The auto-orientation in high magnetic fields of oxidized cytochrome b562 as source of constraints for solution structure determination

¹⁵N-¹H ¹J couplings were measured at 500 MHz and 800 MHz for ¹⁵N enriched oxidized cytochrome b ₅₆₂ from E. coli. The magnetic field dependence of 70 ¹J values, which could be measured without signal overlap, shows that there is a molecular magnetic anisotropy which provides partial molecular orientation in the magnetic field and, consequently, residual dipolar couplings (rdc). The rdc were used as further constraints to improve the existing structure [Arnesano et al. (1999) Biochemistry, 38, 8657–8670] with a protocol which uses the rhombic anisotropy [Banci et al. (1998) J. Am. Chem. Soc., 120, 12903–12909]. The overall large molecular magnetic anisotropy has been found to be determined by both the low spin iron (III) and the four helix bundle structure magnetic susceptibility anisotropy contributions.     

Short cut of protein purification by integration of cell-disrupture and affinity extraction

Screening strategies based on functional genomics require the isolation of gene products of several hundred cDNA clones in a fast and versatile manner. Conventional purification strategies will fail to accomplish this goal within a reasonable time frame. In order to short-cut these procedures, we have developed a combination of cell disintegration and affinity technique for rapid isolation and purification. For our purpose, tagged proteins have been produced in yeast by fusing the FLAG-sequence adjacent to the 5 end of cDNAs coding for the respective protein. The example of an over-expressed FLAG-tagged fusion protein, human serum albumin (HSA), was released into the cytoplasm. Detection and purification of the FLAG-fusion protein were carried out by using a mouse monoclonal antibody directed against the FLAG-peptide. For purification purposes, the antibody was immobilized on PROSEP magnetic glass beads. These magnetic glass beads with 500 m diameter have been investigated for disintegration of yeast and simultaneous capturing of the target protein. After 60 s, 90% of the maximal disintegration level was achieved when a ratio of 20 l yeast cell suspension and 100 l glass are vortexed. After a wash step, the FLAG-fusion proteins have been eluted with chelating agents such as EDTA. The short-cut procedure has been compared to a conventional purification strategy using an affinity chromatography process. Due to the highly favorable binding characteristics of the applied immunoaffinity sorbent the yield observed in batch operation was 90% and purity in the range of 70–80%.     

High affinity nucleocapsid protein binding to the muPsi RNA packaging signal of Rous sarcoma virus

The genomes of all retroviruses contain sequences near their 5' ends that interact with the nucleocapsid domains (NC) of assembling Gag proteins and direct their packaging into virus particles. Retroviral packaging signals often occur in non-contiguous segments spanning several hundred nucleotides of the RNA genome, confounding structural and mechanistic studies of genome packaging. Recently, a relatively short, 82 nucleotide region of the Rous sarcoma virus (RSV) genome, called muPsi, was shown to be sufficient to direct efficient packaging of heterologous RNAs into RSV-like particles. We have developed a method for the preparation and purification of large quantities of recombinant RSV NC protein, and have studied its interactions with native and mutant forms of the muPsi encapsidation element. NC does not bind with significant affinity to truncated forms of muPsi, consistent with earlier packaging and mutagenesis studies. Surprisingly, NC binds to the native muPsi RNA with affinity that is approximately 100 times greater than that observed for other previously characterized retroviral NC-RNA complexes (extrapolated dissociation constant K(d)=1.9 nM). Tight binding with 1:1 NC-muPsi stoichiometry is dependent on a conserved UGCG tetraloop in one of three predicted stem loops, and an AUG initiation codon controvertibly implicated in genome packaging and translational control. Loop nucleotides of other stem loops do not contribute to NC binding. Our findings indicate that the structural determinants of RSV genome recognition and NC-RNA binding differ considerably from those observed for other retroviruses.     

Metabolic comparison of cryopreserved and normal cells from <Emphasis Type="Italic">Tabernaemontana divaricata</Emphasis> suspension cultures

A cryopreservation protocol for Tabernaemontana divaricata suspension cell cultures (6 Div BW 101) was established. Cells were precultured in MS medium supplemented with 0.5 and 0.33 M mannitol for 2 or 3 days following with incubation in MS media with a mixture of 1 M sucrose, 0.5 M glycerol, 0.5 M DMSO, and 0.04 M L-proline as cryoprotectant in an ice bath for 20 min. The cells were transferred into 2 ml cryogenic vials and then, the vials were put into the cryogenic container prior to placing at a −80 °C freezer for 4 h followed by rapid immersion in liquid nitrogen. The cells were transferred without washing a MS medium solidified with 7% (w/v) agarose. Cells that were precultured 3 days after subculturing in MS medium supplemented with 0.5 M mannitol for 3 days, showed growth recovery. Metabolic profiling of control and cryopreserved Tabernaemontana divaricata cells was performed by ¹H-NMR spectroscopy combined with PCA, GC, and HPLC. Differences of metabolic accumulation were found in the level of several amino acids, carbohydrates, and fumaric acid. However, the levels of the main alkaloid precursor tryptamine did not change.     

Analysis of the structure of magnetic fields that induced inhibition of stimulated neurite outgrowth

The important experiments showing nonlinear amplitude dependences of the neurite outgrowth in pheochromocytoma nerve cells due to ELF magnetic field exposure had been carried out in a nonuniform ac magnetic field. The nonuniformity entailed larger than expected variances in magnetic field magnitudes associated with specific levels of biological effects, thereby evoking a question about validity of the interpretations formulated for the case of a uniform field. In this work, we calculate the relative value of nonuniformity and deviations in ac magnetic field. It is shown that these factors do not affect the main conclusion in the original papers about the form of the amplitude dependence of the observed biological effect.     

Exposure to ELF magnetic field tuned to Zn inhibits growth of cancer cells

The effects of ELF alternating magnetic fields tuned to Zn(2+) on the growth of cancer cells with different status of p53 were investigated using a cell proliferation assay. Human cancer cells HeLa (cervix cancer, p53(+/+)), Saos-2 and Saos-2-His-273 (osteosarcoma, p53(-/-) and p53 His-273 mutant, respectively), H1299tTA and H1299tTA-His175 (lung carcinoma, p53(-/-) and p53 His-175 mutant), and normal human fibroblasts VH-10 (p53(+/+)) were used. Exposure parameters were calculated for the first harmonic of Zn(2+) based either on the magnetic parametric resonance (MPR) model of Lednev or the ion parametric resonance (IPR) model of Blanchard and Blackman. ELF exposure was for 72 and 96 h. The vertical alternating field was 20 Hz at amplitudes of either 38.7 or 77.4 microT (peaks, IPR or MPR, respectively). The vertical static magnetic field was 43 microT, and the horizontal static magnetic field was zeroed. Treatments of cells with PRIMA-1 and gamma-rays were used as positive controls. Growth inhibition was observed in cells after exposure to ELF at 38.7 microT. Inhibition of HeLa, VH-10, and Saos-2-His-273 cells was statistically significant, P=0.0003, 0.02, and 0.006, respectively. No consistent ELF effects following exposure 77.4 microT were seen. PRIMA-1 inhibited the growth of all cell lines with the strongest effect in mutant p53-carrying cell line H1299tTA-His175. The effects of gamma-rays were relatively weak, suggesting that the cell proliferation assay under conditions employed in this study is not very sensitive to apoptosis. In conclusion, ELF under conditions of exposure tuned to Zn(2+) according to the IPR model inhibited the growth of cancer and normal cells. No clear relationship of the observed growth inhibition to p53 status was found. Further experiments, using complementary techniques, are required to test whether p53 reactivation by ELF is feasible.     

Magnetic field influence on electrical properties of human blood measured by impedance spectroscopy

The impedance spectroscopy technique (IST) was used for studying the effect of a 0.5 T magnetic field on the electrical properties of whole human blood. A Solartron SI 1260 spectrometer was used to measure the impedance spectra of magnetic field exposed blood samples compared to non-exposed samples. An equivalent electrical circuit model, consisting in a resistance Rs in series with a parallel circuit formed by a constant phase element (CPE) and another resistance Rp, is proposed to fit the data in both cases. The experiment used 3 ml human blood samples from 160 healthy donors. A Wilcoxon matched pairs statistical test was applied to the data. The data analysis seems to show a statistically significant increase of the values of resistance Rp (Z = 5.06, P < 0.001) and capacitance CT (Z = 3.32, P < 0.001) of the blood exposed to magnetic field, by approximately 10.4% and 1.9%, respectively.     

Physical interactions of static magnetic fields with living tissues

Clinical magnetic resonance imaging (MRI) was introduced in the early 1980s and has become a widely accepted and heavily utilized medical technology. This technique requires that the patients being studied be exposed to an intense magnetic field of a strength not previously encountered on a wide scale by humans. Nonetheless, the technique has proved to be very safe and the vast majority of the scans have been performed without any evidence of injury to the patient. In this article the history of proposed interactions of magnetic fields with human tissues is briefly reviewed and the predictions of electromagnetic theory on the nature and strength of these interactions are described. The physical basis of the relative weakness of these interactions is attributed to the very low magnetic susceptibility of human tissues and the lack of any substantial amount of ferromagnetic material normally occurring in these tissues. The presence of ferromagnetic foreign bodies within patients, or in the vicinity of the scanner, represents a very great hazard that must be scrupulously avoided. As technology and experience advance, ever stronger magnetic field strengths are being brought into service to improve the capabilities of this imaging technology and the benefits to patients. It is imperative that vigilance be maintained as these higher field strengths are introduced into clinical practice to assure that the high degree of patient safety that has been associated with MRI is maintained.     

Static magnetic field effects on human subjects related to magnetic resonance imaging systems

Goal: This paper reviews recent studies evaluating human subjects for physiologic or neuro-cognitive function adverse effects resulting from exposure to static magnetic fields of magnetic resonance imaging systems.

Materials and Methods: The results of three studies are summarized. Two studies evaluated exposure to a maximum of 8 Tesla (T). The first series studied 25 normal human subjects’ sequential vital signs (heart rate, blood pressure, blood oxygenation, core temperature, ECG, respiratory rate) measured at different magnetic field strengths to a maximum of 8 T. A second series of 25 subjects were studied at 0.05 and 8 T (out and in the bore of the magnet), performing 12 different standardized neuro-psychological tests and auditory–motor reaction times. The subjects’ comments were recorded immediately following the study and after a three-month interval. The third study contained 17 subjects, placed near the bore of a 1.5 T magnet, and it used six different cognitive, cognitive–motor, or sensory tests.

Results: There were no clinically significant changes in the subjects’ physiologic measurements at 8 T. There was a slight increase in the systolic blood pressure with increasing magnetic field strength. There did not appear to be any adverse effect on the cognitive performance of the subjects at 8 T. A few subjects commented at the time of initial exposure on dizziness, metallic taste in the mouth, or discomfort related to the measurement instruments or the head coil. There were no adverse comments at 3 months. The 1.5 T study had two of the four neuro-behavioral domains exhibiting adverse effects (sensory and cognitive–motor).

Conclusions: These studies did not demonstrate any clinically relevant adverse effects on neuro-cognitive testing or vital sign changes. One short-term memory, one sensory, and one cognitive–motor test demonstrated adverse effects, but the significance is not clear.