Aiolos controls gene conversion and cell death in DT40 B cells.

The Ikaros family transcription factor Aiolos is important for B cell function, since B cells of Aiolos-null mutant mice exhibit an activated phenotype, enhanced B-cell receptor (BCR) signalling response and develop a systemic lupus erythematosus (SLE) type autoimmune disease. Aiolos has also been reported to interact with anti-apoptotic Bcl-2 and Bcl-x(L) in T cells, but whether Aiolos regulates cell death has not been studied in B cells. Here we show that the disruption of Aiolos in the DT40 B cell line induces a cell death sensitive phenotype, as the Aiolos(-/-) cells are more prone to apoptosis by nutritional stress, BCR cross-linking, UV- or gamma-irradiation. Furthermore, the Aiolos(-/-) cells have defective Ig gene conversion providing evidence that Aiolos is needed for the somatic diversification of the BCR repertoire. The re-expression of DNA-binding isoform Aio-1 was able to restore the gene conversion defect of the Aiolos-deficient cells, whereas the introduction of dominant negative isofom Aio-2 had no effect on gene conversion, thus demonstrating the functional importance of alternative splicing within Ikaros family. Although the Aiolos(-/-) cells exhibit reduced expression of activation-induced cytidine deaminase (AID), ectopic AID overexpression did not restore the gene conversion defect in the Aiolos(-/-) cells. Our findings indicate that Aiolos may regulate gene conversion in an AID independent manner.

Proton- and sodium-coupled phosphate transport systems and energy status of Yarrowia lipolytica cells grown in acidic and alkaline conditions.

In this study we have used a newly isolated Yarrowia lipolytica yeast strain with a unique capacity to grow over a wide pH range (3.5-10.5), which makes it an excellent model system for studying H(+)- and Na(+)-coupled phosphate transport systems. Even at extreme growth conditions (low concentrations of extracellular phosphate, alkaline pH values) Y. lipolytica preserved tightly-coupled mitochondria with the fully competent respiratory chain containing three points of energy conservation. This was demonstrated for the first time for cells grown at pH 9.5-10.0. In cells grown at pH 4.5, inorganic phosphate (P(i)) was accumulated by two kinetically discrete H(+)/P(i)-cotransport systems. The low-affinity system is most likely constitutively expressed and operates at high P(i) concentrations. The high-affinity system, subjected to regulation by both extracellular P(i) availability and intracellular polyphosphate stores, is mobilized during P(i)-starvation. In cells grown at pH 9.5-10, P(i) uptake is mediated by several kinetically discrete Na(+)-dependent systems that are specifically activated by Na(+) ions and insensitive to the protonophore CCCP. One of these, a low-affinity transporter operative at high P(i) concentrations is kinetically characterized here for the first time. The other two, high-affinity, high-capacity systems, are derepressible and functional during P(i)-starvation and appear to be controlled by extracellular P(i). They represent the first examples of high-capacity, Na(+)-driven P(i) transport systems in an organism belonging to neither the animal nor bacterial kingdoms. The contribution of the H(+)- and Na(+)-coupled P(i) transport systems in Y. lipolytica cells grown at different pH values was quantified. In cells grown at pH values of 4.5 and 6.0, the H(+)-coupled P(i) transport systems are predominant. The contribution of the Na(+)/P(i) cotransport systems to the total cellular P(i) uptake activity is progressively increased with increasing pH, reaching its maximum at pH 9 and higher.

Regulation of cation-coupled high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae.

Studies of the high-affinity phosphate transporters in the yeast Saccharomyces cerevisiae using mutant strains lacking either the Pho84 or the Pho89 permease revealed that the transporters are differentially regulated. Although both genes are induced by phosphate starvation, activation of the Pho89 transporter precedes that of the Pho84 transporter early in the growth phase in a way which may possibly reflect a fine tuning of the phosphate uptake process relative to the availability of external phosphate.

Fetal sex and prenatal betamethasone therapy.

We examined the influence of fetal sex on the occurrence of respiratory distress syndrome in premature infants after maternal treatment with betamethasone. Among treated infants of 1,251 to 1,750 gm birth weight, the incidence of RDS was 40.9% in 22 males and 7.1% (P = 0.03) in 14 females. Cord serum levels of betamethasone were similar for infants of both sexes, and there was no sex difference in suppression of serum cortisol, dehydroepiandrosterone sulfate, and growth hormone after treatment. These findings suggest that prenatal corticosteroid therapy is less effective in male infants than in female infants. This effect is not due to a difference in transfer or metabolism of betamethasone, nor is it reflected in the responsiveness of the fetal hypothalamic-pituitary-adrenal axis to synthetic glucocorticoid.

Prenatal administration of betamethasone for prevention of respiratory distress syndrome.

The outcome of 114 infants of birth weight 750 to 1,750 gm who received prenatal betamethasone therapy was compared retrospectively to that of 138 infants delivered to untreated women. The incidence of respiratory distress syndrome in all treated infants was 37.7% compared with 50.7% (P = 0.05) in untreated infants. There was no apparent benefit of therapy among infants delivering less than 48 hours after the first dose and among infants less than 750 gm birth weight. Among infants delivering two to ten days after therapy, RDS 25.0 vs 50.7%) and mortality (8.9 vs 22.5%) were significantly reduced. Among surviving infants with RDS, fewer infants in the two to ten-day treated group required oxygen at FIO2 greater than 0.5 for more than 24 hours. Our findings confirm previous reports that prenatal glucocorticoid treatment reduces the incidence of RDS and mortality in premature infants. In addition, they indicate that therapy is more effective when delivery is delayed at least two days, that very small premature infants do not benefit from treatment, and that RDS may be less severe after prenatal exposure to betamethasone.

The role of sulfhydryl groups in the binding of glucocorticoids by cytoplasmic receptors of lung and other mammalian tissues.

We examined the levels and stability of glucocorticoid receptor activity in cytosol preparations of rat lung and other tissues. [3H]Dexamethasome binding capacity at 2 C in lung cytosol decreases with a t1/2 of 40 min in the absence of steroid or a sulfhydryl compound. This rapid inactivation of unbound receptor is prevented and reversed by addition of a sulfhydryl compound (t1/2 = 22h); maximal binding occurs with 2 mM dithiothreitol (DTT), or with 20-25 mM mercaptoethanol, thioglycerol or glutathione. Binding activity is also stabilized by formation of the receptor-steroid complex; dissociation of the complex occurs at the same rate (t1/2 = 18.6h) in both the presence and absence of DTT. In the presence of DTT, cytosol of adult rat lung specifically binds 0.82 pmol of [3H]dexamethasone per mg protein (average 19,000 sites per cell) with an equilibrium dissociation contant of 2.5 nM at 2 C. At 37 C, dispersed lung cells show specific nuclear binding of hormone. DTT also increased dexamethasone binding activity in cytosol prepared from lung of adult hamster, adult mouse, fetal monkey and 11 of 15 tissues of adult rat. There was a minimal effect of DTT in reactions using cytosol of fetal rat lung, fetal and newborn human lung, rabbit lung, and liver, kidney, heart and testis of adult rat. Liver contains a heat-stable factor which mimics that stabilizing effect of DTT. The variable requirement for DTT may reflect in part tissue concentrations of endogenous sulfhydryl compounds. Our findings indicate that glucocorticoid binding activity of lung and many other tissues decays rapidly in vitro due to oxidation of receptor sulfhydryl groups. Maintenance of these groups in the reduced form by endogenous tissue factors, addition of sulfhydryl compounds, or binding of glucocorticoid stabilizes receptor and allows its detection in lung and other tissues previously found to contain little if any activity.