Transduction and transfection of difficult-to-transfect cells: Systematic attempts for the transfection of protozoa Leishmania

Cell-penetrating peptides (CPPs) are used to internalize different cargoes, including DNA, into live mammalian and plant cells. Despite many cells being easily transfected with this approach, other cells are rather “difficult” or “hard to transfect,” including protist cells of the genus Leishmania. Based on our previous results in successfully internalizing proteins into Leishmania tarentolae cells, we used single CPPs and three different DNA-binding proteins to form protein-like complexes with plasmids covered with CPPs. We attempted magnetofection, electroporation, and transfection using a number of commercially available detergents. While complex formation with negatively charged DNA required substantially higher amounts of CPPs than those necessary for mostly neutral proteins, the cytotoxicity of the required amounts of CPPs and auxiliaries was thoroughly studied. We found that Leishmania cells were indeed susceptible to high concentrations of some CPPs and auxiliaries, although in a different manner compared with that for mammalian cells. The lack of successful transfections implies the necessity to accept certain general limitations regarding DNA internalization into difficult-to-transfect cells. Only electroporation allowed reproducible internalization of large and rigid plasmid DNA molecules through electrically disturbed extended membrane areas, known as permeable membrane macrodomains.     

Effects of 45-Hz magnetic fields on the functional state of the human brain

The influence of sinusoidal 45-Hz magnetic fields on the brain functions of 20 volunteers was investigated in a double-blind study using spectral analysis of EEG and measurements of Omega potentials and reaction time (RT). The field strength was 1,000 A/m (1.26 mT) and the duration of exposure was 1 h. Ten volunteers were exposed to a continuous field and ten received an intermittent exposure (1 s on/1 s off). Each person received one real and one sham exposure. One half of the volunteers got the real exposure first and the sham treatment after at least 24 h. For the rest, the sequence was inverse. The measurements of EEG, omega potentials and RT were performed before and after each exposure. Several statistically significant changes were observed, most of them after intermittent exposure. In the EEG, an increase of alpha (7.6–13.9 Hz) activity and a decrease of delta (1.5–3.9 Hz) activity were observed. β waves (14.2–20 Hz) increased in the frontal derivations as did the total power in occipital derivations. The mean and peak frequencies of EEG increased mainly in the frontal derivations. No direct effects on RT were seen. Learning to perform the RT test (decrease of RT in repeated trials), however, seemed to be affected by the exposure. The persons who received real exposure first learned more slowly than those who got sham exposure first. Further experiments are necessary to confirm the findings and for understanding the mechanisms of the effects. © 1993 Wiley-Liss. Inc.     

Pair potentials for protein folding: choice of reference states and sensitivity of predicted native states to variations in the interaction schemes

We examine the similarities and differences between two widely used knowledge-based potentials, which are expressed as contact matrices (consisting of 210 elements) that gives a scale for interaction energies between the naturally occurring amino acid residues. These are the Miyazawa-Jernigan contact interaction matrix M and the potential matrix S derived by Skolnick J et al., 1997, Protein Sci 6:676-688. Although the correlation between the two matrices is good, there is a relatively large dispersion between the elements. We show that when Thr is chosen as a reference solvent within the Miyazawa and Jernigan scheme, the dispersion between the M and S matrices is reduced. The resulting interaction matrix B gives hydrophobicities that are in very good agreement with experiment. The small dispersion between the S and B matrices, which arises due to differing reference states, is shown to have dramatic effect on the predicted native states of lattice models of proteins. These findings and other arguments are used to suggest that for reliable predictions of protein structures, pairwise additive potentials are not sufficient. We also establish that optimized protein sequences can tolerate relatively large random errors in the pair potentials. We conjecture that three body interaction may be needed to predict the folds of proteins in a reliable manner.     

Changes in EEG Power Spectra During Biofeedback of Slow Cortical Potentials in Epilepsy

The goal of the study was to explore parallel changes in EEG spectral frequencies during biofeedback of slow cortical potentials (SCPs) in epilepsy patients. Thirty-four patients with intractable focal epilepsy participated in 35 sessions of SCP self-regulation training. The spectral analysis was carried out for the EEG recorded at the same electrode site (Cz) that was used for SCP feedback. The most prominent effect was the increase in the 2 power (6.0–7.9 Hz) and the relative power decrement in all other frequency bands (particularly 1, 2, and 2) in transfer trials (i.e., where patients controlled their SCPs without continuous feedback) compared with feedback trials. In the second half of the training course (i.e., sessions 21–35) larger power values in the , , and bands were found when patients were required to produce positive versus negative SCP shifts. Both across-subject and across-session (within-subject) correlations between spectral EEG parameters, on the one hand, and SCP data, on the other hand, were low and inconsistent, contrary to high and stable correlations between different spectral variables. This fact, as well as the lack of considerable task-dependent effects during the first part of training, indicates that learned SCP shifts did not directly lead to the specific dynamics of the EEG power spectra. Rather, these dynamics were related to nonspecific changes in patients' brain state.     

A Novel Rieske Iron-Sulfur Protein from the Hyperthermophilic Crenarchaeon Pyrobaculum aerophilum: Sequencing of the Gene, Expression in E. coli and Characterization of the Protein

The crenarchaeon Pyrobaculum aerophilum is with an optimalgrowth temperature of 100 °C one of the most thermophilic organisms knownto possess an aerobic respiratory chain. The analysis of DNA sequences fromthe Pyrobaculum genome project lead to the identification of an openreading frame potentially coding for a Rieske iron-sulfur protein. Thecomplete gene (named parR) was cloned and sequenced. The deducedamino acid sequence displays unusual amino acid exchanges and a so farunknown sequence insertion. The N-terminus shows similarities to bacterialsignal sequences. Several forms of the gene were expressed in E.coli in order to verify the classification as a Rieske protein and tofacilitate biophysical studies. Soluble, thermo-stable proteins withcorrectly inserted iron-sulfur clusters were expressed from two versions ofthe gene. The 1–23 truncated holo-protein is redox active. Itdisplays the typical spectroscopic properties of a Rieske protein. The redoxpotential was determined to be +215 mV at pH 6.5 and is pH dependentabove pH 7.5 revealing the influence of two protonation equilibria with pKavalues of 8.1 and 9.8. Phylogenetic analysis demonstrates that the parRprotein clusters together with the two other available archaeal Rieskesequences from Sulfolobus on a separate branch of the phylogenetictree apart from the proteins from thermophilic bacteria like Aquifexand Thermus.     

Seasonal patterns of tissue water relations in three Mediterranean shrubs co‐occurring at a natural CO2 spring

Seasonal changes in tissue water relations of Erica arborea L., Myrtus communis L. and Juniperus communis L., grown in a Mediterranean environment, were analysed under field conditions over a 12 month period by comparing plants grown in the proximity of a natural CO₂ spring (about 700 μ mol mol ^{? 1} atmospheric CO₂ concentration, [CO₂]) with plants in ambient conditions. Tissue water relations varied in response to changes in water availability, but the seasonal course of tissue water relations parameters was also related to ontogeny. Tissue water relations of these co‐occurring shrubs were not alike. Osmotic potentials and saturated mass/dry mass ratio were lowest during peak drought stress periods. Diurnal changes in osmotic potential at the point of turgor loss were least early in the season, maximal in mid‐season, and decreased again in autumn. Turgor potentials decreased as drought progressed and were highest in late fall and mid‐winter. Symplastic water fraction was highest in mid‐spring for E. arborea and M. communis and decreased during the summer, while the opposite was observed for J. communis. Common to all species, under elevated [CO₂], was an increase of turgor pressure, particularly during the summer months. Other parameters showed species‐specific responses to long‐term elevated [CO₂]. In particular, exposure to elevated [CO₂] increased osmotic potentials in E. arborea under drought, while the opposite was the case for J. communis. Site differences in predawn to midday shifts were not strong in any of the species. Differences in tissue water relations suggest that the coexistence of these shrubs in the same environment with similar water availability are partially based on differential water relations strategies and water use patterns. Regardless of the mechanisms, growth of these shrubs in elevated [CO₂] may be either less, similarly or more affected by drought stress than plants in ambient [CO₂] depending on the species and season.     

Molecular recognition of aldehydes by aldehyde dehydrogenase and mechanism of nucleophile activation

Experimental structural data on the state of substrates bound to class 3 Aldehyde Dehydrogenases (ALDH3A1) is currently unknown. We have utilized molecular mechanics (MM) simulations, in conjunction with new force field parameters for aldehydes, to study the atomic details of benzaldehyde binding to ALDH3A1. Our results indicate that while the nucleophilic Cys243 must be in the neutral state to form what are commonly called near-attack conformers (NACs), these structures do not correlate with increased complexation energy calculated with the MM-Generalized Born Molecular Volume (GBMV) method. The negatively charged Cys243 (thiolate form) of ALDH3A1 also binds benzaldehyde in a stable conformation but in this complex the sulfur of Cys243 is oriented away from benzaldehyde yet yields the most favorable MM-GBMV complexation energy. The identity of the general base, Glu209 or Glu333, in ALDHs remains uncertain. The MM simulations reveal structural and possible functional roles for both Glu209 and Glu333. Structures from the MM simulations that would support either glutamate residue as the general base were further examined with Hybrid Quantum Mechanical (QM)/MM simulations. These simulations show that, with the PM3/OPLS potential, Glu209 must go through a step-wise mechanism to activate Cys243 through an intervening water molecule while Glu333 can go through a more favorable concerted mechanism for the same activation process.     

Relationships between amino acid sequence and backbone torsion angle preferences

Statistical averages and correlations for backbone torsion angles of chymotrypsin inhibitor 2 are calculated by using the Rotational Isomeric States model of chain statistics. Statistical weights of torsional states of phipsi pairs, needed for the statistics of the full chain, are obtained in two different ways: 1) by using knowledge-based pairwise dependent phipsi energy maps from Protein Data Bank (PDB) and 2) by collecting torsion angle data from a large number of random coil configurations of an all-atom protein model with volume exclusion. Results obtained by using PDB data show strong correlations between adjacent torsion angle pairs belonging to both the same and different residues. These correlations favor the choice of the native-state torsion angles, and they are strongly context dependent, determined by the specific amino acid sequence of the protein. Excluded volume or steric clashes, only, do not introduce context-dependent phipsi correlations into the chain that would affect the choice of native-state torsional angles.     

Measurements of protein sequence-structure correlations

Correlations between protein structures and amino acid sequences are widely used for protein structure prediction. For example, secondary structure predictors generally use correlations between a secondary structure sequence and corresponding primary structure sequence, whereas threading algorithms and similar tertiary structure predictors typically incorporate interresidue contact potentials. To investigate the relative importance of these sequence-structure interactions, we measured the mutual information among the primary structure, secondary structure and side-chain surface exposure, both for adjacent residues along the amino acid sequence and for tertiary structure contacts between residues distantly separated along the backbone. We found that local interactions along the amino acid chain are far more important than non-local contacts and that correlations between proximate amino acids are essentially uninformative. This suggests that knowledge-based contact potentials may be less important for structure predication than is generally believed.     

Crystallographic studies of V44 mutants of Clostridium pasteurianum rubredoxin: effects of side-chain size on reduction potential

Understanding the structural origins of differences in reduction potentials is crucial to understanding how various electron transfer proteins modulate their reduction potentials and how they evolve for diverse functional roles. Here, the high-resolution structures of several Clostridium pasteurianum rubredoxin (Cp Rd) variants with changes in the vicinity of the redox site are reported in order to increase this understanding. Our crystal structures of [V44L] (at 1.8 A resolution), [V44A] (1.6 A), [V44G] (2.0 A) and [V44A, G45P] (1.5 A) Rd (all in their oxidized states) show that there is a gradual decrease in the distance between Fe and the amide nitrogen of residue 44 upon reduction in the size of the side chain of residue 44; the decrease occurs from leucine to valine, alanine or glycine and is accompanied by a gradual increase in their reduction potentials. Mutation of Cp Rd at position 44 also changes the hydrogen-bond distance between the amide nitrogen of residue 44 and the sulfur of cysteine 42 in a size-dependent manner. Our results suggest that residue 44 is an important determinant of Rd reduction potential in a manner dictated by side-chain size. Along with the electric dipole moment of the 43-44 peptide bond and the 44-42 NH--S type hydrogen bond, a modulation mechanism for solvent accessibility through residue 41 might regulate the redox reaction of the Rds.