Contrasting modes of natural selection acting on pigmentation genes in the Drosophila dunni subgroup

Genes that encode for divergent adaptive traits may have genealogies that contrast with those from loci that are not functionally involved in differentiation. Here, we examine DNA sequence variation among the species of the eastern Caribbean Drosophila dunni subgroup at two loci, yellow and dopa decaboxylase (Ddc), which both play integral roles in pigmentation patterning of adult Drosophila. Phylogenetic analyses of these loci produce gene genealogies with topologies that mirror those described for other nuclear genes: the six morphologically distinct species within the subgroup are divided into only three lineages, with one lineage containing four species that share extensive ancestral polymorphism. At the Ddc locus these major lineages are delineated only by silent site variation. We observe a significantly higher rate of synonymous site divergence than non-synonymous divergence, consistent with strong purifying selection acting on the locus. In contrast, the yellow locus exhibits patterns of amino acid divergence and nucleotide diversity that are consistent with recent diversifying selection acting in two different lineages. This selection appears to be targeting amino acid variants in the signal sequence of the Yellow protein, a region which is tightly constrained among members of the larger D. cardini radiation. This result highlights not only the potential importance of yellow in the evolution of divergent pigmentation patterns among members of the D. dunni subgroup, but also hints that variation in signal peptide sequences may play a role in phenotypic diversification.     

Identification and initial characterization of a new low-molecular-weight virus-encoded T antigen in a line of simian virus 40-transformed cells.

SV80 cells, a simian virus 40 (SV40)-transformed derivative of a strain of human fibroblasts, synthesize an 8-kilodalton anti-T reactive polypeptide in addition to large T and small t antigens. Although not observed during lytic infection carried out under a variety of conditions, an anti-T reactive molecule which comigrated with the SV80 8-kilodalton protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis was synthesized by one of five other SV40-transformed cell lines studied. The SV40 8-kilodalton protein was present in lysates of cells exposed to a brief pulse of radioactive methionine and did not accumulate during an extended chase period. This polypeptide could not by generated by mixing an unlabeled extract of SV80 cells with a labeled extract of infected monkey cells. The 8-kilodalton molecule reacts with antibody raised against homogeneous large T antigen, is present only in the cytoplasm, is not complexed with T, lacks DNA-binding properties, and is not phosphorylated. This protein could be translated in a cell-free system programmed by SV40-specific mRNA. At least two messenger species (approximately 19S and approximately 22S) directed its synthesis. Tryptic peptide analysis of [35S]methionine-labeled proteins demonstrated that the 8-kilodalton protein contains all eight of the common T/t peptides and one additional peptide not present in the maps of t or T. It lacks both of the t-unique peptides. The organization of the integrated viral sequences which encode this molecule was determined by restriction endonuclease analysis. In particular, SV80 cells contain at least two integrated SV40 genomes which are oriented in tandem, with an intervening cellular sequence..     

Synthesis and initial characterization of FGFR3 transmembrane domain: consequences of sequence modifications

Receptor Tyrosine Kinases (RTKs) conduct biochemical signals via lateral dimerization in the plasma membrane, and defects in their dimerization lead to unregulated signaling and disease. RTK transmembrane (TM) domains are proposed to play an important role in the process, underscored by the finding that single amino acids mutations in the TM domains can induce pathological phenotypes. Therefore, many important questions pertaining to the mode of signal transduction and the mechanism of pathology induction could be answered by studying the chemical-physical basis behind RTK TM domain dimerization and the interactions of RTK TM domains with lipids in model bilayer systems. As a first step towards this goal, here we report the synthesis of the TM domain of fibroblast growth factor receptor 3 (FGFR3), an RTK that is crucial for skeletal development. We have used solid phase peptide synthesis to produce two peptides: one corresponding to the membrane embedded segment and the naturally occurring flanking residues at the N- and C-termini (TMwt), and a second one in which the flanking residues have been substituted with diLysines at the termini (TMKK). We have demonstrated that the hydrophobic FGFR3 TM domain can be synthesized for biophysical studies with high yield. The protocol presented in the paper can be applied to the synthesis of other RTK TM domains. As expected, the Lys flanks decrease the hydrophobicity of the TM domain, such that TMKK elutes much earlier than TMwt during reverse phase HPLC purification. The Lysines have no effect on peptide solubility in SDS and on peptide secondary structure, but they abolish peptide dimerization on SDS gels. These results suggest that caution should be exercised when modifying RTK TM domains to render them more manageable for biophysical studies.     

Blueberry-Enriched Diet Protects Rat Heart from Ischemic Damage

Objectives

to assess the cardioprotective properties of a blueberry enriched diet (BD).

Background

Reactive oxygen species (ROS) play a major role in ischemia-related myocardial injury. The attempts to use synthetic antioxidants to block the detrimental effects of ROS have produced mixed or negative results precipitating the interest in natural products. Blueberries are readily available product with the highest antioxidant capacity among fruits and vegetables.

Methods and Results

Following 3-mo of BD or a regular control diet (CD), the threshold for mitochondrial permeability transition (t_MPT) was measured in isolated cardiomyocytes obtained from young male Fischer-344 rats. Compared to CD, BD resulted in a 24% increase (p<0.001) of ROS indexed t_MPT. The remaining animals were subjected to a permanent ligation of the left descending coronary artery. 24 hrs later resulting myocardial infarction (MI) in rats on BD was 22% less than in CD rats (p<0.01). Significantly less TUNEL(+) cardiomyocytes (2% vs 9%) and 40% less inflammation cells were observed in the myocardial area at risk of BD compared to CD rats (p<0.01). In the subgroup of rats, after coronary ligation the original diet was either continued or switched to the opposite one, and cardiac remodeling and MI expansion were followed by serial echocardiography for 10 weeks. Measurements suggested that continuation of BD or its withdrawal after MI attenuated or accelerated rates of post MI cardiac remodeling and MI expansion.

Conclusion

A blueberry-enriched diet protected the myocardium from induced ischemic damage and demonstrated the potential to attenuate the development of post MI chronic heart failure.     

Conserved Non-Coding Regulatory Signatures in Arabidopsis Co-Expressed Gene Modules

Complex traits and other polygenic processes require coordinated gene expression. Co-expression networks model mRNA co-expression: the product of gene regulatory networks. To identify regulatory mechanisms underlying coordinated gene expression in a tissue-enriched context, ten Arabidopsis thaliana co-expression networks were constructed after manually sorting 4,566 RNA profiling datasets into aerial, flower, leaf, root, rosette, seedling, seed, shoot, whole plant, and global (all samples combined) groups. Collectively, the ten networks contained 30% of the measurable genes of Arabidopsis and were circumscribed into 5,491 modules. Modules were scrutinized for cis regulatory mechanisms putatively encoded in conserved non-coding sequences (CNSs) previously identified as remnants of a whole genome duplication event. We determined the non-random association of 1,361 unique CNSs to 1,904 co-expression network gene modules. Furthermore, the CNS elements were placed in the context of known gene regulatory networks (GRNs) by connecting 250 CNS motifs with known GRN cis elements. Our results provide support for a regulatory role of some CNS elements and suggest the functional consequences of CNS activation of co-expression in specific gene sets dispersed throughout the genome.     

Cytochrome c peroxidase compound I: formation of covalent protein crosslinks during the endogenous reduction of the active site

Cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) was oxidized by hydrogen peroxide in the absence of exogenous electron donor. Higher molecular weight species were observed in the decay products at pH 4.5. Monomer and dimer were separated by gel filtration and purified by anion-exchange chromatography. Peptide mapping of tryptic digests of the dimer indicated a tyrosine crosslink localized between residues 32 and 48 of the native enzyme.     

Triepitopic Antibody Fusions Inhibit Cetuximab-Resistant BRAF and KRAS Mutant Tumors via EGFR Signal Repression

Dysregulation of epidermal growth factor receptor (EGFR) is a hallmark of many epithelial cancers, rendering this receptor an attractive target for cancer therapy. Much effort has been focused on the development of EGFR-directed antibody-based therapeutics, culminating in the clinical approval of the drugs cetuximab and panitumumab. Unfortunately, the clinical efficacy of these drugs has been disappointingly low, and a particular challenge to targeting EGFR with antibody therapeutics has been resistance, resulting from mutations in the downstream raf and ras effector proteins. Recent work demonstrating antibody cocktail-induced synergistic downregulation of EGFR motivated our design of cetuximab-based antibody-fibronectin domain fusion proteins that exploit downregulation-based EGFR inhibition by simultaneously targeting multiple receptor epitopes. We establish that, among our engineered multiepitopic formats, trans-triepitopic antibody fusions demonstrate optimal efficacy, inducing rapid EGFR clustering and internalization and consequently ablating downstream signaling. The combined effects of EGFR downregulation, ligand competition, and immune effector function conspire to inhibit tumor growth in xenograft models of cetuximab-resistant BRAF and KRAS mutant cancers. Our designed triepitopic constructs have the potential to enhance the efficacy and expand the scope of EGFR-directed therapies, and our multiepitopic may be readily applied to other receptor targets to formulate a new class of antibody-based therapeutics.     

A class of parametrically excited calcium oscillation detectors.

Intracellular Ca2+ oscillations are often a response to external signals such as hormones. Changes in the external signal can alter the frequency, amplitude, or form of the oscillations suggesting that information is encoded in the pattern of Ca2+ oscillations. How might a cell decode this signal? We show that an excitable system whose kinetic parameters are modulated by the Ca2+ concentration can function as a Ca2+ oscillation detector. Such systems have the following properties: (1) They are more sensitive to an oscillatory than to a steady Ca2+ signal. (2) Their response is largely independent of the signal amplitude. (3) They can extract information from a noisy signal. (4) Unlike other frequency sensitive detectors, they have a flat frequency response. These properties make a Ca(2+)-sensitive excitable system nearly ideal for detecting and decoding Ca2+ oscillations. We suggest that Ca2+ oscillations, in concert with these detectors, can act as cellular timekeepers to coordinate related biochemical reactions and enhance their overall efficiency.     

Use of Biochronology to Examine Interactions of Freshwater Drum, Walleye and Yellow Perch in the Red Lakes of Minnesota

Growth histories for freshwater drum, Aplodinotus grunniens, walleye, Stizostedion vitreum, and yellow perch, Perca flavescens, were constructed using calcified structures for the period 1947 through 1996 for the Red Lakes, Minnesota. Increased walleye growth and decreased yellow perch growth were observed over the period from 1983 to 1996, which are attributed to intensive fishing resulting in decreased intraspecific competition in walleye and increased intraspecific competition in yellow perch through release from predation. Strong year-classes of yellow perch were positively correlated with walleye growth (r=0.57, p-value=0.042). There was no evidence for interactions of walleye or yellow perch with freshwater drum. Freshwater drum growth (r=0.680, p-value=0.0001) was more highly correlated with temperature than were walleye (r=0.386, p-value=0.006) and yellow perch growth (r=0.303, p-value=0.036).     

Effect of pathogenic cysteine mutations on FGFR3 transmembrane domain dimerization in detergents and lipid bilayers

Mutations in fibroblast growth factor receptors are known as the genetic basis of skeletal growth disorders. The mechanism of pathogenesis, as determined by mutation-induced changes in receptor structure, interactions, and function, is elusive. Here we study three pathogenic Cys mutations, associated with either thanatophoric dysplasia or achondroplasia, in the TM domain of fibroblast growth factor receptors 3 (FGFR3). We characterize the dimerization propensities of the mutant TM domains in detergents and in lipid bilayers, in the presence and absence of reducing agents, and compare them to previous measurements of wild-type. We find that the Cys mutations increase the propensity for dimerization in detergent, with the Cys370 mutant exhibiting the highest propensity for disulfide bond formation, the Cys371 mutant having an intermediate propensity, and Cys375 the lowest. Thus, disulfide bonds readily form in detergents, with efficiency that correlates with the severity of the phenotype. In lipid bilayers, however, the Cys370 mutant, which dimerizes strongly in detergent, behaves as the wild-type, suggesting that Cys370-mediated disulfide bonds do not form between the isolated TM domains in bilayers. Thus, the nature of the hydrophobic environment plays an important role in defining the structure and flexibility of transmembrane dimers. These results and previous findings from cellular studies lead us to propose a conformational flexibility mechanism of receptor stabilization as a basis for disregulated FGFR3 signaling in thanatophoric dysplasia and achondroplasia.