Don Jones

Microwave radiation can alter protein conformation without bulk heating

Exposure to microwave radiation enhances the aggregation of bovine serum albumin in vitro in a time‐ and temperature‐dependent manner. Microwave radiation also promotes amyloid fibril formation by bovine insulin at 60°C. These alterations in protein conformation are not accompanied by measurable temperature changes, consistent with estimates from field modelling of the specific absorbed radiation (15–20 mW kg−1). Limited denaturation of cellular proteins could explain our previous observation that modest heat‐shock responses are induced by microwave exposure in Caenorhabditis elegans. We also show that heat‐shock responses both to heat and microwaves are suppressed after RNA interference ablating heat‐shock factor function.

Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins

BackgroundThe eukaryotic ubiquitin-conjugation system sets the turnover rate of many proteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substrate recognition, respectively. There are also ubiquitin-like proteins with distinct functions, which require their own E1s and E2s for attachment. We describe the results of RNA interference (RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans. We also present a phylogenetic analysis of UBCs.ResultsThe C. elegans genome encodes 20 UBCs and three ubiquitin E2 variant proteins. RNAi shows that only four UBCs are essential for embryogenesis: LET-70 (UBC-2), a functional homolog of yeast Ubc4/5p, UBC-9, an ortholog of yeast Ubc9p, which transfers the ubiquitin-like modifier SUMO, UBC-12, an ortholog of yeast Ubc12p, which transfers the ubiquitin-like modifier Rub1/Nedd8, and UBC-14, an ortholog of Drosophila Courtless. RNAi of ubc-20, an ortholog of yeast UBC1, results in a low frequency of arrested larval development. A phylogenetic analysis of C. elegans, Drosophila and human UBCs shows that this protein family can be divided into 18 groups, 13 of which include members from all three species. The activating enzymes and the ubiquitin-like proteins NED-8 and SUMO are required for embryogenesis.ConclusionsThe number of UBC genes appears to increase with developmental complexity, and our results suggest functional overlap in many of these enzymes. The ubiquitin-like proteins NED-8 and SUMO and their corresponding activating enzymes are required for embryogenesis.

Cell biology: Non-thermal heat-shock response to microwaves

Exposure limits set for microwave radiation assume that any biological effects result from tissue heating: non-thermal effects have been reported but remain controversial. We show here that prolonged exposure to low-intensity microwave fields can induce heat-shock responses in the soil nematode Caenorhabditis elegans. This effect appears to be non-thermal, suggesting that current exposure limits set for microwave equipment may need to be reconsidered.

Feeding is inhibited by sublethal concentrations of toxicants and by heat stress in the nematode Caenorhabditis elegans: relationship to the cellular stress response.

We report that the free-living nematode Caenorhabditis elegans can respond to a variety of stressors (compounds known to induce the production of cellular stress proteins in model biological systems), by ceasing pharyngeal pumping. This phenomenon results in both a reduction in intake of the stressor and a cessation of feeding. The effect of stressors can therefore be conveniently assayed by monitoring the decrease in the density of the bacterial food in liquid cultures of nematodes. A great range of stressors induced this response including alcohols, heavy metals, sulfhydryl-reactive compounds, salicylate, and heat. For several of these stressors, inhibition of pharyngeal pumping occurred at stressor concentrations below the threshold required for the induction of the 16-kDa heat shock proteins. Salicylate, which did not induce 16-kDa heat shock proteins at any concentration, nevertheless inhibited pharyngeal pumping. Heat was also inhibitory, at a temperature where 16-kDa heat shock protein production was near maximal. Some compounds caused only a partial inhibition of feeding while with others the effect was complete. Upon removal of the stressor, the nematodes resumed pharyngeal pumping with a residual inhibitory effect that depended on the concentration and type of stressor that had been applied. A number of C. elegans neurosensory mutant strains also exhibited a cessation of pharyngeal pumping when exposed to stressors suggesting that the mechanism underlying this inhibition was not entirely neurosensory and may be intrinsic to the pharynx. In C. elegans and other invertebrates, stress-induced inhibition of feeding may be an important survival mechanism that limits the intake of toxic solutes.

Transgenic strains of the nematode C. elegans in biomonitoring and toxicology: effects of captan and related compounds on the stress response.

The fungicide, captan, induces a cellular stress response in the soil nematode Caenorhabditis elegans. Transgenic C, elegans, which produce beta-galactosidase as a surrogate stress protein, reveal that captan-induced stress is localized mainly to muscle cells of the pharynx. The stress response is elicited by captan concentrations above 5 ppm and occurs within five hours of the initial exposure to the fungicide. Higher concentrations of captan, up to the solubility limit, increase the intensity of the response. Adult nematodes are significantly more sensitive to captan than are larvae. Captan also inhibits feeding in C. elegans, and nematodes exposed to captan rapidly cease muscular contractions in the pharynx. Stress induction and feeding inhibition are also caused by the related fungicides, captafol and folpet, but not by the parent compounds, phthalimide and tetrahydrophthalimide. The inhibition of feeding caused by compounds which elicit the cellular stress response may be an important survival mechanism for C, elegans.

Transgenic Caenorhabditis elegans strains as biosensors

Toxicity bioassays rely largely on lethality measurements. Such assays are generally lengthy and expensive, and provide little information on mechanisms of toxicity. A desire to understand the mechanisms by which cells respond to physical and chemical stresses has led to interest in measuring stress proteins as toxicological endpoints. Transgenic strains of the nematode Caenorhabditis elegans that carry a reporter enzyme under control of a stress-inducible promoter have been created. The reporter is easily quantified in intact nematodes, and it responds to a wide range of chemical stressors. Therefore, transgenic C. elegans can provide the basis for a wide range of quick, simple and informative bioassays.

UbiA, the major polyubiquitin locus in Caenorhabditis elegans, has unusual structural features and is constitutively expressed

Ubiquitin is a multifunctional 76-amino-acid protein which plays critical roles in many aspects of cellular metabolism. In Caenorhabditis elegans, the major source of ubiquitin RNA is the polyubiquitin locus, UbiA. UbiA is transcribed as a polycistronic mRNA which contains 11 tandem repeats of ubiquitin sequence and possesses a 2-amino-acid carboxy-terminal extension on the final repeat. The UbiA locus possesses several unusual features not seen in the ubiquitin genes of other organisms studied to date. Mature UbiA mRNA acquires a 22-nucleotide leader sequence via a trans-splicing reaction involving a 100-nucleotide splice leader RNA derived from a different chromosome. UbiA is also unique among known polyubiquitin genes in containing four cis-spliced introns within its coding sequence. Thus, UbiA is one of a small class of genes found in higher eucaryotes whose heterogeneous nuclear RNA undergoes both cis and trans splicing. The putative promoter region of UbiA contains a number of potential regulatory elements: (i) a cytosine-rich block, (ii) two sequences resembling the heat shock regulatory element, and (iii) a palindromic sequence with homology to the DNA-binding site of the mammalian steroid hormone receptor. The expression of the UbiA gene has been studied under various heat shock conditions and has been monitored during larval moulting and throughout the major stages of development. These studies indicate that the expression of the UbiA gene is not inducible by acute or chronic heat shock and does not appear to be under nutritional or developmental regulation in C. elegans.

The magnetic behavior of clusters of the type [M3O(RCOO)6]+. An isotropic model involving dynamic distortions

A dynamic distortion model for the magnetic behavior of clusters which contain equilateral triangles of exchange coupled spins is derived using the assumptions of (i) isotropic coupling and (ii) a linear relationship between the coupling constants and interatomic distances. The model is shown to be consistent with experimental data for complexes of the form [M3O(RCOO)6]+, M=Fe,Cr. The spectrum of magnetic states resulting from the dynamic distortion model is found to be very similar to that of the isoceles triangle model of Kambe, but the present model is physically more satisfactory.

An isotropic exchange model for the magnetic behavior of tetranuclear copper complexes of the type Cu4OX6L4

A theory for the magnetic behavior of distorted tetrahedra of magnetically coupled spin 1/2 atoms is derived using the assumptions of (i) isotropic coupling; and (ii) a linear relationship between the coupling constants and interatomic distances. It is found that the theory provides the basis for a model of the magnetic behavior of complexes of the form Cu4OX6L4 which involves dynamic or fluxional distortions. The new model is found to be in several respects more satisfactory than either the anisotropic or the intercluster exchange models proposed previously.

Microwave radiation induces a heat-shock response and enhances growth in the nematode Caenorhabditis elegans

This paper shows that prolonged (overnight) exposure to continuous microwave fields (750 MHz, 0.5 W) can induce both a heat-shock response and enhanced growth in the nematode worm Caenorhabditis elegans. Exposures were conducted in a TEM cell with matched load, producing an E-field of approximately 45 V m/sup -1/ at the center (where test worms are placed). Biomonitoring of heat-shock responses has been simplified by using two transgenic strains (PC72 and PC161), which both carry stress-inducible reporter constructs, respectively, placing lacZ (/spl beta/-galactosidase) and lacZ plus green fluorescent protein expression under the control of C. elegans hsp16-1 promoters. In situ localization of reporter expression reveals a minority of test worms, which respond strongly to microwave exposure. Enzyme activity measurements average these reporter responses across many thousands of individual worms, giving a reliable indication of the overall stress imposed on a population. The temperature profile of reporter responses induced by microwave exposure parallels that induced in controls by heat alone, but is displaced down the temperature scale by some 3/spl deg/C. Length measurements were conducted at intervals in synchronized C. elegans cultures seeded with L1 larvae. Using pooled data from nine separate runs, growth was stimulated by 8.5% after overnight microwave exposure (relative to controls), and this disparity increased to 11% after 24 h of further growth without irradiation. Both heat-shock responses and increased growth would be consistent with a modest increase in temperature, raising the possibility that microwave exposure might cause limited heating in this system. However, there is no detectable rise in the temperature of either medium or worms during overnight exposure under these conditions, discounting both generalized and localized (worm-specific) heating effects. It is concluded that both growth and heat-shock responses are induced by microwave exposure through one or more nonthermal routes.