RESUMEN
The effectiveness of the plantarflexor muscle group to generate desired plantarflexion moments is modulated by the geometry of the Achilles tendon moment arm (ATMA). Children with cerebral palsy (CP) frequently have reduced plantarflexion function, which is commonly attributed to impaired muscle structure and function, however little attention has been paid to the potential contribution of ATMA geometry. The use of musculoskeletal modelling for the simulation of gait and understanding of gait mechanics, rely on accuracy of ATMA estimates. This study aimed to compare 3D in-vivo estimates of ATMA of adults, children with CP and typically developing (TD) children, as well as compare 3D in-vivo estimates to linearly scaled musculoskeletal model estimates. MRI scans for eight children with CP, 11 TD children and nine healthy adults were used to estimate in-vivo 3D ATMA using a validated method. A lower limb musculoskeletal model was linearly scaled to individual tibia length to provide a scaled ATMA estimate. Normalised in-vivo 3D ATMA for children with CP was 17.2%⯱â¯2.0 tibia length, which was significantly larger than for TD children (15.2%⯱â¯1.2, pâ¯=â¯0.013) and adults (12.5%⯱â¯0.8, pâ¯<â¯0.001). Scaled ATMA estimates from musculoskeletal models significantly underestimated in-vivo estimates for all groups, by up to 34.7%. The results of this study show children with CP have larger normalised 3D ATMA compared to their TD counterparts, which may have implications in understanding reduced plantarflexor function and the efficacy of surgical interventions whose aim is to modify the musculoskeletal geometry of this muscle group.
Asunto(s)
Tendón Calcáneo/fisiopatología , Parálisis Cerebral/fisiopatología , Desarrollo Infantil/fisiología , Adulto , Niño , Femenino , Marcha , Humanos , Masculino , Músculo Esquelético/fisiopatologíaRESUMEN
Cancer frequently arises in epithelial tissues subjected to repeated cycles of injury and repair. Improving our understanding of tissue regeneration is, therefore, likely to reveal novel processes with inherent potential for aberration that can lead to carcinoma. These highly conserved regenerative mechanisms are increasingly understood and in the liver are associated with special characteristics that underlie the organ's legendary capacity for restoration of size and function following even severe or chronic injury. The nature of the injury can determine the cellular source of epithelial regeneration and the signalling mechanisms brought to play. These observations are shaping how we understand and experimentally investigate primary liver cancer, in particular cholangiocarcinoma; a highly invasive malignancy of the bile ducts, resistant to chemotherapy and whose pathogenesis has hitherto been poorly understood. Interestingly, signals that drive liver development become activated in the formation of cholangiocarcinoma, such as Notch and Wnt and may be potential future therapeutic targets. In this review, we summarise the work which has led to the current understanding of the cellular source of cholangiocarcinoma, how the tumour recruits, sustains and is educated by its supporting stromal environment, and the tumour-derived signals that drive the progression and invasion of the cancer. With few current treatments of any true efficacy, advances that will improve our understanding of the mechanisms driving this aggressive malignancy are welcome and may help drive therapeutic developments.
RESUMEN
We aimed to describe the experiences of families of potential organ and tissue donors eligible for donation after circulatory death or brain death. Forty-nine family members of potential donors from four Melbourne hospitals were interviewed to assess their experiences of communication, processes and the outcomes of donation. Interviews were recorded, transcribed verbatim and analysed thematically. Families expressed a range of perspectives on themes of communication, hospital processes and care, the processes of consent and donation and reflected on decisions and outcomes. They expressed satisfaction overall with communication when receiving bad news, discussing death and donation. Honest and frank communication and being kept up-to-date and prepared for potential outcomes were important aspects for families, especially those of post circulatory death donors. Participants reported high levels of trust in healthcare professionals and satisfaction with the level of care received. Many donor families indicated the process was lengthy and stressful, but not significantly enough to adversely affect their satisfaction with the outcome. Both the decision itself and knowing others' lives had been saved provided them with consolation. No consenting families, and only some non-consenting families, regretted their decisions. Many expressed they would benefit from a follow-up opportunity to ask questions and clarify possible misunderstandings. Overall, while experiences varied, Australian families valued frank communication, trusted health professionals, were satisfied with the care their family member received and with donation processes, despite some apparent difficulties. Family satisfaction, infrequently assessed, is an important outcome and these findings may assist education for Australian organ donation professionals.
Asunto(s)
Comunicación , Donantes de Tejidos , Obtención de Tejidos y Órganos , Familia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Satisfacción PersonalRESUMEN
Numbers of deceased organ donors in Australia have increased, but rates of consent to donation remain at around 60%. Increasing family consent is a key target for the Australian Organ and Tissue Authority. Reasons for donation decisions have been reported in the international literature, but little is known of reasons for Australian families' decisions. Potential organ donors in four Melbourne hospitals were identified and 49 participants from 40 families (23 consenting and 17 non-consenting) were interviewed to understand reasons for consent decisions. Themes for consent to organ donation included that: donation was consistent with the deceased's explicit wishes or known values, the desire to help others or self-including themes of altruism, pragmatism, preventing others from being in the same position, consolation received from donation and aspects of the donation conversation and care that led families to believe donation was right for them. Themes for non-consent included: lack of knowledge of wishes; social, cultural and religious beliefs; factors related to the donation process and family exhaustion; and conversation factors where negative events influenced decisions. While reasons for consent were similar to those described in international literature, reasons for non-consent differed in that there was little emphasis on lack of trust of the medical profession, concerns regarding level of care provided to the potential donor, preserving the deceased's body, fears of body invasion or organ allocation fairness.
Asunto(s)
Actitud Frente a la Muerte , Toma de Decisiones/fisiología , Familia/psicología , Donantes de Tejidos/psicología , Obtención de Tejidos y Órganos , Altruismo , Australia , Femenino , Humanos , Entrevistas como Asunto/métodos , MasculinoRESUMEN
BACKGROUND: Obtaining family consent to organ donation is a significant obstacle to improving further Australian deceased organ donation rates. Currently, neither the consent rates for donors eligible to donate after circulatory death, nor factors that influence decision to decline or consent to donation in general are known in Australia. METHODS: This study at four university teaching hospitals in Melbourne, Victoria, examined consecutive patients where organ donation was discussed with the family RESULTS: A total of 123 cases were identified; the family consent rate was 52.8%, and 34.1% proceeded to donation. Consent to donation was related to potential donor factors such as country of birth, cultural background in Australia, a non-religious or Christian background and registration on the Australian Organ Donor Register. Family-related factors included being English speaking and having knowledge of the deceased's wishes about organ donation. Family of donation after circulatory death-eligible donors were less likely to consent to donation than the family of donation after brain death-eligible donors, although not reaching statistical significance. Among consented potential donors, those eligible for donation after brain death and with a shorter length of stay were more likely to proceed to donating organs for transplantation. CONCLUSION: Despite a small sample size, these findings describe current consent and donation rates and associated factors and may assist in improving conversations about organ donation.
Asunto(s)
Cultura , Toma de Decisiones , Donantes de Tejidos , Obtención de Tejidos y Órganos/tendencias , Anciano , Familia/psicología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , VictoriaRESUMEN
PURPOSE: The purpose of this study was to evaluate surgical residents' educational experience related to ventral hernias. METHODS: A 16-question survey was sent to all program coordinators to distribute to their residents. Consent was obtained following a short introduction of the purpose of the survey. Comparisons based on training level were made using χ(2) test of independence, Fisher's exact, and Fisher's exact with Monte Carlo estimate as appropriate. A p value <0.05 was considered significant. RESULTS: The survey was returned by 183 residents from 250 surgical programs. Resident postgraduate year (PG-Y) level was equivalent among groups. Preferred techniques for open ventral hernia varied; the most common (32 %) was intra-abdominal placement of mesh with defect closure. Twenty-two percent of residents had not heard of the retrorectus technique for hernia repair, 48 % had not performed the operation, and 60 % were somewhat comfortable with and knew the general categories of mesh prosthetics products. Mesh choices, biologic and synthetic, varied among the different products. The most common type of hernia education was teaching in the operating room in 87 %, didactic lecture 69 %, and discussion at journal club 45 %. Number of procedures, comfort level with open and laparoscopic techniques, indications for mesh use and technique, familiarity and use of retrorectus repair, and type of hernia education varied significantly based on resident level (p < 0.05). CONCLUSION: Exposure to hernia techniques and mesh prosthetics in surgical residency programs appears to vary. Further evaluation is needed and may help in standardizing curriculums for hernia repair for surgical residents.
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Cirugía General/educación , Hernia Ventral/cirugía , Herniorrafia/educación , Educación de Postgrado en Medicina/estadística & datos numéricos , Cirugía General/estadística & datos numéricos , Herniorrafia/estadística & datos numéricos , Humanos , Internado y Residencia/estadística & datos numéricos , Estados Unidos/epidemiologíaRESUMEN
Polycyclic Aromatic Hydrocarbons (PAHs) are ubiquitous in the environment and are produced by both natural and anthropogenic processes, principally from the incomplete combustion of organic matter. Levels of emissions of PAHs from the combustion of fossil fuels have increased rapidly over the last ca. 200 years. As PAHs have detrimental environmental and human health impacts, assessing spatial and temporal variations in environmental loadings has become a pressing issue in many industrialised and industrializing countries. The current paper reports spatial and temporal variations in levels of atmospheric deposition of PAHs recorded in sediment cores from three lakes in Ireland, the locations of which were selected on the basis of known geographic differences in the deposition of atmospheric pollutants. Thirteen PAH compounds were analysed for in samples of lake sediment that were assumed to represent contemporary/recent and historical (possibly reference) levels of deposition. A third sample was selected from each core on the basis of measured levels of spheroidal carbonaceous particles, which are regarded as a direct indicator of depositions from the industrial-level combustion of fossil fuels. Chronological control was provided by the (210)Pb dating technique which also allowed for the calculation of PAH flux. For the most part, and when compared with the limited published data, measured levels of PAH depositions were relatively low. However, levels of deposition of PAHs in the west of Ireland are higher now than previously, which is in contrast to a general trend of decreasing levels in Europe.
Asunto(s)
Contaminantes Ambientales/química , Sedimentos Geológicos/química , Hidrocarburos Policíclicos Aromáticos/química , Monitoreo del Ambiente , Contaminantes Ambientales/análisis , Sedimentos Geológicos/análisis , Historia del Siglo XX , Historia del Siglo XXI , Irlanda , Hidrocarburos Policíclicos Aromáticos/análisis , Hidrocarburos Policíclicos Aromáticos/historiaRESUMEN
OBJECTIVE: To compare the urine protein-creatinine ratio with urinalysis to predict significant proteinuria (>or=300 mg per day). STUDY DESIGN: A total of 116 paired spot urine samples and 24-h urine collections were obtained prospectively from women at risk for preeclampsia. Urine protein-creatinine ratio and urinalysis were compared to the 24-h urine collection. RESULT: The urine protein-creatinine ratio had better discriminatory power than urinalysis: the receiver operating characteristic curve had a greater area under the curve, 0.89 (95% confidence interval (CI) 0.83 to 0.95) vs 0.71 (95% CI 0.64 to 0.77, P<0.001). When matched for clinically relevant specificity, urine protein-creatinine ratio (cutoff >or=0.28) is more sensitive than urinalysis (cutoff >or=1+): 66 vs 41%, P=0.001 (with 95 and 100% specificity, respectively). Furthermore, the urine protein-creatinine ratio predicted the absence or presence of proteinuria in 64% of patients; urinalysis predicted this in only 19%. CONCLUSION: The urine protein-creatinine ratio is a better screening test. It provides early information for more patients.
Asunto(s)
Creatinina/orina , Preeclampsia/diagnóstico , Proteinuria/diagnóstico , Adulto , Algoritmos , Femenino , Humanos , Preeclampsia/orina , Embarazo , Estudios Prospectivos , Curva ROCRESUMEN
Integral components of mammalian cell membranes, glycosphingolipids (GSL) reside in specialized plasma membrane microdomains critical for cell signaling. N-alkylated nojirimycins are compounds developed for GSL substrate deprivation therapy, blocking GSL synthesis by specifically inhibiting an essential enzyme, ceramide glucosyltransferase. Peritoneal macrophages recruited in mice pretreated with an inhibitory N-alkylnojirimycin displayed a reduced capacity to release either TNFalpha or interleukin-6 when re-exposed to whole killed E. coli in vitro. Cell viability and protein content were not affected. A nojirimycin analogue without GSL inhibitory capacity had no effect. The results show inhibition of GSL synthesis in vivo by an N-alkylnojirimycin can reduce the response to an inflammatory stimulus and indicate N-alkylnojirimycins have experimental and potential clinical value for modulating innate immune responses in vivo.
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Inhibidores Enzimáticos/farmacología , Glucosamina/análogos & derivados , Glucosiltransferasas/antagonistas & inhibidores , Glicoesfingolípidos/antagonistas & inhibidores , Inmunidad Innata/efectos de los fármacos , Macrófagos Peritoneales , 1-Desoxinojirimicina/análogos & derivados , Animales , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Glucosamina/farmacología , Glicoesfingolípidos/biosíntesis , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C3HRESUMEN
Inhibitors, activators, and substrates of cyclin-dependent kinases (cdks) utilize a cyclin-binding sequence, known as a Cy or RXL motif, to bind directly to the cyclin subunit. Alanine scanning mutagenesis of the Cy motif of the cdk inhibitor p21 revealed that the conserved arginine or leucine (constituting the conserved RXL sequence) was important for p21's ability to inhibit cyclin E-cdk2 activity. Further analysis of mutant Cy motifs showed, however, that RXL was neither necessary nor sufficient for a functional cyclin-binding motif. Replacement of either of these two residues with small hydrophobic residues such as valine preserved p21's inhibitory activity on cyclin E-cdk2, while mutations in either polar or charged residues dramatically impaired p21's inhibitory activity. Expressing p21N with non-RXL Cy sequences inhibited growth of mammalian cells, providing in vivo confirmation that RXL was not necessary for a functional Cy motif. We also show that the variant Cy motifs identified in this study can effectively target substrates to cyclin-cdk complexes for phosphorylation, providing additional evidence that these non-RXL motifs are functional. Finally, binding studies using p21 Cy mutants demonstrated that the Cy motif was essential for the association of p21 with cyclin E-cdk2 but not with cyclin A-cdk2. Taking advantage of this differential specificity toward cyclin E versus cyclin A, we demonstrate that cell growth inhibition was absolutely dependent on the ability of a p21 derivative to inhibit cyclin E-cdk2.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Alanina/química , Secuencias de Aminoácidos , Ciclo Celular , División Celular , Separación Celular , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/química , Análisis Mutacional de ADN , Escherichia coli/metabolismo , Citometría de Flujo , Concentración 50 Inhibidora , Leucina/química , Mutación , Papillomaviridae/genética , Péptidos/metabolismo , Plásmidos/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
In all eukaryotic organisms, inappropriate firing of replication origins during the G2 phase of the cell cycle is suppressed by cyclin-dependent kinases. Multicellular eukaryotes contain a second putative inhibitor of re-replication called geminin. Geminin is believed to block binding of the mini-chromosome maintenance (MCM) complex to origins of replication, but the mechanism of this inhibition is unclear. Here we show that geminin interacts tightly with Cdt1, a recently identified replication initiation factor necessary for MCM loading. The inhibition of DNA replication by geminin that is observed in cell-free DNA replication extracts is reversed by the addition of excess Cdt1. In the normal cell cycle, Cdt1 is present only in G1 and S, whereas geminin is present in S and G2 phases of the cell cycle. Together, these results suggest that geminin inhibits inappropriate origin firing by targeting Cdt1.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Interfase , Fase S , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/farmacología , Núcleo Celular/metabolismo , Sistema Libre de Células , Cromatina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/farmacología , Evolución Molecular , Fase G1 , Fase G2 , Geminina , Células HeLa , Humanos , Datos de Secuencia Molecular , Peso Molecular , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/metabolismo , Origen de Réplica , Xenopus , Proteínas de XenopusRESUMEN
Overexpressed heat shock protein 70 (Hsp70) is known to be associated with thermoprotection in a number of cell lines and transgenic animals. We hypothesized that because overexpression of Hsp70 protects cells from lethal heat stress, inhibition of expression should make cells susceptible to heat stress. The model used for this study was a stably transfected P-19 carcinoma cell line expressing antisense hsp70 under the control of the hsp70b promoter. The results showed marked inhibition of Hsp70 expression after heat shock correlated with heat-induced cell death. Hsp90 and Hsc70 protein expression were not affected by the antisense construct. Unexpectedly, heme oxygenase (HO-1), another highly inducible heat shock protein, was not induced after heat shock in the antisense hsp70 cell line. Heat shock transcription factor-1 (HSF-1) was in a highly phosphorylated state in the antisense cell line before and after heat shock. This was in contrast to the untransfected control P-19 cells where HSF-1 was primarily highly phosphorylated after heat shock. A control cell line expressing only the vector, pMAMneo, without the antisense construct also showed partial loss of Hsp70 induction but not increased cell death after heat shock. The findings support the role of Hsp70 in thermoresistance.
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Elementos sin Sentido (Genética)/genética , Muerte Celular , Proteínas HSP70 de Choque Térmico/genética , Respuesta al Choque Térmico , Animales , Elementos sin Sentido (Genética)/metabolismo , Northern Blotting , Western Blotting , Carcinoma , Proteínas de Unión al ADN/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico , Humanos , ARN/metabolismo , Ratas , Temperatura , Factores de Transcripción , Transfección , Células Tumorales CultivadasRESUMEN
The excitotoxin, L-alpha-aminoadipic acid (L-AAA), kills primary astrocytes in the brain. The mechanism underlying the induction of cell death is not well understood although many possible mechanisms are theorized. Previous studies have reported that astrocytes die after prolonged exposure to L-AAA suggesting a delayed programmed cell death and apoptosis. In this study rat cortical astrocytes exposed to continuous 1 mM L-AAA exposure for 24-, 48-, or 72 hours demonstrated increased DNA laddering, a characteristic of apoptosis. Unexpectedly, this was not ameliorated by the presence of cycloheximide at 0.1 microg/ml medium. Because of our interest in cytoprotective heat shock proteins induced by excitoxic stress, we studied the effect of prolonged exposure of L-AAA on the synthesis of stress proteins and protein synthesis in rat cortical astrocytes. Protein synthesis as measured by [35S]-methionine labeling showed a marked and significant decrease in incorporation of radiolabel after 24 hours of exposure to L-AAA and prior to induction of significant cell death noted at 48- and 72 hours of L-AAA exposure. The inhibition of protein synthesis was partially reversible at 24 hours if cells were labeled in medium without L-AAA during the radiolabeling period. Heat shock or stress proteins, HSP70 and heme oxygenase-1 (HO-1), were analyzed after a 24 hour exposure to L-AAA and showed no significant induction of HSP70 or HO-1. The findings suggest that the prolonged inhibition of protein synthesis and associated lack of induction of HSP70 and HO-1 synthesis contributed to apoptotic cell death induced by the excitoxin L-AAA.
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Ácido 2-Aminoadípico/farmacología , Astrocitos/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Proteínas HSP70 de Choque Térmico/efectos de los fármacos , Hemo Oxigenasa (Desciclizante)/efectos de los fármacos , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Astrocitos/fisiología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Ratas , Ratas WistarRESUMEN
Spontaneous intracerebral hemorrhage (ICH) is the stroke subtype with highest mortality and morbidity. ICH can also occur following traumatic brain injury and thrombolysis for ischemic stroke and myocardial infarction. Development of ICH-induced hemispheric edema can elevate intracranial pressure and cause death. In survivors, edema-related white matter injury can lead to life-long neurological deficits. At present, there are no scientifically proven treatments for ICH. Heme oxygenase products, particularly iron and bilirubin, can be toxic to cells. In cerebral ischemia models, metalloporphyrins that are potent heme oxygenase inhibitors, reduce edema and infarct size. Tin-mesoporphyrin (SnMP) is a neuroprotectant that has also been used clinically to treat hyperbilirubinemia. Presently, we tested the hypothesis that SnMP treatment would reduce edema development following experimental ICH. We produced hematomas in pentobarbital-anesthetized pigs (9-11 kg) by infusing autologous blood into the frontal white matter. To maximize tissue concentrations, SnMP (87.5 microM in DMSO) or DMSO (vehicle controls) was included in the infused blood. Pig brains were frozen in situ at 24 hrs. following ICH and hematoma and edema volumes were determined on coronal sections by computer-assisted image analysis. We also examined the effects of SnMP in vitro on ferritin iron release, the formation of iron-induced thiobarbituric acid reactive substances (TBARS) and initial clot formation and hemolysis. SnMP treatment significantly reduced intracerebral mass following ICH. This was due to significant decreases in hematoma (0.68+/-0.08 vs. 1.39+/-0.30 cc, vehicle controls p<0.025) and edema volumes (edema = 1. 16+/-0.33 vs. 1.77+/-0.31 cc, p<0.05). In vitro, SnMP did not stabilize ferritin iron against reductive release nor did it decrease iron-induced TBARS formation in brain homogenates. SnMP or DMSO added to pig blood did not alter clot weights. In conclusion, SnMP reduced intracerebral mass in an ICH model by decreasing both hematoma and edema volumes SnMP's mechanism of action is presently unknown but may involve its potent inhibition of heme oxygenase activity. SnMP's effect appears unrelated to ferritin iron release, antioxidant activity or initial clot formation. Since SnMP treatment could be brain protective following ICH, further investigations into neurological and neuropathological outcomes and as well as into its mechanism of action are warranted.
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Antioxidantes/uso terapéutico , Hemorragia Cerebral/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Hematoma/tratamiento farmacológico , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Metaloporfirinas/uso terapéutico , Animales , Coagulación Sanguínea/efectos de los fármacos , Edema Encefálico/sangre , Edema Encefálico/tratamiento farmacológico , Edema Encefálico/metabolismo , Edema Encefálico/fisiopatología , Hemorragia Cerebral/sangre , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/fisiopatología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/metabolismo , Ferritinas/metabolismo , Hematoma/sangre , Hematoma/metabolismo , Hematoma/fisiopatología , Técnicas In Vitro , Hierro/metabolismo , Metaloporfirinas/metabolismo , PorcinosRESUMEN
Increased expression of heme oxygenase-1 (HO-1) is a common feature in a number of neurodegenerative diseases. Interestingly, the spatial distribution of HO-1 expression in diseased brain is essentially identical to that of pathological expression of tau. In this study, we explored the relationship between HO-1 and tau, using neuroblastoma cells stably transfected with sense and antisense HO-1 constructs as well as with the vector alone. In transfected cells overexpressing HO-1, the activity of heme oxygenase was increased, and conversely, the level of tau protein was dramatically decreased when compared with antisense HO-1 or CEP transfected cells. The suppression of tau protein expression was almost completely reversed by zinc-deuteroporphyrin, a specific inhibitor of heme oxygenase activity. The activated forms of ERKs (extracellular signal-regulated kinases) were also decreased in cells overexpressing HO-1 although no changes in the expression of total ERK-1/2 proteins were observed. These data are in agreement with the finding that the expression of tau is regulated through signal cascades including the ERKs, whose activities are modulated by oxidative stresses. The expression of tau and HO-1 may be regulated by oxidative stresses in a coordinated manner and play a pivotal role in the cytoprotection of neuronal cells.
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Hemo Oxigenasa (Desciclizante)/metabolismo , Neuronas/enzimología , Proteínas tau/metabolismo , Enfermedad de Alzheimer/enzimología , Northern Blotting , Supervivencia Celular , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Hemo Oxigenasa (Desciclizante)/genética , Hemo-Oxigenasa 1 , Humanos , Peróxido de Hidrógeno/farmacología , Immunoblotting , Proteínas Quinasas JNK Activadas por Mitógenos , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana , Proteína Quinasa 9 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuroblastoma/enzimología , Estrés Oxidativo , Plásmidos , Proteínas Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteínas tau/genéticaRESUMEN
Tin-mesoporphyrin (tin-mp), a potent inhibitor of heme oxygenase, and manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a potent superoxide dismutase mimetic, reduced H2O2 toxicity in cultures of transformed rat astroglial cells if added 30 min before, or at the same time as, H2O2. Reduced toxicity was not observed if treatment was delayed for 60 min, the time by which H2O2 was essentially eliminated from cultures. Coadministration of tin-mp and MnTMPyP did not increase protection over either compound administered individually. Tin-mp, but not MnTMPyP, was stable in culture. MnCl2 was not protective, suggesting that protection by MnTMPyP was not dependent on manganous ion, a by-product of MnTMPyP breakdown. Protection by tin-mp and MnTMPyP was not associated with metalloporphyrin-mediated induction of heme oxygenase-1 or with changes in heme oxygenase-2 on western blots. Whereas protective concentrations of tin-mp did not have superoxide dismutase-mimetic properties in vitro, protective concentrations of MnTMPyP partially inhibited heme oxygenase. The data support the hypothesis that heme oxygenase inhibition is protective against acute oxidative injury.
Asunto(s)
Astrocitos/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Manganeso/farmacología , Porfirinas/farmacología , Estaño/farmacología , Animales , Astrocitos/ultraestructura , Línea Celular Transformada , Depuradores de Radicales Libres/farmacología , Peróxido de Hidrógeno/antagonistas & inhibidores , Metaloporfirinas/farmacología , Microscopía Electrónica , RatasAsunto(s)
Discitis/microbiología , Osteomielitis/microbiología , Enfermedades de la Columna Vertebral/microbiología , Infecciones Estreptocócicas , Discitis/diagnóstico , Endocarditis Bacteriana/diagnóstico , Endocarditis Bacteriana/microbiología , Humanos , Masculino , Persona de Mediana Edad , Osteomielitis/diagnóstico , Infecciones Estreptocócicas/diagnósticoRESUMEN
In earlier studies, treatment with sodium arsenite was shown to decrease total hepatic CYP in rats. A concomitant increase in heme oxygenase, the rate-limiting step in heme degradation to biliverdin, was considered responsible for the decrease in CYP. Here we investigated the effect of sodium arsenite on induction of CYP2H, CYP1A, and heme oxygenase in primary cultures of chicken embryo hepatocytes. When added simultaneously with inducer, arsenite inhibited phenobarbital-mediated increases in CYP2H and 3-methylcholanthrene-mediated increases in CYP1A, as measured enzymatically and immunochemically. Near maximal decreases were observed in these forms of CYP at a concentration of 2.5 microM sodium arsenite. The concentration-dependent decreases in CYP2H and CYP1A by sodium arsenite were concomitant with increases in heme oxygenase. Sodium arsenite was not toxic at concentrations as high as 10 microM, as indicated by protein synthesis and the reduction of MTT by intact cells. Sodium arsenite had no effect on induction of CYP2H1 mRNA, suggesting that the decreases in this form of CYP occurred post-transcriptionally. Treatment of cells with tin mesoporphyrin (SnMeso), an inhibitor of heme oxygenase, resulted in inhibition of arsenite-induced heme oxygenase. However, SnMeso did not alter the effect of arsenite to prevent phenobarbital-mediated increases in CYP2H protein. SnMeso alone inhibited phenobarbital-mediated increases in CYP2H. Inclusion of 2 or 5 microM exogenous heme with arsenite did not prevent the arsenite-mediated decrease in CYP2H. Combined treatment with heme and phenobarbital induced heme oxygenase to the same extent as treatment with heme, arsenite, and phenobarbital. However, CYP2H activity was decreased only when the treatment included arsenite. These results suggest that elevated levels of heme oxygenase alone are not responsible for arsenite-mediated decreases in CYP2H.
Asunto(s)
Arsenitos/toxicidad , Sistema Enzimático del Citocromo P-450/biosíntesis , Hemo Oxigenasa (Desciclizante)/biosíntesis , Hígado/efectos de los fármacos , Compuestos de Sodio/toxicidad , Animales , Células Cultivadas , Embrión de Pollo , Citocromo P-450 CYP1A1/análisis , Citocromo P-450 CYP1A1/biosíntesis , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/análisis , Inducción Enzimática/efectos de los fármacos , Hemo/farmacología , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hígado/enzimología , Oxidorreductasas N-Desmetilantes/análisis , Oxidorreductasas N-Desmetilantes/biosíntesis , Fenobarbital/farmacología , ARN Mensajero/análisis , RatasRESUMEN
Heme oxygenase-1 (HO-1) is a stress protein inducible in some cells by oxidative stress. The status of heme oxygenase was investigated in a transgenic mouse model of amyotrophic lateral sclerosis (ALS) since oxidative mechanisms are postulated in neuronal injury. Three ALS mice [(SOD1-G93A)1Gur] and three controls [(SOD-1)2Gur] were obtained from The Jackson Laboratory. Behavioral differences suggestive of neurodegeneration in ALS mice developed at 4-5 months of age. All mice were killed at 7-8 months of age. Tissue vacuolation, cell loss, and the presence of GFAP+ cells were noted in the spinal cords of ALS mice. Spinal cord motor neurons in both control and ALS mice stained positive for heme oxygenase-2 (HO-2). While not precluding the presence of low levels of HO-1 neither immunohistochemical staining nor Western blot analysis provided evidence for significant HO-1 induction in degenerating spinal cord.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Hemo Oxigenasa (Desciclizante)/biosíntesis , Médula Espinal/enzimología , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Proteína Ácida Fibrilar de la Glía/análisis , Hemo Oxigenasa (Desciclizante)/análisis , Hemo-Oxigenasa 1 , Humanos , Inmunohistoquímica , Proteínas de la Membrana , Ratones , Ratones Transgénicos , Estrés Oxidativo , Mutación Puntual , Valores de Referencia , Médula Espinal/patología , Superóxido Dismutasa/biosíntesisRESUMEN
PCR technology offers alternatives to conventional diagnosis of Cryptosporidium for both clinical and environmental samples. We compared microscopic examination by a conventional acid-fast staining procedure with a recently developed PCR test that can not only detect Cryptosporidium but is also able to differentiate between what appear to be host-adapted genotypes of the parasite. Examinations were performed on 511 stool specimens referred for screening on the basis of diarrhea. PCR detected a total of 36 positives out of the 511 samples, while routine microscopy detected 29 positives. Additional positives detected by PCR were eventually confirmed to be positive by microscopy. A total of five samples that were positive by routine microscopy at Western Diagnostic Pathology but negative by PCR and by microscopy in our laboratory were treated as false positives. Microscopy therefore exhibited 83.7% sensitivity and 98.9% specificity compared to PCR. PCR was more sensitive and easier to interpret but required more hands-on time to perform and was more expensive than microscopy. PCR, however, was very adaptable to batch analysis, reducing the costs considerably. Bulk buying of reagents and modifications to the procedure would decrease the cost of the PCR test even more. An important advantage of the PCR test, its ability to directly differentiate between different Cryptosporidium genotypes, will assist in determining the source of cryptosporidial outbreaks. Sensitivity, specificity, ability to genotype, ease of use, and adaptability to batch testing make PCR a useful tool for future diagnosis and studies on the molecular epidemiology of Cryptosporidium infections.