RESUMO
The ability of lactobacilli isolated from feedlot cattle environment to differentially modulate the innate immune response triggered by Toll-like receptors (TLRs) activation in bovine intestinal epithelial (BIE) cells was evaluated. BIE cells were stimulated with Lactobacillus mucosae CRL2069, Lactobacillus acidophilus CRL2074, Lactobacillus fermentum CRL2085 or Lactobacillus rhamnosus CRL2084 and challenged with heat-stable pathogen associated molecular patterns (PAMPs) from enterotoxigenic Escherichia coli (ETEC) to induce the activation of TLR4 or with polyinosinic:polycytidylic acid (poly(I:C)) to activate TLR3. Type I interferons, cytokines, chemokines and negative regulators of TLR signalling were studied by RT-PCR. L. mucosae CRL2069 significantly reduced the expression of interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 in BIE cells in the context of TLR3 activation. L. mucosae CRL2069 also reduced the expression of tumour necrosis factor-α, IL-ß, MCP-1, and IL-8 in heat-stable ETEC PAMPs-challenged BIE cells. In addition, reduced expressions of IL-6, MCP-1, and IL-8 were found in BIE cells stimulated with L. rhamnosus CRL2084, although its effect was significantly lower than that observed for the CRL2069 strain. The reduced levels of pro-inflammatory factors in BIE cells induced by the CRL2069 and CRL2085 strains was related to their ability of increasing the expression of TLR negative regulators. L. mucosae CRL2069 significantly improved the expression of A20-binding inhibitor of NFκ-B activation 3 (ABIN-3), interleukin-1 receptor-associated kinase M (IRAK-M) and mitogen-activated protein kinase 1 (MKP-1) while L. rhamnosus CRL2084 augmented ABIN-3 expression in BIE cells. The results of this work suggest that among the studied strains, L. mucosae CRL2069 was able to regulate TLR3-mediated innate immune response and showed a remarkable capacity to modulate TLR4-mediated inflammation in BIE cells. The CRL2069 strain induce the up-regulation of three TLR negative regulators that would influence nuclear factor kB and mitogen-activated protein kinases signalling pathways while reducing the expression of pro-inflammatory cytokines and chemokines. Therefore, L. mucosae CRL2069 is an interesting immunobiotic candidate for the protection of the bovine host against TLR-mediated intestinal inflammatory damage.
Assuntos
Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Imunidade Inata , Intestinos/imunologia , Lactobacillales/imunologia , Probióticos/administração & dosagem , Receptores Toll-Like/imunologia , Animais , Bovinos , Linhagem Celular , Quimiocinas/genética , Quimiocinas/imunologia , Citocinas/genética , Citocinas/imunologia , Inflamação , Mucosa Intestinal/imunologia , Intestinos/citologia , Lactobacillales/isolamento & purificação , Lactobacillus/imunologia , Lactobacillus acidophilus/imunologia , Lacticaseibacillus rhamnosus/imunologia , Transdução de Sinais , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptores Toll-Like/genéticaRESUMO
The medial prefrontal areas 32, 24, 14, and 25 (mPFC) form part of the limbic memory system, but little is known about their functional specialization in humans. To add anatomical precision to structural and functional magnetic resonance imaging (MRI) data, we aimed to identify these mPFC subareas in histological preparations of human brain tissue, determine sulci most consistently related with mPFC areal boundaries, and use these sulci to delineate mPFC areas in MRIs. To achieve this, we obtained three-dimensional MRI data from 11 ex vivo hemispheres and processed them for cyto- and myelo-architectonic analysis. The architectonic boundaries of mPFC areas were identified in histology and cortical surface length and volumes were measured. Unfolded maps of histologically determined boundaries were generated to identify the association of mPFC areal boundaries with sulci across cases. This analysis showed that cingulate and superior rostral were the sulci most consistently related to mPFC areal boundaries. Based on presence/absence and anastomosis between such sulci, 6 sulci patterns in the 11 hemispheres were found. A further analysis of 102 hemispheres of in vivo MRI scans (N = 51 males, mean ± SD 24.1 ± 3.1 years of age) showed similar sulci patterns, which allowed us to delineate the mFPC areas in them. The volumes of mPFC areas across histological, ex vivo and in vivo MRI delineations were comparable and probabilistic maps generated from the MRIs of the102 hemispheres. Probabilistic maps of mPFC areas were registered to MNI space and are available for regional analysis of functional magnetic resonance imaging data.
Assuntos
Mapeamento Encefálico/métodos , Imageamento por Ressonância Magnética/métodos , Córtex Pré-Frontal/anatomia & histologia , Córtex Pré-Frontal/diagnóstico por imagem , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Córtex Pré-Frontal/citologia , Adulto JovemRESUMO
We demonstrated recently that immunization with recombinant Neospora caninum profilin (rNcPRO) induces limited protection and a regulatory T-cell response in mice. The aim of this study was to evaluate the immune response elicited by rNcPRO in cattle and assess a strategy to enhance its immunogenicity, combining the addition of T-cell epitopes and immune modulators. We developed a chimeric recombinant profilin fused to functional T-cell epitopes present in the N-terminal sequence of vesicular stomatitis virus (VSV) glycoprotein G (rNcPRO/G). Groups of three cattle were immunized with two doses (2 weeks apart) of rNcPRO or rNcPRO/G formulated with alum hydroxide or a nanoparticulated soya-based adjuvant enriched with Toll-like receptor (TLR) 2 and TLR9 agonists, aimed to tackle the MyD88 pathway (AVECplus). rNcPRO induced only a primary immune response (IgM mediated), while antibodies in rNcPRO/G-vaccinated animals switched to IgG1 after the booster. The vaccine formulated with rNcPRO/G and AVECplus improved the production of systemic IFN-γ and induced long-term recall B-cell responses. Overall, our study provides data supporting the use of T-cell epitopes from VSV glycoprotein G and TLR agonists to enhance and modulate immunity to peptide antigens in bovines, particularly when using small proteins from parasites for which immune responses are usually feeble.
Assuntos
Doenças dos Bovinos/imunologia , Coccidiose/veterinária , Neospora/fisiologia , Vacinas Protozoárias/imunologia , Receptores Toll-Like/agonistas , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antiprotozoários/imunologia , Linfócitos B/imunologia , Bovinos , Coccidiose/imunologia , Epitopos de Linfócito T , Feminino , Imunoglobulina G , Interferon gama/imunologia , Camundongos , Profilinas/análise , Profilinas/genética , Vacinas Protozoárias/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/metabolismo , Linfócitos T Reguladores/imunologiaRESUMO
Mice immunized with a soluble extract of Neospora caninum tachyzoites (sNcAg) formulated with Providean-AVEC, an aqueous soy-based adjuvant, are fully protected from N. caninum multiplication. Here we evaluated the dose-dependent immunogenicity of this vaccine formulation in cattle. Cattle (N=3 per group) were immunized with two applications (30 days apart) of formulations containing Providean-AVEC and different payloads of sNcAg (100, 50 and 10 µg), that were five to fifty times lower than the only reported study using this same antigen in cattle. Kinetics and magnitude of the vaccine-induced immune responses were dose-dependent. Cattle immunized with 100 µg-sNcAg elicited high-avidity specific antibodies 3 weeks after the primary vaccination while those that received 50 µg of antigen had maximum levels of specific high-avidity antibodies 5 days after the day 30 boost. Vaccination with 10 µg of sNcAg induced comparable antibody responses after 2 weeks post re-vaccination. IgG1 was the predominant isotype in all vaccinated animals. Maximum systemic IFN-γ levels were measured in cattle immunized with 50 and 100 µg-sNcAg (14 ± 2.8 ng/ml). CD4(+)-T cells from vaccinated animals proliferated after sNcAg stimulation in vitro, producing IFN-γ. Recall IFN-γ responses mediated by CD4(+)-T cells were detected up to 140 days post vaccination. Formulations containing Providean-AVEC and 50 µg of sNcAg stimulated broad cellular and humoral immune responses against N. caninum in cattle. The profile and magnitude of the immune response elicited by this vaccine can be modified by the antigen-dose and vaccination schedule. This is the first dose-response study performed in cattle using sNcAg as antigen.
Assuntos
Doenças dos Bovinos/prevenção & controle , Coccidiose/veterinária , Lecitinas/química , Neospora/imunologia , Vacinas Protozoárias/imunologia , beta-Glucanas/química , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Bovinos , Coccidiose/prevenção & controle , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática/veterinária , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Testes SorológicosRESUMO
BACKGROUND: Markers for outcome prediction in bladder cancer are urgently needed. We have previously identified a molecular signature for predicting progression in non-muscle-invasive bladder cancer. ANXA10 was one of the markers included in the signature and we now validated the prognostic relevance of ANXA10 at the protein level. METHODS: We investigated ANXA10 expression by immunohistochemistry using a tissue microarray with 249 Ta and T1 urothelial carcinomas. The expression of ANXA10 was also investigated in an additional set of 97 more advanced tumours. The functional role of ANXA10 in cell lines was investigated by siRNA-mediated ANXA10 knockdown using wound-healing assays, proliferation assays, and ingenuity pathway analysis. RESULTS: Low expression of ANXA10 correlated with shorter progression-free survival in patients with stage Ta and T1 tumours (P<0.00001). Furthermore, patients with more advanced tumours and low ANXA10 expression had an unfavourable prognosis (P<0.00001). We found that ANXA10 siRNA transfected cells grew significantly faster compared with control siRNA transfected cells. Furthermore, a wound-healing assay showed that ANXA10 siRNA transfected cells spread along wound edges faster than control transfected cells. CONCLUSION: We conclude that ANXA10 may be a clinical relevant marker for predicting outcome in both early and advanced stages of bladder cancer.
Assuntos
Anexinas/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Biomarcadores Tumorais/análise , Progressão da Doença , Intervalo Livre de Doença , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Células Neoplásicas Circulantes , PrognósticoRESUMO
BACKGROUND: The KIAA1199 transcript is upregulated in colon adenomas and downregulated upon ß-catenin knockdown. METHODS: Transcript profiling was performed on >500 colon biopsies, methylation profiling data were compared with transcript data. Immunohistochemistry assessed KIAA1199 protein expression in 270 stage II/III tumours (>3 years follow-up). The effects of stable KIAA1199 knockdown in SW480 cells (three different constructs) were studied using transcriptional profiling, proliferation and protein analysis. RESULTS: The KIAA1199 transcript was strongly upregulated in 95% of adenocarcinomas. Absent expression in normal mucosa correlated with KIAA1199 promotor methylation. Nuclear and cytoplasmic KIAA1199 protein expression was identified in colon adenocarcinomas and other types of cancers. A subpopulation of patients with tumours strongly expressing KIAA1199 in the nucleus showed a better outcome with regard to recurrence as lung or liver metastases. The KIAA1199 knockdown affected the cell cycle and the Wnt-signalling pathway. Reduced cellular proliferation and decreased KI67, phosphorylated retinoblastoma, ß-catenin and ASCL2 protein expression supported these findings. Eighteen Wnt-signalling genes differentially expressed upon KIAA1199 knockdown correlated with the KIAA1199 expression profile in clinical specimens. CONCLUSION: The KIAA1199 knockdown attenuates the effects of the Wnt/ß-catenin signalling and it may thus be regarded as a regulatory part of this pathway.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Adesão Celular , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Hialuronoglucosaminidase , Imuno-Histoquímica , Microscopia de Fluorescência , Estadiamento de Neoplasias , Análise Serial de Proteínas , Proteínas/genética , Regulação para CimaRESUMO
This study describes the development and validation of a blocking ELISA that measures avidity of BVDV-specific immunoglobulins (Igs) as an alternative to the classic virus neutralization test. The assay comprises a recombinant soluble E2 glycoprotein as target antigen, a neutralizing serum as detector antibody and a washing-step with a chaotropic agent to determine BVDV-specific Igs avidity. Avidity-Blocking ELISA was validated with 100 negative and 87 positive BVDV-neutralization serum samples from either infected or vaccinated bovines (inactivated commercial vaccines). Specificity and sensitivity of the Avidity-Blocking ELISA were 100% and 98.8%, respectively. The assay was standardized to use a single dilution, so that 90 samples can be tested per plate. Results expressed as Avidity Index (AI) correlated with BVDV neutralizing titers (r=0.94). Unlike the virus neutralization test, the Avidity-Blocking ELISA could discriminate between infected and vaccinated animals (DIVA), suggesting that avidity measurement can be a valuable tool to achieve DIVA compliances. The data show that the avidity of anti BVDV antibodies is related to their capacity to block viral infection in vitro.
Assuntos
Anticorpos Antivirais/sangue , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Técnicas de Laboratório Clínico/métodos , Vírus da Diarreia Viral Bovina/imunologia , Diarreia/virologia , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Ensaio de Imunoadsorção Enzimática/métodos , Testes de Neutralização/métodos , Sensibilidade e EspecificidadeRESUMO
The temporal pole (TP) is the rostralmost portion of the human temporal lobe. Characteristically, it is only present in human and nonhuman primates. TP has been implicated in different cognitive functions such as emotion, attention, behavior, and memory, based on functional studies performed in healthy controls and patients with neurodegenerative diseases through its anatomical connections (amygdala, pulvinar, orbitofrontal cortex). TP was originally described as a single uniform area by Brodmann area 38, and von Economo (area TG of von Economo and Koskinas), and little information on its cytoarchitectonics is known in humans. We hypothesize that 1) TP is not a homogenous area and we aim first at fixating the precise extent and limits of temporopolar cortex (TPC) with adjacent fields and 2) its structure can be correlated with structural magnetic resonance images. We describe here the macroscopic characteristics and cytoarchitecture as two subfields, a medial and a lateral area, that constitute TPC also noticeable in 2D and 3D reconstructions. Our findings suggest that the human TP is a heterogeneous region formed exclusively by TPC for about 7 mm of the temporal tip, and that becomes progressively restricted to the medial and ventral sides of the TP. This cortical area presents topographical and structural features in common with nonhuman primates, which suggests an evolutionary development in human species.
Assuntos
Mapeamento Encefálico/métodos , Imageamento por Ressonância Magnética/métodos , Giro Para-Hipocampal/anatomia & histologia , Lobo Temporal/anatomia & histologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/citologia , Neurônios/fisiologia , Giro Para-Hipocampal/fisiologia , Lobo Temporal/fisiologia , Adulto JovemRESUMO
BACKGROUND: The identification of new prognostic markers in prostate cancer (PC) is essential to improve patient treatment and management. Data suggest that SMARCC1 protein, a part of the intranuclear SWI/SNF complex which enhances the transactivation of the androgen receptor, is upregulated in PC and therefore a possible candidate marker for PC progression. MATERIALS: Expression of SMARCC1 immunostaining was analysed on a tissue microarray containing specimens from 327 patients with prostate cancer and clinical follow-up information. Furthermore, 30 specimens from patients with benign prostate hyperplasia were included as controls as well as 30 specimens of benign prostate tissue from PC patients. Also, 18 specimens from lymph node metastases were analysed. RESULTS: All benign specimens showed no or minimal staining for SMARCC1. In contrast, 20% of the specimens from patients with non-metastatic and non-recurrent disease showed moderate to marked staining. In 31% of the patients with recurrent disease and in 31% of the patients with metastatic disease we found moderate to strong SMARCC1 immunostaining. In total, 23% of lymph node metastases expressed SMARCC1. SMARCC1 expression was also positively correlated to Gleason score (p<0.05), clinical T stage (p<0.01) and time to recurrence (p<0.001). In a logistic regression analysis, patients with a marked SMARCC1 immunostaining had a significantly elevated odds ratio (OR) of 16 for recurrent cancer and an OR of 4.5 for metastatic disease. Conclusions. Our present results demonstrate an increased expression of SMARCC1 protein in prostate cancer and reveal a positive correlation with tumour dedifferentiation, progression, metastasis and time to recurrence.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias da Próstata/metabolismo , Fatores de Transcrição/metabolismo , Adenocarcinoma/secundário , Adenocarcinoma/cirurgia , Animais , Biomarcadores Tumorais/metabolismo , Células COS , Desdiferenciação Celular , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Chlorocebus aethiops , Técnica Direta de Fluorescência para Anticorpo , Humanos , Técnicas Imunoenzimáticas , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Masculino , Recidiva Local de Neoplasia , Razão de Chances , Próstata/metabolismo , Próstata/patologia , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Análise Serial de Tecidos , Regulação para CimaRESUMO
Microarray analysis on pooled samples has previously identified ZDHHC9 (DHHC9) to be upregulated in colon adenocarcinoma compared to normal colon mucosa. Analyses of 168 samples from proximal and distal adenocarcinomas using U133plus2.0 microarrays validated these findings, showing a significant two-fold (log 2) upregulation of DHHC9 transcript (P<10(-6)). The upregulation was more striking in microsatellite stable (MSS), than in microsatellite instable (MSI), tumours. Genes known to interact with DHHC9 as H-Ras or N-Ras did not show expression differences between MSS and MSI. Immunohistochemical analysis was performed on 60 colon adenocarcinomas, previously analysed on microarrays, as well as on tissue microarrays with 40 stage I-IV tumours and 46 tumours from different organ sites. DHHC9 protein was strongly expressed in MSS compared to MSI tumours, readily detectable in premalignant lesions, compared to the rare expression seen in normal mucosa. DHHC9 was specific for tumours of the gastrointestinal tract and localised to the Golgi apparatus, in vitro and in vivo. Overexpression of DHHC9 decreased the proliferation of SW480 and CaCo2 MSS cell lines significantly. In conclusion, DHHC9 is a gastrointestinal-related protein highly expressed in MSS colon tumours. The palmitoyl transferase activity, modifying N-Ras and H-Ras, suggests DHHC9 as a target for anticancer drug design.
Assuntos
Aciltransferases/genética , Neoplasias Colorretais/genética , Divisão Celular , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias Colorretais/classificação , Neoplasias Colorretais/patologia , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Mucosa Intestinal/citologia , Repetições de Microssatélites/genética , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos , RNA Neoplásico/genética , Neoplasias Retais/genética , Neoplasias Retais/patologia , Dedos de Zinco/genéticaRESUMO
OBJECTIVE: We studied a large proportion of the population in our health district who have Down's syndrome to determine the incidence and variety of changes in the spine and to define the guidelines for preventive diagnosis advisable in relation to atlanto-axial instability, a common disorder in these patients. PATIENTS AND METHODS: First phase: a plain X-ray of the cervical spine in a neutral lateral projection and in flexion in 188 patients, measuring the atlanto-odontoid distance. Second phase: computerized tomography (CT) studies and three dimensional reconstructions in 25 patients (13.3%) chosen at random. The axial cuts from the upper portion of C3 to the occiput were 3 mm in thickness with 3 mm intervals and a standard reconstruction algorithm. RESULTS: The incidences of atlanto-axial instability with an atlodontoid distance (3)5 mm were not comparable with the published series. There was a lower incidence (4.2%), with no difference between measurements in flexion and in the neutral lateral views. There was a greater incidence of malformations than in other reports, including a rare case of os odontoideum and also constant asymmetry of the occipital condyles (100%) in the patients of the CT series and consequently instability of the atlas (96%) and off-centered odontoides (84%). CONCLUSIONS: The study showed that there was deficient asymmetrical development of the occipital bone, which caused different heights of the occipital condyles and led to cervico-cranial mal-position. For study of the degree of error of position and congenital anomalies. We recommend replacing plain X-ray studies by CT with three dimensional reconstructions.
Assuntos
Articulação Atlantoaxial/diagnóstico por imagem , Articulação Atlantoccipital/diagnóstico por imagem , Vértebras Cervicais/diagnóstico por imagem , Síndrome de Down/complicações , Processo Odontoide/diagnóstico por imagem , Doenças da Coluna Vertebral/complicações , Doenças da Coluna Vertebral/diagnóstico , Adolescente , Adulto , Articulação Atlantoaxial/anormalidades , Articulação Atlantoaxial/fisiopatologia , Articulação Atlantoccipital/fisiopatologia , Vértebras Cervicais/anormalidades , Vértebras Cervicais/fisiopatologia , Criança , Feminino , Humanos , Masculino , Processo Odontoide/anormalidades , Processo Odontoide/fisiopatologia , Doenças da Coluna Vertebral/fisiopatologia , Tomografia Computadorizada por Raios XRESUMO
Mounier-Kuhn syndrome--or tracheobronchomegaly--is a rare congenital disorder characterized by significant dilation of the trachea and main bronchi. It is accompanied by ineffective cough and is often complicated by recurrent lung infections and bronchiectasis. Clinical presentation varies widely, ranging from forms with scarce involvement of functional capacity to others that progress to respiratory failure that can prove fatal. We report an exceptional case of massive hemoptysis secondary to the presence of pulmonary aspergillomas and bilateral bronchiectasis in which bronchial arteriography with embolization and surgical resection were insufficient for resolving the complications.
Assuntos
Aspergilose/etiologia , Aspergillus niger , Hemoptise/etiologia , Pneumopatias Fúngicas/etiologia , Traqueobroncomegalia/complicações , Doença Aguda , Adulto , Aspergilose/diagnóstico , Aspergilose/cirurgia , Bronquiectasia/diagnóstico , Bronquiectasia/etiologia , Bronquiectasia/cirurgia , Terapia Combinada , Suscetibilidade a Doenças , Evolução Fatal , Hemoptise/diagnóstico , Hemoptise/cirurgia , Humanos , Pneumopatias Fúngicas/diagnóstico , Pneumopatias Fúngicas/cirurgia , Masculino , Pneumonectomia , Traqueobroncomegalia/diagnóstico , Traqueobroncomegalia/cirurgiaRESUMO
In our previous work (Mansilla et al. (1997) Protein Eng. 10, 927-934) we showed that Arg7 of Escherichia coli elongation factor Tu (EF1A) plays an essential role in aminoacyl-tRNA (aa-tRNA) binding. Substitution of Arg7 by Ala or Glu lost this activity. We proposed that Arg7 forms a salt bridge with the charged conserved amino acid Glu272 (Asp284 in Thermus aquaticus) thereby binding the N-terminal region of the protein to domain 2 and thus completing the conformational rearrangement needed for binding aa-tRNA. In this work we have mutated Glu272 to arginine, either alone (Glu272Arg), or in combination with one of the above mentioned mutations (Arg7Glu/Glu272Arg) in order to test this hypothesis. Our results show that, in confirmation of our thesis based on structural knowledge, the substitution of Glu272 (Asp284) decreases the ability of EF1A:GTP to bind aa-tRNA.
Assuntos
Proteínas de Bactérias/metabolismo , Elongação Traducional da Cadeia Peptídica , Fator Tu de Elongação de Peptídeos/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Proteínas de Bactérias/genética , Análise Mutacional de DNA , Escherichia coli , Ácido Glutâmico , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Mutagênese Sítio-Dirigida , Fator Tu de Elongação de Peptídeos/genéticaRESUMO
The function of the N-terminal region of elongation factor Tu is still unexplained. Until recently, it has not been visible in electron density maps from x-ray crystallography studies, but the presence of several well conserved basic residues suggest that this part of the molecule is of structural importance for the factor to function properly. In this study, two lysines at positions 4 and 9 were mutated separately to alanine or glutamate. The resulting four point mutants were expressed and purified using the pGEX system. The untagged products were characterized with regard to guanine-nucleotide interaction, intrinsic GTPase activity, and binding of aminoacyl-tRNA (aa-tRNA). The results show that Lys9 is especially strongly involved in the association with guanine nucleotides and the binding of aa-tRNA. Also Lys4 plays a role in the association of GDP and GTP and is also of some importance in aa-tRNA binding. Our results are discussed in structural terms with the conclusion that a complex network of interactions across the interface between domains 1 and 2 with Lys9 being a key residue seems to be important for the fine tuning of the dimensions of the cleft accommodating the acceptor end of aa-tRNA as well as delineating the structure of the effector region.
Assuntos
Fator Tu de Elongação de Peptídeos/metabolismo , Escherichia coli/genética , Fatores de Elongação Ligados a GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Mutagênese Sítio-Dirigida , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/isolamento & purificação , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
We have mutated lysine 2 and arginine 7 in elongation factor Tu from Escherichia coli separately either to alanine or glutamic acid. The aim of the work was to reveal the possible interactions between the conserved N-terminal part of the molecule, which is rich in basic residues and aminoacyl-tRNA. The enzymatic characterization, comprising GDP and GTP temperature stability assays and measurement of nucleotide dissociation and association rate constants, GTPase activity and aminoacyl-tRNA binding, shows that position 2 is not involved in aminoacyl-tRNA binding, while position 7 is necessary to accomplish this activity. Furthermore, arginine 7 seems to play a role in regulating the binding of GTP. The three-dimensional structure of the ternary complex, EF-Tu:GTP:Phe-tRNAPhe, involving Thermus aquaticus EF-Tu and yeast Phe-tRNA(Phe), shows that Arg7 is in a position which permits salt bridge formation with Asp284, thus binding the N-terminus tightly to domain 2. We propose that this interaction is needed for aminoacyl-tRNA binding, and also for completing the structural rearrangement, which takes place when the factor switches from its GDP to its GTP form.
Assuntos
Análise Mutacional de DNA , Escherichia coli/química , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/genética , Fatores de Elongação Ligados a GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Modelos Moleculares , Mutagênese , Conformação de Ácido Nucleico , Fator Tu de Elongação de Peptídeos/metabolismo , Conformação Proteica , Piridonas/farmacologia , RNA de Transferência de Fenilalanina/metabolismoRESUMO
Posttransplant lymphoproliferative disorders are infrequent tumors related to chronic immunosuppressive therapy. We present a liver transplant recipient who developed such a tumor in the porta hepatis that provoked obstruction of the entire portal triad. Treatment consisted of systemic chemotherapy, percutaneous dilatation, and placement of Wallstent endoprostheses across both biliary and portal vein stenoses. The patient died 3 weeks later of pneumonia and sepsis. At necropsy, the tumor was completely necrosed and the prostheses in both the common bile duct and the portal vein were patent.
Assuntos
Colestase/terapia , Neoplasias Hepáticas/complicações , Transplante de Fígado/efeitos adversos , Linfoma Imunoblástico de Células Grandes/complicações , Veia Porta/patologia , Punções , Stents , Colestase/diagnóstico por imagem , Colestase/etiologia , Constrição Patológica , Feminino , Humanos , Neoplasias Hepáticas/etiologia , Linfoma Imunoblástico de Células Grandes/etiologia , Pessoa de Meia-Idade , Veia Porta/diagnóstico por imagem , Radiografia IntervencionistaRESUMO
Portal vein (PV) thrombosis increases the risk of variceal bleeding in cirrhotic patients with portal hypertension. Its presence also complicates PV access during transjugular porto-caval shunt (TIPS) placement. We overcame this obstacle by using ultrasound (US) guidance for PV entry. Clot disruption by balloon catheters was then performed before placing the vascular endoprostheses for portal-venous shunting. We treated 3 cirrhotic patients in such fashion with good clinical results. Portal thrombi progressively disappeared after shunting due to both balloon disruption and the rise in portal blood flow velocity.
Assuntos
Veia Porta , Derivação Portossistêmica Cirúrgica/métodos , Trombose/terapia , Cateterismo , Varizes Esofágicas e Gástricas/cirurgia , Feminino , Hemorragia Gastrointestinal/cirurgia , Humanos , Hipertensão Portal/complicações , Hipertensão Portal/cirurgia , Masculino , Pessoa de Meia-Idade , Trombose/complicações , Ultrassonografia de IntervençãoRESUMO
From 1982 to 1989, 18 eligible patients (median age 50.5 years, range 30-72 years) with inflammatory breast carcinoma have been treated with neoadjuvant intra-arterial (IA) chemotherapy. The treatment regimen includes IA cisplatin, adriamycin, mytomycin C and thiotepa on Day 1 and intravenous 5-fluorouracil on Days 1 and 2. An objective clinical response rate of 100% (eight complete and 10 partial) has been observed. The median disease-free and overall survivals are 27 months (range 5-85+ months) and 33 months (range 8-85+ months), respectively. With a median follow-up of 21.5 months, six (33.3%) patients remain alive and free of disease and 12 patients have died because of distant metastases. No local recurrences have been observed. Intra-arterial chemotherapy is an attractive technique for the treatment of locally advanced breast carcinoma with mild toxicity and high local control rate.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Adulto , Idoso , Neoplasias da Mama/radioterapia , Neoplasias da Mama/cirurgia , Quimioterapia Adjuvante , Cisplatino/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Fluoruracila/administração & dosagem , Humanos , Infusões Intra-Arteriais , Artéria Torácica Interna , Pessoa de Meia-Idade , Mitomicina/administração & dosagem , Tiotepa/administração & dosagemRESUMO
Direct percutaneous drainage of pancreatic pseudocysts in communication with the duct of Wirsung can lead to pancreaticocutaneous fistula. These patients are safely treated with the percutaneous transgastric approach. In a gastrectomized patient who developed a pseudocyst, we percutaneously placed in internal endoprosthesis from the duct of Wirsung to the afferent loop and gastric pouch.