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.     

Nano-to-micro scale dynamics of P-selectin detachment from leukocyte interfaces. III. Numerical simulation of tethering under flow

Transient capture of cells or model microspheres from flow over substrates sparsely coated with adhesive ligands has provided significant insight into the unbinding kinetics of leukocyte:endothelium adhesion complexes under external force. Whenever a cell is stopped by a point attachment, the full hydrodynamic load is applied to the adhesion site within an exceptionally short time-less than the reciprocal of the hydrodynamic shear rate (e.g., typically <0.01 s). The decay in numbers of cells or beads that remain attached to a surface has been used as a measure of the kinetics of molecular bond dissociation under constant force, revealing a modest increase in detachment rate at growing applied shear stresses. On the other hand, when detached under steady ramps of force with mechanical probes (e.g., the atomic force microscope and biomembrane force probe), P-selectin:PSGL-1 adhesion bonds break at rates that increase enormously under rising force, yielding 100-fold faster off rates at force levels comparable to high shear. The comparatively weak effect of force on tether survival in flow chamber experiments could be explained by a possible partition of the load amongst several bonds. However, a comprehensive understanding of the difference in kinetic behavior requires us to also inspect other factors affecting the dynamics of attachment-force buildup, such as the interfacial compliance of all linkages supporting the adhesion complex. Here, combining the mechanical properties of the leukocyte interface measured in probe tests with single-bond kinetics and the kinetics of cytoskeletal dissociation, we show that for the leukocyte adhesion complex P-selectin:PSGL-1, a detailed adhesive dynamics simulation accurately reproduces the tethering behavior of cells observed in flow chambers. Surprisingly, a mixture of 10% single bonds and 90% dimeric bonds is sufficient to fully match the data of the P-selectin:PSGL-1 experiments, with the calculated decay in fraction of attached cells still appearing exponential.     

Molecular and quantitative genetic divergence among populations of house mice with known evolutionary histories

Evolutionary biologists have long been interested in the processes influencing population differentiation, but separating the effects of neutral and adaptive evolution has been an obstacle for studies of population subdivision. A recently developed method allows tests of whether disruptive (ie, spatially variable) or stabilizing (ie, spatially uniform) selection is influencing phenotypic differentiation among subpopulations. This method, referred to as the F(ST) vs Q(ST) comparison, separates the total additive genetic variance into within- and among-population components and evaluates this level of differentiation against a neutral hypothesis. Thus, levels of neutral molecular (F(ST)) and quantitative genetic (Q(ST)) divergence are compared to evaluate the effects of selection and genetic drift on phenotypic differentiation. Although the utility of such comparisons appears great, its accuracy has not yet been evaluated in populations with known evolutionary histories. In this study, F(ST) vs Q(ST) comparisons were evaluated using laboratory populations of house mice with known evolutionary histories. In this model system, the F(ST) vs Q(ST) comparisons between the selection groups should reveal quantitative trait differentiation consistent with disruptive selection, while the F(ST) vs Q(ST) comparisons among lines within the selection groups should suggest quantitative trait differentiation in agreement with drift. We find that F(ST) vs Q(ST) comparisons generally produce the correct evolutionary inference at each level in the population hierarchy. Additionally, we demonstrate that when strong selection is applied between populations Q(ST) increases relative to Q(ST) among populations diverging by drift. Finally, we show that the statistical properties of Q(ST), a variance component ratio, need further investigation.     

Differential inhibition of inducible T cell cytokine secretion by potent iron chelators

Effector functions and proliferation of T helper (Th) cells are influenced by cytokines in the environment. Th1 cells respond to a synergistic effect of interleukin-12 (IL-12) and interleukin-18 (IL-18) to secrete interferon-gamma (IFN-gamma). In contrast, Th2 cells respond to interleukin-4 (IL-4) to secrete IL-4, interleukin-13 (IL-13), interleukin-5 (IL-5), and interleukin-10 (IL-10). The authors were interested in identifying nonpeptide inhibitors of the Th1 response selective for the IL-12/IL-18-mediated secretion of IFN-gamma while leaving the IL-4-mediated Th2 cytokine secretion relatively intact. The authors established a screening protocol using human peripheral blood mononuclear cells (PBMCs) and identified the hydrazino anthranilate compound 1 as a potent inhibitor of IL-12/IL-18-mediated IFN-gamma secretion from CD3(+) cells with an IC(50) around 200 nM. The inhibitor was specific because it had virtually no effect on IL-4-mediated IL-13 release from the same population of cells. Further work established that compound 1 was a potent intracellular iron chelator that inhibited both IL-12/IL-18- and IL-4-mediated T cell proliferation. Iron chelation affects multiple cellular pathways in T cells. Thus, the IL-12/IL-18-mediated proliferation and IFN-gamma secretion are very sensitive to intracellular iron concentration. However, the IL-4-mediated IL-13 secretion does not correlate with proliferation and is partially resistant to potent iron chelation.     

Insect octopamine receptors: a new classification scheme based on studies of cloned Drosophila G-protein coupled receptors

Summary Insect octopamine receptors are G-protein coupled receptors. They can be coupled to second messenger pathways to mediate either increases or decreases in intracellular cyclic AMP levels or the generation of intracellular calcium signals. Insect octopamine receptors were originally classified on the basis of second messenger changes induced in a variety of intact tissue preparations. Such a classification system is problematic if more than one receptor subtype is present in the same tissue preparation. Recent progress on the cloning and characterization in heterologous cell systems of octopamine receptors from Drosophila and other insects is reviewed. A new classification system for insect octopamine receptors into “α-adrenergic-like octopamine receptors (OctαRs)”, “β-adrenergic-like octopamine receptors (OctβRs)” and “octopamine/tyramine (or tyraminergic) receptors” is proposed based on their similarities in structure and in signalling properties with vertebrate adrenergic receptors. In future studies on the molecular basis of octopamine signalling in individual tissues it will be essential to identify the relative expression levels of the different classes of octopamine receptor present. In addition, it will be essential to identify if co-expression of such receptors in the same cells results in the formation of oligomeric receptors with specific emergent pharmacological and signalling properties.     

Zebrafish Hsp70 is required for embryonic lens formation

Heat shock proteins (Hsps) were originally identified as proteins expressed after exposure of cells to environmental stress. Several Hsps were subsequently shown to play roles as molecular chaperones in normal intracellular protein folding and targeting events and to be expressed during discrete periods in the development of several embryonic tissues. However, only recently have studies begun to address the specific developmental consequences of inhibiting Hsp expression to determine whether these molecular chaperones are required for specific developmental events. We have previously shown that the heat-inducible zebrafish hsp70 gene is expressed during a distinct temporal window of embryonic lens formation at normal growth temperatures. In addition, a 1.5-kb fragment of the zebrafish hsp70 gene promoter is sufficient to direct expression of a gfp reporter gene to the lens, suggesting that the hsp70 gene is expressed as part of the normal lens development program. Here, we used microinjection of morpholino-modified antisense oligonucleotides (MOs) to reduce Hsp70 levels during zebrafish development and to show that Hsp70 is required for normal lens formation. Hsp70-MO-injected embryos exhibited a small-eye phenotype relative to wild-type and control-injected animals, with the phenotype discernable during the second day of development. Histological and immunological analysis revealed a small, underdeveloped lens. Numerous terminal deoxynucleotidyl transferase-mediated dUTP-fluoroscein nick-end labeling (TUNEL)-positive nuclei appeared in the lens of small-eye embryos after 48 hours postfertilization (hpf), whereas they were no longer apparent in untreated embryos by this age. Lenses transplanted from hsp70-MO-injected embryos into wild-type hosts failed to recover and retained the immature morphology characteristic of the small-eye phenotype, indicating that the lens phenotype is lens autonomous. Our data suggest that the lens defect in hsp70-MO-injected embryos is predominantly at the level of postmitotic lens fiber differentiation, a result supported by the appearance of mature lens organization in these embryos by 5 days postfertilization, once morpholino degradation or dilution has occurred.     

Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein

During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and 1H NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.     

Inhibition by calyculin A and okadaic acid of the Ca(2+) release-activated Ca(2+) entry pathway in rat basophilic leukemia cells: evidence for regulation by type 1/2A serine/threonine phosphatase activity

Using a combination of fluorescence measurements of intracellular Ca(2+) ion concentration ([Ca(2+)](i)) and membrane potential we have investigated the sensitivity to serine/threonine phosphatase inhibition of Ca(2+) entry stimulated by activation of the Ca(2+) release-activated Ca(2+) (CRAC) entry pathway in rat basophilic leukemia cells. In both suspension and adherent cells, addition of the type 1/2A phosphatase inhibitor calyculin A, during activation of CRAC uptake, resulted in a fall in [Ca(2+)](i) to near preactivation levels. Pre-treatment with calyculin A abolished the component of the Ca(2+) rise associated with activation of CRAC uptake and inhibited Mn(2+) entry, consistent with a requirement of phosphatase activity for activation of the pathway. Depletion of intracellular Ca(2+) stores is accompanied by a large depolarisation which is absolutely dependent upon Ca(2+) entry via the CRAC uptake pathway. Application of calyculin A or okadaic acid, a structurally unrelated phosphatase antagonist inhibits this depolarisation. Taken in concert, these data demonstrate a marked sensitivity of the CRAC entry pathway to inhibition by calyculin A and okadaic acid.     

Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: the phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron-sulfur cluster F(X)

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A(1), the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron-sulfur centre F(X) and the phylloquinone bound to either the PsaA (A(1A)) or the PsaB (A(1B)) subunit of the reaction centre and the equilibrium between the iron-sulfur centres F(A) and F(B). The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A(1)) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F(X). A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A(1B) quinone and slightly endergonic, in the case of the A(1A) quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A(0) on both electron transfer branches and the reduction of F(A) by F(X).     

Glutamine inhibits cytokine-induced apoptosis in human colonic epithelial cells via the pyrimidine pathway

Glutamine (Gln) prevents apoptosis in intestinal epithelial cells, but the mechanism(s) remain unknown. Gln-derived metabolites include ammonia, glutamate (Glu), glutathione (GSH), and nucleotides. We previously showed that Gln potently inhibited apoptosis in cytokine-treated human colonic HT-29 cells; this effect was specific to Gln, unaffected by Glu, and unrelated to intracellular GSH. The current research examines mechanism(s) for Gln-induced antiapoptotic effects in HT-29 cells treated with TNF-alpha-related apoptosis-inducing ligand (TRAIL). Proliferating cells were treated with Gln or selected Gln metabolites for 24 h. Cells were then treated with TRAIL and Gln or its downstream metabolites, and apoptosis was assessed at 8 h after treatment. The purine and pyrimidine precursors inosine and orotate inhibited TRAIL-induced apoptosis. However, inhibition of purine synthesis with azaserine did not alter the potent antiapoptotic effect of Gln. In contrast, the pyrimidine synthesis inhibitor, acivicin, completely prevented this response. Supplementation with the pyrimidine uracil or the pyrimidine precursor orotate rescued the acivicin-induced blockade of Gln antiapoptotic action. Removal of bicarbonate, a substrate for pyrimidine synthesis, also inhibited the antiapoptotic effects of Gln. Uracil and thymine alone also significantly decreased TRAIL-induced apoptosis. The antiapoptotic effects of Gln were independent of DNA/RNA synthesis as measured by flow cytometry and bromodeoxyuridine incorporation. In conclusion, Gln prevents TRAIL-induced apoptosis in HT-29 cells through a mechanism involving the pyrimidine pathway. Our data also demonstrate the novel antiapoptotic effects of pyrimidine bases and their precursor orotate in these human intestinal cells.