Mutations that cause osteoglophonic dysplasia define novel roles for FGFR1 in bone elongation

Activating mutations in the genes for fibroblast growth factor receptors 1-3 (FGFR1-3) are responsible for a diverse group of skeletal disorders. In general, mutations in FGFR1 and FGFR2 cause the majority of syndromes involving craniosynostosis, whereas the dwarfing syndromes are largely associated with FGFR3 mutations. Osteoglophonic dysplasia (OD) is a "crossover" disorder that has skeletal phenotypes associated with FGFR1, FGFR2, and FGFR3 mutations. Indeed, patients with OD present with craniosynostosis, prominent supraorbital ridge, and depressed nasal bridge, as well as the rhizomelic dwarfism and nonossifying bone lesions that are characteristic of the disorder. We demonstrate here that OD is caused by missense mutations in highly conserved residues comprising the ligand-binding and transmembrane domains of FGFR1, thus defining novel roles for this receptor as a negative regulator of long-bone growth.     

Conditional expression of 15-lipoxygenase-1 inhibits the selenoenzyme thioredoxin reductase: modulation of selenoproteins by lipoxygenase enzymes

The selenoenzyme thioredoxin reductase regulates redox-sensitive proteins involved in inflammation and carcinogenesis, including ribonucleotide reductase, p53, NFkappaB, and others. Little is known about endogenous cellular factors that modulate thioredoxin reductase activity. Here we report that several metabolites of 15-lipoxygenase-1 inhibit purified thioredoxin reductase in vitro. 15(S)-Hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic acid, a metastable hydroperoxide generated by 15-lipoxygenase-1, and 4-hydroxy-2-nonenal, its non-enzymatic rearrangement product inhibit thioredoxin reductase with IC(50) = 13 +/- 1.5 microm and 1 +/- 0.2 microm, respectively. Endogenously generated metabolites of 15-lipoxygenase-1 also inhibit thioredoxin reductase in HEK-293 cells that harbor a 15-LOX-1 gene under the control of an inducible promoter complex. Conditional, highly selective induction of 15-lipoxygenase-1 caused an inhibition of ribonucleotide reductase activity, cell cycle arrest in G(1), impairment of anchorage-independent growth, and accumulation of the pro-apoptotic protein BAX. All of these responses are consistent with inhibition of thioredoxin reductase via 15-lipoxygenase-1 overexpression. In contrast, metabolites of 5-lipoxygenase were poor inhibitors of isolated thioredoxin reductase, and the overexpression of 5-lipoxygenase did not inhibit thioredoxin reductase or cause a G cell cycle arrest. The influences of 15-lipoxygenase-1 on (1)inflammation, cell growth, and survival may be attributable, in part, to inhibition of thioredoxin reductase and several redox-sensitive processes subordinate to thioredoxin reductase.     

Specificity of the MAP kinase ERK2 for phosphorylation of tyrosine hydroxylase

Short-term regulation of catecholamine biosynthesis involves reversible phosphorylation of several serine residues in the N-terminal regulatory domain of tyrosine hydroxylase. The MAP kinases ERK1/2 have been identified as responsible for phosphorylation of Ser31. As an initial step in elucidating the effects of phosphorylation of Ser31 on the structure and activity of tyrosine hydroxylase, the kinetics of phosphorylation of the rat enzyme by recombinant rat ERK2 have been characterized. Complete phosphorylation results in incorporation of 2mol of phosphate into each subunit of tyrosine hydroxylase. The S8A and S31A enzymes only incorporate a single phosphate, while the S19A and S40A enzymes incorporate two. Phosphorylation of S8A tyrosine hydroxylase is nine times as rapid as phosphorylation of the S31A enzyme, consistent with a ninefold preference of ERK2 for Ser31 over Ser8.     

Identification of tyrosine hydroxylase as a physiological substrate for Cdk5

Cyclin-dependent kinase 5 (Cdk5) is emerging as a neuronal protein kinase involved in multiple aspects of neurotransmission in both post- and presynaptic compartments. Within the reward/motor circuitry of the basal ganglia, Cdk5 regulates dopamine neurotransmission via phosphorylation of the postsynaptic signal transduction pathway integrator, DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, M(r) 32,000). Cdk5 has also been implicated in regulating various steps in the presynaptic vesicle cycle. Here we report that Cdk5 phosphorylates tyrosine hydroxylase (TH), the key enzyme for synthesis of dopamine. Using phosphopeptide mapping, site-directed mutagenesis, and phosphorylation state-specific antibodies, the site was identified as Ser31, a previously defined extracellular signal-regulated kinases 1/2 (ERK1/2) site. The phosphorylation of Ser31 by Cdk5 versus ERK1/2 was investigated in intact mouse striatal tissue using a pharmacological approach. The results indicated that Cdk5 phosphorylates TH directly and also regulates ERK1/2-dependent phosphorylation of TH through the phosphorylation of mitogen-activated protein kinase kinase 1 (MEK1). Finally, phospho-Ser31 TH levels were increased in dopaminergic neurons of rats trained to chronically self-administer cocaine. These results demonstrate direct and indirect regulation of the phosphorylation state of a Cdk5/ERK1/2 site on TH and suggest a role for these pathways in the neuroadaptive changes associated with chronic cocaine exposure.     

Male reproductive suppression in the cooperatively breeding fish Neolamprologus pulcher

In most cooperative breeders, dominants suppress the reproductionof subordinates. However, two previous studies of Neolamprologuspulcher, a cooperatively breeding cichlid fish, have suggestedthat socially subordinate helper males sneak fertilizationsfrom dominant breeding males. If such sneaking does occur, boththeoretical work and empirical studies of other fish speciessuggest that sperm competition will select for increased reproductiveinvestment by sneaker males, relative to more dominant males.To address these issues, we quantified gonadal investment andsperm characteristics of 41 N. pulcher male breeders and 62male helpers from 55 groups in Lake Tanganyika. Gonadal investmentfollowed patterns consistent with reproductive suppression,with breeders having considerably larger testes masses thanhelpers. Breeders also had faster and longer swimming spermand a higher percentage of motile sperm compared to helpers.However, sperm characteristics of large helpers were similarto those of breeders, but these same helpers had lower testesmasses. Thus, large helpers had sperm that were physiologicallyequivalent to that of breeders, but their relatively small gonadsimply that they were reproductively suppressed.     

Problem formulation in the environmental risk assessment for genetically modified plants

Problem formulation is the first step in environmental risk assessment (ERA) where policy goals, scope, assessment endpoints, and methodology are distilled to an explicitly stated problem and approach for analysis. The consistency and utility of ERAs for genetically modified (GM) plants can be improved through rigorous problem formulation (PF), producing an analysis plan that describes relevant exposure scenarios and the potential consequences of these scenarios. A properly executed PF assures the relevance of ERA outcomes for decision-making. Adopting a harmonized approach to problem formulation should bring about greater uniformity in the ERA process for GM plants among regulatory regimes globally. This paper is the product of an international expert group convened by the International Life Sciences Institute (ILSI) Research Foundation.     

Analysis of genetic diversity in flowering dogwood natural stands using microsatellites: the effects of dogwood anthracnose

Flowering dogwood (Cornus florida L.) populations recently have experienced severe declines caused by dogwood anthracnose. Mortality has ranged from 48 to 98%, raising the concern that genetic diversity has been reduced significantly. Microsatellite data were used to evaluate the level and distribution of genetic variation throughout much of the native range of the tree. Genetic variation in areas affected by anthracnose was as high as or higher than areas without die-offs. We found evidence of four widespread, spatially contiguous genetic clusters. However, there was little relationship between geographic distance and genetic difference. These observations suggest that high dispersal rates and large effective population sizes have so far prevented rapid loss of genetic diversity. The effects of anthracnose on demography and community structure are likely to be far more consequential than short-term genetic effects.     

Crystal structures of nitroalkane oxidase: insights into the reaction mechanism from a covalent complex of the flavoenzyme trapped during turnover

Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones with the production of H(2)O(2) and nitrite. The flavoenzyme is a new member of the acyl-CoA dehydrogenase (ACAD) family, but it does not react with acyl-CoA substrates. We present the 2.2 A resolution crystal structure of NAO trapped during the turnover of nitroethane as a covalent N5-FAD adduct (ES*). The homotetrameric structure of ES* was solved by MAD phasing with 52 Se-Met sites in an orthorhombic space group. The electron density for the N5-(2-nitrobutyl)-1,5-dihydro-FAD covalent intermediate is clearly resolved. The structure of ES was used to solve the crystal structure of oxidized NAO at 2.07 A resolution. The c axis for the trigonal space group of oxidized NAO is 485 A, and there are six subunits (1(1)/(2) holoenzymes) in the asymmetric unit. Four of the active sites contain spermine (EI), a weak competitive inhibitor, and two do not contain spermine (E(ox)). The active-site structures of E(ox), EI, and ES* reveal a hydrophobic channel that extends from the exterior of the protein and terminates at Asp402 and the N5 position on the re face of the FAD. Thus, Asp402 is in the correct position to serve as the active-site base, where it is proposed to abstract the alpha proton from neutral nitroalkane substrates. The structures for NAO and various members of the ACAD family overlay with root-mean-square deviations between 1.7 and 3.1 A. The homologous region typically spans more than 325 residues and includes Glu376, which is the active-site base in the prototypical member of the ACAD family. However, NAO and the ACADs exhibit differences in hydrogen-bonding patterns between the respective active-site base, substrate molecules, and FAD. These likely differentiate NAO from the homologues and, consequently, are proposed to result in the unique reaction mechanism of NAO.     

Reduction and oxidation of the active site iron in tyrosine hydroxylase: kinetics and specificity

Tyrosine hydroxylase (TyrH) is a pterin-dependent enzyme that catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine. The oxidation state of the active site iron atom plays a central role in the regulation of the enzyme. The kinetics of reduction of ferric TyrH by several reductants were determined by anaerobic stopped-flow spectroscopy. Anaerobic rapid freeze-quench EPR confirmed that the change in the near-UV absorbance of TyrH upon adding reductant corresponded to iron reduction. Tetrahydrobiopterin reduces wild-type TyrH following a simple second-order mechanism with a rate constant of 2.8 +/- 0.1 mM(-)(1) s(-)(1). 6-Methyltetrahydropterin reduces the ferric enzyme with a second-order rate constant of 6.1 +/- 0.1 mM(-)(1) s(-)(1) and exhibits saturation kinetics. No EPR signal for a radical intermediate was detected. Ascorbate, glutathione, and 1,4-benzoquinone all reduce ferric TyrH, but much more slowly than tetrahydrobiopterin, suggesting that the pterin is a physiological reductant. E332A TyrH, which has an elevated K(m) for tetrahydropterin in the catalytic reaction, is reduced by tetrahydropterins with the same kinetic parameters as those of the wild-type enzyme, suggesting that BH(4) does not bind in the catalytic conformation during the reduction. Oxidation of ferrous TyrH by molecular oxygen can be described as a single-step second-order reaction, with a rate constant of 210 mM(-)(1) s(-)(1). S40E TyrH, which mimics the phosphorylated state of the enzyme, has oxidation and reduction kinetics similar to those of the wild-type enzyme, suggesting that phosphorylation does not directly regulate the interconversion of the ferric and ferrous forms.     

Influence of patellofemoral articular geometry and material on mechanics of the unresurfaced patella

Patellar resurfacing during knee replacement is still under debate, with several studies reporting higher incidence of anterior knee pain in unresurfaced patellae. Congruency between patella and femur impacts the mechanics of the patellar cartilage and strain in the underlying bone, with higher stresses and strains potentially contributing to cartilage wear and anterior knee pain. The material properties of the articulating surfaces will also affect load transfer between femur and patella. The purpose of this study was to evaluate the mechanics of the unresurfaced patella and compare with natural and resurfaced conditions in a series of finite element models of the patellofemoral joint. In the unresurfaced analyses, three commercially available implants were compared, in addition to an 'ideal' femoral component which replicated the geometry, but not the material properties, of the natural femur. Hence, the contribution of femoral component material properties could be assessed independently from geometry changes. The ideal component tracked the kinematics and patellar bone strain of the natural knee, but had consistently inferior contact mechanics. In later flexion, compressive patellar bone strain in unresurfaced conditions was substantially higher than in resurfaced conditions. Understanding how femoral component geometry and material properties in unresurfaced knee replacement alters cartilage contact mechanics and bone strain may aid in explaining why the incidence of anterior knee pain is higher in the unresurfaced population, and ultimately contribute to identifying criteria to pre-operatively predict which patients are suited to an unresurfaced procedure and reducing the incidence of anterior knee pain in the unresurfaced patient population.