A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Purification and characterization of a methionine aminopeptidase from Saccharomyces cerevisiae

Methionine aminopeptidase (MAP), which catalyzes the removal of NH2-terminal methionine from proteins, was isolated from Saccharomyces cerevisiae. The enzyme was purified 472-fold to apparent homogeneity. The Mr of the native enzyme was estimated to be 36,000 +/- 5,000 by gel filtration chromatography, and the Mr of the denatured protein was estimated to be 34,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 7.0, and its pI is 7.8 as determined by chromatofocusing on Mono P. The enzyme was inactivated by metalloprotease inhibitors (EDTA, o-phenanthroline and nitrilotriacetic acid), sulfhydryl-modifying reagents (HgCl2 and p-hydroxymercuribenzoic acid), and Zn2+. Yeast MAP failed to cleave methionine p-nitroanilide. Among 11 Xaa-Ala-Ser analogues (Xaa = Ala, Asp, Gln, Glu, Ile, Leu, Lys, Met, Phe, Pro, and Ser), MAP cleaved only Met-Ala-Ser. MAP also cleaved methionine from other tripeptides whose penultimate amino acid residue is relatively small and/or uncharged (e.g. Pro, Gly, Val, Thr, or Ser) but not when bulky and/or charged (Arg. His, Leu, Met, or Tyr). Yeast MAP displayed similar substrate specificities compared with those of Escherichia coli (Ben-Bassat, A., Bauer, K., Chang, S.Y., Myambo, K., Boosman, A., and Chang, S. (1987) J. Bacteriol. 169, 751-757) and Salmonella typhimurium MAP (Miller, C., Strauch, K. L., Kukral, A. M., Miller, J. L., Wingfield, P. T., Mazzei, G. J., Werlen, R. C., Garber, P., and Movva, N. R. (1987) Proc. Natl, Acad. Sci. U.S.A. 84, 2718-2722). In general, the in vitro specificity of yeast MAP is consistent with the specificity observed in previous in vivo studies in yeast (reviewed in Arfin, S. M., and Bradshaw, R. A. (1988) Biochemistry 27, 7979-7984).     

Chick embryo fibroblasts produce two forms of hyaluronidase

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.     

Association between Traffic-Related Air Pollution,Subclinical Inflammation and Impaired Glucose Metabolism: Results from the SALIA Study

Background

Environmental and lifestyle factors regulate the expression and release of immune mediators. It has been hypothesised that ambient air pollution may be such an external factor and that the association between air pollution and impaired glucose metabolism may be attributable to inflammatory processes. Therefore, we assessed the associations between air pollution, circulating immune mediators and impaired glucose metabolism.

Methods

We analysed concentrations of 14 pro- and anti-inflammatory immune mediators as well as fasting glucose and insulin levels in plasma of 363 women from the Study on the influence of Air pollution on Lung function, Inflammation and Aging (SALIA, Germany). Exposure data for a group of pollutants such as nitrogen oxides (NO₂, NO_x) and different fractions of particulate matter were available for the participants'' residences. We calculated the association between the pollutants and impaired glucose metabolism by multiple regression models.

Results

The study participants had a mean age of 74.1 (SD 2.6) years and 48% showed impaired glucose metabolism based on impaired fasting glucose or previously diagnosed type 2 diabetes. Only long-term exposure NO₂ and NO_x concentrations showed positive associations (NO₂: OR 1.465, 95% CI 1.049-2.046, NO_x: OR 1.409, 95% CI 1.010-1.967) per increased interquartile range of NO₂ (14.65 µg/m³) or NO_x (43.16 µg/m³), respectively, but statistical significance was lost after correction for multiple comparisons. Additional adjustment for circulating immune mediators or the use of anti-inflammatory medication had hardly any impact on the observed ORs.

Conclusions

Our results suggest that exposure to nitrogen oxides may contribute to impaired glucose metabolism, but the associations did not reach statistical significance so that further studies with larger sample sizes are required to substantiate our findings. Our data do not preclude a role of inflammatory mechanisms in adipose or other tissues which may not be reflected by immune mediators in plasma.     

A Fungal Sarcolemmal Membrane-Associated Protein (SLMAP) Homolog Plays a Fundamental Role in Development and Localizes to the Nuclear Envelope,Endoplasmic Reticulum,and Mitochondria

Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.     

In vitro stability and content release properties of phosphatidylglyceroglycerol containing thermosensitive liposomes

Recently, we reported that 1,2-dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol (DPPGOG) prolongs the circulation time of thermosensitive liposomes (TSL). Since the only TSL formulation in clinical trials applies DSPE-PEG2000 and lysophosphatidylcholine (P-lyso-PC), the objective of this study was to compare the influence of these lipids with DPPGOG on in vitro stability and heat-induced drug release properties of TSL. The content release rate was significantly increased by incorporating DPPGOG or P-lyso-PC in TSL formulations. DPPC/DSPC/DPPGOG 50:20:30 (m/m) and DPPC/P-lyso-PC/DSPE-PEG2000 90:10:4 (m/m) did not differ significantly in their release rate of carboxyfluorescein with > 70% being released within the first 10s at their phase transition temperature. Furthermore, DPPC/DSPC/DPPGOG showed an improved stability at 37 °C in serum compared to the PEGylated TSL. The in vitro properties of DPPGOG-containing TSL remained unchanged when encapsulating doxorubicin instead of carboxyfluorescein. The TSL retained 89.1 ± 4.0% of doxorubicin over 3 h at 37  °C in the presence of serum. The drug was almost completely released within 120s at 42 °C. In conclusion, DPPGOG improves the in vitro properties in TSL formulations compared to DSPE-PEG2000, since it not only increases the in vivo half-life, it even increases the content release rate without negative effect on TSL stability at 37 °C which has been seen for DSPE-PEG2000/P-lyso-PC containing TSL.     

Diversity of the neurotoxic Conus peptides: a model for concerted pharmacological discovery

Predatory cone snails (genus Conus) produce a rich array of venoms that collectively contain an estimated 100,000 small, disulfide-rich peptides (i.e., conotoxins, or conopeptides). Over the last few decades, the conopeptides have revealed a remarkable diversity of pharmacological function and utility. An evolutionary rationale for the existence of such a large and pharmacologically diverse set of gene products can be premised on the complexity of intra- and interspecies interactions that define the ecology of Conus snails. Insights into these evolutionary trends, moreover, have been exploited with great neuropharmacological success, so that research into the Conus snails effectively recapitulates a new concerted discovery approach, which we discuss here, for developing unique ligands for both laboratory and therapeutic applications. The Conus peptides thus serve as a model system for reaping the pharmacological potential of biodiverse animal lineages.