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1.
Anal Chim Acta ; 1081: 81-92, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31446968

RESUMO

The novel ionic liquids skeleton typed magnetic core-shell molecularly imprinted polymers (Fe3O4-COOH@IL-MIP) were firstly constructed with 1-vinyl-3-aminoformylmethyl imidazolium chloride ionic liquid ([VAFMIM]Cl-IL) modified magnetic particles as the substrate materials, [VAFMIM]Cl-IL as functional monomer, 1,6-hexanediyl-3,3'-bis-1-vinylimidazolium dichloride ionic liquid as cross-linker and Lysozyme (Lys) as template protein via surface-imprinting technique. The structure of Fe3O4-COOH@IL-MIP were confirmed by transmission and scanning electron microscopy, dynamic light scattering, thermo-gravimetric analysis, fourier transform infrared spectrometry and X-ray diffraction. The adsorption mechanism was discussed from the perspective of amino acid residues of Lys. The maximum adsorption capacity of MIPs was 166.36 mg g-1 and imprinting factor was 2.67. The competitive adsorption experiments demonstrated the favorable recognition ability of MIPs toward Lys. Reusability studies indicated MIPs can be reused ten times without obvious loss of rebinding ability. The Lys conformation maintained intact after elution and the elution rate was as high as 74%. The adsorption experiment of egg white manifested that MIPs can effectively separate Lys in practical samples. Only ILs and Fe3O4 were utilized to fabricate MIPs, this strategy realized the goal of energy and cost saving while achieving simple synthesis of imprinted materials, and is expected to provide a new feasible idea to exploit synthetic methods for protein-MIPs.


Assuntos
Líquidos Iônicos/química , Nanopartículas de Magnetita/química , Muramidase/análise , Polímeros/química , Adsorção , Animais , Galinhas , Concentração de Íons de Hidrogênio , Fenômenos Magnéticos , Impressão Molecular/métodos , Muramidase/química
2.
Anal Bioanal Chem ; 411(22): 5799-5807, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31209550

RESUMO

The abnormal concentration of lysozyme in body fluids and tissues is a potential indicator for diseases such as leukemia and meningitis. In this work, by combining the excellent optical properties of carbon dots (CDs) with the favorable selectivity of molecularly imprinted polymer (MIP), a novel fluorescent probe for lysozyme detection and cell imaging was constructed, where silanized CDs with low cytotoxicity (CDs/SiO2) were used as the fluorescence signal reporter and N-isopropylacrylamide (NIPAM) was used as the temperature-sensitive monomer. The as-prepared CDs/SiO2/MIP showed a thermo-sensitive property for the response to lysozyme. Moreover, this probe could be used for quantitative detection of lysozyme, with a wider detection range (0.001~0.01 mg/mL), a low detection limit (0.55 µg/mL), and a high selectivity. Importantly, the MTT assay testified that the fluorescent CDs/SiO2/MIP probe had low cytotoxicity. In addition, human hepatoma carcinoma cells (HepG-2 cells) could be stained by the CDs/SiO2/MIP, and showed a bright intracellular green fluorescence, indicating that the imaging of live cells was possible. This study provides a new way to detect lysozyme in vitro and an attractive perspective to probe intracellular lysozyme in vivo.


Assuntos
Carbono/química , Corantes Fluorescentes/química , Impressão Molecular , Muramidase/análise , Nanopartículas/química , Polímeros/química , Dióxido de Silício/química , Ligação Competitiva , Células Hep G2 , Humanos , Limite de Detecção , Microscopia Eletrônica de Transmissão , Espectrometria de Fluorescência/métodos , Espectrofotometria Ultravioleta
3.
Talanta ; 200: 57-66, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31036225

RESUMO

In our work, aptamers and hemin/G-quadruplex DNAzyme modified sandwich-rod graphene quantum dots @ graphene oxide @ carbon fiber composite (DNAzyme/L-Apt/GQDs@GO@CF) was successfully prepared for sensitive and selective chemiluminescence (CL) detection of lysozyme (LZM). Initially, GQDs@GO@CF was successfully prepared and characterized. Lysozyme aptamers (L-Apt) as a recognition element and hemin/G-quadruplex DNAzyme (DNAzyme) as a catalyst of luminal - H2O2 were modified on the surface of GQDs@GO@CF, sequentially. The immobilization properties of GQDs@GO@CF to L-Apt and the adsorption properties of L-Apt/GQDs@GO@CF to DNAzyme were also researched, respectively. Then, the modified sandwich-rod carbon fiber composite was applied to the construction of CL biosensor for LZM detection. When LZM existed, DNAzyme would be released from the surface of L-Apt/GQDs@GO@CF and catalyzed the reaction of luminal - H2O2. Under optimized conditions, the CL biosensor for LZM detection showed wide linear range of 2.64 × 10-10 to 6.6 × 10-8 g/L and low detection limit of 1.25 × 10-11 g/L (3δ). Finally, the CL biosensor was successfully used for LZM detection in human urine samples and illustrated the potential application in pratical samples.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Carbono/química , DNA Catalítico/química , Hemina/química , Muramidase/análise , Adsorção , DNA Catalítico/metabolismo , Quadruplex G , Luminescência , Muramidase/metabolismo , Propriedades de Superfície
4.
Anal Chim Acta ; 1070: 112-122, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31103164

RESUMO

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has proven to be an effective technique for aptamers selection. In this study, we present an online reaction based convenient single-step CE-SELEX (ssCE-SELEX) mode with human thrombin (H-Thr) as a model target. The selection progress was monitored through bulk Kd analysis, which showed more than a 1000-fold improvement over the initial library after two rounds of selection. Three selected candidate sequences presented high binding affinities against H-Thr with nanomolar (nM) Kd determined by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM, 56.4-177.1 nM) and CE based non-linear fitting (CE-NLF, 98.2-199.7 nM). They also exhibited high specificities towards H-Thr compared with bovine thrombin, IgG, lysozyme, and lactoferrin. Meanwhile, the Kd results by isothermal titration calorimetry (ITC) confirmed the effective CE in measuring the aptamer affinity. In addition, three candidates were applied as aptasensors in the AuNPs based colorimetric assay, which showed visible color change and good linear relationships (R2 > 0.93) with H-Thr concentration. Furthermore, molecular dynamics (MD) simulation was performed to validate the binding of the three candidates with H-Thr by binding sites and binding free energy. The ssCE-SELEX method avoids off-line incubation, saves time and sample, and may provide a universal and convenient method for aptamers selection.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Sistemas On-Line , Técnica de Seleção de Aptâmeros/métodos , Animais , Calorimetria , Bovinos , Eletroforese Capilar , Humanos , Imunoglobulina G/análise , Lactoferrina/análise , Ligantes , Muramidase/análise , Muramidase/metabolismo , Trombina/análise
5.
Talanta ; 199: 507-512, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952291

RESUMO

Black phosphorus quantum dots (BPQDs) can react with Ru(bpy)32+ to generate strong anodic electrogenerated chemiluminescence (ECL). However, the instability and the lack of functional groups on BPQDs limit its further application in the fabrication of ECL biosensor. In the present work, uniform BPQDs-styrene-acrylamide (St-AAm) nanospheres (BSAN) are synthesized by encapsulating BPQDs into St-AAm copolymer nanospheres. Sufficient amount of BPQDs can be embedded into nanospheres, and react with Ru(bpy)32+ to generate strong anodic ECL which is comparable to that of pure BPQDs. Amino group of polymer endows BPQDs the ability to connect with DNA, and can be used to fabricate ECL aptasensor for the sensitive detection of lysozyme. The proposed aptasensor shows high sensitivity, good selectivity and stability for the detection of lysozyme in the range of 0.1-100 pg mL-1 with a detection limit of 0.029 pg mL-1 (3σ). The proposed method reveals the promising ECL sensing application of BP nanomaterials in the detection of various proteins.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Medições Luminescentes , Muramidase/análise , Pontos Quânticos/química , Acrilamida/química , Clara de Ovo/química , Muramidase/metabolismo , Nanoestruturas/química , Fósforo/química , Polímeros/química , Estireno/química
6.
J Colloid Interface Sci ; 546: 312-323, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30927595

RESUMO

An electrostatic nanocomplex between naturally occurring ε-poly-l-lysine (εPL) and ß-cyclodextrin sulphate (sCD) was designed, and its capacity to entrap four model proteins with high or low molecular weight and isoelectric point, i.e., lactoferrin, albumin, actinidin, and lysozyme, was investigated. The optimal formulations gave nanocomplexes with an average diameter around 276 ±â€¯16 nm, a ζ-potential of -39 ±â€¯1.5 mV, and a spherical shape with a core-shell structure. Different strategies were pursued to increase the entrapment efficiency for selected proteins, which led to 40-100% entrapment depending on the protein type. Under simulated gastric conditions with pepsin, the complexes protected lactoferrin and albumin against proteolysis, whereas actinidin and lysozyme were intrinsically stable. In Caco-2 cells, these complexes transiently decreased the trans-epithelial electrical resistance, indicating the potential to enhance the paracellular permeability of bioactive macromolecules. Thus, these εPL-sCD complexes would be a promising system for loading diverse proteins for gastric protection and enhancing intestinal absorption.


Assuntos
Albuminas/metabolismo , Cisteína Endopeptidases/metabolismo , Sistemas de Liberação de Medicamentos , Trato Gastrointestinal/efeitos dos fármacos , Lactoferrina/metabolismo , Muramidase/metabolismo , Polilisina/farmacologia , Substâncias Protetoras/farmacologia , beta-Ciclodextrinas/farmacologia , Albuminas/análise , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cisteína Endopeptidases/análise , Relação Dose-Resposta a Droga , Humanos , Lactoferrina/análise , Estrutura Molecular , Muramidase/análise , Tamanho da Partícula , Polilisina/química , Substâncias Protetoras/química , Relação Estrutura-Atividade , Propriedades de Superfície , beta-Ciclodextrinas/química
7.
Fish Shellfish Immunol ; 88: 17-27, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30831244

RESUMO

Pharmaceuticals and household chemicals are important components of municipal sewage. Many of them are biologically active, disrupting not only hormonal regulation of aquatic animals but also, indirectly, disturbing their immunological protection. In the environment, chemicals rarely act as individual substances, but as elements of mixtures. Therefore, the aim of this study was to check whether the acute laboratory exposure of common carp juveniles to a mixture of ibuprofen, sodium dodecyl sulphate (SDS), dimethyl sulfoxide (DMSO) and 17 α-ethynylestradiol in increasing concentrations, modifies the levels of innate immunity (lysozyme, C-reactive protein) as well as general stress (metallothioneins, heat shock proteins HSP70) markers in brain, liver, gills, spleen and mucus. The levels of the markers were measured by an immunodetection technique. Not only do the pharmaceuticals and household chemicals impair immunological reactions of young carp in various tissues but also do that in a concentration-dependent manner in the liver, gills, spleen and mucus. This has a very important implication, since it may result in higher sensitivity of young fish to pathogens due to energy allocation to defence processes. The comparisons of the pattern of stress reactions in the studied organ samples indicated that mucus appeared to be a good, non-invasive material for monitoring of environmental state and fish conditions.


Assuntos
Carpas/imunologia , Poluentes Químicos da Água/toxicidade , Animais , Biomarcadores , Proteína C-Reativa/análise , Dimetil Sulfóxido/toxicidade , Etinilestradiol/toxicidade , Proteínas de Choque Térmico HSP70/análise , Ibuprofeno/toxicidade , Imunidade Inata , Metalotioneína/análise , Muco/química , Muramidase/análise , Esgotos/química , Dodecilsulfato de Sódio/toxicidade , Estresse Fisiológico , Poluentes Químicos da Água/imunologia
8.
Gen Comp Endocrinol ; 275: 73-81, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30735670

RESUMO

Phenotypic effects of global warming have been documented in many different taxa. However, the importance of transgenerational phenotypic plasticity in these adaptations are seldom studied. In birds, temperature could affect egg characteristics. Higher temperatures during egg-laying may reduce maintenance costs for females and allow a higher investment in reproduction. Yet, females may also use temperatures as a cue for the risk of mismatch latter in the season. Thus, higher temperatures may be correlated to an acceleration of embryonic development (e.g. via hormonal manipulation). We performed an experiment in which night-time temperature was increased in the nestbox by approximately 1 °C throughout the entire laying period in great tits (Parus major). We collected one pre-treatment egg (beginning of the laying sequence) and one post-treatment egg (end of the laying sequence). Egg content (yolk androgens and lysozymes in the albumen), eggshell coloration, eggshell mass, egg mass, and shape were not affected by the treatment. However, last-laid eggs in clutches from control nestboxes had a thicker eggshell than last-laid eggs from heated nestboxes, suggesting a putative slight decrease of maternal investment with the experimental increase of temperature. We also observed effects of the laying sequence on egg characteristics. Eggs that were laid late in the laying sequence were heavier, larger, had larger spots and higher yolk androgens than eggs laid earlier. Lysozyme concentration decrease with the laying sequence in late clutches only. Thus, effects of temperature may also change with the laying sequence and it would be interesting in the future to tests the effects on first-laid eggs.


Assuntos
Casca de Ovo/anatomia & histologia , Gema de Ovo/química , Aquecimento Global , Temperatura Alta/efeitos adversos , Passeriformes , Pigmentação , Androgênios/análise , Animais , Casca de Ovo/citologia , Ovos/análise , Feminino , Comportamento Materno/fisiologia , Muramidase/análise , Comportamento de Nidação/fisiologia , Oviposição/fisiologia , Passeriformes/anatomia & histologia , Passeriformes/embriologia , Passeriformes/fisiologia , Fenótipo , Temperatura Ambiente
9.
Talanta ; 197: 539-547, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30771973

RESUMO

Immobilized enzyme micro-reactors (IMERs) are of vital importance in developing miniaturized bioanalytical systems and have promising applications in various biomanufacturing. An inherent limitation in designing IMERs is the one-dimensional cylindrical geometry of micro-channels that offers limited exposed surface area for molecular reorganization and enzyme immobilization. In this study, we report a robust capillary-IMER based on a three dimensional porous layer open tubular (3D-PLOT) column which is prepared by an easy-to-control surface modification strategy via single-step in situ biphasic reaction. The 3D-PLOT column with highly uniform porous geometry and narrow distribution of porosity can greatly enhance the surface-area-to-volume ratio of the micro-channels, showing the beneficial effects for enzyme immobilization to enhance reaction efficiency and shorten analysis time. Taking trypsin as a model enzyme, enzymatic activities of immobilized enzyme are analyzed. We compare enzyme assays using the proposed 3D-PLOT-IMER with those using normal capillary-IEMR without surface modification as well as free trypsin. The 3D-PLOT-IMER exhibits excellent stability and inter/intra-day reproducibility, and these characteristics imply the reliability of the proposed IMERs for accurate enzyme assay. The feasibility of the proposed method for potential application in biological analysis is demonstrated by coupling the 3D-PLOT-IMER with a nano-LC-MS/MS system for online digestion of standard proteins, cell extraction and living Hela cells. Our study show that the surface modification with the proposed 3D-porous layer is a simple and efficient approach for enzyme immobilization, and could be widely suitable for different kinds of IMERs.


Assuntos
Enzimas Imobilizadas/metabolismo , Muramidase/metabolismo , Dióxido de Silício/química , Cromatografia Líquida de Alta Pressão , Enzimas Imobilizadas/análise , Células HeLa , Humanos , Muramidase/análise , Tamanho da Partícula , Porosidade , Espectrometria de Massas por Ionização por Electrospray , Propriedades de Superfície , Espectrometria de Massas em Tandem
10.
Spectrochim Acta A Mol Biomol Spectrosc ; 213: 127-133, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30684881

RESUMO

Silver nanoparticles (AgNPs) functionalized with glutamic acid (GA) was used as a biochemical sensing probe in colorimetry for detection of lysozyme protein in milk samples. The method is based on the color change of AgNPs/GA from yellow to reddish-yellow differentiated with naked eyes for qualitative determination and red shift of localized surface plasmon resonance (LSPR) absorption signal intensity of AgNPs/GA in visible region used for quantitative determination of lysozyme. The control experiments were performed to demonstrate the electrostatic force of interactions between AgNPs/GA and protein molecule. A wide linear range of 3-150 nM with limit of detection of 1.5 nM was acquired for quantitative determination of lysozyme using AgNPs/GA as a biochemical sensing probe. The advantages of using AgNPs/GA as a biochemical sensing probe are simple, label-free and economic for determination of lysozyme from milk samples.


Assuntos
Técnicas Biossensoriais/métodos , Nanopartículas Metálicas/química , Leite/enzimologia , Muramidase/análise , Prata/química , Animais , Bovinos , Clara de Ovo , Ácido Glutâmico/química , Concentração de Íons de Hidrogênio , Limite de Detecção , Nanopartículas Metálicas/ultraestrutura
11.
Fish Shellfish Immunol ; 86: 653-661, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30502467

RESUMO

Recently, mucosal surfaces, especially fish skin and its secreted mucus, have attracted significant interest from immunologists. Amphiprion clarkii, a member of the family Pomacentridae, lives symbiosis with sea anemones and has a good resistance to common seawater bacterial diseases and parasites owing to the protection from its abundant skin mucus. In the present work, the activity of immune-related enzymes (lysozyme, protease, antiprotease, cathepsin B, alkaline phosphatase and peroxidase), the antibacterial activity against two Gram-positive bacteria and five Gram-negative bacteria, the antiparasitic activity against the pathogen of marine white spot disease (Cryptocaryon irritans theronts) and the physico-chemical stability (to pH and heat) of the skin mucus of A. clarkii were analysed. The results showed that the levels of lysozyme and peroxidase were very similar (from 2 to 4 U mg-1 protein). However, cathepsin B was detected of 63.32 U mg-1 protein and alkaline phosphatase was only 0.12 U mg-1 protein. Moreover, protease showed a higher percentage of activity than antiprotease. A. clarkii skin mucus showed a strong antibacterial activity against Gram-negative bacteria, particularly against Aeromonas hydrophila and Vibrio parahaemolyticus but showed no effect on Gram-positive bacteria at the tested concentrations. The bactericidal activity functioned within a short time in a distinct time- and dose-dependent manner. SEM showed that after treated with A. clarkii skin mucus, the V. parahaemolyticus cells distorted and piled together, and the filaments appeared and became into cotton-shaped or quasi-honeycomb texture to adhere cells. Meanwhile, A. clarkii skin mucus showed an apparent antiparasitic activity against C. irritans theronts with a distinct dose- and time-dependent relationship. LM and SEM observation showed that after treated with skin mucus, the theronts quickly stopped their swimming and cilia movement, cells became rounded, cilia shed, small bubbles formed on the surface, cell nucleolus enlarged, cytoskeleton deformed, cell membranes ruptured and cell content leaked out. Antibacterial activity was not affected by 30-90 °C heat treatment but was slightly suppressed by 100 °C. In the pH treatment groups, antibacterial activity was not affected by the moderate pH treatment of 5.0-8.0, but slightly suppressed by weak acid and weak base. Therefore, we speculated that the skin mucus of A. clarkii might be a potential source of novel antibacterial and antiparasitic components for fish or human health-related applications. This study broadened our understanding of the role of skin mucus in the innate immune system and provided a basis for the further isolation and purification of active substances.


Assuntos
Doenças dos Peixes/enzimologia , Muco/química , Perciformes , Pele/química , Fosfatase Alcalina/análise , Animais , Catepsina B/análise , Doenças dos Peixes/microbiologia , Doenças dos Peixes/parasitologia , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Concentração de Íons de Hidrogênio , Muco/enzimologia , Muramidase/análise , Peroxidase/análise , Estabilidade Proteica , Pele/enzimologia
12.
J Sci Food Agric ; 99(4): 1643-1650, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30198063

RESUMO

BACKGROUND: Lysozyme has been studied as a potential alternative to antibiotics for animals in recent years. The aim of this study was to evaluate the effect of dietary lysozyme on growth performance, serum biochemical parameters, immune response and gut health of growing pigs. RESULTS: A total of 216 growing pigs (19.81 ± 0.47 kg) were fed the diets supplemented with colistin sulfate at 20 mg kg-1 (control), or lysozyme at 50 (L50) or 100 mg kg-1 (L100) diet for 30 days. The results showed that pigs fed with L100 or control had greater average daily gain and gain-to-feed ratio than pigs in the L50 group. Pigs fed with L100 or colistin had greater villus height to crypt depth ratio in jejunum compared with pigs in the L50 group. Pigs fed with L100 had greater serum immunoglobulin A and jejunal secretory immunoglobulin A than control and L50, but lower serum total protein and globulin than control. No differences were observed in the messenger RNA expression of genes related to mucosal cytokines, antioxidant capacity, enzyme activity, and barrier functions among three treatments. The caecal microflora evenness was lower in the L100 group than in the control or L50 group by 16S ribosomal DNA sequencing. Phylogenetic investigation of communities by reconstruction of unobserved states analysis predicted that lysozyme may modify nutrient metabolism by changing intestinal microbial function of pigs. CONCLUSIONS: Pigs supplemented with 100 mg kg-1 lysozyme had similar growth performance and intestinal morphology as pigs fed with colistin. This was likely due to the improved systemic and gut immune responses and the reduced microbiota diversity by feeding 100 mg kg-1 lysozyme. © 2018 Society of Chemical Industry.


Assuntos
Microbioma Gastrointestinal , Intestinos/microbiologia , Muramidase/administração & dosagem , Suínos/crescimento & desenvolvimento , Suínos/imunologia , Ração Animal/análise , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Citocinas/genética , Citocinas/imunologia , Dieta/veterinária , Suplementos Nutricionais/análise , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Imunoglobulina A/imunologia , Intestinos/efeitos dos fármacos , Intestinos/crescimento & desenvolvimento , Intestinos/imunologia , Masculino , Muramidase/análise , Filogenia , Suínos/genética , Suínos/microbiologia
13.
Matern Child Nutr ; 14 Suppl 6: e12566, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30592165

RESUMO

Expressed human milk can be donated or sold through a variety of channels, including human milk banks, corporations or individuals, or peer-to-peer milk sharing. There is a paucity of research regarding the nutrient and bioactive profiles of expressed human milk exchanged through commerce-free scenarios, including peer-to-peer milk sharing. The study objective was to evaluate the macronutrient, antimicrobial protein, and bacteria composition in expressed human milk acquired via commerce-free arrangements. Expressed human milk samples were collected from the following commerce-free scenarios: milk expressed for a mother's or parent's own infant (MOM; N = 30); unpasteurized milk donated to a non-profit milk bank (BANKED; N = 30); milk expressed for peer-to-peer milk sharing (SHARED; N = 31); and health professional-facilitated milk sharing where donors are serologically screened and milk is dispensed raw (SCREENED; N = 30). Analyses were conducted for total protein, lactose, percent fat and water, lysozyme activity, immunoglobulin A (IgA) activity, total aerobic bacteria, coliform, and Staphylococcus aureus. No bacterial growth was observed in 52/121 samples, and 15/121 had growth greater than 5.0 log colony-forming units/mL. There was no evidence of differences by groups (p > .05) in lactose, fat, water, lysozyme activity, sIgA activity, aerobic bacteria, coliforms, and S. aureus. Mean protein values (95% confidence interval) were 1.5 g/dL (1.4, 1.6) for BANKED, 1.4 g/dL (1.3, 1.5) for MOM, 1.6 g/dL (1.5, 1.7) for SCREENED, and 1.5 g/dL (1.4, 1.6) for SHARED, which was not significantly different (p = .081). This research contributes to growing literature on the risks and benefits of uncompensated, peer-to-peer milk sharing.


Assuntos
Anti-Infecciosos/análise , Proteínas do Leite/análise , Leite Humano/química , Leite Humano/microbiologia , Nutrientes/análise , Bactérias Aeróbias/isolamento & purificação , Extração de Leite , Enterobacteriaceae/isolamento & purificação , Gorduras/análise , Feminino , Humanos , Imunoglobulina A/análise , Lactose/análise , Bancos de Leite , Muramidase/análise , Projetos Piloto , Staphylococcus aureus/isolamento & purificação , Doadores de Tecidos
15.
Zhonghua Nan Ke Xue ; 24(2): 109-115, 2018 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-30156068

RESUMO

Objective: To elucidate the possible role of human lysozyme-like protein 4 (LYZL4) in fertilization and characterize its enzymatic properties. METHODS: The localization of LYZL4 in human spermatozoa was investigated by immunofluorescence staining, the sources of LYZL4 on the sperm surface examined by RT-PCR, and the role of LYZL4 in fertilization assessed by the zona-free hamster egg penetration test. The recombinant plasmid pPIC9K-LYZL4 was constructed and its expression induced with methanol after transformed into competent Pichia pastoris GS115. The recombinant LYZL4 protein (rLYZL4) was purified from the fermentation supernatant and subsequently identified by Western blot. The hyaluronan binding ability of rLYZL4 was determined by ELISA and the muramidase activity, hyaluronidase activity, and free radical scavenging ability examined by spectrophotometric methods. RESULTS: Immunodetection with a specific antiserum localized LYZL4 on the acrosomal membrane of mature spermatozoa, which was exclusively secreted from the testis and epididymis as shown by RT-PCR. Immunoneutralization of LYZL4 significantly decreased the number of human spermatozoa bound to zona-free hamster eggs in a dose-dependent manner in vitro. The recombinant protein was expressed successfully by the P. pastoris strain GS115. Purified rLYZL4 exhibited a potent hyaluronan binding ability and a strong free radical scavenging ability but no muramidase or hyaluronidase activity. CONCLUSIONS: LYZL4 secreted from the testis and epididymis is localized on the acrosomal membrane of mature spermatozoa and plays a role in sperm-egg binding as well as in binding hyaluronan and scavenging free radicals, which suggests that it might be a multi-functional molecule contributive to sperm protection and sperm-egg binding.


Assuntos
Acrossomo/enzimologia , Muramidase/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Animais , Western Blotting , Cricetinae , Ensaio de Imunoadsorção Enzimática , Epididimo , Feminino , Fertilização/fisiologia , Depuradores de Radicais Livres/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Masculino , Muramidase/análise , Pichia , Plasmídeos/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Espermatozoides/enzimologia , Testículo
16.
Prog. obstet. ginecol. (Ed. impr.) ; 61(4): 361-364, jul.-ago. 2018. ilus
Artigo em Espanhol | IBECS | ID: ibc-174978

RESUMO

El carcinoma de células acinares primario de mama es un tumor raro que muestra similitudes con neoplasias de las glándulas salivales; y aunque pertenece al grupo de carcinomas de mama "triple negativos", su comportamiento biológico parece ser más favorable. Se presenta el caso de una mujer de 54 años, sin antecedentes oncoginecológicos, remitida a la consulta de patología mamaria para valoración por mamografía de screening que informa de BIRADS IV en mama derecha. A la exploración física, las mamas y axilas son normales a la inspección y a la palpación, tratándose de una lesión no palpable. El estudio citológico tras punción ecoguiada informa de sospecha de malignidad, motivo por el cual se practica biopsia excisional radioguiada que informa de la presencia de un carcinoma de células acinares de mama. Se realizó tratamiento quirúrgico conservador en mama-axila derecha y recibió radioterapia adyuvante. Tras 6 años del diagnóstico, se encuentra libre de enfermedad


Primary acinic cell carcinoma of the breast is a rare tumor that shows similarities with neoplasms of the salivary glands; and although it belongs to the group of "triple negative" breast cancer, its biological behavior seems to be more favorable. We present the case of a 54-year-old woman, with no gynecological cáncer history, referred to the mammary pathology office for evaluation after mammography screening that reports BIRADS IV in the right breast. On physical examination, the breasts and axillas are normal to inspection and palpation, being this the case of a non-palpable lesion. The cytological study after echoguided puncture reports suspicion of malignancy, which is why a radioguided excisional biopsy is performed, which shows the presence of an acinic cell carcinoma of the breast. Conservative surgical treatment is performed in the right breast-axilla, together adjuvant radiotherapy. 6 years after diagnosis, the patient is free of disease


Assuntos
Humanos , Feminino , Pessoa de Meia-Idade , Carcinoma de Células Acinares/patologia , Neoplasias da Mama/patologia , Mastectomia , Muramidase/análise , Metástase Linfática/patologia , Radioterapia Adjuvante , Biópsia Guiada por Imagem/métodos
17.
Protein Sci ; 27(10): 1837-1841, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30056633

RESUMO

A simple "diffusion-to-capture" model is used to estimate the upper limit to the growth rate of macromolecular crystals under conditions when the rate limiting process is the mass transfer of sample from solution to the crystal. Under diffusion-limited crystal growth conditions, this model predicts that the cross-sectional area of a crystal will increase linearly with time; this prediction is validated by monitoring the growth rate of lysozyme crystals. A consequence of this analysis is that when crystal growth is diffusion-limited, micron-sized crystals can be produced in ~1 s, which would be compatible with the turnover time of many enzymes. Consequently, the ability to record diffraction patterns from sub-micron sized crystals by X-ray Free Electron Lasers and micro-electron diffraction technologies opens the possibility of trapping intermediate enzyme states by crystallization.


Assuntos
Muramidase/análise , Cristalização , Humanos , Substâncias Macromoleculares/análise , Substâncias Macromoleculares/síntese química , Substâncias Macromoleculares/metabolismo , Muramidase/síntese química , Muramidase/metabolismo
18.
Anal Sci ; 34(7): 765-770, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29998956

RESUMO

When proteins are attached to microstructures such as a metal mesh device, changes in their optical properties occur. These changes have been characterized based on actual measurements in the infrared region of the spectrum. We have previously theoretically and experimentally demonstrated the optical changes associated with streptavidin. Here, we investigate three types of proteins: avidin, BSA, and lysozyme. The three proteins were adsorbed onto three types of metal mesh devices having different resonant frequencies, and the corresponding spectra were measured in the infrared region. The change in the frequency of the dip point in the spectrum was extracted to quantitatively determine the quantity of protein; these results were correlated with the quantitative measurements obtained by electrophoresis. By examining three types of different proteins, it was verified that a variety of proteins can be measured based on the optical characteristics of metal mesh devices.


Assuntos
Avidina/análise , Metais/química , Muramidase/análise , Soroalbumina Bovina/análise , Animais , Bovinos , Muramidase/metabolismo
19.
Talanta ; 188: 493-498, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30029403

RESUMO

Recently, the separation of proteins has received much attention, although many techniques require expensive instrumentation and trained analysts. In this work, a low-cost, effective, and environmental friendship capillary electrophoresis (CE) for proteins separation was first time introduced. The ZIF-8 with outstanding properties of large surface area, and accessible tunnels and cages were coated the inner surface of silica capillary as a separation media by electrostatic interaction. The fast baseline separation of Lys, CC, BSA and RNase A can be obtained within 10 min using the ZIF-8 nanocrystals coated capillary column under the optimum separation conditions. Meanwhile, this system showed good reproducibility and stability. Using L-glutamic acid as the selector ligand, the D- and L-phenylalanine were successfully separated by the ZIF-8 nanocrystals coated capillary column. Furthermore, the method was also applied to separate egg white proteins, and three main proteins were separated in a single run.


Assuntos
Eletroforese Capilar/métodos , Estruturas Metalorgânicas/química , Proteínas/análise , Zeolitas/química , Animais , Bovinos , Galinhas , Citocromos c/análise , Química Verde/métodos , Imidazóis/síntese química , Imidazóis/química , Estruturas Metalorgânicas/síntese química , Muramidase/análise , Nanopartículas/química , Fenilalanina/análise , Fenilalanina/química , Reprodutibilidade dos Testes , Ribonuclease Pancreático/análise , Soroalbumina Bovina/análise , Estereoisomerismo , Zeolitas/síntese química
20.
ACS Sens ; 3(8): 1480-1488, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-29984996

RESUMO

A unique split-type photoelectrochemical (PEC) immunoassay has been constructed for detection of low-abundance biocompounds (lysozyme, Lyz, used in this case) via a new trigger strategy by disintegrating bioconjugates of dopamine-grafted silica nanospheres (DA@SiO2NSs) for signal amplification. The preferred electron donor assembly of DA@SiO2NSs is first used as a molecular printboard for positioning anti-Lyz secondary antibody (Ab2) through an amide reaction. With specific immunoreactions in a high-binding microplate, a sandwich immunoassay, the DA@SiO2NSs-based bioconjugate is achieved. By initiating the disintegration of the bioconjugates via acid etching, numerous electron donors of DA are released, thus efficiently triggering hole-trapping with amplified signals obtained. The smart integration of ZnIn2S4-based heterojunctions as photoactive material, a split-type detection mode, and a new trigger strategy by disintegrating the DA@SiO2NSs-based bioconjugate offer an attractive high-throughput signal-on PEC immunoassay for detection of Lyz. Such an unusual PEC sensor exhibits an outstanding linear response to the concentration in the range between 0.002 and 500 ng mL-1, and the detection limit is as low as 0.6 ppt ( S/ N = 3). The as-fabricated assay is cost-effective and sensitive. It has been successfully used for measuring Lyz in real samples, which demonstrates great promise for practical applications.


Assuntos
Dopamina/química , Imunoensaio/métodos , Muramidase/análise , Nanosferas/química , Dióxido de Silício/química , Anticorpos Imobilizados/imunologia , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/metabolismo , Técnicas Eletroquímicas , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Muramidase/sangue , Muramidase/imunologia , Óxido de Zinco/química
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