ABSTRACT
Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age. To date, no systematic study of interactions between selenium status parameters (SSPs: serum selenium concentration, plasma glutathione peroxidase, GPX3, plasma selenoprotein P, SELENOP), sex hormones, thyroid function parameters, and other laboratory parameters in patients with PCOS has been undertaken. Therefore we aimed to compare such parameters in women with PCOS and in the control groups, and to investigate the multidimensional interactions between various parameters in PCOS patients and in controls. The subjects were diagnosed either with PCOS (n=28, 25.4±5.2 y) or with PCOS+Hashimoto disease (n=13, 27.3±5.6 y). Female patients having normal menses were recruited into the first control group (n=70, 26.8±7.3 y) or to the second control group comprising women only with Hashimoto disease (n=10, 26.2±6.9 y). No apparent differences in SSPs between control subjects and patients with PCOS, also complicated with Hashimoto disease, were identified, though such differences were noticeable for total testosterone (tT), sex hormone binding globulin, free androgen index, dehydroepiandrosterone sulfate (DHEAS), and insulin profile. The correlation between tT and DHEAS was found the strongest. The other group of mutually highly and positively correlated parameters consisted of GPX3, follicle stimulating hormone, free triiodothyronine and free thyroxine. All the latter parameters correlated negatively with vitamin D3. SSPs took part in interactions with thyroid hormones, sex hormones and some other parameters, but only for GPX3 such interactions were statistically significant. The significance of these findings remains open for further investigation, particularly in patients with PCOS and/or Hashimoto disease.
Subject(s)
Polycystic Ovary Syndrome/blood , Selenium/blood , Adult , Case-Control Studies , Dehydroepiandrosterone Sulfate/blood , Female , Humans , Insulin/blood , Insulin Resistance , Least-Squares Analysis , Sex Hormone-Binding Globulin/metabolism , Testosterone/bloodABSTRACT
We reviewed the long-term clinical and radiological results of 63 uncemented Low Contact Stress (LCS) total knee replacements (TKRs) in 47 patients with rheumatoid arthritis. The mean age of the patients at the time of surgery was 69 years (53 to 81). At a mean follow-up of 22 years (20 to 25), 12 patients were alive (17 TKRs), 27 had died (36 TKRs), and eight (ten TKRs) were lost to follow-up. Revision was necessary in seven patients (seven TKRs, 11.1%) at a mean of 12.1 years (0 to 19) after surgery. In the surviving ten patients who had not undergone revision (15 TKRs), the mean Oxford knee score was 30.2 (16 to 41) at a mean follow-up of 19.5 years (15 to 24.7) and mean active flexion was 105° (90° to 150°). The survival rate was 88.9% at 20 years (56 of 63) and the Kaplan-Meier survival estimate, without revision, was 80.2% (95% confidence interval 37 to 100) at 25 years.
Subject(s)
Arthritis, Rheumatoid/surgery , Arthroplasty, Replacement, Knee/methods , Knee Prosthesis/statistics & numerical data , Prosthesis Design/statistics & numerical data , Reoperation/statistics & numerical data , Aged , Aged, 80 and over , Arthritis, Rheumatoid/mortality , Follow-Up Studies , Humans , Middle Aged , Prospective Studies , Prosthesis Failure , Stress, Mechanical , Survival Analysis , Survival Rate , Treatment OutcomeABSTRACT
Selenium may be beneficial in reducing the risk of cancer incidence and mortality in many cancer types such as liver, prostate, colorectal and lung. However, despite the extensive recent research on selenium and selenium-containing proteins, there are still open questions concerning their expression in certain human cancer types, including colorectal carcinoma. Therefore, the expression level of the selenoproteins thioredoxin reductases 1 and 2 (TRXR-1 and TRXR-2) and glutathione peroxidases 1 and 4 (GPX1 and GPX4) in human colon carcinoma tissues was investigated. Up-regulation of TRXR-1 in the colon carcinoma specimens was found both in disease stage-dependent and independent analyses. No differences were found for TRXR-2 expression levels. GPX1 was up-regulated in carcinoma tissues at both the protein and mRNA levels. GPX4 was also up-regulated at the protein level, except for the samples derived from stage III patients. The expression of TRXR-1, GPX1 and GPX4, but not TRXR-2 is differently regulated in cancer as compared to healthy colonic tissue.
Subject(s)
Colonic Neoplasms/metabolism , Neoplasm Proteins/metabolism , Selenium/analysis , Base Sequence , DNA Primers , Enzyme-Linked Immunosorbent Assay , Humans , Immunohistochemistry , Neoplasm Proteins/chemistry , Polymerase Chain ReactionABSTRACT
The ovarian cancer cell lines A2780 (wild-type p53) and NIHOVCAR3 (mutated p53) showed, respectively, sensitivity and resistance towards several chemotherapy drugs. We hypothesized that the two cell lines differ in their ability to activate the intrinsic death pathway and have, therefore, dissected the lysosome-mitochondrion signalling pathway by pharmacological inhibition or genetic manipulation of key regulators and executioners. Biochemical and morphological confocal fluorescence studies showed that: (1) In A2780 cells bcl-2 is expressed at an undetectable level, whereas Bax is expressed at a rather high level; by contrast, bcl-2 is highly expressed and Bax is expressed at extremely low levels in NIHOVCAR3 cells; (2) Chemotherapy treatment reduced the expression of bcl-2 in NIHOVCAR3 cells, yet these cells resisted to drug toxicity; (3) Cathepsin D (CD), not cathepsin B or L, mediates the activation of the mitochondrial intrinsic death pathway in A2780 cells; (4) Lysosome leakage and cytosolic relocation of CD occurs in the chemosensitive A2780 cells, not in the chemoresistant NIHOVCAR3 cells; (5) Bax is essential for the permeabilization of both lysosomes and mitochondria in A2780 cells exposed to chemotherapy drugs; (6) CD activity is mandatory for the oligomerization of Bax on both mitochondrial and lysosomal membranes; (7) Bax activation did not occur in the resistant NIHOVCAR3 cells despite their high content in CD. The present data are consistent with a model in which on treatment with a cytotoxic drug the activation of a CD-Bax loop leads to the generalized permeabilization of lysosomes and eventually of mitochondria, thus reaching the point of no return, and culminates with the activation of the caspase cascade. Our data also imply that dysfunctional permeabilization of lysosomes contributes to the development of chemoresistance.
Subject(s)
Antineoplastic Agents/pharmacology , Cathepsin D/metabolism , Signal Transduction/drug effects , bcl-2-Associated X Protein/metabolism , Apoptosis/drug effects , Cathepsin D/genetics , Cell Line, Tumor , Cell Survival/drug effects , Cisplatin/pharmacology , Dose-Response Relationship, Drug , Etoposide/pharmacology , Female , Humans , Immunoblotting , Lysosomes/drug effects , Lysosomes/metabolism , Membrane Potential, Mitochondrial/drug effects , Microscopy, Fluorescence , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Paclitaxel/pharmacology , Protein Transport/drug effects , RNA Interference , bcl-2-Associated X Protein/geneticsABSTRACT
In human colorectal DLD1 cancer cells, the dietary bioflavonoid resveratrol (RV) rapidly induced autophagy. This effect was reversible (on removal of the drug) and was associated with increased expression and cytosolic redistribution of the proteins Beclin1 and LC3 II. Supplementing the cells with asparagine (Asn) abrogated the Beclin-dependent autophagy. When applied acutely (2 h), RV was not toxic; however, reiterate chronic (48 h) exposure to RV eventually led to annexin V- and terminal deoxinucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cell death. This toxic effect was autophagy dependent, as it was prevented either by Asn, by expressing a dominant-negative lipid kinase-deficient class III phosphoinositide 3-phosphate kinase, or by RNA interference knockdown of Beclin1. Lamp2b silencing abolished the fusion of autophagosomes with lysosomes and preserved cell viability despite the ongoing formation of autophagosomes in cells chronically exposed to RV. The pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone inhibited RV-induced cell death, but not autophagy. These results uncover a novel pathway of RV cytotoxicity in which autophagy plays a dual role: (i) at first, it acts as a prosurvival stress response and (ii) at a later time, it switches to a caspase-dependent apoptosis pathway. The present data also indicate that genetic or epigenetic inactivation of autophagy proteins in cancer cells may confer resistance to RV-mediated killing.
Subject(s)
Apoptosis , Enzyme Inhibitors/pharmacology , Phagosomes/metabolism , Phosphatidylinositol 3-Kinases/biosynthesis , Phosphotransferases/metabolism , Stilbenes/pharmacology , Autophagy , Cell Line, Tumor , Epigenesis, Genetic , Gene Silencing , Genes, Dominant , Humans , Lipid Metabolism , Lysosomes/metabolism , Phosphatidylinositol 3-Kinases/metabolism , ResveratrolABSTRACT
Hydrogen peroxide, the major oxidoradical species in the central nervous system, has been involved in neuronal cell death and associated neurodegenerative diseases. In this study, we have investigated the involvement of the lysosomal pathway in the cytotoxic mechanism of hydrogen peroxide in human neuroblastoma cells. Alteration of lysosomal and mitochondrial membrane integrity was shown to be an early event in the lethal cascade triggered by oxidative stress. Desferrioxamine (DFO), an iron chelator that abolishes the formation of reactive oxygen species within lysosomes, prevented lysosome leakage, mitochondrial permeabilization and caspase-dependent apoptosis in hydrogen peroxide-treated cells. Inhibition of cathepsin D, not of cathepsin B, as well as small-interference RNA-mediated silencing of the cathepsin D gene prevented hydrogen peroxide-induced injury of mitochondria, caspase activation, and TUNEL-positive cell death. Cathepsin D activity was shown indispensable for translocation of Bax onto mitochondrial membrane associated with oxidative stress. DFO abolished both the cytosolic relocation of Cathepsin D and the mitochondrial relocation of Bax in hydrogen peroxide-treated cells. siRNA-mediated down-regulation of Bax expression protected the cells from oxidoradical injury. The present study identifies the lysosome as the primary target and the axis cathepsin D-Bax as the effective pathway of hydrogen peroxide lethal activity in neuroblastoma cells.
Subject(s)
Cathepsin D/metabolism , Deferoxamine/pharmacology , bcl-2-Associated X Protein/metabolism , Cathepsin D/genetics , Cell Line, Tumor , Cell Survival/drug effects , Humans , Hydrogen Peroxide/pharmacology , Neuroblastoma , Oxidative Stress/drug effects , Pepstatins/pharmacology , RNA, Small Interfering/genetics , Siderophores/pharmacology , Transfection , bcl-2-Associated X Protein/geneticsABSTRACT
Adequate supply of selenium (Se) is critical for synthesis of selenoproteins through selenocysteine insertion mechanism. To explore this process we investigated the expression of the cytosolic and mitochondrial isoenzymes of thioredoxin reductase (TrxR1 and TrxR2) in response to altered Se supply. Rats were fed diets containing different quantities of selenium and the levels of TrxR1 and TrxR2 protein and their corresponding mRNAs were determined in liver and kidney. Expression of the two isoenzymes was differentially affected, with TrxR1 being more sensitive to Se depletion than TrxR2 and greater changes in liver than kidney. In order to determine if the selenocysteine incorporation sequence (SECIS) element was critical in this response liver and kidney cell lines (H4 and NRK-52E) were transfected with reporter constructs in which expression of luciferase required read-through at a UGA codon and which contained either the TrxR1 or TrxR2 3'UTR, or a combination of the TrxR1 5' and 3'UTRs. Cell lines expressing constructs with the TrxR1 3'UTR demonstrated no response to restricted Se supply. In comparison the Se-deficient cells expressing constructs with the TrxR2 3'UTR showed considerably less luciferase activity than the Se-adequate cells. No disparity of response to Se supply was observed in the constructs containing the different TrxR1 5'UTR variants. The data show that there is a prioritisation of TrxR2 over TrxR1 during Se deficiency such that TrxR1 expression is more sensitive to Se supply than TrxR2 but this sensitivity of TrxR1 was not fully accounted for by TrxR1 5' or 3'UTR sequences when assessed using luciferase reporter constructs.
Subject(s)
Cytosol/enzymology , Kidney/enzymology , Liver/enzymology , Mitochondria/enzymology , Rats/metabolism , Selenium/administration & dosage , Thioredoxin-Disulfide Reductase/metabolism , Administration, Oral , Animals , Cells, Cultured , Cytosol/drug effects , Dose-Response Relationship, Drug , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/physiology , Kidney/drug effects , Liver/drug effects , Male , Mitochondria/drug effectsABSTRACT
The precursor of human cathepsin D (CD) is converted into the single-chain and the double-chain active polypeptides by subsequent proteolysis reactions taking place in the endosomal-lysosomal compartment and involving specific aminoacid sequences. We have mutagenized the region of aminoacids (comprising the beta-hairpin loop) involved in the latter proteolytic maturation step and generated a mutant CD that cannot be converted into the mature double-chain form. This mutant CD expressed in rodent cells reaches the lysosome and is stable as single-chain polypeptide, bears high-mannose type sugars, binds to pepstatin A and is enzymatically active, indicating that it is correctly folded. The present work provides new insights on the aminoacid region involved in the terminal processing of human CD and on the function of the processing beta-hairpin loop.
Subject(s)
Cathepsin D/chemistry , Cathepsin D/genetics , Amino Acid Sequence , Animals , CHO Cells , Cathepsin D/antagonists & inhibitors , Cathepsin D/metabolism , Cell Line , Cricetinae , Cricetulus , Humans , Hydrogen-Ion Concentration , Lysosomes/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Pepstatins/metabolism , Pepstatins/pharmacology , Protein Binding , Protein Folding , Protein Processing, Post-Translational , Protein Structure, Quaternary , Protein Transport , Rats , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TransfectionABSTRACT
In human colorectal cancer cells, the polyphenol resveratrol (RV) activated the caspase-dependent intrinsic pathway of apoptosis. This effect was not mediated via estrogen receptors. Pepstatin A, an inhibitor of lysosomal cathepsin D (CD), not (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, an inhibitor of cathepsins B and L, prevented RV cytotoxicity. Similar protection was attained by small interference RNA-mediated knockdown of CD protein expression. RV promoted the accumulation of mature CD, induced lysosome leakage and increased cytosolic immunoreactivity of CD. Inhibition of CD or its post-transcriptional down-regulation precluded Bax oligomerization, permeabilization of mitochondrial membrane, cytosolic translocation of cytochrome c, caspase 3 activation and terminal deoxinucleotidyl transferase-mediated dUTP-biotin nick end labeling positivity occurring in RV-treated cells. The present study identifies the lysosome as a novel target of RV activity and demonstrates a hierarchy of the proteolytic pathways involved in its cytotoxic mechanism in which the lysosomal CD acts upstream of the cytosolic caspase activation. Our data indicate that metabolic, pharmacologic or genetic conditions affecting CD expression and/or activity could reflect on the sensitivity of cancer cells to RV.
Subject(s)
Cathepsin D/metabolism , Colorectal Neoplasms/metabolism , Lysosomes/metabolism , Stilbenes/pharmacology , Caspase Inhibitors , Cathepsin L , Cathepsins/metabolism , Cell Death/drug effects , Cell Line , Colorectal Neoplasms/pathology , Cysteine Endopeptidases/metabolism , Cytochromes c/metabolism , Cytosol/metabolism , Dose-Response Relationship, Drug , HT29 Cells , Humans , Resveratrol , Time FactorsABSTRACT
We analysed the long-term clinical and radiological results of 63 uncemented Low Contact Stress total knee replacements in 47 patients with rheumatoid arthritis. At a mean follow-up of 12.9 years (10 to 16), 36 patients (49 knees) were still alive; three patients (five knees) were lost to follow-up. Revision was necessary in three knees (4.8%) and the rate of infection was 3.2%. The mean clinical and functional Knee Society scores were 90 (30 to 98) and 59 (25 to 90), respectively, at final follow-up and the mean active range of movement was 104 degrees (55 degrees to 120 degrees ). The survival rate was 94% at 16 years but 85.5% of patients lost to follow-up were considered as failures. Radiological evidence of impending failure was noted in one knee.
Subject(s)
Arthritis, Rheumatoid/surgery , Knee Prosthesis , Aged , Aged, 80 and over , Arthritis, Rheumatoid/physiopathology , Arthroplasty, Replacement, Knee/methods , Bone Cements , Cementation , Humans , Knee Joint/physiopathology , Middle Aged , Prosthesis Failure , Range of Motion, Articular , Reoperation , Retrospective Studies , Stress, Mechanical , Treatment OutcomeABSTRACT
Selenium (Se) can protect endothelial cells (EC) from oxidative damage by altering the expression of selenoproteins with antioxidant function such as cytoplasmic glutathione peroxidase (cyGPX), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and thioredoxin reductase (TR). If the role of Se on EC function is to be studied, it is essential that a model system be chosen which reflects selenoprotein expression in human EC derived from vessels prone to developing atheroma. We have used [75Se]-selenite labelling and selenoenzyme measurements to compare the selenoproteins expressed by cultures of EC isolated from different human vasculature with EC bovine and porcine aorta. Only small differences were observed in selenoprotein expression and activity in EC originating from human coronary artery, human umbilical vein (HUVEC), human umbilical artery and the human EC line EAhy926. The selenoprotein profile in HUVEC was consistent over eight passages and HUVEC isolated from four cords also showed little variability. In contrast, EC isolated from pig and bovine aorta showed marked differences in selenoprotein expression when compared to human cells. This study firmly establishes the suitability and consistency of using HUVEC (and possibly the human cell line EAhy926) as a model to study the effects of Se on EC function in relation to atheroma development in the coronary artery. Bovine or porcine EC appear to be an inappropriate model.
Subject(s)
Endothelium, Vascular/metabolism , Protein Biosynthesis , Animals , Aorta , Arteriosclerosis/metabolism , Autoradiography , Cattle , Cell Line , Coronary Vessels , Culture Media , Electrophoresis, Polyacrylamide Gel , Humans , Proteins/analysis , Selenious Acid , Selenium Radioisotopes , Selenoproteins , Umbilical ArteriesABSTRACT
Intramuscular (i.m.) injection of plasmids followed by electropermeabilization is an efficient process to deliver genes into skeletal myofibers that permits proteins to be produced and secreted at therapeutically relevant levels. To further improve skeletal muscle as a bioreactor, we identified a formulation that elevates transgene expression in myofibers after i.m. injection and electroporation. With secreted placental alkaline phosphate (SEAP) as reporter gene, plasmid formulated with poly-L-glutamate produced two- to eight-fold higher levels of SEAP in mouse serum than plasmid in saline. Various concentrations and molecular weights of poly-L-glutamate were similarly effective, but 6 mg/ml of 15-50 kDa poly-L-glutamate consistently yielded the highest expression levels. The poly-L-glutamate formulation was effective in two different muscle groups in mice at various plasmid doses for several transgenes, including an erythropoietin (EPO) gene, for which expression was elevated four- to 12-fold in comparison to animals that received EPO plasmid in saline. Transgene expression was localized to myofibers. Poly-L-glutamate may improve transgene expression in part by increasing plasmid retention in skeletal muscle. Poly-L-glutamate did not enhance gene transfer in the absence of electroporation. Therefore, the polymer is a novel formulation that specifically enhances the transfer and expression of genes delivered with electroporation.
Subject(s)
Electroporation/methods , Genetic Therapy/methods , Muscle, Skeletal/metabolism , Plasmids/administration & dosage , Polyglutamic Acid , Alkaline Phosphatase/genetics , Animals , DNA/analysis , Erythropoietin/analysis , Erythropoietin/genetics , Female , Gene Expression , Genes, Reporter , Hindlimb , Humans , Injections, Intramuscular , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Polymerase Chain Reaction/methods , Regression Analysis , TransgenesABSTRACT
The aim of the present work was to clarify whether the activities of selenoenzymes can serve as markers for different tumors or goiters, as classified by histological criteria. The following parameters were determined: 1) selenium content of plasma (Se), 2) activities of the selenoenzymes: plasma glutathione peroxidase (plGSHPx), cytosolic glutathione peroxidase (cGSHPx), type I and type II iodothyronine deiodinases (ID-I, ID-II), thioredoxin reductase (THRR) in human thyroid tissues. The material came from follicular neoplasm, papillary carcinoma, struma nodosa, struma lymphomatosis Hashimoto, other thyroid surgery specimens, and normal tissues. There was no difference in Se nor in plGSHPx between patients and healthy volunteers. No significant differences were found for any parameter in thyroid carcinoma versus normal or goitrous thyroid tissue. In the whole group of thyroid surgery specimens the statistically significant correlations were found between ID-I and ID-II and between THRR and selenoperoxidases. Principal components analysis confirmed the above correlation and moreover revealed correlation between Se and plGSHPx, but did not detect any clear distinction between patients with the different diagnoses.
Subject(s)
Proteins/analysis , Thyroid Gland/chemistry , Thyroid Gland/enzymology , Thyroid Neoplasms/chemistry , Thyroid Neoplasms/enzymology , Adenoma/chemistry , Adenoma/enzymology , Adult , Aged , Carcinoma, Papillary/chemistry , Carcinoma, Papillary/enzymology , Glutathione Peroxidase/blood , Glutathione Peroxidase/metabolism , Humans , Iodide Peroxidase/metabolism , Middle Aged , Reference Values , Selenium/blood , Selenoproteins , Thioredoxin-Disulfide Reductase/metabolism , Thyroiditis, Autoimmune/enzymology , Thyroiditis, Autoimmune/metabolismABSTRACT
Cytosolic thioredoxin reductase (TR) is an FAD-containing homodimeric selenoenzyme which, together with thioredoxin (Trx) and NADPH, forms a powerful oxidoreductase system. Cytoplasmic glutathione peroxidase (GPX-1) is a selenoprotein with antioxidant activity. The TR/Trx system has been associated with cellular processes including regulation of cell growth, and modification of activity of transcription factors. TR may also act as an antioxidant. We have measured TR activity, TR concentration, and GPX-1 activity in human hepatic cytosols from foetuses and neonates. The concentration of TR was significantly greater (P<0.05) in foetal (43.6, 37.9-50.8 microg/g protein, median, interquartile range) than in neonatal liver (11.6, 8.70-15.0 microg/g). This was also true of TR activity which was 2.1, 1.8-2.5 U/g protein in foetal, and 0.65, 0.44-0.74 U/g protein in neonatal liver (P<0.0005). Similarly, GPX-1 activity was significantly higher (P<0.005) in the foetal (199.7, 144.0-227.9 U/g protein) than in neonatal (77.0, 58.4-110.3 U/g protein) hepatic cytosol. Overall, foetal liver expressed approx. 3-fold higher activities of TR and GPX-1 than neonatal liver.
Subject(s)
Glutathione Peroxidase/metabolism , Liver/enzymology , Thioredoxin-Disulfide Reductase/metabolism , Autopsy , Cytoplasm/enzymology , Cytosol/enzymology , Gestational Age , Humans , Infant, Newborn , Liver/embryology , Liver/growth & development , Oxidative StressABSTRACT
OBJECTIVE: To assess selenium (Se) status of cats in 4 regions of the world and to compare results for Se status with reported incidence of hyperthyroidism in cats in those regions. ANIMALS: 50 cats (30 from 2 regions with an allegedly high incidence of hyperthyroidism and 20 from 2 regions in which the disease is less commonly reported). PROCEDURE: Hematologic samples (heparinized whole blood, plasma, and RBC fractions) were obtained from 43 healthy euthyroid cats and 7 hyperthyroid cats. Plasma concentration of Se and activity of glutathione peroxidase (GPX) in whole blood and plasma were determined. RESULTS: Plasma concentration of Se and GPX activity in whole blood or plasma did not differ significantly among cats from the 4 regions. However, cats had a plasma concentration of Se that was approximately 5 times the concentration reported in rats and humans. The GPX activity in whole blood or plasma in cats generally was higher than values reported in rats or humans. CONCLUSIONS AND CLINICAL RELEVANCE: Cats have higher Se concentrations in plasma, compared with values for other species. However, Se status alone does not appear to affect the incidence of hyperthyroidism in cats. High Se concentrations may have implications for health of cats if such concentrations are influenced by the amount of that micronutrient included in diets.
Subject(s)
Cat Diseases/metabolism , Hyperthyroidism/veterinary , Selenium/metabolism , Animals , Cat Diseases/epidemiology , Cats , Denmark/epidemiology , Female , Glutathione Peroxidase/blood , Hyperthyroidism/epidemiology , Hyperthyroidism/metabolism , Male , Queensland/epidemiology , Scotland/epidemiology , Selenium/blood , Statistics, Nonparametric , Thyroxine/blood , Western Australia/epidemiologyABSTRACT
Gene therapy, as a safe and efficacious treatment or prevention of diseases, is one of the next fundamental medical innovations. Direct injection of plasmid into skeletal muscle is still a relatively inefficient and highly variable method of gene transfer. However, published reports have shown that application of an electric field to the muscle immediately after plasmid injection increases gene expression at least 2 orders of magnitude. Using this methodology, we have achieved potentially therapeutic circulating levels of human factor IX (hF.IX) in mice and dogs. A plasmid encoding hF.IX formulated with a protective, interactive, noncondensing (PINC) polymer was injected into the skeletal muscle followed by administration of multiple electrical pulses (electroporation). In mice long-term expression was achieved and the ability to readminister formulated plasmid was demonstrated. In normal dogs, expression of hF.IX reached 0.5-1.0% of normal levels. The transient response in dogs was due to the development of antibodies against hF.IX. Elevated circulating creatine kinase levels and histological examination indicated transient minor trauma associated with the procedure. These data show that gene delivery using a plasmid formulated with a PINC polymer augmented with electroporation is scalable into large animal models and represents a promising approach for treating patients with hemophilia B.
Subject(s)
Electroporation/methods , Genetic Therapy/methods , Hemophilia B/therapy , Muscles/metabolism , Plasmids/genetics , Polymers/chemistry , Animals , Blotting, Western , Creatine Kinase/metabolism , Dogs , Dose-Response Relationship, Drug , Factor IX/genetics , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Mice, SCID , Muscle, Skeletal/metabolism , Plasmids/metabolism , Time FactorsABSTRACT
Skeletal muscle is established as an ideal tissue for gene delivery to treat systemic diseases. However, the relatively low levels of gene expression obtained from using naturally occurring promoters, including the strong cytomegalovirus (CMV) enhancer/promoter (E/P), have limited the use of muscle as a target tissue. The relatively weak simian virus 40 (SV40) enhancer is known to have dual functions promoting localization of DNA to the nucleus and activating transcription. An SV40 enhancer incorporated either at the 5' end of CMV E/P or the 3' end of the polyadenylation site gave as much as a 20-fold increase in the level of exogenous gene expression in muscle in vivo, compared with expression observed with CMV E/P alone. The minimum requirement for this enhancement is a single copy of a 72-bp element of the SV40 enhancer, in combination with either the CMV E/P or skeletal actin (SkA) promoter. Enhancement of gene expression in muscle by this SV40 enhancer was also observed by using the powerful electroporation delivery. However, the SV40 enhancer does not increase the level of CMV E/P driven reporter gene expression in dividing tumor cells in vivo and in the dividing myoblast cell C2C12 in vitro. The data suggest that including this enhancer in the plasmid will enhance the level of gene production for muscle-based gene therapy.
Subject(s)
Antigens, Polyomavirus Transforming/genetics , Enhancer Elements, Genetic , Genetic Therapy/methods , Interleukin-2/genetics , Muscle, Skeletal/metabolism , Transcription, Genetic , Animals , Electroporation , Gene Expression , Hindlimb , Luciferases/genetics , Mice , Mice, Inbred StrainsABSTRACT
The ability of selenium to protect cultured human coronary artery endothelial cells (HCAEC), human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) from oxidative damage induced by 100 microM t-butyl hydroperoxide (t-BuOOH) was compared. Preincubation of human endothelial cells for 24 h with sodium selenite at concentrations as low as 5 nM provided significant protection against the harmful effects of 100 microM t-BuOOH, with complete protection being achieved with 40 nM selenite. The preincubation period was required for selenite to exert this protective effect on endothelial cells. When compared with selenium-deficient cells, the activities of cytoplasmic glutathione peroxidase (GPX-1), phospholipid hydroperoxide glutathione peroxidase (GPX-4) and thioredoxin reductase (TR) were each induced approx. 3--4-fold by 40 nM selenite. HCAEC and HUVEC showed great similarity in their relative abilities to resist oxidative damage in the presence and absence of selenite, and the activities of TR and the GPXs were also similar in these cell types. BAEC were more susceptible to damage by 100 microM t-BuOOH than were human endothelial cells, and could not be protected completely by incubation with selenite at concentrations up to 160 nM. The activity of TR in human endothelial cells was approx. 25-fold greater than that in BAEC of a similar selenium status, but GPX-1 and GPX-4 activities were not significantly different between the human and bovine cells. These studies, although performed with a small number of cultures, show for the first time that selenium at low doses can provide significant protection of the human coronary artery endothelium against damage by oxidative stress. TR may be an important antioxidant selenoprotein in this regard, in addition to the GPXs. The data also suggest that HUVEC, but not BAEC, represent a suitable model system in which to study the effects of selenium on the endothelium of human coronary arteries.
Subject(s)
Endothelium, Vascular/drug effects , Oxidative Stress/drug effects , Sodium Selenite/pharmacology , Thioredoxin-Disulfide Reductase/metabolism , Animals , Cattle , Cell Culture Techniques , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/enzymology , Glutathione Peroxidase/metabolism , Humans , Phospholipid Hydroperoxide Glutathione Peroxidase , tert-Butylhydroperoxide/antagonists & inhibitors , tert-Butylhydroperoxide/pharmacology , Glutathione Peroxidase GPX1ABSTRACT
Korrigan (kor) is a dwarf mutant of Arabidopsis thaliana (L.) Heynh. that is deficient in a membrane-bound endo-1,4-beta-glucanase. The effect of the mutation on the pectin network has been studied in kor by microscopical techniques associated with various probes specific for different classes of pectic polysaccharides. The localisation of native crystalline cellulose was also examined using the cellobiohydrolase I-gold probe. The investigations were focused on the external cell walls of the epidermis, a cell layer that, in a number of plant species, has been shown to be growth limiting. Anionic sites associated with pectic polymers were quantified using the cationic gold probe. Homogalacturonans were quantified using polyclonal anti-polygalacturonic acid/rhamnogalacturonan I antibodies recognising polygalacturonic acid, and monoclonal JIM7 and JIM5 antibodies recognising homogalacturonans with a high or low degree of methyl-esterification, respectively. Rhamnogalacturonans were quantified with two monoclonal antibodies, LM5, recognising beta-1,4 galactan side chains of rhamnogalacturonan I, and CCRCM2. Our results show a marked increase in homogalacturonan epitopes and a decrease in rhamnogalacturonan epitopes in kor compared to the wild type. A substantial decrease in cellobiohydrolase I-gold labelling was also observed in the mutant cell walls. These findings demonstrate that a deficiency in an endo-1,4-beta-glucanase, which is in principle not directly implicated in pectin metabolism, can induce important changes in pectin composition in the primary cell wall. The changes indicate the existence of feedback mechanisms controlling the synthesis and/or deposition of pectic polysaccharides in primary cell walls.
Subject(s)
Arabidopsis/chemistry , Cell Wall/chemistry , Cellulase/chemistry , Cellulase/deficiency , Pectins/analysis , Antibodies, Monoclonal/pharmacology , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis/metabolism , Carboxymethylcellulose Sodium/pharmacology , Cell Wall/ultrastructure , Cellulase/pharmacology , Cellulose/metabolism , Cellulose 1,4-beta-Cellobiosidase , Epitopes/analysis , Gold/pharmacology , Hypocotyl/chemistry , Hypocotyl/ultrastructure , Immunohistochemistry , In Vitro Techniques , Pectins/metabolism , Plant Epidermis/chemistry , Plant Epidermis/ultrastructure , Polylysine/pharmacology , Polysaccharides/metabolismABSTRACT
The blood selenium (Se) concentration in the U.K. population has declined by approx. 50% between 1974 and 1991, reflecting a large decrease in dietary Se supply, with intakes only half the reference nutrient intake of 1 microg/kg body weight. Tissue levels of Se are readily influenced by dietary intake. Therefore selenoprotein activity may be sub-optimal due to low Se status, and thus compromise normal cell function. To examine the effects of changing Se intake on selenoproteins, we have determined the relative effectiveness of organic selenomethionine and inorganic sodium selenite (50 microg of Se daily for 28 days) in modulating glutathione peroxidase activities in blood cells from 45 healthy men and women, from a U.K. population. Transient and acute changes in lymphocyte, granulocyte and platelet phospholipid-hydroperoxide glutathione peroxidase (GPx4) activity occurred by day 7 or 14 of sodium selenite treatment and by day 7 in lymphocytes from selenomethionine-treated subjects compared with controls taking a placebo. In contrast, GPx4 activity in granulocytes and platelets in the selenomethionine group increased gradually over the 28 days. Cytosolic glutathione peroxidase (GPx1) activity in these blood cells from both treatment groups increased gradually over the 28 days. For each cellular selenoenzyme activity a significant inter-individual difference (P<0.001) in the extent of the response to Se supplementation was observed, but this was not related to blood Se concentrations either before or after treatments. Significant inverse correlations were evident between baseline enzyme activities and percentage change in activity after 28 days of supplementation [e.g. lymphocyte GPx4, r=-0.695 (P<0.001)], indicating that pre-treatment activity may be sub-optimal as a result of poor Se status. The different and contrasting effects that Se supplementation had on blood selenoenzyme activities may be indicative of a difference in metabolic need for Se regulated at the level of Se-dependent cell function.