PDK1 controls upstream PI3K expression and PIP3 generation.

The PI3K/PDK1/Akt signaling axis is centrally involved in cellular homeostasis and controls cell growth and proliferation. Due to its key function as regulator of cell survival and metabolism, the dysregulation of this pathway is manifested in several human pathologies including cancers and immunological diseases. Thus, current therapeutic strategies target the components of this signaling cascade. In recent years, numerous feedback loops have been identified that attenuate PI3K/PDK1/Akt-dependent signaling. Here, we report the identification of an additional level of feedback regulation that depends on the negative transcriptional control of phosphatidylinositol 3-kinase (PI3K) class IA subunits. Genetic deletion of 3-phosphoinositide-dependent protein kinase 1 (PDK1) or the pharmacological inhibition of its downstream effectors, that is, Akt and mammalian target of rapamycin (mTOR), relieves this suppression and leads to the upregulation of PI3K subunits, resulting in enhanced generation of phosphatidylinositol-3,4,5-trisphosphate (PIP3). Apparently, this transcriptional induction is mediated by the concerted action of different transcription factor families, including the transcription factors cAMP-responsive element-binding protein and forkhead box O. Collectively, we propose that PDK1 functions as a cellular sensor that balances basal PIP3 generation at levels sufficient for survival but below a threshold being harmful to the cell. Our study suggests that the efficiency of therapies targeting the aberrantly activated PI3K/PDK1/Akt pathway might be increased by the parallel blockade of feedback circuits.

Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids.

The phagocytic clearance of apoptotic cells is essential to prevent chronic inflammation and autoimmunity. The phosphatidylserine-binding protein milk fat globule-EGF factor 8 (MFG-E8) is a major opsonin for apoptotic cells, and MFG-E8(-/-) mice spontaneously develop a lupus-like disease. Similar to human systemic lupus erythematosus (SLE), the murine disease is associated with an impaired clearance of apoptotic cells. SLE is routinely treated with glucocorticoids (GCs), whose anti-inflammatory effects are consentaneously attributed to the transrepression of pro-inflammatory cytokines. Here, we show that the GC-mediated transactivation of MFG-E8 expression and the concomitantly enhanced elimination of apoptotic cells constitute a novel aspect in this context. Patients with chronic inflammation receiving high-dose prednisone therapy displayed substantially increased MFG-E8 mRNA levels in circulating monocytes. MFG-E8 induction was dependent on the GC receptor and several GC response elements within the MFG-E8 promoter. Most intriguingly, the inhibition of MFG-E8 induction by RNA interference or genetic knockout strongly reduced or completely abolished the phagocytosis-enhancing effect of GCs in vitro and in vivo. Thus, MFG-E8-dependent promotion of apoptotic cell clearance is a novel anti-inflammatory facet of GC treatment and renders MFG-E8 a prospective target for future therapeutic interventions in SLE.

[Recurrent intrahepatic cholestasis of pregnancy and chain-like choledocholithiasis in a female patient with stop codon in the ABDC4-gene of the hepatobiliary phospholipid transporter]. / Rezidivierende Schwangerschaftscholestase und perlenschnurartige Choledocholithiasis bei einer Patientin mit Stoppcodon im ABCB4-Gen des hepatischen Phospholipidtransporters.

We report the case of a 40-years-old female patient with recurrent cholestatic liver disease who presented twice with severe intrahepatic cholestasis of pregnancy and pronounced choledocholithiasis between pregnancies. Bile duct stones were removed endoscopically and a laparoscopic cholecystectomy was performed after the second pregnancy. Liver histology revealed intrahepatic cholestasis with portal inflammation and fibrosis, resembling progressive familial intrahepatic cholestasis (PFIC). Molecular genetic studies identified the heterozygous mutation c.957C > T in the ABCB4 gene encoding the hepatobiliary phospholipid transporter. This is the first report of this mutation that introduces a stop codon in an index patient with intrahepatic cholestasis of pregnancy and multiple bile duct stones. In addition, we detected the ABCB11 polymorphism V 444A, which is associated with a decreased expression of the bile salt export pump. Whereas homozygous carriers of the ABCB4 mutation develop PFIC type 3, the heterozygous ABC transporter mutations represent genetic risk factors for cholelithiasis and recurrent cholestatic hepatitis upon challenge with oral contraceptives or during pregnancy. Of note, the patient presented with normal serum gamma-glutamyltranspeptidase activities during pregnancy-associated cholestatic episodes but normal liver enzymes after delivery, whereas choledocholithiasis was associated with high gamma-glutamyl transpeptidase levels. It is unknown whether ursodeoxycholic acid prevents cholestasis or gallstones in patients with ABCB4 deficiency.

Intrahepatic cholestasis of pregnancy: the severe form is associated with common variants of the hepatobiliary phospholipid transporter ABCB4 gene.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is characterised by troublesome maternal pruritus, raised serum bile acid levels and increased fetal risk. Mutations of the ABCB4 gene encoding the hepatobiliary phospholipid transporter have been identified in a small proportion of patients with cholestasis of pregnancy. In a recent prospective study on 693 patients with cholestasis of pregnancy, a cut-off level for serum bile acid (> or =40 micromol/l) was determined for increased risk of fetal complications. OBJECTIVES: To investigate whether common combinations of polymorphic alleles (haplotypes) of the genes encoding the hepatobiliary ATP-binding cassette (ABC) transporters for phospholipids (ABCB4) and bile acids (ABCB11) were associated with this severe form of cholestasis of pregnancy. METHODS: For genetic analysis, 52 women with bile acid levels > or =40 micromol/l (called cases) and 52 unaffected women (called controls) matched for age, parity and geographical residence were studied. Gene variants tagging common ABCB4 and ABCB11 haplotypes were genotyped and haplotype distributions were compared between cases and controls by permutation testing. RESULTS: In contrast with ABCB11 haplotypes, ABCB4 haplotypes differed between the two groups (p = 0.019), showing that the severe form of cholestasis of pregnancy is associated with the ABCB4 gene variants. Specifically, haplotype ABCB4_5 occurred more often in cases, whereas haplotypes ABCB4_3 and ABCB4_7 were more common in controls. These associations were reflected by different frequencies of at-risk alleles of the two tagging polymorphisms (c.711A: odds ratio (OR) 2.27, p = 0.04; deletion intron 5: OR 14.68, p = 0.012). CONCLUSION: Variants of ABCB4 represent genetic risk factors for the severe form of ICP in Sweden.

Extracellular and membrane-bound beta lactamase of Staphylococcus aureus: their importance for the expression of penicillin resistance.

The synthesis and excretion of beta lactamase by several strains of Staphylococcus aureus from different clinical sources and the ability of both the extracellular and membrane-bound enzyme to mediate penicillin resistance was studied. When beta-lactamase production was maximally induced with penicillin G or ampicillin, about 50% of the beta lactamase was excreted from the cells, the amount of extracellular enzyme correlating well with the degree of resistance established by an in-vitro test model. From penicillin-binding experiments it became apparent, however, that the membrane-bound beta lactamase can also constitute a barrier, strong enough on its own to prevent penicillins from reaching their target. This could be of clinical relevance if, under certain conditions in vivo, the extracellular beta lactamase is insufficient for full protection of the staphylococcal cells.

Suppression of intrinsic resistance to penicillins in Staphylococcus aureus by polidocanol, a dodecyl polyethyleneoxid ether.

With polidocanol, it was possible to reduce the MIC as well as the MBC of methicillin, oxacillin, penicillin G, and ampicillin against resistant staphylococci. The strongest effects were obtained with methicillin and oxacillin. All strains tested could be resensitized to these penicillins independent of the original resistance levels. Polidocanol was not inhibitory by itself for Staphylococcus aureus. Furthermore, it did not inhibit the activity of staphylococcal beta-lactamase. This permits the conclusion that an intrinsic resistance mechanism is affected by this substance. Its action cannot be simply explained by an improved accessibility of the penicillin targets as uptake, and binding of methicillin and penicillin G in resistant cells was not changed by polidocanol. On the other hand, the lysis induced by combinations of this substance with small amounts of a penicillin was antagonized by chloramphenicol. This suggests that autolytic enzymes are involved in the polidocanol effect and possibly in the intrinsic resistance mechanism itself. Before polidocanol can trigger lysis, the penicillin must act first in some way. As could be seen with a susceptible strain, the resulting lysis did not exceed that obtained with penicillins alone. Thus, polidocanol does not exhibit an independent lytic mechanism but obviously is able to substitute penicillins in their lytic action.

Binding characteristics of various penicillins in Staphylococcus aureus.

Binding studies with penicillins other than penicillin G are rare in staphylococci. Therefore the binding of a series of penicillins to the isolated cytoplasmic membrane of S. aureus H was examined indirectly by inhibition of (14C)-penicillin G binding. Biphasic inhibition curves were obtained with the beta-lactamase-insensitive penicillins as well as with ampicillin, ciclacillin, and 6-aminopenicillanic acid (6-APA) indicating that there are at least two types of targets. 60--75% of them were those with a high affinity of 10(7)--10(8) l/mol, the affinities of the second type of binding sites being in the range of 10(4)--10(5) l/mol (except 6-APA: 10(5) l/mol resp.). Monophasic binding curves were produced by penicillin G, penicillin V, propicillin, mezlocillin, azlocillin, carbenicillin, ticarcillin and mecillinam. With both groups of penicillins reversible binding to some extent was found. From comparative analysis of the binding of particular penicillins it can be finally concluded that there exist four types of binding sites in staphylococcal membranes. This agrees with the current state of knowledge in the field of penicillin-binding proteins. A good correlation was found between the 50% binding value and the minimum inhibitory concentration for most of the penicillins. As half saturation also with the "biphasic" penicillins was always achieved by (irreversible) binding to the high-affinity targets, it may be suggested that the low-affinity binding sites are probably not related to the antibacterial action of penicillins in staphylococci.

Mechanism of intrinsic penicillin-resistance in Staphylococcus aureus. Binding of penicillin G to the cytoplasmic membrane of resistant staphylococci.

In staphylococci (and other gram-positive bacteria) the phenotypical expression of intrinsic penicillin resistance as well as the frequency of this resistance are unknown. A permeability barrier in the cell wall, as it was established in gram-negative bacteria, is unlikely in staphylococci and was excluded by experiments with whole cells. Assuming that in staphylococci intrinsic resistance is also located in the cell envelope, the cytoplasmic membrane should be the site of responsibility. Therefore the binding of (14C)-penicillin G to the isolated membrane fraction of four staphylococcal strains resistant to penicillin G was studied. Two strains showed an additional resistance to methicillin, one of them was penicillinase-negative. Compared with two sensitive strains the penicillin binding was greatly reduced in the membranes of the resistant ones. This was not due to a loss of specific receptors. The maximum specific binding capacity of about 20 nmol of (14C)-penicillin G/g membrane protein was found in the resistant strains as well as in the sensitive controls. Furthermore penicillin G irreversibly bound also in the resistant bacteria. Differences, however, were found regarding the affinity of the binding sites for penicillin. In all resistant strains it was reduced by a factor of about 500. Our findings confirm the assumption that in staphylococci the mechanism of intrinsic penicillin resistance is located in the cytoplasmic membrane. They can best be explained when considering that not the penicillin binding components themselves but the membrane structure in their environment is altered thus preventing the access of the antibiotic to its site of action in the membrane. In addition, the homogeneity of our results indicates that intrinsic resistance to penicillin G is, contrary to current opinion, a general phenomenon in resistant staphylococci.