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1.
Anal Chim Acta ; 1093: 160-167, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31735210

RESUMO

In this study, poly(butyl methacrylate-co-ethyleneglycol dimethacrylate) polymeric monoliths were in situ developed within 0.75 mm i.d. poly(ethylene-co-tetrafluoroethylene) (ETFE) tubing by UV polymerization via three different free-radical initiators (α,α'-azobisisobutyronitrile (AIBN), 2,2-dimethoxy-2-phenylacetophenone (DMPA) and 2-methyl-4'-(methylthio)-2-morpholinopropiophenone (MTMPP). The influence of the nature of each photo-initiator and irradiation time on the morphological features of the polymer was investigated by scanning electron microscopy, and the chromatographic properties of the resulting microbore columns were evaluated using alkyl benzenes as test substances. The beds photo-initiated with MTMPP gave the best performance (minimum plate heights of 38 µm for alkyl benzenes) and exhibited a satisfactory reproducibility in the chromatographic parameters (RSD < 11%). These monolithic columns were also successfully applied to the separation of phenylurea herbicides, proteins and a tryptic digest of ß-casein.


Assuntos
Acetofenonas/química , Cromatografia Líquida de Alta Pressão/instrumentação , Morfolinas/química , Nitrilos/química , Ácidos Polimetacrílicos/química , Politetrafluoretileno/análogos & derivados , Propiofenonas/química , Acetofenonas/efeitos da radiação , Caseínas/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Herbicidas/isolamento & purificação , Metacrilatos/química , Morfolinas/efeitos da radiação , Nitrilos/efeitos da radiação , Fragmentos de Peptídeos/isolamento & purificação , Compostos de Fenilureia/isolamento & purificação , Polimerização , Ácidos Polimetacrílicos/síntese química , Politetrafluoretileno/química , Propiofenonas/efeitos da radiação , Raios Ultravioleta
2.
Analyst ; 144(21): 6270-6275, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31566639

RESUMO

Enhanced-fluidity, reversed-phase liquid chromatography was developed using custom instrumentation for separation and characterization of intact KRas proteins and tryptic peptides. The KRas, HRas and NRas function as GDP-GTP regulated binary switches in many signalling pathways, and mutations in Ras proteins are frequently found in human cancers and represent poor prognosis markers for patients. Mutations of the KRas isoform constitute some of the most common aberrations among all human cancers and intensive drug discovery efforts have been directed toward targeting the KRas protein. Separation and characterization of the KRas protein and tryptic peptides are helpful for exploring targeting, which has not been fully investigated using liquid chromatography-tandem mass spectrometry. EFLC-MS provided improved chromatographic performance compared to traditional HPLC-MS in terms of shorter analysis time, increased ion intensity and a shift to higher charge states for intact KRas proteins.


Assuntos
Cromatografia Líquida/métodos , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Tripsina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Solventes/química
3.
Int J Mol Sci ; 20(20)2019 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-31635129

RESUMO

Velvet antler has a long history in traditional medicine. It is also an important healthy ingredient in food as it is rich in protein. However, there has been no report about antioxidant peptides extracted from velvet antler by enzymatic hydrolysis. Thus, the objective of this study was to hydrolyze velvet antler using different commercial proteases (Acalase, Neutrase, trypsin, pepsin, and α-chymotrypsin). Antioxidant activities of different hydrolysates were investigated using peroxyl radical scavenging assay by electron spin resonance spectrometry. Among all enzymatic hydrolysates, Alcalase hydrolysate exhibited the highest peroxyl radical scavenging activity. Alcalase hydrolysate was then purified using ultrafiltration, gel filtration, and reverse-phase high performance liquid chromatography. The purified peptide was identified to be Trp-Asp-Val-Lys (tetrapeptide) with molecular weight of 547.29 Da by Q-TOF ESI mass spectroscopy. This purified peptide exhibited strong scavenging activity against peroxyl radical (IC50 value, 0.028 mg/mL). In addition, this tetrapeptide showed significant protection ability against AAPH-induced oxidative stress by inhibiting of reactive oxygen species (ROS) generation in Chang liver cells in vitro and in a zebrafish model in vivo. This research suggests that the tetrapeptide derived from Alcalase-proteolytic hydrolysate of velvet antler are excellent antioxidants and could be effectively applied as functional food ingredients and pharmaceuticals.


Assuntos
Antioxidantes/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Subtilisinas/química , Animais , Antioxidantes/química , Antioxidantes/isolamento & purificação , Chifres de Veado/química , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/farmacologia , Humanos , Hidrólise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra
4.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370181

RESUMO

The ability to control the glycosylation pattern of recombinant viral glycoproteins represents a major prerequisite before their use as vaccines. The aim of this study consisted of expressing the large soluble ectodomain of glycoprotein B (gB) from Human Cytomegalovirus (HMCV) in Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells and of comparing its glycosylation profile with that of gB produced in Chinese hamster ovary (CHO) cells. gB was secreted in the BY-2 culture medium at a concentration of 20 mg/L and directly purified by ammonium sulfate precipitation and size exclusion chromatography. We then measured the relative abundance of N-glycans present on 15 (BY-2) and 17 (CHO) out of the 18 N-sites by multienzymatic proteolysis and mass spectrometry. The glycosylation profile differed at each N-site, some sites being occupied exclusively by oligomannosidic type N-glycans and others by complex N-glycans processed in some cases with additional Lewis A structures (BY-2) or with beta-1,4-galactose and sialic acid (CHO). The profiles were strikingly comparable between BY-2- and CHO-produced gB. These results suggest a similar gB conformation when glycoproteins are expressed in plant cells as site accessibility influences the glycosylation profile at each site. These data thus strengthen the BY-2 suspension cultures as an alternative expression system.


Assuntos
Fragmentos de Peptídeos/química , Polissacarídeos/química , Proteínas do Envelope Viral/química , Sulfato de Amônio/química , Animais , Células CHO , Sequência de Carboidratos , Precipitação Química , Cromatografia em Gel/métodos , Cricetulus , Galactose/química , Expressão Gênica , Glicosilação , Humanos , Ácido N-Acetilneuramínico/química , Fragmentos de Peptídeos/isolamento & purificação , Células Vegetais/metabolismo , Polissacarídeos/isolamento & purificação , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tabaco/citologia , Tabaco/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
5.
Talanta ; 204: 670-676, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357351

RESUMO

Protein phosphorylation is a reversible and important post-translational modification. Identification of phosphopeptides without enrichment is difficult for the low-abundance of phosphopeptides in real complex biological samples. Therefore, the effective and selective concentration of phosphopeptides prior to proteomic identification by mass spectrometer is necessary. In this study, we synthesized a novel titanium-based immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides. To improve material hydrophilia to the maximum extent, titanium ions were immobilized on the 4-armed Poly(ethylene oxide)(4µ-PEO-Ti4+), a totally soluble polymer with large molecular weight (20000 g/mol). The 4µ-PEO-Ti4+ was used to enrich phosphopeptides from tryptic digests of standard proteins and real complex biological samples, followed by MALDI-TOF MS analysis. In enrichment of phosphopeptides from 4 pmol ß-casein, the 4µ-PEO-Ti4+ performed the best property with starting material of 99-132 µg, loading buffer of 50% ACN/5% TFA (v/v), elution buffer of 10% NH3·H2O (v/v) and elution time of 30 min. The 4µ-PEO-Ti4+ has a superior detection sensitivity as low as 2 fmol for phosphopeptides. The high selectivity of 4µ-PEO-Ti4+ allows a deep enrichment of phosphopeptides of ß-casein from a mixture with BSA of 1000-fold abundant. The 4µ-PEO-Ti4+ shows great stability and endurability and can be recycled up to at least 5 times. In addition, 4µ-PEO-Ti4+ could detect 10 and 15 phosphopeptides from non-fat milk and nonenzymatic human saliva, respectively. In total, 4µ-PEO-Ti4+ is a novel excellent material which shows great sensitive and selective enrichment of low-abundance phosphopeptides in real complex biological samples.


Assuntos
Fosfopeptídeos/isolamento & purificação , Polietilenoglicóis/química , Titânio/química , Sequência de Aminoácidos , Animais , Caseínas/química , Caseínas/isolamento & purificação , Cromatografia de Afinidade/métodos , Feminino , Humanos , Leite/química , Fragmentos de Peptídeos/isolamento & purificação , Proteólise , Saliva/química , Proteínas e Peptídeos Salivares/isolamento & purificação , Tripsina/química
6.
Sensors (Basel) ; 19(11)2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31163612

RESUMO

Surface-plasmon-resonance (SPR) is a quantum-electromagnetic phenomenon arising from the interaction of light with free electrons at a metal-dielectric interface. At a specific angle/wavelength of light, the photon's energy is transferred to excite the oscillation of the free electrons on the surface. A change in the refractive-index (RI) may occur, which is influenced by the analyte concentration in the medium in close contact with the metal surface. SPR has been widely used for the detection of gaseous, liquid, or solid samples. In this study, a functionalized specific SPR chip was designed and used in a novel point-of-care SPR module (PhotonicSys SPR H5) for the detection of the stroke biomarkers NT-proBNP and S100ß. These biomarkers have proven to be good for stroke diagnosis, with sensitivity and specificity of >85%. Specific detection was done by binding a biomolecular-recognizing antibody onto the Au SPR-chip. Detection was tested in water and plasma samples. NT-proBNP and S100ß were detected in a range of concentrations for stroke, from 0.1 ng/mL to 10 ng/mL. The RI of the blank plasma samples was 1.362412, and the lowest concentration tested for both biomarkers showed a prominent shift in the RI signal (0.25 ng/mL NT-proBNP (1.364215) and S100ß (1.364024)). The sensor demonstrated a clinically relevant limit-of-detection of less than ng/mL.


Assuntos
Técnicas Biossensoriais , Peptídeo Natriurético Encefálico/isolamento & purificação , Fragmentos de Peptídeos/isolamento & purificação , Subunidade beta da Proteína Ligante de Cálcio S100/isolamento & purificação , Acidente Vascular Cerebral/diagnóstico , Anticorpos/química , Anticorpos/imunologia , Biomarcadores/química , Ouro/química , Humanos , Peptídeo Natriurético Encefálico/química , Fragmentos de Peptídeos/química , Sistemas Automatizados de Assistência Junto ao Leito , Subunidade beta da Proteína Ligante de Cálcio S100/química , Ressonância de Plasmônio de Superfície
7.
Pharm Biol ; 57(1): 369-379, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31156004

RESUMO

Context: Silk peptide from cocoons of silkworm (Bombyx mori L., Bombycidae) has been employed as a biomedical material and exhibits various bioactivities, including immune-modulating activity. Objective: We analyzed whether silk peptide exerts direct modulating effects on NK cells using an NK cell line in vitro and ex vivo splenocytes. We also attempted to delineate the mechanism underlying the modulation. Material and methods: In vitro activity of silk peptide on NK cells was determined by measurement of cytolytic activity against K562 cells at an effector-to-target ratio of 5:1 after incubation of NK-92MI cells with silk peptide (0-2000 µg/mL) for 48 and 72 h. Ex vivo activity of silk peptide on mouse splenic NK cells was determined similarly by using YAC-1 cells. Results: Treatment of NK-92MI NK cells with silk peptide (500-2000 µg/mL) significantly increased cytolytic activity on target cells by 2- to 4-fold. The same concentrations (500-2000 µg/mL) of silk peptide treatment also significantly enhanced the cytolytic activity of splenic NK cells against YAC-1 cells. Silk peptide treatment of IL-2-stimulated splenocytes induced enhanced expression of Th1, 2 and 17 cytokines including TNF-α, IFN-γ, IL-6, IL-4 and IL-17. Finally, ex vivo treatment with silk peptide on mouse splenocytes significantly enhanced the degree of NK cell maturation in a dose-dependent manner from 3.49 to 23.79%. Discussion and conclusions: These findings suggest that silk peptide stimulates NK cells, thereby influencing systemic immune functions and improving natural immunity. Thus, silk peptide could be useful as a complementary therapy in cancer patients.


Assuntos
Bombyx , Fatores Imunológicos/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Seda/química , Baço/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Citocinas/imunologia , Relação Dose-Resposta a Droga , Humanos , Fatores Imunológicos/isolamento & purificação , Células K562 , Células Matadoras Naturais/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Seda/imunologia , Baço/citologia , Baço/imunologia
8.
Anal Chim Acta ; 1062: 156-164, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-30947992

RESUMO

This study reports on the conception of magneto-Capillary Electrophoresis (magneto-CE), an approach integrating immuno-capture on circulating bio-functionalized magnetic beads into a unique capillary for preconcentration and electrokinetic separation. This hybrid mode is an evolution of in-capillary magnetic bead-based operation from static cluster format to dynamic configuration where beads are allowed to controllably circulate inside a CE capillary for interaction improvement. To implement the magneto-CE operation, a purpose-made instrument was constructed, allowing visual observation of the movement of the magnetic beads. We applied a new methodological strategy for determination of the amyloid ß peptide (Aß 1-42), which is as an established biomarker for molecular diagnosis of Alzheimer's disease (AD). The methodology is based on magneto-immuno-capture of fluorescently labeled Aß 1-42 followed by a chemical elution with a basic solution prior to CE separation with laser induced fluorescent (LIF) detection. The superiority of this dynamic configuration of magneto-CE was demonstrated for this target analyte, with sample pretreatment and separation being performed in-capillary without any delay in between and without any waste of pretreated sample, which otherwise would not be the case with offline/batch-wise operation.


Assuntos
Peptídeos beta-Amiloides/isolamento & purificação , Eletroforese Capilar , Separação Imunomagnética , Campos Magnéticos , Fragmentos de Peptídeos/isolamento & purificação , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/química , Humanos , Fragmentos de Peptídeos/química
9.
Pharm Res ; 36(5): 77, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30937539

RESUMO

PURPOSE: To explore how the natural heterogeneity of human coagulation factor VIII (FVIII) and the processing of its B-domain specifically modulate protein aggregation. METHODS: Recombinant FVIII (rFVIII) molecular species containing 70% or 20% B-domain, and B-domain-deleted rFVIII (BDD-rFVIII), were separated from full-length recombinant FVIII (FL-rFVIII). Purified human plasma-derived FVIII (pdFVIII) was used as a comparator. Heterogeneity and aggregation of the various rFVIII molecular species, FL-rFVIII and pdFVIII were analysed by SDS-PAGE, dynamic light scattering, high-performance size-exclusion chromatography and flow cytometry-based particle analysis. RESULTS: FL-rFVIII and pdFVIII were heterogeneous in nature and demonstrated similar resistance to aggregation under physical stress. Differences were observed between these and among rFVIII molecular species. FVIII molecular species exhibited diverging aggregation pathways dependent on B-domain content. The propensity to form aggregates increased with decreasing proportions of B-domain, whereas the opposite was observed for oligomer formation. Development of cross-ß sheet-containing aggregates in BDD-rFVIII induced effective homologous seeding and faster aggregation. Naturally heterogeneous FL-rFVIII and pdFVIII displayed the lowest propensity to aggregate in all experiments. CONCLUSIONS: These results demonstrate that pdFVIII and FL-rFVIII have similar levels of molecular heterogeneity, and suggest that heterogeneity and the B-domain are involved in stabilising FVIII by modulating its aggregation pathway.


Assuntos
Fator VIII/química , Fragmentos de Peptídeos/química , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Fator VIII/isolamento & purificação , Humanos , Espectrometria de Massas , Fragmentos de Peptídeos/isolamento & purificação , Agregados Proteicos , Estabilidade Proteica , Elementos Estruturais de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
10.
Biosens Bioelectron ; 131: 299-306, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30852306

RESUMO

A sandwich-type photoelectrochemical (PEC) immunosensing platform was designed for detection of amino-terminal pro-B-type natriuretic peptide (NT-pro BNP). Thereinto, flower-like Bi2WO6/Ag2S nanoparticles (F-Bi2WO6/Ag2S) were employed as photoelectrochemical matrix, and graphene oxide and polydopamine composite (GO/PDA) were prepared as signal labels. In this proposal, Ag2S was in-situ growth on the surface of F-Bi2WO6 modified with thioglycolic acid (TGA). Specially, a cascade-like band-edge level between F-Bi2WO6 and Ag2S effectively improved the photocurrent conversion efficiency and enhanced the photocurrent response. Then, the conjugated GO/PDA aimed to further amplify signal because PDA as electron donor could sweep the holes and inhibit the recombination of photogenerated electron-hole pairs, while GO owned brilliant conductivity speeding up the electrons transfer. The photocurrent increased with the amount of GO/PDA conjugates which had positive correlation with the NT-pro BNP. Under optimal experimental conditions, the proposed sandwich-type PEC immunosensor presented a desirable linear relationship ranged from 0.1 pg/mL to 100 ng/mL for NT-pro BNP with the detection limit of 0.03 pg/mL (S/N = 3). The prepared PEC immunosensor exhibited high stability and selectivity, which offered an innovative idea for the detection of other biomolecules.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Imunoensaio , Peptídeo Natriurético Encefálico/isolamento & purificação , Fragmentos de Peptídeos/isolamento & purificação , Compostos de Cádmio/química , Grafite/química , Humanos , Indóis/química , Nanocompostos/química , Peptídeo Natriurético Encefálico/química , Fragmentos de Peptídeos/química , Polímeros/química
11.
Biosens Bioelectron ; 131: 287-298, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30851492

RESUMO

Cardiovascular diseases (CVD) remain the leading cause of death within industrialized nations as well as an increasing cause of mortality and morbidity in many developing countries. Smoking, alcohol consumption and increased level of blood cholesterol are the main CVD risk factors. Other factors, such as the prevalence of overweight/obesity and diabetes, have increased considerably in recent decades and are indirect causes of CVD. Among CVDs, the acute coronary syndrome (ACS) represents the most common cause of emergency hospital admission. Since the prognosis of ACS is directly associated with timely initiation of revascularization, missed, misdiagnosis or late diagnosis have unfavorable medical implications. Early ACS diagnosis can reduce complications and risk of recurrence, finally decreasing the economic burden posed on the health care system as a whole. To decrease the risk of ACS and related CVDs and to reduce associated costs to healthcare systems, a fast management of patients with chest pain has become crucial and urgent. Despite great efforts, biochemical diagnostic approaches of CVDs remain difficult and controversial medical challenges as cardiac biomarkers should be rapidly released into the blood at the time of ischemia and persistent for a sufficient length of time to allow diagnostics, with tests that should be rapid, easy to perform and relatively inexpensive. Early biomarker assessments have involved testing for the total enzyme activity of aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK), which cardiac troponins being the main accepted biomarkers for diagnosing myocardial injury and acute myocardial infarction (AMI). To allow rapid diagnosis, it is necessary to replace the traditional biochemical assays by cardiac biosensor platforms. Among the numerous of possibilities existing today, electrochemical biosensors are important players as they have many of the required characteristics for point-of-care tests. Electrochemical based cardiac biosensors are highly adapted for monitoring the onset and progress of cardiovascular diseases in a fast and accurate manner, while being cheap and scalable devices. This review outlines the state of the art in the development of cardiac electrochemical sensors for the detection of different cardiac biomarkers ranging from troponin to BNP, N-terminal proBNP, and others.


Assuntos
Síndrome Coronariana Aguda/sangue , Biomarcadores/sangue , Técnicas Biossensoriais , Doenças Cardiovasculares/sangue , Humanos , Peptídeo Natriurético Encefálico/sangue , Peptídeo Natriurético Encefálico/isolamento & purificação , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/isolamento & purificação , Prognóstico , Fatores de Risco , Troponina/sangue , Troponina/isolamento & purificação
12.
Curr HIV Res ; 17(1): 33-41, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30843489

RESUMO

BACKGROUND: Several approaches have not been successful to suppress HIV (Human immunodeficiency virus) infection among infected individuals or to prevent it yet. In order to expand strong HIV specific humoral and cellular responses, Virus-like particles (VLPs) as potential vaccines show significant increase in neutralizing antibodies secretion, T-cell count and also secretion of cytokines. OBJECTIVE: This study aimed at immunological evaluation of VLPs harboring high copy of MPERV3 in BALB/c mice. METHODS: Female BALB/c mice were immunized with homologous and heterologous primeboosting regimens of HIV-1 VLPMPER-V3. Their immune responses were evaluated for humoral responses (Total IgG and IgG isotyping) and cellular responses (IFN-γ, IL-5 secretion, in vitro CTL assay and T cell proliferation) and compared in immunized mice. RESULTS: The data showed robust induction of humoral response in mice groups which received different regimens of VLP. Furthermore, analysis of cytokine profile indicated that the highest IL-5 secretion was related to VLP+M50 group and confirmed the dominance of Th2 immunity in this group. CONCLUSION: This study showed that VLP MPER-V3 as a potential vaccine candidate has the potency as an effective prophylactic vaccine and this finding guarantees further investigations to achieve a promising HIV-1 vaccine candidate.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/isolamento & purificação , HIV-1/imunologia , Imunidade Humoral , Fragmentos de Peptídeos/isolamento & purificação , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas contra a AIDS/administração & dosagem , Animais , Feminino , Imunidade Celular , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Células Th2/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem
13.
Electrophoresis ; 40(11): 1550-1557, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30815902

RESUMO

Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Sheathless CE-ESI-MS, combining CE resolution power and low-flow ESI sensitivity, was applied to the analysis of PTH in its native conformation in the presence of related forms. Fused silica and neutral-coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis, field-amplified sample injection (FASI), and electrokinetic supercharging (EKS). The method for the separation of PTH and its variants was first developed using fused-silica capillary with UV detection. An acidic BGE was used to separate 1-84 PTH (full length), 7-84 PTH, and 1-34 PTH. Acetonitrile was added to the BGE to reduce peptide adsorption onto the capillary wall and transient isotachophoresis was used as analyte preconcentration method. The method was then transferred to a sheathless CE-ESI-MS instrument. When using a fused silica capillary, CE-MS was limited to µg/mL levels. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1-84 PTH, 7-84 PTH, and 1-34 PTH were detected at concentrations in the low ng/mL (FASI) or pg/mL (EKS) range.


Assuntos
Eletroforese Capilar/métodos , Hormônio Paratireóideo/isolamento & purificação , Fragmentos de Peptídeos/isolamento & purificação , Desenho de Equipamento , Humanos , Isotacoforese/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos
14.
Methods Mol Biol ; 1855: 327-340, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30426429

RESUMO

Aggregation of beta-amyloid peptides especially Aß1-42 in amyloid plaques is one of the major neuropathological events in Alzheimer's disease. This event is normally accompanied by a relative reduction of the concentration of Aß1-42 in the cerebrospinal fluid (CSF) of patient developing the signs of Alzheimer's disease. Here, we describe methods for isolation and for microchip gel electrophoresis of Aß peptides in polydimethylsiloxane (PDMS) microfluidic chip. The method was applied to compare the relative concentration of Aß1-42 with other Aß peptides, for example, Aß 1-40 in CSF. In order to increase the sensitivity of detection, Aß peptides in the CSF samples were first captured and concentrated using magnetic beads coated with specific anti-Aß antibodies.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Eletroforese em Microchip/métodos , Fragmentos de Peptídeos/líquido cefalorraquidiano , Peptídeos beta-Amiloides/isolamento & purificação , Anticorpos Imobilizados/química , Dimetilpolisiloxanos/química , Eletroforese em Microchip/instrumentação , Desenho de Equipamento , Humanos , Imãs/química , Fragmentos de Peptídeos/isolamento & purificação
15.
Methods Mol Biol ; 1855: 483-490, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30426442

RESUMO

Membrane proteins solubilized in a starting buffer containing high concentration of SDS are directly entrapped and immobilized into gel matrix when the membrane protein solution is absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts are removed by washing, the proteins are subjected to in-gel digestion, and the tryptic peptides are extracted and analyzed by CapLC-MS/MS. The newly developed method not only avoids protein loss and the adverse protein modifications during gel-embedment but also improves the subsequent in-gel digestion and the recovery of tryptic peptides, particularly hydrophobic peptides. Thus, this method facilitates the identification of membrane proteins, especially integral membrane proteins.


Assuntos
Resinas Acrílicas/química , Géis/química , Proteínas de Membrana/análise , Fragmentos de Peptídeos/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Alquilação , Animais , Cromatografia Líquida/métodos , Fígado/química , Fígado/citologia , Proteínas de Membrana/isolamento & purificação , Oxirredução , Fragmentos de Peptídeos/isolamento & purificação , Mapeamento de Peptídeos/métodos , Proteólise , Ratos , Tripsina/química
16.
Funct Integr Genomics ; 19(1): 123-136, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30159724

RESUMO

Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions take place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep) significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T2 generation. Reversed phase high-performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T2 lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.


Assuntos
Proteínas Arqueais/genética , Proteínas de Bactérias/genética , Glutens/metabolismo , Peptídeo Hidrolases/genética , Proteínas de Plantas/genética , Triticum/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Biolística , Doença Celíaca/dietoterapia , Doença Celíaca/imunologia , Chryseobacterium/enzimologia , Chryseobacterium/genética , Expressão Gênica , Engenharia Genética/métodos , Gliadina/imunologia , Gliadina/isolamento & purificação , Gliadina/metabolismo , Gliadina/farmacologia , Glutens/química , Glutens/imunologia , Hordeum/enzimologia , Hordeum/genética , Humanos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteólise , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/genética , Transgenes , Triticum/enzimologia
18.
Pak J Pharm Sci ; 31(6 (Supplementary): 2597-2605, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30587467

RESUMO

Many clinical-pathogens have developed resistance against known antibiotics and there is an urgent need for the discovery of novel antibiotics. In this study, low molecular weight peptides were isolated from seeds/leaves of 20 medicinal plants and tested for their antibacterial activity against laboratory strains of S. aureusand P. aeruginosa. Peptides isolated from Peganum harmala (PhAMP) exhibited maximum activity against laboratory strains. As clinical-isolates are more virulent and resistant to antibiotics, we tested the potential of PhAMP on these bacterial strains isolated from infected wounds. Pathogens isolated from burn-wounds (S. aureus, P. aeruginosa and K. pneumoniae) and surgical-wounds (P. aeruginosa and K. pneumoniae) exhibited zones of inhibition against PhAMP when tested by disc diffusion method. Biofilm formation of wound pathogens in the presence/absence of PhAMP was analyzed to check its effect. Surgical-wound pathogens and K. pneumoniae from burn-wound showed significant reduction in biofilm formation and planktonic bacteria. While biofilms of S. aureus and P. aeruginosa from burn-wound showed resistance against PhAMP. An effective antibiotic treatment should not only inhibit but should also disrupt already developed biofilms. PhAMP was very effective in the disruption of developed biofilm of all pathogens after 36 hours. This data unravels the potential of PhAMP as a novel, natural antibiotic against clinical-pathogens.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Queimaduras/microbiologia , Peganum , Extratos Vegetais/farmacologia , Ferida Cirúrgica/microbiologia , Antibacterianos/isolamento & purificação , Antibacterianos/uso terapêutico , Biofilmes/crescimento & desenvolvimento , Queimaduras/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêutico , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/uso terapêutico , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Proteínas de Plantas/uso terapêutico , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/fisiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/fisiologia , Ferida Cirúrgica/tratamento farmacológico
19.
Sensors (Basel) ; 18(11)2018 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-30441776

RESUMO

A rapid and sensitive sandwich electrochemical immunosensor was developed based on the fabrication of the graphene/polyaniline (GP/PANI) nanocomposite onto screen-printed gold electrode (SPGE) for detection of tuberculosis biomarker 10-kDa culture filtrate protein (CFP10). The prepared GP/PANI nanocomposite was characterized using Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM). The chemical bonding and morphology of GP/PANI-modified SPGE were studied by Raman spectroscopy and FESEM coupled with energy dispersive X-ray spectroscopy, respectively. From both studies, it clearly showed that GP/PANI was successfully coated onto SPGE through drop cast technique. Cyclic voltammetry was used to study the electrochemical properties of the modified electrode. The effective surface area for GP/PANI-modified SPGE was enhanced about five times compared with bare SPGE. Differential pulse voltammetry was used to detect the CFP10 antigen. The GP/PANI-modified SPGE that was fortified with sandwich type immunosensor exhibited a wide linear range (20⁻100 ng/mL) with a low detection limit of 15 ng/mL. This proposed electrochemical immunosensor is sensitive, low sample volume, rapid and disposable, which is suitable for tuberculosis detection in real samples.


Assuntos
Técnicas Biossensoriais , Fragmentos de Peptídeos/isolamento & purificação , Tuberculose/diagnóstico , Compostos de Anilina/química , Diagnóstico Precoce , Técnicas Eletroquímicas/métodos , Grafite/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Microscopia Eletrônica de Varredura/métodos , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/patogenicidade , Fragmentos de Peptídeos/imunologia , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Tuberculose/imunologia
20.
Arch Biochem Biophys ; 657: 78-88, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30222950

RESUMO

Bacillus subtilis 168 EpsC is annotated as "Probable polysaccharide biosynthesis protein" in the SwissProt database. epsC is part of the eps operon, thought to be involved in the biosynthesis of exopolymeric substances (EPS). The present study was undertaken to determine the molecular function of EpsC. Sequence analysis of EpsC suggested the presence of a transmembrane domain. Two N-terminal deletion mutants in which residues 1-89 (EpsC89) and 1-115 (EpsC115) are deleted were cloned and overexpressed. Enzyme activity and substrate preferences were investigated by reverse phase HPLC, surface plasmon resonance (SPR) spectroscopy and absorption spectroscopy. These data show that EpsC has UDP-GlcNAc 4,6-dehydratase activity in vitro. Purified recombinant proteins were found to utilise UDP-Glc and TDP-Glc also as substrates. In addition, EpsC115 could utilise UDP-Gal and UDP-GalNAc as substrates whereas EpsC89 could only bind these two sugar nucleotides. These results show that deletion of a longer N-terminal region broadens substrate specificity. These broadened specificity is perhaps an outcome of the deletion of the putative transmembrane domain and may not be present in vivo. EpsC, together with the aminotransferase EpsN (Kaundinya CR et al., Glycobiology, 2018) and acetyltransferase EpsM (unpublished data), appears to be involved in the biosynthesis of N,N'-diacetylbacillosamine.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Hidroliases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Ensaios Enzimáticos , Escherichia coli/genética , Hidroliases/genética , Hidroliases/isolamento & purificação , Cinética , Mutação , Açúcares de Nucleosídeo Difosfato/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Domínios Proteicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Ressonância de Plasmônio de Superfície
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