RESUMEN
STUDY DESIGN: This is a retrospective longitudinal review. OBJECTIVE: The purpose of this review was to identify predictors of developing clinical scoliosis and compare between traumatic and neurological aetiologies of SCI. SETTING: This study was conducted at the Midland Centre of SCI. METHOD: Case notes of all patients injured at an age up to 18 years and admitted between 1971 and 2013 were reviewed. RESULTS: Sixty-nine individuals were identified, of which seven were excluded: three with pre-existing scoliosis and four with spina bifida. The remaining 62 (44 males, 18 females) had a median age at injury of 17 years (inter quartile range 13-17). Of these, 51 (82%) had traumatic and 11 (18%) had neurological injury. Most (42/51; 82%) of the children who had a traumatic injury were older than 13 years. The risk of developing scoliosis was lower for older patients (RR 0.68 per year, 95% CI 0.52-0.83) or following a traumatic injury (RR 0.36, 95% CI 0.20-0.66). A multivariable analysis based on age and trauma showed that only older age decreased the risk. A robust Receiver Operator Curve analysis suggested 14.6 years as the optimal threshold to predict development of scoliosis within 10 years (Area Under the Curve; AUC 0.83 (95% CI 0.73-0.93), sensitivity 70% (95% CI 50-89%), specificity 89% (95% CI 74-100%). CONCLUSION: Our results suggest that age below 14.6 years was a predictor for scoliosis. Once adjustment is made for age, the incidence of scoliosis does not differ between traumatic and neurological aetiologies of paediatric SCI injury.
Asunto(s)
Escoliosis/etiología , Traumatismos de la Médula Espinal/complicaciones , Adolescente , Factores de Edad , Niño , Femenino , Humanos , Incidencia , Estudios Longitudinales , Masculino , Pronóstico , Estudios Retrospectivos , Riesgo , Escoliosis/diagnóstico , Escoliosis/epidemiología , Sensibilidad y Especificidad , Traumatismos de la Médula Espinal/epidemiología , Traumatismos de la Médula Espinal/etiología , Heridas y Lesiones/complicacionesRESUMEN
STUDY DESIGN: Retrospective review of findings during cystourethroscopic surveillance of symptomatic and asymptomatic patients with indwelling urethral catheters (IUC) and suprapubic catheters (SPC) monitored between January 2003 and December 2008. OBJECTIVES: To audit and compare findings between symptomatic and asymptomatic patients and between SPC and IUC population. To systematically review the literature including the recent National Institute for Health and Clinical Excellence guidelines on cystoscopic surveillance. METHODS: Theater management system and the electronic patient records used to retrieve demographics, injury details and operative findings. RESULTS: Of 925 cystoscopies performed in 507 patients, 449 were performed in 277 patients with IUC/SPC. Only 419 procedures (SPC 264; IUC 155) in 262 patients fit the inclusion criteria. Thirty procedures in fifteen non traumatic patients were excluded. Statistically there was no significant difference in incidence of findings between the symptomatic and asymptomatic group. Recurrent blockage of catheter was predominant in the SPC group and symptomatic urinary tract infections (UTIs) were the most common indications in the IUC group. In the asymptomatic group, there were 44 squamous metaplastic changes in 27 patients. Two of these patients had keratinizing variants. The duration of catheterization ranged from 20 months to 27 years and mean of 13.7 years. The average duration between two cystoscopies in the symptomatic group was 16 months compared with an average 21 months in the asymptomatic group. CONCLUSION: Cystourethroscopic surveillance in high-risk patients with IUC/SPC is essential to diagnose and manage at an early-stage complications associated with IUC/SPC, minimize symptomatology, mitigate aggravation of complications, maintain good health and probably good quality of life.
Asunto(s)
Catéteres de Permanencia/efectos adversos , Cistoscopía , Traumatismos de la Médula Espinal/complicaciones , Neoplasias de la Vejiga Urinaria/diagnóstico , Catéteres Urinarios/efectos adversos , Cistoscopía/estadística & datos numéricos , Humanos , Estudios Retrospectivos , Vejiga Urinaria Neurogénica/etiologíaRESUMEN
Tuberculosis (TB) is an important cause of mortality and morbidity all over the world and is particularly relevant in developing countries like India where the disease is endemic. Female reproductive system is very vulnerable to this infection and clinical presentation of this disease in female reproductive tract is protean in nature and in a large majority of patients could be completely silent. This disease is an important cause of infertility, menstrual irregularity, pregnancy loss, and in association with pregnancy, morbidity to both the mother and child increases. Some of the effects of TB infection on female genital tract could be remote in nature due to infection elsewhere. Medicines used to treat TB infection can also have adverse effects on contraception and other areas of female reproductive health. HIV coinfection and multidrug-resistant tuberculosis (MDR-TB) and increased population migration from developed to developing countries have now added a whole new dimension to this infection. Though new, finer diagnostic tools of detection of TB are increasingly available in the form of bacterial cultures and polymerase chain reaction (PCR) based diagnostics, suspicion by clinicians remains the main tool for diagnosis of the condition. Hence, doctors need to be properly trained to become "Tuberculosis Minded".
Asunto(s)
Países en Desarrollo , Transmisión Vertical de Enfermedad Infecciosa , Tuberculosis/complicaciones , Tuberculosis/transmisión , Antituberculosos/efectos adversos , Femenino , Infecciones por VIH/complicaciones , Humanos , Infertilidad Femenina/etiología , Trastornos de la Menstruación/etiología , Embarazo , Complicaciones Infecciosas del Embarazo/etiología , Tuberculosis/epidemiologíaRESUMEN
Familial hypercholesterolemia (FH) is an inherited disorder in humans that is caused by a deficiency of low density lipoprotein receptors (LDLRs). An animal model for FH, the Watanabe Heritable Hyperlipidemic rabbit, was used to develop an approach for liver-directed gene therapy based on transplantation of autologous hepatocytes that were genetically corrected ex vivo with recombinant retroviruses. Animals transplanted with LDLR-transduced autologous hepatocytes demonstrated a 30 to 50 percent decrease in total serum cholesterol that persisted for the duration of the experiment (122 days). Recombinant-derived LDLR RNA was harvested from tissues with no diminution for up to 6.5 months after transplantation.
Asunto(s)
Terapia Genética , Hipercolesterolemia/genética , Hipercolesterolemia/terapia , Receptores de LDL/genética , Transfección , Animales , Expresión Génica , Hígado/fisiología , Trasplante de Hígado/fisiología , ARN/genética , ARN/aislamiento & purificación , Conejos , Receptores de LDL/análisis , Proteínas Recombinantes/análisis , Albúmina Sérica/análisis , Albúmina Sérica/genética , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
Isolated hepatocytes, harvested from normal rat livers by portal vein collagenase perfusion, can be attached to collagen-coated dextran microcarriers and transplanted by intraperitoneal injection into rats. Survival and function of the transplanted hepatocytes have been demonstrated in mutant rats lacking bilirubin-uridine diphosphate glucuronosyltransferase activity (Gunn strain) and rats with inherited lack of plasma albumin (Nagase analbuminemia rat strain). This simple technique promises to be useful in the treatment of acute liver failure in humans.
Asunto(s)
Trasplante de Hígado , Microesferas , Animales , Bilirrubina/sangre , Colágeno , Dextranos , Inyecciones Intraperitoneales , Hígado/citología , Ratas , Ratas Gunn , Ratas Mutantes , Trasplante HomólogoRESUMEN
A novel feedback regulatory mechanism operating on transcription of the albumin gene is described in the rat. In 1946, it was proposed that circulating colloids, including serum albumin, may affect the synthesis and/or secretion of albumin in the liver. The molecular basis for this proposed regulation has now been investigated by adding oncotically active macromolecules to the circulation of normal or genetically albumin-deficient Nagase analbuminemic rats (NAR) and analyzing the hepatic expression of genes, including albumin after 24 h. The transcription rate of the albumin gene was higher in NAR than in normal rats and was dramatically reduced by raising serum albumin to 1.6 g/dl. Intravenous infusion of albumin into normal rats also decreased transcriptional activity of the albumin gene by 50-60%, and this decrease correlated with changes in serum colloid osmotic pressure after albumin infusion. Inhibition of albumin gene transcription was also observed upon intravenous infusion of other protein or nonprotein macromolecules, such as gamma-globulin and dextran. This down-regulation appears to control the steady-state level of albumin mRNA in the liver. Aside from a concomitant decrease in apo E gene transcription after albumin or macromolecule infusion, there was no change in the transcription rate of other genes, including those exhibiting liver-preferred or -specific expression (e.g., tyrosine amino-transferase, cytochrome P-450, alpha 1-antitrypsin, apolipoproteins A-I and B, and transferrin) or general cellular expression (e.g., alpha-tubulin, pro alpha 2 collagen, and beta-actin). Feedback regulation of albumin gene expression by serum colloids may serve as a specific homeostatic mechanism to maintain the steady-state level of total protein in the circulation.
Asunto(s)
Regulación hacia Abajo , Albúmina Sérica/genética , Animales , Coloides/farmacología , Hígado/metabolismo , Sustancias Macromoleculares , Masculino , Presión Osmótica , Ratas , Ratas Endogámicas , Albúmina Sérica/deficiencia , Albúmina Sérica/fisiologíaRESUMEN
Recombinant adenoviruses (Ads) efficiently transfer foreign genes into hepatocytes in vivo, but the duration of transgene expression is limited by the host immune response which precludes gene expression upon readministration of the virus. To test if this immune response can be abrogated by oral tolerization, we instilled protein extracts of a recombinant adenovirus type-5 via gastroduodenostomy tubes into bilirubin-UDP-glucuronosyltransferase-1 (BUGT1)-deficient jaundiced Gunn rats. Control rats received BSA. Subsequent intravenous injection 5 x 10(9) pfu of a recombinant adenovirus-expressing human BUGT1 (Ad-hBUGT1) resulted in hepatic expression of human BUGT1 (hBUGT1) with reduction of serum bilirubin levels by 70%. After 2 mo serum bilirubin increased gradually. In orally tolerized rats, but not in controls, a second dose of the virus on day 98 markedly reduced serum bilirubin again. In the tolerized rats, the development of antiadenoviral neutralizing antibodies and cytotoxic lymphocytes were markedly inhibited, and transplantation of their splenocytes into naive Gunn rats adoptively transferred the tolerance, indicating a role for regulatory cells. Lymphocytes from the tolerized rats hyperexpressed TGFbeta1, IL2, and IL4 upon exposure to viral antigens, whereas IFNgamma expression became undetectable. Thus, oral tolerization with adenoviral antigens permits long-term gene expression by repeated injections of recombinant adenoviruses.
Asunto(s)
Adenoviridae/inmunología , Regulación Viral de la Expresión Génica , Terapia Genética/métodos , Terapia de Inmunosupresión/métodos , Proteínas Recombinantes/inmunología , Recombinación Genética , Proteínas Virales/inmunología , Adenoviridae/genética , Administración Oral , Traslado Adoptivo , Alanina Transaminasa/metabolismo , Animales , Anticuerpos Antivirales/análisis , Pigmentos Biliares/análisis , Bilirrubina/sangre , Bilirrubina/metabolismo , Western Blotting , Cromatografía Líquida de Alta Presión , Citotoxicidad Inmunológica , ADN/análisis , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos/genética , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Humanos , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-2/biosíntesis , Interleucina-2/inmunología , Interleucina-4/biosíntesis , Interleucina-4/inmunología , Operón Lac , Hígado/metabolismo , Pruebas de Neutralización , Plásmidos , Reacción en Cadena de la Polimerasa , Ratas , Ratas Gunn , Ratas Wistar , Proteínas Recombinantes/administración & dosificación , Albúmina Sérica Bovina/administración & dosificación , Albúmina Sérica Bovina/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T Citotóxicos/inmunología , Trasplante de Tejidos , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/inmunología , Transgenes/genética , Transgenes/inmunología , Proteínas Virales/administración & dosificación , beta-Galactosidasa/metabolismoRESUMEN
Recombinant adenoviruses are highly efficient at transferring foreign genes in vivo. However, duration of gene expression is limited by the host antiviral immune response which precludes expression upon viral readministration. We tested the feasibility of prolonging gene expression by induction of central tolerance to adenoviral antigens in bilirubin-UDP-glucuronosyltransferase-1 (BUGT1)-deficient Gunn rats. Tolerance was induced by intraperitoneal injection of antilymphocyte serum, followed by intrathymic inoculation of one of the following: a recombinant adenovirus (Ad), adenovirus human UDP-glucuronosyltransferase (Ad-hBUGT1) carrying the hBUGT1 gene; a protein extract of the same virus; or viral infected hepatocytes. Controls received intrathymic injections of normal saline. After 12 d all groups were injected intravenously with 5 x 10(9) pfu of either Ad-hBUGT1 or adenovirus beta-galactosidase (Ad-LacZ) (expressing the Escherichia coli beta-galactosidase [LacZ] gene). In all three groups of tolerized rats, hBUGT1 was expressed in the liver after administration of Ad-hBUGT1, with glucuronidation of biliary bilirubin of above 95%. Serum bilirubin levels decreased from 7.2 to 1.8 mg/dl within 1 wk and remained low for 7 wk. Similar findings were observed following repeat injections given on days 45 and 112. In control rats serum bilirubin levels were reduced for only 4 wk, and viral readministration was ineffective. In all tolerized groups, but not in controls, there was a marked inhibition of appearance of neutralizing antibodies and cytotoxic lymphocytes against the recombinant adenovirus. Injection of wild type adenovirus-5 (Ad5) into the tolerized rats elicited a wild type-specific cytotoxic lymphocyte response. This is the first demonstration of Ad-directed long-term correction of an inherited metabolic disease following central tolerization with thymic antigen.
Asunto(s)
Adenoviridae , Terapia Genética , Glucuronosiltransferasa/genética , Kernicterus/terapia , Animales , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Pigmentos Biliares/metabolismo , Escherichia coli , Expresión Génica , Genes Reporteros , Glucuronosiltransferasa/biosíntesis , Humanos , Tolerancia Inmunológica , Operón Lac , Hígado/metabolismo , Ratas , Ratas Gunn , Ratas Wistar , Timo , beta-Galactosidasa/biosíntesisRESUMEN
Bilirubin diglucuronide (BDG) may be formed in vitro by microsomal UDP glucuronosyl transferase (EC 2.4.1.17)-mediated transfer of a second mole of glucuronic acid from UDP-glucuronic acid, or by dismutation of bilirubin monoglucuronide (BMG) to BDG and unconjugated bilirubin, catalyzed by an enzyme (EC 2.4.1.95) that is concentrated in plasma membrane-enriched fractions of rat liver. To evaluate the role of these two enzymatic mechanisms in vivo, [(3)H]bilirubin mono-[(14)C]glucuronide was biosynthesized, purified by thin-layer chromatography, and tracer doses were infused intravenously in homozygous Gunn (UDP glucuronyl transferase-deficient) rats or Wistar rats. Bilirubin conjugates in bile were separated by high-pressure liquid chromatography and (3)H and (14)C were quantitated. In Gunn rats, the (14)C:(3)H ratio in BDG excreted in bile was twice the ratio in injected BMG. In Wistar rats the (14)C:(3)H ratio in biliary BDG was 1.25 +/- 0.06 (mean +/- SEM) times the ratio in injected BMG. When double labeled BMG was injected in Wistar rats after injection of excess unlabeled unconjugated bilirubin (1.7 mumol), the (14)C:(3)H ratio in BDG excreted in bile was identical to the ratio in injected BMG. Analysis of isomeric composition of bilirubin conjugates after alkaline hydrolysis or alkaline methanolysis indicated that the bile pigments retained the IX(alpha) configuration during these experiments. The results indicate that both enzymatic dismutation and UDP glucuronyl transferase function in vivo in BDG formation, and that dismutation is inhibited by a high intrahepatic concentration of unconjugated bilirubin. This hypothesis was supported by infusion of [(3)H]bilirubin-monoglucuronide in isolated perfused homozygous Gunn rat liver after depletion of intrahepatic bilirubin by perfusion with bovine serum albumin (2.5%), and after bilirubin repletion following perfusion with 0.34 mM bilirubin. From 20 to 25% of injected radioactivity was recovered in BDG in bile in the bilirubin-depleted state; only 8-10% of radioactivity was in BDG in bile after bilirubin repletion. After infusion of [(3)H]bilirubin di-[(14)C]glucuronide in homozygous Gunn rats, 5-7% of the injected pigment was excreted in bile as BMG. The (14)C:(3)H ratio in the injected BDG was 10% greater than the (14)C:(3)H ratio in BMG excreted in bile. These results indicate that in vivo, dismutation rather than partial hydrolysis, is responsible for BMG formation. Incubation of [(3)H]bilirubin, BDG and a rat liver plasma membrane preparation resulted in formation of BMG (3.3 nmol/min per mg protein) indicating that dismutation is also reversible in vitro.
Asunto(s)
Bilirrubina/análogos & derivados , Hígado/metabolismo , Animales , Bilis/análisis , Bilis/metabolismo , Bilirrubina/biosíntesis , Bilirrubina/metabolismo , Cromatografía Líquida de Alta Presión , Glucuronosiltransferasa/metabolismo , Hexosiltransferasas/metabolismo , Masculino , Microsomas Hepáticos/enzimología , Ratas , Ratas Gunn , Ratas EndogámicasRESUMEN
The microsomal enzyme uridine diphosphate (UDP) glucuronate glucuronyltransferase (E.C. 2.4.1.17) catalyzes formation of bilirubin mono-glucuronide from bilirubin and UDPglucuronic acid. Bilirubin glucuronoside glucuronosyltransferase (E.C. 2.4.1.95), an enzyme concentrated in plasma membrane-enriched fractions of rat liver, converts bilirubin monoglucuronide to bilirubin diglucuronide. Bilirubin glucuronoside glucuronosyltransferase activity was studied in homogenates of liver biopsy specimens obtained from patients with the Crigler-Najjar syndrome (Type I) and in subcellular liver fractions of rats homozygous for UDP glucuronate glucuronyltransferase deficiency (Gunn strain). In patients with the Crigler-Najjar syndrome (Type I) and in Gunn rats, hepatic UDPglucuronate glucuronyltransferase activity was not measurable; however, bilirubin glucuronoside glucuronosyltransferase activity was similar to that in normal controls. The subcellular distribution of bilirubin glucuronoside glucuronosyltransferase activity in Gunn rat liver was similar to the distribution observed in normal Wistar rat liver.When bilirubin monoglucuronide was infused intravenously into Gunn rats, 29+/-5% of the conjugated bilirubin excreted in bile was bilirubin diglucuronide. After transplantation of normal Wistar rat kidney, which contained UDPglucuronate glucuronyltransferase activity, in Gunn rats, the serum bilirubin concentration decreased by 80% in 4 days. The major route of bilirubin removal was biliary excretion of conjugated bilirubin, approximately 70% of which was bilirubin diglucuronide. Although patients with the Crigler-Najjar syndrome (Type I) and Gunn rats lack UDP glucuronate glucuronyltransferase, their livers enzymatically convert bilirubin monoglucuronide to diglucuronide in vitro. Conversion in bilirubin monoglucuronide to diglucuronide was demonstrated in Gunn rats in vivo.
Asunto(s)
Bilirrubina/metabolismo , Glucuronosiltransferasa/deficiencia , Hexosiltransferasas/metabolismo , Hígado/metabolismo , Adulto , Animales , Síndrome de Crigler-Najjar/metabolismo , Femenino , Glucuronatos/metabolismo , Humanos , Técnicas In Vitro , Lactante , Trasplante de Riñón , Masculino , Ratas , Fracciones Subcelulares/metabolismo , Trasplante HomólogoRESUMEN
Crigler-Najjar (CN) disease is classified into two subtypes, type I and II. The molecular basis for the difference between these types is not well understood. Several mutations in the bilirubin UDP-glucuronosyl-transferase (B-UGT) gene of six CN type I and two CN type II patients were identified. Recombinant cDNAs containing these mutations were expressed in COS cells. B-UGT activity was measured using HPLC and the amount of expressed protein was quantitated using a sandwich ELISA. This enabled us to determine the specific activities of the expressed enzymes. All type I patients examined had mutations in the B-UGT1 gene that lead to completely inactive enzymes. The mutations in the B-UGT1 gene of patients with CN type II only partially inactivated the enzyme. At saturating concentrations of bilirubin (75 microM) CN type II patient A had 4.4 +/- 2% residual activity and CN type II patient B had 38 +/- 2% residual activity. Kinetic constants for the glucuronidation of bilirubin were determined. The affinities for bilirubin of B-UGT1 expressed in COS cells and B-UGT from human liver microsomes were similar with Km of 5.1 +/- 0.9 microM and 7.9 +/- 5.3 microM, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, Km = 56 +/- 23 microM. At physiological concentrations of bilirubin both type II patients will have a strongly reduced conjugation capacity, whereas type I patients have no B-UGT activity. We conclude that CN type I is caused by a complete absence of functional B-UGT and that in CN type II B-UGT activity is reduced.
Asunto(s)
Síndrome de Crigler-Najjar/clasificación , Síndrome de Crigler-Najjar/genética , Glucuronosiltransferasa/genética , Mutación , Adolescente , Animales , Bilirrubina/análogos & derivados , Bilirrubina/biosíntesis , Células Cultivadas , Niño , Preescolar , Síndrome de Crigler-Najjar/enzimología , Ensayo de Inmunoadsorción Enzimática , Femenino , Glucuronosiltransferasa/biosíntesis , Glucuronosiltransferasa/inmunología , Humanos , Lactante , Masculino , Microsomas Hepáticos/enzimología , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , TransfecciónRESUMEN
BACKGROUND: Little is known about the use of antibiotics and the extent of antibiotic-associated diarrhoea (AAD) in patients with spinal cord injuries (SCIs). AIMS: To record the use of antibiotics, establish the prevalence of AAD and Clostridium difficile infection (CDI), and assess if there was any seasonal variation in antibiotic use and incidence of AAD in patients with SCIs. METHODS: A retrospective study was conducted in six European SCI centres between October 2014 and June 2015. AAD was defined as two or more watery stools (Bristol Stool Scale type 5, 6 or 7) over 24 h. FINDINGS: In total, 1267 adults (median age 54 years, 30.7% female) with SCIs (52.7% tetraplegia, 59% complete SCI) were included in this study. Among the 215 (17%) patients on antibiotics, the top three indications for antibiotics were urinary tract infections (UTIs), infected pressure ulcers and other skin infections. Thirty-two of these 215 (14.9%) patients developed AAD and two patients out of the total study population (2/1267; 0.16%) developed CDI. AAD was more common in summer than in spring, autumn or winter (30.3% vs 3.8%, 7.4% and 16.9%, respectively; P<0.01). AAD was associated with age ≥65 years, tetraplegia, higher body mass index, hypoalbuminaemia, polypharmacy, multiple antibiotic use and high-risk antibiotic use. Summer and winter seasons and male sex were identified as independent predictors for the development of AAD. CONCLUSION: This survey found that AAD is common in patients with SCIs, and UTI is the most common cause of infection. Summer and winter seasons and male sex are unique predictors for AAD. Both AAD and UTIs are potentially preventable; therefore, further work should focus on preventing the over-use of antibiotics, and developing strategies to improve hospital infection control measures.
Asunto(s)
Antibacterianos/efectos adversos , Infecciones por Clostridium/epidemiología , Diarrea/epidemiología , Diarrea/microbiología , Traumatismos de la Médula Espinal/complicaciones , Anciano , Antibacterianos/uso terapéutico , Clostridioides difficile/aislamiento & purificación , Infecciones por Clostridium/tratamiento farmacológico , Utilización de Medicamentos , Europa (Continente)/epidemiología , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Uso Excesivo de Medicamentos Recetados , Prevalencia , Estudios Retrospectivos , Factores de Riesgo , Estaciones del Año , Encuestas y Cuestionarios , Reino Unido/epidemiología , Infecciones Urinarias/complicaciones , Infecciones Urinarias/epidemiologíaRESUMEN
Dietary antioxidants protect laboratory animals against the induction of tumors by a variety of chemical carcinogens. Among possible mechanisms, protection against chemical carcinogenesis could be mediated via antioxidant-dependent induction of detoxifying enzymes. Therefore, we investigated the effects of two commonly used food preservatives, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), on the expression of UDP-glucuronosyltransferase isoforms in rat liver. Male Wistar rats were fed a control diet or diets containing BHA (0.75%) or BHT (0.5%) for 2 weeks. BHT and BHA increased UDP-glucuronosyltransferase activities in liver microsomes for p-nitrophenol (236 and 218%, respectively), 3-hydroxybenzo(a)pyrene (246 and 175%, respectively), and androsterone (269 and 152%, respectively). Immunoblots showed changes in the amounts of UDP-glucuronosyltransferase isoforms corresponding to the changes in enzyme activities. Northern blot analysis showed that the concentration of UDP-glucuronosyltransferase mRNA paralleled the concentration of enzyme proteins and their respective levels of enzyme activity. BHT, for example, caused about a 250% increase in mRNA using a probe that recognizes the common 3'-domain of bilirubin/phenol UDP-glucuronosyltransferase mRNAs. In addition to inducing hepatic enzyme activities, BHT and BHA increased the activity of UDP-glucuronosyltransferase in the small intestine and kidney.
Asunto(s)
Hidroxianisol Butilado/farmacología , Hidroxitolueno Butilado/farmacología , Glucuronosiltransferasa/efectos de los fármacos , Microsomas/enzimología , Animales , Glucuronosiltransferasa/metabolismo , Intestino Delgado/enzimología , Riñón/enzimología , Masculino , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
Hepatic uridinediphosphoglucuronate glucuronosyl transferase (UDPglucuronyltransferase, EC 2.4.1.17) is functionally heterogeneous; 4-nitrophenol and bilirubin are representative substrates for two functional forms of the enzyme. UDPglucuronyltransferase activity for bilirubin and 4-nitrophenol was separated from solubilized rat liver microsomes by DEAE-cellulose chromatography and corresponding enzymes were purified. A radioimmunoassay was developed using a rabbit antiserum against purified rat 4-nitrophenol-specific UDPglucuronyltransferase, which precipitated enzyme activities toward both 4-nitrophenol and bilirubin. After treatment with triiodothyronine (T3) (0.55 mg/kg body weight), hepatic microsomal UDPglucuronyltransferase activity for 4-nitrophenol was increased 400% as compared to controls; the enzyme activity for bilirubin was decreased by 80%; the changes in the substrate-specific enzyme activities were reflected in the enzymatically active fractions separated after DEAE-cellulose chromatography. The changes in enzyme activities paralleled changes in the concentrations of the two corresponding UDP glucuronyltransferase proteins in the chromatographic fractions, as measured by radioimmunoassay. The results indicate that the opposite effects of T3 on the two forms of UDPglucuronyltransferase activity is due to its differential effect on corresponding enzyme proteins.
Asunto(s)
Glucuronosiltransferasa/metabolismo , Microsomas Hepáticos/enzimología , Triyodotironina/farmacología , Animales , Bilirrubina/metabolismo , Cromatografía de Afinidad , Cromatografía DEAE-Celulosa , Inmunoquímica , Cinética , Masculino , Nitrofenoles/metabolismo , Ratas , Ratas Endogámicas , Especificidad por SustratoRESUMEN
The end product of the breakdown of the heme group of hemoglobin and other heme-containing proteins is bilirubin. Bilirubin is hydrophobic and cannot be excreted as such. Therefore, mammals have a liver enzyme bilirubin UDP-glucuronosyltransferase (B-UGT), which conjugates bilirubin with glucuronic acid, thereby making the molecule much more water soluble. Bilirubin glucuronides are secreted into bile. Patients with Crigler-Najjar (CN) disease have a deficiency in bilirubin UDP-glucuronosyltransferase and accumulate high serum levels of bilirubin. An animal model for CN disease is the Gunn rat. The obvious target for gene therapy for CN disease is the liver, but because liver cells do only divide infrequently, they are difficult to transduce. To investigate whether cells that are easily transduced can be used to develop gene therapy for CN disease, we have transduced Gunn rat fibroblasts with B-UGT, using a recombinant retrovirus. Gunn rat fibroblasts expressing B-UGT were able to glucuronidate bilirubin present in cell culture media. In this study, we describe the intraperitoneal transplantation of Gunn rats with Gunn rat fibroblasts expressing B-UGT. Transplantation of the fibroblasts corrected the genetic deficiency of the Gunn rats, serum bilirubin concentrations of the transplanted Gunn rats were reduced to normal, and bilirubin glucuronides appeared in bile. However, due to the prolonged period of cell culture, the transplanted fibroblasts were transformed, and the experimental animals developed tumors after transplantation.
Asunto(s)
Terapia Genética , Glucuronosiltransferasa/metabolismo , Trasplante Autólogo , Animales , Bilis/química , Bilis/metabolismo , Bilirrubina/sangre , Western Blotting , Cromatografía Líquida de Alta Presión , Síndrome de Crigler-Najjar/genética , Síndrome de Crigler-Najjar/terapia , Modelos Animales de Enfermedad , Fibroblastos , Expresión Génica/genética , Vectores Genéticos , Glucuronidasa/metabolismo , Inmunohistoquímica , Neoplasias Experimentales/metabolismo , Ratas , Ratas Gunn , Ratas Wistar , Retroviridae/genética , Transformación Genética/genéticaRESUMEN
Uridine-diphosphoglucuronate glucuronosyltransferases (UGTs) are a family of enzymes that conjugate various endogenous and exogenous compounds with glucuronic acid and facilitate their excretion in the bile. Bilirubin-UGT(1) (UGT1A1) is the only isoform that significantly contributes to the conjugation of bilirubin. Lesions in the gene encoding bilirubin-UGT(1), lead to complete or partial inactivation of the enzyme causing the rare autosomal recessively inherited conditions, Crigler-Najjar syndrome type-1 (CN-1) and type 2 (CN-2), respectively. Inactivation of the enzyme leads to accumulation of unconjugated bilirubin in the serum. Severe hyperbilirubinemia seen in CN-1 can cause bilirubin encephalopathy (kernicterus). Kernicterus can be fatal or may leave behind permanent neurological sequelae. Here, we have compiled more than 50 genetic lesions of UGT1A1 that cause CN-1 (including 9 novel mutations) or CN-2 (including 3 novel mutations) and have presented a correlation of structure to function of UGT1A1. In contrast to Crigler-Najjar syndromes, Gilbert syndrome is a common inherited condition characterized by mild hyperbilirubinemia. An insertional mutation of the TATAA element upstream to UGT1A1 results in a reduced level of expression of the gene. Homozygosity for the variant promoter is required for Gilbert syndrome, but not sufficient for manifestation of hyperbilirubinemia, which is partly dependent on the rate of bilirubin production. Several structural mutations of UGT1A1, for example, a G71R substitution, have been reported to cause mild reduction of UGT activity toward bilirubin, resulting in mild hyperbilirubinemia, consistent with Gilbert syndrome. When the normal allele of a heterozygote carrier for a Crigler-Najjar type structural mutation contains a Gilbert type promoter, intermediate levels of hyperbilirubinemia, consistent with the diagnosis of CN-2, may be observed.
Asunto(s)
Bilirrubina/metabolismo , Síndrome de Crigler-Najjar/enzimología , Síndrome de Crigler-Najjar/genética , Enfermedad de Gilbert/enzimología , Enfermedad de Gilbert/genética , Glucuronosiltransferasa/genética , Mutación/genética , Animales , Genotipo , Humanos , Isoenzimas/genética , Datos de Secuencia Molecular , FenotipoRESUMEN
This review represents an update of the nomenclature system for the UDP glucuronosyltransferase gene superfamily, which is based on divergent evolution. Since the previous review in 1991, sequences of many related UDP glycosyltransferases from lower organisms have appeared in the database, which expand our database considerably. At latest count, in animals, yeast, plants and bacteria there are 110 distinct cDNAs/genes whose protein products all contain a characteristic 'signature sequence' and, thus, are regarded as members of the same superfamily. Comparison of a relatedness tree of proteins leads to the definition of 33 families. It should be emphasized that at least six cloned UDP-GlcNAc N-acetylglucosaminyltransferases are not sufficiently homologous to be included as members of this superfamily and may represent an example of convergent evolution. For naming each gene, it is recommended that the root symbol UGT for human (Ugt for mouse and Drosophila), denoting 'UDP glycosyltransferase,' be followed by an Arabic number representing the family, a letter designating the subfamily, and an Arabic numeral denoting the individual gene within the family or subfamily, e.g. 'human UGT2B4' and 'mouse Ugt2b5'. We recommend the name 'UDP glycosyltransferase' because many of the proteins do not preferentially use UDP glucuronic acid, or their nucleotide sugar preference is unknown. Whereas the gene is italicized, the corresponding cDNA, transcript, protein and enzyme activity should be written with upper-case letters and without italics, e.g. 'human or mouse UGT1A1.' The UGT1 gene (spanning > 500 kb) contains at least 12 promoters/first exons, which can be spliced and joined with common exons 2 through 5, leading to different N-terminal halves but identical C-terminal halves of the gene products; in this scheme each first exon is regarded as a distinct gene (e.g. UGT1A1, UGT1A2, ... UGT1A12). When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, sequential naming of the genes is being carried out chronologically as they become characterized. We suggest that the Human Gene Nomenclature Guidelines (http://www.gene.acl.ac.uk/nomenclature/guidelines.html++ +) be used for all species other than the mouse and Drosophila. Thirty published human UGT1A1 mutant alleles responsible for clinical hyperbilirubinemias are listed herein, and given numbers following an asterisk (e.g. UGT1A1*30) consistent with the Human Gene Nomenclature Guidelines. It is anticipated that this UGT gene nomenclature system will require updating on a regular basis.
Asunto(s)
Evolución Molecular , Genes , Glucuronosiltransferasa/genética , Familia de Multigenes , Terminología como Asunto , Secuencia de Aminoácidos , Animales , Glucuronosiltransferasa/química , Humanos , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Brain dopamine influences immune functions and the role of immune functions in tumor growth is well established. Therefore, a study has been carried out to evaluate the correlation, if any, between brain dopamine and tumor growth. MPTP selectively destroys dopaminergic neurons in the brain. In the present study, Ehrlich carcinoma growth was evaluated in MPTP-treated mice. Results showed a correlation between depletion of striatal dopamine and enhanced tumor growth. Since in the present study striatal dopamine depletion in mice was associated with significantly decreased immune responses, the suggested correlation between brain dopamine and tumor growth was possibly mediated by the immune system.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Encéfalo/metabolismo , Carcinoma de Ehrlich/patología , Dopamina/deficiencia , Animales , Formación de Anticuerpos , Carcinoma de Ehrlich/inmunología , Carcinoma de Ehrlich/metabolismo , División Celular/efectos de los fármacos , Inmunoglobulinas/análisis , Células Asesinas Naturales/fisiología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos , Trasplante de Neoplasias , Bazo/inmunología , Bazo/patología , Linfocitos T Citotóxicos/fisiología , MusloRESUMEN
The shortage of human livers available for hepatocyte isolation limits its clinical application. The availability of cloned, conditionally immortalized hepatocytes that could be grown in culture but would lose their transformed phenotype and provide metabolic support upon transplantation would greatly facilitate the treatment of acute liver failure. Toward this goal, we transduced isolated Lewis rat hepatocytes using a replication-defective recombinant retrovirus capable of transferring a gene encoding a thermolabile mutant simian virus 40 T antigen (SV40ts). The cloned, immortalized hepatocytes proliferate at 33 degrees C. At the nonpermissive temperatures (37-39 degrees C), they stop growing and exhibit characteristics of differentiated hepatocytes. These cells did not produce tumors when transplanted in mice with severe combined immunodeficiency disease or in syngeneic rats. To induce acute liver failure, Lewis rats were subjected to 90% hepatectomy (Hpx) and given 5% oral dextrose. All rats that did not undergo hepatocyte transplantation died within 96 hr. Fifty percent of rats that received intrasplenic injection of 10 x 10(6) primary Lewis rat hepatocytes (G2, n=6) or 10 x 10(6) SV40ts-conditionally immortalized (SV40ts-ci) hepatocytes (G3, n=8) 1 day before 90% hepatectomy survived, whereas 80% of rats that received an intraperitoneal injection of 200 x 10(6) primary Lewis rat hepatocytes (G4, n=10) or 200 x 10(6) SV40ts-ci hepatocytes (G5, n=10) on the day of hepatectomy survived. Survival after intraperitoneal injection of a cellular homogenate of 200 x 10(6) primary Lewis rat (G7, n=9) or SV40ts-ci hepatocytes (G8, n=10) on the day of Hpx was 33% and 40%, respectively, whereas survival after intraperitoneal injection of 200 x 10(6) Lewis rat bone marrow cells (G6, n=7) was 29%. Thus, transplanted, conditionally immortalized hepatocytes can be as effective as primary hepatocytes in supporting life during acute liver insufficiency. This work represents the first step in developing an hepatocyte cell line that would partially alleviate the organ-donor shortage and could be of potential clinical value.