Retrograde transport of toxins across the endoplasmic reticulum membrane.

Several protein toxins, including the A chain of the plant protein ricin (RTA), enter mammalian cells by endocytosis and catalytically modify cellular components to disrupt essential cellular processes. In the case of ricin, the process inhibited is protein synthesis. In order to reach their cytosolic substrates, several toxins undergo retrograde transport to the ER (endoplasmic reticulum) before translocating across the ER membrane. To achieve this export, these toxins exploit the ERAD (ER-associated protein degradation) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates in order to intoxicate the cell. Toxins that translocate from the ER have an unusually low lysine content that reduces the likelihood of ubiquitination and ubiquitin-mediated proteasomal degradation. We have changed the two lysyl residues normally present in RTA to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect on the protein, suggesting that the endogenous lysyl residues are not sites for ubiquitin attachment. However, when four additional lysyl residues were introduced into RTA in a way that did not compromise the activity, structure or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasomal inhibitor lactacystin increased the cytotoxicity of the lysine-enriched RTA to a level approaching that of wild-type RTA.

Gene disruption in Schizosaccharomyces pombe using a temperature-sensitive Ura4p.

We have generated a temperature-sensitive form of the Ura4p protein from the fission yeast Schizosaccharomyces pombe. A single T-to-C mutation at nucleotide 782 (relative to the initiator ATG codon of ura4) changes the leucine residue at position 261 in Ura4p to a proline. The mutant Ura4p(ts) supports growth at 30 degrees C but is unable to allow growth at 37 degrees C in the absence of uracil when a single copy of the gene is integrated into the host chromosome. Using the ura4(ts) cassette for gene replacements simplifies the identification of transformants in which the disruption construct has undergone homologous integration into the host chromosome, as these individuals contain a single copy of the ura4(ts) gene and fail to grow when replicated to 37 degrees C in the absence of uracil.

Initial training as a stimulus for optimal physical fitness in firemen.

In this investigation the physical fitness of 34 recruits to the UK Fire Service was assessed before and after their initial training (tests 1-2) and on three occasions (tests 3-5) during the first 18 months of their service (n = 34 + 6 = 40). The initial training (test 2-test 1) resulted in an increase in maximum aerobic power (11% p < 0.05), body mass (2 kg, p < 0.001), lean body mass (2.02 kg, p < 0.001), grip and lifting strengths (p < 0.001), and in calf girth (p < 0.05). But no change in waist girth, maximum anaerobic power or measures of lung function was noted. There was evidence therefore that the men became physically fitter. In the first eighteen months of their service, subjects' maximum oxygen consumption declined to pre-training levels, while body mass continued to increase with a decrease in lean body mass (p < 0.001). Strength remained unaltered or tended to fall with a decrease in calf girth (p < 0.05). These changes reflected a return towards pre-training physical fitness levels. It is suggested that the initial training, while ill-matched to the firemen's habitual activity, produced levels of physical fitness which may approximate more closely to the infrequent peak requirements experienced by the men during actual fire fighting. Consequently it is concluded that the physical training during service was insufficiently intense and that a more effective programme could be designed to maintain an appropriate level of physical fitness.