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PubMed1999

High-dose nitric oxide inhalation increases lung injury after gastric aspiration.

BACKGROUND: Inhaled nitric oxide is often used in patients with adult respiratory distress syndrome. However, nitric oxide also may be significantly toxic, especially if administered concurrently with hyperoxia. The authors evaluated the isolated effect of nitric oxide and the combined effects of nitric oxide and hyperoxia on lung injury in rats after acid aspiration. METHODS: Animals were injured by instillation of 1.2 ml/kg hydrogen chloride in low-pH saline (the acid group) or acidified gastric particles (the casp group) into the lungs under halothane anesthesia via a tracheal catheter. Controls received no injury vehicle but rather underwent the surgical process. After recovery from anesthesia, the animals were exposed to 20% or 90% oxygen with or without 20, 40, or 80 ppm nitric oxide for 5 h. The permeability index, alveolar-arterial oxygen difference, the ratio of oxygen pressure to the inspired fraction of oxygen, and the ratio of wet to dry weight were assessed 5 h after injury as indices of lung injury. Data were assessed using analysis of variance. RESULTS: Each group included 6-10 rats. Exposure to nitric oxide (80 ppm) in air increased protein permeability in the lungs to a permeability index of 1.42+/-0.12 after acid aspiration. The combination of nitric oxide (80 ppm) and hyperoxia further increased protein leakage to a permeability index of 2.1+/-0.25. Exposure to lower concentrations of nitric oxide (e.g., 20 and 40 ppm) increased the permeability index of the lungs (1.44+/-0.21, 1.75+/-0.29, respectively) in the presence of hyperoxia, although it did not affect the permeability index of the lungs during exposure to air. Pretreatment of animals with deferoxamine and methylene blue partially inhibited the adverse effect of hyperoxia and nitric oxide, which suggested a complex underlying mechanism involving both reactive-species generation and pulmonary vasomotor changes. CONCLUSIONS: These results show that inhaled nitric oxide at 80 ppm for a short duration (5 h) increases the severity of the inflammatory microvascular lung injury after acid aspiration. The pulmonary damage is exacerbated further in the presence of high oxygen concentrations. Although lower concentrations of nitric oxide did not increase the extent of lung injury, longer exposure times need to be assessed.

Administration, Inhalation↗
PubMed2000

The survivin-like C. elegans BIR-1 protein acts with the Aurora-like kinase AIR-2 to affect chromosomes and the spindle midzone.

Baculoviral IAP repeat proteins (BIRPs) may affect cell death, cell division, and tumorigenesis. The C. elegans BIRP BIR-1 was localized to chromosomes and to the spindle midzone. Embryos and fertilized oocytes lacking BIR-1 had defects in chromosome behavior, spindle midzone formation, and cytokinesis. We observed indistinguishable defects in fertilized oocytes and embryos lacking the Aurora-like kinase AIR-2. AIR-2 was not present on chromosomes in the absence of BIR-1. Histone H3 phosphorylation and HCP-1 staining, which marks kinetochores, were reduced in the absence of either BIR-1 or AIR-2. We propose that BIR-1 localizes AIR-2 to chromosomes and perhaps to the spindle midzone, where AIR-2 phosphorylates proteins that affect chromosome behavior and spindle midzone organization. The human BIRP survivin, which is upregulated in tumors, could partially substitute for BIR-1 in C. elegans. Deregulation of bir-1 promotes changes in ploidy, suggesting that similar deregulation of mammalian BIRPs may contribute to tumorigenesis.

Animals↗
PubMedDate not supplied

Hormonal regulation of bovine secretory proteins derived from caput and cauda epididymal epithelial cell cultures.

The goal of this study was to investigate the effect of hormones (testosterone, dihydrotestosterone [DHT], and hydrocortisone) on the protein secretion of caput and cauda epididymal epithelial cells cultured in principal cell medium (PCM). A confluent monolayer of caput and cauda epididymal epithelial cells was obtained from serum-containing PCM in the presence or absence of hormones after 7 days of culture at 38.5 degrees C (5% CO(2) in air). The protein secretion of epididymal epithelial monolayers incubated in serum-free PCM for 3 days was examined. The secreted proteins were separated by 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). A comparison of the different protein patterns showed 61 spots, of which 11 were secreted only in the presence of hormones, 3 appeared to show hormone-related changes, and 25 were region-specific. Most of these secreted proteins were low-molecular-weight acidic proteins. To obtain evidence of the epididymal origin of the secreted proteins, proteins present in caput and cauda epididymal plasma were analyzed. In conclusion, our data indicate that hormones influence the synthesis of a number of caput and cauda epididymal proteins. Some of these proteins could be important for improving our understanding of spermatozoa maturation and storage and their acquisition of fertilizing ability.

Animals↗
PubMed1995

Purification and characterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein.

Acetylene hydratase of the mesophilic fermenting bacterium Pelobacter acetylenicus catalyzes the hydration of acetylene to acetaldehyde. Growth of P. acetylenicus with acetylene and specific acetylene hydratase activity depended on tungstate or, to a lower degree, molybdate supply in the medium. The specific enzyme activity in cell extract was highest after growth in the presence of tungstate. Enzyme activity was stable even after prolonged storage of the cell extract or of the purified protein under air. However, enzyme activity could be measured only in the presence of a strong reducing agent such as titanium(III) citrate or dithionite. The enzyme was purified 240-fold by ammonium sulfate precipitation, anion-exchange chromatography, size exclusion chromatography, and a second anion-exchange chromatography step, with a yield of 36%. The protein was a monomer with an apparent molecular mass of 73 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was at pH 4.2. Per mol of enzyme, 4.8 mol of iron, 3.9 mol of acid-labile sulfur, and 0.4 mol of tungsten, but no molybdenum, were detected. The Km for acetylene as assayed in a coupled photometric test with yeast alcohol dehydrogenase and NADH was 14 microM, and the Vmax was 69 mumol.min-1.mg of protein-1. The optimum temperature for activity was 50 degrees C, and the apparent pH optimum was 6.0 to 6.5. The N-terminal amino acid sequence gave no indication of resemblance to any enzyme protein described so far.

Acetaldehyde↗
PubMed1993

Corrinoid-Dependent Methyl Transfer Reactions Are Involved in Methanol and 3,4-Dimethoxybenzoate Metabolism by Sporomusa ovata.

Washed and air-oxidized proteins from Sporomusa ovata cleaved the C-O bond of methanol or methoxyaromatics and transferred the methyl to dl-tetrahydrofolate. The reactions strictly required a reductive activation by titanium citrate, catalytic amounts of ATP, and the addition of dl-tetrahydrofolate. Methylcorrinoid-containing proteins carried the methanol methyl, which was transferred to dl-tetrahydrofolate at a specific rate of 120 nmol h mg of protein. Tetrahydrofolate methylation diminished after the addition of 1-iodopropane or when the methyl donor methanol was replaced by 3,4-dimethoxybenzoate. However, whole Sporomusa cells utilize the methoxyl groups of 3,4-dimethoxybenzoate as a carbon source by a sequential O demethylation to 4-hydroxy-3-methoxybenzoate and 3,4-dihydroxybenzoate. The in vitro O demethylation of 3,4-[4-methoxyl-C]dimethoxybenzoate proceeded via two distinct corrinoid-containing proteins to form 5-[C]methyltetrahydrofolate at a specific rate of 200 nmol h mg of protein. Proteins from 3,4-dimethoxybenzoate-grown cells efficiently used methoxybenzoates with vicinal substituents only, but they were unable to activate methanol. These results emphasized that specific enzymes are involved in methanol activation as well as in the activation of various methoxybenzoates and that similar corrinoid-dependent methyl transfer pathways are employed in 5-methyl-tetrahydrofolate formation from these substrates. Methyl-tetrahydrofolate could be demethylated by a distinct methyl transferase. That enzyme activity was present in washed and air-oxidized cell extracts from methanol-grown cells and from 3,4-dimethoxybenzoate-grown cells. It used cob(I)alamin as the methyl acceptor in vitro, which was methylated at a rate of 48 nmol min mg of protein even when ATP was omitted from the assay mixture. This methyl-cob(III)alamin formation made possible a spectrophotometric quantification of the preceding methyl transfers from methanol or methoxybenzoates to dl-tetrahydrofolate.

Journal Article↗
PubMed2024

Aire mediates tolerance to insulin through thymic trimming of high-affinity T cell clones.

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (Aire) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance.

Animals↗
PubMed1994

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: II. Monolayers of pulmonary surfactant protein SP-C and phospholipids.

The interaction of the hydrophobic pulmonary surfactant protein SP-C with dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and DPPC:DPPG (7:3, mol:mol) in spread monolayers at the air-water interface has been studied. At low concentrations of SP-C (about 0.5 mol% or 3 weight%protein) the protein-lipid films collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At initial protein concentrations higher than 0.8 mol%, or 4 weight%, the isotherms displayed kinks at surface pressures of about 50 mN.m-1 in addition to the collapse plateaux at the higher pressures. The presence of less than 6 mol%, or 27 weight%, of SP-C in the protein-lipid monolayers gave a positive deviation from ideal behavior of the mean areas in the films. Analyses of the mean areas in the protein-lipid films as functions of the monolayer composition and surface pressure showed that SP-C, associated with some phospholipid (about 8-10 lipid molecules per molecule of SP-C), was squeezed out from the monolayers at surface pressures of about 55 mN.m-1. The results suggest a potential role for SP-C to modify the composition of the monolayer at the air-water interface in the alveoli.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2000

Sonic wave separation of invertase from a dilute solution to generated droplets.

It has previously been shown that a droplet fractionation process, simulated by shaking a separatory funnel containing a dilute protein solution, can generate droplets richer in protein than present in the original dilute solution. In this article, we describe an alternative method that can increase the amount of protein transferred to the droplets. The new method uses ultrasonic waves, enhanced by a bubble gas stream to create the droplets. The amount of protein in these droplets increases by about 50%. In this method, the top layer of the dilute protein solution (of the solution-air interface) becomes enriched in protein when air is bubbled into the solution. This concentrating procedure is called bubble fractionation. Once the protein has passed through the initial buildup, this enriched protein layer is transferred into droplets with the aid of a vacuum above the solution at the same time that ultrasonic waves are introduced. The droplets are then carried over to a condenser and coalesced. We found that this new method provides an easier way to remove the protein-enriched top layer of the dilute solution and generates more droplets within a shorter period than the separatory funnel droplet generation method. The added air creates the bubbles and carries the droplets, and the vacuum helps remove the effluent airstream from the condenser. The maximum partition coefficient, the ratio of the protein concentration in the droplets to that in the residual solution (approx 8.5), occurred at pH 5.0.

Glycoside Hydrolases↗
PubMed2001

A novel mutation of the autoimmune regulator gene in an Italian kindred with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, acting in a dominant fashion and strongly cosegregating with hypothyroid autoimmune thyroiditis.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy is a rare autosomal recessive disorder characterized by hypoparathyroidism, adrenal failure, chronic mucocutaneous candidiasis, and ectodermal dystrophies and other organ-specific autoimmune diseases. Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy is caused by mutations of the autoimmune regulator gene. We identified an Italian family with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy and a pattern of inheritance suggestive of a dominant mechanism. Serological and clinical studies showed a high prevalence of hypothyroid autoimmune thyroiditis in affected members with classical autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. Direct sequencing of the entire coding region of the autoimmune regulator gene revealed the presence in the proband of a novel missense (G228W) mutation in exon 6 in a heterozygous state. The same heterozygous mutation was identified in all family members with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy and/or hypothyroid autoimmune thyroiditis. None of the unaffected family members and 50 unrelated Italian controls carried the mutation. In contrast with all other autoimmune regulator mutations reported in families, the novel G228W mutation acts in a dominant fashion in our family, as only one heterozygous mutation was found in the entire coding sequence of the autoimmune regulator gene in the proband. Moreover, analysis of the family tree showed direct transmission of the hypothyroid autoimmune thyroiditis/polyendocrinopathy-candidiasis-ectodermal dystrophy phenotype to the offspring in each generation in the absence of consanguinity, further supporting a dominant inheritance. The G228W closely cosegregated with hypothyroid autoimmune thyroiditis in our family, whereas a low penetrance of the full autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy phenotype was observed. In conclusion, we report a novel mutation of the autoimmune regulator gene in a family with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, closely cosegregating with hypothyroid autoimmune thyroiditis. The G228W mutation acts in a dominant fashion and may shed light on the structure-function relationship of the autoimmune regulator protein.

Adolescent↗
PubMed1999

Indoor air pollutants stimulate interleukin-8-specific mRNA expression and protein secretion of alveolar macrophages.

Indoor air pollutants may cause inflammatory changes of the airways and adjacent pulmonary tissue. After phagocytosis of inhaled particles alveolar macrophages (AM) release chemotactic mediators capable of attracting inflammatory cells into the lung tissue. To evaluate these mechanisms further peripheral blood mononuclear cells (PBMNC) and human AM (freshly recovered from the lower respiratory tract) were exposed to the indoor particles Soot FR 101 and Printex 90, the asbestos fiber Chrysotile B, and titanium dioxide (TiO2) at concentrations of 10 or 50 microg/10(6) cells for up to 8 h. The migration of granulocytes into the conditioned supernatants of AM and PBMNC was quantified by chemotaxis assay in a Boyden chamber. Granulocyte migration increased by 42.3 +/- 25.8% (Chrysotile B), 64. 6 +/- 18.3% (FR 101), 74.2 +/- 16.5% (P 90), and 86.7 +/- 25.6% (TiO2) in AM-conditioned supernatants (p < 0.05). Qualitative, Interleukin (IL)-8 specific reverse transcriptase-polymerase chain reaction was performed after exposure of AM or PBMNC to Chrysotile B, FR 101, P 90, and TiO2 at concentrations of 10 and 50 microg/10(6) cells for 90 min. Each of the tested particles caused an increase in IL-8-specific mRNA expression of AM or PBMNC after particle exposure compared with the unexposed control. To find out if IL-8, the most powerful granulocyte chemokine, is involved, supernatants were preincubated with anti-IL-8. Granulocyte migration decreased by up to 35 +/- 15% (50 ng/ml anti-IL-8) and 41.5 +/- 16% (100 ng/ml anti-IL-8) (p < 0.0625) in AM-conditioned supernatants. Pretreatment of the granulocytes with human IL-8 decreased by up to 59 +/- 18% (10 ng/ml) (p < 0.0625) in AM-conditioned supernatants. Similar reaction patterns were observed using anti-IL-8-pretreated supernatants of particle-exposed PBMNC. In conclusion, indoor air pollutants may promote inflammatory changes in the lung via IL-8 release by alveolar macrophages.

Adult↗
PubMed1985

Resonance Raman study of cytochrome b562-o complex, a terminal oxidase of Escherichia coli in its ferric, ferrous, and CO-ligated states.

Cytochrome b562-o complex, a terminal oxidase in the respiratory chain of aerobically grown Escherichia coli, has been studied by resonance Raman spectroscopy in its air-oxidized, dithionite-reduced, and reduced and CO-ligated states. In the reduced state, with a 406.7-nm excitation, there appeared 1494 and 1473 cm-1 lines, indicating that low spin and high spin components are included in the cytochrome b562-o complex. For the air-oxidized protein, resonance Raman lines were observed at 1372, 1503, and 1580 cm-1 with a 413.1-nm excitation, indicating that there is a ferric low spin heme. In addition, a weak but appreciable Raman line was observed at 1480 cm-1 assignable to a ferric high spin heme. Accordingly, it was concluded that low spin and high spin components are included in the cytochrome b562-o complex in the reduced and the air-oxidized states. In the CO-ligated state, with a defocused laser beam of 413.1 nm, two Raman bands assignable to the Fe-CO stretching mode have been observed at 489 and 523 cm-1, as a major and a minor component, respectively. When the laser beam was focused upon the sample to cause a photodissociation of CO from the heme moiety, the intensity of the major band at 489 cm-1 was reduced as expected. On the other hand, the minor band at 523 cm-1 remained still obvious. It was suggested that the cytochrome b562-o complex may have an additional anomalous site for CO that is resistant to photodissociation.

Carbon Monoxide↗
PubMed1994

Species differences in the biotransformation of ethyl chloride. II. GSH-dependent metabolism.

Groups of male and female F-344 rats and B6C3F1 mice were exposed to 15,000 ppm ethyl chloride (monochloroethane, ECL) or to air for 5 days (6 h/day). In this report, features of GSH-dependent ECL metabolism in the animals are described. A concurrent report describes the features of the cytochrome P450-dependent oxidative ECL metabolism (Fedtke et al. 1994). ECL conjugation to GSH in hepatic cytosolic fractions was catalyzed by GSH S-transferases. The specific activities were 0.16 +/- 0.03 and 0.17 +/- 0.01 nmol ECL conjugated/(min mg protein) in air treated male and female F-344 rats, respectively. These activities were not significantly altered by the ECL treatment. Compared with rats, the GSH-transferase activities towards ECL were generally higher in male and female B6C3F1 mice (0.71 +/- 0.19 and 1.01 +/- 0.19, respectively) and were slightly decreased by ECL treatment. The ECL conjugation to GSH resulted in a marked reduction of the GSH concentration in the lung and the uterus after 5 days of exposure. In contrast, liver and kidney GSH concentrations were affected only to a minor degree. Formed S-ethyl-glutathione was converted to the mercapturic acid S-ethyl-N-acetyl-L-cysteine (SENACys), which was detected in the urine of both species. In addition, the non-acetylated intermediate S-ethyl-L-cysteine (SECys) was excreted in mouse urine but not in rat urine. The cumulative amounts of SENACys and SECys excreted after 5 days were up to fivefold higher in mice than in rats and the excretion kinetics were species specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed1987

Modulation of lymphocyte activating factor activity (interleukin 1 like activity) and acute phase proteins in pertussis-induced air pouch inflammation by muramyl dipeptide.

We have studied the effect of the macrophage activator, muramyl dipeptide (MDP) on immune inflammation induced in the rat six day subcutaneous air pouch. Treated animals received either 100 micrograms or 200 micrograms MDP at the time of challenge and twenty four hours before exudate harvest. Using the thymocyte co-mitogenic assay for lymphocyte activating factor (LAF), 100 micrograms MDP enhanced LAF activity whereas 200 micrograms caused inhibition. Increased dilution of 200 micrograms exudate in this assay removed this inhibition. Similarly, at the lower dose, MDP caused enhanced production of the acute phase protein alpha 1 glycoprotein, whereas the higher dose had no effect. The present study suggests that macrophage activity can be manipulated in vivo to produce LAF and naturally occurring inhibitors of LAF. These studies indicate that the stimulation of LAF inhibitors by MDP may be a potential therapeutic action.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2005

Protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction are enhanced in IVF media: an effect that is not associated with an increase in protein kinase A activation.

Sperm capacitation is a prerequisite for successful in vitro fertilization (IVF) and therefore a focus of sperm preparation in IVF laboratories. The technology of IVF is, therefore, potentially valuable in advancing our understanding of the molecular processes that occur during sperm capacitation. We have investigated sperm capacitation induced by a commercial IVF medium compared to that occurring in standard capacitating medium (CM) typically used in a nonclinical setting. Percoll-washed spermatozoa were resuspended in Cook Sydney IVF medium, Cook Sydney IVF sperm buffer, Earle's balanced salt medium (capacitating medium) or a modified Earle's balanced salt medium [non-capacitating medium (NCM)] for up to 120 min at 37 degrees C and, if applicable, in the presence of 5% CO2 in air. Sperm protein kinase A (PKA) activity, PKA-dependent serine/threonine phosphorylation, tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction were evaluated. IVF medium was shown to accelerate sperm capacitation (compared with capacitating medium) as determined by tyrosine phosphorylation, sperm hyperactivation and progesterone-induced acrosome reaction. This effect was not associated with enhanced activation of PKA or increased levels of serine/threonine phosphorylation. In contrast, IVF sperm buffer (used for sperm preparation) did not stimulate sperm capacitation when incubated for up to 90 min. We have shown that different capacitating media vary strikingly in their efficacy and that this difference reflects activation of a pathway other than the well-characterized activation of soluble adenylyl cyclase/cAMP/PKA.

Acrosome Reaction↗
PubMed2001

Heterologous expression and properties of the gamma-subunit of the Fe-only hydrogenase from Thermotoga maritima.

Thermotoga maritima is a hyperthermophilic bacterium that contains a complex, heterotrimeric (alpha(beta)gamma) Fe-only hydrogenase. Sequence analysis indicates that the gene encoding the smallest subunit (gamma), hydC, contains a predicted iron-sulfur cluster binding motif. However, characterization of the native gamma-subunit has been hampered by interference from and the inability to separate intact gamma-subunit from the other two subunits (alpha and beta). To investigate the function and properties of the isolated gamma-subunit, the gene encoding HydG was expressed in Escherichia coli. Two forms of the recombinant protein were obtained with molecular masses of 10 and 18 kDa, respectively. Both contained a single [2Fe-2S] cluster based on metal analysis, EPR and UV-visible spectroscopy. NH2-terminal sequencing revealed that the 10 kDa protein is a truncated form of the intact gamma-subunit and lacks the first 65 amino acid residues. The midpoint potential of the 18 kDa form was -356 mV at pH 7.0 and 25 degrees C, as measured by direct electrochemistry, and was pH dependent with a pK(ox) of 7.5 and a pK(red) of 7.7. The oxidized, recombinant gamma-subunit was stable at 80 degrees C under anaerobic conditions with a half-life greater than 24 h, as judged by the UV-visible spectrum of the [2Fe-2S] cluster. In the presence of air the protein was less stable and denatured with a half-life of approx. 2.5 h. The recombinant gamma-subunit was electron transfer competent and was efficiently reduced by pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus, with a Km of 5microM and a Vmax of 9 U/mg. In contrast, native T. maritima hydrogenase holoenzyme and its separated alpha-subunit were much less effective electron donors for the gamma-subunit, with a V(max) of 0.01 U/mg and 0.1 U/mg, respectively.

Amino Acid Sequence↗
PubMed2001

Subcellular localization of the autoimmune regulator protein. characterization of nuclear targeting and transcriptional activation domain.

The autoimmune regulator (AIRE) gene, defective in the hereditary autoimmune disease APECED, encodes a transcriptional regulator protein. AIRE is expressed in the medullary epithelial cells and monocyte-dendritic cells of the thymus with lower expression in the spleen, fetal liver, and lymph nodes. At the cellular level, AIRE is located in microtubular structures of the cytoskeleton and in discrete nuclear dots resembling ND10 nuclear bodies. We studied the determinants of the targeting of AIRE into these structures. We report here that the N-terminal HSR domain confers localization to the microtubular network whereas the C-terminal region contains a second nuclear localization signal. We also demonstrate that the consensus nuclear localization signal of AIRE is functional and that the HSR domain harbors a nuclear export signal. Accordingly, the nuclear export inhibitor leptomycin B partially inhibits the nuclear export of AIRE. From a functional standpoint, we show that AIRE can activate the interferon beta minimal promoter in a transfection assay and demonstrate that the transcriptional activating function of AIRE is mediated by its two plant homeodomain (PHD) zinc fingers.

Animals↗
PubMed1994

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: I. Monolayers of pulmonary surfactant protein SP-B and phospholipids.

The effects of pulmonary surfactant protein SP-B on the properties of monolayers of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG), and a mixture of DPPC:DPPG (7:3, mol:mol) were studied using spread films at the air-water interface. The addition of SP-B to the phospholipid monolayers gave positive deviations from additivity of the mean areas in the films. At low protein concentrations (less than 45% amino acid residues which corresponds to 0.5 mol% or 10 weight% SP-B) monolayers of SP-B/DPPC, SP-B/DPPG and SP-B/(DPPC:DPPG) collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At higher concentrations of SP-B in the protein-lipid monolayers, kink points appeared in the isotherms at about 40-45 mN.m-1, implying possible exclusion of material from the films, hence, changes in the original monolayer compositions. Calculated analyses of the monolayer compositions as a function of surface pressure indicated that nearly pure SP-B, associated with small amounts of phospholipid (2-3 lipid molecules per SP-B dimer), was lost from SP-B/DPPC, SP-B/DPPG, and SP-B/(DPPC:DPPG) films at surface pressures higher than 40-45 mN.m-1. The results are consistent with a low effectiveness of SP-B in removing saturated phospholipids, DPPC or DPPG, from the spread SP-B/phospholipid films.

1,2-Dipalmitoylphosphatidylcholine↗