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1.
Food Chem ; 309: 125769, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31734007

RESUMO

Phenolic compounds are known to bind non-covalently with starch, but the impact of this interaction on the stability of the phenolic compounds through processing and digestion has received little attention. In this study, we examined the recovery of intact phenolic compounds (gallic acid-GA, catechin-CAT and epigallocatechin gallate-EGCG) from processed and digested porridges with different formulations (starch or starch/protein). We observed that phenolics were less degraded in presence of starch only than in presence of starch + proteins. This protection seemed to be linked to the ability of the phenolic compounds to form V-type inclusion complexes with starch, with GA, CAT and EGCG in decreasing order of protection. This work could influence formulation of functional cereal-based foods containing phenolic compounds in order to maximize their retention.


Assuntos
Manipulação de Alimentos/métodos , Fenóis/química , Proteínas/química , Amido/química , Catequina/análogos & derivados , Catequina/química , Catequina/metabolismo , Ácido Gálico/química , Ácido Gálico/metabolismo , Gelatina/química , Gelatina/metabolismo , Fenóis/metabolismo , Proteínas/metabolismo , Amido/metabolismo
2.
Mater Sci Eng C Mater Biol Appl ; 104: 110003, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31500012

RESUMO

Hemostatic materials could reduce avertible death from bleeding during surgery and emergency treatment. To this end, silk fibroin (SF) loaded with Ca2+ (1.8, 3.6 5.4, or 7.2%, w:w) was tested as a new hemostatic material (designated as SF1.8, SF3.6, SF5.4, or SF7.2), and the Ca2+ release rate, platelet adhesion, blood coagulation, cytocompatibility, and antimicrobial properties were investigated. Platelet adhesion on SF1.8 was improved significantly compared with pure SF porous material, and increased with increasing Ca2+ concentration. For SF3.6, platelet adhesion was greater than observed for gelatin and calcium alginate porous materials, clotting occurred earlier, and the complete coagulation time was shorter. Additionally, rabbit ear wound studies revealed that the hemostatic time for SF3.6 was significantly shorter than for gelatin, and similar to that for calcium alginate. The shed blood weight was lowest when SF was loaded with 7.2% Ca2+. The SF3.6 porous material displayed no obvious cytotoxicity, and exhibited satisfactory antibacterial activity against Escherichia coli and Staphylococcus aureus.


Assuntos
Alginatos/química , Materiais Biocompatíveis/química , Cálcio/metabolismo , Fibroínas/química , Seda/química , Animais , Antibacterianos/química , Plaquetas/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Gelatina/metabolismo , Hemostáticos/metabolismo , Porosidade/efeitos dos fármacos , Coelhos , Staphylococcus aureus/efeitos dos fármacos , Tecidos Suporte
3.
Amino Acids ; 51(9): 1397-1407, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31471743

RESUMO

Acetoanaerobium sticklandii DSM 519 is a hyper-ammonia-producing anaerobe. It has the ability to produce organic solvents and acids from protein catabolism through Stickland reactions and specialized pathways. Nevertheless, its protein catabolism-directed biofuel production has not yet been understood. The present study aimed to decipher such growth-associated metabolic potential of this organism at different growth phases using metabolic profiling. A seed culture of this organism was grown separately in metabolic assay media supplemented with gelatin and or a mixture of amino acids. The extracellular metabolites produced by this organism were qualitatively analyzed by gas chromatography-mass spectrometry platform. The residual amino acids after protein degradation and amino acids assimilation were identified and quantitatively measured by high-performance liquid chromatography (HPLC). Organic solvents and acids produced by this organism were detected and the quantity of them determined with HPLC. Metabolic profiling data confirmed the presence of amino acid catabolic products including tyramine, cadaverine, methylamine, and putrescine in fermented broth. It also found products including short-chain fatty acids and organic solvents of the Stickland reactions. It reported that amino acids were more appropriate for its growth yield compared to gelatin. Results of quantitative analysis of amino acids indicated that many amino acids either from gelatin or amino acid mixture were catabolised at a log-growth phase. Glycine and proline were poorly consumed in all growth phases. This study revealed that apart from Stickland reactions, a specialized system was established in A. sticklandii for protein catabolism-directed biofuel production. Acetone-butanol-ethanol (ABE), acetic acid, and butyric acid were the most important biofuel components produced by this organism. The production of these components was achieved much more on gelatin than amino acids. Thus, A. sticklandii is suggested herein as a potential organism to produce butyric acid along with ABE from protein-based wastes (gelatin) in bio-energy sectors.


Assuntos
Aminoácidos/metabolismo , Biocombustíveis , Clostridiales/metabolismo , Gelatina/metabolismo , Ácido Acético/metabolismo , Acetona/metabolismo , Aminoácidos/química , Butanóis/metabolismo , Ácido Butírico/metabolismo , Cromatografia Líquida de Alta Pressão , Etanol/metabolismo , Fermentação , Cromatografia Gasosa-Espectrometria de Massas , Metabolômica , Solventes/química , Solventes/metabolismo
4.
Biofabrication ; 11(4): 045020, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31387086

RESUMO

Bioink is of paramount importance in the process of three-dimensional extrusive bioprinting technology. Alginate is extensively used in cell-laden extrusive bioprinters with the advantage of biocompatibility, gelling and crosslinking features; however, the bioinert properties of alginate made it hard to degrade in vivo, and restrict cellular adhesion, extension and migration. In this study, we incorporated two concentrations of alginate lyase (0.5 mU ml-1 and 5 mU ml-1) into alginate/gelatin bioink to improve its degradation properties and effects on cellular behavior. The enzymatically degradable bioink demonstrated lower stiffness and higher porosity. Cellular proliferation, adhesion and extension were facilitated in the degradable bioink without sacrifice of cell viability. Additionally, the property of degradation still worked in vivo, with cellular infiltration and retention being observed in the grafted bioprinted constructs. The results suggest that alginate lyase could be incorporated into alginate/gelatin bioink. Degradation properties and cellular behavior could be promoted both in vitro and in vivo, providing a new avenue for the upgrade and modification of alginate-based bioink for further applications.


Assuntos
Alginatos/metabolismo , Fibroblastos/citologia , Gelatina/metabolismo , Tinta , Polissacarídeo-Liase/metabolismo , Animais , Bioimpressão , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Impressão Tridimensional , Ratos , Tecidos Suporte/química
5.
Food Funct ; 10(8): 4674-4684, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31292590

RESUMO

Wheat grain roller milling disrupts starch containing endosperm cell walls and extracts white flour. Many wheat based food processes involve simultaneous use of heat and water which then cause starch to gelatinize and enhance its digestibility. In this study, the impact of starch enclosure in intact endosperm cell walls on starch physicochemical properties and digestibility was investigated. Wheat kernels milled into coarse farina (average particle size: 705 µm) contained a substantial portion of intact cells and exhibited 15-30% lower Rapid Visco Analyzer peak viscosity readings than flour and fine farina (average particle size: 85 and 330 µm, respectively) since its higher level of intact cell walls limited the swelling of the enclosed starch. Xylanase use in situ substantially degraded coarse farina cell walls and increased their swelling and viscosifying potential. Following full gelatinization of the different samples, the starch in coarse farina was digested at a 40% lower rate in an in vitro gastrointestinal digestion assay, but still to a similar extent to that in fully gelatinized flour. This indicates that while wheat endosperm cell walls are permeable to pancreatic amylase, they can sufficiently slow down enzyme diffusion. When xylanase treatment was performed after starch gelatinization and pasting, the rates of starch digestion were similar for all samples evidencing that cell walls act as physical barriers to enzyme diffusion and thus retard its digestion. The present findings offer ways to produce wheat-based foods with sustained energy release benefits.


Assuntos
Parede Celular/química , Endosperma/química , Manipulação de Alimentos/métodos , Amido/química , Triticum/química , Biocatálise , Parede Celular/metabolismo , Digestão , Endo-1,4-beta-Xilanases/química , Endosperma/metabolismo , Farinha/análise , Gelatina/química , Gelatina/metabolismo , Humanos , Tamanho da Partícula , Sementes/química , Amido/metabolismo , Triticum/metabolismo , Viscosidade , alfa-Amilases/química
6.
BMC Oral Health ; 19(1): 161, 2019 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-31340803

RESUMO

BACKGROUND: Aim of the study was to evaluate the gelatinolytic activity in the saliva and gingival crevicular fluid from a sample group of subjects with Marfan syndrome. METHODS: Two groups were analyzed in this case-control study. A group of 28 subjects with Marfan syndrome (MG) was recruited from the Centre for Rare Disease, Marfan Clinic of Tor Vergata University Hospital. The second sample, 23 subjects, with the same characteristics and without any syndrome, was the control group (CG). Saliva and gingival crevicular fluid were collected and transferred to a sterile test tube and stored frozen at - 20 °C until analysis at the Medical Chemistry Laboratory. Gelatin substrate zymography was used for the evaluation and characterization of saliva and crevicular fluid proteinases. Correlation test and Student's t-test have been used to analyze data. RESULTS: In all samples different gelatin-degrading activities were observed. Two bands, which are related to the molecular weights of pro-MMP-9 and active MMP-9, respectively, were detectable in 100% of Marfan and control samples. MMP-2 activity was higher in Marfan group. Additional bands (55/48 kDa), corresponding to the activated forms of collagenase (MMP-13), were observed in saliva samples of both groups. CONCLUSIONS: The association of an enhanced activity by MMP-13 with an increased amount of active MMP-9 might be an important biomarker for the diagnosis of Marfan syndrome.


Assuntos
Líquido do Sulco Gengival/enzimologia , Síndrome de Marfan/enzimologia , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Saliva/enzimologia , Estudos de Casos e Controles , Criança , Feminino , Gelatina/metabolismo , Humanos , Masculino , Síndrome de Marfan/complicações
7.
Food Chem ; 296: 100-108, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31202293

RESUMO

The effects of the addition of edible gums (xanthan gum (XG), flaxseed gum (FG), konjac glucomannan (KGM) or tamarind seed gum (TSG)) on texture, gelatinisation and retrogradation of selected starches were investigated. XG and KGM significantly (p < 0.05) reduced syneresis of rich starch gels, from 62.5% to 13.1% and 62.5% to 21.2%, respectively. Moreover, the addition of gums resulted in softer binary gels, the hardness of lotus root starch was reduced from 39.07 to 14.08 g with the increase in XG content. Additionally, FG and TSG significantly increased the peak viscosity of mung bean starch (MBS) gels from 4372 to 10,285 cp. This facilitated the use of MBS gels as thickeners in foods. Binary gels improved the heat stability of starch gels, thus, widening its application in the preparation of baked foods. Binary gels could be used as additives in food production to improve the overall quality of foods.


Assuntos
Géis/química , Polissacarídeos Bacterianos/química , Amido/química , Gelatina/química , Gelatina/metabolismo , Lotus/metabolismo , Mananas , Sementes/metabolismo , Paladar , Temperatura Ambiente , Viscosidade
8.
Food Chem ; 295: 484-492, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174785

RESUMO

The eating and cooking quality (ECQ) of stored rice grains is significantly affected by ageing, but the molecular mechanisms for this are not well understood. In the present study, changes of starch hierarchical structures and thermal properties of starch were investigated for rices with up to 12 months storage. For the first time, it was seen that storage resulted in molecular degradation of starch. This mainly involved shorter amylose chains and around (1 → 6)-α glycosidic branching points of amylopectin, which altered the crystalline structure. This resulted in lower gelatinization temperatures and enthalpy but higher crystalline heterogeneity. The ageing effect was varietally-dependent. The information obtained from this study offers improved molecular-level understanding of the effects of ageing process on rice cooking and eating qualities.


Assuntos
Armazenamento de Alimentos , Oryza/química , Amido/química , Amilopectina/química , Amilopectina/metabolismo , Amilose/química , Amilose/metabolismo , Cromatografia em Gel , Culinária , Gelatina/química , Gelatina/metabolismo , Microscopia Eletrônica de Varredura , Estrutura Molecular , Oryza/metabolismo , Espalhamento a Baixo Ângulo , Sementes/química , Amido/metabolismo , Fatores de Tempo , Difração de Raios X
9.
Food Chem ; 295: 569-578, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174797

RESUMO

Fish scales are usually discarded or used to produce fish meal, etc. In order to enhance their utility, we produced the gelatin hydrolysates from fish scales (FSGH) and they were heated with glucose, xylose, and ribose to prepare sugar-FSGH Maillard reaction products (MRPs). The antioxidant capacity and sensory property of MRPs were evaluated. The results showed that ribose-FSGH MRPs exhibited higher antioxidant capacity than glucose- and xylose-FSGH MRPs. After simulated gastrointestinal digestion, the DPPH and ABTS scavenging activity of ribose-FSGH MRPs were 25.32 µM and 193.37 µM Trolox equivalent/g sample, respectively, and the reducing power was 0.509. Flavor compounds (such as butanal, benzaldehyde, 2-methyl-3-furanthiol, and maltol) of ribose-FSGH MRPs were produced in abundance after 5 h of heating and ribose-FSGH MRPs exhibited flavor enhanced effect on caramel-like and mouthfulness sensory attributes. These results suggest that ribose-FSGH MRPs can be potentially used as food antioxidants.


Assuntos
Antioxidantes/química , Carpas/metabolismo , Gelatina/metabolismo , Produtos Finais de Glicação Avançada/química , Compostos Orgânicos Voláteis/análise , Aminoácidos/análise , Animais , Cromatografia Líquida de Alta Pressão , Aromatizantes/química , Aromatizantes/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Concentração de Íons de Hidrogênio , Alimentos Marinhos/análise , Temperatura Ambiente
10.
Prep Biochem Biotechnol ; 49(5): 501-509, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945982

RESUMO

A new collagenase producing a strain of Bacillus cereus, isolated from the pollen of a bee of Amazon Region (Brazil), had its enzyme characterized and the production medium composition and culture conditions enhanced. A two-level design on three factors, namely initial medium pH, the substrate (gelatin) concentration and agitation intensity, allowed identifying the first two variables as the most significant ones, while a central composite design (CCD) was subsequently used to identify their optimal levels. Statistics highlighted maximized collagenolytic activity when substrate concentration and initial medium pH were selected at their highest levels (positive effects), whereas agitation intensity at the lowest (negative effect). Triplicate runs performed under predicted optimal conditions (pH 7.8 and 1.7% gelatin concentration) yielded a collagenolytic activity (305.39 ± 5.15 U) 4.6- to 15-fold those obtained with the preliminary design. The enzyme displayed optimum activity at 45 °C and pH 7.2, was stable over wide ranges of pH values and temperatures (7.2-11.0 and 25-50 °C, respectively) and was strongly inhibited by 10 mM phenylmethylsulphonyl fluoride. The zymogram showed two prominent bands at 50 and 76 kDa. These results are a first attempt to elucidate the features of this new collagenase, its production conditions, and possible scale-up.


Assuntos
Bacillus cereus/enzimologia , Colagenases/química , Animais , Bacillus cereus/genética , Técnicas de Tipagem Bacteriana , Abelhas , Brasil , Colagenases/isolamento & purificação , Meios de Cultura , Precursores Enzimáticos/química , Precursores Enzimáticos/isolamento & purificação , Gelatina/metabolismo , Concentração de Íons de Hidrogênio , Inibidores de Metaloproteinases de Matriz/química , Pólen/microbiologia , RNA Ribossômico 16S/genética , Temperatura Ambiente
11.
Methods Mol Biol ; 1952: 193-199, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825175

RESUMO

To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present an in situ gelatin zymography method that allows for a precise localization of active gelatin-degrading enzymes in a tissue section. In this method, dye-quenched gelatin is put on top of a tissue section. During an incubation period, active gelatinolytic enzymes will degrade the substrate and fluorescent signals are emitted from the locations of these enzymes.


Assuntos
Ensaios Enzimáticos/métodos , Gelatinases/metabolismo , Microscopia de Fluorescência/métodos , Microtomia/métodos , Animais , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Gelatina/análise , Gelatina/metabolismo , Gelatinases/análise , Humanos , Glândulas Mamárias Humanas/química , Glândulas Mamárias Humanas/enzimologia , Glândulas Mamárias Humanas/ultraestrutura , Camundongos , Especificidade por Substrato , Inclusão do Tecido/métodos , Fixação de Tecidos/métodos
12.
Methods Mol Biol ; 1952: 201-210, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825176

RESUMO

To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present a protocol for real-time gelatin zymography that is very useful for the detection of gelatin-degrading proteases in tissue extracts. This method uses fluorescence-labeled gelatin and therefore we also present an easy, fast, and cheap method for labeling gelatin with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF).


Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Ensaios Enzimáticos/métodos , Gelatinases/metabolismo , Animais , Linhagem Celular , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Furanos/análise , Furanos/metabolismo , Gelatina/análise , Gelatina/metabolismo , Gelatinases/análise , Humanos , Rim/química , Rim/enzimologia , Fígado/química , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Pele/química , Pele/enzimologia
13.
PLoS One ; 14(2): e0208658, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30789932

RESUMO

We developed a novel skin regeneration therapy combining nevus tissue inactivated by high hydrostatic pressure (HHP) in the reconstruction of the dermis with a cultured epidermal autograft (CEA). The issue with this treatment is the unstable survival of CEA on the inactivated dermis. In this study, we applied collagen/gelatin sponge (CGS), which can sustain the release of basic fibroblast growth factor (bFGF), to the inactivated skin in order to accelerate angiogenesis. Murine skin grafts from C57BL6J/Jcl mice (8 mm in diameter) were prepared, inactivated by HHP and cryopreserved. One month later, the grafts were transplanted subcutaneously onto the back of other mice and covered by CGS impregnated with saline or bFGF. Grafts were harvested after one, two and eight weeks, at which point the engraftment was evaluated through the histology and angiogenesis-related gene expressions were determined by real-time polymerase chain reaction. Histological sections showed that the dermal cellular density and newly formed capillaries in the bFGF group were significantly higher than in the control group. The relative expression of FGF-2, PDGF-A and VEGF-A genes in the bFGF group was significantly higher than in the control group at Week 1. This study suggested that the angiogenesis into grafts was accelerated, which might improve the engraftment of inactivated dermis in combination with the sustained release of bFGF by CGSs.


Assuntos
Preparações de Ação Retardada/farmacologia , Derme/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Animais , Capilares/efeitos dos fármacos , Capilares/metabolismo , Capilares/fisiologia , Colágeno/metabolismo , Derme/metabolismo , Derme/fisiologia , Gelatina/metabolismo , Pressão Hidrostática , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologia , Regeneração/fisiologia , Transplante de Pele/métodos , Tecidos Suporte
14.
Appl Microbiol Biotechnol ; 103(7): 2973-2984, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30805670

RESUMO

Microbial transglutaminase (mTGase) is commonly known in the food industry as meat glue due to its incredible ability to "glue" meat proteins together. Aside from being widely exploited in the meat processing industries, mTGase is also widely applied in other food and textile industries by catalysing the formation of isopeptide bonds between peptides or protein substrates. The advancement of technology has opened up new avenues for mTGase in the field of biomedical engineering. Efforts have been made to study the structural properties of mTGase in order to gain an in-depth understanding of the structure-function relationship. This review highlights the developments in mTGase engineering together with its role in biomedical applications including biomaterial fabrication for tissue engineering and biotherapeutics.


Assuntos
Bioengenharia/métodos , Terapia Biológica , Streptomyces/enzimologia , Engenharia Tecidual , Transglutaminases/biossíntese , Materiais Biocompatíveis , Quitosana/metabolismo , Colágeno/metabolismo , Indústria Alimentícia , Gelatina/metabolismo , Transglutaminases/genética
15.
Molecules ; 24(2)2019 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-30641905

RESUMO

Studies were undertaken to investigate the effects of ultraviolet (UV) irradiation on the gel strength, color, thermal properties, protein molecular masses, and functional groups of commercially available fish gelatin samples. Commercially available tilapia skin gelatin powder was used as the raw material to investigate the functional properties of fish skin gelatin powder treated with UV irradiation for different durations (0⁻6 h). The functional properties of fish gelatin and the optimum irradiation treatment conditions were determined through gel strength testing, color characterization, differential scanning calorimetry, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Fourier transform infrared (FTIR) spectroscopy, and Raman spectroscopy. UV irradiation treatment increased gel strength and thermal stability, and significantly degraded the macromolecules. FTIR and Raman spectroscopy data indicated that UV irradiation treatment did not significantly change the molecular structure of fish gelatin powder, but these methods could discriminate the molecular structure of gelatin from various sources. Irradiation for 2 h yielded the highest gel strength and melting peak temperature, and the lowest chromatic aberration.


Assuntos
Gelatina/metabolismo , Pele/metabolismo , Pele/efeitos da radiação , Tilápia , Raios Ultravioleta , Animais , Doses de Radiação , Espectroscopia de Infravermelho com Transformada de Fourier , Raios Ultravioleta/efeitos adversos
16.
Mol Biol Cell ; 30(5): 622-635, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30601698

RESUMO

During podosome formation, distinct phosphatidylinositol 3,4,5-trisphosphate lipid (PI(3,4,5)P3) production and F-actin polymerization take place at integrin-mediated adhesions. Membrane-associated actin regulation factors, such as myosin-1, serve as key molecules to link phosphatidylinositol signals to podosome assembly. Here, we report that long-tailed myosin-1e (Myo1e) is enriched at the ventral layer of the podosome core in a PI(3,4,5)P3-dependent manner. The combination of TH1 and TH2 (TH12) of Myo1e tail domains contains the essential motif for PI(3,4,5)P3-dependent membrane association and ventral localization at the podosome. TH12 KR2A (K772A and R782A) becomes dissociated from the plasma membrane. While F-actin polymerizations are initialized from the ventral layer of the podosome, TH12 precedes the recruitment of N-WASP and Arp2/3 in the initial phase of podosome formation. Overexpression of TH12, not TH12 KR2A, impedes PI(3,4,5)P3 signaling, restrains F-actin polymerization, and inhibits podosome formation. TH12 also suppresses gelatin degradation and migration speed of invadopodia-forming A375 melanoma cells. Thus, TH12 domain of Myo1e serves as a regulatory component to connect phosphatidylinositol signaling to F-actin polymerization at the podosome.


Assuntos
Actinas/metabolismo , Miosinas/química , Miosinas/metabolismo , Fosfatidilinositóis/metabolismo , Podossomos/metabolismo , Polimerização , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Gelatina/metabolismo , Humanos , Camundongos , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Domínios Proteicos , Células RAW 264.7 , Ratos
17.
J Biol Chem ; 294(12): 4621-4633, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30659094

RESUMO

Phosphoinositide 3-kinase ß (PI3Kß) is regulated by receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and small GTPases such as Rac1 and Rab5. Our lab previously identified two residues (Gln596 and Ile597) in the helical domain of the catalytic subunit (p110ß) of PI3Kß whose mutation disrupts binding to Rab5. To better define the Rab5-p110ß interface, we performed alanine-scanning mutagenesis and analyzed Rab5 binding with an in vitro pulldown assay with GST-Rab5GTP Of the 35 p110ß helical domain mutants assayed, 11 disrupted binding to Rab5 without affecting Rac1 binding, basal lipid kinase activity, or Gßγ-stimulated kinase activity. These mutants defined the Rab5-binding interface within p110ß as consisting of two perpendicular α-helices in the helical domain that are adjacent to the initially identified Gln596 and Ile597 residues. Analysis of the Rab5-PI3Kß interaction by hydrogen-deuterium exchange MS identified p110ß peptides that overlap with these helices; no interactions were detected between Rab5 and other regions of p110ß or p85α. Similarly, the binding of Rab5 to isolated p85α could not be detected, and mutations in the Ras-binding domain (RBD) of p110ß had no effect on Rab5 binding. Whereas soluble Rab5 did not affect PI3Kß activity in vitro, the interaction of these two proteins was critical for chemotaxis, invasion, and gelatin degradation by breast cancer cells. Our results define a single, discrete Rab5-binding site in the p110ß helical domain, which may be useful for generating inhibitors to better define the physiological role of Rab5-PI3Kß coupling in vivo.


Assuntos
Neoplasias da Mama/patologia , Invasividade Neoplásica , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Sítios de Ligação , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Quimiotaxia , Gelatina/metabolismo , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Mutação , Fosfatidilinositol 3-Quinase/genética , Ligação Proteica
18.
Parasitol Res ; 118(3): 829-835, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30689051

RESUMO

Cathepsin L-like protease is an important member of the papain-like cysteine protease and plays numerous indispensable roles in the biology of parasitic organisms. In a previous study, we identified a gene encoding a cathepsin L-like protease of Clonorchis sinensis (CsCPL) that was detected in the cercaria, metacercaria, and adult worm stages by immunolocalization, suggesting that this cysteine protease may be important and involved in the development of C. sinensis. In this study, the mature domain of CsCPL (CsCPL-m) was cloned and expressed in the form of inclusion bodies in Escherichia coli. After refolding, the recombinant CsCPL-m displayed optimal protease activity towards Z-Phe-Arg-AMC substrates but not towards Z-Arg-Arg-AMC, and the activity of the protease was inhibited completely by the cysteine protease-specific inhibitors E-64 and IAA, which further demonstrated that CsCPL belongs to the cathepsin L-like cysteine protease family. Recombinant CsCPL-m exhibited considerable activity at temperatures ranging from 28 to 42 °C, with the highest activity observed at 42 °C. Furthermore, recombinant CsCPL-m exhibited activity across a broad range of pH values (pH 4.0-8.0), with an optimal pH of 5.5. The Km and Vmax of the recombinant CsCPL-m towards Z-Phe-Arg-AMC were determined to be 5.71 × 10-6 M and 0.6 µM/min, respectively, at 37 °C and pH 5.5. The recombinant CsCPL-m could degrade BSA and gelatine, but could not degrade human hemoglobin and human immunoglobulin G. These results implied that CsCPL might participate in the catabolism of host proteins for nutrition during the parasitic life cycle of C. sinensis; thus, CsCPL could be used as a potential vaccine antigen and drug target against C. sinensis infection.


Assuntos
Catepsina L/metabolismo , Clonorchis sinensis/enzimologia , Cisteína Proteases/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Catepsina L/antagonistas & inibidores , Catepsina L/genética , Clonagem Molecular , Cisteína Proteases/genética , Inibidores de Cisteína Proteinase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Gelatina/metabolismo , Humanos , Dobramento de Proteína , Proteínas Recombinantes/genética , Soroalbumina Bovina/metabolismo
19.
Mol Neurobiol ; 56(6): 4566-4581, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30353492

RESUMO

The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES+/CD133+) and their differentiated (diff) progeny cells (NES-/CD133-). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFß1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53ß in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression.


Assuntos
Astrócitos/metabolismo , Neoplasias Encefálicas/metabolismo , Vesículas Extracelulares/metabolismo , Glioblastoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Idoso , Diferenciação Celular , Linhagem Celular Tumoral , Senescência Celular , Vesículas Extracelulares/ultraestrutura , Gelatina/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Nanopartículas/ultraestrutura , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Tamanho da Partícula , Fenótipo , Podossomos/metabolismo , Isoformas de Proteínas/metabolismo , Proteólise , Proteoma/metabolismo
20.
Methods Mol Biol ; 1894: 133-143, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30547459

RESUMO

Gelatin zymography is a relatively simple, inexpensive, and powerful technique to detect proteolytic enzymes capable of degrading gelatin from various biological sources. It has been used particularly to detect the two members of the matrix metalloproteinase family, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), due to their potent gelatin-degrading activity. MMP-2 and MMP-9 are also able to degrade a number of extracellular matrix molecules including type IV, V, and XI collagens, laminin, and aggrecan core protein, thus making them important in the nanotoxicity research. In this technique, proteins are separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and gelatinases, activated by SDS, digest gelatin embedded in the gel. After staining with Coomassie Brilliant Blue R-250, areas of degradation are visible as clear bands against a blue-stained background. Here, we describe the detailed procedure for gelatin zymography.


Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Ensaios Enzimáticos/métodos , Nanopartículas/toxicidade , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Células Cultivadas , Eletroforese em Gel de Poliacrilamida/instrumentação , Ensaios Enzimáticos/instrumentação , Gelatina/metabolismo , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Corantes de Rosanilina/química , Dodecilsulfato de Sódio/química , Coloração e Rotulagem/métodos
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