Inhibition of ubiquitin-protein ligase (E3) by mono- and bifunctional phenylarsenoxides. Evidence for essential vicinal thiols and a proximal nucleophile.

Trivalent arsenoxides bind to vicinal thiol groups of proteins. We showed previously that the simplest trivalent arsenoxide, inorganic arsenite, inhibits ubiquitin-dependent protein degradation in rabbit reticulocyte lysate (Klemperer, N.S., and Pickart, C.M. (1989) J. Biol. Chem. 264, 19245-19242). We now show that, relative to arsenite, phenylarsenoxides are 10-165-fold more potent inhibitors of protein degradation in the same system (K0.5 for inhibition by p-aminophenylarsenoxide was 3.5-20 microM, depending on the substrate). In the ubiquitin-dependent proteolytic pathway, covalent ligation of ubiquitin to protein substrates targets the latter for degradation. In certain cases, specificity in ubiquitin-substrate conjugation depends critically upon the properties of ubiquitin-protein ligase or E3. Among other effects, p-aminophenylarsenoxide decreased the steady-state level of ubiquitinated human alpha-lactalbumin; this is a substrate which is acted upon directly by ubiquitin-protein ligase-alpha (E3-alpha). This finding suggests that phenylarsenoxides (unlike arsenite) inhibit E3. Several other lines of evidence confirm this conclusion. 1) A complex of E3-alpha and the 14-kDa ubiquitin-conjugating (E2) isozyme binds to phenylarsenoxide-Sepharose resin, with the E3 component of the complex mediating binding. 2) p-Aminophenylarsenoxide inhibited isolated E3 (K0.5 approximately 50 microM); inhibition was readily reversed by addition of dithiothreitol (which contains a competing vicinal thiol group), but not by beta-mercaptoethylamine (a monothiol). 3) A bifunctional phenylarsenoxide (bromoacetylaminophenylarsenoxide) rapidly and irreversibly inactivated E3; bromoacetyl aniline, which lacks an arsenoxide moiety, did not inhibit E3. These results suggest that E3 possesses essential vicinal thiol groups and that there is a reactive nucleophile proximal to the vicinal thiol site. The bifunctional phenylarsenoxide should be a useful tool for probing the relationship between structure and function in E3. As expected from prior results with arsenite, p-aminophenylarsenoxide was also a potent inhibitor of the turnover of ubiquitin-(human) alpha-lactalbumin conjugates.     

Hybridization and speciation

Hybridization has many and varied impacts on the process of speciation. Hybridization may slow or reverse differentiation by allowing gene flow and recombination. It may accelerate speciation via adaptive introgression or cause near‐instantaneous speciation by allopolyploidization. It may have multiple effects at different stages and in different spatial contexts within a single speciation event. We offer a perspective on the context and evolutionary significance of hybridization during speciation, highlighting issues of current interest and debate. In secondary contact zones, it is uncertain if barriers to gene flow will be strengthened or broken down due to recombination and gene flow. Theory and empirical evidence suggest the latter is more likely, except within and around strongly selected genomic regions. Hybridization may contribute to speciation through the formation of new hybrid taxa, whereas introgression of a few loci may promote adaptive divergence and so facilitate speciation. Gene regulatory networks, epigenetic effects and the evolution of selfish genetic material in the genome suggest that the Dobzhansky–Muller model of hybrid incompatibilities requires a broader interpretation. Finally, although the incidence of reinforcement remains uncertain, this and other interactions in areas of sympatry may have knock‐on effects on speciation both within and outside regions of hybridization.     

Neuronal Development in Caenorhabditis elegans Is Regulated by Inhibition of an MLK MAP Kinase Pathway

We show that loss-of-function mutations in kinases of the MLK-1 pathway (mlk-1, mek-1, and kgb-1/jnk) function cell-autonomously in neurons to suppress defects in synapse formation and axon termination caused by rpm-1 loss of function. Our genetic analysis also suggests that the phosphatase PPM-1, like RPM-1, is a potential inhibitor of kinases in the MLK-1 pathway.     

Butterfly response to severe ENSO-induced forest fires in Borneo

Abstract.  1. Little is known about animal community response to severe El Niño Southern Oscillation (ENSO)-induced fire events. Here the response of butterflies to the 1997/98 ENSO-induced fire event in East Kalimantan (Indonesian Borneo) is assessed. In addition to the community-wide study, a detailed assessment of the lycaenid Jamides celeno is made.
2. Species richness declined significantly from 211 species pre-ENSO to 39 species post-ENSO and community composition changed significantly. Along with the decline in species richness there was a marked increase in dominance. Jamides celeno , for example, increased from 3% of the pre-ENSO assemblage to 58% of the post-ENSO assemblage. Like J. celeno , most of the species in the post-ENSO assemblage were generalists; most of the specialist species having disappeared from pre- to post-ENSO.
3. The major host plant used by J. celeno was the abundant resprouting Fordia splendidissima . Furthermore, significantly more eggs were laid on plants with the crazy ant, Anoplolepis gracilipes , present than on plants with other ants or no ant attendance. Caterpillar presence was significantly higher on plants tended by ants than on untended plants.
4. The median distance moved by J. celeno was 30 m with a maximum recorded distance of 290 m.
5. The abundance of J. celeno and other generalists in the post-ENSO assemblage at Wanariset was probably related to their ability to utilise the few available resources after the fire (e.g. F. splendidissima resprouts), their presence in degraded habitats surrounding the Wanariset forest, and their ability to disperse successfully by either being strong fliers (e.g. Euploea spp.) or being able to attain very high population sizes and thereby produce a surplus of dispersers (e.g. J. celeno ).     

Sulphate uptake and xylem loading of young pea (Pisum sativum L.) seedlings

Sulphate uptake and xylem loading was analysed in young pea (Pisum sativum) seedlings. The rate of sulphate uptake into intact 8-days-old pea seedlings (determined by a 1 h exposure to radiolabelled sulphate in the nutrient solution) was 585 nmol sulphate g^–1 root fresh weight h^–1. When the cotyledons were removed on day 6 the 8-days-old seedlings took up only 7% of the controls. Interruption of the phloem transport by steam girdling of the stem or the root (1 h before incubation with radiolabelled sulphate) diminished sulphate uptake by approximately 50%. The addition of sucrose to the nutrient solution during incubation did not restore sulphate uptake rates indicating that the decrease was not due to a lack of energy. Apparently, a signal from the shoot and/or the cotyledons is necessary to stimulate sulphate uptake into the roots of pea seedlings. Glutathione fed to the roots for 3 h prior to incubation with radiolabelled sulphate diminished sulphate uptake by approximately 50%. The relative proportion of the sulphate taken up that was loaded into the xylem remained unchanged (between 7 and 9% of total uptake), even when the stem was girdled above the cotyledons or when the seedlings were pre-exposed to glutathione. Only removal of the cotyledons or girdling of the root below the cotyledons increased the proportion of sulphate loaded into the xylem to 13–15% of total uptake upon exposure to glutathione. Apparently, a signal from the cotyledons represses xylem loading to some extent.