RESUMEN
Ankylosing spondylitis (AS) is the most frequent prototype of spondyloarthritides. Substantial direct costs and productivity losses often arise in young patients. Currently, tumor necrosis factor (TNF) inhibitors are the only approved therapy escalation when usual care (physiotherapy and NSAIDs) proves to be insufficient. Owing to their high medication costs, TNF inhibitors are a target of cost-effectiveness analyses. There is consistent evidence regarding the use of TNF inhibitors according to recommendations in patients with active AS finding TNF inhibitors to be cost effective from a societal perspective. However, there are relevant uncertainties (discontinuation rate and progression rate) in the long-term estimates of the cost-effectiveness analyses analyzed. Whether TNF inhibitors are cost effective from an insurance perspective in the long run will have to be addressed by models based on observational data.
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Factores Inmunológicos/uso terapéutico , Espondilitis Anquilosante/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Costo de Enfermedad , Análisis Costo-Beneficio , Costos de los Medicamentos , Costos de la Atención en Salud , Humanos , Factores Inmunológicos/economía , Espondilitis Anquilosante/economíaRESUMEN
OBJECTIVE: Defensins are immunomodulatory peptides and components of the innate immune response. They have been shown to be modulated in various disease states and in response to inflammatory stimuli. Recently, alpha-defensins have been implicated in the pathogenesis of autoimmune diseases. In order to explore whether these defensins may have a role in the pathogenesis of systemic lupus erythematosus (SLE), we sought to determine whether altered expression can be found in SLE patients. MATERIAL AND METHODS: Serum and EDTA-blood of 50 SLE patients who fulfilled the American College of Rheumatology (ACR) criteria (aged 41.4 ± 13.3 years) and 28 age- and sex-matched healthy controls were collected. Real-time polymerase chain reaction with gene-specific primers for human neutrophil peptides (HNPs), human beta-defensin 2 and 3 (hBD2, 3) in isolated polymorphonuclear cells and enzyme-linked immunosorbent assay (ELISA) in serum samples were performed. Results of SLE patients were compared with the control group and correlated to routine laboratory parameters, clinical data and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). RESULTS: SLE patients were predominantly female (90%) with a mean SLEDAI of 5.7 ± 6.1. In sera, patients displayed higher amounts of hBD2 and HNPs when compared with healthy controls. Furthermore, hBD2 correlated with levels of anti-dsDNA antibodies, erythrocyte count and the SLEDAI. Elevated values were observed in patients with myositis (n = 4). Serum HNPs on the other hand correlated with the neutrophil count and was elevated in patients with a rash (n = 7). Lupus patients suffering from transverse myelitis (n = 3) had raised serum-values of both HNPs and hBD2. While no mRNA of hBD2 or hBD3 was detected in polymorphonuclear cells, HNP mRNA was found in both healthy controls and patients without significant difference. Lupus nephritis and rash were associated with higher amounts of HNP mRNA, and the relative amount of copies correlated positively with the SLEDAI and negatively with C3 measurements. CONCLUSIONS: Serum levels of hBD2 and HNPs are elevated in SLE. The correlations of hBD2 and HNPs to established disease activity parameters and distinct clinical situations suggest that innate immune mechanisms are activated. Defensins may be involved in SLE pathogenesis.
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Regulación de la Expresión Génica , Lupus Eritematoso Sistémico/inmunología , alfa-Defensinas/sangre , beta-Defensinas/sangre , Adulto , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lupus Eritematoso Sistémico/fisiopatología , Nefritis Lúpica/inmunología , Masculino , Persona de Mediana Edad , Miositis/etiología , Miositis/inmunología , Neutrófilos/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Índice de Severidad de la Enfermedad , alfa-Defensinas/genética , beta-Defensinas/genéticaRESUMEN
BACKGROUND: In a multidisciplinary conference patients with advanced non-resectable hepatocellular carcinoma (HCC) were stratified according to their clinical status and tumor extent to different regional modalities or to best supportive care. The present study evaluated all patients who were stratified to repeated transarterial chemoembolization (TACE) from 1999 until 2003 in terms of tumor response, toxicity, and survival. A moderate embolizing approach was chosen using a combination of degradable starch microspheres (DSM) and iodized oil (Lipiodol) in order to combine anti-tumoral efficiency and low toxicity. METHODS: Fourty-seven patients were followed up prospectively. TACE treatment consisted of cisplatin (50 mg/m(2)), doxorubicin (50 mg/m(2)), 450-900 mg DSM, and 5-30 ml Lipiodol. DSM and Lipiodol were administered according to tumor vascularization. Patient characteristics, toxicity, and complications were outlined. In multivariate regression analyses of pre-treatment variables from a prospective database, predictors for tumor response and survival after TACE were determined. RESULTS: 112 TACE courses were performed (2.4+/-1.5 courses per patient). Mean maximum tumor size was 75 (+/-43) mm, in 68% there was bilobar disease. Best response to TACE treatment was: progressive disease (PD) 9%, stable disease (SD) 55%, partial remission (PR) 36%, and complete remission (CR) 0%. Multivariate regression analyses identified tumor size
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Carcinoma Hepatocelular/tratamiento farmacológico , Quimioembolización Terapéutica , Aceite Yodado/administración & dosificación , Neoplasias Hepáticas/tratamiento farmacológico , Adulto , Anciano , Carcinoma Hepatocelular/mortalidad , Quimioembolización Terapéutica/efectos adversos , Cisplatino/administración & dosificación , Doxorrubicina/administración & dosificación , Femenino , Humanos , Neoplasias Hepáticas/mortalidad , Masculino , Microesferas , Persona de Mediana Edad , AlmidónRESUMEN
AIM: To evaluate the treatment effect of percutaneous ethanol injection (PEI) for patients with advanced, non-resectable HCC compared with combination of transarterial chemoembolisation (TACE) and repeated single-session PEI, repeated single-session PEI alone, repeated TACE alone, or best supportive care. METHODS: All patients who received PEI treatment during the study period were included and stratified to one of the following treatment modalities according to physical status and tumor extent: combination of TACE and repeated single-session PEI, repeated single-session PEI alone, repeated TACE alone, or best supportive care. Prognostic value of clinical parameters including Okuda-classification, presence of portal vein thrombosis, presence of ascites, number of tumors, maximum tumor diameter, and serum cholinesterase (CHE), as well as Child-Pugh stage, alpha-fetoprotein (AFP), fever, incidence of complications were assessed and compared between the groups. Survival was determined using Kaplan-Meier and multivariate regression analyses. RESULTS: The 1- and 3-year survival of all patients was 73% and 47%. In the subgroup analyses, the combination of TACE and PEI (1) was associated with a longer survival (1-, 3-, 5-year survival: 90%, 52%, and 43%) compared to PEI treatment alone (2) (1-, 3-, 5-year survival: 65%, 50%, and 37%). Secondary PEI after initial stratification to TACE (3) yielded comparable results (1-, 3-, 5-year survival: 91%, 40%, and 30%) while PEI after stratification to best supportive care (4) was associated with decreased survival (1-, 3-, 5-year survival: 50%, 23%, 12%). Apart from the chosen treatment modalities, predictors for better survival were tumor number (n < 5), tumor size (< 5 cm), no ascites before PEI, and stable serum cholinesterase after PEI (P < 0.05). The mortality within 2 wk after PEI was 2.8% (n = 3). There were 24 (8.9%) major complications after PEI including segmental liver infarction, focal liver necrosis, and liver abscess. All complications could be managed non-surgically. CONCLUSION: Repeated single-session PEI is effective in patients with advanced HCC at an acceptable and manageable complication rate. Patients stratified to a combination of TACE and PEI can expect longer survival than those stratified to repeated PEI alone. Furthermore, patients with large or multiple tumors in good clinical status may also profit from a combination of TACE and reconsideration for secondary PEI.
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Carcinoma Hepatocelular/tratamiento farmacológico , Quimioembolización Terapéutica/métodos , Etanol/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Solventes/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Quimioembolización Terapéutica/efectos adversos , Colinesterasas/sangre , Cisplatino/administración & dosificación , Cisplatino/uso terapéutico , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Doxorrubicina/administración & dosificación , Doxorrubicina/uso terapéutico , Etanol/administración & dosificación , Etanol/efectos adversos , Femenino , Humanos , Inyecciones Intradérmicas , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Solventes/administración & dosificación , Solventes/efectos adversos , Análisis de Supervivencia , Resultado del Tratamiento , alfa-Fetoproteínas/análisisRESUMEN
OBJECTIVE: The purpose was to assess the volumes of the different hepatic territories and especially the drainage of the right paramedian sector in adult living donor liver transplantation (ALDLT). METHODS: CT was performed in 40 potential donors of whom 28 underwent partial living donation. Data sets of all potential donors were postprocessed using dedicated software for segmentation, volumetric analysis and visualization of liver territories. During an initial period, volumes and shapes of liver parts were calculated based on the individual portal venous perfusion areas. After partial hepatic congestion occurring in three grafts, drainage territories with special regard to MHV tributaries from the right paramedian sector, and the IRHV were calculated additionally. Results were visualized three-dimensionally and compared to the intraoperative findings. RESULTS: Calculated graft volumes based on hepatic venous drainage and graft weights correlated significantly (r = 0.86, P < 0.001). Mean virtual graft volume was 930 ml and drained as follows: RHV: 680 ml, IRHV: 170 ml (n = 11); segment 5 MHV tributaries: 100 ml (n = 16); segment 8 MHV tributaries: 110 ml (n = 20). When present, the mean aberrant venous drainage fraction of the right liver lobe was 28%. CONCLUSION: The evaluated protocol allowed a reliable calculation of the hepatic venous draining areas and led to a change in the hepatic venous reconstruction strategy at our institution.
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Hepatectomía , Venas Hepáticas/diagnóstico por imagen , Trasplante de Hígado , Donadores Vivos , Tomografía Computarizada por Rayos X , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Selección de Paciente , Interpretación de Imagen Radiográfica Asistida por ComputadorRESUMEN
Programmes for the eradication and control of infections with bovine viral diarrhea virus (BVDV) concentrate on the identification and elimination of persistently infected (PI) animals. The identification of these animals is mainly based on the detection of viral antigen using ELISA techniques. Protocols detecting viral nucleic acid using RT-PCR have been described recently. Due to high costs the German model recommends screening of animals of 9 up to 36 months of age. Screening of bulk milk samples using RT-PCR technology would allow a system independent of age. The aim of the present study was to test whether bulk milk samples (1433 including max. 50 animals each) collected in four counties of Lower Saxony are suitable for a complementary identification of PI animals via RT-PCR. Thirty-one bulk milk samples derived from 27 dairy herds were BVDV positive, corresponding to 2.3 % of the herds analysed in this study. Two samples first scored doubtful. Follow up tests revealed lactating PI animals in most cases (18). In other cases the epidemiological status of the herd, i.e. high sero-prevalence and/or presence of PI animals among non-lactating cattle, suggested a transient infection detected in the first bulk milk sample. These results demonstrate that monitoring of lactating cattle of any age using RT-PCR is a very sensitive, economically effective additional method for the identification of PI animals.
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Diarrea Mucosa Bovina Viral/diagnóstico , Virus de la Diarrea Viral Bovina/aislamiento & purificación , Leche/virología , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Animales , Diarrea Mucosa Bovina Viral/epidemiología , Bovinos , Virus de la Diarrea Viral Bovina/genética , Femenino , Alemania/epidemiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y EspecificidadRESUMEN
The aim of this study was to evaluate a software tool for non-invasive preoperative volumetric assessment of potential donors in living donated liver transplantation (LDLT). Biphasic helical CT was performed in 56 potential donors. Data sets were post-processed using a non-commercial software tool for segmentation, volumetric analysis and visualisation of liver segments. Semi-automatic definition of liver margins allowed the segmentation of parenchyma. Hepatic vessels were delineated using a region-growing algorithm with automatically determined thresholds. Volumes and shapes of liver segments were calculated automatically based on individual portal-venous branches. Results were visualised three-dimensionally and statistically compared with conventional volumetry and the intraoperative findings in 27 transplanted cases. Image processing was easy to perform within 23 min. Of the 56 potential donors, 27 were excluded from LDLT because of inappropriate liver parenchyma or vascular architecture. Two recipients were not transplanted due to poor clinical conditions. In the 27 transplanted cases, preoperatively visualised vessels were confirmed, and only one undetected accessory hepatic vein was revealed. Calculated graft volumes were 1110 +/- 180 ml for right lobes, 820 ml for the left lobe and 270 +/- 30 ml for segments II+III. The calculated volumes and intraoperatively measured graft volumes correlated significantly. No significant differences between the presented automatic volumetry and the conventional volumetry were observed. A novel image processing technique was evaluated which allows a semi-automatic volume calculation and 3D visualisation of the different liver segments.
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Imagenología Tridimensional , Trasplante de Hígado , Hígado/irrigación sanguínea , Hígado/diagnóstico por imagen , Donadores Vivos , Intensificación de Imagen Radiográfica , Tomografía Computarizada Espiral , Adulto , Aorta Abdominal/diagnóstico por imagen , Arteria Celíaca/diagnóstico por imagen , Femenino , Arteria Hepática/diagnóstico por imagen , Humanos , Masculino , Cómputos Matemáticos , Arteria Mesentérica Superior/diagnóstico por imagen , Persona de Mediana Edad , Cuidados Preoperatorios , Sensibilidad y Especificidad , Estadística como AsuntoRESUMEN
BACKGROUND: Thiamin pyrophosphokinase (TPK) catalyzes the transfer of a pyrophosphate group from ATP to vitamin B1 (thiamin) to form the coenzyme thiamin pyrophosphate (TPP). Thus, TPK is important for the formation of a coenzyme required for central metabolic functions. TPK has no sequence homologs in the PDB and functions by an unknown mechanism. The TPK structure has been determined as a significant step toward elucidating its catalytic action. RESULTS: The crystal structure of Saccharomyces cerevisiae TPK complexed with thiamin has been determined at 1.8 A resolution. TPK is a homodimer, and each subunit consists of two domains. One domain resembles a Rossman fold with four alpha helices on each side of a 6 strand parallel beta sheet. The other domain has one 4 strand and one 6 strand antiparallel beta sheet, which form a flattened sandwich structure containing a jelly-roll topology. The active site is located in a cleft at the dimer interface and is formed from residues from domains of both subunits. The TPK dimer contains two compound active sites at the subunit interface. CONCLUSIONS: The structure of TPK with one substrate bound identifies the location of the thiamin binding site and probable catalytic residues. The structure also suggests a likely binding site for ATP. These findings are further supported by TPK sequence homologies. Although possessing no significant sequence homology with other pyrophospokinases, thiamin pyrophosphokinase may operate by a mechanism of pyrophosphoryl transfer similar to those described for pyrophosphokinases functioning in nucleotide biosynthesis.
Asunto(s)
Saccharomyces cerevisiae/genética , Tiamina Pirofosfoquinasa/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Tiamina Pirofosfoquinasa/metabolismo , Tiamina/metabolismoRESUMEN
Thiamin pyrophosphate (TPP) is a coenzyme derived from vitamin B1 (thiamin). TPP synthesis in eukaryotes requires thiamin pyrophosphokinase (TPK), which catalyzes the transfer of a pyrophosphate group from ATP to thiamin. TPP is essential for central metabolic processes, including the formation of acetyl CoA from glucose and the Krebs cycle. Deficiencies in human thiamin metabolism result in beriberi and Wernicke encephalopathy. The crystal structure of mouse TPK was determined by multiwavelength anomalous diffraction at 2.4 A resolution, and the structure of TPK complexed with thiamin has been refined at 1.9 A resolution. The TPK polypeptide folds as an alpha/beta-domain and a beta-sandwich domain, which share a central ten-stranded mixed beta-sheet. TPK subunits associate as a dimer, and thiamin is bound in the dimer interface. Despite lacking apparent sequence homology with other proteins, the alpha/beta-domain resembles the Rossman fold and is similar to other kinase structures, including another pyrophosphokinase and a thiamin biosynthetic enzyme. Comparison of mouse and yeast TPK structures reveals differences that could be exploited in developing species-specific inhibitors of potential use as antimicrobial agents.
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Tiamina Pirofosfoquinasa/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Diseño de Fármacos , Ratones , Modelos Moleculares , Conformación Proteica , Subunidades de Proteína , Saccharomyces cerevisiae/enzimología , Especificidad de la Especie , Tiamina Pirofosfoquinasa/metabolismo , Tiamina/metabolismoRESUMEN
The mouse major urinary proteins are pheromone-binding proteins that function as carriers of volatile effectors of mouse physiology and behavior. Crystal structures of recombinant mouse major urinary protein-I (MUP-I) complexed with the synthetic pheromones, 2-sec-butyl-4,5-dihydrothiazole and 6-hydroxy-6-methyl-3-heptanone, have been determined at high resolution. The purification of MUP-I from mouse liver and a high-resolution structure of the natural isolate are also reported. These results show the binding of 6-hydroxy-6-methyl-3-heptanone to MUP-I, unambiguously define ligand orientations for two pheromones within the MUP-I binding site, and suggest how different chemical classes of pheromones can be accommodated within the MUP-I beta-barrel.
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Feromonas/metabolismo , Proteínas/química , Proteínas/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Hígado/química , Ratones , Modelos Moleculares , Feromonas/síntesis química , Feromonas/química , Unión Proteica , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by a deficiency of the enzyme involved in the last step of tyrosine degradation, fumarylacetoacetate hydrolase (FAH). Thus far, 34 mutations in the FAH gene have been reported in various HT1 patients. Site-directed mutagenesis of the FAH cDNA was used to investigate the effects of eight missense mutations found in HTI patients on the structure and activity of FAH. Mutated FAH proteins were expressed in Escherichia coli and in mammalian CV-1 cells. Mutations N16I, F62C, A134D, C193R, D233V, and W234G lead to enzymatically inactive FAH proteins. Two mutations (R341W, associated with the pseudo-deficiency phenotype, and Q279R) produced proteins with a level of activity comparable to the wild-type enzyme. The N16I, F62C, C193R, and W234G variants were enriched in an insoluble cellular fraction, suggesting that these amino acid substitutions interfere with the proper folding of the enzyme. Based on the tertiary structure of FAH, on circular dichroism data, and on solubility measurements, we propose that the studied missense mutations cause three types of structural effects on the enzyme: 1) gross structural perturbations, 2) limited conformational changes in the active site, and 3) conformational modifications with no significant effect on enzymatic activity.
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Hidrolasas/genética , Mutación Missense , Tirosinemias/genética , Animales , Secuencia de Bases , Línea Celular , Dicroismo Circular , Cartilla de ADN , ADN Complementario , Humanos , Hidrolasas/química , Hidrolasas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Tirosinemias/enzimologíaRESUMEN
Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to yield fumarate and acetoacetate as the final step of Phe and Tyr degradation. This unusual reaction is an essential human metabolic function, with loss of FAH activity causing the fatal metabolic disease hereditary tyrosinemia type I (HT1). An enzymatic mechanism involving a catalytic metal ion, a Glu/His catalytic dyad, and a charged oxyanion hole was previously proposed based on recently determined FAH crystal structures. Here we report the development and characterization of an FAH inhibitor, 4-(hydroxymethylphosphinoyl)-3-oxo-butanoic acid (HMPOBA), that competes with the physiological substrate with a K(i) of 85 microM. The crystal structure of FAH complexed with HMPOBA refined at 1.3-A resolution reveals the molecular basis for the competitive inhibition, supports the proposed formation of a tetrahedral alkoxy transition state intermediate during the FAH catalyzed reaction, and reveals a Mg(2+) bound in the enzyme's active site. The analysis of FAH structures corresponding to different catalytic states reveals significant active site side-chain motions that may also be related to catalytic function. Thus, these results advance the understanding of an essential catabolic reaction associated with a fatal metabolic disease and provide insight into the structure-based development of FAH inhibitors.
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Acetoacetatos/farmacología , Inhibidores Enzimáticos/farmacología , Hidrolasas/metabolismo , Compuestos Organofosforados/farmacología , Animales , Sitios de Unión , Calcio/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Hidrolasas/antagonistas & inhibidores , Hidrolasas/química , Magnesio/metabolismo , Ratones , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación ProteicaRESUMEN
The 2.5 A X-ray crystal structure of the nucleosome core particle presented here provides significant additions to the understanding of the nucleosome, the fundamental unit of chromatin structure. Extensions are made to the structure of the N-terminal histone tails and details are provided on hydration and ion binding. The structure is composed of twofold symmetric molecules, native chicken histone octamer cores and the DNA palindrome, which were expected to form a perfectly twofold symmetric nucleosome core particle. In fact, the result is asymmetric owing to the binding of the DNA to the protein surface and to the packing of the particles in the crystal lattice. An analysis is made of the asymmetries by comparisons both within the nucleosome core particle and to the structure of the histone octamer core of the nucleosome.
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ADN/química , Histonas/química , Nucleosomas/química , Secuencia de Aminoácidos , Animales , Pollos , Cristalografía por Rayos X , Proteínas de Unión al ADN , Histonas/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Conformación Proteica , Pliegue de Proteína , Ciclo del SustratoRESUMEN
Alkaptonuria (AKU), the prototypic inborn error of metabolism, was the first human disease to be interpreted as a Mendelian trait by Garrod and Bateson at the beginning of last century. AKU results from impaired function of homogentisate dioxygenase (HGO), an enzyme required for the catabolism of phenylalanine and tyrosine. With the novel 7 AKU and 22 fungal mutations reported here, a total of 84 mutations impairing this enzyme have been found in the HGO gene from humans and model organisms. Forty-three of these mutations result in single amino acid substitutions. This mutational information is analysed here in the context of the HGO structure and function using kinetic assays performed using purified AKU mutant enzymes and the crystal structure of human HGO. HGO is a topologically complex structure which assembles as a functional hexamer arranged as a dimer of trimers. We show how the intricate pattern of intra- and inter-subunit interactions and the extensive surfaces required for subunit folding and association of this oligomeric enzyme can be inactivated at multiple levels by single-residue substitutions. This explains, in part, the predominance of missense mutations (67%) in AKU.
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Alcaptonuria/genética , Dioxigenasas , Oxigenasas/genética , Alcaptonuria/metabolismo , Alcaptonuria/patología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Dominio Catalítico , Homogentisato 1,2-Dioxigenasa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oxigenasas/química , Oxigenasas/metabolismo , Conformación Proteica , Pliegue de Proteína , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
Homogentisate dioxygenase (HGO) cleaves the aromatic ring during the metabolic degradation of Phe and Tyr. HGO deficiency causes alkaptonuria (AKU), the first human disease shown to be inherited as a recessive Mendelian trait. Crystal structures of apo-HGO and HGO containing an iron ion have been determined at 1.9 and 2.3 A resolution, respectively. The HGO protomer, which contains a 280-residue N-terminal domain and a 140-residue C-terminal domain, associates as a hexamer arranged as a dimer of trimers. The active site iron ion is coordinated near the interface between subunits in the HGO trimer by a Glu and two His side chains. HGO represents a new structural class of dioxygenases. The largest group of AKU associated missense mutations affect residues located in regions of contact between subunits.
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Alcaptonuria/enzimología , Dioxigenasas , Oxigenasas/química , Alcaptonuria/genética , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Dimerización , Homogentisato 1,2-Dioxigenasa , Humanos , Hierro/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oxigenasas/deficiencia , Oxigenasas/genética , Oxigenasas/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Primary extraosseous osteosarcoma of the thyroid gland is a very rare tumor with a poor prognosis. Only few cases have been reported in the literature. Metastases of osteosarcoma in the thyroid gland were also found but are very seldom. A primary thyroid tumor with osteosarcomatous differentiation in a 82-year-old man is presented and the histopathological classification, biological features and possible mechanisms of the occurrence of such thyroid tumors are briefly discussed.
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Osteosarcoma/cirugía , Neoplasias de la Tiroides/cirugía , Anciano , Anciano de 80 o más Años , Estudios de Seguimiento , Humanos , Escisión del Ganglio Linfático , Masculino , Osteosarcoma/patología , Glándula Tiroides/patología , Neoplasias de la Tiroides/patología , TiroidectomíaRESUMEN
BACKGROUND: Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of tyrosine and phenylalanine catabolism, the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate, to yield fumarate and acetoacetate. FAH has no known sequence homologs and functions by an unknown mechanism. Carbon-carbon hydrolysis reactions are essential for the human metabolism of aromatic amino acids. FAH deficiency causes the fatal metabolic disease hereditary tyrosinemia type I. Carbon-carbon bond hydrolysis is also important in the microbial metabolism of aromatic compounds as part of the global carbon cycle. RESULTS: The FAH crystal structure has been determined by rapid, automated analysis of multiwavelength anomalous diffraction data. The FAH polypeptide folds into a 120-residue N-terminal domain and a 300-residue C-terminal domain. The C-terminal domain defines an unusual beta-strand topology and a novel 'mixed beta-sandwich roll' structure. The structure of FAH complexed with its physiological products was also determined. This structure reveals fumarate binding near the entrance to the active site and acetoacetate binding to an octahedrally coordinated calcium ion located in close proximity to a Glu-His dyad. CONCLUSIONS: FAH represents the first structure of a hydrolase that acts specifically on carbon-carbon bonds. FAH also defines a new class of metalloenzymes characterized by a unique alpha/beta fold. A mechanism involving a Glu-His-water catalytic triad is suggested based on structural observations, sequence conservation and mutational analysis. The histidine imidazole group is proposed to function as a general base. The Ca(2+) is proposed to function in binding substrate, activating the nucleophile and stabilizing a carbanion leaving group. An oxyanion hole formed from sidechains is proposed to stabilize a tetrahedral alkoxide transition state. The proton transferred to the carbanion leaving group is proposed to originate from a lysine sidechain. The results also reveal the molecular basis for mutations causing the hereditary tyrosinemia type 1.
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Carbono/química , Hidrolasas/química , Aminoácidos/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Dimerización , Histidina , Humanos , Hidrolasas/metabolismo , Hidrólisis , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , AguaRESUMEN
Additional examples of successful application of macromolecular crystal annealing are presented. A qualitative evaluation of variables related to the annealing process was conducted using a variety of macromolecular crystals to determine in which cases parameters may be varied and in which cases the original macromolecular crystal annealing protocol is preferred. A hypothesis is presented relating the solvent content of the crystal to the specific protocol necessary for the successful application of annealing.
Asunto(s)
Proteínas/química , Animales , Estudios de Evaluación como AsuntoRESUMEN
Although cryogenic data collection has become the method of choice for macromolecular crystallography, the flash-cooling step can dramatically increase the mosaicity of some crystals. Macromolecular crystal annealing significantly reduces the mosaicity of flash-cooled crystals without affecting molecular structure. The process, which cycles a flash-cooled crystal to ambient temperature and back to cryogenic temperature, is simple, quick and requires no special equipment. The annealing process has been applied to crystals of several different macromolecules grown from different precipitants and using a variety of cryoprotectants. The protocol for macromolecular crystal annealing also has been applied to restore diffraction from flash-cooled crystals that were mishandled during transfer to or from cryogenic storage. These results will be discussed in relation to crystal mosaicity and effects of radiation damage in flash-cooled crystals.
Asunto(s)
Cristalografía por Rayos X/métodos , Cristalización , Congelación , Histonas/química , Humanos , Muramidasa/química , Nucleosomas/químicaRESUMEN
A crystal structure of the serine protease, mouse glandular kallikrein 13 (mGK-13) has been determined at 2.6-A resolution. This enzyme, isolated from the mouse submandibular gland, is also known as prorenin-converting enzyme and cleaves submandibular gland Ren-2 prorenin to yield active renin. The mGK-13 structure is similar to other members of the mammalian serine protease family, having five conserved disulfide bonds and an active site located in the cleft between two beta-barrel domains. The mGK-13 structure reveals for the first time an ordered kallikrein loop conformation containing a short 3(10) helix. This loop is disordered in the related porcine pancreatic kallikrein and rat submandibular tonin structures. The kallikrein loop is in close spatial proximity to the active site and is also involved in a dimeric arrangement of mGK-13. The catalytic specificity of mGK-13 for Ren-2 prorenin was studied by modeling a prorenin-derived peptide into the active site of mGK-13. This model emphasizes two electronegative substrate specificity pockets on the mGK-13 surface, which could accommodate the dibasic P2 and P1 residues at the site of prorenin cleavage by mGK-13.