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1.
Mol Reprod Dev ; 75(7): 1176-84, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18161793

ABSTRACT

Cdc25C is a dual specificity phosphatase essential for dephosphorylation and activation of cyclin-dependent kinase 1 (cdk1), a prerequisite step for mitosis in all eucaryotes. Cdc25C activation requires phosphorylation on at least six sites including serine 214 (S214) which is essential for metaphase/anaphase transit. Here, we have investigated S214 phosphorylation during human meiosis with the objectives of determining if this mitotic phosphatase cdc25C participates in final meiotic divisions in human oocytes. One hundred forty-eight human oocytes from controlled ovarian stimulation protocols were stained for immunofluorescence: 33 germinal vesicle (GV), 37 metaphase stage I (MI), and 78 unfertilized metaphase stage II (MII). Results were stage dependent, identical, independent of infertility type, or stimulation protocol. During GV stages, phospho-cdc25C is localized at the oocyte periphery. During early meiosis I (MI), phosphorylated cdc25C is no longer detected until onset of meiosis I. Here, phospho-cdc25C localizes on interstitial microtubules and at the cell periphery corresponding to the point of polar body expulsion. As the first polar body reaches the periphery, phosphorylated cdc25C is localized at the junction corresponding to the mid body position. On polar body expulsion, the interior signal for phospho-cdc25C is lost, but remains clearly visible in the extruded polar body. In atresic or damaged oocytes, the polar body no longer stains for phospho-cdc25C. Human cdc25C is both present and phosphorylated during meiosis I and localizes in a fashion similar to that seen during human mitotic divisions implying that the involvement of cdc25C is conserved and functional in meiotic cells.


Subject(s)
Cell Cycle/physiology , Metaphase/physiology , Oocytes/cytology , cdc25 Phosphatases/metabolism , Amino Acid Sequence , Animals , Conserved Sequence , Female , Humans , Meiosis , Mitosis , Molecular Sequence Data , Oocytes/enzymology , Phosphorylation , Phosphoserine/metabolism , Rabbits , Sequence Alignment , Sequence Homology, Amino Acid , cdc25 Phosphatases/chemistry , cdc25 Phosphatases/genetics
2.
J Biol Chem ; 276(11): 8173-9, 2001 Mar 16.
Article in English | MEDLINE | ID: mdl-11087731

ABSTRACT

Insulin-like growth factors positively regulate muscle differentiation through activation of the phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling pathway. Here, we compare the role of the two closely related alpha (Akt1) and beta (Akt2) isoforms of PKB in muscle differentiation. During differentiation of C2.7 or L6D2 myoblasts, PKBbeta was up-regulated whereas expression of PKBalpha was unaltered. Although the two isoforms were found active in both myoblasts and myotubes, cell fractionation experiments indicated that they displayed distinct subcellular localizations in differentiated cells with only PKBbeta localized in the nuclei. In a transactivation assay, PKBbeta (either wild-type or constitutively active) was more efficient than PKBalpha in activating muscle-specific gene expression. Moreover, microinjection of specific antibodies to PKBbeta inhibited differentiation of muscle cells, whereas control or anti-PKBalpha antibodies did not. On the other hand, microinjection of the anti-PKBalpha antibodies caused a block in cell cycle progression in both non muscle and muscle cells, whereas anti-PKBbeta antibodies had no effect. Taken together, these results show that PKBbeta plays a crucial role in the commitment of myoblasts to differentiation that cannot be substituted by PKBalpha.


Subject(s)
Cell Differentiation , Muscles/cytology , Muscles/enzymology , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/physiology , Gene Expression Regulation, Enzymologic , Humans , Microinjections , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt , Transcriptional Activation
3.
Crit Care Med ; 28(7): 2271-6, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10921552

ABSTRACT

OBJECTIVES: Cardiopulmonary bypass (CPB) surgery is often associated with mild lung injury and in some patients leads to acute lung injury and acute respiratory distress syndrome (ARDS). Aberrant plasma iron chemistry (increased iron loading of transferrin and/or the presence of redox-active low molecular mass iron) and increased plasma thiol levels are features of this type of surgery and represent a potential pro-oxidant risk for oxidative damage. Oxidative damage is a feature of ARDS, and we hypothesized that pro-oxidant forces may contribute to the onset and progression of ARDS. DESIGN: Prospective, single center, observational study. SETTING: University-affiliated tertiary referral cardiothoracic center. PATIENTS: A total of 19 patients with ARDS secondary to CPB surgery and 64 patients with ARDS secondary to a variety of other predisposing causes. INTERVENTIONS: Supportive techniques appropriate to the treatment of ARDS. MEASUREMENTS AND MAIN RESULTS: Blood samples were collected into lithium heparin tubes for all patient groups on the first day of the admission of patients to the intensive care unit immediately after the diagnosis of ARDS. Plasma was immediately assayed for thiol content and total protein and albumin levels. Plasma from patients with ARDS secondary to CPB surgery was also assayed for changes in iron chemistry. Nonsurviving patients with ARDS secondary to CPB surgery displayed significantly greater levels of aberrant iron chemistry (elevated levels of iron saturation of transferrin) with decreased iron-binding antioxidant protection and elevated plasma thiol levels than did survivors. Plasma thiol levels in patients with ARDS secondary to other predisposing causes were (with the exception of lung-surgery patients) significantly elevated in survivors compared with those in nonsurvivors of the syndrome. CONCLUSIONS: Increased levels of plasma thiol appear to be associated with mortality in patients with ARDS secondary to CPB surgery.


Subject(s)
Cardiopulmonary Bypass/adverse effects , Iron/metabolism , Respiratory Distress Syndrome/blood , Respiratory Distress Syndrome/etiology , Sulfhydryl Compounds/blood , Transferrin/metabolism , APACHE , Adult , Aged , Blood Proteins , Coronary Artery Bypass , Humans , Intensive Care Units , Iron/blood , Middle Aged , Postoperative Complications/blood , Prospective Studies , Respiratory Distress Syndrome/mortality , Serum Albumin
4.
Curr Biol ; 10(9): 543-6, 2000 May 04.
Article in English | MEDLINE | ID: mdl-10801445

ABSTRACT

During skeletal muscle differentiation, a subset of myoblasts remains quiescent and undifferentiated but retains the capacity to self-renew and give rise to differentiating myoblasts [1] [2] [3]: this sub-population of muscle cells was recently termed 'reserve cells' [3]. In order to characterise genes that can regulate the ratio between reserve cells and differentiating myoblasts, we examined members of the retinoblastoma tumor suppressor family - Rb, p107 and p130 - an important family of negative regulators of E2F transcription factors and cell cycle progression [4]. Although pRb and p107 positively regulate muscle cell differentiation [5] [6] [7], the role of p130 in muscle cells remains unknown. We show here that p130 (protein and mRNA), but neither pRb nor p107, preferentially accumulates during muscle differentiation in reserve cells. Also, p130 is the major Rb-family protein present in E2F complexes in this sub-population of cells. Although forced expression of either p130 or pRb in mouse C2 myoblasts efficiently blocked cell cycle progression, only p130 inhibited the differentiation program. Furthermore, muscle cells overexpressing p130 had reduced levels of the muscle-promoting factor MyoD. In addition, p130 repressed the transactivation capacity of MyoD, an effect abolished by co-transfection of pRb. Thus, we propose that p130, by blocking cell cycle progression and differentiation, could be part of a specific pathway that defines a pool of reserve cells during terminal differentiation.


Subject(s)
Carrier Proteins , Cell Cycle Proteins , DNA-Binding Proteins , Muscle, Skeletal/cytology , Phosphoproteins/metabolism , Proteins , Animals , Cell Cycle , Cell Differentiation , Cell Line , E2F Transcription Factors , Gene Expression Regulation , Mice , MyoD Protein/genetics , Phosphoproteins/genetics , Retinoblastoma-Binding Protein 1 , Retinoblastoma-Like Protein p130 , Stem Cells/cytology , Transcription Factor DP1 , Transcription Factors/metabolism , Transcriptional Activation
5.
Virology ; 266(2): 246-56, 2000 Jan 20.
Article in English | MEDLINE | ID: mdl-10639311

ABSTRACT

Activation of the HIV-1 promoter by the virally encoded Tat protein is characterized by efficient processive transcription, mediated by host cell factors that are tethered to the promoter with the Tat-TAR RNA complex. Importantly, viral gene activation has been shown to be stimulated in mitogenically induced cells, although the link between cell cycle regulation and viral gene activation is unclear. We reported a Tat-associated CAK/CTD kinase from mitogenically induced primary human T-cells (TTK) (S. Nekhai et al., 1997, J. Virol. 71, 7436-7441). Here, biological activity of the kinase has been studied by direct microinjection at the individual-cell level. The TTK-dependent Tat response is maximal during G1 phase as shown by co-injection with Tat protein in cells synchronized at the various stages of the cell cycle. The cell cycle dependence of the Tat response was confirmed by inhibiting G0 --> G1 progression with the expression of dominant negative mutant Ras(Asn17) or the cyclin-dependent kinase CDK4. The results support a mechanism whereby transactivation of the HIV promoter is regulated by cell growth signal transduction pathways that target the Tat cofactor.


Subject(s)
Cell Cycle Proteins , Gene Products, tat/metabolism , HIV-1/genetics , HIV-1/metabolism , Promoter Regions, Genetic , Receptor Protein-Tyrosine Kinases/metabolism , Cell Cycle , Cell Line , Discoidin Domain Receptor 1 , G1 Phase , Humans , Plasmids/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases , Repetitive Sequences, Nucleic Acid , Signal Transduction , Transcriptional Activation , tat Gene Products, Human Immunodeficiency Virus
6.
Biochem J ; 344 Pt 1: 153-8, 1999 Nov 15.
Article in English | MEDLINE | ID: mdl-10548545

ABSTRACT

Haem oxygenase-1 (HO-1) is a highly inducible stress protein that removes haem from cells with the release of biliverdin, carbon monoxide and low-molecular-mass iron (LMrFe). Several antioxidant functions have been ascribed to HO; its induction is considered to be a protective event. However, LMrFe produced during haem catabolism might elicit a pro-oxidant response, with deleterious consequences. We therefore investigated the delicate balance between pro-oxidant and antioxidant events with the use of a microsomal lipid peroxidation (LPO) system. By using microsomal-bound HO in an NADPH-dependent LPO system, we assessed the pro-oxidant nature of the released LMrFe and the antioxidant effect of the released bilirubin. Hb, a biologically relevant substrate for HO, was included with the microsomes to supplement the source of haem iron and to promote LPO. We found significant increases in microsomal LPO, by using the thiobarbituric acid (TBA) test, after incubation with Hb. This Hb-stimulated peroxidation was inhibited by HO inhibitors and by iron chelators, suggesting a HO-driven, iron-dependent mechanism. GLC-MS was employed to measure the specific LPO product 4-hydroxy-2-nonenal and to confirm our TBA test results. A HO inhibitor attenuated an increase in intracellular LMrFe that occurred after treatment of rat pulmonary artery smooth-muscle cells with Hb. Additionally, exogenously added bilirubin at an equimolar concentration to the LMrFe present in both microsomal and liposomal systems was unable to prevent the pro-oxidant effect of the iron. Under certain circumstances HO can act as a pro-oxidant and seems to have a role in stimulating microsomal LPO.


Subject(s)
Heme Oxygenase (Decyclizing)/metabolism , Iron/metabolism , Microsomes, Liver/metabolism , Animals , Bilirubin/metabolism , Hemoglobins/metabolism , In Vitro Techniques , Iron/chemistry , Lipid Peroxidation , Male , Malondialdehyde/metabolism , Molecular Weight , Oxidants/metabolism , Rats , Rats, Wistar , Thiobarbituric Acid Reactive Substances/metabolism
7.
Crit Care Med ; 27(9): 1738-44, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10507592

ABSTRACT

OBJECTIVE: To assess the degree, source, and patterns of oxidative damage to bronchoalveolar lavage proteins as a modification of amino acid residues in patients with acute respiratory distress syndrome (ARDS). DESIGN: Prospective, controlled study. SETTING: Adult intensive care unit of a postgraduate teaching hospital. PATIENTS: Twenty-eight patients with established ARDS were studied and compared with six ventilated patients without ARDS and 11 normal healthy controls. INTERVENTIONS: Supportive techniques appropriate to ARDS. MEASUREMENTS AND MAIN RESULTS: Evidence of oxidative modification of bronchoalveolar lavage fluid protein, indicative of the production of specific reactive oxidizing species, was sought using a high-performance liquid chromatography technique. Bronchoalveolar lavage fluid samples from patients with ARDS, ventilated intensive care controls, and normal healthy controls were analyzed. Concentrations of orthotyrosine were significantly higher in the ARDS group than in either control group (7.98 + 3.78 nmol/mg for ARDS, 0.67 + 0.67 for ventilated controls, and 0.71 + 0.22 for healthy controls; p < .05). Chlorotyrosine concentrations were also significantly increased in the ARDS group over either control group (4.82 + 1.07 nmol/mg for ARDS, 1.55 + 1.34 for ventilated controls, and 0.33 + 0.12 for healthy controls; p < .05). Nitrotyrosine concentrations were similarly significantly increased in the ARDS groups compared with each control group (2.21 + 0.65 nmol/mg for ARDS, 0.29 + 0.29 for ventilated controls, and 0.06 + 0.03 for healthy controls; p < .05). Chlorotyrosine and nitrotyrosine concentrations showed significant correlations with myeloperoxidase concentrations in bronchoalveolar lavage fluid, measured using an enzyme-linked immunosorbent assay in patients with ARDS. These findings suggest a possible relationship between inflammatory cell activation, oxidant formation, and damage to proteins in the lungs of these patients CONCLUSIONS: Overall, our data strongly suggest heightened concentrations of oxidative stress in the lungs of patients with ARDS that lead to significantly increased oxidative protein damage.


Subject(s)
Bronchoalveolar Lavage Fluid/chemistry , Neutrophils/metabolism , Oxidative Stress/immunology , Proteins/metabolism , Respiratory Distress Syndrome/metabolism , Adolescent , Adult , Aged , Biomarkers , Case-Control Studies , Chromatography, High Pressure Liquid , Female , Humans , Hydroxyl Radical/metabolism , Hydroxylation , Hypochlorous Acid/metabolism , Linear Models , Male , Middle Aged , Neutrophil Activation , Nitrates/metabolism , Oxidants/metabolism , Proteins/immunology , Respiration, Artificial , Respiratory Distress Syndrome/immunology , Respiratory Distress Syndrome/therapy , Statistics, Nonparametric , Tyrosine/analogs & derivatives , Tyrosine/metabolism
8.
Am J Respir Crit Care Med ; 160(3): 1031-4, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10471637

ABSTRACT

Inhaled nitric oxide (.NO) is used to improve gas exchange and reduce pulmonary vascular resistance (PVR) in patients with the acute respiratory distress syndrome (ARDS). Although controlled studies have shown no survival benefit, some investigators have suggested that inhaled.NO may have antiinflammatory properties under these circumstances. In contrast, others have speculated that.NO given by inhalation could be cytotoxic, as it combines with superoxide at near diffusion-limited rates to produce the highly reactive oxidant peroxynitrite (ONOO(-)). We therefore quantified levels of 3-nitrotyrosine, a marker for ONOO(-) formation, in bronchoalveolar lavage fluid (BAL) from patients with ARDS receiving inhaled.NO, and from patients with comparable lung injury who were not so treated. We also measured levels of 3-chlorotyrosine as an index of neutrophil activation to assess indirectly the effects of inhaled.NO on lung inflammation. Patients receiving .NO had increased levels of 3-nitrotyrosine (6.76 +/- 2.79 versus 0.4 +/- 0.15 nmol/mg of protein, p < 0.05) and 3-chlorotyrosine (7.97 +/- 2.74 versus 1. 53 +/- 1.09 nmol/mg of protein, p < 0.05) in BAL protein compared with controls. In patients with ARDS, inhaled.NO increases the formation of 3-nitrotyrosine and is accompanied by an increase in levels of 3-chlorotyrosine (a marker of neutrophil activation). The possible long-term consequences of these observations remain to be evaluated.


Subject(s)
Bronchoalveolar Lavage Fluid/chemistry , Bronchodilator Agents/administration & dosage , Nitric Oxide/administration & dosage , Respiratory Distress Syndrome/drug therapy , Tyrosine/analogs & derivatives , Adult , Aged , Cell Count , Chromatography, High Pressure Liquid , Female , Humans , Male , Middle Aged , Neutrophil Activation/drug effects , Respiratory Distress Syndrome/metabolism , Tyrosine/analysis
9.
Am J Respir Cell Mol Biol ; 21(1): 105-10, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10385598

ABSTRACT

Pulmonary hypertension is characterized by hypertrophy and hyperplasia of vascular smooth muscle occurring via an unknown mechanism. Cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) are expressed under inflammatory conditions and produce mediators that regulate growth in some tissues. We have therefore addressed the question of COX-2 and iNOS involvement in proliferation of human and rat pulmonary artery (PA) smooth-muscle cells (SMC). Interleukin (IL)-1beta suppressed proliferation of both human and rat PA SMC. Moreover, IL-1beta induced COX-2 expression in both cell types. By contrast, IL-1beta stimulated the expression of iNOS protein in rat cells only. COX-2 induced in human cells inhibited proliferation, whereas COX-2 products in rat cells were without affect. However, iNOS activity in rat cells suppressed their proliferation. We conclude that human and rat evolution has diverged such that COX-2 and iNOS, although induced by the same mediator, have different levels of activity and functions in the two species. In humans, induction of COX-2 during pulmonary hypertension may be beneficial for long-term treatment of this disease.


Subject(s)
Interleukin-1/pharmacology , Isoenzymes/metabolism , Muscle, Smooth, Vascular/enzymology , Nitric Oxide Synthase/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Pulmonary Artery/enzymology , Animals , Cell Division/drug effects , Cells, Cultured , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/pharmacology , Dinoprostone/pharmacology , Epoprostenol/analogs & derivatives , Epoprostenol/pharmacology , Guanidines/pharmacology , Humans , Indans/pharmacology , Indomethacin/pharmacology , Membrane Proteins , Muscle, Smooth, Vascular/drug effects , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase Type II , Rats
10.
Mol Biol Cell ; 10(6): 1997-2015, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10359611

ABSTRACT

The intermediate filament protein vimentin is a major phosphoprotein in mammalian fibroblasts, and reversible phosphorylation plays a key role in its dynamic rearrangement. Selective inhibition of type 2A but not type 1 protein phosphatases led to hyperphosphorylation and concomitant disassembly of vimentin, characterized by a collapse into bundles around the nucleus. We have analyzed the potential role of one of the major protein phosphatase 2A (PP2A) regulatory subunits, B55, in vimentin dephosphorylation. In mammalian fibroblasts, B55 protein was distributed ubiquitously throughout the cytoplasm with a fraction associated to vimentin. Specific depletion of B55 in living cells by antisense B55 RNA was accompanied by disassembly and increased phosphorylation of vimentin, as when type 2A phosphatases were inhibited using okadaic acid. The presence of B55 was a prerequisite for PP2A to efficiently dephosphorylate vimentin in vitro or to induce filament reassembly in situ. Both biochemical fractionation and immunofluorescence analysis of detergent-extracted cells revealed that fractions of PP2Ac, PR65, and B55 were tightly associated with vimentin. Furthermore, vimentin-associated PP2A catalytic subunit was displaced in B55-depleted cells. Taken together these data show that, in mammalian fibroblasts, the intermediate filament protein vimentin is dephosphorylated by PP2A, an event targeted by B55.


Subject(s)
Intermediate Filaments/metabolism , Phosphoprotein Phosphatases/metabolism , Pyrans , Spiro Compounds , Vimentin/metabolism , Antifungal Agents/pharmacology , Cytoplasm/metabolism , Enzyme Inhibitors/pharmacology , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Intermediate Filaments/ultrastructure , Interphase/physiology , Okadaic Acid/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/genetics , Phosphorylation , Protein Phosphatase 2 , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Vimentin/genetics , Vimentin/ultrastructure
11.
FEBS Lett ; 447(1): 111-4, 1999 Mar 19.
Article in English | MEDLINE | ID: mdl-10218593

ABSTRACT

Haem oxygenase-1 is upregulated by numerous insults, including oxidative stress, and under such circumstances it is considered to be a protective stratagem. We have measured the haem oxygenase-1 expression in heart, lung and liver tissues of control and iron-overloaded rats. Lung tissue from iron-overloaded rats displayed a significant increase in the haem oxygenase-1 protein but no changes in haem oxygenase-1 mRNA. Conversely, heart tissue showed a significant increase in haem oxygenase-1 mRNA but no changes in haem oxygenase-1 protein. We conclude that during oxidative stress caused by iron overload, lung tissue responds with a rapid upregulation of haem oxygenase-1 levels.


Subject(s)
Heme Oxygenase (Decyclizing)/biosynthesis , Iron Overload/metabolism , Lung/metabolism , Oxidative Stress/physiology , Animals , Blood Pressure , Gene Expression Regulation, Enzymologic , Heme Oxygenase (Decyclizing)/genetics , Heme Oxygenase-1 , Liver/metabolism , Lung/enzymology , Male , Myocardium/metabolism , RNA, Messenger/analysis , Rats , Rats, Wistar , Up-Regulation
12.
J Cell Biol ; 142(6): 1447-59, 1998 Sep 21.
Article in English | MEDLINE | ID: mdl-9744876

ABSTRACT

The muscle regulators MyoD and Myf-5 control cell cycle withdrawal and induction of differentiation in skeletal muscle cells. By immunofluorescence analysis, we show that MyoD and Myf-5 expression patterns become mutually exclusive when C2 cells are induced to differentiate with Myf-5 staining present in cells which fail to differentiate. Isolation of these undifferentiated cells reveals that upon serum stimulation they reenter the cell cycle, express MyoD and downregulate Myf-5. Similar regulations of MyoD and Myf-5 were observed using cultured primary myoblasts derived from satellite cells. To further analyze these regulations of MyoD and Myf-5 expression, we synchronized proliferating myoblasts. Analysis of MyoD and Myf-5 expression during cell cycle progression revealed distinct and contrasting profiles of expression. MyoD is absent in G0, peaks in mid-G1, falls to its minimum level at G1/S and reaugments from S to M. In contrast, Myf-5 protein is high in G0, decreases during G1 and reappears at the end of G1 to remain stable until mitosis. These data demonstrate that the two myogenic factors MyoD and Myf-5 undergo specific and distinct cell cycle-dependent regulation, thus establishing a correlation between the cell cycle-specific ratios of MyoD and Myf-5 and the capacity of cells to differentiate: (a) in G1, when cells express high levels of MyoD and enter differentiation; (b) in G0, when cells express high levels of Myf-5 and fail to differentiate.


Subject(s)
Cell Cycle , DNA-Binding Proteins , Muscle Proteins/biosynthesis , Muscles/metabolism , MyoD Protein/biosynthesis , Trans-Activators , Animals , Cell Differentiation , Cell Division , Cell Line , Cells, Cultured , Methionine/metabolism , Mice , Mice, Inbred BALB C , Muscles/cytology , Myogenic Regulatory Factor 5
13.
Free Radic Res ; 28(3): 319-22, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9688217

ABSTRACT

Ferric ions bind to citrate and undergo an autoreduction to form a ferrous-citrate complex, greatly increasing the redox activity of the iron complex. Ferrous ions and citrate are also essential for the enzymic activity of aconitase. Aconitase, with its iron-sulphur cluster has a versatile structure which allows it to act as an iron regulatory protein (IRP-1). The purpose of this study was to see whether iron binding, and its autoreduction by citrate, could play a physiological signalling role in iron regulation. Significant amounts of ferrous ions were associated with citrate, when measured using ferrozine, however, these did not appear to activate iron-requiring aconitase.


Subject(s)
Aconitate Hydratase/metabolism , Citric Acid/metabolism , Ferric Compounds/metabolism , Iron-Sulfur Proteins/metabolism , RNA-Binding Proteins/metabolism , Animals , Chlorides , Iron Regulatory Protein 1 , Iron-Regulatory Proteins , Oxidation-Reduction , Receptors, Transferrin/metabolism
14.
Exp Cell Res ; 238(2): 305-16, 1998 Feb 01.
Article in English | MEDLINE | ID: mdl-9473338

ABSTRACT

The cyclic AMP-dependent protein kinase (PKA) type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs). A full-length human clone encoding AKAP95 was identified and sequenced, and revealed a 692-amino acid open reading frame that was 89% homologous to the rat AKAP95 (V. M. Coghlan, L. K. Langeberg, A. Fernandez, N. J. Lamb, and J. D. Scott (1994) J. Biol. Chem. 269, 7658-7665). The gene encoding AKAP95 was mapped to human chromosome 19p13.1-q12 using somatic cell hybrids and PCR. A fragment covering amino acids 414-692 of human AKAP95 was expressed in Escherichia coli and shown to bind RIIalpha. Competition with a peptide covering the RII-binding domain of AKAP Ht31 abolished RIIalpha binding to AKAP95. Immunofluorescence studies in quiescent human Hs-68 fibroblasts showed a nuclear localization of AKAP95, whereas RIIalpha was excluded from the nucleus. In contrast, during mitosis AKAP95 staining was markedly changed and appeared to be excluded from the condensed chromatin and localized outside the metaphase plate. Furthermore, the subcellular localizations of AKAP95 and RIIalpha overlapped in metaphase but started to segregate in anaphase and were again separated as AKAP95 reentered the nucleus in telophase. Finally, RIIalpha was coimmunoprecipitated with AKAP95 from HeLa cells arrested in mitosis, but not from interphase HeLa cells, demonstrating a physical association between these two molecules during mitosis. The results show a distinct redistribution of AKAP95 during mitosis, suggesting that the interaction between AKAP95 and RIIalpha may be cell cycle-dependent.


Subject(s)
Cell Cycle/genetics , Chromosomes, Human, Pair 19/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA, Complementary/genetics , DNA-Binding Proteins/genetics , Nuclear Proteins/genetics , Amino Acid Sequence , Base Sequence , Cell Line , Cell Nucleus/chemistry , Chromosome Mapping , Cloning, Molecular , Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit , Cyclic AMP-Dependent Protein Kinase Type II , Cyclic AMP-Dependent Protein Kinases/analysis , DNA-Binding Proteins/analysis , DNA-Binding Proteins/metabolism , Fibroblasts , HeLa Cells , Humans , Interphase/genetics , Intracellular Signaling Peptides and Proteins , Mitosis/genetics , Molecular Sequence Data , Nuclear Proteins/analysis , Nuclear Proteins/metabolism , Organ Specificity , RNA, Messenger/analysis , Sequence Homology, Amino Acid , Zinc Fingers/genetics
15.
Am J Respir Crit Care Med ; 157(3 Pt 1): 970-7, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9517619

ABSTRACT

Ozone is an environmental pollutant with potent oxidizing properties. We investigated whether exposure to ozone-induced cell proliferation in the lungs of rats, and determined the effect of an antioxidant and of a glucocorticosteroid in Brown-Norway (BN) rats. Following single ozone exposure (0.5, 1.0, or 3.0 ppm for 6 h), proliferating cell nuclear antigen (PCNA) expression, as determined with immunohistochemistry, was significantly increased in the bronchial epithelium and alveolar epithelium as compared with controls exposed to filtered air with a maximal effect at 24 to 48 h (p < 0.001). Apocynin (5 mg/kg, orally), a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, reduced the PCNA index in bronchial epithelium induced by ozone (3 ppm, 6 h) from 11.5 +/- 1.3% (percent of nuclear cells expressing PCNA) to 4.4 +/- 1.3% (mean +/- SEM; p < 0.05). Dexamethasone (3 mg/kg, intraperitoneally) also reduced the PCNA index in bronchial epithelium, from 19.2 +/- 2.3% to 10.9 +/- 2.6% (p < 0.05). Dexamethasone but not apocynin inhibited ozone-induced neutrophil influx. Rats exposed repeatedly to ozone (3.0 ppm, 3 h, on three occasions 48 h apart) expressed a lower PCNA index in bronchial epithelium than did rats exposed only once at 1.9 +/- 0.7% versus 6.0 +/- 0.9%, respectively (p < 0.05). The proliferative epithelial response following a single exposure to ozone is modulated through oxidative and inflammatory mechanisms probably involving neutrophils.


Subject(s)
Acetophenones/pharmacology , Air Pollutants/adverse effects , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anti-Inflammatory Agents/pharmacology , Antioxidants/pharmacology , Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Lung/drug effects , Oxidants, Photochemical/adverse effects , Ozone/adverse effects , Acetophenones/administration & dosage , Administration, Oral , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Antioxidants/administration & dosage , Bronchi/drug effects , Bronchi/pathology , Cell Division/drug effects , Chemotaxis, Leukocyte/drug effects , Dexamethasone/administration & dosage , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacology , Epithelium/drug effects , Epithelium/pathology , Gene Expression Regulation , Glucocorticoids/administration & dosage , Immunohistochemistry , Inflammation , Injections, Intraperitoneal , Lung/pathology , Male , NADP/antagonists & inhibitors , Neutrophils/drug effects , Oxidation-Reduction , Proliferating Cell Nuclear Antigen/analysis , Proliferating Cell Nuclear Antigen/genetics , Pulmonary Alveoli/drug effects , Pulmonary Alveoli/pathology , Rats , Rats, Inbred BN , Time Factors
16.
Eur J Biochem ; 248(1): 200-8, 1997 Aug 15.
Article in English | MEDLINE | ID: mdl-9310379

ABSTRACT

All protein phosphatase 2A (PP2A) holoenzymes contain a 36-kDa catalytic subunit (PP2Ac) and a regulatory subunit of 65 kDa (PR65). We have studied the interaction between PP2Ac and PR65 in an in vitro system, using PP2Ac isolated from rabbit skeletal muscle and recombinant PR65alpha expressed in bacteria or insect cells. Bacterially expressed PR65alpha exhibited identical biochemical properties to the protein expressed and isolated from the baculoviral expression system. The association of recombinant PR65 with PP2Ac was very tight (K(D)app = 85 pM) and led to a suppression of PP2A activity, which was maximal (70-80%) when phosphoproteins were used as substrates. When less-structured or smaller substrates (such as phosphopeptides) were used, this inhibition was only 30%. PR65 stimulated PP2Ac activity when the assays were performed in the presence of polycations. This indicates that the PR65 not only serves the previously predicted structural role as a molecular scaffold, but also allosterically modulates the enzymatic properties of PP2Ac. Furthermore, we identified a site of interaction between PP2Ac and PR65alpha by disruption of a stretch of basic amino acids by introduction of a glutamate at position 416. This produced an almost 100-fold reduced affinity for PP2Ac and indicated that this basic motif is an important determinant for the interaction of PR65 and PP2Ac.


Subject(s)
Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/metabolism , Amino Acid Sequence , Animals , Baculoviridae/genetics , Binding Sites , Cell Line , Dimerization , Escherichia coli/genetics , Humans , In Vitro Techniques , Molecular Sequence Data , Molecular Weight , Mutagenesis, Site-Directed , Phosphoprotein Phosphatases/genetics , Protein Conformation , Protein Phosphatase 2 , Rabbits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spodoptera , Substrate Specificity
17.
J Cell Sci ; 110 ( Pt 10): 1251-60, 1997 May.
Article in English | MEDLINE | ID: mdl-9191048

ABSTRACT

We have examined the expression, activity and localization of cyclin dependent kinase 5 (cdk5), during myogenesis. Cdk5 protein was found expressed in adult mouse muscle. In murine C2 cells, both the protein level and kinase activity of cdk5 showed a marked increase during early myogenesis with a peak between 36 and 48 hours of differentiation, decreasing as myotubes fuse after 60 to 72 hours. This increase in cdk5 protein level was specific for differentiation and not simply related to cell cycle arrest since it was not observed in fibroblasts grown for 48 hours in low serum medium. Indirect immunofluorescence using monospecific purified anti-cdk5 antibodies showed a low level cytoplasmic staining in proliferative myoblasts, a rapid increase in nuclear staining during the initial 12 hours of differentiation and a predominant nuclear staining in myotubes. Microinjection of plasmids encoding wild-type cdk5 into C2 myoblasts enhanced differentiation as assessed by both myogenin and troponin T expression after 48 hours of differentiation. In contrast, microinjection of plasmids encoding a dominant negative mutant of cdk5 inhibited the onset of differentiation. These data imply a previously unsuspected role for cdk5 protein kinase as a positive modulator of early myogenesis.


Subject(s)
Cyclin-Dependent Kinases , Muscle Development , Muscle, Skeletal/enzymology , Muscle, Skeletal/growth & development , Protein Serine-Threonine Kinases/metabolism , Animals , Base Sequence , Cell Differentiation , Cell Line , Cell Nucleus/enzymology , Cyclin-Dependent Kinase 5 , Cytoplasm/enzymology , DNA Primers/genetics , Gene Expression , Immunohistochemistry , Mice , Muscle, Skeletal/metabolism , Mutagenesis, Site-Directed , Myogenin/metabolism , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Rats , Subcellular Fractions/enzymology , Troponin/metabolism , Troponin T
18.
Am J Respir Crit Care Med ; 155(2): 479-84, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9032182

ABSTRACT

Acute respiratory distress syndrome in adults (ARDS) carries a high mortality. Patients with ARDS experience severe oxidative stress from neutrophil activation, and from treatment with high inspired oxygen concentrations (F(I)O2). Oxidative stress arises from an increased generation of reactive oxygen species (ROS) which overwhelm existing antioxidant defenses. Patients who do not survive ARDS sustain much greater levels of oxidative molecular damage, suggesting that they are less able to protect themselves against increased oxidative stress. We measured plasma levels of pro-oxidant substrates for xanthine oxidase, namely hypoxanthine and xanthine, and correlated them with the loss of plasma protein thiol groups. All patients with ARDS had higher levels of hypoxanthine (37.48 +/- 3.1 microM in nonsurvivors, 15.24 +/- 2.09 microM in survivors) compared with patients undergoing pulmonary resection (9.22 +/- 1.89 microM), patients in intensive care with sepsis but no lung injury (1.12 +/- 0.69 microM) and normal healthy control subjects (1.43 +/- 0.38 microM). The difference in plasma hypoxanthine levels between survivors and nonsurvivors of ARDS was highly significant (p < 0.001) and showed a negative correlation with loss of protein thiol groups. Xanthine levels were also higher in patients with ARDS but were not significantly different between ARDS survivors and nonsurvivors. Nonsurvivors of ARDS appear to experience higher levels of oxidative stress and damage than do survivors.


Subject(s)
Hypoxanthine/blood , Oxidative Stress , Respiratory Distress Syndrome/blood , Xanthines/blood , APACHE , Adolescent , Adult , Aged , Bronchoalveolar Lavage Fluid/chemistry , Child , Chromatography, High Pressure Liquid , Female , Humans , Male , Middle Aged , Oxygen Consumption , Reactive Oxygen Species/metabolism , Respiratory Distress Syndrome/mortality , Survival Analysis , Xanthine
20.
J Biol Chem ; 272(50): 31515-24, 1997 Dec 12.
Article in English | MEDLINE | ID: mdl-9395488

ABSTRACT

We have investigated the role of subcellular localization in the regulation of protein kinase B (PKB) activation. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N terminus of protein kinase B to alter its subcellular location. Myristoylated/palmitylated (m/p)-PKBalpha was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKBalpha was 60-fold higher compared with the unstimulated wild-type enzyme, and could not be stimulated further by growth factors or phosphatase inhibitors. In vivo 32P labeling and mutagenesis demonstrated that m/p-PKBalpha activity was due to phosphorylation on Thr308 and Ser473, that are normally induced on PKB following stimulation of the cells with insulin or insulin-like growth factor-1 (IGF-1). A dominant negative form of phosphoinositide 3-kinase (PI3-K) did not affect m/p-PKBalpha activity. The pleckstrin homology (PH) domain of m/p-PKBalpha was not required for its activation or phosphorylation on Thr308 and Ser473, suggesting that this domain may serve as a membrane-targeting module. Consistent with this view, PKBalpha was translocated to the plasma membrane within minutes after stimulation with IGF-1. This translocation required the PH domain and was sensitive to wortmannin. Our results indicate that PI3-K activity is required for translocation of PKB to the plasma membrane, where its activation occurs through phosphorylation of the same sites that are induced by insulin or IGF-1. Following activation the kinase detached from the membrane and translocated to the nucleus.


Subject(s)
Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Amino Acid Substitution , Biological Transport , Cell Membrane/metabolism , Cells, Cultured , Chromones/pharmacology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Humans , Insulin-Like Growth Factor I/pharmacology , Morpholines/pharmacology , Phosphorylation , Protein Sorting Signals/metabolism , Proto-Oncogene Proteins c-akt , Serine/metabolism , Threonine/metabolism
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