RESUMEN
BACKGROUND: To review and discuss important topics regarding periodontal treatment pre- and post-radiotherapy for head and neck cancer in human patients; to discuss the references for adequate techniques, the appropriate moment for tooth extractions and periodontal management; and to discuss the prevention of osteoradionecrosis. MATERIAL AND METHODS: Thirty-nine studies including original studies, randomized clinical trials (RCTs) and reviews were searched in online databases MEDLINE (PubMed) and the Cochrane library. No year of publication restriction was applied. RESULTS: Language was restricted to English, and the following Medical Subject Heading terms were used: radiotherapy, radiation therapy and periodontal treatment. Studies regarding periodontal treatment and tooth extraction that involved clinical management of irradiated patients were selected. CONCLUSIONS: The treatment of periodontal diseases before radiotherapy is mainly required to avoid future dental extraction and to reduce the development of osteoradionecrosis. Periodontal treatment in irradiated patients mostly includes scaling and root planing, extraction of condemned teeth and topical and systemic antimicrobial therapy. Tooth removal should be planned at least 14 days before the first day of radiation treatment. Particular care and mouthwashes should be taken during and after radiation. CLINICAL SIGNIFICANCE: The management of irradiated patients represents a challenge for health professionals, including dentists. It is important to establish recommendations for clinicians concerning dental and periodontal management in irradiated patients before, during and after treatment.
Asunto(s)
Neoplasias de Cabeza y Cuello/radioterapia , Enfermedades Periodontales/terapia , Atención Odontológica , Humanos , Osteorradionecrosis/prevención & controlRESUMEN
Irrigation with treated wastewater (TWW) is a vital alternative for arid and semi-arid lands but it poses pollution-risk to soil, vegetation and groundwater. Therefore, in the present study, in vitro bioassays were used to evaluate the adverse effects of TWW and irrigated-soil extract sample, on mammalian cells, with respect to heavy metal--Ni, Cd, Pb, Fe, Al-content. The heat shock protein (HSP) 47, E-screen, and transepithelial electrical resistance (TEER) assays served to investigate the stress response of treated-HSP47-transfected Chinese hamster ovary (CHO) cells, the estrogenic activity of the samples in MCF-7 breast cancer cells, and the barrier function (BF) of Caco-2 cells. Furthermore, proteomics analyses were performed to shed light on involved mechanisms and to establish pollution biomarkers. Results showed that the TWW elicited a stress response on HSP cells from 0.1% concentration while soil extract samples exhibited a stress at 1%. TWW induced an estrogenic activity at 10%; up-regulating cell proliferation and tumor-related proteins. Soil extract triggered the enhanced expression of HSP70 family proteins as survival mechanisms against their cytotoxicity toward MCF-7 cells. Moreover, depending on the concentration, 1% of soil extract from 20 cm depth (T20) resulted in a disruption of BF in Caco-2 cells involving cell metabolism, protein synthesis and tumor marker proteins, whereas, 5% of T20 induced the expression of BF-related proteins associated to heat shock, oxidative stress, cell proliferation and glycolytic metabolic pathway. These biological techniques were found to be extremely useful to evaluate the impact of wastewater reuse and to establish specific biomarkers that are common proteins for humans, other mammals and plants. Future studies should focus on exposure quantifications.
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Biomarcadores/análisis , Monitoreo del Ambiente , Regulación de la Expresión Génica/efectos de los fármacos , Metales Pesados/toxicidad , Contaminantes del Suelo/toxicidad , Suelo/química , Aguas Residuales/toxicidad , Animales , Células CACO-2 , Proliferación Celular/efectos de los fármacos , Cricetinae , Cricetulus , Impedancia Eléctrica , Perfilación de la Expresión Génica , Humanos , Metales Pesados/análisis , Plantas/efectos de los fármacos , Plantas/metabolismo , Contaminantes del Suelo/análisis , Aguas Residuales/químicaRESUMEN
The simple method of evaluating highly marbled beef was examined by image analysis. The images of the cross section at the 6 to 7th rib were obtained from 82 carcasses of Wagyu cattle. By using an overall trace method, the surrounding edges of the longissimus thoracis and three muscles were traced automatically and manually with image analysis. In a spot method, 3 to 5 locations (2.5 or 3.0 cm in diameter) for each muscle were rapidly selected with no manual trace. The images were flattened, binarized, and the ratio of fat area to muscle area was determined. The correlation coefficients for marbling between different muscles, and between the overall trace and the spot methods were 0.55 to 0.81 between different muscles and 0.89 to 0.97, respectively. These results suggested that the simple spot method is speedy and almost as useful as the overall trace method as a measuring technique for beef marbling in loin muscles, especially for highly marbled beef.
RESUMEN
The present study aimed to verify the changes in the expression levels of 13 candidate genes associated with chemotherapy resistance and to construct a scoring system to predict resistance to these drugs. The expression levels of the 13 candidate genes were compared between 20 dogs with lymphoma that were sensitive to drugs used in CHOP-based protocol and 16 dogs with lymphoma that were resistant to these drugs. The expression levels of six genes; ASNS, CCR3, CALCA, FCER1A, LOC448801, and EDNRB were significantly different between the two groups. A scoring system to predict resistance to cyclophosphamide, doxorubicin and vincristine, which are used in CHOP-based protocol, was constructed based on expression levels of the six genes in these 36 dogs using logistic regression models. After internal validation, sensitivity and specificity of the scoring system were 0.759 and 0.853, respectively. External validation was conducted in another cohort of 33 dogs with lymphoma, and sensitivity and specificity of the scoring system were 0.800 and 0.696, respectively. In conclusion, this study identified six genes associated with resistance to drugs used in CHOP-based protocol in canine lymphoma and proposed a novel scoring system to predict resistance to these drugs. This system might be beneficial in selecting the most appropriate chemotherapy protocol for individual dogs with lymphoma.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Enfermedades de los Perros/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Linfoma/veterinaria , Transcriptoma , Animales , Estudios de Cohortes , Ciclofosfamida/uso terapéutico , Perros , Doxorrubicina/uso terapéutico , Femenino , Linfoma/tratamiento farmacológico , Masculino , Prednisona/uso terapéutico , Proyectos de Investigación , Vincristina/uso terapéuticoRESUMEN
The surface morphology of a diarylethene single crystal [1,2-bis(2,4-dimethyl-5-phenyl-3-thienyl)perfluorocyclopentene] determined by atomic force microscopy changed reversibly upon photoirradiation. The crystal underwent a thermally irreversible but photochemically reversible color change (colorless to blue) upon alternate irradiation with ultraviolet (wavelength lambda = 366 nm) and visible (lambda > 500 nm) light that drove reversible photocyclization reactions. Upon irradiation with 366-nm light, new steps appeared on the (100) single-crystalline surface that disappeared upon irradiation with visible light (lambda > 500 nm). The step height, about 1 nm, corresponds to one molecular layer. Irradiation with 366-nm light formed valleys on the (010) surface that also disappeared by bleaching upon irradiation with visible light (lambda > 500 nm). The surface morphological changes can be explained by the molecular structural changes of diarylethenes regularly packed in the single crystal. These crystals could potentially be used as photodriven nanometer-scale actuators.
RESUMEN
PSD-95 is a component of postsynaptic densities in central synapses. It contains three PDZ domains that localize N-methyl-D-aspartate receptor subunit 2 (NMDA2 receptor) and K+ channels to synapses. In mouse forebrain, PSD-95 bound to the cytoplasmic COOH-termini of neuroligins, which are neuronal cell adhesion molecules that interact with beta-neurexins and form intercellular junctions. Neuroligins bind to the third PDZ domain of PSD-95, whereas NMDA2 receptors and K+ channels interact with the first and second PDZ domains. Thus different PDZ domains of PSD-95 are specialized for distinct functions. PSD-95 may recruit ion channels and neurotransmitter receptors to intercellular junctions formed between neurons by neuroligins and beta-neurexins.
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Proteínas Quinasas Dependientes de Calcio-Calmodulina , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Prosencéfalo/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Moléculas de Adhesión Celular Neuronal , Línea Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Homólogo 4 de la Proteína Discs Large , Guanilato-Quinasas , Uniones Intercelulares/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/química , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Nucleósido-Fosfato Quinasa/metabolismo , Canales de Potasio/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Supresoras de TumorRESUMEN
This study was designed to investigate whether different vascular endothelial growth factor (VEGF) genotypes are associated with ascites formation in cirrhotic patients. Seventy cirrhotic patients were included in the study: 25 cirrhotic patients with ascites and 45 cirrhotic patients without ascites. Patient characteristics were investigated and compared between the two groups. With regard to VEGF genotype, 42 patients were C/C and 28 patients were T/T or C/T. The genotypes T/T or C/T were observed in 23 cases (51%) among the non-ascites group, but in only five cases (20%) among the ascites group. Serum levels of albumin and creatinine, and the VEGF genotypes were significantly different between the two groups. Multiple regression analysis showed that serum levels of creatinine and the VEGF genotypes were significantly correlated with ascites formation. Thus, it can be concluded that VEGF genotyping might be a valuable susceptibility marker for ascites formation in cirrhotic patients.
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Ascitis/complicaciones , Ascitis/genética , Predisposición Genética a la Enfermedad , Cirrosis Hepática/complicaciones , Cirrosis Hepática/genética , Factor A de Crecimiento Endotelial Vascular/genética , Ascitis/sangre , Femenino , Humanos , Cirrosis Hepática/sangre , Masculino , Persona de Mediana Edad , Análisis de Regresión , Factor A de Crecimiento Endotelial Vascular/sangreRESUMEN
The fat quality is an important aspect, especially for Wagyu beef. A handheld fiber-optic near-infrared spectrometer for on-site evaluation of beef fat quality was developed, and the interactance spectra of the intermuscular fat from 833 Wagyu carcasses at 12 markets were measured. The calibration model was transferred to five slave instruments using twenty-six block samples. The performance of one slave instrument was verified at five meat markets (n=360). The coefficients of determination of the slave instrument for monounsaturated, oleic, and saturated fatty acid compositions determined by gas chromatography and near-infrared measurements were 0.69, 0.64, and 0.67, respectively. The standard error of prediction for the slave instrument was approximately 2%. The fiber-optic near-infrared spectrometers were highly accurate in the fat quality evaluation of Wagyu carcasses based on monounsaturated, oleic, and saturated fatty acid composition with easy calibration model transfer.
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Tejido Adiposo/química , Bovinos , Ácidos Grasos/análisis , Carne Roja/análisis , Espectroscopía Infrarroja Corta/métodos , Animales , Cromatografía de Gases , Femenino , Masculino , Carne Roja/normasRESUMEN
The effects of a humidity-stabilizing sheet containing glycerol, on the color and K value of beef stored at cold temperatures were investigated in this study. A drip-absorbing sheet seems to be effective for the preservation of meat quality, while a humidity-stabilizing sheet containing glycerol prevents the increase of metmyoglobin in cold stored beef. Maintaining samples wrapped in humidity-stabilizing sheets containing glycerol at low temperature for 7days was a functional method for conserving the concentration of inosine monophosphate. Beef samples wrapped in sheets containing glycerol had lower K values than samples wrapped in sheets not containing glycerol. When the surface of the meat starts to desiccate, the humidity-stabilizing sheet releases moisture that has been absorbed from the beef in the early stages of storage. Thus, glycerol could potentially play a role as a humidity-stabilizing controller and color preservative. This research suggests that a humidity-stabilizing sheet containing glycerol is a useful moisture controller and prevents deterioration of meat quality, discoloration, and desiccation.
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The imprinted region on mouse distal chromosome 12 covers about 1 Mb and contains at least three paternally expressed genes (Pegs: Peg9/Dlk1, Peg11/Rtl1, and Dio3) and four maternally expressed genes (Megs: Meg3/Gtl2, antiPeg11/antiRlt1, Meg8/Rian, and Meg9/Mirg). Gtl2(lacZ) (Gene trap locus 2) mice have a transgene (TG) insertion 2.3 kb upstream from the Meg3/Gtl2 promoter and show about 40% growth retardation when the TG-inserted allele is paternally derived. Quantitative RT-PCR experiments showed that the expression levels of Pegs in this region were reduced below 50%. These results are consistent with the observed phenotype in Gtl2lacZ mice, because at least two Pegs(Peg9/Dlk1 and Dio3) have growth-promoting effects. The aberrant induction of Megs from silent paternal alleles was also observed in association with changes in the DNA methylation level of a differentially methylated region (DMR) located around Meg3/Gtl2 exon 1. Interestingly, a 60 approximately 80% reduction in all Megs was observed when the TG was maternally derived, although the pups showed no apparent growth or morphological abnormalities. Therefore, the paternal or maternal inheritance of the TG results in the down-regulation in cis of either Pegs or Megs, respectively, suggesting that the TG insertion influences the mechanism regulating the entire imprinted region.
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Impresión Genómica , Proteínas/genética , Animales , Secuencia de Bases , Aberraciones Cromosómicas , Mapeo Cromosómico , Cartilla de ADN , Femenino , Regulación de la Expresión Génica , Trastornos del Crecimiento/genética , Masculino , Ratones , Ratones Transgénicos , Mutagénesis Insercional , ARN Largo no Codificante , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , beta-Galactosidasa/genéticaRESUMEN
Differential scanning calorimetry (DSC) was used for the direct analysis of melting properties in porcine subcutaneous, intermuscular, and kidney leaf adipose tissue by heating at a constant ratio of +5°C/min from 4 to 90°C. Melting curves for adipose tissues as well as fat extracted from the associated tissues by chloroform-methanol were generated using DSC. Major peaks in DSC curves were similar among types of adipose tissue but the temperatures of the melting peak and conclusion point differed among types of adipose tissues. From the visual appearance of fat samples it appeared that the major DSC peak corresponded to phase transition of the fat. The direct DSC analysis of porcine adipose tissues may be useful to determine melting properties.
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PURPOSE: Sirolimus (SRL) is used to treat pulmonary lymphangioleiomyomatosis (P-LAM). There is limited evidence that SRL has systemic efficacy for the patients with extrapulmonary lymphangioleiomyomatosis (E-LAM) remaining after lung transplantation (LT) for P-LAM. This report examines the efficacy of SRL treatment for the patient with E-LAM remaining after an LT for P-LAM. CASE SUMMARY: The course of the patient's recovery from an LT for P-LAM was complicated by lymphedema in the left femoral region that was caused by two E-LAM lesions remaining in the left pelvic cavity and in the retroperitoneal area. After the LT was performed, the patient started SRL treatment to reduce the E-LAM lesions. The daily SRL dose, selected based on the standard SRL dose for P-LAM, was initiated at 1 mg/d and was maintained at 2 mg/d. The remaining E-LAM lesions and lymphedema in the left femoral region improved in approximately 9 months after the LT with the administration of both SRL and the standard immunosuppressive therapy used by Okayama University Hospital, including tacrolimus, mycophenolate mofetil, and prednisolone. The SRL and tacrolimus trough concentrations in whole blood were maintained within the therapeutic window for the next 1.5 years after initiation of SRL treatment. The patient experienced no severe adverse events that required discontinuation of the SRL treatment during this time. CONCLUSION: The patients with remaining E-LAM lesions may receive SRL treatment to improve the quality of life after LT for P-LAM as effective therapy in cases where the patient's recovery is complicated by E-LAM lesions.
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Inmunosupresores/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Trasplante de Pulmón , Linfangioleiomiomatosis/tratamiento farmacológico , Sirolimus/uso terapéutico , Abdomen/patología , Adulto , Femenino , Humanos , Terapia de Inmunosupresión/métodos , Neoplasias Pulmonares/patología , Linfangioleiomiomatosis/patología , Linfangioleiomiomatosis/cirugía , Linfedema/tratamiento farmacológico , Linfedema/etiología , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/uso terapéutico , Neoplasia Residual , Pelvis/patología , Prednisolona/uso terapéutico , Calidad de Vida , Tacrolimus/uso terapéuticoRESUMEN
In Caenorhabditis elegans, the vulval induction is mediated by the let-23 receptor tyrosine kinase (RTK)/ Ras signaling pathway. The precise localization of the let-23 RTK at the epithelial junctions is essential for the vulval induction, and requires three genes including lin-2, -7, and -10. The mammalian homologue of lin-2 has been identified as a protein interacting with a neuronal adhesion molecule, neurexin, and named CASK. CASK has recently been reported to interact with syndecans and an actin-binding protein, band 4.1, at epithelial and synaptic junctions, and to play central roles in the formation of cell-cell junctions. The product of C. elegans lin-7 directly interacts with let-23 RTK and localize it at epithelial junctions. Here, we report three rat homologues of lin-7 ubiquitously expressed in various tissues. These homologues are accumulated at the junctional complex region in cultured Madin-Darby canine kidney cells, and are also localized at the synaptic junctions in neurons. The mammalian homologues of lin-7 may be implicated in the formation of cell-cell junctions.
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Proteínas de Caenorhabditis elegans/genética , Moléculas de Adhesión Celular Neuronal/genética , Proteínas del Helminto/genética , Uniones Intercelulares/genética , Proteínas de la Membrana/genética , Neuronas/metabolismo , Secuencia de Aminoácidos , Animales , Células COS/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular , Perros , Proteínas del Helminto/metabolismo , Uniones Intercelulares/metabolismo , Riñón/citología , Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Neuronas/citología , Conejos , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/genética , Sinapsis/metabolismo , TransfecciónRESUMEN
The interaction of adenylyl-3',5'-cytidine (ApC) with ribonuclease-A (RNAase-A) was studied by steady-state kinetics and ultraviolet difference spectroscopy. X-ray difference Fourier synthesis at 4 A resolution was also used to study the binding of ApC to RNAase-S. Unlike well-studied compounds like uridylyl-3',5'-adenosine, ApC binds in an unique way: (1) the cytidine moiety is bound to the B1 and R1 sites, (2) the adenosine moiety protrudes to the solution and is not fixed spatially and (3) the phosphate group is bound to the non-specific site (the "Po site") previously postulated (Sawada, F. and Irie, M. (1969) J. Biochem. (Tokyo) 66, 415--418) as the binding site for the 5'-phosphate of uridine 2',5'-diphosphate or uridine 3',5'-diphosphate. This conclusion is consistent with that derived for adenylyl-3',5' -4-thiouridine based on CD difference spectroscopy (White, M.D., Keren-Zur, M. and Lapidot, Y. (1977) Nucleic Acid Res. 4, 843--851). The "Po site" is most likely the epsilon-amino group of Lys 66.
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Nucleótidos de Adenina/metabolismo , Ribonucleasas/metabolismo , Sitios de Unión , Citidina/metabolismo , Cinética , Nucleótidos/metabolismo , Nucleótidos/farmacología , Unión Proteica , Ribonucleasas/antagonistas & inhibidores , Espectrofotometría Ultravioleta , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
Kinetic constants for the transesterification of eight dinucleoside phosphates CpX and UpX by bovine and turtle pancreatic ribonuclease were determined. Both ribonucleases have a preference for purine nucleotides at the position X. However, bovine ribonuclease, like other mammalian ribonucleases, prefers 6-amino bases at this site, while turtle ribonuclease prefers 6-keto bases. This difference in specificity at the B2 site may be explained by the substitution of glutamic acid at position 111 by valine in turtle ribonuclease. These results have been confirmed by inhibition studies with the four nucleoside triphosphates. Inhibition studies with pT and pTp showed that a cationic binding group (P0) for the 5'-phosphate of the pyrimidine nucleotides bound at the primary B1 site is present in turtle ribonuclease, although lysine at position 66 in bovine ribonuclease is absent in turtle ribonuclease. However, the side chain of lysine 122 in turtle ribonuclease is probably located in the correct position to take over the role as cationic P0 site.
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Páncreas/enzimología , Ribonucleasas/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , Esterificación , Cinética , Modelos Moleculares , Ribonucleasa Pancreática/metabolismo , Especificidad de la Especie , Especificidad por Sustrato , TortugasRESUMEN
It is surprising that only relatively recently has attention been directed to the characterization of the properties of acid ribonucleases (RNases), leading to some understanding of their biochemistry and their functional roles. The present review summarizes current progress in this field under the following general topics: (1) the wide distribution of acid RNases in organisms from viruses to animals; (2) recent findings concerning their primary and three-dimensional structure; (3) the structure-function relationship of acid RNases, with a fungal RNase from Rhizopus niveus as a model enzyme; (4) the unique localization of acid RNases in the periplasm of bacteria, vacuoles in plants, and lysosomes of animals and protozoa; and (5) the diversity of physiological roles, depending on the organism, such as self-incompatibility factors and defense proteins in some plants, the surface protein of an animal virus related to pathogenicity, and possible relationship to human cancer.
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Ribonucleasas/química , Ribonucleasas/fisiología , Secuencia de Aminoácidos , Animales , Bacterias/enzimología , Hongos/enzimología , Humanos , Conformación Molecular , Datos de Secuencia Molecular , Plantas/enzimología , Relación Estructura-Actividad , Virus/enzimologíaRESUMEN
The crystals of a complex between ribonuclease Ms, the extracellular ribonuclease from Aspergillus saitoi, and 3'-guanylic acid were obtained from 2-methyl-2,4-pentanediol solution by vapor diffusion technique in the hanging drop mode. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with dimensions a = 47.0 A, b = 62.8 A, c = 37.9 A. The crystals diffract strongly up to at least 2.0 A resolution.
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Aspergillus , Endorribonucleasas , Nucleótidos de Guanina , Guanosina Monofosfato , Cristalización , Sustancias Macromoleculares , Difracción de Rayos XRESUMEN
Crystals of ribonuclease Rh, a new class of microbial ribonuclease from Rhizopus niveus, were obtained from polyethylene glycol 8000 solution by a vapour diffusion technique in the hanging drop mode. Two crystal forms, type I and type II, were obtained from the same droplet solution. Both forms belong to the space group P2(1)2(1)2(1), but their cell dimensions are markedly different: a = 68.3 A, b = 73.0 A, c = 50.0 A for type I and a = 67.5 A, b = 72.3 A, c = 44.2 A for type II. The type I crystals diffract beyond 2.0 A resolution and are suitable for X-ray structure analysis at high resolution.
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Rhizopus/enzimología , Ribonucleasas , Cristalización , Difracción de Rayos XRESUMEN
Ribonuclease LE (RNase LE) from cultured tomato (Lycopersicon esculentum) cells is a member of the RNase T(2) family showing broad base specificity. The crystal structure of RNase LE has been determined at 1.65 A resolution. The structure consists of seven alpha-helices and seven beta-strands, belonging to an alpha+beta type structure. Comparison of the structure of RNase LE with that of RNase Rh, a microbial RNase belonging to the RNase T(2) family, reveals that while the overall folding topologies are similar to each other, major insertions and deletions are found at the N-terminal regions. The structural comparison, an amino acid sequence alignment of the RNase T(2) enzymes, and comparison of the disulfide-bonding pattern of these enzymes show that the structure of RNase LE shown here is the basic framework of the animal/plant subfamily of RNase T(2) enzymes (including a self-incompatibility protein called S-RNase), and the structure of RNase Rh is that of the fungal subfamily of RNase T(2) enzymes (including RNase T(2)). Subsequently, we superposed the active-site of the RNase LE with that of RNase Rh and found that (1) His39, Trp42, His92, Glu93, Lys96, and His97 of RNase LE coincided exactly with His46, Trp49, His104, Glu105, Lys108, and His109, respectively, of RNase Rh, and (2) two conserved water molecules were found at the putative P(1) sites of both enzymes. These facts suggest that plant RNase LE has a very similar hydrolysis mechanism to that of fungal RNase Rh, and almost all the RNase T(2) enzymes widely distributed in various species share a common catalytic mechanism. A cluster of hydrophobic residues was found on the active-site face of the RNase LE molecule and two large hydrophobic pockets exist. These hydrophobic pockets appear to be base binding sites mainly by hydrophobic interactions and are responsible for the base non-specificity of RNase LE.
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Endorribonucleasas/química , Proteínas de Plantas/química , Solanum lycopersicum/enzimología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Secuencia Conservada , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/clasificación , Proteínas de Unión al ADN/metabolismo , Disulfuros/metabolismo , Endorribonucleasas/clasificación , Endorribonucleasas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/clasificación , Proteínas de Plantas/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Relación Estructura-Actividad , Especificidad por Sustrato , Agua/metabolismoRESUMEN
The three-dimensional structure of ribonuclease Rh (RNase Rh), a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.0 A resolution. The overall structure of RNase Rh is completely different from those of other previously studied RNases, such as RNase A from bovine pancreas and RNase T1 from Aspergillus oryzae. In the structure of RNase Rh, two histidine residues (His46 and His109) and one glutamic acid residue (Glu105), which were predicted to be critical to the activity from the chemical modification and mutagenesis experiments, are found to be located close together, constructing the active site. The indole ring of Trp49 plays an important role in preserving the active site structure by its stacking interactions with the imidazole ring of His 109, and by hydrogen bonding with the carboxyl group of Glu105. There exists a hydrophobic pocket around the active site, which contains the aromatic side-chain of Trp49 and Tyr57. The results of mutagenesis studies suggest that this pocket is the base binding site of the substrate.