RESUMEN

B7H1 is consistently associated with inhibition of the immune system in many solid tumors. However, there is no report about its impact on differentiated thyroid carcinoma (DTC) presentation, aggressiveness, or evolution. Aiming to investigate the role of B7H1 in DTC and correlate this protein with other tumor-infiltrating immune cells, we studied 407 thyroid nodule tissue samples including 293 from DTC patients, all managed according to a same standard protocol. In addition, we obtained 5 normal and 114 benign thyroid lesions. Eighteen out of the 253 papillary thyroid carcinomas were paired with respective metastatic lymph node tissues. B7H1 (CD274) protein expression was assessed by immunohistochemistry and the gene expression was quantified by real-time PCR. Malignant tissues displayed a more intense B7H1 staining and higher mRNA levels than benign tissues (both P<0.0001). We observed a positive linear correlation between higher age at diagnosis and B7H1 mRNA levels (P=0.02896). Elevated levels of B7H1 protein were associated with the presence of CD4+, CD8+, CD20+, and FoxP3+ lymphocytes (all P<0.05); tumor-associated macrophages (P<0.0001); and the presence of myeloid-derived suppressor cells (P=0.03256). Stage II-IV patients presented higher B7H1 mRNA levels than stage I cases (P=0.03522). On the contrary, a decreased expression of B7H1 protein was observed in lymph node metastasis (P=0.0152). In conclusion, our data demonstrate that B7H1 expression is associated with features of aggressiveness, suggesting that this is an immune evasion mechanism of DTC cells.

Asunto(s)

Antígeno B7-H1/metabolismo , Carcinoma Papilar/metabolismo , Diferenciación Celular , Linfocitos Infiltrantes de Tumor/inmunología , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Adulto , Anciano , Antígeno B7-H1/genética , Western Blotting , Carcinoma Papilar/genética , Carcinoma Papilar/inmunología , Femenino , Humanos , Técnicas para Inmunoenzimas , Metástasis Linfática , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándula Tiroides/inmunología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/inmunología , Adulto Joven

RESUMEN

Iron (Fe) is an essential nutrient for plants, but it can generate oxidative stress at high concentrations. In this study, Coffea arabica L. cell suspension cultures were exposed to excess Fe (60 and 240 µM) to investigate changes in the gene expression of ferritin and antioxidant enzymes. Iron content accumulated during cell growth, and Western blot analysis showed an increase of ferritin in cells treated with Fe. The expression of two ferritin genes retrieved from the Brazilian coffee EST database was studied. CaFER1, but not CaFER2, transcripts were induced by Fe exposure. Phylogenetic analysis revealed that CaFER1 is not similar to CaFER2 or to any ferritin that has been characterised in detail. The increase in ferritin gene expression was accompanied by an increase in the activity of antioxidant enzymes. Superoxide dismutase, guaiacol peroxidase, catalase, and glutathione reductase activities increased in cells grown in the presence of excess Fe, especially at 60 µM, while the activity of glutathione S-transferase decreased. These data suggest that Fe induces oxidative stress in coffee cell suspension cultures and that ferritin participates in the antioxidant system to protect cells against oxidative damage. Thus, cellular Fe concentrations must be finely regulated to avoid cellular damage most likely caused by increased oxidative stress induced by Fe. However, transcriptional analyses indicate that ferritin genes are differentially controlled, as only CaFER1 expression was responsive to Fe treatment.

Asunto(s)

Antioxidantes/metabolismo , Café/metabolismo , Ferritinas/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Catalasa/genética , Catalasa/metabolismo , Café/efectos de los fármacos , Etiquetas de Secuencia Expresada , Ferritinas/clasificación , Ferritinas/genética , Glutatión Reductasa/genética , Glutatión Reductasa/metabolismo , Datos de Secuencia Molecular , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/genética , Peroxidasa/metabolismo , Filogenia , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo

RESUMEN

Human EFHC1 is a member of the EF-hand superfamily of Ca(2+)-binding proteins with three DM10 domains of unclear function. Point mutations in the EFHC1 gene are related to juvenile myoclonic epilepsy, a fairly common idiopathic generalized epilepsy. Here, we report the first structural and thermodynamic analyses of the EFHC1C-terminus (residues 403-640; named EFHC1C), comprising the last DM10 domain and the EF-hand motif. Circular dichroism spectroscopy revealed that the secondary structure of EFHC1C is composed by 34% of alpha-helices and 17% of beta-strands. Size exclusion chromatography and mass spectrometry showed that under oxidizing condition EFHC1C dimerizes through the formation of disulfide bond. Tandem mass spectrometry (MS/MS) analysis of peptides generated by trypsin digestion suggests that the Cys575 is involved in intermolecular S-S bond. In addition, DTNB assay showed that each reduced EFHC1C molecule has one accessible free thiol. Isothermal titration calorimetry (ITC) showed that while the interaction between Ca(2+) and EFHC1C is enthalpically driven (DeltaH=-58.6 to -67 kJ/mol and TDeltaS=-22.5 to -31 kJ/mol) the interaction between Mg(2+) and EFHC1C involves an entropic gain, and is approximately 5 times less enthalpically favorable (DeltaH=-11.7 to -14 kJ/mol and TDeltaS=21.9 to 19 kJ/mol) than for Ca(2+) binding. It was also found that under reducing condition Ca(2+) or Mg(2+) ions bind to EFHC1C in a 1/1 molar ratio, while under oxidizing condition this ratio is reduced, showing that EFHC1C dimerization blocks Ca(2+) and Mg(2+) binding.

Asunto(s)

Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Magnesio/metabolismo , Epilepsia Mioclónica Juvenil/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Western Blotting , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Cromatografía en Gel , Cartilla de ADN , Dimerización , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Epilepsia Mioclónica Juvenil/genética , Unión Proteica , Estructura Secundaria de Proteína

RESUMEN

Mutations in LGI1 were described in patients with autosomal dominant partial epilepsy with auditory features (ADPEAF), and recent clinical findings have implicated LGI1 in human brain development. However, the precise role of LGI1 in epileptogenesis remains largely unknown. The objective of this study was to determine the expression pattern of Lgi1 in mice brain during development and in adult animals. Real-time polymerase chain reaction (PCR) quantification and Western blot experiments showed a relative low expression during intrauterine stages, increasing until adulthood. In addition, we did not find significant differences between left and right hemispheres. The hippocampus presented higher levels of Lgi1 expression when compared to the neocortex and the cerebellum of adult animals; however, these results did not reach statistical significance. This study was the first to determine a specific profile of Lgi1 gene expression during central nervous system development, which suggests a possible inhibitory function in latter stages of development. In addition, we did not find differences in hemispheric expression that could explain the predominance of left-sided abnormalities in patients with ADPEAF.

Asunto(s)

Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas/genética , Proteínas/metabolismo , Envejecimiento/genética , Animales , Western Blotting , Encéfalo/metabolismo , Cerebelo/embriología , Cerebelo/crecimiento & desarrollo , Cerebelo/metabolismo , Lateralidad Funcional/fisiología , Hipocampo/embriología , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos BALB C , Neocórtex/embriología , Neocórtex/crecimiento & desarrollo , Neocórtex/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.

Asunto(s)

ADN Protozoario/química , ADN Protozoario/genética , Evolución Molecular , Telómero , Trypanosomatina/química , Trypanosomatina/genética , Animales , Especificidad de la Especie , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genética

RESUMEN

The Leishmania amazonensis telomerase gene was cloned by a polymerase chain reaction-based strategy using primers designed from a Leishmania major sequence that shared similarities with conserved telomerase motifs. The genes from three other species were cloned for comparative purposes. A ClustalW multiple-sequence alignment demonstrated that the Leishmania telomerases show greater homology with each other than with the proteins of other kinetoplastids and eukaryotes. Characterization experiments indicated that the putative Leishmania telomerase gene was probably in single copy and located in the largest chromosomes. A single messenger ribonucleic acid transcript was found in promastigotes. Phylogenetic analysis suggested that Leishmania telomerase might represent a liaison between the oldest and the newest branches of telomerases.

Asunto(s)

Clonación Molecular , Leishmania mexicana/enzimología , Telomerasa/genética , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Cromosomas/genética , Cartilla de ADN/genética , ADN Protozoario/química , ADN Protozoario/genética , Evolución Molecular , Leishmania major/genética , Leishmania mexicana/genética , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , ARN Mensajero/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Telomerasa/química

RESUMEN

The mtDNA control region (CR) and flanking genes of the blowflies Chrysomya albiceps, Chrysomya megacephala and Chrysomya chloropyga (Calliphoridae) were characterized. The most unusual feature found was the presence of duplicated tRNA sequences corresponding to trnI and a portion of trnQ. The partially duplicated trnQ was very likely a pseudogene since most of the sequence of the typical insect trnQ gene was missing. In contrast, the trnI gene had a conserved primary sequence following the duplication event and may represent a functional copy. These results demonstrate the plasticity of the mtDNA molecule in Chrysomya, especially for tRNA genes and the adjacent control region sequences.

Asunto(s)

ADN Mitocondrial/genética , Dípteros/genética , Genes Duplicados/genética , ARN de Transferencia/genética , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada/genética , ADN/química , ADN/genética , ADN Mitocondrial/química , Datos de Secuencia Molecular , Seudogenes/genética , ARN de Transferencia de Glutamina/genética , ARN de Transferencia de Isoleucina/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico

RESUMEN

The chromosomal ends of Leishmania (Leishmania) amazonensis contain conserved 5'-TTAGGG-3' telomeric repeats. Protein complexes that associate in vitro with these DNA sequences, Leishmania amazonensis G-strand telomeric protein (LaGT1-3), were identified and characterized by electrophoretic mobility shift assays and UV cross-linking using protein fractions purified from S100 and nuclear extracts. The three complexes did not form (a) with double-stranded DNA and the C-rich telomeric strand, (b) in competition assays using specific telomeric DNA oligonucleotides, or (c) after pretreatment with proteinase K. LaGT1 was the most specific and did not bind a Tetrahymena telomeric sequence. All three LaGTs associated with an RNA sequence cognate to the telomeric G-rich strand and a complex similar to LaGT1 is formed with a double-stranded DNA bearing a 3' G-overhang tail. The protein components of LaGT2 and LaGT3 were purified by affinity chromatography and identified, after renaturation, as approximately 35 and approximately 52 kDa bands, respectively. The

Asunto(s)

Leishmania/genética , Proteínas Protozoarias/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas de Unión a Telómeros/metabolismo , Telómero/genética , Animales , Composición de Base , Secuencia de Bases , Fraccionamiento Celular , ADN Protozoario/genética , ADN Protozoario/aislamiento & purificación , ADN Protozoario/metabolismo , Humanos , Leishmania/fisiología , Sustancias Macromoleculares , Espectrometría de Masas , Mapeo Peptídico , Proteínas Protozoarias/aislamiento & purificación , Sales (Química)/química , Proteínas de Unión a Telómeros/aislamiento & purificación