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1.
J Dent Res ; 100(13): 1492-1500, 2021 12.
Article in English | MEDLINE | ID: mdl-33978512

ABSTRACT

The parotid, submandibular, and sublingual glands represent a trio of oral secretory glands whose primary function is to produce saliva, facilitate digestion of food, provide protection against microbes, and maintain oral health. While recent studies have begun to shed light on the global gene expression patterns and profiles of salivary glands, particularly those of mice, relatively little is known about the location and identity of transcriptional control elements. Here we have established the epigenomic landscape of the mouse submandibular salivary gland (SMG) by performing chromatin immunoprecipitation sequencing experiments for 4 key histone marks. Our analysis of the comprehensive SMG data sets and comparisons with those from other adult organs have identified critical enhancers and super-enhancers of the mouse SMG. By further integrating these findings with complementary RNA-sequencing based gene expression data, we have unearthed a number of molecular regulators such as members of the Fox family of transcription factors that are enriched and likely to be functionally relevant for SMG biology. Overall, our studies provide a powerful atlas of cis-regulatory elements that can be leveraged for better understanding the transcriptional control mechanisms of the mouse SMG, discovery of novel genetic switches, and modulating tissue-specific gene expression in a targeted fashion.


Subject(s)
Epigenomics , Submandibular Gland , Animals , Mice , Parotid Gland , Salivary Glands , Sublingual Gland
2.
Clin Genet ; 94(1): 174-178, 2018 07.
Article in English | MEDLINE | ID: mdl-29652076

ABSTRACT

As genomic sequencing expands, so does our knowledge of the link between genetic variation and disease. Deeper catalogs of variant frequencies improve identification of benign variants, while sequencing affected individuals reveals disease-associated variation. Accumulation of human genetic data thus makes reanalysis a means to maximize the benefits of clinical sequencing. We implemented pipelines to systematically reassess sequencing data from 494 individuals with developmental disability. Reanalysis yielded pathogenic or likely pathogenic (P/LP) variants that were not initially reported in 23 individuals, 6 described here, comprising a 16% increase in P/LP yield. We also downgraded 3 LP and 6 variants of uncertain significance (VUS) due to updated population frequency data. The likelihood of identifying a new P/LP variant increased over time, as ~22% of individuals who did not receive a P/LP variant at their original analysis subsequently did after 3 years. We show here that reanalysis and data sharing increase the diagnostic yield and accuracy of clinical sequencing.


Subject(s)
Developmental Disabilities/diagnosis , Developmental Disabilities/genetics , Genetic Variation , Genomics , Intellectual Disability/diagnosis , Intellectual Disability/genetics , Alleles , DNA Copy Number Variations , Gene Frequency , Genetic Testing , Genomics/methods , Genotype , Humans , Polymorphism, Single Nucleotide , Exome Sequencing , Whole Genome Sequencing
3.
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
4.
Biochem Soc Trans ; 34(Pt 4): 578-80, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16856865

ABSTRACT

Advancing maternal age has long been identified as the primary risk factor for human chromosome trisomy. More recently, altered patterns of meiotic recombination have been found to be associated with non-disjunction. We have used trisomy 21 as a model for human non-disjunction that occurs during the formation of oocytes to understand the role of maternal age and recombination. Patterns of recombination that increase the risk for non-disjunction of chromosome 21 include absence of any exchange, an exchange near the centromere or a single, telomeric exchange. Our recent work has shown that different susceptibility patterns are associated with the origin of the meiotic error and maternal age. For MI (meiosis I) errors, the proportion of oocytes with susceptible recombination patterns is highest among young mothers and decreases significantly in the oldest age group. In fact, the pattern of exchanges among the oldest age group mimics the pattern observed among normally disjoining chromosomes 21. These results suggest that oocytes of younger women, with functional meiotic apparatus and/or robust ovarian environment, are able to properly resolve all but the most susceptible exchange patterns. As women age, however, meiotic mechanisms erode, making it difficult to resolve even stable exchange events. Interestingly, our preliminary recombination results on MII errors reveal the opposite relationship with maternal age: susceptible pericentromeric exchanges occur most often in the older age group compared with the younger age group. If confirmed, we will have further evidence for multiple risk factors for non-disjunction that act at different times in the meiotic process.


Subject(s)
Chromosomes, Human, Pair 21/genetics , Maternal Age , Recombination, Genetic/genetics , Humans , Meiosis
5.
Cytogenet Genome Res ; 111(3-4): 250-5, 2005.
Article in English | MEDLINE | ID: mdl-16192701

ABSTRACT

Within the last decade, aberrant meiotic recombination has been confirmed as a molecular risk factor for chromosome nondisjunction in humans. Recombination tethers homologous chromosomes, linking and guiding them through proper segregation at meiosis I. In model organisms, mutations that disturb the recombination pathway increase the frequency of chromosome malsegregation and alterations in both the amount and placement of meiotic recombination are associated with nondisjunction. This association has been established for humans as well. Significant alterations in recombination have been found for all meiosis I-derived trisomies studied to date and a subset of so called "meiosis II" trisomy. Often exchange levels are reduced in a subset of cases where the nondisjoining chromosome fails to undergo recombination. For other trisomies, the placement of meiotic recombination has been altered. It appears that recombination too near the centromere or too far from the centromere imparts an increased risk for nondisjunction. Recent evidence from trisomy 21 also suggests an association may exist between recombination and maternal age, the most widely identified risk factor for aneuploidy. Among cases of maternal meiosis I-derived trisomy 21, increasing maternal age is associated with a decreasing frequency of recombination in the susceptible pericentromeric and telomeric regions. It is likely that multiple risk factors lead to nondisjunction, some age dependent and others age independent, some that act globally and others that are chromosome specific. Future studies are expected to shed new light on the timing and placement of recombination, providing additional clues to the link between altered recombination and chromosome nondisjunction.


Subject(s)
Aneuploidy , Meiosis , Recombination, Genetic , Female , Humans , Male , Nondisjunction, Genetic , Ovum/physiology , Spermatozoa/physiology
6.
Cytogenet Genome Res ; 111(3-4): 273-80, 2005.
Article in English | MEDLINE | ID: mdl-16192705

ABSTRACT

The leading cause of Down syndrome (DS) is nondisjunction of chromosome 21 occurring during the formation of gametes. In this review, we discuss the progress made to identify risk factors associated with this type of chromosome error occurring in oogenesis and spermatogenesis. For errors occurring in oocytes, the primary risk factors are maternal age and altered recombination. We review the current progress made with respect to these factors and briefly outline the potential environmental and genetic influences that may play a role. Although the studies of paternal nondisjunction are limited due to the relatively small proportion of errors of this type, we review the potential influence of paternal age, recombination and other environmental and genetic factors on susceptibility. Although progress has been made to understand the mechanisms and risk factors that underlie nondisjunction, considerably more research needs to be conducted to dissect this multifactorial trait, one that has a considerable impact on our species.


Subject(s)
Down Syndrome/epidemiology , Down Syndrome/genetics , Adolescent , Adult , Female , Humans , Male , Maternal Age , Middle Aged , Models, Genetic , Nondisjunction, Genetic , Pregnancy , Risk Factors
7.
Bioinformatics ; 20(5): 808-11, 2004 Mar 22.
Article in English | MEDLINE | ID: mdl-14751982

ABSTRACT

UNLABELLED: GermOnline is a web-accessible relational database that enables life scientists to make a significant and sustained contribution to the annotation of genes relevant for the fields of mitosis, meiosis, germ line development and gametogenesis across species. This novel approach to genome annotation includes a platform for knowledge submission and curation as well as microarray data storage and visualization hosted by a global network of servers. AVAILABILITY: The database is accessible at http://www.germonline.org/. For convenient world-wide access we have set up a network of servers in Europe (http://germonline.unibas.ch/; http://germonline.igh.cnrs.fr/), Japan (http://germonline.biochem.s.u-tokyo.ac.jp/) and USA (http://germonline.yeastgenome.org/). SUPPLEMENTARY INFORMATION: Extended documentation of the database is available through the link 'About GermOnline' at the websites.


Subject(s)
Database Management Systems , Databases, Genetic , Documentation/methods , Germ Cells/cytology , Germ Cells/physiology , Information Storage and Retrieval/methods , User-Computer Interface , Animals , Artificial Intelligence , Cell Differentiation/genetics , Gene Expression Profiling/methods , Humans , Information Dissemination/methods , Internet , Oligonucleotide Array Sequence Analysis/methods , Species Specificity
8.
Nucleic Acids Res ; 32(Database issue): D560-7, 2004 Jan 01.
Article in English | MEDLINE | ID: mdl-14681481

ABSTRACT

GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on approximately 700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subject-oriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics.


Subject(s)
Cell Differentiation/genetics , Databases, Genetic , Gene Expression Profiling , Germ Cells/cytology , Germ Cells/metabolism , Animals , Computational Biology , Genomics , Humans , Information Storage and Retrieval , Internet , Meiosis/genetics , Mitosis/genetics , Oligonucleotide Array Sequence Analysis , Proteins/metabolism , Proteome , Proteomics , Rats
10.
Am J Hum Genet ; 69(6): 1255-65, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11704925

ABSTRACT

Trisomy is the most common genetic abnormality in humans and is the leading cause of mental retardation. Although molecular studies that use a large number of highly polymorphic markers have been undertaken to understand the recombination patterns for chromosome abnormalities, there is a lack of multilocus approaches to incorporating crossover interference in the analysis of human trisomy data. In the present article, we develop two statistical methods that simultaneously use all genetic information in trisomy data. The first approach relies on a general relationship between multilocus trisomy probabilities and multilocus ordered-tetrad probabilities. Under the assumption that no more than one chiasma exists in each marker interval, we describe how to use the expectation-maximization algorithm to examine the probability distribution of the recombination events underlying meioses that lead to trisomy. One limitation of the first approach is that the amount of computation increases exponentially with the number of markers. The second approach models the crossover process as a chi(2) model. We describe how to use hidden Markov models to evaluate multilocus trisomy probabilities. Our methods are applicable when both parents are available or when only the nondisjoining parent is available. For both methods, genetic distances among a set of markers can be estimated and the pattern of overall chiasma distribution can be inspected for differences in recombination between meioses exhibiting trisomy and normal meioses. We illustrate the proposed approaches through their application to a set of trisomy 21 data.


Subject(s)
Chromosome Mapping/methods , Down Syndrome/genetics , Algorithms , Computer Simulation , Crossing Over, Genetic/genetics , Female , Genetic Markers/genetics , Humans , Likelihood Functions , Male , Markov Chains , Meiosis/genetics , Models, Genetic , Nondisjunction, Genetic
11.
Org Lett ; 3(7): 1021-3, 2001 Apr 05.
Article in English | MEDLINE | ID: mdl-11277785

ABSTRACT

[structure: see text]. A general approach to functionalized hexahydroanthracene dione skeletons is described. The planar dicarbonyl triene 15 cyclized spontaneously upon oxidation of the precursor diol 14 to the tricyclicdiketone 16. The adducts 16 and 2 were further transformed to the corresponding epoxides and oxetanes as a model for the D ring of taxoids.

12.
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
13.
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
14.
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
15.
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
16.
Oncogene ; 18(46): 6222-32, 1999 Nov 04.
Article in English | MEDLINE | ID: mdl-10597220

ABSTRACT

Cell cycle modulation of cyclin A expression is due to the periodic relief of a transcriptional repression mediated by a bipartite negative DNA regulatory region. The 5' element (Cell Cycle Responsive Element: CCRE; cell Cycle Dependent Element: CDE) is clearly occupied in a cyclic manner in vivo, whereas the 3' element, whose sequence is shared by B-myb, cdc25C and cdc2 genes (cell Cycle gene Homology Region: CHR), is involved in more subtle interactions. Mutation of either element results in complete deregulation of cyclin A promoter activity. Whereas some reports claim that E2F/DP can bind to the CCRE/CDE, the nature of the protein(s) interacting with the CHR is unknown. In the present work we have characterized an activity present in quiescent cells and absent in cells blocked in S phase, which binds specifically to cyclin A CHR, but not to B-myb, or to cdc25C, or to cdc2 CHRs. A 90 kD protein, named CHF (cyclin A CHR binding factor), has been identified through preparative electrophoresis and UV crosslinking experiments. In order to address in more functional terms the binding of CHF to cyclin A CHR, we developed in vitro and in vivo oligonucleotide competition assays. Both in vitro transcription and in vivo microinjection experiments demonstrate that a functional difference exists between the composite CCRE/CDE-CHR repressor regions of cell cycle regulated genes such as cyclin A and cdc25C.


Subject(s)
Cell Cycle/genetics , Cyclin A/genetics , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , T-Lymphocytes/metabolism , Transcription Factors/isolation & purification , Transcription, Genetic , Animals , Base Sequence , Binding Sites , Binding, Competitive , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Humans , Hydroxyurea/pharmacology , Macromolecular Substances , Mice , Microinjections , Molecular Sequence Data , Molecular Weight , Resting Phase, Cell Cycle , S Phase/drug effects , T-Lymphocytes/drug effects , Transcription Factors/metabolism
17.
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
18.
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
19.
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
20.
Oncogene ; 18(30): 4357-63, 1999 Jul 29.
Article in English | MEDLINE | ID: mdl-10439043

ABSTRACT

The p16-cyclin D-pRB-E2F pathway is frequently deregulated in human tumors. This critical regulatory pathway controls the G1/S transition of the mammalian cell cycle by positive and negative regulation of E2F-responsive genes required for DNA replication. To assess the value of the transcription factors E2Fs as targets for antiproliferative strategies, we have initiated a program aiming to develop inhibitors targeting specifically these proteins in vitro and in vivo. The cellular activity of E2F is the result of the heterodimeric association of two families of proteins, E2Fs and DPs, which then bind DNA. Here, we use a two hybrid approach to isolate from combinatorial libraries peptide aptamers that specifically interact with E2Fs DNA binding and dimerization domains. One of these is a potent inhibitor of E2F binding activity in vitro and in mammalian fibroblasts, blocks cells in G1, and the free variable region from this aptamer has the same effect. Our experiments argue that the variable region of this aptamer is structured, and that it functions by binding E2F with a motif that resembles a DP heterodimerization region, and blocking E2F's association with DP. These results show that cell proliferation can be inhibited using genetically-selected synthetic peptides that specifically target protein-protein interaction motifs within cell cycle regulators. These results also emphasize the critical role of the E2F pathway for cell proliferation and might allow the design of novel antiproliferative agents targeting the cyclin/CDK-pRB-E2F pathway.


Subject(s)
Carrier Proteins , Cell Cycle Proteins , Cell Division , DNA-Binding Proteins , Growth Inhibitors/pharmacology , Peptides/pharmacology , Transcription Factors/antagonists & inhibitors , Amino Acid Sequence , Animals , Cell Line , Cricetinae , Dose-Response Relationship, Drug , Drug Design , E2F Transcription Factors , Fibroblasts , Fluorescent Antibody Technique , G1 Phase , Humans , Models, Biological , Molecular Sequence Data , Retinoblastoma-Binding Protein 1 , S Phase , Thioredoxins/chemistry , Transcription Factor DP1
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