Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field

New approaches to increase the efficiency of non-viral gene delivery are still required. Here we report a simple approach that enhances gene delivery using permanent and pulsating magnetic fields. DNA plasmids and novel DNA fragments (PCR products) containing sequence encoding for green fluorescent protein were coupled to polyethylenimine coated superparamagnetic nanoparticles (SPIONs). The complexes were added to cells that were subsequently exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency 40 times more than in cells not exposed to the magnetic field. The transfection efficiency was highest when the nanoparticles were sedimented on the permanent magnet before the application of the pulsating field, both for small (50 nm) and large (200–250 nm) nanoparticles. The highly efficient gene transfer already within 5 min shows that this technique is a powerful tool for future in vivo studies, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.     

Enhancement of albumin expression in bone tissues with healing rat fractures

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral-diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [2002] Int. J. Mol. Med. 9:503-508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral-diaphyseal tissues with fracture healing were cultured for 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N-terminal sequencing of 66 kDa protein indicated that its N-terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral-diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or zinc acexamate (10(-4) M), medium albumin was significantly increased in the presence of those bone-stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral-diaphyseal and -metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral-diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components.     

Enhancement in activity of an invertase from the thermophilic fungus Thermomyces lanuginosus by exogenous proteins

A highly purified invertase from a thermophilic fungus Thermomyces lanuginosus showed enhanced activity when incubated with exogenous proteins. These proteins also stabilized the invertase when incubated at 50°C, 4°C and –20°C. However, none of these proteins stabilized the invertase at or above 55°C, the temperature of inactivation. This property was found to be specific for the thermophilic invertase, as no such activation was observed for the mesophilic invertases from yeasts.     

Spreadsheet method for calculating the induced currents in bone-fracture healing by a low-frequency magnetic field

A commercially available spreadsheet program is used on a microcomputer to calculate the induced current density and electric field patterns produced in a nonhomogeneous, anisotropic model of tissue by a localized, low-frequency magnetic field source. Specific application is made to coils used to promote the healing of bone fractures in limbs. The variation of the conductivity of the fracture gap during healing causes the induced current density pattern to change correspondingly, whereas the induced electric field remains relatively unchanged. Use of more simplified, isotropic models for the bone and for the soft tissue leads to results that differ significantly from those obtained from the full model. The magnetic field beyond the region of the coils contributes little to the induced currents in the fracture gap if the gap is located near the center of the coils. © 1994 Wiley-Liss, Inc.     

Magnetization of activated sludge by an external magnetic field

Magnetization of acclimated activated sludge with the application of an external magnetic field was observed using a magnetic force microscope and can be expressed as the visual magnetic flux density graph. FeCl₃ addition up to 0.1% (w/v) into the sludge, which was acclimated with synthetic sewage wastewater, enhanced the magnetization. Such magnetization was also observed in the activated sludge obtained from the practical wastewater treatment plants of a food processing plant and sewage wastewater. FeCl₃ addition also enhanced the sludge magnetization. The possibility is suggested that this magnetization causes the flock to enlarge and enhance sedimentation of the activated sludge on application of an external magnetic field.     

Possible cardiac driving by an external rotating magnetic field

The possible role of a rotating magnetic field as a potential "driver" for experimentally drug-induced failing heart systems was investigated. In two experiments, rats that had been exposed during successive doses of pentobarbital for approximately four hours to a rotating magnetic field of 0.5 Hz, 1–20 gauss, showed contraction rates within the field frequency range significantly longer than either control field or sham-field rats before cessation of QRS. However no significant differences were found between the groups in SGOT, serumiron or oxyhemoglobin levels. Implications of the results are discussed in relation to natural ELF cardiofrequency signals and failing cardiac systems.

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Zusammenfassung In zwei Experimenten mit Ratten wurde untersucht ob rotierende magnetische Felder als Steuerfaktor bei barbituratinduziertem Nachlassen der Herzfunktion eine Rolle spielen. Tiere, die in 30 Min Abständen Na-Pentobarbiturat erhielten und während 4 Stunden einem rotierenden magnetischen Feld der Stärke von 0,5 Hz 10–20 Gauss ausgesetzt waren, zeigten signifikant längere Herzkontraktionsraten innerhalb des Feldfrequenzbereiches vor dem Stillstand des QRS als Kontrollgruppen. Im Serumtransaminase- (GOT), Serumeisen- und Oxyhämoglobinspiegel waren am Versuchsende keine signifikanten Unterschiede zwischen den Gruppen feststellbar. Die Bedeutung der Befunde wird unter Bezug auf natürliche niederfrequente elektromagnetische Herzfrequenzimpulse und geschwächte Herzfunktion besprochen.

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Resume L'auteur a étudié les possibilités d'effets d'un champ magnétique tournant agissant comme "stimulateur" possible des systèmes cardiaques défaillants par suite de l'influence de drogues administrées expérimentalement. Lors de deux expériences, des rats, sous l'influence de doses successives de pentobarbital, ont été exposés pendant approximativement quatre heures à un champ tournant de 0.5 Hz et 1–20 gauss. Ils ont alors montré des rythmes de contractions comprises dans les limites de l'échelle de fréquence du champ, de durée significativement plus longue avant la cessation du QRS, que ceux de rats exposés à un champ de contrôle ou à un champ simulé. Cependant, les groupes n'ont pas montré de différences marquantes en ce qui concerne les niveaux de la transaminase du sérum, du taux de fer ou de l'oxyhémoglobine. Les implications des résultats sont étudiées dans la relation existant entre les signaux naturels de fréquence cardiaque IF et les systèmes cardiaques défaillants.

     

Enhancement of natural killer cell cytotoxicity by using static magnetic field to increase their viability

Natural killer (NK) cells are innately immune to the body’s immune system and can actively recognize and kill cancer cells. This study explores the potential for enhancing the killing ability of NK cells by co-culturing the NK cells with the target cells under a static magnetic field (SMF). In this study, NK92-MI cell lines were cultured in the presence of a 0.4-T SMF. The effect of the SMF on NK cell viability was evaluated by means of an MTT assay. Culturing tests were performed with inhibitors of the DAG/IP3, STAT3, ERK, JNK and p38 pathways in order to examine the possible signaling cascade responsible for the SMF effect on the NK92-MI cell viability. Finally, the effect of the SMF on the cytotoxicity of the NK92-MI cells was evaluated by co-culturing the NK cells with K562 leukemia cell lines. The results showed that the application of a 0.4-T SMF significantly increased (p < 0.05) the viability of the NK92-MI cells. Furthermore, the inhibitor tests indicated that the SMF affected cell viability by activating multiple MAPK signaling pathways (ERKs, JNKs, and p38-MAPK). Finally, SMF pre-exposure for 48 hr significantly improved the killing activity of the NK92-MI cells (p < 0.05). That is, pre-exposure to SMF increased the viability of the NK92-MI cells and improved their killing ability against K562 tumor cells. In general, the present results suggest that NK cells pre-exposed to 0.4-T SMF show potential as a tool for immune-therapy treatment of cancer.     

Enhancement of germination, growth, and photosynthesis in soybean by pre-treatment of seeds with magnetic field

Experiments were conducted to study the effect of static magnetic fields on the seeds of soybean (Glycine max (L.) Merr. var: JS-335) by exposing the seeds to different magnetic field strengths from 0 to 300 mT in steps of 50 mT for 30, 60, and 90 min. Treatment with magnetic fields improved germination-related parameters like water uptake, speed of germination, seedling length, fresh weight, dry weight and vigor indices of soybean seeds under laboratory conditions. Improvement over untreated control was 5-42% for speed of germination, 4-73% for seedling length, 9-53% for fresh weight, 5-16% for dry weight, and 3-88% and 4-27% for vigor indices I and II, respectively. Treatment of 200 mT (60 min) and 150 mT (60 min), which were more effective than others in increasing most of the seedling parameters, were further explored for their effect on plant growth, leaf photosynthetic efficiency, and leaf protein content under field conditions. Among different growth parameters, leaf area, and leaf fresh weight showed maximum enhancement (more than twofold) in 1-month-old plants. Polyphasic chlorophyll a fluorescence (OJIP) transients from magnetically treated plants gave a higher fluorescence yield at the J-I-P phase. The total soluble protein map (SDS-polyacrylamide gel) of leaves showed increased intensities of the bands corresponding to a larger subunit (53 KDa) and smaller subunit (14 KDa) of Rubisco in the treated plants. We report here the beneficial effect of pre-sowing magnetic treatment for improving germination parameters and biomass accumulation in soybean.     

Enhancement of NaCl tolerance in Arabidopsis thaliana by exogenous L-asparagine and D-asparagine

The tolerances of Columbia Arabidopsis thaliana (L.) Heynh. to NaCl, L-asparagine (L-Asn) and D-asparagine (D-Asn) during seedling establishment on sterile agar medium were determined. Germination and the establishment of upright seedlings with expanded green cotyledons were increasingly inhibited by NaCl concentrations from 20 to 180 m M and radicle growth was prevented at 225 m M NaCl. Tolerance of established seedlings to NaCl was similar at these concentrations. Seedling establishment was prevented at 20 m M L-Asn and 60 m M D-Asn, but L-Asn was not toxic to established seedlings. At lower concentrations, exogenous L- and D-Asn enhanced NaCl tolerance during germination and seedling establishment. Inhibition of seedling establishment by NaCl concentrations below 225 m M was reduced by the addition of L- and D-Asn to the medium. Maximal reduction of NaCl inhibition occurred between 2 and 4 m M for both L- and D-Asn. Higher concentrations of NaCl prevented establishment whether exogenous Asn was present or not. Reduction of NaCl inhibition occurred to the same extent whether L-Asn was presented simultaneously with the NaCl or preloaded for up to 24 h. The total seedling content of Na⁺ increased about 4-fold to 55 μg (mg dry weight)⁻¹ as the medium concentration of NaCl was increased from 9 μ M to 150 m M NaCl. Total K⁺ content declined about 80% from about 34 μg (mg dry weight)⁻¹ over the same range of NaCl concentrations. The Na⁺ uptake and K⁺ efflux by whole seedlings were similar whether or not NaCl tolerance was increased by exogenous Asn.     

Enhancement of the protective efficacy of an oprF DNA vaccine against Pseudomonas aeruginosa

The outer membrane protein F gene (oprF) of Pseudomonas aeruginosa was recently shown by us to protect mice from P. aeruginosa chronic pulmonary infection when used as a DNA vaccine administered by three biolistic (gene gun) intradermal inoculations given at 2-week intervals. In the present study, we used two different strategies to improve the protective efficacy of the DNA vaccine. In the first strategy, mice were primed with two biolistic intradermal inoculations with the oprF vaccine and then were given a final intramuscular booster immunization containing either a synthetic peptide-keyhole limpet hemocyanin (KLH) conjugate or a chimeric influenza virus. Both the synthetic peptide conjugate and the chimeric virus contained peptide 10, a previously identified immunoprotective epitope of protein F. The second strategy involved the addition of a second outer membrane protein to the vaccine. DNA encoding a fusion protein comprised of the C-terminal half of protein F fused to OprI was administered by three biolistic intradermal inoculations. Challenge with P. aeruginosa in a chronic pulmonary infection model demonstrated that boosting with the chimeric virus (but not with peptide-KLH) or adding oprI to the DNA vaccine significantly enhanced protection as compared to that afforded by the oprF vaccine given alone. Thus, both strategies appear to augment the protection afforded by an oprF-only DNA vaccine.     

Enhancement of DNA vaccine potency by electroporation in vivo

The potential of electric current-mediated delivery technology to enhance DNA delivery and DNA vaccine potency was evaluated. Higher levels of reporter gene expression were observed in muscle cells of mice inoculated with luciferase or beta-galactosidase DNA followed by the application of electrical current, compared with DNA injected with no current. Similarly, substantially higher levels of immune responses (up to 20-fold) were demonstrated in mice vaccinated with HIV gag DNA and electric current. These enhanced responses were observed after one or two inoculations, and were maintained for at least 12 weeks. Therefore, the present studies demonstrate the utility of electroporation for enhancement of DNA vaccine potency in animals.     

Immunolocalization of myostatin (GDF-8) following musculoskeletal injury and the effects of exogenous myostatin on muscle and bone healing

The time course and cellular localization of myostatin expression following musculoskeletal injury are not well understood; therefore, the authors evaluated the temporal and spatial localization of myostatin during muscle and bone repair following deep penetrant injury in a mouse model. They then used hydrogel delivery of exogenous myostatin in the same injury model to determine the effects of myostatin exposure on muscle and bone healing. Results showed that a "pool" of intense myostatin staining was observed among injured skeletal muscle fibers 12-24 hr postsurgery and that myostatin was also expressed in the soft callus chondrocytes 4 days following osteotomy. Hydrogel delivery of 10 or 100 μg/ml recombinant myostatin decreased fracture callus cartilage area relative to total callus area in a dose-dependent manner by 41% and 80% (p<0.05), respectively, compared to vehicle treatment. Myostatin treatment also decreased fracture callus total bone volume by 30.6% and 38.8% (p<0.05), with the higher dose of recombinant myostatin yielding the greatest decrease in callus bone volume. Finally, exogenous myostatin treatment caused a significant dose-dependent increase in fibrous tissue formation in skeletal muscle. Together, these findings suggest that early pharmacological inhibition of myostatin is likely to improve the regenerative potential of both muscle and bone following deep penetrant musculoskeletal injury.     

Influence of pulsed magnetic field on the bone growth plates in rabbits

Repeated in situ exposure of the rabbit knee joint to a pulsed magnetic field (1.5 T) is shown to suppress the functional activity of bone growth plates.     

Enhancement of enzyme reaction of magnetically anisotropic polyacrylamide gel rods immobilized with ferromagnetic powder and beta-D-galactosidase in an alternating magnetic field

An immobilized polyacrylamide gel containing beta-D-galactosidase and Sr-Ba-ferrite was magnetized in a static magnetic field. The gel rods (10 mm long, O 2 mm) exhibiting magnetic anisotropy could move at lower than 100 Hz but not at higher than 250 Hz in an alternating magnetic field of 200 Oe. In case of immovability of gel rods, the apparent enzymic activity increased 3 times higher under exposure of an alternating magnetic field of 500 Oe (570 Hz). It could be explained that the ferromagnetic powder inside the gel might vibrate under the influence of elasticity of gel in the alternating magnetic field of 100 or 500 Oe and 0.2-12 kHz. This might facilitate faster diffusion of the substance inside the gel and transportation of the substrate and the product through the surface of gel. Consequently, the enzyme reaction was apparently activated.     

Inflammatory phase of bone healing initiates the regenerative healing cascade

Bone healing commences with an inflammatory reaction which initiates the regenerative healing process leading in the end to reconstitution of bone. An unbalanced immune reaction during this early bone healing phase is hypothesized to disturb the healing cascade in a way that delays bone healing and jeopardizes the successful healing outcome. The immune cell composition and expression pattern of angiogenic factors were investigated in a sheep bone osteotomy model and compared to a mechanically-induced impaired/delayed bone healing group. In the impaired/delayed healing group, significantly higher T cell percentages were present in the bone hematoma and the bone marrow adjacent to the osteotomy gap when compared to the normal healing group. This was mirrored in the higher cytotoxic T cell percentage detected under delayed bone healing conditions indicating longer pro-inflammatory processes. The highly activated periosteum adjourning the osteotomy gap showed lower expression of hematopoietic stem cell markers and angiogenic factors such as heme oxygenase and vascular endothelial growth factor. This indicates a deferred revascularization of the injured area due to ongoing pro-inflammatory processes in the delayed healing group. Results from this study suggest that there are unfavorable immune cells and factors participating in the initial healing phase. In conclusion, identifying beneficial aspects may lead to promising therapeutical approaches that might benefit further by eliminating the unfavorable factors.     

Raising the efficiency of a plasma opening switch by applying an external magnetic field

The influence of an external magnetic field on the performance of a high-impedance plasma opening switch is studied experimentally. A 1.5-fold increase in the output voltage of a plasma opening switch operating in the erosion mode is achieved by applying an external magnetic field. The magnetic field strength and the parameters of the plasma opening switch at which the maximum output voltage is attained are determined. It is shown experimentally that the predicted dependence of the maximum output voltage on the Marx generator voltage, U _POS [MV]=3.6 (U _MG [MV])^4/7, is confirmed experimentally.     

Effect of exogenous fibronectin on the healing of skin wounds as evidenced by tensiometric data

Fibronectin (FN) solution purified from human blood plasma significantly increased the breaking strength of skin wounds in rats, when applied locally and/or intravenously. The best effect (more than 2-fold) was revealed after combined local and i. v. application of FN. Maximum stimulating activity was observed on the 3rd postoperative day, directly indicating the importance of FN for the early phases of wound healing. The data suggest the possibility of using FN preparations for the stimulation of tissue repair processes.