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PubMed1999

Intracellular accumulation of secreted proteoglycans inhibits cationic lipid-mediated gene transfer. Co-transfer of glycosaminoglycans to the nucleus.

Molecules secreted by potential target cells may interfere with cationic lipid-mediated gene transfer. This has been studied using human lung fibroblasts and human epidermoid lung cancer cells. Secreted cell medium components caused a substantial decrease both in the uptake of cationic lipid-DNA complexes (2-4-fold) and in reporter gene expression (100-1000-fold). Metabolic labeling of the cell medium showed that especially [35S]sulfate-labeled macromolecules competed with DNA for binding to the cationic lipid. Release of DNA from the cationic lipid by cell medium components was demonstrated by an ethidium bromide intercalation assay. In the presence of the cationic lipid, the secreted macromolecules were internalized by the cells. By enzymatic digestions, it was shown that the competing macromolecules consist of chondroitin/dermatan sulfate and heparan sulfate proteoglycans and that the effects on transfection were mediated by the polyanionic glycosaminoglycan portion of the proteoglycan. Accordingly, pretreatment of cell medium with the polycationic peptide protamine sulfate abrogated the inhibitory effects on gene transfer. Fluorescence microscopy studies revealed that heparan sulfate, internalized as a complex with cationic lipids, accumulated in the cell nuclei. These results support the view that the lack of specificity of this type of gene transfer vehicle is a major hindrance to efficient and safe in vivo administration.

Biological Transport↗

PubMed1984

DNA-mediated gene transfer into human diploid fibroblasts derived from normal and ataxia-telangiectasia donors: parameters for DNA transfer and properties of DNA transformants.

We investigate the feasibility of DNA-mediated gene transfer into human diploid fibroblasts derived from patients with the radiation sensitive syndrome ataxia-telangiectasia (A-T) and from a normal donor. Although they are markedly different in their growth characteristics, both normal and A-T strains give similar frequencies for DNA transfer in a model system using the recombinant plasmid pSV2 -gpt. pSV2 -gpt DNA transformants arise with a frequency between 10(-5) and 10(-4) per viable cell. Analysis of such transformants, although possible, is severely handicapped by the limited clonal life span of diploid human cells. Despite these problems it may be concluded that diploid human fibroblasts are competent recipients for DNA-mediated gene transfer and the putative repair deficiency of A-T does not markedly effect the efficiency of this process.

Ataxia Telangiectasia↗

PubMed1954

Studies on the transfer of lymph node cells. IV. Effects of X-irradiation of recipient rabbits on the appearance of antibody after cell transfer.

Cells of the popliteal lymph node were teased 3 days after the injection of Shigella paradysenteriae into the hind foot pads of rabbits. These cells were transferred to normal and x-irradiated recipients. It was noted that the serum titers of dysentery agglutinins in irradiated recipients were higher than in normal recipients. This was represented both in a higher peak: titer and a tendency to remain higher for a longer period than in normal animals. Recipients were x-irradiated within 1 hour after receiving cells of the lymph node prepared as indicated above. The serum titers of these recipients were markedly reduced in comparison with those of non-irradiated control animals. If the irradiation of the recipients followed the transfer of cells by a day, however, this difference was much smaller and in the case of a 2 day interval after the transfer of the lymph node cells the irradiation appeared to have no effect on the resulting serum titer.

Animals↗

PubMed1956

Studies on the transfer of lymph node cells. VII. Transfer of cells incubated in vitro with filtrates of trypsin-treated suspensions of Shigella paradysenteriae.

Shigella paradysenteriae organisms were incubated in a solution of trypsin and then removed from the suspension by Seitz filtration. Serologic tests with anti whole Shigella serum indicated that the filtrate contained antigenic material derived from the organisms. When lymph node cells from rabbits not previously injected with Shigella were incubated in vitro with such filtrates and then transferred to irradiated recipients, agglutinins to Shigella appeared in the sera of the latter. The transfer of heated suspensions of cells was not followed by the appearance of agglutinins in irradiated recipient rabbits. Non-irradiated recipients of lymph node cells incubated with the filtrate also developed agglutinin titers, to a lower level than irradiated animals. Agglutinins did not appear in the sera of the majority of non-irradiated recipients of heated cells. The addition of excess antiserum to either the filtrate or the cell suspension before the incubation of these two materials resulted in a marked reduction in the subsequent agglutinin titers of recipient animals. However, if antiserum was added at the conclusion of the usual 30 minute incubation of cells and filtrate, or even after 5 minutes of incubation, there was no reduction in the agglutinin titers of the recipients. Cytologic examination of aliquots of a number of the suspensions of cells prepared for transfer revealed that approximately 98 to 99 per cent of the cells belonged to the lymphocytic series.

Animals↗

PubMed1956

Studies on the transfer of lymph node cells. VIII. The transfer of cells from blood and peritoneal exudates incubated in vitro with antigenic material derived from Shigella paradysenteriae.

Leucocytes were obtained from the blood of rabbits, incubated in vitro with soluble antigenic material derived from Shigella paradysenteriae, washed, and transferred to irradiated recipient animals. On the 4th day after cell transfer, agglutinins to Shigella appeared in the sera of the majority of such recipients. The distribution of maximum agglutinin titers was substantially lower in these animals than in recipients of lymph node cells similarly incubated, as reported in earlier studies. Recipients of iodoacetate-treated cells showed no agglutinins. Similar results were obtained with cells of peritoneal exudates obtained 9 days after the intraperitoneal injection of lanolin and light mineral oil. When cells of peritoneal exudates obtained 1 or 2 days after the injection of this irritant were incubated with the antigenic material and transferred to irradiated recipients, agglutinins did not appear in the sera of the latter. Similar results were obtained with cells from peritoneal exudates obtained a few days after the intraperitoneal injection of heavy mineral oil. The preponderant cell types in the suspensions from these two sources were the polymorphonuclear neutrophils and monocytes, respectively.

Animals↗

PubMed1993

Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression.

Striated muscle is the only tissue found to be capable of taking up and expressing reporter genes that are transferred in the form of plasmid DNA. Thus, direct gene transfer is a potential method of gene therapy for the primary inherited myopathies. However, results to date have had insufficient and too variable expression to consider using direct gene transfer in human trials. We have determined that much of the variability of expression is due to nonuniform distribution of substances injected into skeletal muscle in vivo, and have developed a model to ameliorate this. Preinjection of muscles with a relatively large volume of hypertonic sucrose improves the distribution of injected substances and results in significantly less variable expression of reporter genes for luciferase or beta-galactosidase; the coefficient of variation for mean luciferase activity was reduced from about 120% to 25%. Expression is not directly proportional to dose, but is more so if the muscles are preinjected with sucrose than not. Expression is higher and less variable if DNA is injected in a larger than a smaller volume. The choice of promoter appears to be particularly important. Luciferase reporter gene expression from the SV40 promoter was transient and low, whereas expression driven by the Rous sarcoma virus (RSV) promoter was high and sustained, such that a 1,000-fold difference in expression could be observed. The mechanism of gene uptake is still unknown, but our findings indicate that fibers damaged by the injection procedure do not take up and express plasmid DNA.

Animals↗

PubMed2004

Theory of current-driven domain wall motion: spin transfer versus momentum transfer.

A self-contained theory of the domain wall dynamics in ferromagnets under finite electric current is presented. The current has two effects: one is momentum transfer, which is proportional to the charge current and wall resistivity (rho(w)); the other is spin transfer, proportional to spin current. For thick walls, as in metallic wires, the latter dominates and the threshold current for wall motion is determined by the hard-axis magnetic anisotropy, except for the case of very strong pinning. For thin walls, as in nanocontacts and magnetic semiconductors, the momentum-transfer effect dominates, and the threshold current is proportional to V(0)/rho(w), V0 being the pinning potential.

Journal Article↗

PubMed1992

Conformation of MgATP bound to nucleotidyl and phosphoryl transfer enzymes 1H-transferred NOE measurements on complexes of methionyl tRNA synthetase and pyruvate kinase.

The conformations of MgATP bound to a nucleotidyl transfer enzyme, methionyl tRNA synthetase and a phosphoryl transfer enzyme, pyruvate kinase, were studied by transferred NOE (TRNOE) measurements in 1H NMR. The experiments were performed on D2O solutions at 276 MHz and 300 MHz, and 10 degrees C in the presence of approximately a tenfold excess of substrate over the enzyme (sites). Selective inversion of chosen resonances was accomplished with an appropriately tailored DANTE sequence consisting of 100 phase-alternating hard 1.8 degree pulses. NOE measurements were made in terms of difference spectra (with and without inversion) at 6-8 delay times ranging from 10-500 ms following the DANTE sequence. A full complement of ten NOE build-up curves obtained for each enzyme complex was analyzed by using the complete relaxation-matrix method (which includes all the non-exchangeable protons in MgATP) suitably modified to include exchange between bound and free substrate. Molecular mechanics computations were used to examine the energetic implications of the NOE-determined structure. The final structures obtained for MgATP bound to the two enzymes were very similar to each other, with a 3'-endo sugar pucker and an anti conformation with a glycosidic torsional angle (O'4-C'1-N9-C8) of 39 degrees +/- 4 degrees. Both enzymes contain multiple binding sites for MgATP and hence the structure obtained in each case represents an average due to chemical exchange. However, TRNOE experiments performed on a tryptic fragment of methionyl tRNA synthetase which has a single MgATP binding site, show that the same structure fits these measurements as well. This evidence, coupled with the striking similarity of the structures deduced, for the two enzyme complexes, and the reciprocal sixth-power dependence of NOE on interproton distance, strongly suggests that the conformations at the individual binding sites of both the enzymes are virtually identical. This conclusion is in contrast with multiple conformations of MgATP bound to pyruvate kinase, proposed by Rosevear, P.R., Fox, T.L. & Mildvan, A.S. (1987) Biochemistry 26, 3487-3493.

Adenine↗

PubMed2001

Contribution of heat transfer to turbine blades and vanes for high temperature industrial gas turbines. Part 2: Heat transfer on serpentine flow passage.

The improvement of the heat transfer coefficient of the 1st row blades in high temperature industrial gas turbines is one of the most important issues to ensure reliable performance of these components and to attain high thermal efficiency of the facility. This paper deals with the contribution of heat transfer to increase the turbine inlet temperature of such gas turbines in order to attain efficient and environmentally benign engines. Following the experiments described in Part 1, a set of trials was conducted to clarify the influence of the blade's rotating motion on the heat transfer coefficient for internal serpentine flow passages with turbulence promoters. Test results are shown and discussed in this second part of the contribution.

Journal Article↗

PubMed1994

C9-mediated killing of bacterial cells by transferred C5b-8 complexes: transferred C5b-9 complexes are nonbactericidal.

The formation of the C5b-9 complex on the outer membrane of complement-sensitive cells of Escherichia coli results in inhibition of inner membrane function and the death of the cell. Cells bearing a precursor of the C5b-9 site, the C5b-8 complex, suffer no loss in viability. Antibiotic-sensitive, complement-sensitive donor cells bearing precursor C5b-8 complexes were incubated with equal numbers of antibiotic-resistant, complement-sensitive acceptor cells that had not been exposed to a complement source. This cell mixture was incubated with 5 mM EDTA for 5 min and then with calcium chloride (20 mM) for various times. The excess calcium ion concentration was effectively reduced with additional EDTA, and the cell mixture was washed and resuspended in buffer. The viability of the acceptor cells was assayed by plating on antibiotic-containing media. C9 was added to the mixture, and the mixture was incubated for 10 min at 37 degrees C and then plated as described above. It was found that the acceptor cells were killed by the addition of purified C9 only after incubation with donor cells bearing C5b-8 sites during the transfer procedure. This indicates that precursor C5b-8 sites that support C9-mediated killing could be transferred between cells. No loss in viability was detected for acceptor cells subjected to the procedure described above in the presence of donor cells bearing complete C5b-9 complexes, formed prior to mixing with acceptor cells for the transfer procedure.

Bacteria↗

PubMed1984

Conjugal transfer of bacterial chromosomes mediated by the RK2 plasmid transfer origin cloned into transposon Tn5.

We report here a novel system for the conjugal transfer of bacterial chromosomes which utilizes the transfer origin (oriT) of plasmid RK2 cloned into transposon Tn5. Tn5 with oriT was inserted by transposition into the chromosomes of Escherichia coli and Rhizobium meliloti. The oriT sequence then served as the origin of high-frequency chromosome transfer when a helper RK2 plasmid was present in the same cell. The broad host range features of RK2 make this system of oriented chromosome mobilization applicable to most gram-negative bacteria.

Anti-Bacterial Agents↗

PubMed1987

Plasmid transfer in Streptomyces lividans: identification of a kil-kor system associated with the transfer region of pIJ101.

The 8.9-kilobase Streptomyces plasmid pIJ101 is self-transmissible at high frequency into recipient strains. By genetic analysis of the transfer region of the plasmid, we identified six plasmid-encoded loci involved in gene transfer and the associated pocking phenomenon. Two loci, kilA and kilB, could not be cloned into Streptomyces lividans on a minimal pIJ101-based replicon unless suitable kil-override (kor) genes were present, either in cis or in trans. korA could control the lethal effects of both kilA and kilB, whereas korB could control only the effects of kilB. KilB mutants were defective in their pocking reaction; kilA mutants produced no visible pocks whatsoever. Mutations in two other loci, tra and spd, produced no pocks and defective pocks, respectively. These results suggest that kilA, kilB, tra, and spd are intimately involved in plasmid transfer and that the actions of kilA and kilB are regulated by the products of the korA and korB genes.

Cloning, Molecular↗

PubMed1997

Regulation of transfer of the Enterococcus faecalis pheromone-responding plasmid pAD1: temperature-sensitive transfer mutants and identification of a new regulatory determinant, traD.

The enterococcal, conjugative, cytolysin plasmid pAD1 confers a mating response to the peptide sex pheromone cAD1 secreted by plasmid-free strains of Enterococcus faecalis. Cells carrying pAM714, a pAD1::Tn917 derivative with wild-type conjugation properties, were mutagenized with ethyl methanesulfonate to obtain variants that were induced (in the absence of pheromone) to transfer plasmid DNA upon shifting from 32 to 42 degrees C. Of 31 such mutants generated, the results of analyses of 7 are presented in detail. All seven strains were thermosensitive in the E. faecalis host FA2-2; colony morphology, clumping, and DNA transfer correlated well with each other at the two temperatures. In the nonisogenic host E. faecalis OG1X, however, only one derivative (pAM2725) exhibited correlation of all three traits at both temperatures. Three (pAM2700, pAM2703, and pAM2717) clumped and had colonies characteristic of pheromone-induced cells at 32 degrees C but transferred plasmid DNA at a higher frequency only at the elevated temperature. The other three (pAM2708, pAM2709, and pAM2712) were derepressed at both temperatures for all three characteristics. Four of the mutations, including that of pAM2725, mapped within the traA determinant, whereas two mapped identically in a previously unnoted open reading frame (designated traD) putatively encoding a short (23-amino-acid) peptide downstream of the inhibitor peptide determinant iad and in the opposite orientation. One mutant could not be located in the regions sequenced. Studies showed that the traA and traD mutations could be complemented in trans with a DNA fragment carrying the corresponding regions.

Amino Acid Sequence↗

PubMedDate not supplied

1H and 13C NMR study of proton transfer in azoles. Mechanism of proton transfer in pyrazole in solutions containing bis-(acetylacetonato) nickel (II).

Proton transfer in pyrazole systems involving interaction with a paramagnetic transition metal complex has been studied with 1H and 13C NMR techniques. Kinetics of the ligand exchange in the pyrazole complex of bis-(acetylacetonato)nickel (II) has been investigated, and hyperfine coupling constants for the interaction of the nickel unpaired electron with the ligand nuclei in the complex have been determined. Catalytic admixtures of bis-(acetylacetonato)nickel (II) are shown to modify the mechanism of the proton transfer in pyrazole. The spectral data are discussed in terms of outer-sphere coordination and cooperative proton transfer effects.

Chemical Phenomena↗

PubMed2004

Electroneutrality coupling of electron transfer at an electrode surface and ion transfer across the interface between thin-layer of 1-octyl-3-methylimidazolium bis(perfluoroalkylsulfonyl)imide covering the electrode surface and an outer electrolyte solution.

The electrode reaction of decamethylferrocene (DMFc) dissolved in a thin layer of a room-temperature molten salt (RTMS), 1-octyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (C8mimC1C1N) or 1-octyl-3-methylimidazolium bis(pentafluoroethylsulfonyl)imide (C8mimC2C2N), on a self-assembled monolayer-modified gold electrode is coupled with the ion transfer across the interface between the RTMS and the outer aqueous solution (W) to give a voltammogram whose shape resembles a voltammogram of a simple one-electron transfer process. The electroneutrality of the RTMS layer during the oxidation of DMFc to decamethylferricenium ion is maintained by the concomitant dissolution of C8mim+ ion from the RTMS phase to the W phase, and the reduction of decamethylferricenium ion to DMFc is accompanied by the transfer of either C1C1N- or C2C2N- from RTMS to W. The midpoint potential of the voltammogram varies with the concentration of the salt in the aqueous phase, C8mimCl or LiCnCnN (n = 1 or 2), in a Nernstian manner, showing that the phase-boundary potential between the RTMS and the W is controlled by the partition of these ions. Although the phase-boundary potential across the RTMS / W interface is Nernstian with respect to the ions common to both phases at the equilibrium, the polarization at the RTMS / W interface under current flow distorts the shape of the voltammograms, resulting in a wider peak separation in the voltammogram.

Journal Article↗

PubMed2005

Is the transfer factor a relevant tool to assess the soil-to-plant transfer of radionuclides under field conditions?

The radiological impact of radionuclides released to the terrestrial environment is usually predicted with mathematical models in which the transfer of radionuclides from soil to the plant is described with the transfer factor (TF). This paper questions the validity of the protocols proposed by the International Atomic Energy Agency to measure TF in the field and in greenhouses conditions. We grew maize (Zea mays L.) both in the field after a surface application of radionuclides ((54)Mn, (57)Co, (65)Zn, and (134)Cs) and in a greenhouse with the same soil that has received the same fertilization and that had been previously sieved and homogeneously labeled with the same radionuclides before being repacked in pots. The analysis of the displacement of radionuclides in the field soil profile showed a higher concentration of the surface-applied radionuclides in the preferential flow path (PFP) in comparison to the soil matrix indicating that they infiltrated heterogeneously in the soil profile due to the structure-induced non-uniform water flow. A significantly higher recovery of (57)Co and (134)Cs was observed in the plants grown in the field soil, whereas no differences in the recovery of (54)Mn and (65)Zn between the two experiments were detected. These results suggest that (i) under field conditions the soil-to-plant transfer of radionuclides that co-exist as stable elements present at low concentrations in the soil and in the plant is higher than that measured under greenhouse conditions and (ii) the implicit assumption made when calculating the TF (that radionuclides are homogeneously distributed in the soil profile) is not valid, thereby preventing the calculation of an average concentration to obtain the TF parameter.

Models, Theoretical↗

PubMed1991

[Transfer of a very low birth weight newborn infant to the intensive therapy center at birth. Transfer "in utero" or after delivery?].

During the 1975-1984 period, 93 infants weighing 1500 g or less were transferred to the Magenta Perinatal Center Hospital. The survival rate at discharge was 82.9% for infants transferred in utero and 63.5% for infants transferred postnatally (p less than 0.05). The incidence of major neurological sequelae was 3.1% and 23.3% respectively (p less than 0.02). This experience confirms that the high risk pregnancies should be referred to the Perinatal Center for optimal care of the mother, fetus and newborn infant.

Adult↗

PubMed1990

DNA strand transfer protein beta from yeast mitotic cells differs from strand transfer protein alpha from meiotic cells.

We have purified to homogeneity an activity from mitotic cell extracts of the yeast Saccharomyces cerevisiae, which promotes the transfer of a strand from a duplex linear DNA molecule to a complementary circular single strand. This activity does not require any nucleotide cofactor and is greatly stimulated by yeast single-stranded DNA-binding protein. It consists of a single polypeptide of an apparent molecular mass of 180 kDa as determined by SDS-polyacrylamide gel electrophoresis. This activity, which we call DNA strand transfer protein beta (STP beta), has reaction properties similar to those of DNA strand transfer protein alpha (STP alpha) purified from crude extracts of yeast meiotic cells (Sugino, A., Nitiss, J., and Resnick, M. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3683-3687). However, STP beta differs from STP alpha in its molecular weight and column chromatographic behavior as well as by immunological comparison. Furthermore, the STP beta polypeptide remains in cells in which the STP alpha gene has been disrupted. Thus, we conclude the STP beta activity is encoded by a gene different from that for STP alpha. Although STP beta was isolated from mitotic cells, the amount of STP beta increases severalfold during meiosis. STP beta also appears to differ in molecular weight from similar activities described by other groups and may be an intact form of their activities.

Bacteriophage phi X 174↗