NMR structure and mutagenesis of the FADD (Mort1) death-effector domain

When activated, membrane-bound receptors for Fas and tumour-necrosis factor initiate programmed cell death by recruiting the death domain of the adaptor protein FADD to the membrane. FADD then activates caspase 8 (also known as FLICE or MACH) through an interaction between the death-effector domains of FADD and caspase 8. This ultimately leads to the apoptotic response. Death-effector domains and homologous protein modules known as caspase-recruitment domains have been found in several proteins and are important regulators of caspase (FLICE) activity and of apoptosis. Here we describe the solution structure of a soluble, biologically active mutant of the FADD death-effector domain. The structure consists of six antiparallel, amphipathic alpha-helices and resembles the overall fold of the death domains of Fas and p75. Despite this structural similarity, mutations that inhibit protein-protein interactions involving the Fas death domain have no effect when introduced into the FADD death-effector domain. Instead, a hydrophobic region of the FADD death-effector domain that is not present in the death domains is vital for binding to FLICE and for apoptotic activity.     

Risk factors associated with incisor injury in elementary school children

This study examined risk factors associated with incisor injury in 3396 third and fourth grade school children in Alachua County, Florida. One of six orthodontists completed a standardized examination form for each child to assess severity of incisor injury, gender, age, race, skeletal relationships, morphologic malocclusion, incisor exposure, interlabial gap, TMJ sounds, chin trauma, and history of lower facial trauma. One in five (19.2%) exhibited some degree of incisor injury. This was limited to a single tooth in 73.1% of those with injury, while enamel injury predominated (89.4%). The majority of the injuries (75.4%) were localized in the maxillary arch, with central incisors the most frequently traumatized. Chi-square tests of association indicated that gender, race, school, orthodontist, history of lower facial trauma, chin trauma, profile, and maxillary and mandibular horizontal positions were associated with incisor injury (P < 0.05). Wilcoxon rank sum tests identified differences in age, overjet, time of screening, and interlabial gap between those with and without injury (P < 0.05). Results of logistic regression analyses indicated risk of incisor injury was greater for children who had a prognathic maxilla, a history of trauma, were older, were male, and had greater overjet and mandibular anterior spacing.     

Ferritin in cultured human cytotrophoblasts: synthesis and subunit distribution

The present study aims at the role of ferritin in the regulation of syncytiotrophoblast free iron levels. The differentiated cytotrophoblast cell in culture is used as a model for this maternal-fetal interface. Cytotrophoblast cells isolated from term placentae are cultured in iron-poor (Medium 199), iron-depleted [desferrioxamine(DFO)] and iron-supplemented [diferric transferrin (hTF-2Fe), ferric ammonium citrate (FAC)] medium. Distribution and de novo synthesis of isoferritins is studied, together with the cellular iron concentration and the ferritin iron saturation. Compared to ferritin isolated from total placenta, ferritin obtained from villous tissue is enriched with acidic isoforms. This observation is in agreement with measured light (L) to heavy (H) subunit ratios < 1 of de novo synthesized ferritin in cultured cytotrophoblast cells. Neither iron-poor culture medium, nor hTf-2Fe supplemented medium affects the cellular iron or ferritin concentration. FAC increased the cellular ferritin iron saturation and (by synthesis) the acidic isoferritin concentrations. The results strongly suggest, that the term syncytiotrophoblast is able to balance transferrin-mediated iron uptake and iron release. In case of FAC supplementation, the syncytiotrophoblast is unable to keep intracellular iron low, and ferritin synthesis is stimulated. The predominance of acidic ferritins and the preferential synthesis of H subunits can be functionally explained by the established fact that iron incorporation in acidic ferritins is faster due to the presence of ferroxidase centres. Damage by free iron catalysed hydroxyl radical formation is therefore minimized.     

Altered glycosylation of the env-sea oncoprotein inhibits intracellular transport and transformation

The transforming gene product of the S13 avian erythroblastosis virus, env-sea, is a member of the growth factor receptor class of tyrosine kinases. The env-sea precursor protein gp155env-sea is proteolytically processed into the mature cleavage products gp85env and gp70env-sea, which are subsequently terminally glycosylated and transported to the cell surface. Previous studies have shown that the abnormal glycosylation of gp155env-sea induced by the carbohydrate processing inhibitor castanospermine blocks the proteolytic cleavage of gp155env-sea and impairs its transforming ability. We have shown recently that an uncleaved but fully glycosylated sea-encoded protein retains the ability to transform chicken embryo fibroblasts, indicating that proteolytic processing is not essential for transformation by the env-sea tyrosine kinase. To address the question of how castanospermine blocks transformation by env-sea, differential sucrose gradient centrifugation was performed on env-sea-transformed cells treated with the inhibitor. This report shows that no surface forms of env-sea could be detected in inhibitor-treated cells, suggesting that castanospermine acts by blocking the transport of sea-encoded proteins to the cell surface.     

Preservation of myocyte contractile function after hypothermic, hyperkalemic cardioplegic arrest with 2, 3-butanedione monoxime

One proposed contributory mechanism for depressed ventricular performance after hypothermic, hyperkalemic cardioplegic arrest is a reduction in myocyte contractile function caused by alterations in intracellular calcium homeostasis. Because 2,3-butanedione monoxime decreases intracellular calcium transients, this study tested the hypothesis that 2,3-butanedione monoxime supplementation of the hyperkalemic cardioplegic solution could preserve isolated myocyte contractile function after hypothermic, hyperkalemic cardioplegic arrest. Myocytes were isolated from the left ventricles of six pigs. Magnitude and velocity of myocyte shortening were measured after 2 hours of incubation under normothermic conditions (37 degrees C, standard medium), hypothermic, hyperkalemic cardioplegic arrest (4 degrees C in Ringer's solution with 20 mEq potassium chloride and 20 mmol/L 2,3-butanedione monoxime). Because beta-adrenergic agonists are commonly employed after cardioplegic arrest, myocyte contractile function was examined in the presence of the beta-agonist isoproterenol (25 nmol/L). Hypothermic, hyperkalemic cardioplegic arrest and rewarming reduced the velocity (32%) and percentage of myocyte shortening (27%, p < 0.05). Supplementation with 2,3 butanedione monoxime normalized myocyte contractile function after hypothermic, hyperkalemic cardioplegic arrest. Although beta-adrenergic stimulation significantly increased myocyte contractile function under normothermic conditions and after hypothermic, hyperkalemic cardioplegic arrest, contractile function of myocytes exposed to beta-agonist after hypothermic, hyperkalemic cardioplegic arrest remained significantly reduced relative to the normothermic control group. Supplementation with 2,3-butanedione monoxime restored beta-adrenergic responsiveness of myocytes after hypothermic, hyperkalemic cardioplegic arrest. Thus, supplementation of a hyperkalemic cardioplegic solution with 2,3-butanedione monoxime had direct and beneficial effects on myocyte contractile function and beta-adrenergic responsiveness after cardioplegic arrest. A potential mechanism for the effects of 2,3-butanedione monoxime includes modulation of intracellular calcium transients or alterations in sensitivity to calcium. Supplementation with 2,3-butanedione monoxime may have clinical utility in improving myocardial contractile function after hypothermic, hyperkalemic cardioplegic arrest.     

Familial hypokalemic periodic paralysis. Clinical, diagnostic and therapeutic aspects

A model of induced lactation was modified to examine the effects of bovine prolactin (bPRL) and bovine placental lactogen (bPL) on mammary growth and differentiation. Thirty-two peripubertal, non-pregnant Holstein heifers were given daily s.c. injections of oestradiol (0.05 mg/kg) and progesterone (0.25 mg/kg) for 7 days to initiate mammary growth. Treatment with bromocriptine (40 mg/3 days) reduced serum PRL concentrations to approximately 25% of pretreatment levels, for the duration of the study. On the day following the last steroid injection, groups of eight heifers were given twice daily s.c. injections of either saline (negative control), recombinant bPRL (rbPRL; 80 mg/day) or recombinant bPL (rbPL; 80 and 160 mg/day) for 7 days. At the end of this period (day 15), growth and differentiation of the mammary glands were assessed. Treatment with rbPL increased total mammary DNA above control value by 50 and 60% for the 80 and 160 mg/day doses respectively. However, total DNA was not different for the control and rbPRL-treated groups. The blood serum concentration of alpha-lactalbumin was measured daily throughout the study and used as an index of mammary differentiation. Both rbPRL and rbPL stimulated mammary differentiation (i.e. induction of milk synthesis), although rbPRL appeared to be more potent than rbPL. These results indicate that rbPL is lactogenic in vivo and strongly suggest that bPL is a mammary mitogen.