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1.
Rev Peru Med Exp Salud Publica ; 36(2): 275-280, 2019.
Artigo em Espanhol | MEDLINE | ID: mdl-31460641

RESUMO

The aim of this study was to compare different methods of concentration to recover the largest number of Giardia spp. cysts from coprological samples. One hundred (100) samples from national reference hospitals were analyzed and four parasitological methods were applied: spontaneous tube sedimentation concentration (TSET), Faust, single-phase sucrose gradient, and two-phase sucrose gradient. The two-phase sucrose gradient method was found to achieve significantly better results in cyst concentration (121,903 cysts/ml) and amount of debris (6%), compared to Faust methods (35,355 cysts/ml), spontaneous tube sedimentation concentration (20,145 cysts/ml), and single-phase sucrose gradient (18,702 cysts/ml). It is concluded that the most effective method for the concentration and purification of Giardia spp. cysts from coprological samples is the two-phase sucrose gradient method, which would facilitate in vitro culture of Giardia spp.


Assuntos
Centrifugação com Gradiente de Concentração/métodos , Fezes/parasitologia , Giardia/isolamento & purificação , Humanos , Peru
2.
Pediatr Blood Cancer ; 66(11): e27936, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31390164

RESUMO

Bone marrow-derived mesenchymal stem and stromal cells (BM-MSCs) protect malignant cells from chemotherapy and are important potential therapeutic targets. Isolating primary BM-MSCs for research traditionally requires the sacrifice of valuable cell populations from within the same sample. To avoid this, we report here a resource for isolating patient-derived BM-MSCs from the red blood cell layer of ficoll gradients of bone marrow aspirates, a resource that has until now been universally discarded. This resource yields BM-MSCs nearly identical to those obtained conventionally and includes cells with a more stem-cell like nature. Obtaining primary BM-MSCs in this way will likely expand opportunities to study this important cell population.


Assuntos
Células da Medula Óssea , Separação Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Células-Tronco Mesenquimais , Adipogenia , Células da Medula Óssea/citologia , Exame de Medula Óssea/métodos , Técnicas de Cultura de Células , Divisão Celular , Células Cultivadas , Eritrócitos , Ficoll , Humanos , Leucemia/patologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Neoplásicas/citologia , Paracentese
3.
Hematology ; 24(1): 533-537, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31280705

RESUMO

OBJECTIVE: Buffy coat and ficoll of bone marrow (BM) are viable options for the study of minimal residual disease (MRD) in multiple myeloma (MM). As yet, there is no data about the superiority of either sample types. Herein, we aimed to address this issue. METHODS: Forty pairs of ficolled BMs and BM buffy coats of 19 MM patients were studied for MRD by allele-specific oligonucleotide real-time quantitative PCR, with patient-specific primers/probes whenever appropriate. RESULTS: There were 41 pairs of MRD data for comparison analysis due to one patient with biclonal disease. MRD levels in ficolls and buffy coats were highly concordant (rs = 0.936, P < 0.0001), with 31 (76%) and seven (17%) pairs being concomitantly MRD-positive or -negative. On the other hand, apart from the 16 pairs being both MRD-negative, or -positive but not quantifiable in ficolls and buffy coats, majority (n = 22, 88%) had higher MRD levels in ficolled BMs than BM buffy coats. Furthermore, in 17 pairs, in which MRD was quantifiable in both, MRD levels in ficolled BMs were 3.1 times those of BM buffy coats (median, 567/105 vs. 184/105, P = 0.001). CONCLUSION: Taken together, ficolled BM is more sensitive than BM buffy coat for MRD detection in MM, hence should be recommended.


Assuntos
Exame de Medula Óssea/métodos , Separação Celular/métodos , Centrifugação/métodos , Ficoll , Mieloma Múltiplo/patologia , Centrifugação com Gradiente de Concentração/métodos , Células Clonais , Primers do DNA , DNA de Neoplasias/análise , DNA de Neoplasias/isolamento & purificação , Rearranjo Gênico , Genes de Imunoglobulinas , Humanos , Leucócitos Mononucleares , Mieloma Múltiplo/genética , Neoplasia Residual , Células-Tronco Neoplásicas , Plasmócitos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Manejo de Espécimes
4.
Mater Sci Eng C Mater Biol Appl ; 103: 109817, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31349423

RESUMO

In this research, silver nanoparticles were synthesized by chemical precipitation method and stabilized by chitosan biopolymer in the range of 15 to 235 nm. Then, the silver nanoparticles were separated by density gradient centrifugation method at different gradients and centrifuged at various duration and speed. The best separation was done with the gradient chosen at 10%, 20%, 30% and 40% for a duration of 2 h at 6000 rpm. The UV-visible spectra demonstrated the proper synthesis of silver nanoparticles, while FTIR spectrum and XRD data revealed the structure of prepared Ag-NPs. The FESEM and TEM analysis were used to check for the exact shape and size of Ag-NPs, respectively. However, the separation of silver nanoparticles was assessed making use of DLS analysis.


Assuntos
Centrifugação com Gradiente de Concentração/métodos , Nanopartículas Metálicas/química , Prata/química , Coloides/química , Difusão Dinâmica da Luz , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
5.
Eur J Obstet Gynecol Reprod Biol ; 240: 74-79, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31234060

RESUMO

RESEARCH QUESTION: The purposes of this study are to evaluate the performance of the novel SpermVD device on vitrification of a few human sperms, and determine whether PICSI dish and density gradient centrifugation can improve the quality of sperm after thawing. DESIGN AND METHODS: In order to determine the suitable preparation methods, both washed sperm and neat sperm were selected in ICSI dish and PICSI dish, and frozen with the novel SpermVD device. The selected sperms were transferred to freezing droplets with 1 µl droplet of a 50/50 v/v mixture of QA Sperm Freezing Medium and QA Medium w/Hepes on the SpermVD wells. And the device was exposed to vapor of liquid nitrogen for 5 min and then placed into LN2 immediately. The sperms were thawed in a 37℃ oil filled dish which contained QA Medium w/Hepes droplets and they were searched and revaluated immediately. The frozen effects of sperm were evaluated by progressive motility, motility, viability and recovery rates of freezing-thawing sperm. RESULTS: After freezing-thawing, the overall sperm recovery rate was 94.2% with 70% viability, 20.7% progressive motility, and 36.2% motility. The progressive motility, motility, viability and recovery rates of washed sperm were lower than that of the neat sperm, with a significant difference (P <  0.01) at both viability and motility rates. In addition, the viability rate was significantly higher in PICSI dish group than that of ICSI dish group (P <  0.01) and the motility, recovery and progressive motility rates were not significantly different between the two groups (P > 0.05). CONCLUSIONS: The spermVD device was one of the effective platforms for freezing a few human sperms and using PICSI dish to select mature neat sperms could improve the quality of sperm after thawing. Density gradient centrifugation might be not required or suitable sperm preparation methods before freezing.


Assuntos
Preservação do Sêmen/métodos , Espermatozoides/citologia , Centrifugação com Gradiente de Concentração , Criopreservação/métodos , Humanos , Masculino , Vitrificação
6.
Methods Mol Biol ; 1988: 1-14, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31147928

RESUMO

Proteasomes are the main cytosolic proteases responsible for generating peptides for antigen processing and presentation in the MHC (major histocompatibility complex) class-I pathway. Purified 20S and 26S proteasomes have been widely used to study both specificity and efficiency of antigen processing. Here, we describe the purification of active human 20S and 26S proteasomes from human erythrocytes by DEAE-ion exchange chromatography, ammonium sulfate precipitation, glycerol density gradient centrifugation, and Superose-6 size exclusion chromatography and their characterization using fluorogenic substrates and specific inhibitors.


Assuntos
Bioensaio/métodos , Citosol/enzimologia , Complexo de Endopeptidases do Proteassoma/isolamento & purificação , Centrifugação com Gradiente de Concentração , Precipitação Química , Cromatografia em Gel , Cromatografia por Troca Iônica , Eritrócitos/enzimologia , Corantes Fluorescentes/metabolismo , Humanos , Inibidores de Proteassoma/farmacologia , Especificidade por Substrato/efeitos dos fármacos
7.
Methods Mol Biol ; 1982: 461-472, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172489

RESUMO

Reactive oxygen species (ROS) convey signals essential for proliferation, maintenance, and senescence of a growing list of cell types. Compartmentalization of these signals is integral to cell viability as well as the signaling pathways ROS direct. Redox-active endosomes (redoxosomes) are formed downstream of several ligand-activated receptors. NADPH oxidase (NOX) is a main component of redoxosomes, which recruits multiple proteins (Rac1, NOX2, p67phox, SOD1). Isolation of redoxosomes and evaluation of how superoxide (O2˙-) production directs receptor signaling at the level of the endosome have enabled a better understanding of biologic processes controlled by ROS. In this chapter, we will first review the major signaling pathways that utilize redoxosomes and components that control its redox-dependent functions. We will then outline biochemical and biophysical methods for the isolation and characterization of redoxosome properties.


Assuntos
Fracionamento Celular , Endossomos/metabolismo , NADPH Oxidases/metabolismo , Oxirredução , Fracionamento Celular/métodos , Linhagem Celular , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Ativação Enzimática , Humanos , Espécies Reativas de Oxigênio/metabolismo
8.
mBio ; 10(2)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040239

RESUMO

The tissue cyst of Toxoplasma gondii, found in latent infection, serves a critical role in both transmission and reactivation of this organism. Within infected cells, slowly replicating parasites (bradyzoites) are surrounded by a cyst matrix, cyst wall, and cyst membrane. The cyst wall is clearly delineated by ultrastructural analysis; however, the composition and function of this layer in host-parasite interactions are not fully understood. In order to understand the composition of the cyst wall, a proteomic analysis of purified cyst wall fragments, that were enriched with Percoll gradients and subsequently immunoprecipitated with CST1 antibody, was performed. Known cyst wall proteins, such as CST1, BPK1, MCP4, MAG1, GRA2, GRA3, and GRA5, were identified in this preparation by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, dense granule proteins (GRAs) not previously shown to associate with the cyst wall, as well as uncharacterized hypothetical proteins, were identified in this cyst wall preparation. Several of these hypothetical cyst wall (CST) proteins were epitope tagged, and immunofluorescence assays confirmed their localization as novel cyst matrix and cyst wall proteins. Expression of two of these newly identified cyst wall proteins was eliminated by gene knockout (CST2-KO and CST3-KO). CST2-KO parasites were highly attenuated in virulence and did not establish detectable cyst burdens. This targeted proteomic approach allowed the identification of new components of the cyst wall that probably have roles in the parasite/host interface.IMPORTANCE Toxoplasma gondii is a highly prevalent parasite worldwide that presents life-threatening risks to immunocompromised and pregnant individuals. Whereas the life stage responsible for acute infection can be treated, the life stage responsible for chronic infection is refractory to currently available therapeutics. Little is known about the protein composition of the cyst wall, an amorphous structure formed by parasites that is suspected to facilitate persistence within muscle and nervous tissue during chronic (latent) infection. By implementing a refined approach to selectively purify cyst wall fragments, we identified several known and novel cyst wall proteins from our sample preparations. We confirmed the localizations of several proteins from this data set and identified one that is involved in parasite virulence. These data will propel further studies on cyst wall structure and function, leading to therapeutic strategies that can eliminate the chronic infection stage.


Assuntos
Parede Celular/química , Proteoma/análise , Proteínas de Protozoários/análise , Esporos de Protozoários/química , Toxoplasma/química , Animais , Células Cultivadas , Centrifugação com Gradiente de Concentração , Cromatografia Líquida , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Humanos , Imunoprecipitação , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Proteômica , Proteínas de Protozoários/genética , Espectrometria de Massas em Tandem , Toxoplasma/genética , Toxoplasmose/parasitologia , Toxoplasmose/patologia , Virulência
9.
J Assist Reprod Genet ; 36(7): 1413-1421, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31089933

RESUMO

PURPOSE: TUNEL assay is the most common, direct test for sperm chromatin integrity assessment. But, lack of standardized protocols makes interlaboratory comparisons impossible. Consequently, clinical thresholds to predict the chance of a clinical pregnancy also vary with the technique adopted. This prospective study was undertaken to assess the incidence of sperm DNA fragmentation in a subfertile population and to establish threshold values of normality as compared to a fertile cohort, both before and after density gradient centrifugation in the total and vital fractions. METHOD: Men presenting at a university hospital setup for infertility treatment. DNA damage via TUNEL assay was validated on fresh semen samples, as conventional semen parameters, to reduce variability of results. RESULTS: Total DNA fragmentation in the neat semen was significantly higher in the subfertile group, but the vital fraction was not significantly different between the two cohorts. After gradient centrifugation, DNA fragmentation increased significantly in the total fraction of the subfertile group but decreased significantly in the vital fraction. In the fertile cohort, there was a non-significant increase in total fragmentation and in the vital fraction the trend was unclear. CONCLUSIONS: Estimating total and vital sperm DNA fragmentation, after density gradient centrifugation, increased both the sensitivity and the specificity, thereby lowering the number of false negatives and false positives encountered. These findings provide opportunities to investigate the significance of the total and the vital fractions after different assisted reproductive technologies.


Assuntos
Centrifugação com Gradiente de Concentração , Fragmentação do DNA , Fertilidade/genética , Infertilidade Masculina/terapia , Adulto , Sobrevivência Celular/genética , Cromatina/genética , Dano ao DNA/genética , Feminino , Fertilização In Vitro , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Masculino , Pessoa de Meia-Idade , Gravidez , Técnicas de Reprodução Assistida , Sêmen/química , Sêmen/metabolismo , Análise do Sêmen , Espermatozoides/química , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/patologia
10.
Mol Cell ; 75(1): 184-199.e10, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31076284

RESUMO

The comprehensive but specific identification of RNA-binding proteins as well as the discovery of RNA-associated protein functions remain major challenges in RNA biology. Here we adapt the concept of RNA dependence, defining a protein as RNA dependent when its interactome depends on RNA. We converted this concept into a proteome-wide, unbiased, and enrichment-free screen called R-DeeP (RNA-dependent proteins), based on density gradient ultracentrifugation. Quantitative mass spectrometry identified 1,784 RNA-dependent proteins, including 537 lacking known links to RNA. Exploiting the quantitative nature of R-DeeP, proteins were classified as not, partially, or completely RNA dependent. R-DeeP identified the transcription factor CTCF as completely RNA dependent, and we uncovered that RNA is required for the CTCF-chromatin association. Additionally, R-DeeP allows reconstruction of protein complexes based on co-segregation. The whole dataset is available at http://R-DeeP.dkfz.de, providing proteome-wide, specific, and quantitative identification of proteins with RNA-dependent interactions and aiming at future functional discovery of RNA-protein complexes.


Assuntos
Centrifugação com Gradiente de Concentração/métodos , Mapas de Interação de Proteínas , Proteoma/genética , Proteínas de Ligação a RNA/genética , RNA/genética , Fatores de Transcrição/genética , Centrifugação com Gradiente de Concentração/instrumentação , Cromatina/química , Cromatina/metabolismo , Regulação da Expressão Gênica , Ontologia Genética , Células HeLa , Humanos , Disseminação de Informação , Internet , Anotação de Sequência Molecular , Ligação Proteica , Proteoma/classificação , Proteoma/metabolismo , Proteômica/métodos , RNA/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismo
11.
Chemistry ; 25(43): 10026-10032, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980567

RESUMO

Gradient centrifugation is an important technique in chemistry, biology, materials science and engineering. It has big potential beyond the well-known centrifugation for separation of molecules and particles. Various possibilities for special analysis and separation of particles, but also preparative applications like the production of gradient materials and controlled polymerizations exist. In all examples, a gradient of physical and/or chemical properties is generated by centrifugation and used for the further application. In this Concept article, selected examples of gradient centrifugation are presented, to show important developments in the field and discuss their applications, potential, and limitations. It concludes by analysing future trends of gradient centrifugation that are relevant for academic and industrial usage.


Assuntos
Centrifugação com Gradiente de Concentração/métodos , Ouro/química , Nanopartículas Metálicas/química , Polímeros/química
12.
PLoS One ; 14(4): e0215324, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30973950

RESUMO

Ultracentrifugation (UC) is recognized as a robust approach for the isolation of extracellular vesicles (EVs). However, recent studies have highlighted limitations of UC including low recovery efficiencies and aggregation of EVs that could impact downstream functional analyses. We tested the benefit of using a liquid cushion of iodixanol during UC to address such shortcomings. In this study, we compared the yield and purity of EVs isolated from J774A.1 macrophage conditioned media by conventional UC and cushioned-UC (C-UC). We extended our study to include two other common EV isolation approaches: ultrafiltration (UF) and polyethylene glycol (PEG) sedimentation. After concentrating EVs using these four methods, the concentrates underwent further purification by using OptiPrep density gradient ultracentrifugation (DGUC). Our data show that C-DGUC provides a two-fold improvement in EV recovery over conventional UC-DGUC. We also found that UF-DGUC retained ten-fold more protein while PEG-DGUC achieved similar performance in nanoparticle and protein recovery compared to C-DGUC. Regarding purity as assessed by nanoparticle to protein ratio, our data show that EVs isolated by UC-DGUC achieved the highest purity while C-DGUC and PEG-DGUC led to similarly pure preparations. Collectively, we demonstrate that the use of a high-density iodixanol cushion during the initial concentration step improves the yield of EVs derived from cell culture media compared to conventional UC. This enhanced yield without substantial retention of protein contaminants and without exposure to forces causing aggregation offers new opportunities for the isolation of EVs that can subsequently be used for functional studies.


Assuntos
Fracionamento Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Vesículas Extracelulares/ultraestrutura , Animais , Linhagem Celular , Meios de Cultivo Condicionados , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Nanopartículas/metabolismo , Nanopartículas/ultraestrutura , Polietilenoglicóis , Proteínas/metabolismo , RNA/metabolismo , Ácidos Tri-Iodobenzoicos
13.
Fish Shellfish Immunol ; 89: 361-367, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974218

RESUMO

The circulating hemocytes of cultivated marine mussel (Mytilus galloprovincialis) were investigated using light microscopy and flow cytometry. In mussels two cell types, granulocytes and agranulocytes, were identified based on the existence of two subpopulations of cells differing by size and granularity level on light-scattered plots. Light microscopic observation confirmed the presence of cells with cytoplasmic granules and cells without granulation in hemolymph of mussels. The main type of cells in hemolymph were agranular cells amounting 78.4 ±â€¯8.9% in mussels. Flow cytometry showed that the agranular hemocytes of the mollusks produce significantly less reactive oxygen species compared to granulocytes. Mussel were exposed for 24 h of hypoxia and immune functions including hemocyte mortality, proliferation and reactive oxygen species (ROS) production were analysed using flow cytometric methods. Granulocyte number was higher at low oxygen concentration than that at normoxia; agranulocytes number decreased, in contrast. The ROS production after hypoxic treatment was decreased compared to normoxia level. No significant changes in hemocyte mortality and proliferation were observed.


Assuntos
Proliferação de Células , Hemócitos/fisiologia , Mytilus/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Anaerobiose , Animais , Centrifugação com Gradiente de Concentração , Citometria de Fluxo
14.
Methods Mol Biol ; 1936: 37-63, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820892

RESUMO

Molecular characterization of myelin is a prerequisite for understanding the normal structure of the axon/myelin-unit in the healthy nervous system and abnormalities in myelin-related disorders. However, reliable molecular profiles necessitate very pure myelin membranes, in particular when considering the power of highly sensitive "omics"-data acquisition methods. Here, we recapitulate the history and recent applications of myelin purification. We then provide our laboratory protocols for the biochemical isolation of a highly pure myelin-enriched fraction from mouse brains and for its proteomic analysis. We also supply methodological modifications when investigating posttranslational modifications, RNA, or myelin from peripheral nerves. Notably, technical advancements in solubilizing myelin are beneficial for gel-based and gel-free myelin proteome analyses. We conclude this article by exemplifying the exceptional power of label-free proteomics in the mass-spectrometric quantification of myelin proteins.


Assuntos
Proteínas da Mielina/metabolismo , Proteômica/métodos , Animais , Centrifugação com Gradiente de Concentração , Espectrometria de Massas , Camundongos , Processamento de Proteína Pós-Traducional
15.
Andrologia ; 51(5): e13249, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30873668

RESUMO

Intracytoplasmic sperm injection (ICSI) is a technique developed to help attain successful fertilisation for couples with severe male factor. However, a small percentage of couples confront low or failed fertilisation, mainly due to failed oocyte activation. Several studies have introduced phospholipase Cζ (PLCζ) as the main sperm factor inducing oocyte activation and thereby has the potential to act as a biomarker for the prediction of ICSI fertilisation outcome. On the other hand, researchers have focused on novel sperm selection procedures based on cellular characteristics of spermatozoa such as surface electrical charge (Zeta potential) to isolate normal sperm subpopulation with intact chromatin. Therefore, we aimed to compare PLCζ between Zeta method and routine sperm preparation procedure: density gradient centrifugation (DGC). Our results showed that number of PLCζ-positive spermatozoa was significantly low in the Zeta method, but the intensity of PLCζ protein in such spermatozoa was significantly higher than DGC procedure. Therefore, the combination of DGC with Zeta procedure may allow selecting the population of spermatozoa with a high percentage of PLCζ which may also contain a high amount of PLCζ and with intact chromatin. This sperm selection procedure can open a new approach for infertile men with previously failed fertilisation.


Assuntos
Oócitos/fisiologia , Fosfoinositídeo Fosfolipase C/metabolismo , Análise do Sêmen/métodos , Espermatozoides/metabolismo , Centrifugação com Gradiente de Concentração , Humanos , Infertilidade Masculina/terapia , Masculino , Fosfoinositídeo Fosfolipase C/análise , Injeções de Esperma Intracitoplásmicas , Eletricidade Estática
16.
Biochemistry (Mosc) ; 84(1): 40-46, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30927524

RESUMO

Protein synthesis in mitochondria is generally organized in a bacterial-like manner but, at the same time, possesses several unique traits. Translation initiation in mitochondria is regulated by two protein factors, mtIF2 and mtIF3. Previously we demonstrated that Saccharomyces cerevisiae Aim23 protein is an ortholog of IF3 in budding yeast. However, the data on the interactions between Aim23p and other proteins are limited. Here, we demonstrated that Aim23p interacts with the yeast mitochondrial ribosomal small subunit both in vivo and in vitro using co-immunoprecipitation and density gradient sedimentation.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Subunidades Ribossômicas Menores/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Centrifugação com Gradiente de Concentração , Imunoprecipitação , Proteínas Mitocondriais , Ribossomos Mitocondriais , Fator de Iniciação 2 em Procariotos , Fator de Iniciação 3 em Procariotos , Proteínas Ribossômicas/metabolismo
17.
Int J Biol Macromol ; 130: 786-797, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30831171

RESUMO

ß-Amylase has been de novo synthesized from germinating fenugreek seeds. Enzyme has been isolated and purified from 36 h germinated seeds with 226-fold purification and specific activity of 763 U/mg. Homogeneity of the purified ß-amylase has been confirmed with size-exclusion chromatography, SDS-PAGE and MALDI MS/MS analysis. The isoelectric point, optimum pH and temperature of the enzyme were found to be pH 5.2, 5.7 and 57 °C, respectively. The enzyme was specific for soluble starch with Km and Vmax of 2.4 mg/mL and 833.3 U/mg, respectively. Maltose was found to be competitive inhibitor of the enzyme with inhibition constant (Ki) of 14 mM. However, metallic ions like Ag+ and Hg2+ were found to be non-competitive inhibitors of the enzyme. Thermodynamic parameters like Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes have further revealed that thermal denaturation of the enzyme has followed first-order with the enzyme unfolding rather an aggregation with the process being irreversible. The activation energy of ß-amylase during thermal activation and denaturation were 27.5 kJ/mol and 145.23 kJ/mol, respectively at R2 > 0.92. Thus, the enzyme was stable even at higher temperature with ability of undergoing catalysis making it commercially exploitable, particularly in food and pharmaceutical industries.


Assuntos
Fenômenos Químicos , Termodinâmica , Trigonella/enzimologia , beta-Amilase/química , Centrifugação com Gradiente de Concentração , Cromatografia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Biossíntese de Proteínas/efeitos dos fármacos , Sementes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Temperatura Ambiente , beta-Amilase/biossíntese , beta-Amilase/isolamento & purificação
18.
Proc Natl Acad Sci U S A ; 116(13): 6319-6328, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850549

RESUMO

Lipoprotein lipase (LPL), the enzyme that hydrolyzes triglycerides in plasma lipoproteins, is assumed to be active only as a homodimer. In support of this idea, several groups have reported that the size of LPL, as measured by density gradient ultracentrifugation, is ∼110 kDa, twice the size of LPL monomers (∼55 kDa). Of note, however, in those studies the LPL had been incubated with heparin, a polyanionic substance that binds and stabilizes LPL. Here we revisited the assumption that LPL is active only as a homodimer. When freshly secreted human LPL (or purified preparations of LPL) was subjected to density gradient ultracentrifugation (in the absence of heparin), LPL mass and activity peaks exhibited the size expected of monomers (near the 66-kDa albumin standard). GPIHBP1-bound LPL also exhibited the size expected for a monomer. In the presence of heparin, LPL size increased, overlapping with a 97.2-kDa standard. We also used density gradient ultracentrifugation to characterize the LPL within the high-salt and low-salt peaks from a heparin-Sepharose column. The catalytically active LPL within the high-salt peak exhibited the size of monomers, whereas most of the inactive LPL in the low-salt peak was at the bottom of the tube (in aggregates). Consistent with those findings, the LPL in the low-salt peak, but not that in the high-salt peak, was easily detectable with single mAb sandwich ELISAs, in which LPL is captured and detected with the same antibody. We conclude that catalytically active LPL can exist in a monomeric state.


Assuntos
Lipase Lipoproteica/química , Lipase Lipoproteica/isolamento & purificação , Animais , Células CHO , Bovinos , Centrifugação com Gradiente de Concentração/métodos , Cromatografia de Afinidade , Cromatografia em Agarose , Cricetulus , Epitopos , Heparina , Humanos , Lipase Lipoproteica/sangue , Receptores de Lipoproteínas/sangue , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/isolamento & purificação , Sefarose/análogos & derivados , Triglicerídeos/metabolismo , Ultracentrifugação
19.
PLoS One ; 14(2): e0211679, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30785892

RESUMO

In 50% of all infertility cases, the male is subfertile or infertile, however, the underlying mechanisms are often unknown. Even when assisted reproductive procedures such as in vitro fertilization and intracytoplasmic sperm injection are performed, the causes of male factor infertility frequently remain elusive. Since the overall activity of cells is closely linked to their metabolic capacity, we analyzed a panel of 180 metabolites in human sperm and seminal plasma and elucidated their associations with spermiogram parameters. Therefore, metabolites from a group of 20 healthy donors were investigated using a targeted LC-MS/MS approach. The correlation analyses of the amino acids, biogenic amines, acylcarnitines, lysophosphatidylcholines, phosphatidylcholines, sphingomyelins and sugars from sperm and seminal plasma with standard spermiogram parameters revealed that metabolites in sperm are closely related to sperm motility, whereas those in seminal plasma are closely related to sperm concentration and morphology. This study provides essential insights into the metabolome of human sperm and seminal plasma and its associations with sperm functions. This metabolomics technique could be a promising screening tool to detect the factors of male infertility in cases where the cause of infertility is unclear.


Assuntos
Metaboloma , Sêmen/química , Espermatozoides/química , Adulto , Aminoácidos/análise , Aminas Biogênicas/análise , Carnitina/análogos & derivados , Carnitina/análise , Centrifugação com Gradiente de Concentração , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Lisofosfatidilcolinas/análise , Masculino , Fosfatidilcolinas/análise , Sêmen/metabolismo , Análise do Sêmen , Espermatozoides/metabolismo , Esfingomielinas/análise , Açúcares/análise
20.
Methods Mol Biol ; 1920: 265-275, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30737696

RESUMO

The Balbiani body (Bb) is a large membrane-less organelle, densely packed with mitochondria, endoplasmic reticulum, proteins, and RNA. The Bb is present in many vertebrate female gametes. In frogs, the Bb is established early during oogenesis and operates as a maternal inherited embryonic determinant that specifies germline identity through the formation of germplasm. We describe here two techniques to isolate the Bb/germplasm from Xenopus laevis primary oocytes.


Assuntos
Fracionamento Celular , Oócitos/metabolismo , Oogênese , Organelas/metabolismo , Xenopus laevis , Animais , Fracionamento Celular/métodos , Centrifugação com Gradiente de Concentração , Células Germinativas/metabolismo , Mitocôndrias/metabolismo , Oogênese/genética
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