ABSTRACT
BACKGROUND: Antimicrobial stewardship initiatives in secondary care depend on clinicians undertaking antibiotic prescription reviews but decisions to limit antibiotic treatment at review are complex. AIM: To assess the feasibility and acceptability of implementing ARK (Antibiotic Review Kit), a behaviour change intervention made up of four components (brief online tool, prescribing decision aid, regular data collection and feedback process, and patient leaflet) to support stopping antibiotic treatment when it is safe to do so among hospitalized patients; before definitive evaluation through a stepped-wedge cluster-randomized controlled trial. METHODS: Acceptability of the different intervention elements was assessed for a period of 12 weeks by uptake of the online tool, adoption of the decision aid into prescribing practice, and rates of decisions to stop antibiotics at review (assessed through repeated point-prevalence surveys). Patient perceptions of the information leaflet were assessed through a brief questionnaire. FINDINGS: All elements of the intervention were successfully introduced into practice. A total of 132 staff encompassing a broad range of prescribers and non-prescribers completed the online tool (19.4 per 100 acute beds), including 97% (32/33) of the pre-specified essential clinical staff. Among 588 prescription charts evaluated in seven point-prevalence surveys over the 12-week implementation period, 82% overall (76-90% at each survey) used the decision aid. The median antibiotic stop rate post implementation was 36% (range: 29-40% at each survey) compared with 9% pre implementation (P < 0.001). CONCLUSION: ARK provides a feasible and acceptable mechanism to support stopping antibiotics safely at post-prescription reviews in an acute hospital setting.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Antimicrobial Stewardship/methods , Behavior Therapy/methods , Patient Acceptance of Health Care , Attitude of Health Personnel , Feasibility Studies , Hospitals , HumansABSTRACT
BACKGROUND: Hospital antimicrobial stewardship strategies, such as 'Start Smart, Then Focus' in the UK, balance the need for prompt, effective antibiotic treatment with the need to limit antibiotic overuse using 'review and revise'. However, only a minority of review decisions are to stop antibiotics. Research suggests that this is due to both behavioural and organizational factors. OBJECTIVES: To develop and optimize the Antibiotic Review Kit (ARK) intervention. ARK is a complex digital, organizational and behavioural intervention that supports implementation of 'review and revise' to help healthcare professionals safely stop unnecessary antibiotics. METHODS: A theory-, evidence- and person-based approach was used to develop and optimize ARK and its implementation. This was done through iterative stakeholder consultation and in-depth qualitative research with doctors, nurses and pharmacists in UK hospitals. Barriers to and facilitators of the intervention and its implementation, and ways to address them, were identified and then used to inform the intervention's development. RESULTS: A key barrier to stopping antibiotics was reportedly a lack of information about the original prescriber's rationale for and their degree of certainty about the need for antibiotics. An integral component of ARK was the development and optimization of a Decision Aid and its implementation to increase transparency around initial prescribing decisions. CONCLUSIONS: The key output of this research is a digital and behavioural intervention targeting important barriers to stopping antibiotics at review (see http://bsac-vle.com/ark-the-antibiotic-review-kit/ and http://antibioticreviewkit.org.uk/). ARK will be evaluated in a feasibility study and, if successful, a stepped-wedge cluster-randomized controlled trial at acute hospitals across the NHS.
Subject(s)
Anti-Bacterial Agents/administration & dosage , Antimicrobial Stewardship/methods , Drug Prescriptions/statistics & numerical data , General Practice/methods , Health Personnel/education , Anti-Bacterial Agents/standards , Drug Prescriptions/standards , General Practice/education , General Practice/standards , Health Plan Implementation/methods , Health Plan Implementation/standards , Hospitals/statistics & numerical data , Humans , Qualitative Research , Stakeholder Participation , United KingdomABSTRACT
Long-term potentiation (LTP) and long-term depression represent important processes that modulate synaptic transmission that carries out a key role in neural mechanisms of memory. Many studies give strong evidences on a role of the reactive oxygen species in the induction of LTP in CA1 region of hippocampal slices that was inhibited by adding the scavenger enzyme superoxide dismutase (SOD1). Previous data showed that SOD1 is secreted by many cellular lines, including neuroblastoma SK-N-BE cells through microvesicles by an ATP-dependent mechanism; moreover, it has been shown that SOD1 interacts with human neuroblastoma cell membranes increasing intracellular calcium levels via a phospholipase C-protein kinase C pathway activation. The aim of this study was to investigate the effect of intracerebral injection of SOD1 or the inactive form of enzyme (ApoSOD) on the modulation of synaptic transmission in dentate gyrus of the hippocampus in urethane anesthetized rats. The results of the present research showed that intracerebral injection of SOD1 and ApoSOD in the dentate gyrus of the rat hippocampal formation inhibits LTP induced by high-frequency stimulation of the perforant path. This result cannot be only explained by the dismutation of oxygen radical induced by SOD1 since also ApoSOD, that lacks the enzymatic activity, carries out the same inhibitory effect on LTP induction.
Subject(s)
Gene Expression/drug effects , Long-Term Potentiation/drug effects , Receptor, Muscarinic M1/metabolism , Superoxide Dismutase/metabolism , Synaptic Transmission , Animals , Cell Line, Tumor , Dentate Gyrus/metabolism , Humans , Male , Neuroblastoma/metabolism , Oligodendroglia/cytology , Oligodendroglia/metabolism , Rats , Rats, Sprague-Dawley , Superoxide Dismutase/administration & dosage , Superoxide Dismutase/chemistry , Synaptic Transmission/drug effectsABSTRACT
To identify the transductional mechanisms responsible for the neuroprotective effect of nitric oxide (NO) during ischemic preconditioning (IPC), we investigated the effects of this gaseous mediator on mitochondrial Mn-superoxide dismutase (Mn-SOD) expression and activity. In addition, the possible involvement of Ras/extracellular-regulated kinase (ERK) ERK1/2 pathway in preserving cortical neurons exposed to oxygen and glucose deprivation (OGD) followed by reoxygenation was also examined. Ischemic preconditioning was obtained by exposing neurons to a 30-min sublethal OGD (95% N(2) and 5% CO(2)). Then, after a 24-h interval, neurons were exposed to 3 h of OGD followed by 24 h of reoxygenation (OGD/Rx). Our results revealed that IPC reduced cytochrome c (cyt c) release into the cytosol, improved mitochondrial function, and decreased free radical production. Moreover, it induced an increase in nNOS expression and NO production and promoted ERK1/2 activation. These effects were paralleled by an increase in Mn-SOD expression and activity that persisted throughout the following OGD phase. When the neurons were treated with L-NAME, a well known NOS inhibitor, the increase in Mn-SOD expression occurring during IPC was reduced and, as a result, IPC-induced neuroprotection was prevented. Similarly, when ERK1/2 was inhibited by its selective inhibitor PD98059, the increase in Mn-SOD expression observed during IPC was almost completely abolished. As a result, its neuroprotective effect on cellular survival was thwarted. The present findings indicate that during IPC the increase in Mn-SOD expression and activity are paralleled by NO production. This suggests that NO neuroprotective role occurs through the stimulation of Mn-SOD expression and activity. In particular, NO via Ras activation stimulates downstream ERK1/2 cascade. This pathway, in turn, post-transcriptionally activates Mn-SOD expression and activity, thus promoting neuroprotection during preconditioning.
Subject(s)
Ischemic Preconditioning , MAP Kinase Signaling System/physiology , Mitochondrial Proteins/metabolism , Mitogen-Activated Protein Kinase 1/physiology , Mitogen-Activated Protein Kinase 3/physiology , Nitric Oxide/physiology , Superoxide Dismutase/metabolism , ras Proteins/physiology , Animals , Cell Survival/physiology , Cells, Cultured , Enzyme Activation/physiology , Gene Expression Regulation/physiology , Ischemic Preconditioning/methods , Mitochondrial Proteins/biosynthesis , Mitochondrial Proteins/genetics , Neuroprotective Agents/metabolism , Rats , Rats, Wistar , Superoxide Dismutase/biosynthesis , Superoxide Dismutase/geneticsABSTRACT
Cyclic adenosine 3'5' monophosphate (cAMP) and protein kinase A (PKA) cooperate with phosphatidylinositol 3' kinase (PI3K) signals in the control of growth and survival. To determine the molecular mechanism(s) involved, we identified and mutagenized a specific serine (residue 83) in p85alpha(PI3K), which is phosphorylated in vivo and in vitro by PKA. Expression of p85alpha(PI3K) mutants (alanine or aspartic substitutions) significantly altered the biological responses of the cells to cAMP. cAMP protection from anoikis was reduced in cells expressing the alanine version p85alpha(PI3K). These cells did not arrest in G1 in the presence of cAMP, whereas cells expressing the aspartic mutant p85D accumulated in G1 even in the absence of cAMP. S phase was still efficiently inhibited by cAMP in cells expressing both mutants. The binding of PI3K to Ras p21 was greatly reduced in cells expressing p85A in the presence or absence of cAMP. Conversely, expression of the aspartic mutant stimulated robustly the binding of PI3K to p21 Ras in the presence of cAMP. Mutation in the Ser 83 inhibited cAMP, but not PDGF stimulation of PI3K. Conversely, the p85D aspartic mutant amplified cAMP stimulation of PI3K activity. Phosphorylation of Ser 83 by cAMP-PKA in p85alpha(PI3K) was also necessary for estrogen signaling as expression of p85A or p85D mutants inhibited or amplified, respectively, the binding of estrogen receptor to p85alpha and AKT phosphorylation induced by estrogens. The data presented indicate that: (1) phosphorylation of Ser 83 in p85alpha(PI3K) is critical for cAMP-PKA induced G1 arrest and survival in mouse 3T3 fibroblasts; (2) this site is necessary for amplification of estrogen signals by cAMP-PKA and related receptors. Finally, these data suggest a general mechanism of PI3K regulation by cAMP, operating in various cell types and under different conditions.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/pharmacology , Estrogens/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Animals , Cell Proliferation/drug effects , Cell Survival/genetics , Cells, Cultured , Cytoprotection , Estrogens/metabolism , G1 Phase/drug effects , G1 Phase/genetics , Humans , Mutation , Phosphatidylinositol 3-Kinases/genetics , Phosphorylation , Platelet-Derived Growth Factor/pharmacology , Proto-Oncogene Proteins p21(ras)/metabolism , Serine/genetics , Serine/metabolismABSTRACT
The addition of epinephrine to solutions containing fentanyl and bupivacaine for epidural infusion has been shown to improve the quality of analgesia. However, this admixture is not available commercially in the United Kingdom. Moreover, stability data applicable to UK practice for this admixture are limited. This study investigated the stability of fentanyl 2 microg.ml(-1) plus bupivacaine 1 mg.ml(-1) in PVC bags with and without epinephrine 2 microg.ml(-1) over a period of 184 days both at room temperature and at 4 degrees C. All infusions were found to be stable over the study period (> 90% remaining) using stability-indicating High Performance Liquid Chromatography (HPLC) methods, with no changes in physical appearance or pH (range 4.5-4.2). The infusions were prepared using standard pharmaceutical products, so facilitating the batch preparation of epinephrine, fentanyl and bupivacaine epidural solutions by hospital pharmacy departments.
Subject(s)
Analgesia, Epidural/methods , Analgesics/chemistry , Anesthetics, Local/chemistry , Drug Stability , Bupivacaine/chemistry , Chromatography, High Pressure Liquid/methods , Drug Combinations , Drug Storage/methods , Epinephrine/chemistry , Fentanyl/chemistry , Humans , Hydrogen-Ion Concentration , TemperatureABSTRACT
The cytotoxicity of dental monomers has been widely investigated, but the underlying mechanisms have not been elucidated. We studied the molecular mechanisms involved in cell death induced by HEMA. In human primary fibroblasts, HEMA induced a dose-dependent apoptosis that was confirmed by the activation of caspases-8, -9, and -3. We found an increase of reactive oxygen species (ROS) and NF-kappaB activation after HEMA exposure. Blocking of ROS production by anti-oxidants had no direct influence on apoptosis caused by HEMA, but inhibition of NF-kappaB increased the fraction of apoptotic cells. Accordingly, mouse embryonic fibroblasts (MEF) from p65-/- mice were more susceptible to HEMA-induced apoptosis than were wild-type controls. Our results indicate that exposure to HEMA triggers apoptosis and that this mechanism is not directly dependent upon redox signaling. Nevertheless, ROS induction by HEMA activates NF-kappaB, which exerts a protective role in counteracting apoptosis.
Subject(s)
Apoptosis/drug effects , Fibroblasts/drug effects , Methacrylates/toxicity , NF-kappa B/physiology , Analysis of Variance , Animals , Blotting, Western , Caspases/metabolism , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Fibroblasts/cytology , Humans , Mice , Reactive Oxygen Species/metabolism , Skin/cytologyABSTRACT
Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.
Subject(s)
Genes, ras/physiology , Signal Transduction/physiology , 3T3 Cells , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Mice , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Oxidation-Reduction , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Rats , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolismABSTRACT
The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen-1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.
Subject(s)
Endosomes/metabolism , Protein Transport/physiology , rab4 GTP-Binding Proteins/metabolism , Cell Compartmentation/drug effects , Cell Compartmentation/physiology , Endocytosis/drug effects , Epidermal Growth Factor/metabolism , Gene Expression , Genes, Dominant , HeLa Cells , Humans , Iodine Radioisotopes , Ligands , Lipoproteins, LDL/metabolism , Lipoproteins, LDL/pharmacokinetics , Microtubules/metabolism , Mutagenesis, Site-Directed , Protein Transport/drug effects , Receptors, Transferrin/metabolism , Transfection , Transferrin/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , rab GTP-Binding Proteins/pharmacology , rab4 GTP-Binding Proteins/genetics , rab4 GTP-Binding Proteins/pharmacology , rab5 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/metabolism , rab5 GTP-Binding Proteins/pharmacology , rab7 GTP-Binding ProteinsABSTRACT
It has previously been demonstrated that CuZn-superoxide dismutase (SOD) is secreted by several human cell lines. This suggests that the circulating enzyme derives from both hemolysis and peripheral tissues as a result of cellular secretion. In the present report, we evaluated the presence of CuZn-SOD in human serum lipoproteins by both enzyme-linked immunosorbent assay and Western blot analysis of immunoprecipitated lipoprotein samples. The distribution of CuZn-SOD activity among the different lipoprotein fractions was also determined by the xanthine/xanthine oxidase method. The results demonstrated that CuZn-SOD is noticeably present in serum lipoproteins and mainly in low and high density lipoproteins (LDL and HDL). Moreover, experiments performed by incubating CuZn-SOD with a lipid emulsion and subsequent separation of the lipid fraction by ultracentrifugation showed that this enzyme associates in a saturable manner with lipids. The CuZn-SOD bound to LDL and HDL could exert a physiological protective role against oxidative damage of these lipoprotein classes that carry out a crucial role in the cholesterol transport.
Subject(s)
Lipoproteins/blood , Lipoproteins/metabolism , Superoxide Dismutase/blood , Superoxide Dismutase/metabolism , Adult , Biological Transport , Blotting, Western , Carrier Proteins/blood , Carrier Proteins/chemistry , Carrier Proteins/classification , Carrier Proteins/metabolism , Emulsions , Enzyme-Linked Immunosorbent Assay , Female , Humans , Lipoproteins/chemistry , Lipoproteins/classification , Lipoproteins, HDL/blood , Lipoproteins, HDL/chemistry , Lipoproteins, HDL/metabolism , Lipoproteins, LDL/blood , Lipoproteins, LDL/chemistry , Lipoproteins, LDL/metabolism , Male , Middle Aged , Protein Binding , Superoxide Dismutase/analysis , UltracentrifugationABSTRACT
Apolipoprotein E (apo E) exerts a protective effect against atherosclerosis, related to its role in intracellular cholesterol removal and remnants clearance. In this study we investigated the effect of dietary and hypothyroid hypercholesterolemia, induced respectively by a high cholesterol diet and by propylthiouracil, on hepatic apo E expression in Wistar male rats. The Northern and Western blot analysis of hepatic mRNA and protein levels showed a 2-3-fold increase of apo E in hypercholesterolemic rats compared to controls. The incubation of FAO rat hepatoma cells with 25-OH cholesterol and mevalonate led to a three-fold increase of apo E mRNA, demonstrating a direct role of cholesterol on apo E expression. This effect was completely abolished by elevating intracellular cAMP levels with forskolin. Immunoblot and immunofluorescence analysis revealed that 25-OH cholesterol/mevalonate strongly increased also apo E protein synthesis and secretion in FAO cells. Our data demonstrate that hypercholesterolemia, apart of the cause (diet or hypothyroidism) induces liver apo E expression in the rat and that this effect can be directly related, via cAMP, to cholesterol.
Subject(s)
Apolipoproteins E/biosynthesis , Cholesterol, Dietary/metabolism , Hypercholesterolemia/metabolism , Liver/metabolism , Animals , Blotting, Northern , Blotting, Western , Cell Membrane/metabolism , Densitometry , Hypercholesterolemia/chemically induced , Male , Microscopy, Fluorescence , Propylthiouracil , RNA, Messenger/metabolism , Rats , Rats, Wistar , Tumor Cells, CulturedABSTRACT
BACKGROUND: We have previously shown that 63.3% of colorectal cancer neoplastic specimens did not express the Low Density Lipoprotein Receptor (LDLR) protein and that the absence of LDLR predicted shorter survival. We now report the findings of a preliminary study on HMG-CoA reductase (HMG-CoAR) activity in neoplastic tissue specimens of human colorectal cancers (CRC) expressing or not expressing LDLR. MATERIALS AND METHODS: The tissue specimens were obtained from 16 patients (10 males and 6 females) undergoing surgical resection for CRC, and previously characterized for LDLR (7 not expressing LDLR and 9 expressing LDLR). HMG-CoAR activity was measured by radiochemical assay using 14C-HMG-CoA as substrate. RESULTS: HMG-CoAR activity was significantly higher in specimens not expressing LDLR than in those expressing LDLR [8.3 pmol/min/mg prot (2.4-16) vs 3.9 pmol/min/mg prot (1.2-7.8), data expressed as median value and the range, p = 0.02, Wilcoxon Rank sum test)]. CONCLUSIONS: The cholesterol requirement in CRC not expressing LDLR may be met by increasing endogenous synthesis. For this reason, the use of HMG-CoA R inhibitors for the treatment of CRC expressing high HMG-CoAR activity-dependence for growth may be clinically important.
Subject(s)
Colorectal Neoplasms/enzymology , Hydroxymethylglutaryl CoA Reductases/metabolism , Receptors, LDL/analysis , Adult , Aged , Aged, 80 and over , Cholesterol/blood , Colorectal Neoplasms/pathology , Female , Humans , Male , Middle AgedABSTRACT
Rab proteins are small molecular mass GTP-ases involved in the regulation of vescicular transport. The ability of rab proteins to carry out their role in intracellular membrane traffic requires the post-translational attachment to their C-terminus of a geranylgeranyl group, an isoprenoid lipid moiety derived from mevalonate. Here we report that depletion of intracellular mevalonate by lovastatin in FRTL-5 thyroid cells specifically resulted in a four-fold increase of Rab5 and Rab7 protein levels. This increase was reversed within 4 h upon addition of mevalonate. Similarly lovastatin also induced, at same extent, mRNA levels. Lovastatin effect was not common to other prenylated proteins. Moreover incubation with cycloheximide abolished the observed increase in lovastatin treated cells, suggesting that the effect is mediated by newly synthesized protein. These findings demonstrate that Rab5 and Rab7 expression are regulated by the isoprenoid pathway.
Subject(s)
GTP-Binding Proteins/biosynthesis , Gene Expression Regulation , Lovastatin/pharmacology , rab GTP-Binding Proteins , Animals , Cell Line , Cycloheximide/pharmacology , GTP Phosphohydrolases/metabolism , Gene Expression Regulation/drug effects , Kinetics , Mevalonic Acid/metabolism , Protein Prenylation , RNA, Messenger/biosynthesis , Rats , Thyroid Gland , Time Factors , Transcription, Genetic/drug effects , rab5 GTP-Binding Proteins , rab7 GTP-Binding ProteinsABSTRACT
It has been shown that dietary fatty acids affect serum low density lipoprotein (LDL) levels, but the mechanism responsible for this effect is still under debate. Here we investigate the effect of different free fatty acids on LDL receptor activity in BHK-21 cells. These cells possess a classical LDL receptor strongly regulated by substances like 25-OH-cholesterol or lovastatin. Preincubation of cells for 24 h with both oleic (cis 18:1) and its trans counterpart, elaidic acid, enhanced 125I-LDL binding, internalization and degradation, being oleic acid more effective than elaidic acid. Among polyunsaturated fatty acids (PUFA) of the n-6 series arachidonic acid (20:4) enhanced LDL receptor activity more than linoleic acid (18:2), and among PUFA of the n-3 series docosahexaenoic (22:6) and eicosapentaenoic acids (20:5) were more effective compared to alpha-linolenic acid (18:3). Conversely, preincubation of cells with saturated fatty acids, palmitic (16:0) and stearic (18:0) acids, decreased binding, internalization and degradation of 125I-LDL. Scatchard analysis of binding data obtained with palmitic and oleic acids showed that these two fatty acids affect LDL receptor number without altering receptor affinity. The regulatory effect of free fatty acids on LDL receptor activity in BHK-21 cells is consistent with the hypothesis that the ability of fatty acids to modulate LDL-cholesterol levels in vivo is mediated, at least in part, by an action on receptor-dependent uptake of LDL.
Subject(s)
Fatty Acids, Nonesterified/pharmacology , Kidney/drug effects , Receptors, LDL/metabolism , Animals , Blotting, Western , Cell Line , Cells, Cultured , Cricetinae , Fatty Acids, Monounsaturated/pharmacology , Fatty Acids, Unsaturated/pharmacology , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Hydroxycholesterols/pharmacology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Kidney/cytology , Kidney/metabolism , L-Lactate Dehydrogenase/metabolism , Lovastatin/pharmacology , Receptors, LDL/drug effectsABSTRACT
CuZn superoxide dismutase (SOD) secretion was detected in media of [35S]cysteine-labeled human neuroblastoma SK-N-BE cells precipitated with antihuman CuZn SOD antibodies. The ability of Fe2+/ascorbate oxidative stress to induce CuZn SOD in SK-N-BE cells was evaluated by Western blot analysis. The results showed that, like human hepatocarcinoma cells and human fibroblasts, SK-N-BE cells secrete CuZn SOD. In addition, the CuZn SOD concentration was higher in cells subjected to oxidative stress than in unstressed cells. The secretion of CuZn SOD and the ability of Fe2+/ascorbate to increase its protein content in SK-N-BE cells indicates that this enzyme protects the brain from damage induced by oxidative stress.
Subject(s)
Neuroblastoma/enzymology , Oxidative Stress/physiology , Superoxide Dismutase/biosynthesis , Ascorbic Acid/pharmacology , Ferrous Compounds/pharmacology , Free Radicals/metabolism , Humans , Kinetics , L-Lactate Dehydrogenase/analysis , Malondialdehyde/metabolism , Superoxide Dismutase/metabolism , Thiobarbituric Acid Reactive Substances/analysis , Tumor Cells, CulturedABSTRACT
N6-Isopentenyladenosine (i6A), an adenosine and mevalonate derivative, inhibits, like adenosine, TSH-induced cAMP increase and its related events (I- uptake and DNA synthesis) in FRTL-5 cells. This inhibition is dose-dependent and is measurable at 10(-8) M. However, unlike adenosine, i6A prevents TSH-promoted microfilament disassembly. The effect of i6A on cytoskeletal structure is antagonized by pertussis toxin and could be assigned to its N6 substitution since it can be mimicked by other synthetic N6-adenosine derivatives. It is suggested that a step beyond cAMP is involved, since i6A prevents also microfilament disassembly induced by 8-bromo-cAMP. This is the first demonstration that an adenosine derivative, which is also an end-product of the isoprenoid pathway, affects cAMP-dependent microfilament organization.
Subject(s)
Actin Cytoskeleton/drug effects , Cyclic AMP/metabolism , Isopentenyladenosine/pharmacology , Thyroid Gland/cytology , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Actin Cytoskeleton/metabolism , Animals , Cattle , Cells, Cultured , DNA/biosynthesis , Fluorescent Dyes , Iodine/pharmacokinetics , Rats , Thyroid Gland/metabolism , Thyrotropin/pharmacologyABSTRACT
Rab7 is a small GTPase localized to the late endosomal compartment. Its function was investigated by overexpressing dominant negative or constitutively active mutants in BHK-21 cells. The effects of such overexpression on the internalization and/or degradation of different endocytic markers and on the morphology of the late endosomal compartment were analyzed. We observed a marked inhibition of the degradation of 125I-low density lipoproteins in cells transfected with the Rab7 dominant negative mutants while the rate of internalization was not affected. Moreover in these cells there was an accumulation of many small vesicles scattered throughout the cytoplasm. In contrast, overexpression of the activating mutants led to the appearance of atypically large endocytic structures and caused a dramatic change in the distribution of the cation-independent mannose 6-phosphate receptor. Our data indicate that the Rab7 protein in mammalian cells is present on a late endosomal compartment much larger than the compartment labeled by the cation-independent mannose 6-phosphate receptor. Rab7 also appears to play a fundamental role in controlling late endocytic membrane traffic.
Subject(s)
Endocytosis , GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/metabolism , rab GTP-Binding Proteins , Animals , Cell Compartmentation , Cell Line , Cricetinae , Fluorescent Antibody Technique , GTP-Binding Proteins/genetics , Horseradish Peroxidase/metabolism , Humans , Hydrolysis , Iodine Radioisotopes , Isoquinolines/metabolism , Lipoproteins, LDL/metabolism , Mutagenesis , Receptor, IGF Type 2/metabolism , Transferrin/metabolism , rab7 GTP-Binding ProteinsABSTRACT
Tumor resistance to oxidative stress prevents the efficacy of cancer therapy based upon a free radical-mediated mechanism. K-ras transformed NIH 3T3 cells (E32-4-2) showed, under oxidative stress, reactive oxygen species (ROS) levels 10-fold lower and lipid peroxide levels 56% lower, compared to their nontransformed counterpart. Since p21(ras) activity depends upon farnesylation, we tested the effect of the inhibitors of farnesylation lovastatin and (alpha-hydroxyfarnesyl) phosphonic acid on susceptibility to oxidative stress in these cells. Preincubation of cells for 24 h with 10 microM lovastatin resulted in a 10-fold increase of ROS levels and a 50% increase of lipid peroxide levels measured under pro-oxidant conditions. Similarly, preincubation of cells with 100 microM (alpha-hydroxyfarnesyl) phosphonic acid for 24 h enhanced stress-induced levels of either ROS (7.5-fold) or lipid peroxides (33%). The effect of lovastatin and (alpha-hydroxyfarnesyl) phosphonic acid is specifically due to their ability to inhibit p21(ras) activity. In fact, inhibition of p21(ras) by transfecting E32-4-2 cells with the transdominant negative mutant of H-ras (L61, S186) led, analogously to lovastatin or (alpha-hydroxyfarnesyl) phosphonic acid treatment, to a strong increase of stress-induced ROS levels. These results suggest that farnesylation inhibitors could be used as an adjuvant therapy to improve the tumoricidal effect of cancer treatment based upon free-radical production in ras-dependent tumors.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Transformation, Neoplastic/metabolism , Genes, ras , Lovastatin/pharmacology , Organophosphonates/pharmacology , Protein Prenylation/drug effects , 3T3 Cells , Animals , Mice , Oxidative StressABSTRACT
The role so far ascribed to intracellular CuZn superoxide dismutase is that of an intracellular scavenger of oxygen radicals. However, other functions of cytosolic CuZn superoxide dismutase have been hypothesized. For example, CuZn superoxide dismutase incubated with rat hepatocyte cells in culture inhibits 3-hydroxy-3methylglutaryl CoA reductase, thereby reducing cholesterol synthesis. We recently demonstrated the presence of surface membrane receptors for CuZn superoxide dismutase, suggesting possible autocrine or paracrine activities. The aim of the present study was to investigate whether cytosolic CuZn superoxide dismutase can be secreted by human hepatocarcinoma and fibroblast cells lines. Proteins in human hepatocellular carcinoma (Hep G2) cells and human fibroblasts were biosynthetically labelled with [35S]-cysteine; then cell lysates and media were immunoprecipitated with rabbit polyclonal anti-human CuZn superoxide dismutase antibodies and separated by 12% polyacrylamide gel electrophoresis. Both Hep G2 cells and human fibroblasts produce and secrete CuZn superoxide dismutase which was detectable in cells and medium as a single protein band with the same electrophoretic mobility as human erythrocyte CuZn superoxide dismutase. These data suggest that CuZn superoxide dismutase, an enzyme thus far considered to be located exclusively intracellularly is secreted by at least two cell lines. This is consistent with autocrine or paracrine roles for CuZn superoxide dismutase.
Subject(s)
Liver/metabolism , Superoxide Dismutase/metabolism , Animals , Carcinoma, Hepatocellular , Fibroblasts/enzymology , Fibroblasts/metabolism , Humans , L-Lactate Dehydrogenase/metabolism , Liver/cytology , Liver/enzymology , Rats , Sulfur Radioisotopes , Tumor Cells, CulturedABSTRACT
We have recently shown that ascorbate has a hypocholesterolemic and hypotriglyceridemic effect on rats fed a diet enriched with 1.5% cholesterol and 25% hydrogenated coconut oil (Nath diet). In this study we evaluated the effect of intraperitoneal ascorbate administration on susceptibility to lipoperoxidation either in rats fed standard or Nath diet. In normal rats ascorbate treatment decreased (p<0.05) the susceptibility to lipoperoxidation induced by incubation of serum for 24 hours with 2.2 mM Cu++, without altering the normal serum fatty acid profile. In rats fed Nath diet we observed a reduced susceptibility of serum to CU++-induced lipoperoxidation (36%), according with their low levels of serum unsaturated fatty acids (40% less than rats fed standard diet). In these animals ascorbate administration affects serum fatty acid profile leading to a decrease of S/U ratio from 1.6 to 1.2 without significantly modifying the susceptibility of serum to lipoperoxidation. Moreover, the production of spontaneous lipid peroxides in liver homogenates, measured as TBARS levels, was strongly inhibited by ascorbate (p<0.01) in rats fed either standard or Nath diet. These data indicate that ascorbate administration exerts an antioxidant effect and that in hypercholesterolemic rats, in addition to a lipid lowering effect, ascorbate exerts a protective role against the peroxidative damage of lipids.