Bex1, a novel interactor of the p75 neurotrophin receptor, links neurotrophin signaling to the cell cycle

A screening for intracellular interactors of the p75 neurotrophin receptor (p75NTR) identified brain-expressed X-linked 1 (Bex1), a small adaptor-like protein of unknown function. Bex1 levels oscillated during the cell cycle, and preventing the normal cycling and downregulation of Bex1 in PC12 cells sustained cell proliferation under conditions of growth arrest, and inhibited neuronal differentiation in response to nerve growth factor (NGF). Neuronal differentiation of precursors isolated from the brain subventricular zone was also reduced by ectopic Bex1. In PC12 cells, Bex1 overexpression inhibited the induction of NF-kappaB activity by NGF without affecting activation of Erk1/2 and AKT, while Bex1 knockdown accelerated neuronal differentiation and potentiated NF-kappaB activity in response to NGF. Bex1 competed with RIP2 for binding to the p75NTR intracellular domain, and elevating RIP2 levels restored the ability of cells overexpressing Bex1 to differentiate in response to NGF. Together, these data establish Bex1 as a novel link between neurotrophin signaling, the cell cycle, and neuronal differentiation, and suggest that Bex1 may function by coordinating internal cellular states with the ability of cells to respond to external signals.     

Identification of members of the Bex gene family as olfactory marker protein (OMP) binding partners

Olfactory marker protein (OMP) expression is a hallmark of mature vertebrate olfactory receptor neurons (ORNs). Evidence for OMP function derives from altered behavioral and electrophysiological activities of OMP-KO mice. The molecular basis for the altered phenotype following the deletion of OMP is still unclear. Recent structural studies predict the involvement of OMP in protein-protein interaction. Here we report the identification of an OMP partner, Bex2, by phage-display screening of an olfactory mucosal cDNA-library. In situ hybridization demonstrates cellular co-localization of OMP mRNA with mRNAs for Bex1, Bex2, and Bex3 in ORNs of olfactory tissue of the mouse. The OMP/Bex interaction has been confirmed by demonstrating the chemical cross-linking of recombinant rat OMP with a synthetic peptide derived from the Bex amino acid sequence. The subcellular localization of Bex and OMP proteins was evaluated in transfected HEK293 cells. Bex is visualized in the nucleus and cytoplasm. Following co-transfection we observed the unexpected presence of some OMP in the nucleus along with Bex. Together, these data argue convincingly that we have identified Bex as an OMP partner whose further characterization will provide insight to the role of OMP and to the mechanism of the OMP/Bex interaction in ORN differentiation and function.     

Characterization of the Bex gene family in humans, mice, and rats

To better understand the development of ventral mesencephalic dopamine neurons, we performed subtractive hybridization screens to find ventral mesencephalic genes expressed at rat embryonic day 10 when these neurons begin to differentiate. The most commonly identified genes in these screens were members of the Bex (Brain expressed X-linked) gene family, rat Bex1 (Rex3), and a novel gene, rat Bex4. After identifying these genes, we then sought to characterize the Bex gene family. Two additional novel Bex genes (human Bex5 and mouse Bex6) were discovered through genomic databases. Bex5 is present in humans and monkeys, but not rodents, while Bex6 exists in mice, but not humans. Bex4 and Bex5 are localized to the X chromosome, are expressed in brain, and are similar in sequence. Bex4 and Bex5 are 54% and 56% identical to human Bex3 (pHGR74, NADE). Mouse Bex6 is on chromosome 16 and is 67% identical to mouse Bex4. Human Bex gene expression was studied with tissue expression arrays probed with specific oligonucleotides. Human Bex1 and Bex2 have similar expression patterns in the central nervous system with high levels in pituitary, cerebellum, and temporal lobe, and Bex1 is widely expressed outside of the central nervous system with high expression in the liver. Human Bex4 is highly expressed in heart, skeletal muscle, and liver, while Bex3 and Bex5 are more widely expressed. The subcellular localization of the Bex proteins varies from nuclear (rat Bex1) to cytoplasmic (rat Bex3, human Bex5, and mouse Bex6) and to both nuclear and cytoplasmic (rat Bex2 and rat Bex4). Rat Bex3, rat Bex4, human Bex5, and mouse Bex6 are degraded by the proteasome, while rat Bex1 or Bex2 are not. Rat Bex3 protein can likely bind transition metals through a histidine-rich domain. Because this gene family was originally named Bex and because these genes are unified by sequence similarity and gene structure, we believe the Bex nomenclature should prevail over nomenclature based on function (NADE) that has not been extended to the other Bex genes. We conclude that the Bex gene family members are highly homologous but differ in their expression patterns, subcellular localization, and degradation by the proteasome.     

Gene Targeting Study Reveals Unexpected Expression of Brain-expressed X-linked 2 in Endocrine and Tissue Stem/Progenitor Cells in Mice

Identification of genes specifically expressed in stem/progenitor cells is an important issue in developmental and stem cell biology. Genome-wide gene expression analyses in liver cells performed in this study have revealed a strong expression of X-linked genes that include members of the brain-expressed X-linked (Bex) gene family in stem/progenitor cells. Bex family genes are expressed abundantly in the neural cells and have been suggested to play important roles in the development of nervous tissues. However, the physiological role of its individual members and the precise expression pattern outside the nervous system remain largely unknown. Here, we focused on Bex2 and examined its role and expression pattern by generating knock-in mice; the enhanced green fluorescence protein (EGFP) was inserted into the Bex2 locus. Bex2-deficient mice were viable and fertile under laboratory growth conditions showing no obvious phenotypic abnormalities. Through an immunohistochemical analysis and flow cytometry-based approach, we observed unique EGFP reporter expression patterns in endocrine and stem/progenitor cells of the liver, pyloric stomach, and hematopoietic system. Although Bex2 seems to play redundant roles in vivo, these results suggest the significance and potential applications of Bex2 in studies of endocrine and stem/progenitor cells.     

Effects of growth medium and cyclic AMP analogues on the cAMP-induced differentiation of F9 teratocarcinoma cells

Summary F9 teratocarcinoma cells differentiate into parietal endodermlike cells when treated with retinoic acid (RA) and cyclic AMP (cAMP). We have previously found that F9 cells can be induced to differentiate by treatment with cAMP in the absence of RA. In the course of determining why other investigators had failed to observe cAMP-induced differentiation, we found that the growth medium played an important role in determining the response of F9 cells to differentiating agents. When F9 cells were grown in minimal essential medium (MEM) and treated with either 8-bromo-cAMP (8BrcA) + 1-methyl, 3-isobutylxanthine (MIX), or dibutyryl cAMP (DBcA) + theophylline (T), they differentiated to parietal endodermlike cells without any requirement for exogenous RA. However, when F9 cells were grown in Dulbecco’s modified Eagle’s medium (DME), DBcA/T failed to induce differentiation alone and required exogenous RA to induce formation of parietal endoderm-like cells. 8BrcA/MIX alone was still able to induce some differentiation, although the extent was not as great as those cells grown in MEM. These results could not be explained by the different growth-promoting properties of the two culture media because there was no difference in the doubling time of F9 cells grown in either medium. Likewise, RA and cAMP both inhibited growth to the same extent in either medium. Inasmuch as almost all published reports on F9 cell differentiation have used DME as a growth medium, and RA with or without DBcA/T as the differentiating agents, these studies would not have had the appropriate conditions to detect the cAMP-induced differentiation of F9 cells.     

Non-haem iron and the dissociation of piericidin A sensitivity from site 1 energy conservation in mitochondria from Torulopsis utilis

1. The aerobic incubation of iron-deficient Torulopsis utilis cells for 12h under non-growing conditions results in the recovery by mitochondria of the previously absent site 1 energy conservation and sensitivity to piericidin A. 2. The recovery of piericidin A sensitivity but not site 1 is prevented by the presence of cycloheximide (100mug/ml) in the medium used for aerobic incubation of the cells. Rotenone sensitivity behaved similarly. 3. Chloramphenicol, erythromycin and tetracycline were without effect on the recovery of site 1 and piericidin A sensitivity. 4. Inclusion of (59)Fe in the growth medium can be used as the basis for a highly sensitive assay for non-haem iron. 5. Iron-limited growth of T. utilis lowers the concentration of both non-haem iron and acid-labile sulphide of submitochondrial particles by over 20-fold compared with the ;normal' situation with iron-supplemented glycerol-limited growth. 6. Increases in the non-haem iron and acid-labile sulphide concentrations of submitochondrial particles occur when site 1 and piericidin A sensitivity are recovered. The increase is approximately halved by the presence of cycloheximide. 7. The non-haem iron of T. utilis submitochondrial particles does not exchange with added iron. 8. Continuous culture of T. utilis at the transition between glycerol- and iron-limitation results in cells where mitochondria possess site 1 energy conservation but lack piericidin A sensitivity. 8. It is concluded, in contrast with widely held views to the opposite, that energy conservation at site 1 does not require electron flow to proceed through a piericidin A- or rotenone-sensitive route. 9. Restriction of the iron supplied to growing T. utilis to a concentration just above that required for growth limitation demonstrates that a 10- to 20-fold decrease of the ;normal' non-haem iron concentration of both cells and mitochondria is without effect on the growth yield per unit of carbon source. Submitochondrial particles prepared from such iron-restricted but otherwise functionally normal cells have a non-haem iron concentration of about 0.5-0.8ng-atom/mg of protein. It is concluded that the concentration of iron-sulphur protein required for normal function by the respiratory chain is close to the concentrations of cytochromes and flavoproteins.     

A mutation inactivating the mitochondrial inner membrane folate transporter creates a glycine requirement for survival of chinese hamster cells

A mutant Chinese hamster ovary cell line, glyB, that required exogenous glycine for survival and growth was reported previously (Kao, F., Chasin, L., and Puck, T. T. (1969) Proc. Natl. Acad. Sci. U. S. A. 64, 1284-1291). We now report that the defect in glyB cells causative of this phenotype is a point mutation in an inner mitochondrial membrane protein required for transport of folates into mitochondria. The CHO mitochondrial folate transporter (mft) was sequenced and compared with that from glyB cells. The hamster sequence was nearly identical to that of the recently reported human mitochondrial folate transporter. The corresponding cDNA from glyB cells contained a single nucleotide change that introduced a glutamate in place of the glycine in wild-type hamster MFT at codon 192 in a predicted transmembrane domain. Transfection of the wild-type hamster cDNA into glyB cells allowed cell survival in the absence of glycine and the accumulation of folates in mitochondria, whereas transfection of the Glu-192 cDNA did not. Genomic sequence analysis and fluorescence in situ hybridization demonstrated a single mutated allele of the mft gene in glyB cells, whereas there were two alleles in CHO cells. We conclude that we have defined the cause of the glyB auxotrophy and that the glyB mft mutation identified a region of this mitochondrial folate carrier vital to its transport function.     

A monoclonal antibody with IL-3-like activity blocks IL-3 binding and stimulates tyrosine phosphorylation

IL-3 is a growth factor for multi-potential hemopoietic cells. A panel of mAb with IL-3-like activities was recently derived from the autoimmune mouse MRL/1 pr. We present detailed evidence that one of these monoclonal antibodies, M7B1-5.1-F9 (F9), interacts with the mouse IL-3 receptor or with part of an IL-3R complex. F9 is a full agonist (80 to 100%) of IL-3 in proliferation assays, with a half-maximum effective concentration (EC50) of 0.2 to 2.0 nM. However, in the variant cell line, NFS60.8, the EC50 for F9 is 30 nM. The decreased sensitivity to the antibody is also paralleled by an increased requirement (EC50) for IL-3. Two stromal cell lines also show increased requirements for IL-3 and F9. F9 stimulates the tyrosine phosphorylation of the same set of proteins phosphorylated after IL-3 interaction with the IL-3R, suggesting that IL-3 and F9 activate the same tyrosine kinase. F9 specifically inhibits 125I-IL-3 binding at a concentration (IC50) of about 300 nM, two log10 orders of magnitude higher than that required for its agonistic effects, suggesting that spare receptors may exist. In cross-linking assays, F9 blocks the specific binding of 125I-IL-3 to proteins of Mr 140, 130, and 70 kDa. Thus, F9 interacts with the IL-3R at or near the binding site, which leads to the stimulation of a tyrosine kinase and cell proliferation.     

Mitochondrial morphogenesis and ultrastructure of basidiomycetes from genera Agaricus and Pleurotus

Supravital study of rhodamine-stained mitochondria in cells of aerial and submerged micelium of 31 strains from 9 species from genus Agaricus (A. arvensis Schaeff., A. bisporus (Lange) Imbach, A. bitorquis (Quel.) Sacc., A. campestris L., A. excellens, (F.H. Müller) F.H. Müller, A. macrocarpus (F.H. Müller) F.H. Müller, A. silvaticus Schaeff., A. silvicola (Vittad.) Peck, A. xanthodermus Genev) and of 2 strains from 2 species from genus Pleurotus (P. ostreatus (Jacg.) P. Kumm., P. pulmonarius (Fr.) Quel.) was carried out. Mitochondrial morphogenesis in micelial cells of species from Agaricus and Pleurotus genera has many common features in distribution of mitochondria in micelial cells both under the favorable growth conditions and during chondriom reorganization (fission and fragmentation of mitochondria to small subunits) under unfarovable growth conditions and during aging. During the balanced growth of mycelium of heterokaryotic strains from genera Agaricus and Pleurotus for 7–14 days on agar media as well as during cultivation of mycelium of oyster mushroom and several strains of field mushroom in submerged culture, distribution of mitochondria by the type 1 (small granular mitochondria in the apical zone—1, long rod-like mitochondria in the subapical zone—2 and short rod-like and granular mitochondria formed as a result of fragmentation of rod-like mitochondria in mature mycelium cells—3) was observed. Under unfarovable growth conditions (starvation), in mycelium cells of homokaryotic strains of champignon, during long cultivation of all strains and species from the studied genera and during cultivation on liquid wort, for majority of champignon, distribution of mitochondria by the type 2 was observed (sphere-like mitochondria in all cells of the studied mycelium zones). Mitochondrial profiles at ultrastructural level had specific features, such as associations of the outer mitochondrial membrane with cytoplasmic ribosomes and changes in cristae structure. The preliminary test for apoptosis-like phenotype of the submerged mycelium cells of Bs94 champignon that hardly grows in the submerged culture gave positive result. Thus, mitochondrial morphogenesis in mycelium cells of Agaricus and Pleurotus species under different conditions and terms of cultivation occurs similarly and is determined by a complex of physiological and biochemical processes reflecting the state of mycelium cells.     

Caspases are not localized in mitochondria during life or death

Caspases are crucial for the initiation, propagation and execution of apoptosis. They normally exist as proenzymes, which can be activated through recruitment into activating complexes and by proteolytic cleavage by other caspases or proteases. Perturbation of organelles such as nuclei, endoplasmatic reticulum and mitochondria results in the activation of caspases. A number of caspases (-2, -3, -8 and -9) were published as being localized in the intermembrane space of mitochondria. However, in three different models of apoptosis (anti-Fas-induced cell death in murine hepatocytes, Fas ligand-induced apoptosis in Jurkat cells and apoptosis induced by growth factor withdrawal in Ba/F3 cells) we could not identify a mitochondrial location of caspases, neither under control nor under apoptotic conditions. In all three apoptotic models caspases were found in the cytosolic (caspases-2, -3, -6, -7, -8, -9) and nuclear subcellular fractions (caspases-2, -3). In another approach we treated isolated liver mitochondria with truncated Bid. Although tBid-dependent release of Cytochrome c, AIF, adenylate kinase, Smac/DIABLO and Omi/HtrA2 could be demonstrated, none of the caspases were detectable both in the supernatant and the mitochondrial fraction after treatment. Our results demonstrate that, in contrast to previous studies, no caspases-2, -3, -8 and -9 are associated with the mitochondrial fraction. These findings support the concept of a separate compartmentalization between proapoptotic cofactors in the mitochondria and silent precursor caspases in the cytosol.     

Apoptosis of metastatic prostate cancer cells by a combination of cyclin-dependent kinase and AKT inhibitors

Effective treatments for advanced prostate cancer are much needed. Toward this goal, we show apoptosis and impaired long-term survival of androgen-independent prostate cancer cells (PC3 and PC3 derivatives) co-treated with the cyclin-dependent kinase (CDK) inhibitor roscovitine and an AKT inhibitor (LY294002 or API-2). Apoptosis of PC3 cells by the drug combination required caspase-9 but not caspase-8 activity and thus is mitochondria-dependent. Roscovitine reduced amounts of the caspase inhibitor XIAP, and API-2 increased amounts of the BH3-only protein Bim. PC3 cells apoptosed when co-treated with API-2 and either cdk9 siRNA, dominant-negative cdk9, or the cdk9 inhibitor DRB; they did not apoptose when co-treated with API-2 and XIAP siRNA. Bax accumulated in mitochondria in response to API-2, whereas release of cytochrome c from mitochondria required both API-2 and roscovitine. We suggest that roscovitine elicits events that activate Bax once it translocates to mitochondria and that inactivation of cdk9 signals these events and the down-regulation of XIAP. Collectively, our data show apoptosis of prostate cancer cells by a drug combination and identify Bax activation as a basis of cooperation.     

The location and expression of fibroblast growth factor (FGF) in F9 visceral and parietal embryonic cells after retinoic acid-induced differentiation

It is well-established that fibroblast growth factors (FGFs) participate in mesoderm formation and patterning in the developing embryo. To identify cells in mammalian embryos that produce and/or respond to FGFs, we utilized the F9 teratocarcinoma cell system. Undifferentiated F9 cells resemble inner cell mass (ICM) cells of the mouse blastocyst by several criteria including having a characteristic high nuclear to cytoplasmic ratio and by their expression of stage-specific embryonic antigens. F9 stem cells differ from ICM cells by their low spontaneous rate of differentiation and their differentiation potential. ICM cells are heterogeneous with a proportion of the cells maintaining totipotency. In contrast, F9 stem cells appear capable of forming only endodermal derivatives. Retinoic acid (RA) treatment of F9 stem cells is required for them to differentiate, and under different culturing conditions the F9 cells will form either extraembryonic parietal or visceral endoderm. We have previously shown that FGF is synthesized by F9 parietal endoderm, but not by F9 stem cells. Our present study demonstrates that F9 aggregate cultures that contain visceral endoderm cells produce cell-associated-heparin-binding mitogens for 3T3 and endothelial cells, factors with characteristics of FGFs. Furthermore, our studies detect endothelial cell-mitogens within the extracellular matrix (ECM) of F9 parietal endoderm cells, not detected within F9 stem cell 'matrices'. Parietal endoderm cell matrix mitogens could be removed by prior treatment of the ECM with buffers containing heparin or 2 M NaCl, and could be neutralized by basic FGF antibodies.     

Expression of bovine F1-ATPase with functional complementation in yeast Saccharomyces cerevisiae

The mitochondrial F(1)F(0)-ATP synthase is a multimeric enzyme complex composed of at least 16 unique peptides with an overall molecular mass of approximately 600 kDa. F(1)-ATPase is composed of alpha(3)beta(3)gammadeltaepsilon with an overall molecular mass of 370 kDa. The genes encoding bovine F(1)-ATPase have been expressed in a quintuple yeast Saccharomyces cerevisiae deletion mutant (DeltaalphaDeltabetaDeltagammaDeltadeltaDeltaepsilon). This strain expressing bovine F(1) is unable to grow on medium containing a non-fermentable carbon source (YPG), indicating that the enzyme is non-functional. However, daughter strains were easily selected for growth on YPG medium and these were evolved for improved growth on YPG medium. The evolution of the strains was presumably due to mutations, but mutations in the genes encoding the subunits of the bovine F(1)-ATPase were not required for the ability of the cell to grow on YPG medium. The bovine enzyme expressed in yeast was partially purified to a specific activity of about half of that of the enzyme purified from bovine heart mitochondria. These results indicate that the molecular machinery required for the assembly of the mitochondrial ATP synthase is conserved from bovine and yeast and suggest that yeast may be useful for the expression, mutagenesis, and analysis of the mammalian F(1)- or F(1)F(0)-ATP synthase.     

Trophectoderm-specific expression of the X-linked Bex1/Rex3 gene in preimplantation stage mouse embryos

The Bex1/Rex3 gene was recently identified as an X-linked gene that is differentially expressed between parthenogenetic and normal fertilized, preimplantation stage mouse embryos. The Bex1/Rex3 gene appears to be expressed preferentially from the maternal X chromosome in blastocysts, but from either X chromosome in later stage embryonic tissues and adult tissues. To investigate whether differential expression of the Bex1/Rex3 gene between normal and parthenogenetic blastocyst stage embryos reflects genomic imprinting at the Bex1/Rex3 locus itself, or instead is the result of preferential inactivation of the paternal X chromosome or differences in timing of cellular differentiation, we examined in detail the expression pattern of the Bex1/Rex3 mRNA in normal preimplantation stage embryos, and compared its expression between androgenetic, gynogenetic, and normal fertilized embryos. Expression data reveal that the Bex1/Rex3 gene is initially transcribed at the 2-cell stage, transiently induced at the 8-cell stage, and then increases in expression again at the blastocyst stage. Very little expression is observed in isolated inner cell masses, indicating selective expression in the trophectoderm. Comparisons of Bex1/Rex3 mRNA expression between male and female androgenetic and control embryos and gynogenetic embros failed to reveal any significant difference in expression between the different classes of embryos at the 8-cell stage, or the expanding blastocyst stage (121 hr post-hCG). At the late blastocyst stage (141 hr post-hCG), expression was significantly lower in XY control embryos as compared with XX controls. Bex1/Rex3 mRNA expression did not differ between XX and XY androgenones at the blastocyst stage or between gynogenones and XX control embryos. Thus, the Bex1/Rex3 gene does not appear to be regulated directly by genomic imprinting during the preimplantation period, just as it is not regulated by imprinting at later stages. Apparent differences in gene expression may arise through the effects of trophectoderm-specific expression coupled with differences in timing of trophectoderm differentiation between the different classes of embryos and effects of preferential paternal X chromosome inactivation (XCI).     

Rhodamine 123 inhibits bioenergetic function in isolated rat liver mitochondria

Rhodamine 123 accumulates in the mitochondria of living cells and exhibits selective anticarcinoma activity. The biochemical basis of toxicity was investigated by testing the effect of the dye on isolated rat liver mitochondria. Much lower concentrations of rhodamine 123 were required to inhibit ADP-stimulated respiration and ATP synthesis in well-coupled energized mitochondria than were required to inhibit uncoupled respiration and uncoupler-stimulated ATP hydrolysis. The amount of rhodamine 123 associated with the mitochondria was several-fold greater under energized as compared to non-energized conditions, which may explain why coupled functions appeared to be more sensitive than uncoupled functions to inhibition at low concentrations of rhodamine 123. It was concluded that the site of rhodamine 123 inhibition is most likely the F0F1 ATPase complex and possibly electron transfer reactions as well.