Cytosolic Receptor Protein Research Articles

Human Endometrium Derived Mesenchymal Stem Cells with Aberrant NOD1 Expression Are Associated with Ectopic Endometrial Lesion Formation.

Nucleotide-binding oligomerization domain 1 (NOD1), a cytosolic pattern recognition receptor protein, plays a crucial role in innate immune responses. However, the functional expression of NOD1 in mesenchymal stem cells (MSCs) derived from endometriosis remains unclear. The aim of this study was to explore the functions of NOD1 in ectopic endometrial lesions. Tissues and MSCs were isolated from both normal endometrium and endometriosis. Immunohistochemistry and real time quantitative polymerase chain reaction (RT-qPCR) were used to determine the expression of NOD1 in the tissues/MSCs. Quantification of various cytokines was performed using RT-qPCR and enzyme-linked immunosorbent assay. To confirm the proliferation, invasion/migration, and apoptotic viabilities of the samples, Cell Counting Kit-8, clonogenic formation, transwell assays, and apoptotic experiments were conducted. Higher levels of NOD1 expression were detected in the ectopic-MSCs obtained from endometriosis compared to those from the endometrium. The expression of interleukin-8 was higher in the ectopic-MSCs than in the eutopic-MSCs. Pretreatment with NOD1 agonist significantly enhanced the proliferation and invasion/migration of eutopic-MSCs. Additionally, the NOD1 inhibitor ML-130 significantly reduced the proliferation, clone formation, invasion, and migration abilities of the ectopic-MSCs, having no effect on their apoptosis capacity. Our findings suggest that the expression of NOD1 in ectopic-MSCs may contribute to the progression of ectopic endometrial lesions.

NLRP11 is a pattern recognition receptor for bacterial lipopolysaccharide in the cytosol of human macrophages.

Endotoxin-bacterial lipopolysaccharide (LPS)-is a driver of lethal infection sepsis through excessive activation of innate immune responses. When delivered to the cytosol of macrophages, cytosolic LPS (cLPS) induces the assembly of an inflammasome that contains caspases-4/5 in humans or caspase-11 in mice. Whereas activation of all other inflammasomes is triggered by sensing of pathogen products by a specific host cytosolic pattern recognition receptor protein, whether pattern recognition receptors for cLPS exist has remained unclear, because caspase-4, caspase-5, and caspase-11 bind and activate LPS directly in vitro. Here, we show that the primate-specific protein NLRP11 is a pattern recognition receptor for cLPS that is required for efficient activation of the caspase-4 inflammasome in human macrophages. In human macrophages, NLRP11 is required for efficient activation of caspase-4 during infection with intracellular Gram-negative bacteria or upon electroporation of LPS. NLRP11 could bind LPS and separately caspase-4, forming a high-molecular weight complex with caspase-4 in HEK293T cells. NLRP11 is present in humans and other primates but absent in mice, likely explaining why it has been overlooked in screens looking for innate immune signaling molecules, most of which have been carried out in mice. Our results demonstrate that NLRP11 is a component of the caspase-4 inflammasome activation pathway in human macrophages.

Tuning TPC2

• Combined addition of TPC2-A1-N and TPC2-A1-P increases the permeability for Ca2+ions of two-pore channel 2 (TPC2). • TPC2 can be tuned to more rapid and larger Ca2+ signals by combined stimulation of TPC2 using TPC2-A1-N and TPC2-A1-P. • Mechanism of action of TPC2-A1-N needs clarification regarding cytosolic NAADP receptor proteins.

The role of Peroxin 7 during Drosophila embryonic development.

Peroxisomes are organelles in eukaryotic cells responsible for processing several types of lipids and management of reactive oxygen species. A conserved family of peroxisome biogenesis (Peroxin, Pex) genes encode proteins essential to peroxisome biogenesis or function. In yeast and mammals, PEROXIN7 (PEX7) acts as a cytosolic receptor protein that targets enzymes containing a peroxisome targeting signal 2 (PTS2) motif for peroxisome matrix import. The PTS2 motif is not present in the Drosophila melanogaster homologs of these enzymes. However, the fly genome contains a Pex7 gene (CG6486) that is very similar to yeast and human PEX7. We find that Pex7 is expressed in tissue-specific patterns analogous to differentiating neuroblasts in D. melanogaster embryos. This is correlated with a requirement for Pex7 in this cell lineage as targeted somatic Pex7 knockout in embryonic neuroblasts reduced survival. We also found that Pex7 over-expression in the same cell lineages caused lethality during the larval stage. Targeted somatic over-expression of a Pex7 transgene in neuroblasts of Pex7 homozygous null mutants resulted in a semi-lethal phenotype similar to targeted Pex7 knockout. These findings suggest that D. melanogaster has tissue-specific requirements for Pex7 during embryo development.

NLRC4 Mutation in flagellin-derived peptide CBLB502 ligand-binding domain reduces the inflammatory response but not radioprotective activity.

ABSTRACTBacterial flagellin is a pathogen-associated molecular pattern recognized by surface-localized Toll-like receptor 5 (TLR5) and cytosolic NOD-like receptor protein 4 (NLRC4). CBLB502, derived from Salmonella flagellin, exhibits high radioprotective efficacy in mice and primates by regulating TLR5 and the nuclear factor kappa B (NF-κB) signaling pathway. In this study, we examined the effects of CBLB502 and mutations in its NLRC4- and TLR5-binding domains on radioprotective efficacy and the immune inflammatory response. The results showed that CBLB502 mutation with I213A in the TLR5-binding domain significantly reduced NF-κB activity and radioprotective activity, whereas CBLB502 mutation with L292A in NLRC4-binding domain did not. Additionally, CBLB502 with both mutations greatly reduced NF-κB activity and eliminated radioprotection in mice. In contrast, NLRC4-binding domain mutation reduced the secretion of inflammatory interleukin-1β and interleukin-18. CBLB502 exerts its radioprotective effects through both the TLR5 and NLRC4 pathways. Additionally, deletion in the NLRC4-binding domain did not reduce radioprotective activity but reduced the inflammatory response.

Aryl Hydrocarbon Receptor Modulates the Expression of TNF-α and IL-8 in Human Sebocytes via the MyD88-p65NF-κB/p38MAPK Signaling Pathways

Activation of Toll-like receptor (TLR)-2 and subsequent inflammatory response contribute to lesion development in acne vulgaris. A cross-talk between aryl hydrocarbon receptor (AhR), a cytosolic receptor protein that responds to environmental and physiological stress, and TLRs has recently been reported. In this study, we explored the possible role of AhR in the effects induced on cultured human SZ95 sebocytes by peptidoglycan (PGN), a classic TLR2 agonist. PGN-induced secretion of inflammatory factors TNF-α and IL-8 in human SZ95 sebocytes was suppressed after knockdown of AhR and pretreatment with the AhR antagonist CH223191. In addition, the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhanced TNF-α and IL-8 secretion in PGN-pretreated sebocytes. Furthermore, PGN-induced expression of myeloid differentiation factor 88 (MyD88), phospho-p38MAPK (p-p38MAPK), and p-p65NF-κB was strengthened by TCDD and repressed by CH223191. AhR inhibition by transfecting shRNA blocked the ability of PGN to stimulate phosphorylation of p38MAPK and p65NF-κB in SZ95 sebocytes. Overall, these data demonstrate that AhR is able to modulate PGN-induced expression of TNF-α and IL-8 in human SZ95 sebocytes involving the MyD88-p65NF-κB/p38MAPK signaling pathway, which probably indicates a new mechanism in TLR2-mediated acne.

Activation of NLRC4 downregulates TLR5-mediated antibody immune responses against flagellin.

Bacterial flagellin is a unique pathogen-associated molecular pattern (PAMP), which can be recognized by surface localized Toll-like receptor 5 (TLR5) and the cytosolic NOD-like receptor (NLR) protein 4 (NLRC4) receptors. Activation of the TLR5 and/or NLRC4 signaling pathways by flagellin and the resulting immune responses play important roles in anti-bacterial immunity. However, it remains unclear how the dual activities of flagellin that activate the TLR5 and/or NLRC4 signaling pathways orchestrate the immune responses. In this study, we assessed the effects of flagellin and its mutants lacking the ability to activate TLR5 and NLRC4 alone or in combination on the adaptive immune responses against flagellin. Flagellin that was unable to activate NLRC4 induced a significantly higher antibody response than did wild-type flagellin. The increased antibody response could be eliminated when macrophages were depleted in vivo. The activation of NLRC4 by flagellin downregulated the flagellin-induced and TLR5-mediated immune responses against flagellin.

Comparative profiling of the peroxisomal proteome of wildtype and Pex7 knockout mice by quantitative mass spectrometry

We present a label-free quantitative proteomic approach for the study of kidney peroxisomes of Pex7 knockout mice which is a bona-fide model for the human disease rhizomelic chondrodysplasia punctata (RCDP). RCDP is an autosomal recessive human disorder caused by mutations in the PEX7 gene encoding for Pex7, the cytosolic receptor protein that is essential for the import of proteins containing a functional peroxisomal targeting signal (PTS)-type 2. In this work, we quantitatively followed hundreds of proteins through high density gradient fractions of wildtype (WT) and Pex7 knockout (Pex7−/−) mice by high resolution mass spectrometry. A set of candidate proteins with altered abundance was defined via statistical and quantitative assessment of protein profiles obtained from WT and Pex7−/− mice. The results obtained demonstrate the feasibility of this approach to identify proteins specifically affected in abundance by the deletion of Pex7. All three known PTS2 proteins, including acetyl-Coenzyme A acyltransferase, alkylglycerone phosphate synthase and phytanoyl-CoA hydroxylase were determined to be virtually absent in these fractions whereas KIAA0564, a so far uncharacterized protein, was barely detectable in peroxisomal fractions of Pex7−/− mice. Furthermore, we report numerous PTS1 proteins with increased abundance levels in Pex7−/− mice that fulfill essential functions in the β-oxidation of very long-chain fatty acids or the biosynthesis of ether-phospholipids in peroxisomes.

Genotype-phenotype correlation in PEX5-deficient peroxisome biogenesis defective cell lines

Proteins destined for the peroxisomal matrix are targeted by virtue of a peroxisomal targeting sequence type 1 (PTS1) or type 2 (PTS2). In humans, targeting of either class of proteins relies on a cytosolic receptor protein encoded by the PEX5 gene. Alternative splicing of PEX5 results in two protein variants, PEX5S and PEX5L. PEX5S is exclusively involved in PTS1 protein import, whereas PEX5L mediates the import of both PTS1 and PTS2 proteins. Genetic complementation testing with over 500 different fibroblast cell lines from patients diagnosed with a peroxisome biogenesis disorder (PBD) identified 11 cell lines with a defect in PEX5. The aim of this study was to characterize these cell lines at a biochemical and genetic level. To this end, the cultured fibroblasts were analyzed for very long chain fatty acid (VLCFA) concentrations, peroxisomal beta-and alpha-oxidation, dihydroxyacetone-phosphate acyltransferase (DHAPAT) activity, peroxisomal thiolase, and catalase immunofluorescence. Mutation analysis of the PEX5 gene revealed 11 different mutations, eight of which are novel. PTS1- and PTS2-protein import capacity was assessed by transfection of the cells with green fluorescent protein (GFP) tagged with either PTS1 or PTS2. Six cell lines showed a defect in both PTS1 and PTS2 protein import, whereas four cell lines only showed a defect in PTS1 protein import. The location of the different mutations within the PEX5 amino acid sequence correlates rather well with the peroxisomal protein import defect observed in the cell lines.

Innate Immunity Minireview Series: Making Biochemical Sense of Nucleic Acid Sensors That Trigger Antiviral Innate Immunity

Innate Immunity Minireview Series: Making Biochemical Sense of Nucleic Acid Sensors That Trigger Antiviral Innate Immunity

An improved genetic system for detection and analysis of protein nuclear import signals

BackgroundNuclear import of proteins is typically mediated by their physical interaction with soluble cytosolic receptor proteins via a nuclear localization signal (NLS). A simple genetic assay to detect active NLSs based on their function in the yeast Saccharomyces cerevisiae has been previously described. In that system, a chimera consisting of a modified bacterial LexA DNA binding domain and the transcriptional activation domain of the yeast Gal4 protein is fused to a candidate NLS. A functional NLS will redirect the chimeric fusion to the yeast cell nucleus and activate transcription of a reporter gene.ResultsWe have reengineered this nuclear import system to expand its utility and tested it using known NLS sequences from adenovirus E1A. Firstly, the vector has been reconstructed to reduce the level of chimera expression. Secondly, an irrelevant "stuffer" sequence from the E. coli maltose binding protein was used to increase the size of the chimera above the passive diffusion limit of the nuclear pore complex. The improved vector also contains an expanded multiple cloning site and a hemagglutinin epitope tag to allow confirmation of expression.ConclusionThe alterations in expression level and composition of the fusions used in this nuclear import system greatly reduce background activity in β-galactosidase assays, improving sensitivity and allowing more quantitative analysis of NLS bearing sequences.

LetterEvidence of Association between FKBP1B and Thyroid Autoimmune Disorders in a Large Tunisian Family

FKBP1B belongs to immunophilins superfamily and functions as a cytosolic receptor protein of FK506. The role of FKBP1B in the immunosuppressive pathway of FK506 is well established. Previously, we reported a strong evidence of linkage between D2S171 microsatellite marker (located in vicinity of FKBP1B gene) and susceptibility to autoimmune thyroid diseases (AITDs). In this study, we report linkage disequilibrium between the dimorphism (C/T) in the 3′ untranslated region (3′ UTR) of FKBP1B gene and susceptibility to AITDs.DNAs were extracted from a large Tunisian family affected with Graves' disease (GD) and Hashimoto's thyroiditis (HT) and analysed by PCR–RFLP using DraIII restriction enzyme. Our results showed an excess of transmission of the allele C from heterozygous parents to affected offspring (transmission disequilibrium test (TDT)=4.76; p=0.012). This suggests a linkage disequilibrium of 3′ UTR (C/T) SNP with AITDs. Moreover, The FBAT analysis gives a significant association with the C allele under the recessive model (χ2=5.50; p=0.018). These results support the involvement of FKBP1B gene in the genetic susceptibility to the AITDs development in the studied family.

Genomic organization and chromosomal localization of the importin α1 gene in the mouse

Importin α1 (also referred to as NPI1 or importin αS1) gene encodes a member of the cytosolic receptor protein superfamily that recognizes classical monopartite and bipartite basic type nuclear localization signals and mediates nuclear protein import via an importin β-dependent pathway. Here we report on the organization of the importin α1 locus in the mouse genome. The gene is approximately 40 kb in length from the translation initiation codon to the poly(A) additional site. The translated region of the gene is comprised of 13 coding exons and the exon-intron boundaries conform to the GT/AG rule. Importin α1 was mapped to a middle region of mouse chromosome 16 by fluorescence in situ hybridization analysis. Moreover, it was found by reverse transcriptase polymerase chain reaction analysis that importin α1 is widely expressed in various tissues in adult mice and at various stages during embyogenesis. This study is the first example that provides detailed genomic information on nuclear transport factors such as importins and exportins and provides a basis for further studies such as the generation of mutants in mice for purposes of investigating the role of importin α1 in development and differentiation.

Rapamycin induces apoptosis in monocyte- and CD34-derived dendritic cells but not in monocytes and macrophages

AbstractRapamycin (Rapa), a recently introduced immunosuppressive drug, seems to be effective in preventing acute allograft rejection. Although its antiproliferative effect on T lymphocytes has been investigated extensively, its effect on the initiators of the immune response, the dendritic cells (DCs), is not known. Therefore, the effect of Rapa on monocyte- (mo-DCs) and CD34+-derived DCs in vitro but also on other myeloid cell types, including monocytes and macrophages, was examined. The present study shows that Rapa does not affect phenotypic differentiation and CD40L-induced maturation of mo-DCs. However, Rapa dramatically reduced cell recovery (40%-50%). Relatively low concentrations of Rapa (10−9 M) induced apoptosis in both mo-DCs and CD34+-derived DCs, as visualized by phosphatidylserine exposure, nuclear condensation and fragmentation, and DNA degradation. In contrast, Rapa did not affect freshly isolated monocytes, macrophages, or myeloid cell lines. The sensitivity to Rapa-induced apoptosis was acquired from day 2 onward of mo-DC differentiation. Rapa exerts its apoptotic effect via a reversible binding to the cytosolic receptor protein FKBP-12, as demonstrated in competition experiments with FK506, which is structurally related to Rapa. Partial inhibition of Rapa-induced apoptosis was obtained by addition of ZVAD-fmk, which implies caspase-dependent and caspase-independent processes. The fact that Rapa exerts a specific effect on DCs but not on monocytes and macrophages might contribute to the unique actions of Rapa in the prevention of allograft rejection and other immune responses.

Ligand-induced differentiation of glucocorticoid receptor (GR) trans-repression and transactivation.

Inhaled glucocorticosteroids (GCs) are the most effective therapy for the control of asthma and are now advocated as first line therapy for most patients. Steroids reduce airway hyper-responsiveness and suppress the airway inflammatory response in asthma, but their exact mechanisms and cellular targets in the lung are uncertain 111. Classically GCs bind to a cytosolic 94kDa receptor protein, that is held in an inactive, non-DNA binding state by a 9OkDa heat shock protein (hsp90). With lioand binding, the DNA binding site is revealed and enhancement of the transcriptional response occurs following the translocation of active receptor to the nucleus. The GR binds to a consensus cis-acting DNA sequence AGAACAnnnTGTTCT (GRE) in the 5’ upstream controlling region of the genes under positive control by GCs causing an increase in gene transcription [2, 31. The mechanisms involved in gene repression are less well understood. In some instances, this may be because GR binding to a negative GRE results in steric hindrance to the binding of transcription factors that may induce the same gene. GR may also form complexes with activating transcription factors in the nucleus to inhibit their effect and so modulate genes which contain no GREs. Pro-inflammatory transcription factors, such as AP-1 and NF-KB, can form complexes with activated GR. causing mutual repression of DNA binding or affects interaction with the basal transcription apparatus thereby reducing steroid responsiveness [4-6]. Furthermore, GCs can upregulate the synthesis of I-KB, the cytosolic inhibitor of NF-KB [7]. Conversely, GCs may inhibit the effects of those cytokines that produce their effects on gene transcription via activation of AP-1 or NF-KB. For example, in human lung and in peripheral blood monocytes, TNFa and phorbol esters increase AP-1 and NF-KB binding to DNA and this is inhibited by dexamethasone [6, 81. Cytokines play an important role in chronic inflammation and the pattern of cytokine expression largely determines the nature and persistence of the inflammatory response. GCs have potent inhibitory effects on cytokine mediated inflammation and inhibit the transcription of several cytokines that are relevant in chronic inflammation, including interleukin-1 (IL-l), TNFa, GM-CSF, 11-3, 11-4, IL-5, 11-6 and IL-8 [3]. Other members of the steroid superfamily also act in an analogous manner and, in the case of retinoids, the ligand is able to modulate the ability of the receptor to bind to DNA and to AP-1 [9]. We have, therefore, investigated the ability of two glucocorticoids, dexamethasone and fluticasone, to influence GR-induced inhibition of NF-KB and AP-1 DNA binding and on the release of GM-CSF, an NF-KB-inducible gene in A549 cells. GM-CSF release was measured following stimulation with lng/ml IL-lp. Furthermore, we examined the ability of GR to bind to its DNA binding motif. Lung epithelium-like A549 cells were cultured in Dulbecco’s Modified Eagles medium (DMEM) in the presence of 2% foetal calf serum (FCS) until 70-80% confluent. FCS was removed from the culture medium and cells cultured for a further 2 days. Equal numbers of A549 cells were incubated with a range of concentrations of dexamethasone (Dex) and fluticasone propionate (FP) for 1-24 hours in the presence or absence of lnglml of IL-lp. Control experiments using cells incubated in serum free media alone were run in parallel. At every time point cells were collected by microcentrifugation and nuclear protein extracted [6]. Double stranded oligonucleotides encoding the consensus target sequence for GRE, AP-1 and NF-KB were end-labelled using t2P]-ATP and T4 polynucleotide kinase. l pg of nuclear protein from each sample was incubated with 50,000 cpm of labelled oligonucleotide. Protein-DNA complexes were separated on 6% polyacrylamide gels. The retarded band was detected by autoradiogaphy and quantified by laser densitometry. All band density measurements were then expressed as a percentage of initial binding. Specificity was determined by addition of excess unlabelled double stranded oligonucleotides. GM-CSF concentrations in culture supernatants were measured using a specific GM-CSF ELISA calibrated with human recombinant GM-CSF (0-2000pg/ml). Sensitivity of the assay was 8pglml. FP and Dex both caused a time and dose dependent inhibition of IL1p-stimulated GM-CSF release. Inhibition was first detected at 4 hrs post-steroid treatment and was at maximal levels at all time points from 10-24 hrs. At lOhrs FP caused a dose-dependent inhibition of IL-1pstimulated GM-CSF release with complete inhibition at lO-”M (EC= 5.7il.8 x lo’”), whilst Dex inhibition of IL-1p-stimulated GM-CSF release was found to be maximal at 10aM (EC= 1.2iO.5 x 10.9. This showed a 21 1-fold shift in responsiveness in spite of there being only an Figure 1. The effects of dexamethasone (Dex) and Fluticasone Proprionate (FP) on GMCSF release into the Supernatant of A549 cells.

Complete structural characterisation of the human aryl hydrocarbon receptor gene

Aims-To clone and characterise the complete structural gene for the human aryl hydrocarbon receptor (AhR). This gene, located on chromosome 7, encodes a cytosolic receptor protein which, upon activation by various xenobiotic ligands, translocates to the nucleus, where it acts as a specific transcription factor.Methods-Primers, based on the AhR cDNA sequence, were used in conjunction with recently developed long range PCR techniques to amplify contiguous sections of the cognate gene. The amplicons produced were then cloned and characterised. A cDNA probe was also used to screen a human P1 library.Results-Using the cDNA primers, DNA fragments which mapped the entire coding region of the gene were amplified and cloned. All but one of these fragments were amplified directly from human genomic DNA. The remaining fragment was amplified using DNA prepared from a P1 clone as the PCR template. This P1 clone, obtained by screening a human P1 library, also contained the entire Ah locus. Characterisation of amplified and cloned DNA fragments provided sufficient information for the construction of a complete structural map of the gene. This also included 150 base pairs of nucleotide sequence data at all intronic termini.Conclusions-These data indicate that the human AhR gene is about 50 kilobases long and contains 11 exons. The overall intron/exon structure of the human gene is homologous to that of the previously characterised mouse gene; however, it is probably some 20 kilobases larger. These results demonstrate the need for further characterisation and provide the data to facilitate this.

Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope

Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60kD importin protein (importin 60) as a single polypeptide. We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)-nitrolotriacetic acid-Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin-agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors. Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.

Mechanism of action of rapamycin: new insights into the regulation of G1-phase progression in eukaryotic cells.

The immunosuppressant drug, rapamycin (RAP), is a potent inhibitor of IL-2-dependent T-cell proliferation. The antiproliferative effect of RAP is mediated through the formation of an active complex with its cytosolic receptor protein, FKBP12. The molecular target of the FKBP12.RAP complex is a putative lipid kinase termed the mammalian Target Of Rapamycin (mTOR). This review will discuss recent findings suggesting that mTOR is a novel regulator of G1- to S-phase progression in eukaryotic cells.

Effect of dexamethasone and oxygen exposure on neonatal rat lung retinoic acid receptor proteins

Retinol deficiency in animal models results in histopathologic airway changes that appear similar to those found in human premature infants with bronchopulmonary dysplasia (BPD). Dexamethasone (DEX), a steroid now often used in the treatment of BPD, might potentially affect lung vitamin A homeostasis since it alters serum and liver retinoid stores in certain models. Our objective was to determine the effect of DEX on neonatal rat lung retinoid status and the binding of retinoic acid (RA) to cytosolic and nuclear receptor proteins. We examined this effect both in room air and when the animals breathed 95% oxygen (O2). Twenty-four 1-day-old rat pups received either 1 microgram/g DEX subcutaneously, an equal volume of normal saline (NS) subcutaneously at 0 (start experiment time), 24, and 48 hours, or no injection at all, and were sacrificed at 72 hours. Twelve rats in each treatment group were housed in room air and 12 in each group were exposed to > 95% O2 for the 3 day period. Lung and liver were analyzed for retinyl palmitate (RP). Nuclear retinoic acid receptor (RAR) and cellular retinoic acid binding protein (CRABP) were measured by specific binding assays. DEX decreased liver RP by 33-55% and rat pup lung RP by over 60%; it also decreased lung RAR binding (mean dpm/microgram protein +/- SEM) in both room air and oxygen groups: Air (11.2 +/- 1.0) vs. Air/DEX (4.6 +/- 1.3, n = 6; P < 0.01), and O2 (18.2 +/- 0.6) vs. O2/DEX (3.2 +/- 0.6, n = 6; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of [Ah] gene battery enzymes and glutathione levels by 5,10-dihydroindeno[1,2-b]indole in mouse hepatoma cell lines.

The murine aromatic hydrocarbon ([Ah]) gene battery consists of at least six genes that code for two functionalizing (Phase I) enzymes and four non-functionalizing (Phase II) enzymes. These enzymes are induced by compounds such as aromatic hydrocarbons and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) that bind to the cytosolic Ah receptor protein. Studies in rodents indicate that certain enzymes of this battery, namely cytochrome P4501A1 (CYP1A1), UDP-glucuronosyltransferase (UGT1*06) and NAD(P)H: quinone acceptor oxidoreductase (NMO1) are induced by the synthetic antioxidant 5,10-dihydroindeno[1,2-b]indole (DHII). The induction of [Ah] gene battery enzymes and the levels of reduced glutathione (GSH) were examined in mouse Hepa-1c1c7 hepatoma wild-type cells (wt), a CYP1A1 metabolism-deficient mutant (c37) and an Ah receptor nuclear translocation-defective mutant (c4). DHII and TCDD increased the activities of ethoxyresorufin O-deethylase, an indicator of CYP1A1 activity, as well as NMO1, UGT1*06, cytosolic aldehyde dehydrogenase class 3 and glutathione S-transferase form A1 in wt cells, but had little or no induction effect in c37 or c4 cells. DHII and TCDD differed in their effects on GSH levels; while DHII increased GSH levels 3-fold in wt, but not at all in c37 or c4 cells, TCDD had no effect on GSH levels in any cell type. However, GSH levels were enhanced in both wt and c4 cells by tert-butyl hydroquinone (TBHQ). L-Buthionine S,R-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, prevented DHII-induced increases in wt cell GSH. The increase in GSH levels occurred after 8 h, while the induction of enzymes occurred within 4 h. The induction of the higher GSH levels in wt cells by DHII and TBHQ correlated with increases in intracellular levels of the GSH precursor thiol cysteine, as well as with increased activities of gamma-glutamylcysteine synthetase, the rate-limiting enzyme of GSH synthesis. However, TBHQ-mediated GSH increases in c4 cells were accompanied by increased gamma-glutamylcysteine synthetase activity with no change in intracellular cysteine concentration. The results suggest that DHII induction of [Ah] gene battery enzymes requires a functional Ah receptor, but not the functional gene product CYP1A1. Furthermore, metabolism, possibly via CYP1A1, appears to be required for DHII to enhance intracellular levels of cysteine and GCS activity that result in higher GSH levels.