The role of infectious disease in the evolution of females: Evidence from anther‐smut disease on a gynodioecious alpine carnation*

In flowering plants, the evolution of females is widely hypothesized to be the first step in the evolutionary pathway to separate male and female sexes, or dioecy. Natural enemies have the potential to drive this evolution if they preferentially attack hermaphrodites over females. We studied sex‐based differences in exposure to anther‐smut (Microbotryum), a sterilizing pollinator‐transmitted disease, in Dianthus pavonius, a gynodioecious perennial herb. We found that within a heavily diseased population, females consistently had lower levels of Microbotryum spore deposition relative to hermaphrodites and that this difference was driven by rapid floral closing in females following successful pollination. We further show that this protective closing behavior is frequency dependent; females close faster when they are rare. These results indicate that anther‐smut disease is an important source of selection for females, especially since we found in a common garden experiment no evidence that females have any inherent fecundity advantages over hermaphrodites. Finally, we show that among populations, those where anther‐smut is present have a significantly higher frequency of females than those where the disease is absent. Taken together our results indicate that anther‐smut disease is likely an important biotic factor driving the evolution and maintenance of females in this gynodioecious species.     

Circular permutation of granulocyte colony-stimulating factor

The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.     

Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability

Src kinase activity was found to protect endothelial cells from apoptosis during vascular endothelial growth factor (VEGF)-, but not basic fibroblast growth factor (bFGF)-, mediated angiogenesis in chick embryos and mice. In fact, retroviral targeting of kinase-deleted Src to tumor-associated blood vessels suppressed angiogenesis and the growth of a VEGF-producing tumor. Although mice lacking individual Src family kinases (SFKs) showed normal angiogenesis, mice deficient in pp60c-src or pp62c-yes showed no VEGF-induced vascular permeability (VP), yet fyn-/- mice displayed normal VP. In contrast, inflammation-mediated VP appeared normal in Src-deficient mice. Therefore, VEGF-, but not bFGF-, mediated angiogenesis requires SFK activity in general, whereas the VP activity of VEGF specifically depends on the SFKs, Src, or Yes.     

X-ray crystal structure of human dopamine sulfotransferase, SULT1A3. Molecular modeling and quantitative structure-activity relationship analysis demonstrate a molecular basis for sulfotransferase substrate specificity

Humans are one of the few species that produce large amounts of catecholamine sulfates, and they have evolved a specific sulfotransferase, SULT1A3 (M-PST), to catalyze the formation of these conjugates. An orthologous protein has yet to be found in other species. To further our understanding of the molecular basis for the unique substrate selectivity of this enzyme, we have solved the crystal structure of human SULT1A3, complexed with 3'-phosphoadenosine 5'-phosphate (PAP), at 2.5 A resolution and carried out quantitative structure-activity relationship (QSAR) analysis with a series of phenols and catechols. SULT1A3 adopts a similar fold to mouse estrogen sulfotransferase, with a central five-stranded beta-sheet surrounded by alpha-helices. SULT1A3 is a dimer in solution but crystallized with a monomer in the asymmetric unit of the cell, although dimer interfaces were formed by interaction across crystallographic 2-fold axes. QSAR analysis revealed that the enzyme is highly selective for catechols, and catecholamines in particular, and that hydrogen bonding groups and lipophilicity (cLogD) strongly influenced K(m). We also investigated further the role of Glu(146) in SULT1A3 using site-directed mutagenesis and showed that it plays a key role not only in defining selectivity for dopamine but also in preventing many phenolic xenobiotics from binding to the enzyme.     

Effects of protein deficient diets on the developmental toxicity of inorganic arsenic in mice

BACKGROUND: Inorganic arsenic, when given by injection to pregnant laboratory animals (mice, rats, hamsters), has been shown to induce malformations. Arsenic methylation may be a detoxification step, and diets deficient in protein are a poor source of methyl donors and may possibly result in impaired arsenic methylation. Human health effects from chronic arsenic exposure have been reported mainly in populations with low socioeconomic status. Individuals in such populations are likely to suffer from malnutrition, which can compromise embryonic/fetal development and diminish arsenic methylating capacity. We sought to determine if dietary protein deficiency affects the developmental toxicity of inorganic arsenic. METHODS: Mated females were randomly assigned to one of 12 treatment groups. Experimental groups received either AsIII or AsV i.p. on Gestation Day 8 (GD 8, plug=GD 0) and were maintained on a 5%, 10%, or 20% protein custom mixed diet from GD 1 until sacrifice. Controls received the custom diets alone, were given AsIII or AsV i.p. on GD 8 with Teklad LM-485 rodent diet, or were fed the LM-485 diet alone. Test females were sacrificed on GD 17, and their litters were examined for mortality and developmental defects. RESULTS: Arsenic plus dietary protein deficiency decreased maternal weight gain and increased the incidences of exencephaly, ablepharia, and skeletal defects, such as malformed vertebral centra, fused ribs, and abnormal sternebrae (bipartite, rudimentary, or unossified). CONCLUSIONS: These results demonstrate that dietary protein deficiency enhances the developmental toxicity of inorganic arsenic, possibly by impairment of arsenic methylation.