Direct proteasome inhibition by clasto-lactacystin beta-lactone permits the detection of ubiquitinated p21(waf1) in ML-1 cells.
(25/1100)
The ubiquitin proteasome pathway regulates the expression of major cellular regulatory proteins. The ubiquitin proteasome system has been demonstrated to be involved in the expression of the cyclin kinase inhibitor, p21. Ubiquitinated p21 is degraded immediately by 26S proteasome, therefore, the detection of p21 is difficult. We report here an improvement for the detection of ubiquitinated p21 using a proteasome inhibitor, clasto-lactacystin beta-lactone. A p21-enriched cell lysate is obtained by pretreating the cells with deferoxamine to induce p21 mRNA expression followed by treatment with 1x10(-6) M beta-lactone. The concentration of p21 from the cell lysate was performed using an anti-p21 antibody crosslinked to protein G Sepharose. Ubiquitinated p21 was detected on Western blots of the concentrated sample using an anti-ubiquitin antibody. This detection system will be used for further analysis of the regulation of p21 ubiquitination. (+info)
Role of reactive oxygen metabolites in organophosphate-bidrin-induced renal tubular cytotoxicity.
(26/1100)
Due to low toxicity to nontarget species and rapid degradation after its application, organophosphate (OP) remains a widely used class of pesticide. Suicidal or accidental overdose of OP can result in acute tubular necrosis. Experimental evidence shows little correlation between the renal tubular necrosis and the degree of OP-induced acetylcholinesterase inhibition, the main mechanism of OP's toxicity, suggesting the involvement of alternate mechanisms. Since reactive oxygen species (ROS) are known mediators of many toxin-induced renal injuries, this study was conducted to investigate whether ROS play a role in Bidrin (BD)-induced renal tubular epithelial cell (LLC-PK1) toxicity. BD is an OP insecticide formulation with dicrotophos as the active ingredient. LLC-PK1 cell death, determined by lactate dehydrogenase (LDH) release (% of total), rose concentration- and time-dependently after exposure of the cells to 1000, 1250, 1500, 1750, and 2000 ppm of BD for 6, 12, 24, and 48 h. Antioxidants 2-methylaminochroman (2-MAC; 0.3 to 2.5 microM) and desferrioxamine (DFO; 0.25 to 2 mM) reduced cell damage induced by 1250 ppm of BD over a 24-h incubation in a concentration-related manner. The greatest reductions in % LDH were produced by DFO 2 mM and 2-MAC 2.5 microM, both significantly lower than BD alone. H2O2 levels (micromol/mg protein per h) were significantly elevated after exposure to 1250 ppm of BD. Significantly increased malondialdehyde formation (nmol/mg protein) compared with control was also found in BD-exposed cells indicating enhanced lipid peroxidation. Malondialdehyde generation was significantly suppressed by 2-MAC and DFO. These results demonstrate that the organophosphate BD can cause direct tubular cytotoxicity, and implicate, at least in part, a role for ROS and accompanying lipid peroxidation in cytotoxicity. Based on these direct in vitro findings, it is hypothesized that, besides hypotension that often accompanies OP intoxication, OP-induced oxidative stress at the tubular level may play a role in the pathogenesis of acute tubular necrosis. (+info)
Mucormycosis in allogeneic bone marrow transplant recipients: report of five cases and review of the role of iron overload in the pathogenesis.
(27/1100)
In a 10-year consecutive series of 263 allogeneic bone marrow transplant recipients, we identified five cases (1.9%) of invasive mucormycosis. Only one infection occurred within the first 100 days after transplantation, while the remainder complicated the late post-transplant course (median day of diagnosis: 343). Sites of infection were considered 'non-classical' and included pulmonary, cutaneous and gastric involvement. No case of fungal dissemination was observed. Mucormycosis was the primary cause of death in three of the five patients. Corticosteroid-treated graft-versus-host disease, either acute or chronic, or severe neutropenia were present in all cases. However, compared with a matched control population, the most striking finding was the demonstration of severe iron overload in each of the mucormycosis patients. The mean level of serum ferritin, transferrin saturation and number of transfused units of red cells (2029 microg/l, 92% and 52 units, respectively) in the study group is significantly higher compared with the control group (P < 0.05). The difference with other risk groups for mucormycosis, including deferoxamine-treated dialysis patients and acidotic diabetics, was analyzed in view of the possible pathogenic role of iron. Although these infections are often fatal, limited disease may have a better prognosis if diagnosed early and treated aggressively. (+info)
Inhibitors of poly (ADP-ribose) synthetase protect rat proximal tubular cells against oxidant stress.
(28/1100)
BACKGROUND: The generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of renal ischemia-reperfusion injury. ROS produce DNA strand breaks that lead to the activation of the DNA-repair enzyme poly (ADP-ribose) synthetase (PARS). Excessive PARS activation results in the depletion of its substrate, nicotinamide adenine dinucleotide (NAD) and subsequently of adenosine 5'-triphosphate (ATP), leading to cellular dysfunction and eventual cell death. The aim of this study was to investigate the effect of various PARS inhibitors on the cellular injury and death of rat renal proximal tubular (PT) cells exposed to hydrogen peroxide (H2O2). METHODS: Rat PT cell cultures were incubated with H2O2 (1 mM) either in the presence or absence of the PARS inhibitors 3-aminobenzamide (3-AB, 3 mM), 1,5-dihydroxyisoquinoline (0.3 mM) or nicotinamide (Nic, 3 mM), or increasing concentrations of desferrioxamine (0.03 to 3 mM) or catalase (0.03 to 3 U/ml). Cellular injury and death were determined using the MTT and lactate dehydrogenase (LDH) assays, respectively. H2O2-mediated PARS activation in rat PT cells and the effects of PARS inhibitors on PARS activity were determined by measurement of the incorporation of [3H]NAD into nuclear proteins. RESULTS: Incubation of rat PT cells with H2O2 significantly inhibited mitochondrial respiration and increased LDH release, respectively. Both desferrioxamine and catalase reduced H2O2-mediated cellular injury and death. All three PARS inhibitors significantly attenuated the H2O2-mediated decrease in mitochondrial respiration and the increase in LDH release. Incubation with H2O2 produced a significant increase in PARS activity that was significantly reduced by all PARS inhibitors. 3-Aminobenzoic acid (3 mM) and nicotinic acid (3 mM), structural analogs of 3-AB and Nic, respectively, which did not inhibit PARS activity, did not reduce the H2O2-mediated injury and necrosis in cultures of rat PT cells. CONCLUSION: We propose that PARS activation contributes to ROS-mediated injury of rat PT cells and, therefore, to the cellular injury and cell death associated with conditions of oxidant stress in the kidney. (+info)
Beta-thalassemia and pulmonary function.
(29/1100)
BACKGROUND AND OBJECTIVE: The survival of patients with beta-thalassemia major and intermedia has improved considerably. This has focused attention on the long-term sequelae of the disease itself and its treatment. The effect of hemosiderosis in major organs (heart, liver, etc) are well-recognized, but the pathophysiology of any lung damage is less clearly understood. We studied lung function changes in 32 patients with beta-thalassemia. DESIGN AND METHODS: Respiratory function tests, CO diffusion and arterial blood gas analysis were performed on 19 patients with beta-thalassemia major (9 F, 10 M) and 13 with beta-thalassemia intermedia (6 M, 7 F). All investigations were performed 24 hours before the patients received a blood transfusion or when they were in a stable state hematologic condition. Echocardiography was performed in all patients and the ejection fraction was employed as a measure of cardiac function. RESULTS: No patient had clinical signs of pulmonary dysfunction. Pulmonary function tests, however, showed a reduction of all main parameters (TLC, FVC, FEV1 and RV) in most patients with beta-thalassemia major, indicating a restrictive type of dysfunction. The pulmonary function of patients with beta-thalassemia intermedia seemed to be preserved. Arterial blood gas values were within the normal range, while in some subjects CO diffusion approached the lower limits of normality. There was no evidence that the observed abnormalities in pulmonary function were secondary to congestive heart failure. INTERPRETATION AND CONCLUSIONS: Iron deposition due to repeated blood transfusions may play a central role in determining lung alterations although the majority of patients are well chelated, suggesting that more than one causal mechanisms could be involved. (+info)
The cellular labile iron pool and intracellular ferritin in K562 cells.
(30/1100)
The labile iron pool (LIP) harbors the metabolically active and regulatory forms of cellular iron. We assessed the role of intracellular ferritin in the maintenance of intracellular LIP levels. Treating K562 cells with the permeant chelator isonicotinoyl salicylaldehyde hydrazone reduced the LIP from 0.8 to 0.2 micromol/L, as monitored by the metalo-sensing probe calcein. When cells were reincubated in serum-free and chelator-free medium, the LIP partially recovered in a complex pattern. The first component of the LIP to reappear was relatively small and occurred within 1 hour, whereas the second was larger and relatively slow to occur, paralleling the decline in intracellular ferritin level (t1/2= 8 hours). Protease inhibitors such as leupeptin suppressed both the changes in ferritin levels and cellular LIP recovery after chelation. The changes in the LIP were also inversely reflected in the activity of iron regulatory protein (IRP). The 2 ferritin subunits, H and L, behaved qualitatively similarly in response to long-term treatments with the iron chelator deferoxamine, although L-ferritin declined more rapidly, resulting in a 4-fold higher H/L-ferritin ratio. The decline in L-ferritin, but not H-ferritin, was partially attenuated by the lysosomotrophic agent, chloroquine; on the other hand, antiproteases inhibited the degradation of both subunits to the same extent. These findings indicate that, after acute LIP depletion with fast-acting chelators, iron can be mobilized into the LIP from intracellular sources. The underlying mechanisms can be kinetically analyzed into components associated with fast release from accessible cellular sources and slow release from cytosolic ferritin via proteolysis. Because these iron forms are known to be redox-active, our studies are important for understanding the biological effects of cellular iron chelation. (+info)
Promotion of copper excretion from the isolated rat heart attenuates postischemic cardiac oxidative injury.
(31/1100)
This study examined the role of Cu as a mediator of cardiac postischemic oxidative injury. Isolated rat hearts were subjected to 20 min of normothermic global ischemia, followed by 30 min of reperfusion; after 20 min of preischemic loading with Krebs-Henseleit buffer +/- 20 or 30 microM zinc-bis-histidinate (Zn-His2), 0.5 mM deferoxamine (DEF) or 42 microM neocuproine (NEO). Postischemic developed systolic pressure and rate-pressure product were highest and postischemic end-diastolic pressure was lowest in hearts treated with 20 or 30 microM Zn-His2 and 0.5 mM DEF. Cu efflux was significantly increased by 225 and 290% (end of preischemic loading), and 325 and 375% (immediate postischemic period) of control basal rates in hearts treated with 30 microM Zn-His2 and 0.5 mM DEF, respectively. NEO did not effect any of these parameters. By the end of ischemia, protein carbonyls were lowest in Zn-His2-treated hearts and highest in DEF-treated hearts when compared with control hearts. The results of this study suggest that removal of redox-active Cu before ischemia has beneficial effects, indicating a mediatory role in postischemic cardiac oxidative injury. (+info)
Interaction of aluminum with PHFtau in Alzheimer's disease neurofibrillary degeneration evidenced by desferrioxamine-assisted chelating autoclave method.
(32/1100)
To demonstrate that aluminum III (Al) interacts with PHFtau in neurofibrillary degeneration (NFD) of Alzheimer's disease (AD) brain, we developed a "chelating autoclave method" that allows Al chelation by using trivalent-cationic chelator desferrioxamine. Its application to AD brain sections before Morin histochemistry for Al attenuated the positive fluorescence of neurofibrillary tangles, indicating Al removal from them. This method, applied for immunostaining with phosphorylation-dependent anti-tau antibodies, significantly enhanced the PHFtau immunoreactivity of the NFD. These results suggest that each of the phosphorylated epitopes in PHFtau are partially masked by Al binding. Incubation of AD sections with AlCl(3) before Morin staining revealed Al accumulation with association to neurofibrillary tangles. Such incubation before immunostaining with the phosphorylation-dependent anti-tau antibodies abolished the immunolabeling of the NFD and this abolition was reversed by the Al chelation. These findings indicate cumulative Al binding to and thereby antigenic masking of the phosphorylated epitopes of PHFtau. Al binding was further documented for electrophoretically-resolved PHFtau on immunoblots, indicating direct Al binding to PHFtau. In vitro aggregation by AlCl(3) was observed for PHFtau but was lost on dephosphorylation of PHFtau. Taken together, phosphorylation-dependent and direct PHFtau-Al interaction occurs in the NFD of the AD brain. (+info)