The iron chelator desferrioxamine inhibits atherosclerotic lesion development and decreases lesion iron concentrations in the cholesterol-fed rabbit

Several epidemiological studies have suggested that increased iron stores are associated with increased atherosclerotic events. In order to test the hypothesis that decreasing the vascular level of iron slows lesion growth, we examined the effects of the iron chelator Desferal (72 mg/kg/day, 5 days/week) on atherosclerosis and lesion iron content in cholesterol-fed New Zealand White rabbits. Rabbits were fed with a 1% w/w cholesterol diet for either 8 weeks (and for the last 5 weeks injected daily with Desferal) or 12 weeks (and for the last 9 weeks injected with Desferal). Controls were injected with saline. A significant reduction in average lesion area (p = 0.038) was observed in the 12-week treated animals compared with the 12-week controls. The average lesion iron level of the 12-week treated animals (58 ppm dry wt) was also significantly lower (p = 0.030) than in 12-week control animals (95 ppm dry wt), as measured using nuclear microscopy with the combination of scanning transmission ion microscopy, Rutherford back-scattering spectroscopy, and particle-induced X-ray emission. No reduction in lesion area or iron content was observed in the 8-week treated animals compared with controls, and no change in lesion zinc concentration was observed for either group. Our data strengthen the concept that iron contributes to the early stages of the development of atherosclerosis.     

Impact of nutrition on oocyte quality: cumulative effects of body composition and diet leading to hyperinsulinemia in cattle

The present study sought to assess the combined effects of body composition and diet (level of feeding) on the postfertilization developmental potential of oocytes recovered from heifers using ultrasound-guided transvaginal follicular aspiration and to relate oocyte quality to the metabolic status of these animals. By collecting oocytes on repeated occasions spanning several weeks, it was possible to assess the cumulative effects of changes in nutritional status on oocyte quality over this period. Twenty-four heifers of low and moderate body condition were placed on one of two levels of feeding (equivalent to once or twice the maintenance requirements of these animals). Oocytes were recovered at two defined time points within each of three successive estrous cycles and were matured, fertilized, and cultured to the blastocyst stage in vitro. The results show that the effect of feeding level on oocyte quality is dependent on the body condition of the animal, with the high level of feeding being beneficial to oocytes from animals of low body condition but detrimental to oocytes from animals of moderately high body condition. Furthermore, the effects of high levels of feeding on oocyte quality were cumulative, with blastocyst yields for relatively fat heifers on twice the maintenance requirement deteriorating with time relative to yields for relatively thin heifers on the same level of feeding. Finally, a significant proportion of the moderately fat animals on the high level of feeding were hyperinsulinemic, and we show, to our knowledge for the first time in ruminants, that this condition is associated with impaired oocyte quality.     

Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy

Global analysis of fluorescence and associated anisotropy decays of intrinsic tissue fluorescence offers a sensitive and non-invasive probe of the metabolically critical free/enzyme-bound states of intracellular NADH in neural tissue. Using this technique, we demonstrate that the response of NADH to the metabolic transition from normoxia to hypoxia is more complex than a simple increase in NADH concentration. The concentration of free NADH, and that of an enzyme bound form with a relatively low lifetime, increases preferentially over that of other enzyme bound NADH species. Concomitantly, the intracellular viscosity is reduced, likely due to the osmotic swelling of mitochondria. These conformation and environmental changes effectively decrease the tissue fluorescence average lifetime, causing the usual total fluorescence increase measurements to significantly underestimate the calculated concentration increase. This new discrimination of changes in NADH concentration, conformation, and environment provides the foundation for quantitative functional imaging of neural energy metabolism.     

Agonist-induced up-regulation of human somatostatin receptor type 1 is regulated by beta-arrestin-1 and requires an essential serine residue in the receptor C-tail

We have previously shown that the human somatostatin receptor type 1 (hSSTR1) does not undergo agonist-induced internalization, but is instead up-regulated at the membrane upon prolonged somatostatin (SST) exposure. The deletion of the carboxyterminal C-tail of the receptor completely abolishes up-regulation. To identify molecular signals that mediate hSSTR1 up-regulation, we created mutant receptors with progressive C-tail deletions. Up-regulation was found to be absent in mutants lacking residues Lys359-Ser360-Arg361. Moreover, point mutation of Ser360 to Ala completely abolished up-regulation. The coexpression of wild type hSSTR1 with V53D, a dominant negative mutant of beta-arrestin-1, completely blocked hSSTR1 up-regulation. Further analysis demonstrated that calcium-calmodulin (CaM) dependent kinases were essential for the SST-induced up-regulation response. Like wild type receptors, all mutants failed to internalize after agonist exposure and were able to inhibit forskolin-stimulated cAMP accumulation. Taking these data together, we suggest that SST-induced hSSTR1 up-regulation is critically dependent upon a specific Lys-Ser-Arg sequence in the C-tail of the receptor, with Ser360 being essential. Up-regulation also requires the participation of CaM protein kinases and interactions with beta-arrestins. In contrast, coupling to adenyl cyclase (AC) and internalization occur independently of molecular signals in the receptor's C-tail.     

Stress coping style predicts aggression and social dominance in rainbow trout

Social stress is frequently used as a model for studying the neuroendocrine mechanisms underlying stress-induced behavioral inhibition, depression, and fear conditioning. It has previously been shown that social subordination may result in increased glucocorticoid release and changes in brain signaling systems. However, it is still an open question which neuroendocrine and behavioral differences are causes, and which are consequences of social status. Using juvenile rainbow trout of similar size and with no apparent differences in social history, we demonstrate that the ability to win fights for social dominance can be predicted from the duration of a behavioral response to stress, in this case appetite inhibition after transfer to a new environment. Moreover, stress responsiveness in terms of confinement-induced changes in plasma cortisol was negatively correlated to aggressive behavior. Fish that exhibited lower cortisol responses to a standardized confinement test were markedly more aggressive when being placed in a dominant social position later in the study. These findings support the view that distinct behavioral-physiological stress coping styles are present in teleost fish, and these coping characteristics influence both social rank and levels of aggression.     

Dual mechanisms for shedding of the cellular prion protein

The cellular prion protein (PrP(C)) is essential for the pathogenesis and transmission of prion diseases. Whereas the majority of PrP(C) is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor, a secreted form of the protein has been identified. Here we show that PrP(C) can be shed into the medium of human neuroblastoma SH-SY5Y cells by both protease- and phospholipase-mediated mechanisms. The constitutive shedding of PrP(C) was inhibited by a range of hydroxamate-based zinc metalloprotease inhibitors in a manner identical to the alpha-secretase-mediated shedding of the amyloid precursor protein, indicating a proteolytic shedding mechanism. Like amyloid precursor protein, this zinc metalloprotease-mediated shedding of PrP(C) could be stimulated by phorbol myristate acetate and by copper ions. The lipid raft-disrupting agents filipin and methyl-beta-cyclodextrin promoted the shedding of PrP(C) via a distinct mechanism that was not inhibited by hydroxamate-based inhibitors. Filipin-mediated shedding of PrP(C) is likely to occur via phospholipase cleavage of the GPI anchor, since a transmembrane polypeptide-anchored PrP construct was not shed in response to filipin treatment. Collectively, our data indicate that shedding of PrP(C) can occur via both secretase-like proteolytic cleavage of the protein and phospholipase cleavage of the GPI anchor moiety.     

The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells

We describe a novel murine progenitor cell population localised to a previously uncharacterised region between sebaceous glands and the hair follicle bulge, defined by its reactivity to the thymic epithelial progenitor cell marker MTS24. MTS24 labels a membrane-bound antigen present during the early stages of hair follicle development and in adult mice. MTS24 co-localises with expression of alpha6-integrin and keratin 14, indicating that these cells include basal keratinocytes. This novel population does not express the bulge-specific stem cell markers CD34 or keratin 15, and is infrequently BrdU label retaining. MTS24-positive and -negative keratinocyte populations were isolated by flow cytometry and assessed for colony-forming efficiency. MTS24-positive keratinocytes exhibited a two-fold increase in colony formation and colony size compared to MTS24-negative basal keratinocytes. In addition, both the MTS24-positive and CD34-positive subpopulations were capable of producing secondary colonies after serial passage of individual cell clones. Finally, gene expression profiles of MTS24 and CD34 subpopulations were compared. These results showed that the overall gene expression profile of MTS24-positive cells resembles the pattern previously reported in bulge stem cells. Taken together, these data suggest that the cell-surface marker MTS24 identifies a new reservoir of hair follicle keratinocytes with a proliferative capacity and gene expression profile suggestive of progenitor or stem cells.     

Rate of iron transfer through the horse spleen ferritin shell determined by the rate of formation of Prussian Blue and Fe-desferrioxamine within the ferritin cavity

Iron (2+ and 3+) is believed to transfer through the three-fold channels in the ferritin shell during iron deposition and release in animal ferritins. However, the rate of iron transit in and out through these channels has not been reported. The recent synthesis of [Fe(CN)6]3-, Prussian Blue (PB) and desferrioxamine (DES) all trapped within the horse spleen ferritin (HoSF) interior makes these measurements feasible. We report the rate of Fe2+ penetrating into the ferritin interior by adding external Fe2+ to [Fe(CN)6]3- encapsulated in the HoSF interior and measuring the rate of formation of the resulting encapsulated PB. The rate at which Fe2+ reacts with [Fe(CN)6]3- in the HoSF interior is much slower than the formation of free PB in solution and is proceeded by a lag period. We assume this lag period and the difference in rate represent the transfer of Fe2+ through the HoSF protein shell. The calculated diffusion coefficient, D approximately 5.8x10(-20) m2/s corresponds to the measured lag time of 10-20 s before PB forms within the HoSF interior. The activation energy for Fe2+ transfer from the outside solution through the protein shell was determined to be 52.9 kJ/mol by conducting the reactions at 10 approximately 40 degrees C. The reaction of Fe3+ with encapsulated [Fe(CN)6]4- also readily forms PB in the HoSF interior, but the rate is faster than the corresponding Fe2+ reaction. The rate for Fe3+ transfer through the ferritin shell was confirmed by measuring the rate of the formation of Fe-DES inside HoSF and an activation energy of 58.4 kJ/mol was determined. An attempt was made to determine the rate of iron (2+ and 3+) transit out from the ferritin interior by adding excess bipyridine or DES to PB trapped within the HoSF interior. However, the reactions are slow and occur at almost identical rates for free and HoSF-encapsulated PB, indicating that the transfer of iron from the interior through the protein shell is faster than the rate-limiting step of PB dissociation. The method described in this work presents a novel way of determining the rate of transfer of iron and possibly other small molecules through the ferritin shell.     

Evidence for a synergistic salt-protein interaction -- complex patterns of activation vs. inhibition of nitrogenase by salt

The molybdenum nitrogenase enzyme system, comprised of the MoFe protein and the Fe protein, catalyzes the reduction of atmospheric N(2) to NH(3). Interactions between these two proteins and between Fe protein and nucleotides (MgADP and MgATP) are crucial to catalysis. It is well established that salts are inhibitors of nitrogenase catalysis that target these interactions. However, the implications of salt effects are often overlooked. We have reexamined salt effects in light of a comprehensive framework for nitrogenase interactions to offer an in-depth analysis of the sources of salt inhibition and underlying apparent cooperativity. More importantly, we have identified patterns of salt activation of nitrogenase that correspond to at least two mechanisms. One of these mechanisms is that charge screening of MoFe protein-Fe protein interactions in the nitrogenase complex accelerates the rate of nitrogenase complex dissociation, which is the rate-limiting step of catalysis. This kind of salt activation operates under conditions of high catalytic activity and low salt concentrations that may resemble those found in vivo. While simple kinetic arguments are strong evidence for this kind of salt activation, further confirmation was sought by demonstrating that tight complexes that have previously displayed little or no activity due to the inability of Fe protein to dissociate from the complex are activated by the presence of salt. This occurs for the combination Azotobacter vinelandii MoFe protein with: (a) the L127Delta Fe protein; and (b) Clostridium pasteurianum Fe protein. The curvature of activation vs. salt implies a synergistic salt-protein interaction.