Rapid Nonsynonymous Evolution of the Iron-Sulfur Protein in Anthropoid Primates

Cytochrome c (CYC) and 9 of the 13 subunits of cytochrome c oxidase (complex IV; COX) were previously shown to have accelerated rates of nonsynonymous substitution in anthropoid primates. Cytochrome b, the mtDNA encoded subunit of ubiquinol-cytochrome c reductase (complex III), also showed an accelerated nonsynonymous substitution rate in anthropoid primates but rate information about the nuclear encoded subunits of complex III has been lacking.We now report that phylogenetic and relative rates analysis of a nuclear encoded catalytically active subunit of complex III, the ironsulfur protein (ISP), shows an accelerated rate of amino acid replacement similar to cytochrome b. Because both ISP and subunit 9, whose function is not directly related to electron transport, are produced by cleavage into two subunits of the initial translation product of a single gene, it is probable that these two subunits of complex III have essentially identical underlying rates of mutation. Nevertheless, we find that the catalytically active ISP has an accelerated rate of amino acid replacement in anthropoid primates whereas the catalytically inactive subunit 9 does not.     

Variation in elemental intensities among teeth and between pre- and postnatal regions of enamel

Microspatial analyses of the trace element composition of dental enamel are made possible using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Fine spatial resolution, multielement capabilities, and minimal sample destruction make this technique particularly well-suited for documenting the distribution of elements in sequentially calcifying layers of enamel. Because deciduous enamel forms from week 13 in utero up to 9 months postnatally (thereafter essentially becoming inert), the application of LA-ICP-MS allows for the retrospective measurement of prenatal and early postnatal trace-element uptake during a critical period of child development. In this study, we compared intra- and intertooth intensities of 25Mg, 57Fe, 66Zn, 68Zn, 88Sr, 138Ba, and 208Pb via LA-ICP-MS of 38 exfoliated deciduous incisors and canines donated by 36 participants in the Solís Valley Mexico Nutrition Collaborative Research Support Program (NCRSP). Pre- and postnatal comparisons within teeth showed significant increases (P < 0.001) and greater variation in the abundance of all isotopes in postnatal enamel, with the exception of a decrease in 25Mg (P < 0.001) and constant values for 88Sr (P = 0.681). Conversely, comparisons by tooth type and mouth quadrant revealed few significant differences between teeth of the same individual. We argue that more variation in the trace element composition of teeth occurs across developmental areas within a tooth than among different teeth of the same person. This study further demonstrates that sequentially calcifying areas of enamel have different chemical concentrations. The results support the use of microspatial analyses of enamel for understanding changes in nutrition, pollution, and residence.     

Heme oxygenase-1 prevents superoxide anion-associated endothelial cell sloughing in diabetic rats

Heme oxygenase-1 (HO-1) represents a key defense mechanism against oxidative injury. Hyperglycemia has been linked to increased oxidative stress, leading to endothelial dysfunction, delayed cell replication, and enhanced apoptosis. The effect of streptozotocin (STZ)-induced diabetes on HO activity, HO-1 promoter activity, superoxide anion (O*-2, and the number of circulating endothelial cells was measured. The expression of HO-1/HO-2 protein was unchanged, but HO activity was decreased in aortas of diabetic rats compared with control (p < 0.05). High glucose decreased HO-1 promoter activity (p < 0.05). Hyperglycemia increased O*-2 and this increase was augmented with HO-1 inhibition and diminished with HO-1 upregulation (p < 0.05). Circulating endothelial cells were significantly higher in diabetic rats and were decreased or increased with administration of the HO-1 inducer (CoPP) or inhibitor (SnMP), respectively (p<0.05). In conclusion, HO-1 upregulation in diabetic rats brings about an increase in serum bilirubin, a reduction in O*-2 production, and a decrease in endothelial cell sloughing.     

Mutations in multiple domains activate paramyxovirus F protein-induced fusion

SER virus, a paramyxovirus that is closely related to simian virus 5 (SV5), is unusual in that it fails to induce syncytium formation. The SER virus F protein has an unusually long cytoplasmic tail (CT), and it was previously observed that truncations or specific mutations of this domain result in enhanced syncytium formation. In addition to the long CT, the SER F protein has nine amino acid differences from the F protein of SV5. We previously observed only a partial suppression of fusion in a chimeric SV5 F protein with a CT derived from SER virus, indicating that these other amino acid differences between the SER and SV5 F proteins also play a role in regulating the fusion phenotype. To examine the effects of individual amino acid differences, we mutated the nine SER residues individually to the respective residues of the SV5 F protein. We found that most of the mutants were expressed well and were transported to the cell surface at levels comparable to that of the wild-type SER F protein. Many of the mutants showed enhanced lipid mixing, calcein transfer, and syncytium formation even in the presence of the long SER F protein CT. Some mutants, such as the I310 M, T438S, M489I, T516V, and N529K mutants, also showed fusion at lower temperatures of 32, 25, and 18 degrees C. The residue Asn529 plays a critical role in the suppression of fusion activity, as the mutation of this residue to lysine caused a marked enhancement of fusion. The effect of the N529K mutation on the enhancement of fusion by a previously described mutant, L539,548A, as well as by chimeric SV5/SER F proteins was also dramatic. These results indicate that activation to a fusogenic conformation is dependent on the interplay of residues in the ectodomain, the transmembrane domain, and the CT domain of paramyxovirus F proteins.     

The Wingless morphogen gradient is established by the cooperative action of Frizzled and Heparan Sulfate Proteoglycan receptors

We have examined the respective contribution of Heparan Sulfate Proteoglycans (HSPGs) and Frizzled (Fz) proteins in the establishment of the Wingless (Wg) morphogen gradient. From the analysis of mutant clones of sulfateless/N-deacetylase-sulphotransferase in the wing imaginal disc, we find that lack of Heparan Sulfate (HS) causes a dramatic reduction of both extracellular and intracellular Wg in receiving cells. Our studies, together with others [Kirkpatrick, C.A., Dimitroff, B.D., Rawson, J.M., Selleck, S.B., 2004. Spatial regulation of Wingless morphogen distribution and signalling by Dally-like protein. Dev. Cell (in press)], reveals that the Glypican molecule Dally-like Protein (Dlp) is associated with both negative and positive roles in Wg short- and long-range signaling, respectively. In addition, analyses of the two Fz proteins indicate that the Fz and DFz2 receptors, in addition to transducing the signal, modulate the slope of the Wg gradient by regulating the amount of extracellular Wg. Taken together, our analysis illustrates how the coordinated activities of HSPGs and Fz/DFz2 shape the Wg morphogen gradient.     

The peroxisome deficient Arabidopsis mutant sse1 exhibits impaired fatty acid synthesis

The Arabidopsis Shrunken Seed 1 (SSE1) gene encodes a homolog of the peroxisome biogenesis factor Pex16p, and a loss-of-function mutation in this gene alters seed storage composition. Two lines of evidence support a function for SSE1 in peroxisome biogenesis: the peroxisomal localization of a green fluorescent protein-SSE1 fusion protein and the lack of normal peroxisomes in sse1 mutant embryos. The green fluorescent protein-SSE1 colocalizes with the red fluorescent protein (RFP)-labeled peroxisomal markers RFP-peroxisome targeting signal 1 and peroxisome targeting signal 2-RFP in transgenic Arabidopsis. Each peroxisomal marker exhibits a normal punctate peroxisomal distribution in the wild type but not the sse1 mutant embryos. Further studies reported here were designed toward understanding carbon metabolism in the sse1 mutant. A time course study of dissected embryos revealed a dramatic rate decrease in oil accumulation and an increase in starch accumulation. Introduction of starch synthesis mutations into the sse1 background did not restore oil biosynthesis. This finding demonstrated that reduction in oil content in sse1 is not caused by increased carbon flow to starch. To identify the blocked steps in the sse1 oil deposition pathway, developing sse1 seeds were supplied radiolabeled oil synthesis precursors. The ability of sse1 to incorporate oleic acid, but not pyruvate or acetate, into triacylglycerol indicated a defect in the fatty acid biosynthetic pathway in this mutant. Taken together, the results point to a possible role for peroxisomes in the net synthesis of fatty acids in addition to their established function in lipid catabolism. Other possible interpretations of the results are discussed.     

Comparison of crenarchaeal consortia inhabiting the rhizosphere of diverse terrestrial plants with those in bulk soil in native environments

To explore whether the crenarchaeal consortium found in the rhizosphere is distinct from the assemblage of crenarchaeotes inhabiting bulk soil, PCR-single-stranded-conformation polymorphism (PCR-SSCP) profiles were generated for 76 plant samples collected from native environments. Divergent terrestrial plant groups including bryophytes (mosses), lycopods (club mosses), pteridophytes (ferns), gymnosperms (conifers), and angiosperms (seed plants) were collected for this study. Statistical analysis revealed significant differences between rhizosphere and bulk soil PCR-SSCP profiles (Hotelling paired T(2) test, P < 0.0001), suggesting that a distinct crenarchaeal consortium is associated with plants. In general, phylotype richness increased in the rhizosphere compared to the corresponding bulk soil, although the range of this increase was variable. Examples of a major change in rhizosphere (versus bulk soil) PCR-SSCP profiles were detected for all plant groups, suggesting that crenarchaeotes form associations with phylogenetically diverse plants in native environments. In addition, examples of minor to no detectable difference were found for all terrestrial plant groups, suggesting that crenarchaeal associations with plants are mediated by environmental conditions.     

Accelerated evolution of the electron transport chain in anthropoid primates

Mitochondria are both the power plant of the cell and a central integrator of signals that govern the lifespan, replication and death of the cell. Perhaps as a consequence, genes that encode components of the mitochondrial electron transport chain (ETC) are generally conserved. Therefore, it is surprising that many of these genes in anthropoid primates (New World monkeys, Old World monkeys and apes, including humans) have been major targets of darwinian positive selection. Sequence comparisons have provided evidence that marked increases of non-synonymous substitution rates occurred in anthropoid ETC genes that encode subunits of Complex III and IV, and the electron carrier molecule cytochrome c (CYC). Two important questions are: (i) how has evolution altered ETC function? and; (ii) how might functional changes in the ETC be linked to evolution of an expanded neocortical brain?