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1.
Int J Nanomedicine ; 11: 4509-4519, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27660443

RESUMO

Titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) are promising candidates for numerous applications in consumer products. This will lead to increased human exposure, thus posing a threat to human health. Both these types of NPs have been studied for their cell toxicity, immunotoxicity, and genotoxicity. However, effects of these NPs on epigenetic modulations have not been studied. Epigenetics is an important link in the genotype and phenotype modulation and misregulation can often lead to lifestyle diseases. In this study, we have evaluated the DNA methylation-based epigenetic changes upon exposure to various concentrations of NPs. The investigation was designed to evaluate global DNA methylation, estimating the corresponding methyltransferase activity and expression of Dnmt gene using lung fibroblast (MRC5) cell line as lungs are the primary route of entry and target of occupational exposure to TiO2 and ZnO NPs. Enzyme-linked immunosorbent assay-based immunochemical assay revealed dose-related decrease in global DNA methylation and DNA methyltransferase activity. We also found direct correlation between the concentration of NPs, global methylation levels, and expression levels of Dnmt1, 3A, and 3B genes upon exposure. This is the first study to investigate effect of exposure to TiO2 and ZnO on DNA methylation levels in MRC5 cells. Epigenetic processes are known to play an important role in reprogramming and adaptation ability of an organism and can have long-term consequences. We suggest that changes in DNA methylation can serve as good biomarkers for early exposure to NPs since they occur at concentrations well below the sublethal levels. Our results demonstrate a clear epigenetic alteration in response to metal oxide NPs and that this effect was dose-dependent.

2.
ACS Appl Mater Interfaces ; 6(16): 14679-89, 2014 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-25046687

RESUMO

This study reports a facile biomineralization route for gold microplates (GMPs) synthesis using bovine serum albumin (BSA) as a reductant and stabilizing agent. Adding BSA to HAuCl4 solution yields spontaneous versatile anisotropic and partially hollow GMPs upon aging. We hypothesize that the instantaneous protein denaturation at low pH enabled access to serine and threonine hydroxyl, and sulfhydryl groups of BSA, which act as a reductant and stabilizer, respectively. This reaction could be hastened by increasing the temperature well beyond 65 °C. Transmission electron microscopy/X-ray diffraction studies revealed highly crystalline and anisotropic structures (triangle, pentagon, and rectangle). Atomic force microscopy/scanning electron microscopy analyses demonstrated unique morphology of microplates with a partially void core and BSA mineralized edge structure. RAW 264.7 mice peritoneal macrophage-microplate interaction studies using live cell confocal imaging reveal that cells are capable of selectively internalizing smaller GMPs. Large GMPs are preferentially picked with sharp vertices but cannot be internalized and exhibit frustrated phagocytosis-like phenomenon. We explored particle phagocytosis as an actin mediated process that recruits phagosome-like acidic organelles, shown by a lysosensor probe technique. The biocompatible GMPs exhibited ∼70% paclitaxel (PCL) loading and sustained release of PCL, showing antitumor activity with the MCF-7 cell line, and could be a novel drug carrier for breast cancer therapy.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Macrófagos/metabolismo , Paclitaxel/química , Fagocitose/fisiologia , Animais , Bovinos , Linhagem Celular , Camundongos , Paclitaxel/administração & dosagem , Soroalbumina Bovina/química
3.
J Appl Microbiol ; 115(6): 1357-67, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23957303

RESUMO

AIM: Potential for xylitol production from xylose and corn cob hydrolysate by a tropical mangrove yeast. METHODS AND RESULTS: In the present study, 21 fungi were isolated from detritus-based mangrove wetlands along the Indian west coast. Of these, one yeast isolate had the ability to grow and assimilate xylose producing significant amounts of xylitol (38·63 g l(-1) ). A maximum yield of 0·54 g g(-1) was obtained after 144 h of growth on xylose (150 g l(-1) ) and corn cob hydrolysate (CCH, containing 65 g l(-1) xylose). Using biochemical and molecular methods, the yeast was identified as Cyberlindnera (Williopsis) saturnus. Preliminary characterization of enzymes in the cell-free extract revealed that while xylose reductase (XR) preferred NADPH to NADH as cofactor, xylitol dehydrogenase (XDH) was NAD specific. CONCLUSIONS: Significant amounts of xylitol could be produced on CCH using C. saturnus isolated from tropical mangrove wetlands. The yeast has the potential to assimilate rather than ferment xylose as its XR has a preference for NADPH. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbes offer an economically viable and green approach for production of xylitol, an industrially important compound. A mangrove ecosystem with its battery of lignocellulolytic enzymes is an ideal location for isolating fungi capable of producing xylitol from agroindustrial waste such as CCH.

4.
J Nanosci Nanotechnol ; 13(1): 77-85, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23646700

RESUMO

The nanoscale interaction of bacterial cells with solid surfaces is a key issue in biomedicine because it constitutes the first pathogenic event in the complex series of biofilm development on prosthetic devices. We report on an Atomic Force Microscopy study of the interaction of Escherichia coli and Pseudomonas aeruginosa bacterial cells with nanostructured titania thin films with controlled and reproducible nanometer-scale morphology, produced by assembling Ti clusters from the gas phase in a Supersonic Cluster Beam Deposition apparatus. The results demonstrate that bacterial adhesion and biofilm formation are significantly influenced by a pure physical stimulus, that is, the nanoscale variation of surface topography. The increase of nanoscale film roughness promotes bacterial adhesion with respect to flat substrates; remarkably, Pseudomonas aeruginosa cells lose their flagella on nanostructured TiO2 thin films upon adhesion, as opposed to same bacteria onto reference smooth glass substrates. Further, we have observed increased cell biovolume and other biofilm properties on nanostructured substrates in comparison with smooth glasses. These findings suggest that the design of innovative biomaterials with a suitable patterning of biomaterials surfaces can be an effective approach to control the adhesion of microorganisms to in vivo implant surfaces with active biological functionalities.


Assuntos
Aderência Bacteriana/fisiologia , Biofilmes/crescimento & desenvolvimento , Microscopia de Força Atômica/métodos , Nanoestruturas/química , Nanoestruturas/microbiologia , Titânio/química , Teste de Materiais , Nanoestruturas/ultraestrutura , Tamanho da Partícula , Propriedades de Superfície
5.
J Biomed Mater Res A ; 101(10): 3019-32, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23559501

RESUMO

The current nano-biotechnologies interfacing synthetic materials and cell biology requires a better understanding of cell-surface interactions on the micro-to-nanometer scale. Cell-substrate interactions are mediated by the presence of proteins adsorbed from biological fluids to the substrate. The effect of nanotopography and surface chemistry on protein adsorption as well as the mediation effect on subsequent cellular communication with substratum is not well documented. This review discusses the role of physicochemical properties of cell-surface interactions and state-of-the-art methods currently available for micro-nanoscale surface fabrication and patterning. We also briefly discuss the current surface patterning techniques that allow the combination of a fine and independent control on nanotopography and chemistry to understand the effect of surface nanoscale substrate morphology on cell-surface interactions which has never been realized in previous reports. In addition, we discuss the influence of various chemical patterning and modulation of the nano-topography of surfaces on cell functionality and phenotype.


Assuntos
Comunicação Celular , Membrana Celular/metabolismo , Fenômenos Químicos , Microtecnologia/métodos , Nanotecnologia/métodos , Animais , Humanos , Nanoestruturas/química
6.
J Mater Sci Mater Med ; 24(6): 1483-90, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23494617

RESUMO

Silica-coated, silicon nanotubes (SCSNTs) and silica-coated, silicon nanoparticles (SCSNPs) have been synthesized by catalyst-free single-step gas phase condensation using the arc plasma process. Transmission electron microscopy and scanning tunneling microscopy showed that SCSNTs exhibited a wall thickness of less than 1 nm, with an average diameter of 14 nm and a length of several 100 nm. Both nano-structures had a high specific surface area. The present study has demonstrated cheaper, resistance-free and effective antibacterial activity in silica-coated silicon nano-structures, each for two Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration (MIC) was estimated, using the optical densitometric technique, and by determining colony-forming units. The MIC was found to range in the order of micrograms, which is comparable to the reported MIC of metal oxides for these bacteria. SCSNTs were found to be more effective in limiting the growth of multidrug-resistant Staphylococcus aureus over SCSNPs at 10 µg/ml (IC 50 = 100 µg/ml).


Assuntos
Anti-Infecciosos/administração & dosagem , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Nanopartículas/administração & dosagem , Dióxido de Silício/administração & dosagem , Silício/administração & dosagem , Anti-Infecciosos/síntese química , Sobrevivência Celular/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/administração & dosagem , Materiais Revestidos Biocompatíveis/síntese química , Relação Dose-Resposta a Droga , Gases/química , Teste de Materiais , Staphylococcus aureus Resistente à Meticilina/citologia , Nanopartículas/química , Transição de Fase , Silício/química , Dióxido de Silício/química
7.
Biofabrication ; 4(2): 025001, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22406493

RESUMO

The contamination of implant devices as a result of biofilm formation through bacterial infection has instigated major research in this area, particularly to understand the mechanism of bacterial cell/implant surface interactions and their preventions. In this paper, we demonstrate a controlled method of nanostructured titanium oxide surface synthesis using supersonic cluster beam depositions. The nanoscale surface characterization using atomic force microscopy and a profilometer display a regulated evolution in nanomorphology and physical properties. X-ray photoelectron spectroscopy analyses display a stoichiometric nanostructured TiO(2) film. Measurement of the water contact angle shows a nominal increase in the hydrophilic nature of ns-TiO(2) films, whereas the surface energy increases with decreasing contact angle. Bacterial species Staphylococcus aureus and Escherichia coli interaction with nanostructured surfaces shows an increase in adhesion and biofilm formation with increasing nanoscale morphological properties. Conversely, limiting ns-TiO(2) film distribution to micro/nanopatterned designed substrates integrated with bovine serum albumin functionalization leads to a reduction in biofilm formations due to a globally decreased bacterial cell-surface interaction area. The results have potential implications in inhibiting bacterial colonization and promoting mammalian cell-implant interactions.


Assuntos
Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Nanoestruturas/química , Nanotecnologia/métodos , Titânio/farmacologia , Animais , Bovinos , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Microscopia , Nanoestruturas/ultraestrutura , Impressão , Soroalbumina Bovina/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Propriedades de Superfície , Titânio/química
8.
Autoimmune Dis ; 2012: 160830, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23346386

RESUMO

Multiple Sclerosis is a multifactorial disease with several pathogenic mechanisms and pathways. Successful MS management and medical care requires early accurate diagnosis along with specific treatment protocols based upon multifunctional nanotechnology approach. This paper highlights advances in nanotechnology that have enabled the clinician to target the brain and CNS in patient with multiple sclerosis with nanoparticles having therapeutic and imaging components. The multipartite theranostic (thera(py) + (diag)nostics) approach puts forth strong implications for medical care and cure in MS. The current nanotheranostics utilize tamed drug vehicles and contain cargo, targeting ligands, and imaging labels for delivery to specific tissues, cells, or subcellular components. A brief overview of nonsurgical nanorepair advances as future perspective is also described. Considering the potential inflammatory triggers in MS pathogenesis, a multifunctional nanotechnology approach will be needed for the prognosis.

9.
Colloids Surf B Biointerfaces ; 69(2): 239-45, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19131222

RESUMO

Foams of bovine serum albumin (BSA) have been utilized for synthesizing in situ protein capped Ag-Pt alloy nanoparticles. The in vitro cytotoxicity and the rate of proliferation of human gingival fibroblasts (HGFs) in presence of the above synthesized alloy nanoparticles is investigated. Expression profile of protein involved in detoxification, i.e. metallotheonein (MT) were assayed by ELISA and expression of mRNA transcripts by reverse transcription polymerase chain reaction (RT-PCR). Cytotoxicity results suggested that protein capped nano-alloys might be promising candidates for implants and prosthetic material. RT-PCR and ELISA confirmed the expression of MT, in cells treated with the alloy nanoparticles. Morphology variation studied by SEM also confirms that cells treated with alloy nanoparticles present an intact morphology.


Assuntos
Ligas/síntese química , Materiais Biocompatíveis/síntese química , Fibroblastos/efeitos dos fármacos , Nanopartículas Metálicas , Platina , Prata , Adolescente , Adulto , Ligas/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Gengiva/citologia , Humanos , Masculino , Metalotioneína/biossíntese , Platina/química , Próteses e Implantes , Soroalbumina Bovina , Prata/química
10.
Clin Exp Allergy ; 37(8): 1239-49, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17651155

RESUMO

BACKGROUND: Approximately 20% of the world's asthmatics are suffering from Aspergillus fumigatus (Afu)-induced allergies. The characterization of specific IgE-inducing allergens in allergic aspergillosis patients is fundamental for clinical diagnosis and for immunotherapy. METHODS: Immunoproteomics combined with mass spectrometric analysis was used to identify proteins of third-week culture filtrate (3wcf) potentially responsible for Afu-specific IgE immunoreactivity, using pooled sera from Afu-sensitized asthmatics. Their allergenic potential was also tested against patients with allergic bronchopulmonary aspergillosis (ABPA), by two-dimensional (2-D) gel electrophoresis immunoblotting of 3wcf proteins with individual sera from such patients. This helped us to establish a set of candidate allergens, which could be explored further for diagnostic application in allergic aspergillosis asthmatics including ABPA. RESULTS: Peptide mass fingerprint using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and/or de novo sequencing by MS/MS analysis of the protein spots from 2-D gels led to the identification of a total of 16 allergens of Afu. Eleven of them are being reported as allergens for the first time and five had been reported earlier. Putative isoforms of the proteins Asp f 13 and chitosanase have been observed for the first time. When studied for reactivity of these proteins among patients with ABPA using their individual sera, these patients exhibited sensitization although the pattern was varying. Taken together, these proteins could thus be considered as potential allergens even among patients with ABPA. Three of these proteins viz. the hypothetical protein (# spot no. 5), extracellular arabinase (# spot no. 6) and chitosanase (# spot no. 11) could be major allergens with specific IgE immunoreactivity with six out of eight patients' sera. CONCLUSIONS: The immunoproteomic approach applied to the analysis of culture filtrate proteins resulted in the identification of several candidate allergens, many of them novel, contributing to the catalogue of Afu allergenic proteins, which would facilitate improved serodiagnosis for allergic aspergillosis. In addition, the immunoreactivity of these proteins observed among the patients with ABPA may be potentially useful for its serodiagnosis and opens up further opportunities for the development of personalized immunotherapeutics for patients with ABPA.


Assuntos
Alérgenos/imunologia , Aspergilose Broncopulmonar Alérgica/imunologia , Aspergillus fumigatus/imunologia , Asma/imunologia , Proteínas Fúngicas/imunologia , Imunoglobulina E/imunologia , Adulto , Aspergilose Broncopulmonar Alérgica/diagnóstico , Aspergilose Broncopulmonar Alérgica/enzimologia , Aspergilose Broncopulmonar Alérgica/terapia , Aspergillus fumigatus/enzimologia , Asma/diagnóstico , Asma/enzimologia , Asma/terapia , Eletroforese em Gel Bidimensional , Feminino , Glicosídeo Hidrolases/imunologia , Humanos , Masculino , Proteômica , Testes Sorológicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
J Biotechnol ; 128(3): 444-51, 2007 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-17210198

RESUMO

The present study describes response of a bacterial strain isolated from a polluted river to heavy metal toxicity. The bacterium was identified to be Klebsiella pneumoniae by biochemical tests using API 20E strips and 16S ribotyping. The isolate was studied for its tolerance to two heavy metals, i.e., cobalt (Co(2+)) and lead (Pb(2+)) by growing it in citrate mineral medium (CMM). Proteomic approach involving two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometry (MS) was used to identify the differentially expressed proteins under heavy metal stress. Two of the differentially expressed proteins were identified to be l-isoaspartate protein carboxymethyltransferase type II and DNA gyrase A. To our knowledge, this is for the first time that K. pneumoniae has been reported to be present in metal contaminated site and l-isoaspartate protein carboxymethyltransferase type II protein to be over expressed under heavy metal stress. The role of these proteins in metal tolerance is discussed.


Assuntos
Proteínas de Bactérias/análise , Farmacorresistência Bacteriana , Metais Pesados/metabolismo , Proteômica/métodos , Rios/microbiologia , Sequência de Bases , Cobalto/metabolismo , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/metabolismo , Chumbo/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mapeamento de Peptídeos , RNA Ribossômico 16S/química , Poluentes Químicos da Água/metabolismo
12.
J Biotechnol ; 126(3): 374-82, 2006 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-16787678

RESUMO

The genus Pseudomonas is a group of gram-negative, motile, rod-shaped bacteria known for their metabolic versatility. One of the species is Pseudomonas fluorescens, which has an efficient system for detoxification of industrial waste. Other aspects include, catabolic versatility, excellent root colonizing abilities and capacity to produce a wide range of antifungal metabolites. They are also known for their resistance and survival in the presence of several organic and inorganic pollutants. P. fluorescens has also been isolated from metal polluted water and soils but the elucidation of proteins responsible for its survival is still not clear. The aim of the study was to elucidate the differential protein expression of this bacterium when exposed to heavy metal stress, using two-dimensional electrophoresis. The proteins spo VG and enolase showed upregulation during the bacterial exposure to lead and copper. Hypothetical protein showed downregulation when bacterium was exposed to cobalt. Some proteins like xylosyltransferase, ORF 18 phage phi KZ, OMP H1 and translational elongation factor EF-Tu appeared only during their exposure to cobalt. These were absent in the control condition. Analysis of the differentially expressed proteins as well as the newly synthesized proteins along with the results obtained growth and enzyme activity indicate the involvement of all these factors in the survival of this organism in the presence of heavy metals.


Assuntos
Proteínas de Bactérias/metabolismo , Metais Pesados/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Proteoma/metabolismo , Pseudomonas fluorescens/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/fisiologia , Pseudomonas fluorescens/efeitos dos fármacos
13.
Indian J Clin Biochem ; 21(2): 12-9, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23105607

RESUMO

Allergenic/antigenic proteins are known to induce Type I and Type III hypersensitivity reactions leading to allergic bronchopulmonary aspergillosis (ABPA) in immunocompetent host. The common structural features or intrinsic properties of the allergens/antigens leading to allergenicity in a host are not well understood. In the current report, comparative analysis of proteins on two dimensional gel electrophoresis (2D-3) and specific IgE immunoblots ofA. fumigatus secretory proteins (1(st), 2(nd) and 3(rd) week culture filtrate proteins) was carried out. We observed a total of 159 proteins in 1(st), 2(nd) and 3(rd) week culture filtrates ofA. fumigatus. Specific IgE immunoreactivity was observed in 75 proteins with different intensity. Third week culture filtrate showed maximum number of proteins, 142, and specific IgE immunoreactive proteins, 65. MALDI-TOF analysis resulted in putative identification of two allergens as hypothetical protein YBL057c fromSaccharomyces cereviseae and unnamed protein product fromDebaryomyces hansenii (similar to IPF14568 ofCandida albicans). Identification of a repertoire of specific IgE immunoreactive proteins will facilitate the studies on structure-function relationship of these proteins relevant for diagnosis and pathogenesis.

14.
Biochim Biophys Acta ; 1747(2): 151-9, 2005 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-15698949

RESUMO

Anthrax toxin produced by Bacillus anthracis is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-binding component, which facilitates the entry of LF or EF into the cytosol. EF is a calmodulin-dependent adenylate cyclase that causes edema whereas LF is a zinc metalloprotease and leads to necrosis of macrophages. It is also important to note that the exact mechanism of LF action is still unclear. With this view in mind, in the present study, we investigated a proteome wide effect of anthrax lethal toxin (LT) on mouse macrophage cells (J774A.1). Proteome analysis of LT-treated and control macrophages revealed 41 differentially expressed protein spots, among which phosphoglycerate kinase I, enolase I, ATP synthase (beta subunit), tubulin beta2, gamma-actin, Hsp70, 14-3-3 zeta protein and tyrosine/tryptophan-3-monooxygenase were found to be down-regulated, while T-complex protein-1, vimentin, ERp29 and GRP78 were found to be up-regulated in the LT-treated macrophages. Analysis of up- and down-regulated proteins revealed that primarily the stress response and energy generation proteins play an important role in the LT-mediated macrophage cell death.


Assuntos
Antígenos de Bactérias/toxicidade , Bacillus anthracis/química , Toxinas Bacterianas/toxicidade , Ativação Enzimática/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteoma/análise , Trifosfato de Adenosina/metabolismo , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Citoesqueleto/metabolismo , Regulação para Baixo , Edema/imunologia , Edema/metabolismo , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática/fisiologia , Proteínas de Choque Térmico/metabolismo , Dose Letal Mediana , Macrófagos/metabolismo , Metaloendopeptidases/metabolismo , Camundongos , Proteoma/metabolismo , Regulação para Cima
15.
Prep Biochem Biotechnol ; 34(2): 145-52, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15195709

RESUMO

The principle of the immobilized metal affinity chromatography (IMAC) is based on the differences in the affinity of proteins for metal ions bound in a 1:1 complex of iminodiacetic acid (IDA) immobilized on a chromatographic support. A single step purification was carried out for luteinizing hormone (LH) on Cu2+, Zn2+, Ni2+, and Co2+ IDA Sepharose affinity columns. Highly purified LH was obtained with a Cu2+ IDA Sepharose column. SDS-PAGE and Western blot analysis were done to confirm the purity of the hormone. Biological activity has been evaluated by radio-immunoassay. This method was found economically viable and suitable for the recovery of biologically active hormone.


Assuntos
Cromatografia de Afinidade/métodos , Histidina/química , Iminoácidos/química , Hormônio Luteinizante/isolamento & purificação , Metais/química , Hipófise/química , Animais , Western Blotting , Bovinos , Eletroforese em Gel de Poliacrilamida
16.
Prep Biochem Biotechnol ; 32(1): 39-49, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11934076

RESUMO

Peroxidase from turnip roots was purified using metal affinity chromatography up to a specific activity of 337 units/mg protein with 3.02 RZ and 63.5% recovery. After purification, the enzyme showed 2-3 bands on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was found to be 37-39 kD with matrix assisted laser desorption ionization mass spectrometer (MALDI-MS). The enzyme showed maximum activity in phosphate buffer, pH 6.0, and lowest activity in borate buffer at the same pH. The Km of the enzyme was found to be 7.07 x 104 mM. Turnip peroxidase also contains an iron moiety which is found to be about 0.28%. The enzyme showed 50% inhibition of its specific activity with ethylene diamine tetraacetic acid (EDTA).


Assuntos
Brassica napus/enzimologia , Peroxidase/isolamento & purificação , Peroxidase/metabolismo , Soluções Tampão , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Ferro/análise , Cinética , Peso Molecular , Peroxidase/química , Plantas/enzimologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Temperatura
17.
J Ethnopharmacol ; 75(1): 25-32, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11282439

RESUMO

Upregulation of expression of cell adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin, is important for immune surveillance. Extravasation and migration of body's effector cells to the site of immune activation is controlled by the expression of cell adhesion molecules on endothelial cells. We demonstrate here that an aqueous extract prepared from Curcuma longa (ClAqE), a dietary component, promotes the adhesion of peripheral neutrophils to human umbilical vein endothelial cells. To delineate the mechanism of increased adhesion, we investigated the possibility that ClAqE induces the expression of ICAM-1 and E-selectin on endothelial cells. ClAqE increases the steady state transcript levels of ICAM-1, VCAM-1, and E-selectin as determined by RT-PCR. We also show that ClAqE activates nuclear transcription factor NF-kappaB, a major transcription factor involved in the transcription of genes encoding ICAM-1, VCAM-1 and E-selectin. These results have implications for the usage of aqueous preparation of C. longa for upregulation of cell adhesion molecule expression and/or NF-kappaB.


Assuntos
Adesão Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Zingiberales/química , Sequência de Bases , Primers do DNA , Selectina E/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismo
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