Redox reactivity of animal apoferritins and apoheteropolymers assembled from recombinant heavy and light human chain ferritins

The redox reactivities of air-oxidized apo horse spleen ferritin (HoSF) and apo rat liver ferritin (RaF) were examined by microcoulometry and reductive optical titrations. Microcoulometry on several independent lots of commercial HoSF revealed two distinct types of redox activity: one requiring 3-4 electrons and one requiring 6-7 electrons for full reduction of the protein shell. ApoRaF required 8-9 electrons to fully reduce the oxidized form. Reductive optical titrations confirmed the microcoulometric reduction stoichiometry and, in addition, showed that the spectra of both oxidized and reduced apoHoSF were distinct and possessed absorbances tailing into the visible region. The redox reactivity of both apoRaF and apoHoSF correlated with their H-subunit composition. Identical microcoulometric and optical experiments were conducted with recombinant apo human liver heavy (rHuHF) and light (rHuLF) ferritins, but neither was redox-active. These results suggest that the redox reactivity of native ferritins is due to their heteropolymeric nature. This was confirmed by mixing various proportions of rHuHF and rHuLF, dissociating the 24-mers into individual subunits with guanidine hydrochloride at pH 3.5, and renaturing to form heteropolymeric 24-mers. Microcoulometric measurements of these apoheteropolymers reassembled in vitro showed that they were redox-active like their native apoheteropolymer counterparts. The redox activity of these apoheteropolymers increased with H-subunit composition, reached a maximum near 12 H- and 12 L-subunits, and then declined to zero with increasing L-subunit composition. The decline in redox reactivity at high L-subunit concentrations indicates that both H- and L-subunits are involved in forming the observed redox centers. Apoheteropolymers formed from rHuLF and W93F (an H-chain mutant) were redox-inactive, suggesting that the conserved tryptophan is necessary for redox center formation.     

In vivo selection of RNA-binding peptides from combinatorial libraries.

We have used a two step procedure to identify peptides that bind strongly to the Rev-response element (RRE) of HIV. In the first step, RRE-binding peptides were screened from a combinatorial peptide library generated by "randomization" using a small subset of the 20 amino acids. In the second step, one such RRE-binding peptide, RSG-1, was "evolved" into an even stronger RRE-binding peptide using a codon-based mutagenesis procedure. After 2 rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity than wild-type Rev peptide.     

Biological effects of power frequency magnetic fields: Neurochemical and toxicological changes in developing chick embryos

BACKGROUND: There are several reports that indicate a linkage between exposure to power frequency (50 - 60 Hz) magnetic fields with abnormalities in the early embryonic development of the chicken. The present study was designed to understand whether power frequency electromagnetic fields could act as an environmental insult and invoke any neurochemical or toxicological changes in developing chick embryo model. METHODS: Fertilized chicken eggs were subjected to continuous exposure to magnetic fields (50 Hz) of varying intensities (5, 50 or 100 microT) for a period of up to 15 days. The embryos were taken out of the eggs on day 5, day 10 and day 15. Neurochemical (norepinephrine and 5-hydroxytryptamine) and amino acid (tyrosine, glutamine and tryptophan) contents were measured, along with an assay of the enzyme glutamine synthetase in the brain. Preliminary toxicological investigations were carried out based on aminotransferases (AST and ALT) and lactate dehydrogenase activities in the whole embryo as well as in the liver. RESULTS: The study revealed that there was a significant increase (p < 0.01 and p < 0.001) in the level of norepinephrine accompanied by a significant decrease (p < 0.01 and p < 0.001) in the tyrosine content in the brain on day 15 following exposure to 5, 50 and 100 microT magnetic fields. There was a significant increase (p < 0.001) in glutamine synthetase activity resulting in the significantly enhanced (p < 0.001) level of glutamine in the brain on day 15 (for 100 microT only). The possible mechanisms for these alterations are discussed. Further, magnetic fields had no effect on the levels of tryptophan and 5-hydroxytryptamine in the brain. Similarly, there was no effect on the activity of either aminotransferases or lactate dehydrogenase in the whole embryo or liver due to magnetic field exposure. CONCLUSIONS: Based on these studies we conclude that magnetic field-induced changes in norepinephrine levels might help explain alterations in the circadian rhythm, observed during magnetic field stress. Also, the enhanced level of glutamine can act as a contributing factor for developmental abnormalities.     

Recognition of nucleic acid bases and base-pairs by hydrogen bonding to amino acid side-chains

Sequence-specific protein-nucleic acid recognition is determined, in part, by hydrogen bonding interactions between amino acid side-chains and nucleotide bases. To examine the repertoire of possible interactions, we have calculated geometrically plausible arrangements in which amino acids hydrogen bond to unpaired bases, such as those found in RNA bulges and loops, or to the 53 possible RNA base-pairs. We find 32 possible interactions that involve two or more hydrogen bonds to the six unpaired bases (including protonated A and C), 17 of which have been observed. We find 186 "spanning" interactions to base-pairs in which the amino acid hydrogen bonds to both bases, in principle allowing particular base-pairs to be selectively targeted, and nine of these have been observed. Four calculated interactions span the Watson-Crick pairs and 15 span the G:U wobble pair, including two interesting arrangements with three hydrogen bonds to the Arg guanidinum group that have not yet been observed. The inherent donor-acceptor arrangements of the bases support many possible interactions to Asn (or Gln) and Ser (or Thr or Tyr), few interactions to Asp (or Glu) even though several already have been observed, and interactions to U (or T) only if the base is in an unpaired context, as also observed in several cases. This study highlights how complementary arrangements of donors and acceptors can contribute to base-specific recognition of RNA, predicts interactions not yet observed, and provides tools to analyze proposed contacts or design novel interactions.     

Sequence and structure space of RNA-binding peptides

Studies of RNA-binding peptides, and recent combinatorial library experiments in particular, have demonstrated that diverse peptide sequences and structures can be used to recognize specific RNA sites. The identification of large numbers of sequences capable of binding to a particular site has provided extensive phylogenetic information used to deduce basic principles of recognition. The high frequency at which RNA-binding peptides are found in large sequence libraries suggests plausible routes to evolve sequence-specific binders, facilitating the design of new binding molecules and perhaps reflecting characteristics of natural evolution.