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1.
Ultramicroscopy ; 209: 112883, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31739191

RESUMO

Cryo-electron microscopy (cryo-EM) has become the method of choice in the field of structural biology, owing to its unique ability to deduce structures of vitreous ice-embedded, hydrated biomolecules over a wide range of structural resolutions. As cryo-transmission electron microscopes (cryo-TEM) become increasingly specialised for high, near-atomic resolution studies, operational complexity and associated costs serve as significant barriers to widespread usability and adoptability. To facilitate the expansion and accessibility of the cryo-EM method, an efficient, user-friendly means of imaging vitreous ice-embedded biomolecules has been called for. In this study, we present a solution to this issue by integrating cryo-EM capabilities into a commercial scanning electron microscope (SEM). Utilising the principle of low-energy in-line electron holography, our newly developed hybrid microscope permits low-to-moderate resolution imaging of vitreous ice-embedded biomolecules without the need for any form of sample staining or chemical fixation. Operating at 20 kV, the microscope takes advantage of the ease-of-use of SEM-based imaging and phase contrast imaging of low-energy electron holography. This study represents the first reported successful application of low-energy in-line electron holographic imaging to vitreous ice-embedded small biomolecules, the effectiveness of which is demonstrated here with three morphologically distinct specimens.

2.
In Vitro Cell Dev Biol Anim ; 55(7): 559-566, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31144267

RESUMO

A novel cell line of canine medullary thyroid carcinoma (MTC) was established from the neck mass, diagnosed histopathologically and immunohistochemically as ectopic MTC. The neoplastic cells arranging trabecular structures were characterized as pleomorphic cells with eosinophilic cytoplasm and nucleus, containing often clear nucleolus. These tumor cells were immuno-positive for calcitonin gene-related protein (CGRP), somatostatin, and chromogranin A. In addition, 8th passaged cultured cells were also immuno-positive for CGRP, somatostatin, and chromogranin A. The cloned tumor cells showed logarithmic cell growth with a doubling time of 33.3 h. From the results of DNA sequencing of rearranged during transfection (RET) proto-oncogene, the cloned tumor cells had four single base substitution, including exon 5 codon 82, exon 16 codon 750, exon 17 codon 777, and exon 24 codon 1085, all of which were single nucleotide polymorphism reported in RET gene of dogs. After the xenotransplantation into severe combined immunodeficiency (SCID) mice, the cloned cells showed tumorigenicity potentials. The morphological and immunohistochemical features of the xenotransplanted tumor were almost in conformity with those of the original tumor, including positive immunoreactivity for calcitonin, CGRP, and chromogranin A. To our knowledge, this is the first report of canine MTC cell line, which provides useful in vitro tool for understanding oncogenic mechanism and pathophysiological state of MTC in dogs.


Assuntos
Carcinoma Neuroendócrino/genética , Carcinoma Neuroendócrino/patologia , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Animais , Sequência de Bases , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células , Cromogranina A/metabolismo , Cães , Feminino , Camundongos , Camundongos SCID , Transplante de Neoplasias , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA , Somatostatina/metabolismo , Transplante Heterólogo
3.
J Struct Biol ; 204(1): 75-79, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30030043

RESUMO

For many macromolecular complexes, the inability to uniformly disperse solubilized specimen particles within vitreous ice films precludes their analysis by cryo-electron microscopy (cryo-EM). Here, we introduce a sample preparation process using "perpetually-hydrated" graphene oxide flakes as particle support films, and report vastly improved specimen dispersion. The new method introduced in this study incorporates hydrated graphene oxide flakes into a standard sample preparation regime, without the need for additional tools or devices, making it a cost-effective and easily adoptable alternative to currently available sample preparation approaches.


Assuntos
Microscopia Crioeletrônica/métodos , Grafite/química , Manejo de Espécimes/métodos
4.
Ultramicroscopy ; 188: 31-40, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29544194

RESUMO

A new SEM-based in-line electron holography microscope has been under development. The microscope utilizes conventional SEM and BF-STEM functionality to allow for rapid searching of the specimen of interest, seamless interchange between SEM, BF-STEM and holographic imaging modes, and makes use of coherent low-energy in-line electron holography to obtain low-dose, high-contrast images of light element materials. We report here an overview of the instrumentation and first experimental results on gold nano-particles and carbon nano-fibers for system performance tests. Reconstructed images obtained from the holographic imaging mode of the new microscope show substantial image contrast and resolution compared to those acquired by SEM and BF-STEM modes, demonstrating the feasibility of high-contrast imaging via low-energy in-line electron holography. The prospect of utilizing the new microscope to image purified biological specimens at the individual particle level is discussed and electron optical issues and challenges to further improve resolution and contrast are considered.

5.
J Struct Biol ; 2018 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-29551713

RESUMO

For many macromolecular complexes, the inability to uniformly disperse solubilized specimen particles within vitreous ice films precludes their analysis by cryo-electron microscopy (cryo-EM). Here, we introduce a sample preparation process using "perpetually-hydrated" graphene oxide flakes as particle support films, and report vastly improved specimen dispersion. Furthermore, we provide evidence that the presence of graphene oxide flakes in vitreous ice results in a significant reduction in electron beam-induced specimen decomposition. The new method introduced in this study incorporates hydrated graphene oxide flakes into a standard sample preparation regime, without the need for additional tools or devices, making it a cost-effective and easily adoptable alternative to currently available sample preparation approaches.

6.
Pest Manag Sci ; 74(3): 672-681, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29024365

RESUMO

BACKGROUND: A new generation of more active succinate dehydrogenase (Sdh) inhibitors (SDHIs) is currently widely used to control Septoria leaf blotch in northwest Europe. Detailed studies were conducted on Zymoseptoria tritici field isolates with reduced sensitivity to fluopyram and isofetamid; SDHIs which have only just or not been introduced for cereal disease control, respectively. RESULTS: Strong cross-resistance between fluopyram and isofetamid, but not with other SDHIs, was confirmed through sensitivity tests using laboratory mutants and field isolates with and without Sdh mutations. The sensitivity profiles of most field isolates resistant to fluopyram and isofetamid were very similar to a lab mutant carrying SdhC-A84V, but no alterations were found in SdhB, C and D. Inhibition of mitochondrial Sdh enzyme activity and control efficacy in planta for those isolates was severely impaired by fluopyram and isofetamid, but not by bixafen. Isolates with similar phenotypes were not only detected in northwest Europe but also in New Zealand before the widely use of SDHIs. CONCLUSION: This is the first report of SDHI-specific non-target site resistance in Z. tritici. Monitoring studies show that this resistance mechanism is present and can be selected from standing genetic variation in field populations. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.


Assuntos
Ascomicetos/efeitos dos fármacos , Resistência a Medicamentos/genética , Fungicidas Industriais/farmacologia , Variação Genética , Succinato Desidrogenase/antagonistas & inibidores , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle
7.
PLoS One ; 12(11): e0186899, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29099843

RESUMO

We have performed open cell transmission electron microscopy experiments through pure water vapor in the saturation pressure regime (>0.6 kPa), in a modern microscope capable of sub-Å resolution. We have systematically studied achievable pressure levels, stability and gas purity, effective thickness of the water vapor column and associated electron scattering processes, and the effect of gas pressure on electron optical resolution and image contrast. For example, for 1.3 kPa pure water vapor and 300kV electrons, we report pressure stability of ± 20 Pa over tens of minutes, effective thickness of 0.57 inelastic mean free paths, lattice resolution of 0.14 nm on a reference Au specimen, and no significant degradation in contrast or stability of a biological specimen (M13 virus, with 6 nm body diameter). We have also done some brief experiments to confirm feasibility of loading specimens into an in situ water vapor ambient without exposure to intermediate desiccating conditions. Finally, we have also checked if water experiments had any discernible impact on the microscope performance, and report pertinent vacuum and electron optical data, for reference purposes.


Assuntos
Microscopia Eletrônica de Transmissão/métodos , Água/química
8.
Microscopy (Oxf) ; 66(4): 261-271, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28453814

RESUMO

The scanning transmission electron microscopy (STEM) mode of today's field emission scanning electron microscopes enables sub-nanometer resolution imaging. Graphene is a single-atom thick, electrically conductive material, making it an excellent specimen support for the low voltage STEM imaging of nanometer-sized objects such as viruses. Here we present low voltage STEM images of bacteriophage T4 recorded on highly cleaned graphene films. The results show that ultrathin graphene support films markedly improve image signal at low accelerating voltages. Staining with a low atomic number methylamine vanadate stain combined with the graphene support film enables the clear visualization of the fine structure of the T4 tail by the low voltage STEM technique. Despite the advantages of graphene support films, difficulties are often encountered in placing hydrophilic biological samples on hydrophobic graphene electron microscopy grids. We employed a spin sedimentation sample loading method to overcome this problem.

9.
J Biol Chem ; 289(33): 23177-88, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24958725

RESUMO

In Escherichia coli, the biogenesis of both cytochrome bd-type quinol oxidases and periplasmic cytochromes requires the ATP-binding cassette-type cysteine/GSH transporter, CydDC. Recombinant CydDC was purified as a heterodimer and found to be an active ATPase both in soluble form with detergent and when reconstituted into a lipid environment. Two-dimensional crystals of CydDC were analyzed by electron cryomicroscopy, and the protein was shown to be made up of two non-identical domains corresponding to the putative CydD and CydC subunits, with dimensions characteristic of other ATP-binding cassette transporters. CydDC binds heme b. Detergent-solubilized CydDC appears to adopt at least two structural states, each associated with a characteristic level of bound heme. The purified protein in detergent showed a weak basal ATPase activity (approximately 100 nmol Pi/min/mg) that was stimulated ∼3-fold by various thiol compounds, suggesting that CydDC could act as a thiol transporter. The presence of heme (either intrinsic or added in the form of hemin) led to a further enhancement of thiol-stimulated ATPase activity, although a large excess of heme inhibited activity. Similar responses of the ATPase activity were observed with CydDC reconstituted into E. coli lipids. These results suggest that heme may have a regulatory role in CydDC-mediated transmembrane thiol transport.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Adenosina Trifosfatases/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Heme/química , Multimerização Proteica , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Transporte Biológico Ativo/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Heme/genética , Heme/metabolismo , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade
10.
J Vet Diagn Invest ; 22(3): 438-41, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20453222

RESUMO

It is well known that tumor-infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) from patients with advanced-stage cancer have a poor immune response. Regulatory T cells (Tregs), characterized by the expression of a cluster of differentiation 4 and intracellular FoxP3 markers, can inhibit antitumor immunoresponse. In the present study, the prevalence of Tregs in peripheral blood and tumor tissue from dogs with oral malignant melanoma was evaluated by triple-color flow cytometry. The percentage of Tregs in the peripheral blood of the dogs with malignancy was significantly increased compared with healthy control dogs, and the percentage of Tregs within tumors was significantly increased compared with Tregs in peripheral blood of dogs with oral malignant melanoma. This finding suggests that the presence of tumor cells induced either local proliferation or selective migration of Tregs to tumor-infiltrated sites. A better understanding of the underlying mechanisms of Treg regulation in patients with cancer may lead to an effective anticancer immunotherapy against canine malignant melanoma and possibly other tumors.


Assuntos
Doenças do Cão/imunologia , Melanoma/veterinária , Neoplasias Bucais/veterinária , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Cães , Citometria de Fluxo , Humanos , Melanoma/sangue , Melanoma/imunologia , Neoplasias Bucais/sangue , Neoplasias Bucais/imunologia , Valores de Referência
11.
Microbiol Immunol ; 54(3): 152-9, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20236425

RESUMO

Recent data suggest a decreased prevalence of IFN-gamma-producing T lymphocytes (Type 1 T cells) in tumor-bearing hosts. Moreover, it has been reported that Treg have a strong impact on the activation and proliferation of CD4 (+) and CD8 (+) lymphocytes; however, no previous reports have described the relationship between Treg and the progression of tumor, or Type 1 T cell populations in dogs with malignant tumor. In this study, the percentage of Treg, Th1, and Tc1 in the peripheral blood of dogs with oral malignant melanoma and healthy dogs was measured and compared. Although the percentages of Th1 and Tc1 in dogs with oral malignant melanoma were less than those in healthy dogs (Th1: P < 0.01, Tc1: P < 0.05), the percentage of Treg was increased (P < 0.01). A significant inverse correlation between the percentage of Tc1 and the clinical tumor stage (P < 0.01), and a significant correlation between that of Treg and the clinical tumor stage (P < 0.001) was found. Moreover, there was a significant inverse correlation between the percentages of Treg and Th1 (P < 0.05) or Tc1 (P < 0.001). In conclusion, the percentage of Treg increases with the tumor stage in the peripheral blood of dogs with oral malignant melanoma. In dogs, Treg appears to suppress Type 1 immunity, which may be responsible for anti-tumor responses.


Assuntos
Doenças do Cão/imunologia , Melanoma/veterinária , Neoplasias Bucais/veterinária , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Estudos de Casos e Controles , Doenças do Cão/patologia , Cães , Melanoma/imunologia , Melanoma/patologia , Neoplasias Bucais/imunologia , Neoplasias Bucais/patologia
12.
Microbiol Immunol ; 53(8): 468-74, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19659931

RESUMO

It is well known that lymphocytes from patients with advanced-stage cancer have impaired immune responsiveness and that type1 T lymphocyte subsets in tumor bearing hosts are suppressed. Treg have been reported to comprise a subgroup which inhibits T cell mediated immune responses. In the present study, the percentage of Treg, Th1 and Tc1 in the peripheral blood of tumor bearing dogs with or without metastases was evaluated. The percentages of Th1 and Tc1 in dogs with metastatic tumor were significantly less, and that of Treg was significantly greater, than those of dogs without metastatic tumor. The percentage of Treg showed an inverse correlation with that of Th1 and Tc1 in tumor bearing dogs. It was concluded that an increase in Treg in the peripheral blood of dogs with metastatic tumor may induce suppression of tumor surveillance by the Type1 immune response and lead to metastasis of tumor[0][0].[0].


Assuntos
Sangue/imunologia , Doenças do Cão/imunologia , Metástase Neoplásica , Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Animais , Células Cultivadas , Doenças do Cão/patologia , Cães , Leucócitos Mononucleares/imunologia , Neoplasias/patologia , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia
14.
Biosci Biotechnol Biochem ; 72(7): 1722-30, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18603802

RESUMO

The cysteinyl residue at the active site of L-methionine gamma-lyase from Pseudomonas putida (MGL_Pp) is highly conserved among the heterologous MGLs. To determine the role of Cys116, we constructed 19 variants of C116X MGL_Pp by saturation mutagenesis. The Cys116 mutants possessed little catalytic activity, while their affinity for each substrate was almost the same as that of the wild type. Especially, the C116S, C116A, and C116H variants composed active site catalytic function as measured by the kinetic parameter k(cat) toward L-methionine. Furthermore, the mutagenesis of Cys116 also affected the substrate specificity of MGL_Pp at the active center. Substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a decrease in that toward L-methionine. Propargylglycine inactivated the WT MGL, C116S, and C116A mutants. Based on these results, we postulate that Cys116 plays an important role in the gamma-elimination reaction of L-methionine and in substrate recognition in the MGLs.


Assuntos
Liases de Carbono-Enxofre/metabolismo , Cisteína , Pseudomonas putida/enzimologia , Antineoplásicos , Sítios de Ligação , Liases de Carbono-Enxofre/genética , Catálise , Metionina , Mutagênese Sítio-Dirigida , Especificidade por Substrato/genética
15.
J Biochem ; 141(4): 535-44, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17289792

RESUMO

l-Methionine gamma-lyase (EC 4.4.1.11, MGL_Pp) from Pseudomonas putida is a multifunctional enzyme, which belongs to the gamma-family of pyridoxal-5'-phosphate (PLP) dependent enzymes. In this report, we demonstrate that the three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method to an R-factor of 20.4% at 1.8 A resolution. Detailed information of the overall structure of MGL_Pp supplies a clear picture of the substrate- and PLP-binding pockets. Tyr59 and Arg61 of neighbouring subunits, which are strongly conserved in other gamma-family enzymes, contact the phosphate group of PLP. These residues are important as the main anchor within the active site. Lys240, Asp241 and Arg61 of one partner monomer and Tyr114 and Cys116 of the other partner monomer form a hydrogen-bond network in the MGL active site which is specific for MGLs. It is also suggested that electrostatic interactions at the subunit interface are involved in the stabilization of the structural conformation. The detailed structure will facilitate the development of MGL_Pp as an anticancer drug.


Assuntos
Liases de Carbono-Enxofre/química , Pseudomonas putida/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Liases de Carbono-Enxofre/metabolismo , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência
18.
Paediatr Anaesth ; 15(10): 858-61, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16176314

RESUMO

BACKGROUND: Ultrasound guidance for cannulation of the internal jugular vein has been shown to increase the success rate and reduce the incidence of complications in infants and children. We compared the use of a small caliber audio-Doppler probe with an ultrasound scanner for cannulation of a central venous (CV) line via the right internal jugular vein in infants and children. METHODS: Fifty-two infants and 29 children scheduled for open-heart surgery were enrolled. Cannulation was guided using a small caliber audio-Doppler probe (the AU group, n = 42), or an ultrasound scanner image (the US group, n = 39). Ultimate success rate, success rate at the first attempt, success rate within 5 min, and complications were compared for the two groups. RESULTS: In children (>12 months), both methods were equally efficient. But in infants (<12 months), success rate at the first attempt using audio-Doppler was worse than the rate using an ultrasound scanner and there were more complications when audio-Doppler was used. CONCLUSIONS: We conclude that application of both the audio-Doppler and the ultrasound scanner is useful in children over 1 year of age for access to the internal jugular vein. However, in infants and neonates, the ultrasound scanner would be more useful than the audio-Doppler.


Assuntos
Cateterismo Venoso Central/métodos , Veias Jugulares/diagnóstico por imagem , Anestesia Geral , Procedimentos Cirúrgicos Cardíacos , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Ultrassonografia
19.
Paediatr Anaesth ; 15(1): 47-9, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15649163

RESUMO

BACKGROUND: There are suggested radiographic landmarks for the lower margin of the superior vena cava (SVC), but none for the cephalad origin of the SVC in children. Therefore, we determined the cephalad origin of the SVC in relation to the level of thoracic vertebrae in children. METHODS: Sixty-five patients (2-96 months) scheduled for routine diagnostic cardiac catheterization were the subjects of the study. Vena cavogram was obtained. The crossing point between the SVC and the innominate vein was considered as the cephalad origin of the SVC, then this point was related to the level of the thoracic vertebra as a radiographic landmark. RESULTS: In approximately 90% of the patients studied the proximal origin of the SVC was situated above the level of Th4/5 interspace. CONCLUSION: We would like to suggest that the position of the tip of central venous line, when inserted via the right internal jugular vein, should optimally be at the level of Th4/5 interspace on the postoperative chest radiograph.


Assuntos
Cateterismo Venoso Central/métodos , Radiografia Torácica/métodos , Vértebras Torácicas/diagnóstico por imagem , Veia Cava Superior/diagnóstico por imagem , Criança , Pré-Escolar , Sedação Consciente , Feminino , Cardiopatias Congênitas/diagnóstico por imagem , Cardiopatias Congênitas/cirurgia , Humanos , Lactente , Masculino , Flebografia , Postura
20.
Paediatr Anaesth ; 14(9): 744-7, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15330956

RESUMO

BACKGROUND: We evaluated an audio-Doppler with a small-caliber probe as a guide for central venous cannulation (CVC) via the internal jugular vein (IJV) in infants and children. METHODS: The right IJV was located with a small-caliber (2.0 mm in diameter) audio-Doppler probe using 10 MHz ultrasound. The probe was placed on the neck about the level of sixth cervical vertebra and was moved until the crisp pulsatile sound of the carotid artery was identified. Then the probe was moved laterally to identify the low-pitched venous hum of the right IJV. After marking the puncture site on the skin, a sterile cannulation procedure was performed. Ultimate success rate, cannulation time < 10 min, successful cannulation within three punctures, and complications were recorded. RESULTS: Ultimate success rate was 65.6% (42/64) in infants (< 12 m), and 94.7% (72/76) in children (12 m or older). Cannulation time < 10 min was 48.4% in infants, and 85.5% in children. Successful cannulation within three punctures was 45.3% in infants and 82.8% in children. Three carotid arterial punctures occurred. CONCLUSIONS: We were not able to demonstrate absolute superiority of the results utilizing this device over the reported results of traditional landmark techniques for CVC via the right IJV. However, this device may contribute to reducing complications and be of value in teaching residents where to insert a needle for an internal jugular puncture.


Assuntos
Cateterismo Venoso Central/métodos , Veias Jugulares/diagnóstico por imagem , Punções/instrumentação , Ultrassonografia Doppler/instrumentação , Ultrassonografia Doppler/métodos , Anestesia Geral , Procedimentos Cirúrgicos Cardíacos/métodos , Pré-Escolar , Decúbito Inclinado com Rebaixamento da Cabeça/fisiologia , Humanos , Lactente , Intubação Intratraqueal
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