RESUMO
The objective of this investigation was to explore the association of OPCML with gastric cancer and its clinical significance. The expression of OPCML was detected by immunohistochemistry in 118 cases of gastric carcinoma. The OPCML expression in the normal tissues and 7 kinds of gastric cells was assessed by RT-PCR. The recombinant plasmid pcDNA3.1-OPCML was constructed and transfected into AGS cells. CCK8 and colony formation assay were employed to analyze the effect of OPCML on AGS. Immunohistochemistry results showed that the expression of OPCML in gastric cancer was 68.6% and the expression of OPCML was negatively correlated with the depth of tumor invasion and tumor differentiation degree (P < 005); OPCML expression, depth of tumor invasion, lymph node metastasis and distant metastasis were important factors affecting the prognosis of the survival of the patients (P <0.05). OPCML m-RNA expression in the gastric cancer cells was significantly lower than that in the normal gastric mucosa. RT-PCR showed that the expression of OPCML was aberrantly increased in the cells transfected with pcDNA3.1-OPCML. CCK8 and colony formation assay showed that OPCML significantly inhibited the growth, proliferation, and colony formation of the AGS cells. OPCML plays an important role in gastric cancer, and may be a new prognostic indicator of gastric cancer.
Assuntos
Moléculas de Adesão Celular/fisiologia , Neoplasias Gástricas/patologia , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Proteínas Ligadas por GPI/análise , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/fisiologia , Humanos , Imuno-Histoquímica , Masculino , Neoplasias Gástricas/mortalidade , Taxa de SobrevidaRESUMO
Objective: To investigate the clinical effects of expanded frontal flap and flip scar flap in repairing partial nasal defect. Methods: A retrospective observational study was conducted. From January 2012 to January 2022, 26 patients with partial nasal defects who met the inclusion criteria were admitted to the First Affiliated Hospital of Air Force Medical University, including 19 males and 7 females, aged 5 to 61 years. The surgery was performed in 4 stages. In the first stage, a rectangular skin and soft tissue expander (hereinafter referred to as expander) with suitable rated capacity was planted in frontal region and expanded by injecting water regularly. In the second stage, flip scar flap was grafted to reconstruct nasal inner lining, whose area was about 10% larger than the area of defect. The expanded frontal flap with pedicle was transferred to repair the nasal defect, whose pedicle was supraorbital vessel or supratrochlear vessel on the contralateral side of the defect, and the area of expanded flap was 20% larger than the nasal defect area after resection and flipping of scar flap. The donor site of expanded flap was sutured directly. After 3 weeks of flap transferring, the flap was delayed in the third stage. After 1 week of delaying operation, the pedicle of flap was cut off in the fourth stage. The number, rated capacity, injection volume, and expansion time of embedded expanders were recorded. The occurrences of complications including infection, hematoma, ulceration of expanded flap after the first stage operation, and blood supply disorder or necrosis of flap after operation in the second and fourth stages were observed. All the patients were followed up for 1 year at least, and the color of flap, scar of frontal donor site, symmetry of bilateral eyebrows, and the nasal appearance and ventilated function of external nasal tract were observed. Results: A total of 26 expanders were embedded in 26 patients. The rated capacity of expanders ranged from 100 to 300 mL. The injection volume was 1.0 to 1.5 times of the rated capacity of expanders. The expansion time ranged from 2.5 to 4.0 months, with an average time of 3 months. There were no complications occurred after each operation. The follow-up showed that the color of flap was similar to the normal nasal skin, the scar of frontal region was not obvious, the bilateral eyebrows were basically symmetrical, the nose had excellent appearance, ventilation function of external nasal tract was not affected, while some of the patients had downward rotation or unapparent tip-defining point of nose. Conclusions: Using the flip scar flap to reconstruct the nasal inner lining and pre-expanded frontal flap to reconstruct the nasal skin, without free cartilage transplantation to repair the partial nasal defects can achieve satisfied nasal appearance post operation, without abnormal external nasal ventilation function.
Assuntos
Retalho Perfurante , Procedimentos de Cirurgia Plástica , Lesões dos Tecidos Moles , Masculino , Feminino , Humanos , Cicatriz/cirurgia , Transplante de Pele , Retalhos Cirúrgicos/irrigação sanguínea , Pele , Resultado do Tratamento , Lesões dos Tecidos Moles/cirurgiaRESUMO
The aim of the present study was to identify and validate the biological significance of new genes/proteins involved in the development of allergic airway disease in a murine asthma model. Gene microarrays were used to identify genes with at least a two-fold increase in gene expression in lungs of two separate mouse strains with high and low allergic susceptibility. Validation of mRNA data was obtained by western blotting and immunohistochemistry, followed by functional analysis of one of the identified genes in mice with targeted disruption of specific gene expression. Expression of two antioxidant enzymes, glutathione peroxidase-2 (GPX2) and glutathione S-transferase omega (GSTO) 1-1 was increased in both mouse strains after induction of allergic airway disease and localised in lung epithelial cells. Mice with targeted disruption of the Gpx-2 gene showed significantly enhanced airway inflammation compared to sensitised and challenged wild-type mice. Our data indicate that genes encoding the antioxidants GPX2 and GSTO 1-1 are common inflammatory genes expressed upon induction of allergic airway inflammation, and independently of allergic susceptibility. Furthermore, we provide evidence to illustrate the importance of a single antioxidant enzyme, GPX2, in protection from allergen-induced disease.
Assuntos
Asma/enzimologia , Asma/genética , Asma/imunologia , Proteínas de Transporte/genética , Glutationa Peroxidase/genética , Glutationa Transferase/genética , Alérgenos/imunologia , Animais , Western Blotting , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Feminino , Expressão Gênica , Imunoglobulina E/sangue , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Fenótipo , Pletismografia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Regulação para CimaRESUMO
Virus-transformed fibroblasts show an increased production of proteases as well as loss of extracellular adhesive proteins. To determine whether these transformation-associated events are related, we investigated the capacity of Rous sarcoma virus-transformed cells (embryonic chick fibroblasts and mouse BALB/c 3T3) to degrade fibronectin by using a novel cross-linked protein substratum: fluorescence-labeled or radiolabeled fibronectin covalently linked to the surface of a fixed gelatin film. In serum-containing medium, the coupled fibronectin was not released when incubated without cells, and only a small amount was released when incubated with nontransformed cells. However, when transformed cells were seeded on the radiolabeled fibronectin-coupled substratum, there was a threefold increase in the time-dependent release of radioactivity into the medium. The released material was characterized as peptides with molecular sizes of less than 30,000 daltons. Correspondingly, growth of transformed cells on the rhodamine-fibronectin substratum resulted in the appearance of discrete negative fluorescent spots beneath the cells and along their migratory paths, whereas a uniform fluorescent carpet was detected with nontransformed cells. The release of radioactivity was partially inhibited by protease inhibitors, including alpha 2-macroglobulin, leupeptin, and benzamidine, but the negative fluorescent spots appeared unaffected by any of these inhibitors. However, both the release of radiolabeled peptides and the appearance of fluorescence-negative spots were inhibited by 1,10-phenanthroline at concentrations that did not affect cellular attachment and protein synthesis, thus supporting a role for proteases in localized degradation of fibronectin substratum. These fluorescence-negative spots coincided with sites of fibronectin disappearance as judged by indirect labeling with antibodies to cellular fibronectin. In addition, immunofluorescent analyses showed a correlation between vinculin localization and the negative fibronectin spots found under transformed cells, indicating that degradation occurs at cell substratum contact sites. These results can be correlated with other transformation-associated phenotypic changes, and are discussed in terms of the invasion of tumor cells into the extracellular matrix.
Assuntos
Transformação Celular Viral , Fibronectinas/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Vírus do Sarcoma Aviário/genética , Células Cultivadas , Embrião de Galinha , Fibroblastos , Imunofluorescência , Gelatina , Histocitoquímica , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Inibidores de Proteases/farmacologiaRESUMO
The aryl hydrocarbon (Ah) receptor binds various environmental pollutants, such as polycyclic aromatic hydrocarbons, heterocyclic amines, and polychlorinated aromatic compounds (dioxins, dibenzofurans, and biphenyls), and mediates the carcinogenic effects of these agents. The complementary DNA and part of the gene for an 87-kilodalton human protein that is necessary for Ah receptor function have been cloned. The protein is not the ligand-binding subunit of the receptor but is a factor that is required for the ligand-binding subunit to translocate from the cytosol to the nucleus after binding ligand. The requirement for this factor distinguishes the Ah receptor from the glucocorticoid receptor, to which the Ah receptor has been presumed to be similar. Two portions of the 87-kilodalton protein share sequence similarities with two Drosophila proteins, Per and Sim. Another segment of the protein shows conformity to the consensus sequence for the basic helix-loop-helix motif found in proteins that bind DNA as homodimers or heterodimers.
Assuntos
Proteínas de Ligação a DNA , Dibenzodioxinas Policloradas/metabolismo , Proteínas/genética , Receptores de Droga/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Translocador Nuclear Receptor Aril Hidrocarboneto , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Clonagem Molecular , Citosol/metabolismo , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Sondas de Oligonucleotídeos , Proteínas/metabolismo , RNA Mensageiro/genética , Receptores de Hidrocarboneto Arílico , Receptores de Droga/genética , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
Objective: To explore the clinical effects of expanded forehead flaps in repairing midfacial defects. Methods: From January 2003 to December 2018, 19 patients with midfacial defects were admitted to our unit, including 8 males and 11 females, aged 7 to 52 years. One cylindrical expander with rated capacity ranged from 100 to 170 mL was placed in the forehead of patients in the first stage of expansion, and the total water injection volume was about 2 times of the rated capacity of the expander during 1 to 2 months. The area of midfacial defects was 4 cm×2 cm to 9 cm×5 cm after resection in the second stage surgery. Expanded forehead flaps with vascular pedicle of supratrochlear vessels or frontal branch of superficial temporal vessels were used to repair the midfacial defects, with flap size ranging from 5 cm×2 cm to 16 cm×6 cm. The donor sites were closed by direct suturing. Three weeks later, the pedicle was divided. The complications, blood supply after flap transfer and pedicle division, and the treatment effects during follow-up were observed. Results: Among the patients, flaps of 11 patients had vascular pedicle of supratrochlear vessels; flaps of 8 patients had vascular pedicle of frontal branch of superficial temporal vessels. All the flaps survived with no complications and good blood supply after flap transfer and pedicle division. During the follow-up of 6 to 12 months after the third stage surgery of pedicle division of 12 patients, no lower eyelid ectropion occurred, the appearance of the flaps was similar to the surrounding tissue with no swelling. Conclusions: The application of expanded forehead flaps can not only repair the defects but also effectively avoid the complication of lower eyelid ectropion, which is a promising method in repairing midfacial defects.
Assuntos
Testa , Retalhos Cirúrgicos , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Procedimentos de Cirurgia Plástica , Transplante de Pele , Adulto JovemRESUMO
A dominant mutant of Hepa-1 cells, c31, expresses a repressor that prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent stimulation of Cyp1a1 transcription. The repressor acts via the xenobiotic-responsive elements (XREs), which are the DNA-binding sites for the aryl hydrocarbon (Ah) receptor-TCDD complex during transcriptional activation of the gene. High-salt nuclear extracts prepared from c31 cells grown with TCDD contained normal levels of the Ah receptor which bound the XRE with normal affinity, as judged by in vitro gel mobility shift assays. Furthermore, extracts prepared from these cells, grown either with or without TCDD, contained no novel XRE-binding proteins compared with extracts from wild-type Hepa-1 cells. However, in vivo genomic footprinting demonstrated that TCDD treatment leads to binding of the Ah receptor to the XREs in Hepa-1 but not mutant cells. This finding suggests that the repressor associates with the Ah receptor to prevent its binding to the XREs and that high-salt treatment either causes dissociation of the receptor/repressor complex or fails to extract the repressor from nuclei. The results underscore the importance of using both in vivo and in vitro assays for analyzing DNA-protein interactions.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Dibenzodioxinas Policloradas/farmacologia , Receptores de Droga/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica/efeitos dos fármacos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Quimera , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Indução Enzimática , Repressão Enzimática , Genes Dominantes , Neoplasias Hepáticas Experimentais , Camundongos , Dados de Sequência Molecular , Mutação , Dibenzodioxinas Policloradas/metabolismo , Ratos , Receptores de Hidrocarboneto Arílico , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , TransfecçãoRESUMO
In estrogen receptor (ER)-positive breast cancer cell lines, very low expression of glutathione peroxidase-1 (GPX-1) activity and hgpx1 mRNA has been observed. Such cell lines have been used as models in studies of resistance to redox cycling anticancer drugs. In particular, large increases in GPX-1 activity levels by expression of transfected GPX-1 cDNA have been shown to confer some resistance to such drugs. It has never been determined that such low GPX-1 expression is a common feature of breast cancer. Based on previous limited surveys of breast cancer cell lines, it has been suggested that there may be an inverse correlation between ER status and GPX-1 production. Here we report the results from a larger survey of breast cancer cell lines, including six recently isolated cell lines. A near absence of hgpx1 mRNA expression was observed in 3 of 13 ER-negative cell lines; 1 of 4 ER-positive cell lines had high production of GPX-1. Both observations weaken the proposed inverse correlation between ER status and GPX-1 production. We have evidence to suggest that one cell line, COH-BR-5 (ER-negative), lacked hgpx1 gene expression prior to culture. This is based on the finding of stable hgpx1 gene expression during serial culture of ER-negative breast cancer cell lines newly isolated from malignant effusion and absence of hgpx1 mRNA expression in COH-BR-5. Expression of hgpx2 mRNA (producing GPXGI, the GI tract GPX) was detected in several long and newly established, ER-negative breast cancer cell lines. Cell lines, COH-BR-5 and MDA-MB-175, expressed only hgpx2 mRNA. The hgpx2 mRNA was detected in COH-BR-5 and COH-BR-7 at low passage number, suggesting that hgpx2 gene expression occurs in breast cancer malignant effusion. Thus, studies of the role of GPX in redox drug resistance may account for changes in hgpx2 gene expression. Phospholipid hydroperoxide GPX activity was not found to be generally elevated above normal tissue levels in newly established breast cancer-derived cell lines.
Assuntos
Neoplasias da Mama/enzimologia , Glutationa Peroxidase/metabolismo , Isoenzimas/metabolismo , Selênio/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Expressão Gênica , Glutationa Peroxidase/genética , Humanos , Isoenzimas/genética , RNA Mensageiro/genética , Receptores de Estrogênio/fisiologia , Células Tumorais CultivadasRESUMO
We investigated the expression of the genes for several antioxidant and xenobiotic-detoxifying enzymes in the multidrug-resistant variant of the human breast cancer cell line MCF-7, MCF-7/Dox. MCF-7/Dox is greater than 500-fold resistant to doxorubicin by clonogenic assay. Enzyme activity determinations in the cytoplasmic compartment of MCF-7/Dox revealed a 25-fold increase in glutathione peroxidase level compared to the parent line (mean +/- SD, 10 +/- 2.8 versus 0.4 +/- 0.24 nmol/min/mg; P less than 0.005). The activity of the other major hydrogen peroxide-detoxifying enzyme, catalase, was diminished in MCF-7/Dox (2.0 +/- 0.4 versus 4.8 +/- 1.4 mumol/min/mg; P less than 0.025 compared to MCF-7). Superoxide dismutase activity did not differ between the two cell lines. The specific activity of the xenobiotic-detoxifying enzyme DT-diaphorase was 4-fold lower in MCF-7/Dox compared to MCF-7 (DT-diaphorase, 117 +/- 45 versus 509 +/- 123 nmol/min/mg; P less than 0.005). Daunorubicinol-producing carbonyl reductase activity was equal in the two lines. Northern blot analysis demonstrated a 0.9-kilobase band of glutathione peroxidase mRNA in MCF-7/Dox; no glutathione peroxidase mRNA was detected in MCF-7. A 2.4-kilobase catalase and 0.7- and 1.4-kilobase superoxide dismutase mRNAs were detectable in MCF-7/Dox and MCF-7. When normalized to 28S RNA, no difference in the mRNA levels of catalase and superoxide dismutase in MCF-7/Dox and MCF-7 could be determined. DT-diaphorase mRNAs of 1.4 and 2.7 kilobases were found in both MCF-7/Dox and MCF-7 cells. A 1.2-kilobase mRNA homologous to the putative carbonyl reductase cDNA was also easily detectable in both MCF-7 and MCF-7/Dox. The amount of mRNA for both xenobiotic-detoxifying enzymes was decreased 2- to 4-fold in the doxorubicin-resistant cells. Southern blot analysis of PstI- and MspI-restricted genomic DNA revealed no evidence for amplification or rearrangement of the glutathione peroxidase gene. These results indicate that, in addition to the previously described overexpression of anionic glutathione S-transferase in MCF-7/Dox cells, an augmented glutathione peroxidase mRNA level is the major alteration in antioxidant and xenobiotic-detoxifying enzyme expression that could contribute to doxorubicin insensitivity in these multidrug-resistant breast cancer cells.
Assuntos
Oxirredutases do Álcool/genética , Neoplasias da Mama/genética , Doxorrubicina/metabolismo , Regulação Enzimológica da Expressão Gênica , Glutationa Peroxidase/genética , Quinona Redutases/genética , RNA Mensageiro/análise , Neoplasias da Mama/enzimologia , Resistência a Medicamentos , Glutationa Peroxidase/metabolismo , Humanos , Quinona Redutases/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Células Tumorais CultivadasRESUMO
We have established a variant of the human breast cancer cell line MCF7, designated MCF7/H2O2, which is 5-fold resistant to H2O2 by clonogenic assay. The specific activity of the H2O2 disposal enzyme catalase was elevated 3-fold in MCF7/H2O2; activities of other antioxidant enzymes, including glutathione peroxidase and superoxide dismutase, were not increased. The steady-state level of catalase mRNA was only slightly elevated (approx. 1.6-fold) in MCF7/H2O2 cells; however, degradation of catalase mRNA was markedly retarded in MCF-7/H2O2 compared to MCF-7 (82% of catalase mRNA remained 24 h after inhibition of RNA synthesis by actinomycin D in MCF-7/H2O2 vs. 32% in MCF7). The degradation rates of superoxide dismutase mRNA and 28 S ribosomal RNA were not reduced in MCF-7/H2O2; however, the rate of degradation of another mRNA species, beta-actin, was also significantly decreased. These data suggest that resistance to H2O2 in MCF7/H2O2 cells is mediated by elevated catalase activity which can be explained by stabilization of certain mRNA species, including catalase mRNA.
Assuntos
Neoplasias da Mama/enzimologia , Catalase/genética , Peróxido de Hidrogênio/farmacologia , RNA Mensageiro/metabolismo , Actinas/genética , Catalase/metabolismo , Dactinomicina/farmacologia , Resistência a Medicamentos , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Meia-Vida , Humanos , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Células Tumorais CultivadasRESUMO
Gpx2 mRNA, encoding a selenium-dependent glutathione peroxidase (GPX-GI), has been found to be highly expressed in the gastrointestinal tract (GI) mucosal epithelium. In this study, we show that GPX-GI is produced in the mucosal epithelium of the adult rat GI tract and that the activity levels are comparable to that from GPX-1. Post-mitochondrial supernatant GPX activity from the mucosal epithelium of the complete length of the small intestine was partially purified. A sample enriched for putative GPX-GI was fractionated by SDS-polyacrylamide gel electrophoresis. Polypeptides of 21 kDa and 22 kDa were digested with trypsin. After resolving the tryptic peptides by high pressure liquid chromatography (HPLC), the major peaks were analyzed for their amino acid sequence by Microflow-HPLC-Tandem Mass Spectrometry and automated Edman degradation sequencing. Both methods revealed that the 21-kDa sample contained rat GPX-GI determined by the sequence homology with the deduced mouse GPX-GI polypeptide sequence. Rat GPX-1 was also detected in the samples. AntiGPX-GI and antiGPX-1 antibodies were used to determine the distribution of the respective isoenzyme activities along the length of the intestine and with respect to the crypt to villus axis in rats. GPX-GI and GPX-1 activities were uniformly distributed in the middle and lower GI tract and with respect to the crypt to villus axis. GPX-GI activity accounted nearly the same percentage of the total GPX activity as GPX-1 in all of the these compartments. Studies on the distal ileum segment of wildtype and Gpx1 gene knockout mice showed that GPX-GI activity was also at parity with GPX-1 in the mucosal epithelium of this segment.
Assuntos
Glutationa Peroxidase/metabolismo , Mucosa Intestinal/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , Epitélio/enzimologia , Expressão Gênica , Glutationa Peroxidase/genética , Glutationa Peroxidase/imunologia , Imuno-Histoquímica , Intestino Delgado/enzimologia , Isoenzimas/genética , Isoenzimas/imunologia , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Selênio/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Recent studies have suggested that the selenoenzyme glutathione peroxidase, in the presence of reducing equivalents from the tripeptide glutathione, is responsible for detoxifying hydrogen peroxide and lipid hydroperoxides generated as a consequence of the cyclic reduction and oxidation of quinone-containing anticancer agents including doxorubicin, daunorubicin, mitomycin C, diaziquone, and menadione. Alterations in the intracellular levels of glutathione peroxidase or glutathione can significantly affect the activity of these drugs against human tumor cells and the expression of their normal tissue toxicity, especially with respect to the heart. Furthermore, augmentation of the glutathione peroxidase pathway appears to render certain human tumor cells relatively resistant to the anticancer quinones; therefore, the glutathione peroxidase system may, at least in part, modulate certain forms of acquired drug resistance in man. Thus, the glutathione peroxidase cycle appears to play a central role in maintaining intracellular peroxide homeostasis during quinone-induced oxidative stress.
Assuntos
Antineoplásicos/farmacologia , Glutationa Peroxidase/metabolismo , Glutationa/metabolismo , Quinonas/farmacologia , Animais , Glutationa/fisiologia , Glutationa Peroxidase/fisiologia , HumanosRESUMO
A human cDNA that encodes a polypeptide that has 94% deduced amino-acid sequence identity to porcine phospholipid hydroperoxide glutathione peroxidase was cloned from a testis library. The sequence shows preservation of the UGA selenocysteine codon, putative active-site Trp and Glu residues and a Tyr residue that is phosphorylated in the porcine protein. The 3'-UTR shows some conservation of sequences implicated in the insertion of selenocysteine at an opal codon in human glutathione peroxidase-1.
Assuntos
Glutationa Peroxidase/genética , Testículo/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , DNA Complementar , Glutationa Peroxidase/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , FosforilaçãoRESUMO
The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX; GPX4) plays a key role in eukaryotic defense against potentially lethal peroxidative injury and also regulation of physiological peroxide tone. In this work we focused on the cytoprotective antiperoxidant effects of GPX4, using a breast tumor epithelial cell line that over-expresses the enzyme. Wild-type COH-BR1 cells, which exhibit little (if any) GPX4 activity, were transfected with a construct encoding the mitochondrion-targeted long (L) form of the enzyme. Several transfectant clones were selected which expressed relatively large amounts of GPX4, as determined by both Northern and Western analysis. Enzyme activity ranged from 15-fold to 190-fold greater than that of wild-type or null-transfected cells. The functional ramifications of GPX4 overexpression were tested by challenging cells with photochemically generated cholesterol hydroperoxides (ChOOHs) in liposomal form. Compared with vector controls, overexpressing clones were found to be substantially more resistant to ChOOH-induced killing, as determined by annexin-V (early apoptotic) and thiazolyl blue (mitochondrial dehydrogenase) reactivity. Concomitantly, the clones exhibited a striking hyper-resistance to free radical-mediated lipid peroxidation, as assessed by labeling cell membranes with [(14)C]cholesterol and measuring a family of radiolabeled oxidation products (ChOX). L-form GPX4's antiperoxidant and cytoprotective effects could reflect its ability to detoxify ChOOHs as they enter cells and/or cell-derived lipid hydroperoxides arising from ChOOH one-electron turnover.
Assuntos
Apoptose/fisiologia , Colesterol/análogos & derivados , Colesterol/metabolismo , Glutationa Peroxidase/metabolismo , Peroxidação de Lipídeos/fisiologia , Estresse Oxidativo/fisiologia , Animais , Neoplasias da Mama/fisiopatologia , Glutationa Peroxidase/genética , Humanos , Isoenzimas/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Ratos , Transfecção/métodos , Células Tumorais Cultivadas/metabolismo , Regulação para Cima/fisiologiaRESUMO
GPX-GI is a cytosolic tetrameric Se-dependent glutathione peroxidase, similar in properties to GPX-1. Unlike the almost ubiquitous GPX-1, GPX-GI is mainly expressed in the epithelium of gastrointestinal tract. GPX-GI contributes to at least fifty percent of GPX activity in rodent small intestinal epithelium. The total GPX activity consists of at least 70% of selenium-dependent GPX activity in this compartment. By analyzing a panel of mouse interspecies DNA from the Jackson Laboratory's backcross resource, we mapped Gpx2 gene to mouse chromosome 12 between D12Mit4 and D12Mit5, near the Ccs1 locus which contains a colon cancer susceptibility gene. A pseudogene, Gpx2-ps is mapped to mouse chromosome 7. Comparison of Gpx2 gene expression in three pairs of C57BL/6Ha and ICR/Ha mice which are respectively resistant and sensitive to dimethylhydrazine-induced colon cancer, we found a higher Gpx2 mRNA level in C57BL/6Ha colon than ICR/Ha colon. Interestingly, a lower level of GPX activity is found in the resistant strain of mice. Because GPX-1 has three times higher specific activity than GPX-GI, our data suggest that the decreased GPX activity may result from a higher level of Gpx2 gene expression in those cells co-express Gpx1 gene.
Assuntos
Sistema Digestório/enzimologia , Glutationa Peroxidase/genética , Sequência de Aminoácidos , Animais , Glutationa Peroxidase/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
cDNA probes of human glutathione peroxidase (GSHPx) genes, including the classic GPX1 (GSHPx-1), the newly characterized GPX2 (GSHPx-GI), the plasma enzyme GPX3 (GSHPx-P), and the phospholipid hydroperoxide glutathione peroxidase GPX4 (PHGPX), were hybridized to Southern blots containing genomic DNA from human x hamster somatic cell hybrids. GPX2 was mapped to chromosome 14, GPX3 to chromosome 5 and GPX4 to chromosome 19. Additionally, human chromosomes 3 and 21 and the X chromosome were shown to contain sequences homologous to GPX1, as reported previously.
Assuntos
Cromossomos Humanos Par 14 , Cromossomos Humanos Par 19 , Cromossomos Humanos Par 5 , Glutationa Peroxidase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA , Humanos , Dados de Sequência MolecularRESUMO
The half-lives of turnover of plasma membrane proteins in rat hepatoma tissue, culture cells, and in primary cultures of rat hepatocytes have been analyzed after resolution by two-dimensional gel electrophoresis. Cell membranes were externally labeled via iodination catalyzed by lactoperoxidase and glucose oxidase. A bimodal pattern of turnover was found for the externally oriented plasma membrane proteins of rat hepatoma cells. Three glycoproteins analyzed in these cells had an average t 1/2 of 22 h while eight proteins which did not bind to concanavalin A had an average t 1/2 of 80 h. In contrast, more heterogeneous rates of turnover were found for the externally oriented plasma membrane proteins of primary cultures of hepatocytes. Most, if not all, of the membrane proteins accessible to iodination in these cells were glycoproteins. Among the glycoproteins resolved by two-dimensional polyacrylamide electrophoresis, the receptors for asialoglycoproteins had the shortest half-lives (18 h). Other glycoproteins, mostly with higher molecular weights and different isoelectric points, showed a spectrum of half-lives ranging from 16 to 99 h. The turnover rates of membrane proteins of primary cultures of rat hepatocytes were also determined with [3H]- and [35S]methionine labeling of cells. Heterogeneous rates of turnover again were found among the labeled glycoproteins and nonglycoproteins. Among the 10 glycoproteins individually analyzed, the half-lives range from 17 to 67 h. Among the 21 proteins which do not bind to concanavalin A, the half-lives range from 18 h to more than 100 h. Three proteins analyzed showed an apparent biphasic pattern of turnover, having a fast phase with a half-life of 4-6 h and a slow phase with a half-life of 15-29 h. Several nonglycoproteins, including clathrin and actin associated with membrane vesicles had extremely long half-lives. The more than 5-fold difference in the half-life between clathrin and the receptors for asialoglycoproteins, which coexist in coated pits indicates that intrinsic proteins of the coated pits turn over at a different rate than peripheral components.
Assuntos
Neoplasias Hepáticas Experimentais/metabolismo , Fígado/citologia , Proteínas de Membrana/metabolismo , Actinas/análise , Animais , Membrana Celular/metabolismo , Células Cultivadas , Clatrina/análise , Concanavalina A/metabolismo , Glicoproteínas/metabolismo , Meia-Vida , Metionina/metabolismo , Microscopia Eletrônica , Ratos , Ratos Endogâmicos BUFRESUMO
We have previously identified and characterized GSHPx-GI, which is a cellular selenium-dependent glutathione peroxidase (GSHPx) distinct from the classic GSHPx-1 and phospholipid hydroperoxide glutathione peroxidase (PHGPX). We have determined the level of GSHPx-GI mRNA expression in the rat gastrointestinal tract from esophagus to colon. Although GSHPx-GI mRNA is readily detectable throughout the GI tract, the highest level is detected in the ileum and cecum. We have also determined the levels of GSHPx-GI mRNA expression and several antioxidant enzyme activities along the villus-to-crypt axis in the rat small intestine by cell fractionation. GSHPx-GI mRNA is present at a similar level in all of the epithelial fractions, whereas GSHPx-1 mRNA is detectable only in the remnant. This suggests that GSHPx-GI is the major cellular tetrameric GSHPx expressed in intestinal epithelium, and the expression of GSHPx-GI in the GI tract is not likely regulated differentially through maturation of epithelial cells. In terms of enzymatic activity, although we detected lower glutathione S-transferase activity in the crypt epithelium, there was a marginal increase of PHGPX activity, a twofold increase of GSHPx activity, and a three- to fivefold increase of catalase activity in the crypt relative to the distal villus. Thus, the crypt epithelial cells may be better protected from peroxidative damage.
Assuntos
Glutationa Peroxidase/metabolismo , Mucosa Intestinal/enzimologia , Animais , Epitélio/enzimologia , Expressão Gênica , Glutationa Transferase/metabolismo , RNA Mensageiro/genética , Ratos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Balb/c 3T3, Swiss 3T3 and Rous sarcoma virus transformed Balb/c 3T3 mouse embryonic fibroblasts produced ceruloplasmin in vitro, whereas primary cultures prepared from the Balb/c mouse embryos did not produce ceruloplasmin. The amount of ceruloplasmin synthesis by the Balb/c 3T3 cell line is enhanced by Rous sarcoma virus-transformation (1.5-3 fold) and by treatment with dexamethasone (about 2.4 fold). The protein was identified as ceruloplasmin by immunoprecipitation with ceruloplasmin-specific polyclonal antibody, and by similarity of peptide maps, and subunit molecular weight (135,000 dalton) to that of authentic ceruloplasmin from primary cultures of mouse hepatocytes.