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1.
Braz. j. med. biol. res ; 51(1): e6536, 2018. tab, graf
Article in English | LILACS | ID: biblio-889004

ABSTRACT

Kidney stone disease is a major cause of chronic renal insufficiency. The role of long non-coding RNAs (lncRNAs) in calcium oxalate-induced kidney damage is unclear. Therefore, we aimed to explore the roles of lncRNAs in glyoxylate-exposed and healthy mouse kidneys using microarray technology and bioinformatics analyses. A total 376 mouse lncRNAs were differentially expressed between the two groups. Using BLAST, 15 lncRNA homologs, including AU015836 and CHCHD4P4, were identified in mice and humans. The AU015836 expression in mice exposed to glyoxylate and the CHCHD4P4 expression in human proximal tubular epithelial (HK-2) cells exposed to calcium oxalate monohydrate were analyzed, and both lncRNAs were found to be upregulated in response to calcium oxalate. To further evaluate the effects of CHCHD4P4 on the cell behavior, we constructed stable CHCHD4P4-overexpressing and CHCHD4P4-knockdown HK-2 cells. The results showed that CHCHD4P4 inhibited cell proliferation and promoted the epithelial-mesenchymal transition in kidney damage and fibrosis caused by calcium oxalate crystallization and deposition. The silencing of CHCHD4P4 reduced the kidney damage and fibrosis and may thus be a potential molecular target for the treatment of kidney stones.


Subject(s)
Humans , Animals , Rabbits , Kidney Calculi/genetics , Mitochondrial Membrane Transport Proteins/physiology , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , RNA, Long Noncoding/physiology , Fibrosis , Calcium Oxalate , Kidney Calculi/physiopathology , Up-Regulation , Cell Fractionation , Cell Line , Blotting, Western , Microarray Analysis , Cell Proliferation/physiology , Epithelial-Mesenchymal Transition/physiology , Real-Time Polymerase Chain Reaction
2.
Immune Network ; : 373-380, 2016.
Article in English | WPRIM | ID: wpr-199704

ABSTRACT

Peroxiredoxin-3 (Prdx3) is a mitochondrial protein of the thioredoxin family of antioxidant peroxidases and is the principal peroxidase responsible for metabolizing mitochondrial hydrogen peroxide. Recent reports have shown that mitochondrial reactive oxygen species (mROS) contribute to macrophage-mediated bactericidal activity in response to Toll-like receptors. Herein, we investigated the functional effect of Prdx3 in bactericidal activity. The mitochondrial localization of Prdx3 in HEK293T cells was confirmed by cell fractionation and confocal microscopy analyses. To investigate the functional role of Prdx3 in bactericidal activity, Prdx3-knockdown (Prdx3(KD)) THP-1 cells were generated. The mROS levels in Prdx3(KD) THP-1 cells were significantly higher than those in control THP-1 cells. Moreover, the mROS levels were markedly increased in response to lipopolysaccharide. Notably, the Salmonella enterica serovar Typhimurium infection assay revealed that the Prdx3(KD) THP-1 cells were significantly resistant to S. Typhimurium infection, as compared with control THP-1 cells. Taken together, these results indicate that Prdx3 is functionally important in bactericidal activity through the regulation of mROS.


Subject(s)
Cell Fractionation , Humans , Hydrogen Peroxide , Lipopolysaccharides , Microscopy, Confocal , Mitochondrial Proteins , Peroxidase , Peroxidases , Reactive Oxygen Species , Salmonella enterica , Serogroup , Thioredoxins , Toll-Like Receptors
3.
Dental press j. orthod. (Impr.) ; 20(2): 119-125, Mar-Apr/2015. graf
Article in English | LILACS | ID: lil-745849

ABSTRACT

INTRODUCTION: The finite element method (FEM) is an engineering resource applied to calculate the stress and deformation of complex structures, and has been widely used in orthodontic research. With the advantage of being a non-invasive and accurate method that provides quantitative and detailed data on the physiological reactions possible to occur in tissues, applying the FEM can anticipate the visualization of these tissue responses through the observation of areas of stress created from applied orthodontic mechanics. OBJECTIVE: This article aims at reviewing and discussing the stages of the finite element method application and its applicability in Orthodontics. RESULTS: FEM is able to evaluate the stress distribution at the interface between periodontal ligament and alveolar bone, and the shifting trend in various types of tooth movement when using different types of orthodontic devices. Therefore, it is necessary to know specific software for this purpose. CONCLUSIONS: FEM is an important experimental method to answer questions about tooth movement, overcoming the disadvantages of other experimental methods. .


INTRODUÇÃO: o Método de Elementos Finitos (MEF) é um recurso da Engenharia empregado para calcular o estresse e a deformação de estruturas complexas, e tem sido amplamente utilizado nas pesquisas em Ortodontia. Apresenta a vantagem de ser um método não-invasivo e preciso, que fornece dados quantitativos e detalhados acerca das reações fisiológicas que podem ocorrer nos tecidos. OBJETIVO: esse artigo pretende realizar uma revisão da literatura sobre as etapas para realização do Método de Elementos Finitos, bem como de sua aplicabilidade na Ortodontia. RESULTADOS: o MEF é capaz de avaliar a distribuição do estresse na interface entre o ligamento periodontal e o osso alveolar, bem como a tendência de deslocamento em diversos tipos de movimentos dentários, quando utilizados diferentes tipos de aparelhos. Para tanto, é necessário conhecimento de softwares específicos para esse fim. CONCLUSÕES: o MEF é um importante método experimental que pode esclarecer questionamentos acerca da movimentação dentária, superando as desvantagens de outros métodos experimentais. .


Subject(s)
Cell Fractionation/methods , Saccharomyces cerevisiae/chemistry , Centrifugation, Density Gradient , Organelles/chemistry , Reproducibility of Results , Subcellular Fractions , Time Factors
4.
Dental press j. orthod. (Impr.) ; 20(1): 45-51, Jan-Feb/2015. tab, graf
Article in English | LILACS | ID: lil-741446

ABSTRACT

INTRODUCTION: The consensus about the relationship between TMD and orthodontic treatment has gone from a cause and effect association between TMD and orthodontic treatment to the idea that there is no reliable evidence supporting this statement. OBJECTIVE: To assess the beliefs, despite scientific evidence, of Brazilian orthodontists about the relationship between TMD and orthodontic treatment with regards to treatment, prevention and etiology of TMD. METHODS: A survey about the relationship between TMD and orthodontic treatment was prepared and sent to Brazilian orthodontists by e-mail and social networks. Answers were treated by means of descriptive statistics and strong associations between variables were assessed by qui-square test. RESULTS: The majority of orthodontists believe that orthodontic treatment not only is not the best treatment option for TMD, but also is not able to prevent TMD. Nevertheless, the majority of orthodontists believe that orthodontic treatment can cause TMD symptoms. CONCLUSION: This study suggests that orthodontists' beliefs about the relationship between orthodontic treatment and TMD are in accordance with scientific evidence only when referring to treatment and prevention of TMD. The majority of orthodontists believe that, despite scientific evidence, orthodontic treatment can cause TMD. .


INTRODUÇÃO: o consenso sobre a relação entre DTM e tratamento ortodôntico foi de uma associação de causa e efeito à ideia de que não há evidências confiáveis que suportem essa afirmação. OBJETIVO: avaliar as crenças, sem considerar as evidências, de ortodontistas brasileiros sobre a relação entre DTM e tratamento ortodôntico com relação ao tratamento, prevenção e etiologia da DTM. MÉTODOS: um questionário sobre a relação entre DTM e tratamento ortodôntico foi preparado e enviado a ortodontistas brasileiros por meio de e-mail e mídias sociais. As respostas foram analisadas por estatística descritiva, e fortes associações entre as variáveis foram verificadas pelo teste χ2. RESULTADOS: a maioria dos ortodontistas acredita que o tratamento ortodôntico não é o melhor tratamento para DTM. Além disso, acreditam que não é a melhor forma para sua prevenção. Também, a maioria dos ortodontistas acredita que o tratamento ortodôntico pode causar sintomas de DTM. CONCLUSÃO: este estudo sugere que as crenças dos ortodontistas sobre a relação entre tratamento ortodôntico e DTM estão de acordo com as evidências científicas apenas quando se trata do tratamento e da prevenção de DTM. A maioria dos ortodontistas acredita que, apesar das evidências científicas, o tratamento ortodôntico pode causar DTM. .


Subject(s)
Humans , Cell Cycle Proteins/metabolism , DNA Replication/genetics , Forkhead Transcription Factors/metabolism , G1 Phase/physiology , Gene Expression Regulation/genetics , Protein Serine-Threonine Kinases/metabolism , Replication Origin/genetics , Signal Transduction/genetics , Blotting, Western , Cell Fractionation , Cell Line , Cell Cycle Proteins/genetics , /metabolism , DNA Primers/genetics , Fluorescent Antibody Technique , Forkhead Transcription Factors/genetics , Immunoblotting , Immunoprecipitation , Intercellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-myc/metabolism , Reverse Transcriptase Polymerase Chain Reaction , RNA Interference
5.
Article in Korean | WPRIM | ID: wpr-49419

ABSTRACT

PURPOSE: Poncirus trifoliata has been reported to have anti-inflammatory, antioxidant, and immune activities. However, its anti-obesity activity and the mechanism by which the water extract of dried, immature fruit of Poncirus trifoliata (PF-W) acts are not clear. This study suggests a potential mechanism associated with the anti-obesity activity of PF-W. METHODS: We measured the effect of PF-W on lipoprotein lipase (LPL) regulation using enzyme-linked immunosorbent assay (ELISA) and an activity assay. The LPL regulation mechanism was examined by reverse transcription polymerase chain reaction (RT-PCR) to measure the mRNA expression of biomarkers related to protein transport and by western blot for analysis of the protein expression of the transcription factor CCAAT-enhancer-binding protein (C/EBPbeta) RESULTS: The total polyphenol and flavonoid content of PF-W was 52.15 +/- 4.02 and 6.56 +/- 0.47 mg/g, respectively. PF-W treatment decreased LPL content in media to 58 +/- 5% of that in control adipocyte media, and increased LPL content to 117 +/- 3.5% of that in control adipocytes, but did not affect the mRNA expression of LPL. PF-W also increased the mRNA expression of sortilin-related receptor (SorLA), a receptor that induces endocytosis and intracellular trafficking of LPL, in a concentration- and time-dependent manner. Finally, cell fractionation revealed that PF-W treatment induced the expression of C/EBPbeta, a SorLA transcription factor, in the nuclei of 3T3-L1 adipocytes. CONCLUSION: The LPL secretion and activity assay showed PF-W to be an LPL secretion inhibitor, and these results suggest the potential mechanism of PF-W involving inhibition of LPL secretion through C/EBPbeta-mediated induction of SorLA expression.


Subject(s)
Adipocytes , Biomarkers , Blotting, Western , CCAAT-Enhancer-Binding Proteins , Cell Fractionation , Endocytosis , Enzyme-Linked Immunosorbent Assay , Fruit , Lipoprotein Lipase , Polymerase Chain Reaction , Poncirus , Protein Transport , Reverse Transcription , RNA, Messenger , Transcription Factors , Water
6.
Rio de Janeiro; s.n; 2015. ix, 104 p. ilus, tab, graf.
Thesis in Portuguese | LILACS | ID: biblio-971487

ABSTRACT

A Leishmania (Leishmania) amazonensis apresenta mecanismos de adaptação guiados porsuas proteinases. Neste contexto, destacam-se as cisteína proteinases (CPs) onde acisteína proteinase B (CPB) é a CP mais estudada dentre as CPs deste parasito. O focodeste trabalho é a região COOH-terminal da CPB (cyspep), gerada a partir doprocessamento final desta proteinase. O estudo foi conduzido em cinco fases: obtenção dacyspep recombinante (rcyspep); avaliação da antigenicidade da rcyspep em camundongosexperimentalmente infectados com L.(L.) amazonensis; diferenciação in vitro dospromastigotas em amastigotas; avaliação da expressão do gene cpb ao longo dadiferenciação do parasito; e detecção da cyspep nas formas promastigotas e amastigotas doparasito. O polipeptídeo rcyspep, de 14 kDa, foi obtida em sistema pET28-a e purificadopara posterior produção de anticorpos policlonais específicos em camundongos. Observouseque, durante a infecção em camundongos, há uma resposta imune humoral contra orcyspep, caracterizada por um aumento da detecção de anti-cyspep quando comparado comanimais de controle (p <0,05). Nos ensaios de diferenciação constatamos três padrõesmorfológicos ao logo de 96 horas de análise, em uma cinética temporal: formas alongadascom flagelo livre, arredondados com flagelo e arredondadas sem flagelo por microscopia.Adicionalmente a cultura foi analisada por citometria de fluxo demonstrando uma migraçãogradual da população da região R1 para região R2 do dotplot indicando uma diminuição notamanho e aumento da granulosidade celular. Observamos que ao longo destadiferenciação ocorre um aumento da quantidade de transcritos do gene cpb, nos tempos de48 horas (1,3 ×), 72 horas (3,2 ×) e 96 horas (3,4 ×) nas preparações de cDNA dosparasitos, quando comparada com os resultados de quantificação realizados compreparações de cDNA dos promastigotas...


Leishmania (Leishmania) amazonensis presents adaptive mechanisms guided by theirproteinases. In this context, we highlight the cysteine proteinases (CPs) where the cysteineproteinase B (CPB) is the most studied CP among these parasites. The focus of this work isthe COOH-terminal region of CPB (cyspep), generated during the final processing stage ofthis proteinase. Our study comprises five main phases: obtaining of a recombinant cyspeppolypeptide (rcyspep); evaluation of the antigenicity of rcyspep in mice experimentallyinfected with L. (L.) amazonensis; in vitro differentiation of promastigotes into amastigotes;evaluation of cpb gene expression throughout morphological differentiation of the parasite;and detection of cyspep in promastigotes and amastigotes of L. (L.) amazonensis. Thercyspep polypeptide, with 14 kDa, was obtained using a pET28-a system and, subsequently,specific polyclonal antibodies were produced by inoculation in mice. We observed that,during mice infection, there is a humoral immune response against rcyspep, characterized byan increase in detection of anti-cyspep when compared to control animals (p<0.05). Duringdifferentiation assays three morphological patterns could be observed over 96 hours ofobservation: elongated shapes with free flagellum, rounded shapes with free flagellum androunded shapes without visible flagellum. The data point to a gradual migration of thepopulation of parasites from region R1 to region R2 into the dotplot indicating a decrease insize and increase in cell granularity. We observed that alongside the morphologicaldifferentiation there is an increase in the amount of cpb gene transcripts, in time of 48 hours(1.3 ×), 72 hours (3.2 ×) and 96 hours (× 3.4), compared with quantitation results obtainedwith cDNA of promastigotes...


Subject(s)
Humans , Leishmania , Cysteine Proteases , Cell Fractionation , Surface Plasmon Resonance
7.
Braz. dent. j ; 25(5): 447-450, Sep-Oct/2014. graf
Article in English | LILACS | ID: lil-731048

ABSTRACT

The radicular cyst is an inflammatory odontogenic cyst of endodontic origin. Radiographically, the lesion appears as a periapical radiolucent image. This report describes a very rare case of a mixed periapical radiographic image diagnosed as a radicular cyst. A 37-year-old female patient presented a mixed, well-circumscribed image located in the periapical region of the left maxillary central incisor, which presented unsatisfactory endodontic treatment. Microscopic examination revealed a cavity lined by non-keratinized squamous epithelium and extensive calcifications in the cystic lumen and lining epithelium. Diagnosis of radicular cyst with extensive calcifications was established. Endodontic retreatment was performed and no radiographic signs of recurrence were observed 18 months after treatment. Although very rare, a radicular cyst should be considered in the differential diagnosis of a mixed periapical image associated to teeth with pulp necrosis.


O cisto radicular é um cisto odontogênico inflamatório de origem endodôntica. Radiograficamente, a lesão se apresenta como uma imagem radiolúcida periapical. Este relato descreve um caso muito raro de uma imagem radiográfica periapical mista diagnosticada como cisto radicular. Uma paciente de 37 anos de idade, do gênero feminino, apresentava uma imagem mista, bem circunscrita, localizada na região periapical do incisivo central superior esquerdo, que apresentava tratamento endodôntico insatisfatório. Avaliação microscópica revelou uma cavidade revestida por epitélio escamoso não-queratinizado e calcificações extensas na cavidade cística e revestimento epitelial. O diagnóstico de cisto radicular com extensas calcificações foi estabelecido. Retratamento endodôntico foi realizado e não foram observados sinais radiográficos de recorrência da lesão após 18 meses de tratamento. Embora muito raro, um cisto radicular deve ser considerado no diagnóstico diferencial de uma imagem periapical mista associada a dentes com necrose pulpar.


Subject(s)
Animals , Mice , Cellular Senescence/physiology , Genes, ras/genetics , MAP Kinase Signaling System/physiology , Nuclear Proteins , /metabolism , Cell Fractionation , Cells, Cultured , Colony-Forming Units Assay , Cell Cycle/physiology , Enzyme Activation , Embryo, Mammalian/physiology , Fibroblasts/cytology , Fibroblasts/metabolism , MAP Kinase Kinase 1 , Mice, Knockout , Microscopy, Fluorescence , Mitogen-Activated Protein Kinase Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Temperature , /metabolism , ras Proteins/metabolism
8.
Electron. j. biotechnol ; 16(3): 9-9, May 2013. ilus, tab
Article in English | LILACS | ID: lil-684007

ABSTRACT

Background: Recombinant proteins, including antibodies and antibody fragments, often contain disulfide bond bridges that are necessary for their folding, stability and function. Production of disulfide-bond-containing proteins in the periplasm of Escherichia coli has been very useful, due to unique characteristics of the periplasm, for obtaining fully active and correctly folded products and for alleviating downstream processing. Results: In this study, fed-batch cultivation of Escherichia coli (E. coli) for production of Fab D1.3, which is an anti-hen egg white lysozyme (HEWL) antibody fragment was carried out at 37ºC, and the bacterial cells were induced by adding 0.1 mM IPTG to the culture medium. Fermentor was sampled over the course of fermentation; the bacterial cells were centrifugally separated from the culture broth and subjected to osmotic shock (with excluding HEWL) and sonication procedures. The resulting fractions were analysed for Fab using a combination of ELISA, SDS-PAGE and Western blotting and changes in product titre, location, and form was assessed throughout growth. It was shown that osmotic shock released the Fab from the periplasm very efficiently and its efficacy was 20-45% more than sonication. This study demonstrates that, at high cell density cultivation in fermentor, target product can appear inside and outside the cells, depending on the time of induction. The maximum amount of Fab (47 mg/l) in the periplasm was reached at 14 hrs cultivation (4 hrs post induction), being suitable time for cell harvest, selective periplasmic extraction and downstream capture. The Fab increasingly leaked into the culture medium, and reached its maximum culture medium titre of ~78 mg/l after 6 hrs post induction. After 16 hrs cultivation (6 hrs post induction) the amount of Fab remained constant in different locations within and outside the cells. Western blot analysis of cell fractions showed that certain amount of the Fab was also produced in the cells as insoluble form. Conclusions: In this work we showed that the production of Fab in the periplasm during high cell density cultivation of E. coli in fermentor can be challenging as the product may appear in various locations within and outside the cells. To exploit the advantages of the periplasmic expression systems for purification in downstream processing, bacterial cells should be harvested when they maintain the majority of the target protein in their periplasmic space (i.e. 4 hrs post induction).


Subject(s)
Immunoglobulin Fragments/biosynthesis , Escherichia coli/metabolism , Recombinant Proteins/biosynthesis , Enzyme-Linked Immunosorbent Assay , Cell Fractionation , Blotting, Western , Biomass , Electrophoresis, Polyacrylamide Gel/methods , Fermentation , Batch Cell Culture Techniques
9.
São Paulo; s.n; s.n; 2013. 198 p. tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-846927

ABSTRACT

Algumas das estratégias utilizadas para entender a biologia de células tronco embrionária (CTE) são baseadas na identificação de cascatas de sinalização que induzem a diferenciação e auto-renovação das CTE através da interferência seletiva de processos específicos. A família das proteínas quinase C (PKC) é conhecida por participar dos processos de auto-renovação e diferenciação celular em CTE, entretanto, o papel específico das diferentes isoenzimas das PKCs ainda precisa ser elucidado. Desta forma investigamos. o papel das PKCs atípicas (aPKCs) em CTE indiferenciadas utilizando um inibidor específico para estas serina/ treonina quinases, o peptídeo pseudossubstrato das aPKCs, e fosfoproteômica. A maioria das proteinas identificadas cuja fosforilação reduziu após o tratamento com o inibidor das aPKC, são proteínas envolvidas com o metabolismo principalmente com a via glicolítica. Além disso, a inibição das aPKCs levou a redução do consumo de glicose, secreção de lactato, acompanhada da redução da atividade da lactato desidrogenase, e aumento da fosforilação oxidativa, sendo analisada através do consumo de oxigênio após o tratamento com oligomicina e FCCP. Verificamos também que as aPKCs são capazes de fosforilar diretamente a piruvato quinase. A glicólise aeróbica parece ser fundamental para a manutenção da indiferenciação das CTE, e demonstramos que as aPKCs participam deste processo auxiliando na auto-renovação das CTE indiferenciadas. Também observamos que as aPKCs assim como a PKCßI modulam a fosforilação da α-tubulina, porém ao passo que as aPKCs interagem com a α-tubulina durante a interfase, a PKCßI interage com a mesma apenas durate a mitose. Estes resultados motivaram a segunda parte da tese, na qual o papel da fosforilação da α-tubulina pela PKCßI foi investigado. O resíduo de treonina 253, conservado em diversas espécies de vertebrados e localizado na interface de polimerização entre a α- e a ß-tubulina foi identificado, como um novo sítio de fosforilação da α-tubulina pela PKCßI. Este sítio não está em um consenso linear para a PKC, entretanto é um consenso formado estruturalmente, onde aminoácidos básicos distantes na sequência linear se tornam justapostos na estrutura terciária da proteína. Estudos de simulação por dinâmica molecular demonstraram que a interação entre a α e ß-tubulina aumenta após esta fosforilação, uma vez que T253 fosforilada passa a interagir com K105, um residuo conservado na ß-tubulina. A fosforilação in vitro de α-tubulina aumenta a taxa de polimerização da tubulina e a inibição da PKCßI em células reduziu a taxa de repolimerização do microtubulo após o tratamento com nocodazol. Além disso, a importância da fosforilação deste sítio foi demonstrada pelo fato de que um mutante fosfomimético GFP-α-tubulina, T253E ser mais incorporado no fuso mitótico ao passo que T253A foi menos incorporado do que a proteína selvagem. Nossos dados suportam a hipótese que os consensos estruturais formados podem ser importantes sítios de reconhecimento pelas quinases e que a fosforilação de T253 da α-tubulina afeta a estabilidade do polímero. Em conclusão, utilizando métodos de fosfoproteômica e interferência seletiva de vias de sinalização, combinados a validações experimentais dos alvos identificados podemos propor a importância funcional das aPKCs e PKCßI em CTE indiferenciadas


Some of the strategies used to understand stem cell biology are based on the identification of signalling cascades that lead to differentiation and self-renewal of embryonic stem cells (ESC) by selective interference of specific signalling processes. The protein kinase C (PKC) family is known to participate in ESC self-renewal and differentiation, however, the specific role of the different PKC isoenzymes in these cells remains to be determined. Therefore, we investigated the role of atypical PKCs (aPKC) in undifferntiated ESC using a specific inhibitor for these serine/ threonine kinases, pseudo-substrate peptide of aPKCs, and phosphoproteomics. The majority of proteins whose phosphorylation decreased upon aPKC inhibition, are proteins involved in metabolism in particular with the glycolytic pathway. Besides that, inhibiton of aPKCs led to a decrease in glucose uptake and lactate secretion, followed by a decrease in lactate dehydrogenase activity, and an increase in mitochondrial activity as measured by oxygen consumption after treatment with olygomycin and a chemical uncoupler. We also verified that aPKCs are able to directly phosphorylated pyruvate kinase. Aerobic glicolysis seems to be fundamental for the maintainance of undifferentiated ESC, and we demonstrated that aPKCs participte in these processes helping to maintain self-renewal of undifferentiated ESC. We also observed that aPKCs as PKCßI modulate the phosphorylation of α-tubulin, however, while aPKCs interact with α-tubulin during interfase PKCßI interacts with α-tubulin only during mitosis. These results lead to the second part of this thesis. We investigated the role of α-tubulina phosphorylation by PKCßI. Indentifying threonine 253, a conserved residue in several vertebrate species, of localized at the polymerization interface between α- and ß-tubulin, as a phosphorylation site of α-tubulin by PKCßI. This site is not in a linear consensus for PKC, however, it is in a structuraly formed consensus, where basic aminoacids distant in the linear sequence are juxtaposed in the three dimentional protein structure. Simulation studies by molecular dynamics show that the interaction between α and ß-tubulin increases upon this phosphorylation, once, phosphorylated T253 interacts with com K105, a conserved residue in ß-tubulin. The in vitro phosphorylation of α-tubulin increased tubulin polymerization rate and inhibiton of PKCßI in cells reduced repolimeration rate of microtubles upon treatment with nocodazole. Besides that, the importance of this phosphorylation site were demonstrated by the fact that a phosphomimetic mutant GFP-α-tubulina, T253E is more incorporated in mitotic fuses while T253A is less than wild type. Our data support the hypothesis that structural consensus may be important sites recognized and that T253 phosphorylation of α-tubulin afects the polymer stability. In conclusion, using phosphoproteomics methods and selective interference of signal transduction pathways combined with experimental validation studies of the identified targets we can propose roles for aPKCs and PKCßI in undifferentiated ESC


Subject(s)
Embryonic Stem Cells/classification , Protein Kinase C beta/analysis , Validation Study , Cell Fractionation/methods , Metabolism/genetics , Nocodazole/analysis , Phosphorylation/genetics , Protein Kinase C/analysis , Usage Remodeling , Tubulina/growth & development , Two-Dimensional Difference Gel Electrophoresis/methods
10.
Chinese Journal of Biotechnology ; (12): 532-535, 2013.
Article in Chinese | WPRIM | ID: wpr-233223

ABSTRACT

Cell-free protein expression system is a new method to express target protein in vitro and has been widely applied to the study of protein structure, protein function and other related fields. Preparation of cell extract is one of the key factors that affect the efficiency of the cell-free system. To improve the efficiency and economical feasibility of cell-free protein synthesis, we discussed the parameters during the preparation of the cell extract. These parameters include centrifugation speed, pre-incubation, and dialysis. We used the green fluorescent protein as the reporter protein, and obtained a simple procedure for the preparation of Escherichia coli cell extract. A simple centrifugation step (12 000 x g, 10 min) followed by a brief incubation was sufficient for the preparation of an active cell extract to support protein expression with higher productivity (209 microg/mL). Compared to the traditional E. coli S30 procedure, the processing time was reduced by 62%, and the productivity was increased by 2.6 times. The new procedure will make the advantage of cell-free technology more obvious, and promote its wider application.


Subject(s)
Cell Fractionation , Methods , Cell-Free System , Escherichia coli , Cell Biology , Genetics , Metabolism , Escherichia coli Proteins , Chemistry , Green Fluorescent Proteins , Metabolism
11.
Article in English | WPRIM | ID: wpr-50000

ABSTRACT

In the previous review, the physical aspect of heavy particles, with a focus on the carbon beam was introduced. Particle beam therapy has many potential advantages for cancer treatment without increasing severe side effects in normal tissue, these kinds of radiation have different biologic characteristics and have advantages over using conventional photon beam radiation during treatment. The relative biological effectiveness (RBE) is used for many biological, clinical endpoints among different radiation types and is the only convenient way to transfer the clinical experience in radiotherapy with photons to another type of radiation therapy. However, the RBE varies dependent on the energy of the beam, the fractionation, cell types, oxygenation status, and the biological endpoint studied. Thus this review describes the concerns about RBE related to particle beam to increase interests of the Korean radiation oncologists' society.


Subject(s)
Carbon , Cell Fractionation , Oxygen , Photons , Population Characteristics , Protons , Relative Biological Effectiveness
12.
Braz. j. med. biol. res ; 44(11): 1156-1163, Nov. 2011. ilus
Article in English | LILACS | ID: lil-604283

ABSTRACT

We evaluated the potential neuroprotective effect of 1-100 µM of four organoselenium compounds: diphenyl diselenide, 3’3-ditri-fluoromethyldiphenyl diselenide, p-methoxy-diphenyl diselenide, and p-chloro-diphenyl diselenide, against methylmercury-induced mitochondrial dysfunction and oxidative stress in mitochondrial-enriched fractions from adult Swiss mouse brain. Methylmercury (10-100 µM) significantly decreased mitochondrial activity, assessed by MTT reduction assay, in a dose-dependent manner, which occurred in parallel with increased glutathione oxidation, hydroperoxide formation (xylenol orange assay) and lipid peroxidation end-products (thiobarbituric acid reactive substances, TBARS). The co-incubation with diphenyl diselenide (100 µM) completely prevented the disruption of mitochondrial activity as well as the increase in TBARS levels caused by methylmercury. The compound 3’3-ditrifluoromethyldiphenyl diselenide provided a partial but significant protection against methylmercury-induced mitochondrial dysfunction (45.4 ± 5.8 percent inhibition of the methylmercury effect). Diphenyl diselenide showed a higher thiol peroxidase activity compared to the other three compounds. Catalase blocked methylmercury-induced TBARS, pointing to hydrogen peroxide as a vector during methylmercury toxicity in this model. This result also suggests that thiol peroxidase activity of organoselenium compounds accounts for their protective actions against methylmercury-induced oxidative stress. Our results show that diphenyl diselenide and potentially other organoselenium compounds may represent important molecules in the search for an improved therapy against the deleterious effects of methylmercury as well as other mercury compounds.


Subject(s)
Animals , Male , Mice , Brain/drug effects , Membrane Potential, Mitochondrial/drug effects , Mercury Poisoning, Nervous System/prevention & control , Methylmercury Compounds/toxicity , Mitochondria/drug effects , Neuroprotective Agents/pharmacology , Organoselenium Compounds/pharmacology , Oxidative Stress/drug effects , Analysis of Variance , Benzene Derivatives/pharmacology , Cell Fractionation , Models, Animal , Neuroprotective Agents/classification , Organoselenium Compounds/chemistry
13.
Rev. argent. microbiol ; 42(1): 57-62, feb. 2010. graf, tab
Article in English | LILACS | ID: lil-634647

ABSTRACT

Polygalacturonase (PG) production by Fomes sclerodermeus using solid-state fermentation (SSF) was carried out. Maximal PG activity (26 U/gdw) was obtained between days 11 and 13 at the end of exponential growth. PG activity in the crude extract was more stable at pH 5-6 and 30 °C and had optimum activity at pH 5 and 50 °C. Optimal conditions for PG extraction were: one time extraction with Na2SO4 as solvent with 10 min. of agitation. In a scale-up system, PG activity per gram of dry substrate decreased about 60% compared with the activity obtained in an Erlenmeyer flask; however, high total PG activity was obtained.


Se estudió la producción de poligalacturonasa (PG) por Fomes sclerodermeus usando técnicas de fermentación en estado sólido. La actividad PG máxima (26 U/g ps) fue observada entre los días 11 y 13. La actividad PG en los extractos crudos fue más estable a pH 5-6 y 30 °C, con una actividad óptima a pH 5 y a 50 °C. Las condiciones óptimas para la extracción de PG se lograron con una única extracción empleando Na2SO4 como solvente, con 10 minutos de agitación. En el escalado del sistema, la actividad PG por gramo de peso seco de sustrato disminuyó cerca de 60% comparada con la obtenida en frascos Erlenmeyer, pero la actividad total fue mayor.


Subject(s)
Coriolaceae/enzymology , Fungal Proteins/isolation & purification , Polygalacturonase/isolation & purification , Cell Fractionation/methods , Coriolaceae/growth & development , Fermentation , Hydrogen-Ion Concentration , Mycology/methods , Solvents , Temperature
14.
J. appl. oral sci ; 17(2): 113-115, Mar.-Apr. 2009. ilus
Article in English | LILACS | ID: lil-503988

ABSTRACT

This study evaluated quantitatively and qualitatively the effect of the storage time of samples before the application of the cell lysis solution (CLS) for extracting DNA from buccal cells (BC). BC from the upper and lower gutter region were collected from 5 volunteers using special cytobrushes (Gentra), totaling 3 collections for each individual. In the control group (n=10), CLS was applied soon after BC collection. In the other two groups, samples were stored at room temperature (n=10) or at 4°C (n=10). After CLS application, DNA was extracted according to the manufacturer's instructions (Puregene DNA Buccal Cell Kit; Gentra Systems, Inc.). The DNA obtained was evaluated by two calibrated blind examiners using spectrophotometry and analysis of DNA bands (0.8 percent agarose gel electrophoresis). The obtained data were submitted to one-way ANOVA. The means and standard deviations for DNA extracted under immediate, room temperature and cooling temperature conditions were 3.5 ± 0.7, 3.0 ± 0.6 and 4.1 ± 1.8 µg, respectively (p=0.385). No significant differences were found in relation to the amount of DNA for the different storage conditions. However, in the visual analysis of the DNA bands, no trace of DNA degradation was detected when CSL was applied soon after DNA collection, while DNA bands with degradation could be observed in the other groups. Within the limitations of the study, it may be concluded that CLS should be applied soon after DNA collection in order to obtain high-quality DNA from BC.


Subject(s)
Humans , DNA , Mouth Mucosa/cytology , Tissue Preservation/methods , Cell Fractionation/methods , DNA Degradation, Necrotic , Specimen Handling/methods , Temperature , Time Factors
15.
Article in English | WPRIM | ID: wpr-151021

ABSTRACT

Malaria parasites adapt to the oxidative stress during their erythrocytic stages with the help of vital thioredoxin redox system and glutathione redox system. Glutathione reductase and thioredoxin reductase are important enzymes of these redox systems that help parasites to maintain an adequate intracellular redox environment. In the present study, activities of glutathione reductase and thioredoxin reductase were investigated in normal and Plasmodium berghei-infected mice red blood cells and their fractions. Activities of glutathione reductase and thioredoxin reductase in P. berghei-infected host erythrocytes were found to be higher than those in normal host cells. These enzymes were mainly confined to the cytosolic part of cell-free P. berghei. Full characterization and understanding of these enzymes may promise advances in chemotherapy of malaria.


Subject(s)
Animals , Antioxidants/isolation & purification , Cell Fractionation , Cytosol/enzymology , Erythrocytes/parasitology , Glutathione Reductase/isolation & purification , Mice , Plasmodium berghei/enzymology , Thioredoxin-Disulfide Reductase/isolation & purification
16.
Asian Pac J Allergy Immunol ; 2008 Jun-Sep; 26(2-3): 129-36
Article in English | IMSEAR | ID: sea-36988

ABSTRACT

Several proteins of rubber latex have been recognized as allergens causing immediate hypersensitivity in humans. In this study, a bottom fraction membrane (BFM) protein preparation from Hevea brasiliensis trees grown in southern Thailand was used to detect specific IgE in four groups of serum samples. The first group included 170 samples of latex glove factory workers (LGWs); group 2 consisted of the sera of 35 health care workers (HCWs) who were repeatedly exposed to powdered latex gloves; groups 3 and 4 were 31 positive and 22 negative sera, respectively, obtained from Johns Hopkins University School of Medicine, Baltimore, USA, tested for IgE to latex allergen. It was found that 56/170 (33%), 5/35 (14%), 11/31 (35.5%) and 1/22 (4.5%) samples of the LGWs, HCWs, CAP+ and CAP- groups had significant IgE to the BFM proteins, respectively. However, of all subjects only one subject of group 1 had experienced allergic morbidity consisting of eczema, conjunctivitis and asthma. The IgE of this subject bound to a 55 kDa component in the rubber latex BFM preparation. Thus, this protein may be regarded as a novel, although minor, latex allergen. Further investigation is needed to characterize the component and to pinpoint its allergenic role.


Subject(s)
Allergens/immunology , Cell Fractionation , Gloves, Protective/adverse effects , Health Care Sector , Health Personnel , Hevea , Humans , Hydrophobic and Hydrophilic Interactions , Immunoglobulin E/blood , Latex Hypersensitivity/blood , Membranes , Occupational Exposure , Plant Proteins/immunology , Rubber/adverse effects
17.
Arq. bras. med. vet. zootec ; 59(3): 730-739, jun. 2007. graf, tab
Article in Portuguese | LILACS | ID: lil-461151

ABSTRACT

Avaliaram-se as frações de carboidratos dos capins setária (Setaria anceps Stapf), hemarthria (Hemarthria altissima [Poir] Stapf. & Hubbard), angola (Brachiaria purpurascens [Raddi] Henr.) e acroceres (Acroceras macrum Stapf.) adubados com 0, 100, 200, 300 e 400kg de N/ha na forma de sulfato de amônio e colhidos aos 28, 42, 56 e 70 dias de idade. Os cortes foram realizados a 20cm do solo para o capim-setária e a 10cm para as demais espécies. As frações dos carboidratos das forrageiras foram influenciadas pela idade de corte, observando-se com avanço da idade elevação dos teores de carboidratos não fibrosos, especialmente aos 56 dias de idade para os capins-hemarthria e angola e aos 70 dias para setária e acroceres. Os maiores teores de carboidratos fibrosos foram obtidos aos 42 dias em todas as forrageiras. A inclusão de níveis crescentes de adubação nitrogenada contribuiu para redução dos teores de carboidratos fibrosos da parede celular, porém os carboidratos não fibrosos não apresentaram respostas evidentes quanto à adubação nitrogenada.


The experiment was carried out to evaluate the levels of 0, 100, 200, 300 and 400kg of nitrogen/ha fertilization and the cutting ages at 28, 42, 56 and 70 days on the carbohydrate fractions of the tropical grasses Setaria (Setaria anceps Stapf), Limpo (Hemarthria altissima [Poir] Stapf. & Hubbard), California (Brachiaria purpurascens [Raddi] Henr.) and Nilo (Acroceras macrum Stpaf). The cuts were made 20cm upper from the ground for Setaria and 10cm for the other species. The fractions of the carbohydrates of the grasses were affected by the age of cut, as the older the forage the higher the non-fibrous carbohydrate content, especially at 56 days for Limpo grass and California grass and at 70 days for Setaria and Nilo grasses. The highest percentages of fibrous carbohydrate were observed at 42 days for all the grasses. The increasing levels of nitrogen fertilization contributed for the reduction of the fibrous carbohydrate percentages of the cellular wall, but no response of non-fibrous carbohydrate was observed in function of the nitrogenous fertilization.


Subject(s)
Cell Fractionation , Carbohydrates/analysis , Fertilizers , Poaceae
18.
Article in English | IMSEAR | ID: sea-32655

ABSTRACT

Cell disruption or lysis is a crucial step to obtain cellular components for various biological studies. We subjected different concentrations of Candida albicans to 5, 10, 15 and 20 cycles of disruption. The degree of cell lysis was observed using light microscopy and the yields obtained were measured and analysed. The optimum extraction with 1 x 10(10) yeast cells/ml was achieved after 5 cycles of disruption with 1.0 mm diameter glass beads at 5,000 rpm. Approximately 80% of the cells were lysed and the protein yield was 6,000 microg/ml. SDS-PAGE analysis revealed approximately 25 distinct protein bands with molecular weights ranging from 8 kDa to 220 kDa. We conclude that this mechanical disruption of fungal cells is a rapid, efficient and inexpensive technique for extracting whole cell proteins from yeast cells.


Subject(s)
Candida albicans/chemistry , Cell Fractionation/methods , Fungal Proteins/isolation & purification , Malaysia , Vibration
19.
Indian J Exp Biol ; 2007 Apr; 45(4): 338-46
Article in English | IMSEAR | ID: sea-56866

ABSTRACT

T3 (3,3', 5-triiodo-L-thyronine; 20 microg/100 g body weight/day in 0.01 N NaOH, i.p for 1, 3 and 5 days) treatment modulated reduced (GSH) and oxidized (GSSG) glutathione contents along with the activities of its metabolizing enzymes (such as glutathione peroxidase, glutathione reductase and glutathione S-transferase) in the testis of Wistar rats. However, the magnitude and nature of changes in the above biochemical parameters in response to T3 treatment were noticed to be different between mitochondrial and post-mitochondrial fractions. This was accompanied with elevated levels of lipid hydroperoxide and ascorbic acid in the crude homogenate of testis. The level of hydrogen peroxide in the post-mitochondrial fractions of testes did not change on first day, decreased on 3rd day and increased on 5th day of the hormone treatment when compared to respective controls. Nevertheless, its content in mitochondria was significantly elevated in response to all the three durations of the hormone treatment having the highest induction on 3rd day. The changes observed in the levels of GSH and GSSG and its metabolizing enzymes in response to T3 treatment reflect an alteration in the redox state of testis, which may be a causative factor for the impairment of testicular physiology as a consequence of oxidative stress.


Subject(s)
Animals , Cell Fractionation , Glutathione/metabolism , Glutathione Disulfide/analysis , Glutathione Peroxidase/metabolism , Glutathione Reductase/metabolism , Glutathione Transferase/metabolism , Hydrogen Peroxide/metabolism , Male , Mitochondria/enzymology , Oxidation-Reduction , Oxidative Stress , Rats , Rats, Wistar , Testis/drug effects , Triiodothyronine/pharmacology
20.
Iranian Journal of Radiation Research. 2007; 5 (2): 53-61
in English | IMEMR | ID: emr-102121

ABSTRACT

Study in design to incorporate accelerated proliferation correction factors into linearquadratic and multiple-component models. Accelerated proliferation rate correction factor has been incorporated into the linearquadratic and the multiple component models by applying accelerated exponential cell growth to explain the tumor cell kinetics and estimates proper treatment results. Biological effectiveness and tumor control probability, in terms of BED [LQ model], BRD [MC model], TCP[LQ model] and TCP[MC model], were computed for three conventional and two accelerated hyperfractionated radiation therapy treatment schedules with using a range of accelerated proliferation rate constants to demonstrate the effect of the proliferation process. The results of the study show that the accelerated proliferation rate reduces the effectiveness of a treatment schedule delivered in a prolonged period of time. Care should be taken in the selection of a treatment protocol for a patient of head and neck cancer with an account of the cell kinetics of the tumor


Subject(s)
Humans , Cell Division , Cell Fractionation , Radiotherapy Dosage , Linear Models , Models, Biological , Cells/radiation effects , Radiobiology , Physiological Effects of Drugs
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