Structural and biophysical characterization of human myo-inositol oxygenase

Altered inositol metabolism is implicated in a number of diabetic complications. The first committed step in mammalian inositol catabolism is performed by myo-inositol oxygenase (MIOX), which catalyzes a unique four-electron dioxygen-dependent ring cleavage of myo-inositol to D-glucuronate. Here, we present the crystal structure of human MIOX in complex with myo-inosose-1 bound in a terminal mode to the MIOX diiron cluster site. Furthermore, from biochemical and biophysical results from N-terminal deletion mutagenesis we show that the N terminus is important, through coordination of a set of loops covering the active site, in shielding the active site during catalysis. EPR spectroscopy of the unliganded enzyme displays a two-component spectrum that we can relate to an open and a closed active site conformation. Furthermore, based on site-directed mutagenesis in combination with biochemical and biophysical data, we propose a novel role for Lys(127) in governing access to the diiron cluster.     

The profilin:actin complex localizes to sites of dynamic actin polymerization at the leading edge of migrating cells and pathogen-induced actin tails

A unique set of affinity-purified anti-profilin and anti-actin antibodies generated against a covalently coupled version of the profilin:actin complex was used to assess the distribution of profilin and non-filamentous actin in mouse melanoma cells. In agreement with the profilin:actin complex being the principal source of actin for filament formation, we observed extensive co-distribution of both antibody preparations with vasodilator-stimulated phosphoprotein (VASP) and the p34 subunit of the Arp2/3 complex, both of which are components of actin polymer-forming protein complexes in the cell. This suggests that the localization of profilin and actin revealed with these antibodies in fact reflects the distribution of the profilin:actin complex rather than the two proteins separately. Significantly, protruding lamellipodia and filopodia showed intensive labeling. The two antibody preparations were also used to stain HeLa cells infected with Listeria monocytogenes or vaccinia virus. In both cases, the pattern of antibody staining of the pathogen-induced microfilament arrangement differed, suggesting a varying accessibility for the antibody-binding epitopes.     

The effect of ACTH stimulation on cortisol and progesterone concentrations in intact and ovariohysterectomized domestic cats

The objective was to evaluate the adrenocortical capacity for cortisol and progesterone production in female cats, both while intact and after ovariohysterectomy. Five privately owned female cats, 1-3 years old, were used in two trials while intact at an inactive stage of the cycle, and again in two trials, 2 weeks after ovariohysterectomy. The four trials were: intact saline injection control trial; intact ACTH injection (0.125 mg); ovariohysterectomized saline injection control trial; and ovariohysterectomized ACTH injection. Blood samples were obtained by an indwelling cephalic vein catheter at -30 and 0 min (immediately before injections) and at 60, 90, 120 and 180 min after injection. The mean basal pre-treatment concentrations of cortisol in the intact and ovariohysterectomized cats were 33 +/- 19 and 32 +/- 19 nmol/L, respectively; the corresponding values for progesterone were 1.1 +/- 0.6 and 0.7 +/- 0.6 nmol/L, respectively. Saline did not alter the serum cortisol or progesterone concentrations. In contrast, both cortisol and progesterone were elevated after ACTH, with peak values at 90 min and returned to basal levels at approximately 180 min. There was a positive correlation between cortisol and progesterone concentrations (r = 0.8, P < 0.05). In some instances, the procedure used to restrain the cats during blood collection induced increases in cortisol and progesterone of the same magnitude as when the ACTH was administered; these effects of restraint could alter the results of assisted reproduction efforts.     

NO binding to naphthalene dioxygenase

Nitric oxide (NO) is commonly used as an analogue for dioxygen in structural and spectroscopic studies of oxygen binding and oxygen activation. In this study, crystallographic structures of naphthalene dioxygenase (NDO) in complex with nitric oxide are reported. In the presence of the aromatic substrate indole, NO is bound end-on to the active-site mononuclear iron of NDO. The structural observations correlate well with spectroscopic measurements of NO binding to NDO in solution. However, the end-on binding of NO is in contrast to the recently reported structure of oxygen to the active-site iron of NDO that binds side-on. While NO is a good oxygen analogue with many similarities to O₂, the different binding mode of NO to the active-site iron atom leads to different mechanistic implications. Hence, caution needs to be used in extrapolating NO as an analogue to O₂ binding.     

Seasonal polyphenism and developmental trade-offs between flight ability and egg laying in a pierid butterfly

Butterflies have competing demands for flight ability depending on, for example, mating system, predation pressure, the localization of host plants and dispersal needs. The flight apparatus, however, is costly to manufacture and therefore trade-offs are expected since resources are limited and must be allocated between flight ability and other functions, such as reproduction. Trade-offs between flight and reproduction may be difficult to reveal since they interact with other factors and can be confounded by differences in resource consumption. Previous studies have shown that adults of the summer generation of Pieris napi have relatively larger thoraxes compared with the spring generation. To study whether difference in thorax size results in a trade-off between flight ability and reproduction among the two generations, we conducted a split-brood experiment under common garden conditions. Our results show that summer generation adults have a higher dispersal capacity measured as flight duration in five different temperatures. Reproductive output differed between the two developmental pathways; spring generation females had a significantly higher output of eggs compared with summer generation females. We suggest that this is a consequence of a resource-allocation trade-off made during pupal development implemented by different demands for flight between the spring and summer generations. The significance of this finding is discussed in relation to reproduction and mobility in butterflies.     

Material properties of the ovine mitral valve anterior leaflet in vivo from inverse finite element analysis

We measured leaflet displacements and used inverse finite-element analysis to define, for the first time, the material properties of mitral valve (MV) leaflets in vivo. Sixteen miniature radiopaque markers were sewn to the MV annulus, 16 to the anterior MV leaflet, and 1 on each papillary muscle tip in 17 sheep. Four-dimensional coordinates were obtained from biplane videofluoroscopic marker images (60 frames/s) during three complete cardiac cycles. A finite-element model of the anterior MV leaflet was developed using marker coordinates at the end of isovolumic relaxation (IVR; when the pressure difference across the valve is approximately 0), as the minimum stress reference state. Leaflet displacements were simulated during IVR using measured left ventricular and atrial pressures. The leaflet shear modulus (G(circ-rad)) and elastic moduli in both the commisure-commisure (E(circ)) and radial (E(rad)) directions were obtained using the method of feasible directions to minimize the difference between simulated and measured displacements. Group mean (+/-SD) values (17 animals, 3 heartbeats each, i.e., 51 cardiac cycles) were as follows: G(circ-rad) = 121 +/- 22 N/mm2, E(circ) = 43 +/- 18 N/mm2, and E(rad) = 11 +/- 3 N/mm2 (E(circ) > E(rad), P < 0.01). These values, much greater than those previously reported from in vitro studies, may result from activated neurally controlled contractile tissue within the leaflet that is inactive in excised tissues. This could have important implications, not only to our understanding of mitral valve physiology in the beating heart but for providing additional information to aid the development of more durable tissue-engineered bioprosthetic valves.     

Optimized in vitro and in vivo expression of proteorhodopsin: a seven-transmembrane proton pump

Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro production of pR using a commercial cell-free synthesis system yielding 1.0mg purified protein per milliliter of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purification yielding 5mg of essentially pure functional protein per liter of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for commercial biotechnological applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural determination of pR are discussed.     

Real-time viability assay based on 51Cr retention in adherent cells

The chromium (51Cr) release assay has been widely used for viability measurements, even though it has major disadvantages such as high manual workload and poor time resolution. By the use of LigandTracer 51Cr release viability measurements on adherent cells can be significantly simplified and improved. LigandTracer enables a time-resolved detection of 5SCr in target cells, with the result that the effect of toxic material is updated continuously throughout the experiment. Here we explain the principle behind this novel real-time viability assay and show viability curves for known toxic compounds on A431 and U343MGaCl2:6 cell lines.     

Lipid droplet changes in proliferating and quiescent 3T3 fibroblasts

Lipid droplets (LDs) are fat-storing organelles present in virtually all eukaryotic cells and involved in many aspects of cell biology related to lipid metabolism and cholesterol homeostasis. In this study, we investigated the presence of LDs in proliferating and quiescent (contact-inhibited) 3T3 fibroblasts to verify a correlation with cell growth. LDs were characterized by Nile red staining, positivity to adipophilin and negativity to perilipin. LDs were numerous in proliferating cells, but very few in quiescent cells. However, the fraction of quiescent cells, which resumed proliferation after scratch-wound assay, also resumed the formation of LDs. In proliferating cells, the number of LDs correlated with the DNA content, suggesting a continuous accumulation of LDs during cell growth. These findings were supported by biochemical data showing much higher rates of cholesterol esterification and triglyceride synthesis in proliferating cells. Both filipin staining and the fluorescent cholesterol analog dehydroergosterol revealed the presence of an intense traffic of free cholesterol, mediated by acidic vesicles, in proliferating cells. Nile red ratiometric measurements revealed a different lipid composition of LDs in proliferating and quiescent cells. Changes in the number and composition of LDs were also found in growing cells treated with inhibitors of cholesterol esterification (Sandoz 58-035), endosomal cholesterol efflux (U18666A) and V-ATPase (bafilomycin-A1).     

Reduced levels of mitochondrial DNA increases the toxicity of 9-beta-D-arabinofuranosylguanine (araG)

Incubation of cells with thymidine (dThd) is known to cause dNTP pool imbalance as well as deletions and depletion of the mtDNA. In order to gain further understanding in the events involved in dThd toxicity over time, H9 cells were cultured for 20 months in the presence or absence of 1 micro M dThd. The level of mtDNA was reduced by 90% in the cells grown in dThd as compared to the control cells. The H9/dThd cells also showed a 100-fold increased sensitivity towards the cytotoxicity of the antileukemic compound 9-beta-D-arabinofuranolsylguanine (araG).