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1.
Commun Biol ; 3(1): 350, 2020 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620935

RESUMO

Entire reconstitution of tRNAs for active protein production in a cell-free system brings flexibility into the genetic code engineering. It can also contribute to the field of cell-free synthetic biology, which aims to construct self-replicable artificial cells. Herein, we developed a system equipped only with in vitro transcribed tRNA (iVTtRNA) based on a reconstituted cell-free protein synthesis (PURE) system. The developed system, consisting of 21 iVTtRNAs without nucleotide modifications, is able to synthesize active proteins according to the redesigned genetic code. Manipulation of iVTtRNA composition in the system enabled genetic code rewriting. Introduction of modified nucleotides into specific iVTtRNAs demonstrated to be effective for both protein yield and decoding fidelity, where the production yield of DHFR reached about 40% of the reaction with native tRNA at 30°C. The developed system will prove useful for studying decoding processes, and may be employed in genetic code and protein engineering applications.

2.
Oncol Rep ; 43(5): 1580-1590, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32323826

RESUMO

Glioblastoma is a malignant brain tumor exhibiting highly aggressive proliferation and invasion capacities. Despite treatment by aggressive surgical resection and adjuvant therapy including temozolomide and radiation therapy, patient prognosis remains poor. Lenalidomide, a derivative of thalidomide, is known to be an immunomodulatory agent that has been used to treat hematopoietic malignancies. There are numerous studies revealing an antitumor effect of lenalidomide in hematopoietic cells, but not in glioma cells. The present study aimed to demonstrate the antitumor effect of lenalidomide on malignant glioma cell lines. The growth inhibition of malignant glioma cells (A­172, AM­38, T98G, U­138MG, U­251MG, and YH­13) by lenalidomide was assessed using a Coulter counter. The mechanism of the antitumor effect of lenalidomide was examined employing a fluorescence­activated cell sorter, western blot analysis, and quantitative real­time reverse transcriptional polymerase chain reaction (RT­qPCR) in malignant glioma cell lines (A­172, AM­38). The results revealed that the number of malignant glioma cells was decreased in a concentration­dependent manner by lenalidomide. DNA flow cytometric analysis demonstrated an increase in the ratio of cells at the G0/G1 phase following lenalidomide treatment. Western blot analysis and RT­qPCR revealed that p53 activation and the expression of p21 were increased in glioma cells treated with lenalidomide. Western blot analysis revealed that cleavage of PARP did not occur; however, increased expression of Bax protein, cleavage of caspase­9 and cleavage of caspase­3 were confirmed. Analysis by FACS also supported the conclusion that little apoptosis induction occurred following lenalidomide treatment of malignant glioma cell lines. In conclusion, lenalidomide exerts an antitumor effect on glioma cells due to alterations in cell cycle distribution.

3.
Commun Biol ; 3(1): 142, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32214223

RESUMO

In vitro reconstitution is a powerful tool for investigating ribosome functions and biogenesis, as well as discovering new ribosomal features. In this study, we integrated all of the processes required for Escherichia coli small ribosomal subunit assembly. In our method, termed fully Recombinant-based integrated Synthesis, Assembly, and Translation (R-iSAT), assembly and evaluation of the small ribosomal subunits are coupled with ribosomal RNA (rRNA) synthesis in a reconstituted cell-free protein synthesis system. By changing the components of R-iSAT, including recombinant ribosomal protein composition, we coupled ribosomal assembly with ribosomal protein synthesis, enabling functional synthesis of ribosomal proteins and subsequent subunit assembly. In addition, we assembled and evaluated subunits with mutations in both rRNA and ribosomal proteins. The study demonstrated that our scheme provides new ways to comprehensively analyze any elements of the small ribosomal subunit, with the goal of improving our understanding of ribosomal biogenesis, function, and engineering.

4.
Epileptic Disord ; 22(1): 90-102, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32031531

RESUMO

To retrospectively evaluate the pharmacological profiles of antiepileptic drugs (AEDs) in epilepsy patients during haemodialysis using therapeutic drug monitoring data. The serum concentration of AEDs was collected before and after haemodialysis, and the clearance rate and concentration-to-dose ratio were calculated as pharmacological parameters. Thirty-six patients were enrolled in the study (25 males, 11 females; age: 65.3 ± 14.8 years). In 24 of the 36 patients, epilepsy was associated with cerebrovascular disorders, and diabetes was the most common reason for haemodialysis in 16 patients. With regards to seizure type, focal aware seizures were less frequent than focal impaired awareness seizures and focal-to-bilateral tonic-clonic seizures. Interictal EEG showed intermittent rhythmic slow waves and intermittent slow waves more often than spikes or sharp waves. Levetiracetam was the most commonly used AED and led to the highest percentage of responders (80%; 16/20 patients). However, the clearance rate of levetiracetam during dialysis was highest among the antiepileptic drugs used, requiring supplementary doses after haemodialysis in all 20 patients. Valproic acid was not effective for focal epilepsy for patients on haemodialysis, and non-responders to phenytoin had low serum concentration of phenytoin both before and after haemodialysis. The pre-haemodialysis concentration of levetiracetam tended to be higher than the reference range, suggesting a potential risk of overdosing before haemodialysis. The pre- and post-haemodialysis concentrations of valproic acid tended to be lower than the reference range, suggesting a potential risk of underdosing. The concentration-to-dose ratios for levetiracetam, valproic acid, phenytoin, and carbamazepine were significantly lower after than before haemodialysis. The majority of patients with epilepsy on haemodialysis had cerebrovascular diseases, and therapeutic drug monitoring for levetiracetam, valproic acid, and phenytoin, before and after haemodialysis, is needed to ensure proper dosing.

5.
Mol Phylogenet Evol ; 146: 106755, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32028028

RESUMO

Freshwater mussels (Bivalvia: Unionidae) is a diverse family with around 700 species being widespread in the Northern Hemisphere and Africa. These animals fulfill key ecological functions and provide important services to humans. Unfortunately, populations have declined dramatically over the last century, rendering Unionidae one of the world's most imperiled taxonomic groups. In Far East Asia (comprising Japan, Korea, and Eastern Russia), conservation actions have been hindered by a lack of basic information on the number, identity, distribution and phylogenetic relationships of species. Available knowledge is restricted to studies on national and sub-national levels. The present study aims to resolve the diversity, biogeography and evolutionary relationships of the Far East Asian Unionidae in a globally comprehensive phylogenetic and systematic context. We reassessed the systematics of all Unionidae species in the region, including newly collected specimens from across Japan, South Korea, and Russia, based on molecular (including molecular species delineation and a COI + 28S phylogeny) and comparative morphological analyses. Biogeographical patterns were then assessed based on available species distribution data from the authors and previous reference works. We revealed that Unionidae species richness in Far East Asia is 30% higher than previously assumed, counting 43 species (41 native + 2 alien) within two Unionidae subfamilies, the Unioninae (32 + 1) and Gonideinae (9 + 1). Four of these species are new to science, i.e. Beringiana gosannensissp. nov., Beringiana fukuharaisp. nov., Buldowskia kamiyaisp. nov., and Koreosolenaia sitgyensisgen. & sp. nov. We also propose a replacement name for Nodularia sinulata, i.e. Nodularia breviconchanom. nov. and describe a new tribe (Middendorffinaiini tribe nov.) within the Unioninae subfamily. Biogeographical patterns indicate that this fauna is related to that from China south to Vietnam until the Mekong River basin. The Japanese islands of Honshu, Shikoku, Kyushu, Hokkaido, and the Korean Peninsula were identified as areas of particularly high conservation value, owing to high rates of endemism, diversity and habitat loss. The genetically unique species within the genera Amuranodonta, Obovalis, Koreosolenaiagen. nov., and Middendorffinaia are of high conservation concern.

6.
Oncol Rep ; 42(6): 2635-2643, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31638255

RESUMO

Tumor necrosis factor­related apoptosis­inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) family, induces apoptosis in cancer cells by binding to its receptors, death receptor 4 (DR4) and DR5, without affecting normal cells, and is therefore considered to be a promising antitumor agent for use in cancer treatment. However, several studies have indicated that most glioma cell lines display resistance to TRAIL­induced apoptosis. To overcome such resistance and to improve the efficacy of TRAIL­based therapies, identification of ideal agents for combinational treatment is important for achieving rational clinical treatment in glioblastoma patients. The main aim of this study was to investigate whether interferon­ß (IFN­ß) (with its pleiotropic antitumor activities) could sensitize malignant glioma cells to TRAIL­induced apoptosis using glioma cell lines. TRAIL exhibited a dose­dependent antitumor effect in all of the 7 types of malignant glioma cell lines, although the intensity of the effect varied among the cell lines. In addition, combined treatment with TRAIL (low clinical dose: 1 ng/ml) and IFN­ß (clinically relevant concentration: 10 IU/ml) in A­172, AM­38, T98G, U­138MG and U­251MG demonstrated a more marked antitumor effect than TRAIL alone. Furthermore, the antitumor effect of the combined treatment with TRAIL and IFN­ß may be enhanced via an extrinsic apoptotic system, and upregulation of DR5 was revealed to play an important role in this process in U­138MG cells. These findings provide an experimental basis to suggest that combined treatment with TRAIL and IFN­ß may offer a new therapeutic strategy for malignant gliomas.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Interferon beta/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Interferon beta/uso terapêutico , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Regulação para Cima/efeitos dos fármacos
7.
J Biol Chem ; 294(49): 18898-18908, 2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31662434

RESUMO

Bacterial membrane proteins are integrated into membranes through the concerted activities of a series of integration factors, including membrane protein integrase (MPIase). However, how MPIase activity is complemented by other integration factors during membrane protein integration is incompletely understood. Here, using inverted inner-membrane vesicle and reconstituted (proteo)liposome preparations from Escherichia coli cells, along with membrane protein integration assays and the PURE system to produce membrane proteins, we found that anti-MPIase IgG inhibits the integration of both the Sec-independent substrate 3L-Pf3 coat and the Sec-dependent substrate MtlA into E. coli membrane vesicles. MPIase-depleted membrane vesicles lacked both 3L-Pf3 coat and MtlA integration, indicating that MPIase is involved in the integration of both proteins. We developed a reconstitution system in which disordered spontaneous integration was precluded, which revealed that SecYEG, YidC, or both, are not sufficient for Sec-dependent and -independent integration. Although YidC had no effect on MPIase-dependent integration of Sec-independent substrates in the conventional assay system, YidC significantly accelerated the integration when the substrate amounts were increased in our PURE system-based assay. Similar acceleration by YidC was observed for MtlA integration. YidC mutants with amino acid substitutions in the hydrophilic cavity inside the membrane were defective in the acceleration of the Sec-independent integration. Of note, MPIase was up-regulated upon YidC depletion. These results indicate that YidC accelerates the MPIase-dependent integration of membrane proteins, suggesting that MPIase and YidC function sequentially and cooperatively during the catalytic cycle of membrane protein integration.

8.
Pathol Int ; 69(9): 526-535, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31559671

RESUMO

Invasive mucinous adenocarcinoma (IMA) is a newly classified variant of lung adenocarcinoma. The aim of this study was to examine the correlation between the proportion of goblet cells and the clinicopathological characteristics of IMA. Ninety-nine patients with stage I IMA were included in this study. We estimated prognostic impact of goblet cell proportion. We classified them into two groups: the cases with a high goblet cell proportion (HGP, goblet cell proportion ≥80%) and the cases with a low goblet cell proportion (LGP, goblet cell proportion ≤30%), and compared the expression levels of five cancer progression markers and the number of tumor-promoting stromal cells between the two groups. Univariate and multivariate analysis revealed that the goblet cell proportion was a prognostic factor for recurrence free survival (P < 0.01) and overall survival (P = 0.01). The expression levels of the cancer stem cell-related marker, ALDH-1, and proliferation-related marker, geminin were significantly higher in the LGP group than in the HGP group. CD204+ tumor-associated macrophages were significantly more in the LGP stroma than the HGP stroma. Our current study indicated that the proportion of goblet cells was correlated with the malignant potential in surgically resected IMA.


Assuntos
Adenocarcinoma de Pulmão/patologia , Adenocarcinoma Mucinoso/patologia , Biomarcadores Tumorais/metabolismo , Neoplasias Pulmonares/patologia , Adenocarcinoma de Pulmão/diagnóstico , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma Mucinoso/diagnóstico , Adenocarcinoma Mucinoso/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células , Intervalo Livre de Doença , Feminino , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Humanos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Células Estromais/patologia
9.
Artigo em Inglês | MEDLINE | ID: mdl-31321610

RESUMO

Most pulmonary ground-glass opacity (GGO) nodules are pathologically well differentiated adenocarcinomas. We performed a limited resection trial of GGO lesions 2 cm or smaller from 2003 to 2009, in which 95 patients were accumulated. We confirmed negative surgical cut-end during surgery by margin lavage cytology. In the trial, a 51-year-old man underwent right lower lobe wedge resection for a 1.7 cm mixed GGO lesion. The tumor was papillary predominant adenocarcinoma, pT1NxM0. The resection scar became thicker and was diagnosed as adenocarcinoma by needle biopsy 10 years after the initial surgery. We performed a right lower lobectomy and lymph node dissection. Pathologically, the second tumor was adenocarcinoma similar to the initial one, papillary predominant, and was diagnosed as cut-end recurrence. Small papillary predominant adenocarcinoma might develop delayed cut-end recurrence more than 5 years after limited resection. Careful follow-up with special attention to the cut-end is necessary ideally for 10 years.

10.
Endocr Connect ; 8(5): 454-461, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30959487

RESUMO

Objective: Regional differences in cardiac magnetic resonance, which can reveal catecholamine-induced myocardial injury in patients with pheochromocytoma, have not yet been assessed using 3T magnetic resonance imaging. We evaluated these differences using myocardial T1-mapping and strain analysis. Design and Methods: We retrospectively reviewed 16 patients newly diagnosed with catecholamine-producing tumors (CPT group) and 16 patients with essential hypertension (EH group), who underwent cardiac magnetic resonance imaging between May 2016 and March 2018. We acquired 3T magnetic resonance cine and native T1-mapping images and performed feature-tracking-based strain analysis in the former. Results: Global cardiac function, morphology, global strain and peak strain rate were similar, but end-diastolic wall thickness differed between groups (CPT vs EH: 10.5 ± 1.7 vs 12.6 ± 2.8 mm; P < 0.05). Basal, but not apical, circumferential strain was significantly higher in the CPT than the EH group (19.4 ± 3.2 vs 16.8 ± 3.6 %; P < 0.05). Native T1 values were significantly higher in CPT than in EH patients, in both the basal septum (1307 ± 48 vs 1241 ± 45 ms; P < 0.01) and the apical septum (1377 ± 59 vs 1265 ± 58 ms; P < 0.01) mid-walls. In the CPT, but not in the EH group, native T1 values in the apical wall were significantly higher than those in the basal wall (P < 0.01). Conclusion: 3T magnetic resonance-based T1-mapping can sensitively detect subclinical catecholamine-induced myocardial injury; the influence of catecholamines may be greater in the apical than in the basal wall.

11.
Nat Commun ; 10(1): 1325, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30902985

RESUMO

Attempts to construct an artificial cell have widened our understanding of living organisms. Many intracellular systems have been reconstructed by assembling molecules, however the mechanism to synthesize its own constituents by self-sufficient energy has to the best of our knowledge not been developed. Here, we combine a cell-free protein synthesis system and small proteoliposomes, which consist of purified ATP synthase and bacteriorhodopsin, inside a giant unilamellar vesicle to synthesize protein by the production of ATP by light. The photo-synthesized ATP is consumed as a substrate for transcription and as an energy for translation, eventually driving the synthesis of bacteriorhodopsin or constituent proteins of ATP synthase, the original essential components of the proteoliposome. The de novo photosynthesized bacteriorhodopsin and the parts of ATP synthase integrate into the artificial photosynthetic organelle and enhance its ATP photosynthetic activity through the positive feedback of the products. Our artificial photosynthetic cell system paves the way to construct an energetically independent artificial cell.


Assuntos
Células Artificiais/metabolismo , Fotossíntese , Biossíntese de Proteínas , Trifosfato de Adenosina/metabolismo , Células Artificiais/efeitos dos fármacos , Metabolismo Energético/efeitos da radiação , Luz , Organelas/metabolismo , Organelas/efeitos da radiação , Fotossíntese/efeitos da radiação , Biossíntese de Proteínas/efeitos da radiação , Lipossomas Unilamelares/metabolismo
12.
Int J Oncol ; 54(5): 1864-1874, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864696

RESUMO

Malignant melanoma is a highly aggressive skin cancer that is highly resistant to chemotherapy. Adjuvant therapy is administered to patients with melanoma that possess no microscopic metastases or have a high risk of developing microscopic metastases. Methylating agents, including dacarbazine (DTIC) and temozolomide (TMZ), pegylated interferon (IFN)­α2b and interleukin­2 have been approved for adjuvant immuno­chemotherapy; however, unsatisfactory results have been reported following the administration of methylating agents. IFN­ß has been considered to be a signaling molecule with an important therapeutic potential in cancer. The aim of the present study was to elucidate whether antitumor effects could be augmented by the combination of TMZ and IFN­ß in malignant melanoma. We evaluated the efficacy of TMZ and IFN­ß by comparing O6­methylguanine­DNA transferase (MGMT)­proficient and ­deficient cells, as MGMT has been reported to be associated with the resistance to methylating agents. Cell viability was determined by counting living cells with a Coulter counter, and apoptosis was analyzed by dual staining with Annexin V Alexa Fluor® 488 and propidium iodide. The expression of proteins involved in the cell cycle, apoptosis and autophagy was evaluated by western blot analysis. The combined treatment with TMZ and IFN­ß suppressed cell proliferation and induced cell cycle arrest. We also demonstrated that a combination of TMZ and IFN­ß enhanced apoptosis and autophagy more efficiently compared with TMZ treatment alone. These findings suggest that antitumor activity may be potentiated by IFN­ß in combination with TMZ.


Assuntos
Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Interferon beta/farmacologia , Melanoma/genética , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/genética , Autofagia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo
13.
RNA ; 25(4): 472-480, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30705137

RESUMO

In vitro reconstitution studies have shown that ribosome assembly is highly cooperative and starts with the binding of a few ribosomal (r-) proteins to rRNA. It is unknown how these early binders act. Focusing on the initial stage of the assembly of the large subunit of the Escherichia coli ribosome, we prepared a 79-nucleotide-long region of 23S rRNA encompassing the binding sites of the early binders uL4 and uL24. Force signals were measured in a DNA/RNA dumbbell configuration with a double optical tweezers setup. The rRNA fragment was stretched until unfolded, in the absence or in the presence of the r-proteins (either uL4, uL24, or both). We show that the r-proteins uL4 and uL24 individually stabilize the rRNA fragment, both acting as molecular clamps. Interestingly, this mechanical stabilization is enhanced when both proteins are bound simultaneously. Independently, we observe a cooperative binding of uL4 and uL24 to the rRNA fragment. These two aspects of r-proteins binding both contribute to the efficient stabilization of the 3D structure of the rRNA fragment under investigation. We finally consider implications of our results for large ribosomal subunit assembly.


Assuntos
RNA Bacteriano/química , RNA Ribossômico 23S/química , Proteínas Ribossômicas/genética , Ribossomos/química , Pareamento de Bases , Sequência de Bases , Fenômenos Biomecânicos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Pinças Ópticas , Biogênese de Organelas , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
14.
Sci Rep ; 9(1): 1372, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718729

RESUMO

MPIase is a glycolipid that is involved in membrane protein integration. Despite evaluation of its functions in vitro, the lack of information on MPIase biosynthesis hampered verification of its involvement in vivo. In this study, we found that depletion of CdsA, a CDP-diacylglycerol synthase, caused not only a defect in phospholipid biosynthesis but also MPIase depletion with accumulation of the precursors of both membrane protein M13 coat protein and secretory protein OmpA. Yeast Tam41p, a mitochondrial CDP-diacylglycerol synthase, suppressed the defect in phospholipid biosynthesis, but restored neither MPIase biosynthesis, precursor processing, nor cell growth, indicating that MPIase is essential for membrane protein integration and therefore for cell growth. Consistently, we observed a severe defect in protein integration into MPIase-depleted membrane vesicles in vitro. Thus, the function of MPIase as a factor involved in protein integration was proven in vivo as well as in vitro. Moreover, Cds1p, a eukaryotic CdsA homologue, showed a potential for MPIase biosynthesis. From these results, we speculate the presence of a eukaryotic MPIase homologue.

15.
J Cell Physiol ; 234(8): 13510-13524, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30613977

RESUMO

Interferon ß (IFN-ß) is considered a signaling molecule with important therapeutic potential in cancer since IFN-ß-induced gene transcription mediates antiproliferation and cell death induction. Whereas, TNF-related apoptosis inducing ligand/Apo2 ligand (TRAIL/Apo2L) has emerged as a promising anticancer agent because it induces apoptosis specifically in cancer cells. In this study, we elucidated that IFN-ß augments TRAIL-induced apoptosis synergistically using five human malignant melanoma cells. All of these cells were induced apoptosis by TRAIL. Whereas, the response against IFN-ß was different in amelanotic cells (A375 and CRL1579) and melanotic cells (G361, SK-MEL-28, and MeWo). The responsibility of amelanotic cells against IFN-ß was higher than those of melanotic cells. The synergism of IFN-ß and TRAIL were correlated with the responsibilities of the cells against IFN-ß. The synergistic interaction was confirmed by a combination index based on the Chou-Talalay method. The upregulation of apoptosis in amelanotic cells was caused by very low doses of IFN-ß (over 0.1 IU/ml). Both of p53-mediated intrinsic pathway and Fas-related extrinsic pathway were activated by IFN-ß alone and combination with TRAIL. Further, TRAIL death receptors (DR4 and DR5) were upregulated by a low-dose IFN-ß (over 0.1 IU/ml) and the expression was more promoted by the combination with TRAIL. It was clarified that the upregulation of DR5 is associated with the declination of viability.

16.
Proteins ; 87(3): 226-235, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30520515

RESUMO

Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the products of defective translation, to recycle the tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its activity is essential for bacterial viability. Here, we have determined the crystal structure of Pth from Thermus thermophilus (TtPth) at 1.00 Å resolution. This is the first structure of a Pth from a thermophilic bacterium and the highest resolution Pth structure reported so far. The present atomic resolution data enabled the calculation of anisotropic displacement parameters for all atoms, which revealed the directionality of the fluctuations of key regions for the substrate recognition. Comparisons between TtPth and mesophilic bacterial Pths revealed that their structures are similar overall. However, the structures of the N- and C-terminal, loop-helix α4, and helix α6 regions are different. In addition, the helix α1 to strand ß4 region of TtPth is remarkably different from those of the mesophilic bacterial Pths, because this region is 9 or 10 amino acid residues shorter than those of the mesophilic bacterial Pths. This shortening seems to contribute to the thermostability of TtPth. To further understand the determinants for the thermostability of TtPth, we compared various structural factors of TtPth with those of mesophilic bacterial Pths. The data suggest that the decreases in accessible surface area and thermolabile amino acid residues, and the increases in ion pairs, hydrogen bonds, and proline residues cooperatively contribute to the thermostability of TtPth.


Assuntos
Hidrolases de Éster Carboxílico/química , Conformação Proteica , Aminoacil-RNA de Transferência/química , Thermus thermophilus/química , Sequência de Aminoácidos , Cristalografia por Raios X , Ligação de Hidrogênio , Ligação Proteica , RNA de Transferência , Aminoacil-RNA de Transferência/genética , Especificidade por Substrato
17.
J Magn Reson Imaging ; 50(1): 153-163, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30565346

RESUMO

BACKGROUND: Excessive trabeculation is present in isolated left ventricular noncompaction (LVNC) and dilated cardiomyopathy (DCM), which sometimes makes the differentiation between these two difficult. Fractal dimension (FD) is a unitless measure value of how completely the object fills space, which can assess the extent of myocardial trabeculae quantitatively. PURPOSE: To compare the trabeculae features and myocardial strain derived from cardiac MR between LVNC and DCM. STUDY TYPE: Respective case-control series. POPULATION: In all, 35 LVNC patients and 30 DCM patients were enrolled, and 20 healthy volunteers were selected as a control group. FIELD STRENGTH/SEQUENCE: 5 T with 8-channel phased-array cardiac receiver coil including steady-state free precession cine imaging. ASSESSMENT: The degree of left ventricular trabeculation was evaluated by a semiautomatic tool based on fractal analysis. Myocardial deformation was assessed by feature tracking. STATISTICAL TESTS: Independent samples Student's t-test, Mann-Whitney U-test, receiver operating characteristics (ROC) curves, and Spearman's rank coefficient were conducted. RESULTS: Max apical FD and mean global FD were higher in the LVNC group than in the DCM group (1.433 ± 0.074 vs. 1.341 ± 0.062, P < 0.001; 1.323 ± 0.036 vs. 1.267 ± 0.041, P < 0.001, respectively). For diagnosing LVNC, max apical FD was 1.392 (area under the curve [AUC] = 0.881, 95% confidence interval [CI]: 0.804-0.957), and the cutoff value of mean global FD was 1.283 (AUC = 0.895, 95% CI: 0.828-0.961). The global peak longitudinal strain value of the left ventricle (GPLS) showed significant differences between the LVNC group and DCM group [-6.49 (-11.41, -4.90) vs. -4.61 (-5.87, -3.61), P = 0.006]. The diagnostic accuracy for LVNC is highest when using FDs in coordination with GPLS (AUC = 0.93, 95% CI: 0.86-0.98, P < 0.001). DATA CONCLUSION: Fractal analysis provides a quantitative measurement of myocardial trabeculation. The combination of fractal analysis with myocardial strain provides a novel biomarker in distinguishing LVNC from DCM. LEVEL OF EVIDENCE: 3 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2019;50:153-163.

18.
Lung Cancer ; 126: 162-169, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30527182

RESUMO

BACKGROUND: Lung cancer with usual interstitial pneumonia (UIP) pattern is a disease with poor prognosis. This study aimed to characterize the tumor microenvironment of lung adenocarcinoma associated with UIP (UIP-ADC). METHODS: A total of 1341 consecutive patients with ADC who had undergone complete surgical resection were enrolled in this study, and the clinicopathological features of UIP-ADC were examined. Further, we selected 17 cases of UIP-ADC and non-UIP ADC each (adjusted for age, smoking status, pathological stage, and invasive size of lesion) for immunohistochemical analysis, and the biological differences between UIP-ADC and non-UIP ADC groups were analyzed. RESULTS: UIP-ADC was detected in 18 patients (1.3%). Patients with UIP-ADC had shorter cancer-specific survival (CSS) (5 yrs CSS; UIP-ADC 52.9% vs non-UIP ADC 81.8%, p < 0.01). Evaluation of tumor-infiltrating lymphocytes (TILs) in cancer stroma showed that the number of CD8+ TILs in UIP-ADC group was significantly lower than that in the non-UIP ADC group (median number 91 vs 121, p < 0.01). In contrast, levels of Foxp3+ TILs were not significantly different between the two groups. The CD8+/Foxp3+ T cell ratio was significantly lower in UIP-ADC than in the non-UIP ADC population (1.9 vs 2.7, p < 0.01). Additionally, among UIP-ADC patients, the CD8+/Foxp3+ T cell ratio was significantly higher in the non-cancerous UIP lesions than in the cancer stroma from the same patient (2.4 vs 1.7, p < 0.01). CONCLUSION: In the current study, we have demonstrated that the tumor microenvironment of UIP-ADC acquires an immunosuppressive state, and this could be one of the possible explanations for poor prognosis of this disease.


Assuntos
Adenocarcinoma/imunologia , Fibrose Pulmonar Idiopática/imunologia , Neoplasias Pulmonares/imunologia , Microambiente Tumoral/imunologia , Adenocarcinoma/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Feminino , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Fibrose Pulmonar Idiopática/complicações , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/complicações , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adulto Jovem
19.
Life (Basel) ; 8(4)2018 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-30400226

RESUMO

Membrane proteins are important drug targets which play a pivotal role in various cellular activities. However, unlike cytosolic proteins, most of them are difficult-to-express proteins. In this study, to synthesize and produce sufficient quantities of membrane proteins for functional and structural analysis, we used a bottom-up approach in a reconstituted cell-free synthesis system, the PURE system, supplemented with artificial lipid mimetics or micelles. Membrane proteins were synthesized by the cell-free system and integrated into lipid bilayers co-translationally. Membrane proteins such as the G-protein coupled receptors were expressed in the PURE system and a productivity ranging from 0.04 to 0.1 mg per mL of reaction was achieved with a correct secondary structure as predicted by circular dichroism spectrum. In addition, a ligand binding constant of 27.8 nM in lipid nanodisc and 39.4 nM in micelle was obtained by surface plasmon resonance and the membrane protein localization was confirmed by confocal microscopy in giant unilamellar vesicles. We found that our method is a promising approach to study the different classes of membrane proteins in their native-like artificial lipid bilayer environment for functional and structural studies.

20.
RNA ; 24(11): 1512-1519, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30076205

RESUMO

Reconstitution of ribosomes in vitro from individual ribosomal proteins provides a powerful tool for understanding the ribosome assembly process including the sequential incorporation of ribosomal proteins. However, conventional assembly methods require high-salt conditions for efficient ribosome assembly. In this study, we reconstituted 30S ribosomal subunits from individually purified ribosomal proteins in the presence of ribosome biogenesis factors. In this system, two GTPases (Era and YjeQ) facilitated assembly of a 30S subunit exhibiting poly(U)-directed polyphenylalanine synthesis and native protein synthesis under physiological conditions. This in vitro system permits a study of the assembly process and function of ribosome biogenesis factors, and it will facilitate the generation of ribosomes from DNA without using cells.


Assuntos
Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/metabolismo , Evolução Biológica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Proteínas Recombinantes
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