than the sets technique. This possibility deserves further exploration and could affect future recommendations.

Adult↗

PubMed2001

High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation.

A highly efficient method for transformation of the fission yeast Schizosaccharomyces pombe by electroporation has been developed. Significantly higher transformation efficiency was obtained when intact cells grown in SD medium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose) were pretreated with thiol compounds before an electric pulse was applied to the cells. Among the thiol compounds tested, dithiothreitol (DTT) was the most effective for pretreatment. A high transformation efficiency was obtained when the cells were pretreated with 25 mM DTT at 30 degrees C for 15 min in an osmotically adjusted buffer, since the cells were sensitive to osmotic pressure. It was important to exclude glucose from the DTT pretreatment buffer, as it caused a drastic decrease in efficiency. The optimal cell concentration and amount of DNA during the electric pulse were 1x10(9) cells/ml and 10 ng, respectively. The maximum transformation efficiency, 1.2x10(7) transformants/microg plasmid DNA, was obtained when an electric pulse of 11.0 kV/cm was applied for 5 ms. Furthermore, the high competency of cells pretreated with DTT was maintained by freezing them in a non-permeating cryoprotectant such as sorbitol.

Cryopreservation↗

PubMed1994

A method to increase efficiency and minimize anomalous electrophoretic transfer in protein blotting.

We find that in protein blotting, the electrophoretic transfer efficiency of a given protein can be markedly affected by other components in the sample. Using a variety of electrophoretic elution protocols, we obtained very inefficient transfer of purified 125I-labeled 275-kDa cation-independent mannose 6-phosphate receptor from a sodium dodecyl sulfate-polyacrylamide gel to a membrane. Surprisingly, the transfer efficiency increased significantly if other proteins that migrated in the vicinity of the receptor were present. This may be a generally unappreciated but potentially widespread phenomenon as other but not all radiolabeled proteins examined behaved similarly. This carrier effect occurs under conditions typically used in protein blotting experiments and can lead to large errors in quantitative analysis. We developed two methods to minimize the carrier effect. First, addition of excess carrier protein to samples prior to electrophoresis saturated the carrier effect so that different samples had similar transfer efficiencies. Second, addition of either bovine serum albumin or sodium deoxycholate to the cathodic electro-elution buffer markedly increased transfer efficiency of radiolabeled proteins that exhibited poor transfer efficiency and minimized transfer differences between samples that contained or lacked carrier. These methods should be generally applicable to standard protein blotting and analysis protocols.

Electrophoresis, Polyacrylamide Gel↗