Robert E. Thach

Invasive honeysuckle eradication reduces tick-borne disease risk by altering host dynamics

Despite the ubiquity of invasive organisms and their often deleterious effects on native flora and fauna, the consequences of biological invasions for human health and the ecological mechanisms through which they occur are rarely considered. Here we demonstrate that a widespread invasive shrub in North America, Amur honeysuckle (Lonicera maackii), increases human risk of exposure to ehrlichiosis, an emerging infectious disease caused by bacterial pathogens transmitted by the lone star tick (Amblyomma americanum). Using large-scale observational surveys in natural areas across the St. Louis, Missouri region, we found that white-tailed deer (Odocoileus virginianus), a preeminent tick host and pathogen reservoir, more frequently used areas invaded by honeysuckle. This habitat preference translated into considerably greater numbers of ticks infected with pathogens in honeysuckle-invaded areas relative to adjacent honeysuckle-uninvaded areas. We confirmed this biotic mechanism using an experimental removal of honeysuckle, which caused a decrease in deer activity and infected tick numbers, as well as a proportional shift in the blood meals of ticks away from deer. We conclude that disease risk is likely to be reduced when honeysuckle is eradicated, and suggest that management of biological invasions may help ameliorate the burden of vector-borne diseases on human health.

Role of the formylmethionine codon AUG in phasing translation of synthetic messenger RNA

The coding specificity of the triplets AUG and UUG was measured in the binding assay of Nirenberg & Leder (1964) , as a function of the magnesium ion and oligonucleotide concentration. AUG was extremely active in promoting the binding of formylmethionine-sRNA under all conditions tested, while UUG was active only at very high magnesium ion and oligonucleotide concentration. These results suggest that AUG is the correct formylmethionine codon. When the AUG codon is incorporated into longer polynucleotide chains, it suppresses the reading of codons which partially overlap its sequence, and promotes the reading of the adjacent 3′ codon. For example, ApUpG ( pU ) 15 ¯ stimulates the binding of Met- and Phe-sRNA, but not of Val-sRNA; in contrast, ApUpGpG ( pU ) 16 ¯ codes for Met- and Val-sRNA, whereas the binding of Phe-sRNA is reduced to a low level, comparable to oligo U. The triplets ACG, UAG and AAG do not show this phasing activity. AUG is capable of fixing the reading frame of the message regardless of its position within the polynucleotide chain. Thus ApApApUpG stimulates strongly the binding of Met-sRNA, but not of Lys-sRNA. On the other hand, ApApApCpG codes very well for Lys-sRNA. The phasing activity of AUG is a maximum at about 0·009 M -Mg2+ , and decreases sharply at higher concentrations, where the selection of a reading frame is made more or less at random. Since the initiation of polypeptide synthesis directed by natural messengers also occurs best at a low magnesium ion concentration, it is possible that the AUG phaser may be a part of the natural initiation mechanism.

Blood meal analysis to identify reservoir hosts for Amblyomma americanum ticks

Blood meal analysis identified white-tailed deer as hosts for ticks that carry zoonotic pathogens.

Involvement of Heme in the Degradation of Iron-regulatory Protein 2

Iron-regulatory proteins (IRPs) recognize and bind to specific RNA structures called iron-responsive elements. Mediation of these binding interactions by iron and iron-containing compounds regulates several post-transcriptional events relevant to iron metabolism. There are two known IRPs, IRP1 and IRP2, both of which can respond to iron fluxes in the cell. There is ample evidence that IRP1 is converted by iron to cytoplasmic aconitase in vivo. It has also been shown that, under certain conditions, a significant fraction of IRP1 is degraded in cells exposed to iron or heme. Studies have shown that the degradation of IRP1 that is induced by iron can be inhibited by either desferrioxamine mesylate (an iron chelator) or succinyl acetone (an inhibitor of heme synthesis), whereas the degradation induced by heme cannot. This suggests that heme rather than iron is responsible for this degradation. Several laboratories have shown that IRP2 is also degraded in cells treated with iron salts. We now show evidence suggesting that this IRP2 degradation may be mediated by heme. Thus, in experiments analogous to those used previously to study IRP1, we find that IRP2 is degraded in rabbit fibroblast cells exposed to heme or iron salts. However, as shown earlier with IRP1, both desferrioxamine mesylate and succinyl acetone will inhibit the degradation of IRP2 induced by iron but not that induced by heme.

Damage to Biological Samples Caused by the Electron Beam during Electron Microscopy

A new method has been developed which measures directly the beam damage suffered by biological specimens in the electron microscope. This method involves the use of radioautography to measure specific radioactivity of labeled specimens, either exposed or unexposed to the beam. Using this technique, it has been found that macromolecular samples such as ribosomes and R17 virions are severely damaged during standard electron microscopic operations: from 15 to 40% of the mass of the sample may be lost in a 30 sec exposure to the beam.

Mechanism of viral replication. I. Structure of replication complexes of R17 bacteriophage.

The replication of R17 bacteriophage in Escherichia coli MRE-600 cells was investigated using a new electron microscopic technique. The structures of replicating ribonucleoprotein complexes, as well as of purified replicative intermediate and replicative form, were studied. These structures are identical to those predicted by the model of Weissmann et al. (1968). From this it may be concluded that replication proceeds through essentially single-stranded inter-mediates and that double-stranded structures are either by-products or artifacts.

[26] Isolation and purification of initiation factors f1 and f2

Publisher Summary Initiation of protein synthesis in the bacterium Escherichia coli involves three protein factors, f1, f2, and f3, and when, synthetic oligonucleotides containing the initiator codon AUG are employed as messenger RNA, f1 and f2 alone stimulate protein synthesis maximally. This chapter describes a simple and convenient way to prepare pure f1 and f2 and discusses the preparation of a radioactive factor, f1. Initiation factors f1 and f2 stimulate the binding of fMet-tRNA to 30 S ribosomes in the presence of GTP and synthetic oligonucleotides containing the codon AUG. The 30 S initiation complex then joins a 50 S ribosomal subunit to produce the 70 S complex. Factor f1 is released from the complex during the junction step; while, the fate of f2 is not yet known. The degree of stimulation of fMet-tRNA binding by f1 depends on the temperature of incubation, and excessive amounts of f2, fMet-tRNA or ribosomes seem to reduce the degree of f1 stimulation. Fractionation of crude initiation factors employs phosphocellulose for the first step in a purification scheme because of its high capacity, and results in the complete separation of f1 and f2 activities, especially a 25-fold purification of f1.

Specificity of the induction of ferritin synthesis by hemin

We have previously reported that hemin derepresses ferritin mRNA translation in vitro. As noted earlier, pre-incubation of a 90 kDa ferritin repressor protein (FRP) with hemin prevented subsequent repression of ferritin synthesis in a wheat germ extract. The significance of this observation has been investigated further. Evidence is presented here that this inactivation of FRP is temperature dependent. Neither FeCl3, Fe3+ chelated with EDTA, nor protoporphyrin IX caused significant inactivation of FRP under comparable conditions, whereas Zn2(+)-protoporphyrin IX produced an intermediate degree of inhibition. The presence of a glutathione redox buffer (GSB), which was previously shown to minimize non-specific side-effects of hemin, was not necessary for the derepression reaction. Inclusion of mannitol, a free radical scavenger, did not alter the inactivation caused by hemin. Calculation of the expected ratio of hemin monomers to dimers suggests that the active species is the monomer.

Binding of formylmethionyl-tRNA and aminoacyl-tRNA to ribosomes.

Inhibition of aminoacyl-tRNA binding by the purified initiation factors f1 and f2 could be regarded as a mechanism for ensuring that only F-met-tRNA binds initially to the entry site, thereby preventing random or non-specific binding by aminoacyl-tRNA. This inhibition would be relieved by the hydrolysis of the GTP molecule normally involved in the binding of F-met-tRNA.

Effects of the Ferritin Open Reading Frame on Translational Induction by Iron.

Publisher Summary Ferritin—a multimeric iron-storage protein—is evolutionarily conserved from prokaryotes to eukaryotes. It has been proposed that ferritin acts both to store iron for later use and to defend the cytosol against the generation of potentially toxic free radicals via Fenton oxygen chemistry. The regulation of ferritin synthesis is tightly coupled to changes in intracellular iron concentrations in both cultured cells and model vertebrate organisms. Cells in vertebrate organisms respond to excess iron chiefly by post-transcriptional mechanisms. Factors that modulate the effect of iron on ferritin expression are cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), nitric oxide (NO), and oxidative agents. Cytokines such as IL-1β modulate the expression of ferritin in the presence of iron. The regulation is accomplished both by transcriptional and translational mechanisms. There have been numerous reports demonstrating that the effect of ferritin iron-responsive element (IRE) on the iron inducibility of heterologous open reading frames is not as potent as on that of endogenous ferritin mRNAs. The difference in inducibility is accounted for by sequences downstream from the IRE and probably within the ferritin open reading frame (ORF). However, mRNAs that contain the ferritin ORF but lack IRE have little or no iron inducibility. This indicates a requirement for iron-responsive protein (IRP) to bind the IRE, and this interaction serves as the fundamental iron-responsive component of the translational regulatory system for ferritin.