1 Department of Mathematics, University of Michigan, Ann Arbor, Michigan
2 Institute of Gerontology, University of Michigan, Ann Arbor, Michigan
3 Department of Pathology, University of Michigan, Ann ...Arbor, Michigan
4 Geriatrics Center, University of Michigan, Ann Arbor, Michigan
5 Ann Arbor Veterans Affairs Medical Center, University of Michigan, Ann Arbor, Michigan
6 Department of Human Genetics, University of Michigan, Ann Arbor, Michigan
A previous analysis of serum insulin-like growth factor I (IGF-I) levels in a mouse population ( n = 961) derived from a cross of (BALB/cJ x C57BL/6J) F 1 females and (C3H/HeJ x DBA/2J) F 1 males documented quantitative trait loci (QTL) on chromosomes 1, 10, and 17. We employed a newly developed, random walk-based method to search for three- and four-way allelic combinations that might influence IGF-I levels through nonadditive (conditional or epistatic) interactions among 185 genotyped biallelic loci and with significance defined by experiment-wide permutation ( P < 0.05). We documented a three-locus combination in which an epistatic interaction between QTL on paternal-derived chromosomes 5 and 18 had an opposite effect on the phenotype based on the allele inherited at a third locus on maternal-derived chromosome 17. The search also revealed three four-locus combinations that influence IGF-I levels through nonadditive genetic interactions. In two cases, the four-allele combinations were associated with animals having high levels of IGF-I, and, in the third case, a four-allele combination was associated with animals having low IGF-I levels. The multiple-locus genome scan algorithm revealed new IGF-I QTL on chromosomes 2, 4, 5, 7, 8, and 12 that had not been detected in the single-locus genome search and showed that levels of this hormone can be regulated by complex, nonadditive interactions among multiple loci. The analysis method can detect multilocus interactions in a genome scan experiment and may provide new ways to explore the genetic architecture of complex physiological phenotypes.
quantitative trait loci; epistasis; gene interactions
The natterjack toad Bufo calamita is rare in Britain, which is at the northwestern edge of its biogeographical range. We investigated the level of genetic differentiation amongst almost all (34 out ...of 38) of the surviving British populations of this species, and among six new populations established by translocations during the 1980s. For eight microsatellite loci, allele sizes and frequencies were analysed using samples from each of these populations. The populations clustered into three robustly differentiated groups, each of which corresponded with a geographical region (east/southeast England, Merseyside and Cumbria). The Cumbrian populations showed a further weak geographical substructuring into northern and southern clades. The populations in south Cumbria were genetically more diverse than those in any of the other regions, as judged by the mean numbers of alleles per locus and the mean heterozygosity estimates. The translocated populations clustered close to their founders and, with one exception, did not differ significantly with respect to mean allele numbers, heterozygosity or polymorphism level. However, significant genetic differentiation (as measured by unbiased RST) was found between all but one of the founder‐translocation pairs. The implications of this phylogeographic study for the future conservation of B. calamita in Britain are discussed.
Development of phosphotyrosyl (pTyr) mimetics which are stable to protein−tyrosine phosphatases (PTPs), yet can retain biological potency when incorporated into peptides, is an active area of drug ...development. Since a majority of pTyr mimetics derive their “phosphofunctionality” from phosphorus-containing moieties, such as phosphonates, evolution of new inhibitors and modes of prodrug derivatization have been restricted to chemistries appropriate for phosphorus-containing moieties. A new, nonphosphorus-containing pTyr mimetic has recently been reported, l-O-(2-malonyl)tyrosine (OMT, 5), which can be incorporated into peptides that exhibit good PTP and Src homology 2 (SH2) domain inhibitory potency. For phosphonate-based pTyr mimetics such as phosphonomethyl phenylalanine (Pmp, 2), introduction of fluorines α to the phosphorus has provided higher affinity pTyr mimetics. This strategy has now been applied to OMT, and herein is reported 4‘-O-2-(2-fluoromalonyl)-l-tyrosine (FOMT, 6), a new fluorine-containing nonphosphorus pTyr mimetic. Incorporation of FOMT into appropriate peptides results in good inhibition of both PTP and SH2 domains. In an assay measuring the inhibition of PTP 1B-mediated dephosphorylation of phosphorylated insulin receptor, the peptide Ac-D-A-D-E-X-L-amide exhibited a 10-fold enhancement in inhibitory potency for X = FOMT (19) (IC50 = 1 μM) relative to the unfluorinated peptide, X = OMT (18) (IC50 = 10 μM). Molecular modeling indicated that this increased affinity may be attributable to new hydrogen-bonding interactions between the fluorine and the enzyme catalytic site, and not due to lowering of pK a values. In a competition binding assay using the p85 PI 3-kinase C-terminal SH2 domain GST fusion construct, the inhibitory peptide, Ac-D-X-V-P-M-L-amide, showed no enhancement of inhibitory potency for X = FOMT (22) (IC50 = 18 μM) relative to the unfluorinated peptide, X = OMT (21) (IC50 = 14 μM). The use of FOMT would therefore appear to have particular potential for the development of PTP inhibitors.
The binding of several phosphonodifluoromethyl phenylalanine (F2Pmp)-containing peptides to protein-tyrosine phosphatase 1B (PTP1B) and its substrate-trapping mutants (C215S and D181A) has been ...studied using isothermal titration calorimetry. The binding of a high affinity ligand, Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2, to PTP1B (Kd = 0.24 μm) is favored by both enthalpic and entropic contributions. Disruption of ionic interactions between the side chain of Arg-47 and the N-terminal acidic residues reduces the binding affinity primarily through the reduction of the TΔS term. The role of Arg-47 may be to maximize surface contact between PTP1B and the peptide, which contributes to high affinity binding. The active site Cys-215 → Ser mutant PTP1B binds ligands with the same affinity as the wild-type enzyme. However, unlike wild-type PTP1B, peptide binding to C215S is predominately driven by enthalpy change, which likely results from the elimination of the electrostatic repulsion between the thiolate anion and the phosphonate group. The increased enthalpic contribution is offset by reduction in the binding entropy, which may be the result of increased entropy of the unbound protein caused by this mutation. The general acid-deficient mutant D181A binds the peptide 5-fold tighter than the C215S mutant, consistent with the observation that the Asp to Ala mutant is a better “substrate-trapping” reagent than C215S. The increased binding affinity for D181A as compared with the wild-type PTP1B results primarily from an increase in the ΔH of binding in the mutant, which may be related to decreased electrostatic repulsion between the phosphate moiety and PTP1B. These results have important implications for the design of high affinity PTP1B inhibitors.
The phosphonodifluoromethyl phenylalanine (F2Pmp) is superior to phosphonomethyl phenylalanine (Pmp) as a non-hydrolyzable phosphotyrosine (pTyr) mimetic. The difluoromethyl moiety increases the ...inhibitory potency of a F2Pmp-containing peptide over a Pmp-containing counterpart by 1000-fold toward the protein tyrosine phosphatase (PTPase), PTP1. Fluorine substitution at the methylene carbon have the double effect of lowering the phosphonate pKa2 as well as introducing hydrogen bonding interactions similar to the phosphate ester oxygen in pTyr. The inhibition of PTP1-catalyzed dephosphorylation reaction by both the F2Pmp and Pmp-containing peptides did not vary as a function of pH. The data indicate that both the monoanion and the dianion forms of the phosphonate bind PTP1 with equal efficiency. Thus, the better binding by the F2Pmp-peptide as compared to the Pmp-peptide is not due to the difference in pKa2. Taken together, these results offer an explanation for the increased affinity of F2Pmp for PTP1. The two fluorine atoms in F2Pmp may be able to interact with active site residues in PTP1 in a fashion analogous to that involving the phenolic oxygen and side chains in the active site of PTP1. Ki measurements for a simple phosphonic acid, Pmp- and F2Pmp-containing peptides suggest that although the principal recognition element is F2Pmp itself, the surrounding amino acids are required for high affinity binding. Comparative analysis of the inhibition of PTP1, PTPα and LAR by F2Pmp-containing peptides suggests that selective, tight-binding PTPase inhibitors can be developed.
Abstract The aim of this study was to examine effects of polymorphic genes on vertebral bone morphology and mechanical properties. Genotypes from 525 18-month-old female mice were compared to ...geometric traits obtained from micro-computed tomography and mechanical properties from compression testing. Genetic markers were associated with traits on at least 13 different chromosomes, demonstrating the complexity of genetic control over vertebral form, function and aging.
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