ACTN3 genotype is associated with human elite athletic performance

There is increasing evidence for strong genetic influences on athletic performance and for an evolutionary "trade-off" between performance traits for speed and endurance activities. We have recently demonstrated that the skeletal-muscle actin-binding protein alpha-actinin-3 is absent in 18% of healthy white individuals because of homozygosity for a common stop-codon polymorphism in the ACTN3 gene, R577X. alpha-Actinin-3 is specifically expressed in fast-twitch myofibers responsible for generating force at high velocity. The absence of a disease phenotype secondary to alpha-actinin-3 deficiency is likely due to compensation by the homologous protein, alpha-actinin-2. However, the high degree of evolutionary conservation of ACTN3 suggests function(s) independent of ACTN2. Here, we demonstrate highly significant associations between ACTN3 genotype and athletic performance. Both male and female elite sprint athletes have significantly higher frequencies of the 577R allele than do controls. This suggests that the presence of alpha-actinin-3 has a beneficial effect on the function of skeletal muscle in generating forceful contractions at high velocity, and provides an evolutionary advantage because of increased sprint performance. There is also a genotype effect in female sprint and endurance athletes, with higher than expected numbers of 577RX heterozygotes among sprint athletes and lower than expected numbers among endurance athletes. The lack of a similar effect in males suggests that the ACTN3 genotype affects athletic performance differently in males and females. The differential effects in sprint and endurance athletes suggests that the R577X polymorphism may have been maintained in the human population by balancing natural selection.     

Achieving selectivity between highly homologous tyrosine kinases: a novel selective erbB2 inhibitor

The discovery of small molecule kinase inhibitors for use as drugs is a promising approach for the treatment of cancer and other diseases, but the discovery of highly specific agents is challenging because over 850 kinases are expressed in mammalian cells. Systematic modification of the 4-anilino functionality of a selective quinazoline inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase can invert selectivity to favor inhibition of the highly homologous erbB2 tyrosine kinase. The selectivity pattern was demonstrated in assays of recombinant kinases and recapitulated in measures of kinase activity in intact cells. The most potent and selective erbB2 inhibitor of the analog series has anti-proliferative activity against an erbB2-overexpressing cell line that was lacking in the original EGFR-selective compound. Subtle changes to the molecular structure of ATP-competitive small molecule inhibitors of tyrosine kinases can yield dramatic changes in potency and selectivity. These results suggest that the discovery of highly selective small molecule inhibitors of very homologous kinases is achievable.     

Characterization of chicken TNFR superfamily decoy receptors,DcR3 and osteoprotegerin

Tumor necrosis factor (TNF) family ligands bind to death domain-containing TNF receptors (death receptors), which can subsequently activate intracellular signaling pathways to initiate caspase activity and apoptotic cell death. Decoy receptors, without intracellular death domains, have been reported to prevent cytotoxic effects by binding to and sequestering such ligands, or by interfering with death receptor trimerization. The chicken death receptors, Fas, TNFR1, DR6, and TVB, are constitutively expressed in a relatively wide variety of hen tissues. In this study, two chicken receptors with sequence homology to the mammalian decoys, DcR3 and osteoprotegerin, were identified and their pattern of expression was characterized. Unlike the previously identified chicken death receptors, the newly characterized decoy receptors show comparatively limited expression among tissues, suggesting a tissue-specific function. Finally, characterization of these chicken receptors further contributes to understanding the evolutionary divergence of TNFR superfamily members among vertebrate species.     

Testing of the additivity-based protein sequence to reactivity algorithm

The standard free energies of association (or equilibrium constants) are predicted for 11 multiple variants of the turkey ovomucoid third domain, a member of the Kazal family of protein inhibitors, each interacting with six selected enzymes. The equilibrium constants for 38 of 66 possible interactions are strong enough to measure, and for these, the predicted and measured free energies are compared, thus providing an additional test of the additivity-based sequence to reactivity algorithm. The test appears to be unbiased as the 11 variants were designed a decade ago to study furin inhibition and the specificity of furin differs greatly from the specificities of our six target enzymes. As the contact regions of these inhibitors are highly positive, nonadditivity was expected. Of the 11 variants, one does not satisfy the restriction that either P(2) Thr or P(1)' Glu should be present and all three measurable results on it, as expected, are nonadditive. For the remaining 35 measurements, 22 are additive, 12 are partially additive, and only one is (slightly) nonadditive. These results are comparable to those obtained for a set of 398 equilibrium constants for natural variants of ovomucoid third domains. The expectation that clustering of charges would be nonadditive is modified to the expectation that major nonadditivity will be observed only if the combining sites in both associating proteins involve large charge clusters of the opposite sign. It is also shown here that an analysis of a small variant set can be accomplished with a smaller subset, in this case 13 variants, rather than the whole set of 191 members used for the complete algorithm.     

Characterization of a lactose permease mutant that binds IIAGlc in the absence of ligand

Enzyme IIA(Glc) of the Escherichia coli phosphoenolpyruvate:glucose phosphotransferase system plays a direct role in regulating inducible transport systems. Dephosphorylated IIA(Glc) binds directly to lactose permease in a reaction that requires binding of a galactosidic substrate. A double-Cys mutation (Ile129 --> Cys/Lys131 --> Cys) was introduced into helix IV of the permease near the IIA(Glc) binding site in cytoplasmic loop IV/V and in the vicinity of the galactoside binding site at the interface of helices IV, V, and VIII. The mutant no longer requires galactoside for IIA(Glc) binding as demonstrated by both a [(125)I]IIA(Glc) binding assay and a newly developed fluorescence anisotropy assay. Further characterization of the mutant shows that it binds substrate with high affinity, but is almost completely defective in all modes of translocation across the cytoplasmic membrane. The data are consistent with the interpretation that the double mutant is locked in an inward-facing conformation.     

Does alligator testis produce estradiol? A comparison of ovarian and testicular aromatase

Testicular secretion of estradiol is necessary for normal spermatogenesis and male reproductive physiology in humans and rodents. The role of estradiol in nonmammalian vertebrates remains unknown, but elevated circulating estradiol has been reported in male lizards, alligators, and various bird species. We have been unable to detect circulating estradiol in male alligators; therefore, we reexamined the question of testicular production of estradiol in alligators using more rigorous assay procedures. A large pool of plasma from a male alligator was extracted and run through an HPLC column. Immunoreactive estradiol-like material eluted coincident with authentic estradiol. By using an ultrasensitive RIA and processing large volumes of male plasma (1000 microl), we were able to measure estradiol. Estradiol in male alligators ranged from 0.23 to 3.14 pg/ml, whereas estradiol in immature female alligators ranged from 14 to 66 pg/ml. Aromatase activity in microsomes from adult alligator ovarian tissue was 36.2 +/- 1.6 pmol mg-1 h-1, whereas activity in testicular microsomes ranged between 0.92 and 2.38 pmol mg-1 h-1. Ovarian aromatase activity was inhibited in a concentration-dependent fashion by Fadrozole, but the essentially background activity of testicular aromatase was not inhibited at any concentration of Fadrozole. Likewise, a comparison of alligator testicular and ovarian aromatase mRNA expression gave a similar result: the ovarian expression was 600-fold higher and brain tissue was 10-fold higher than that of the testis. Circulating estradiol in male alligators is probably of extragonadal origin, and the testis produces little if any of this steroid.     

Interferon-tau stimulates secretion of macrophage migration inhibitory factor from bovine endometrial epithelial cells

During early pregnancy in ruminants, the embryo not only prevents prostaglandin F2alpha release, but it also modifies protein synthesis in the endometrium. This is accomplished by the secretion of interferon-tau (IFN-tau) from the embryo. The objective of this study was to identify and characterize specific proteins secreted from endometrial epithelial cells in response to IFN-tau that could be important for endometrial function and/or embryo development. The epithelial cells were prepared and cultured to confluence and then incubated with or without 100 ng/ml IFN-tau. At the end of the incubation, the proteins in the medium were analyzed by two-dimensional PAGE. The result showed that two major protein spots were induced by IFN-tau. One has a molecular mass of approximately 12 kDa and an isoelectric point (pI) of 6.7; the other has a molecular mass of 76 kDa and pI of 4.8. Protein sequence analysis showed that the 12-kDa protein contained a partial amino acid sequence that corresponded to macrophage migration inhibitory factor (MIF). To determine whether MIF is expressed in endometrial cells, isolated stromal or epithelial cells were incubated with or without 100 ng/ml IFN-tau for 0, 3, 6, 12, 24, and 48 h. After incubation, the MIF protein in cells was examined by Western blotting analysis, and the steady-state mRNA for MIF was examined by Northern analysis. Results showed that MIF protein and mRNA were present in the epithelial cells but not the stromal cells. The presence of MIF in the luminal epithelium of endometrial tissue was confirmed by immunohistochemistry. However, there was no effect of IFN-tau on MIF expression in the epithelial cells. The concentration of MIF in the medium was quantified by Western blotting analysis to determine if IFN-tau altered MIF protein secretion from the epithelial cells. The results showed that IFN-tau significantly stimulated the secretion of MIF protein from the cells. These data show that MIF is expressed in the epithelial, but not the stromal, cells of the endometrium and that MIF secretion from the epithelial cells is stimulated by IFN-tau. It is therefore likely that MIF plays a role in early embryo development, and further characterization of MIF expression and its regulation in the endometrium will add significantly to our understanding of early embryo-uterine interactions.     

SLLP1, a unique,intra-acrosomal,non-bacteriolytic,c lysozyme-like protein of human spermatozoa

We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm. C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by beta-1,4 glycosidic bonds. Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively. The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88. The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm. As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity. A significant decrease (54%, P < or = 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, approximately 1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.     

Sequence and structure space of RNA-binding peptides

Studies of RNA-binding peptides, and recent combinatorial library experiments in particular, have demonstrated that diverse peptide sequences and structures can be used to recognize specific RNA sites. The identification of large numbers of sequences capable of binding to a particular site has provided extensive phylogenetic information used to deduce basic principles of recognition. The high frequency at which RNA-binding peptides are found in large sequence libraries suggests plausible routes to evolve sequence-specific binders, facilitating the design of new binding molecules and perhaps reflecting characteristics of natural evolution.