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1.
J Virol ; 94(20)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32796061

RESUMO

Retroviral envelope glycoprotein (Env) is essential for the specific recognition of the host cell and the initial phase of infection. As reported for human immunodeficiency virus (HIV), the recruitment of Env into a retroviral membrane envelope is mediated through its interaction with a Gag polyprotein precursor of structural proteins. This interaction, occurring between the matrix domain (MA) of Gag and the cytoplasmic tail (CT) of the transmembrane domain of Env, takes place at the host cell plasma membrane. To determine whether the MA of Mason-Pfizer monkey virus (M-PMV) also interacts directly with the CT of Env, we mimicked the in vivo conditions in an in vitro experiment by using a CT in its physiological trimeric conformation mediated by the trimerization motif of the GCN4 yeast transcription factor. The MA protein was used at the concentration shifting the equilibrium to its trimeric form. The direct interaction between MA and CT was confirmed by a pulldown assay. Through the combination of nuclear magnetic resonance (NMR) spectroscopy and protein cross-linking followed by mass spectrometry analysis, the residues involved in mutual interactions were determined. NMR has shown that the C terminus of the CT is bound to the C-terminal part of MA. In addition, protein cross-linking confirmed the close proximity of the N-terminal part of CT and the N terminus of MA, which is enabled in vivo by their location at the membrane. These results are in agreement with the previously determined orientation of MA on the membrane and support the already observed mechanisms of M-PMV virus-like particle transport and budding.IMPORTANCE By a combination of nuclear magnetic resonance (NMR) and mass spectroscopy of cross-linked peptides, we show that in contrast to human immunodeficiency virus type 1 (HIV-1), the C-terminal residues of the unstructured cytoplasmic tail of Mason-Pfizer monkey virus (M-PMV) Env interact with the matrix domain (MA). Based on biochemical data and molecular modeling, we propose that individual cytoplasmic tail (CT) monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that the membrane-bound MA of Gag recruits Env through interaction with the full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively.


Assuntos
Produtos do Gene env/química , Produtos do Gene gag/química , Vírus dos Macacos de Mason-Pfizer/química , Produtos do Gene env/genética , Produtos do Gene gag/genética , Vírus dos Macacos de Mason-Pfizer/genética , Domínios Proteicos
2.
Materials (Basel) ; 17(4)2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38399166

RESUMO

In vitro testing is the first important step in the development of new biomaterials. The human fetal osteoblast cell line hFOB 1.19 is a very promising cell model; however, there are vast discrepancies in cultivation protocols, especially in the cultivation temperature and the presence of the selection reagent, geneticin (G418). We intended to use hFOB 1.19 for the testing of Zn-based degradable metallic materials. However, the sensitivity of hFOB 1.19 to zinc ions has not yet been studied. Therefore, we compared the toxicity of zinc towards hFOB 1.19 under different conditions and compared it with that of the L929 mouse fibroblast cell line. We also tested the cytotoxicity of three types of Zn-based biomaterials in two types of media. The presence of G418 used as a selection reagent decreased the sensitivity of hFOB 1.19 to Zn2+. hFOB 1.19 cell line was more sensitive to Zn2+ at elevated (restrictive) temperatures. hFOB 1.19 cell line was less sensitive to Zn2+ than L929 cell line (both as ZnCl2 and extracts of alloys). Therefore, the appropriate cultivation conditions of hFOB 1.19 during biomaterial testing should be chosen with caution.

3.
Biochim Biophys Acta ; 1823(4): 911-9, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22306003

RESUMO

Small ubiquitin-related modifiers 1, 2 and 3 (SUMO-1, -2, -3), members of the ubiquitin-like protein family, can be conjugated to various cellular proteins. Conjugates of SUMO-2 and SUMO-3 (SUMO-2/3) accumulate in cells exposed to various stress stimuli or to MG132 treatment. Although the proteins modified by SUMO-2/3 during heat shock or under MG132 treatment have been identified, the significance of this modification remains unclear. Our data show that the inhibition of translation by puromycin or cycloheximide blocks both the heat shock and MG132 induced accumulation of SUMO-2/3 conjugates in HEK 293T and U2OS cells. However, the heat shock induced accumulation of SUMO-2/3 conjugates was restored by proteasome inhibition, which suggests that the inhibition of translation did not abolish SUMOylation itself. Furthermore, we show that some of the proteins truncated due to the treatment by low concentration of puromycin are SUMOylated in HEK 293T cells. We suggest that the SUMO-2/3 conjugates accumulating under the heat shock or MG132 treatment result largely from new protein synthesis and that portion of them is incorrectly folded.


Assuntos
Resposta ao Choque Térmico/efeitos dos fármacos , Leupeptinas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Benzoquinonas/farmacologia , Cicloeximida/farmacologia , Células HEK293 , Células HeLa , Humanos , Lactamas Macrocíclicas/farmacologia , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Sumoilação/efeitos dos fármacos
4.
Biochem Biophys Res Commun ; 436(2): 240-5, 2013 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-23726919

RESUMO

UBL5 protein, a structural homologue of ubiquitin, was shown to be involved in pre-mRNA splicing and transcription regulation in yeast and Caenorhabditis elegans, respectively. However, role of the UBL5 human orthologue is still elusive. In our study, we observed that endogenous human UBL5 that was localized in the nucleus, partially associates with Cajal bodies (CBs), nuclear domains where spliceosomal components are assembled. Simultaneous expression of exogenous UBL5 and coilin resulted in their nuclear colocalization in HeLa cells. The ability of UBL5 to interact with coilin was proved by GST pull-down assay using coilin that was either in vitro translated or extracted from HEK293T cells. Further, our results showed that the UBL5-coilin interaction was not influenced by coilin phosphorylation. These results suggest that UBL5 could be targeted to CBs via its interaction with coilin. Relation between human UBL5 protein and CBs is in the agreement with current observations about yeast orthologue Hub1 playing important role in alternative splicing.


Assuntos
Corpos Enovelados/metabolismo , Proteínas do Olho/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitinas/metabolismo , Proteínas do Olho/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopia de Fluorescência , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Ubiquitinas/genética
5.
Toxins (Basel) ; 15(4)2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-37104201

RESUMO

(1) Background: The detection of DNA double-strand breaks in vitro using the phosphorylated histone biomarker (γH2AX) is an increasingly popular method of measuring in vitro genotoxicity, as it is sensitive, specific and suitable for high-throughput analysis. The γH2AX response is either detected by flow cytometry or microscopy, the latter being more accessible. However, authors sparsely publish details, data, and workflows from overall fluorescence intensity quantification, which hinders the reproducibility. (2) Methods: We used valinomycin as a model genotoxin, two cell lines (HeLa and CHO-K1) and a commercial kit for γH2AX immunofluorescence detection. Bioimage analysis was performed using the open-source software ImageJ. Mean fluorescent values were measured using segmented nuclei from the DAPI channel and the results were expressed as the area-scaled relative fold change in γH2AX fluorescence over the control. Cytotoxicity is expressed as the relative area of the nuclei. We present the workflows, data, and scripts on GitHub. (3) Results: The outputs obtained by an introduced method are in accordance with expected results, i.e., valinomycin was genotoxic and cytotoxic to both cell lines used after 24 h of incubation. (4) Conclusions: The overall fluorescence intensity of γH2AX obtained from bioimage analysis appears to be a promising alternative to flow cytometry. Workflow, data, and script sharing are crucial for further improvement of the bioimage analysis methods.


Assuntos
Dano ao DNA , Microscopia , Humanos , Projetos Piloto , Valinomicina/toxicidade , Reprodutibilidade dos Testes , Células HeLa , Biomarcadores/análise
6.
Sci Rep ; 13(1): 18536, 2023 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-37898679

RESUMO

Lilial (also called lysmeral) is a fragrance ingredient presented in many everyday cosmetics and household products. The concentrations of lilial in the final products is rather low. Its maximum concentration in cosmetics was limited and recently, its use in cosmetics products was prohibited in the EU due to the classification as reproductive toxicant. Additionally, according to the European Chemicals Agency, it was under assessment as one of the potential endocrine disruptors, i.e. a substance that may alter the function of the endocrine system and, as a result, cause health problems. Its ability to act as an androgen receptor agonist and the estrogenic and androgenic activity of its metabolites, to the best of our knowledge, have not yet been tested. The aim of this work was to determine the intestinal absorption, cytotoxicity, nephrotoxicity, mutagenicity, activation of cellular stress-related signal pathways and, most importantly, to test the ability to disrupt the endocrine system of lilial and its Phase I metabolites. This was tested using set of in vitro assays including resazurin assay, the CHO/HPRT mutation assay, γH2AX biomarker-based genotoxicity assay, qPCR and in vitro reporter assays based on luminescence of luciferase for estrogen, androgen, NF-κB and NRF2 signalling pathway. It was determined that neither lilial nor its metabolites have a negative effect on cell viability in the concentration range from 1 nM to 100 µM. Using human cell lines HeLa9903 and MDA-kb2, it was verified that this substance did not have agonistic activity towards estrogen or androgen receptor, respectively. Lilial metabolites, generated by incubation with the rat liver S9 fraction, did not show the ability to bind to estrogen or androgen receptors. Neither lilial nor its metabolites showed a nephrotoxic effect on human renal tubular cells (RPTEC/TERT1 line) and at the same time they were unable to activate the NF-κB and NRF2 signalling pathway at a concentration of 50 µM (HEK 293/pGL4.32 or pGL4.37). Neither lilial nor its metabolites showed mutagenic activity in the HPRT gene mutation test in CHO-K1 cells, nor were they able to cause double-strand breaks in DNA (γH2AX biomarker) in CHO-K1 and HeLa cells. In our study, no negative effects of lilial or its in vitro metabolites were observed up to 100 µM using different in vitro tests.


Assuntos
Hipoxantina Fosforribosiltransferase , NF-kappa B , Humanos , Ratos , Animais , Células HeLa , Células HEK293 , Fator 2 Relacionado a NF-E2 , Estrogênios/toxicidade , Estrogênios/metabolismo , Androgênios , Biomarcadores
7.
J Ethnopharmacol ; 312: 116484, 2023 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-37044231

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia officinalis L., Sambucus nigra L., Matricaria chamomilla L., Agrimonia eupatoria L., Fragaria vesca L. and Malva sylvestris L. are plants that have a long tradition in European folk medicine. To this day, they are part of medicinal teas or creams that help with the healing of skin wounds and the treatment of respiratory or intestinal infections. However, so far these plants have not been investigated more deeply than in their direct antibacterial effect. AIM OF THE STUDY: Our research is focused on adjuvants that inhibit the mechanism of antibiotic resistance or modulate bacterial virulence. Based on a preliminary screening of 52 European herbs, which commonly appear as part of tea blends or poultice. Six of them were selected for their ability to revert the resistant phenotype of nosocomial bacterial strains. METHODS: Herbs selected for this study were obtained from commercially available sources. For the extraction of active compounds ethanol was used. Modulation of virulence was observed as an ability to inhibit bacterial cell-to-cell communication using two mutant sensor strains of Vibrio campbellii. Biofilm formation, and planktonic cell adhesion was measured using a static antibiofilm test. Ethidium bromide assay was used to checked the potential of inhibition bacterial efflux pumps. The antibacterial activities of the herbs were evaluated against resistant bacterial strains using macro dilution methods. RESULTS: Alcohol extracts had antibacterial properties mainly against Gram-positive bacteria. Of all of them, the highest antimicrobial activity demonstrated Malva sylvestris, killing both antibiotic resistant bacteria; Staphylococcus aureus with MIC of 0.8 g/L and Pseudomonas aeruginosa 0.7 g/L, respectively. Fragaria vesca extract (0.08 g/L) demonstrated strong synergism with colistin (4 mg/L) in modulating the resistant phenotype to colistin of Pseudomonas aeruginosa. Similarly, the extract of S. officinalis (0.21 g/L) reverted resistance to gentamicin (1 mg/L) in S. aureus. However, Sambucus nigra and Matricaria chamomilla seem to be a very promising source of bacterial efflux pump inhibitors. CONCLUSION: The extract of F. vesca was the most active. It was able to reduce biofilm formation probably due to the ability to decrease bacterial quorum sensing. On the other hand, the activity of S. nigra or M. chamomilla in reducing bacterial virulence may be explained by the ability to inhibit bacterial efflux systems. All these plants have potential as an adjuvant for the antibiotic treatment.


Assuntos
Plantas Medicinais , Staphylococcus aureus , Extratos Vegetais/farmacologia , Virulência , Colistina/farmacologia , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Bactérias , Pseudomonas aeruginosa , Biofilmes
8.
J Agric Food Chem ; 70(38): 11833-11843, 2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36103343

RESUMO

Potatoes (Solanum tuberosum) are one of the most important crops worldwide. However, its production and nutrient content are endangered by both biotic and abiotic stresses. The main yield losses are caused by pest damage (e.g., Colorado potato beetle and aphids), virus disease (e.g., Potato leafroll virus and Potato viruses Y and X), or oomycete pathogens (like Phytophthora infestans), which also significantly affect the production of antinutrients and toxic metabolites of plants. Therefore, the use of genetic engineering could be an efficient tool, not harmful to the environment, and beneficial to the consumer. In this review, we focus on the main sources of problems in the field of potato production according to approved genetic modifications, their traditional solution and positive impact of gene transfection reducing economic losses, use of insecticides, and improving the nutritional properties of potatoes. We summarize all transgenic events that have been performed on potatoes and have been approved for cultivation and/or direct use or processing as feed or food.


Assuntos
Inseticidas , Phytophthora infestans , Solanum tuberosum , Animais , Inocuidade dos Alimentos , Phytophthora infestans/genética , Doenças das Plantas/genética , Doenças das Plantas/prevenção & controle , Plantas Geneticamente Modificadas/genética , Solanum tuberosum/genética
9.
Materials (Basel) ; 15(21)2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36363162

RESUMO

In the field of magnesium-based degradable implantable devices, the Mg-Y-RE-Zr alloying system (WE-type) has gained popularity due to its satisfying degradation rate together with mechanical strength. However, utilization of RE and Zr in the WE-type alloys was originally driven to improve Mg-based alloys for high-temperature applications in the industry, while for medical purposes, there is a question of whether the amount of alloying elements may be further optimized. For this reason, our paper presents the Mg-3Y (W3) magnesium alloy as an alternative to the WE43 alloy. This study shows that the omission of RE and Zr elements did not compromise the corrosion resistance and the degradation rate of the W3 alloy when compared with the WE43 alloy; appropriate biocompatibility was preserved as well. It was shown that the decrease in the mechanical strength caused by the omission of RE and Zr from the WE43 alloy could be compensated for by severe plastic deformation, as achieved in this study, by equal channel angular pressing. Ultrafine-grained W3 alloy exhibited compression yield strength of 362 ± 6 MPa and plastic deformation at maximum stress of 18 ± 1%. Overall, the early results of this study put forward the motion of avoiding RE elements and Zr in magnesium alloy as a suitable material for biodegradable applications and showed that solo alloying of yttrium is sufficient for maintaining desirable properties of the material at once.

10.
Biomed Pharmacother ; 149: 112806, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35303568

RESUMO

Antibiotic resistance is currently a serious health problem. Since the discovery of new antibiotics no longer seems to be a sufficient tool in the fight against multidrug-resistant infections, adjuvant (combination) therapy is gaining in importance as well as reducing bacterial virulence. Silymarin is a complex of flavonoids and flavonolignans known for its broad spectrum of biological activities, including its ability to modulate drug resistance in cancer. This work aimed to test eleven, optically pure silymarin flavonolignans for their ability to reverse the multidrug resistance phenotype of Staphylococcus aureus and reduce its virulence. Silybin A, 2,3-dehydrosilybin B, and 2,3-dehydrosilybin AB completely reversed antibiotic resistance at concentrations of 20 µM or less. Both 2,3-dehydrosilybin B and AB decreased the antibiotic-induced gene expression of representative efflux pumps belonging to the major facilitator (MFS), multidrug and toxic compound extrusion (MATE), and ATP-binding cassette (ABC) families. 2,3-Dehydrosilybin B also inhibited ethidium bromide accumulation and efflux in a clinical isolate whose NorA and MdeA overproduction was induced by antibiotics. Most of the tested flavonolignans reduced cell-to-cell communication on a tetrahydrofuran-borate (autoinducer-2) basis, with isosilychristin leading the way followed by 2,3-dehydrosilybin A and AB, which halved communication at 10 µM. Anhydrosilychristin was the only compound that reduced communication based on acyl-homoserine lactone (autoinducer 1), with an IC50 of 4.8 µM. Except for isosilychristin and anhydrosilychristin, all of the flavonolignans inhibited S. aureus surface colonization, with 2,3-dehydrosilybin A being the most active (IC50 10.6 µM). In conclusion, the selected flavonolignans, particularly derivatives of 2,3-dehydrosilybin B, 2,3-dehydrosilybin AB, and silybin A are non-toxic modulators of S. aureus multidrug resistance and can decrease the virulence of the bacterium, which deserves further detailed research.


Assuntos
Silimarina , Infecções Estafilocócicas , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Humanos , Silibina/farmacologia , Silimarina/química , Silimarina/farmacologia , Staphylococcus aureus , Virulência
11.
Protein Expr Purif ; 79(1): 122-7, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21640189

RESUMO

Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus budding. We used Escherichia coli, carrying N-myristoyltransferase gene, for the expression of the myristoylated His-tagged matrix protein of Mason-Pfizer monkey virus. An efficient, single-step purification procedure eliminating all contaminating proteins including, importantly, the non-myristoylated matrix protein was designed. The comparison of NMR spectra of matrix protein with its myristoylated form revealed substantial structural changes induced by this fatty acid modification.


Assuntos
Aciltransferases/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Vírus dos Macacos de Mason-Pfizer/genética , Ácido Mirístico/química , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Aciltransferases/química , Aciltransferases/isolamento & purificação , Expressão Gênica , Vírus dos Macacos de Mason-Pfizer/química , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas da Matriz Viral/isolamento & purificação
12.
Proc Natl Acad Sci U S A ; 105(30): 10565-70, 2008 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-18647839

RESUMO

Despite extensive data demonstrating that immature retroviral particle assembly can take place either at the plasma membrane or at a distinct location within the cytoplasm, targeting of viral precursor proteins to either assembly site still remains poorly understood. Biochemical data presented here suggest that Tctex-1, a light chain of the molecular motor dynein, is involved in the intracellular targeting of Mason-Pfizer monkey virus (M-PMV) polyproteins to the cytoplasmic assembly site. Comparison of the three-dimensional structures of M-PMV wild-type matrix protein (wt MA) with a single amino acid mutant (R55F), which redirects assembly from a cytoplasmic site to the plasma membrane, revealed different mutual orientations of their C- and N-terminal domains. This conformational change buries a putative intracellular targeting motif located between both domains in the hydrophobic pocket of the MA molecule, thereby preventing the interaction with cellular transport mechanisms.


Assuntos
Membrana Celular/metabolismo , Membrana Celular/virologia , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Nucleares/fisiologia , Retroviridae/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , Humanos , Vírus dos Macacos de Mason-Pfizer/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fenótipo , Estrutura Terciária de Proteína , Região do Complexo-t do Genoma
13.
Sci Rep ; 11(1): 6628, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758226

RESUMO

In vitro cytotoxicity testing is an indispensable part of the development of new biomaterials. However, the standard ISO 10993-5 enables variability in the testing conditions, which makes the results of the test incomparable. We studied the influence of media composition on the results of the cytotoxicity test. Solutions of ZnCl2 served as simulated extracts and we also used extracts of three types of Zn-based and Mg-based degradable metals. We incubated the cells with the solutions prepared in two types of media with two concentrations of serum (5 and 10%). We compared the toxic effect of the extracts on L929 murine fibroblast-derived cell line, which is recommended by ISO standard and on "osteoblast-like cells" U-2 OS. We also compared two methods of exposition: solutions were added either to a sub-confluent layer or to the cell suspension. We evaluated the metabolic activity of the cells using the resazurin test. We found out that in vitro cytotoxicity is dramatically influenced by the concentration of serum and by the type of the medium as well as by the type of exposition and type of cells. Therefore, when performing in vitro cytotoxicity testing of biomaterials, the authors should carefully specify the conditions of the test and comparison of different studies should be carried out with caution.


Assuntos
Materiais Biocompatíveis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Ligas , Animais , Células Cultivadas , Técnicas In Vitro , Teste de Materiais , Camundongos
14.
Pharmaceuticals (Basel) ; 14(1)2020 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-33375355

RESUMO

Selaginella P. Beauv. is a group of vascular plants in the family Selaginellaceae Willk., found worldwide and numbering more than 700 species, with some used as foods and medicines. The aim of this paper was to compare methanolic (MeOH) and dichloromethane (DCM) extracts of eight Selaginella species on the basis of their composition and biological activities. Six of these Selaginella species are underinvestigated. Using ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) analysis, we identified a total of 193 compounds among the tested Selaginella species, with flavonoids predominating. MeOH extracts recovered more constituents that were detected, including selaginellins, the occurrence of which is only typical for this plant genus. Of all the tested species, Selaginella apoda contained the highest number of identified selaginellins. The majority of the compounds were identified in S. apoda, the fewest compounds in Selaginella cupressina. All the tested species demonstrated antioxidant activity using oxygen radical absorption capacity (ORAC) assay, which showed that MeOH extracts had higher antioxidant capacity, with the half maximal effective concentration (EC50) ranging from 12 ± 1 (Selaginella myosuroides) to 124 ± 2 (Selaginella cupressina) mg/L. The antioxidant capacity was presumed to be correlated with the content of flavonoids, (neo)lignans, and selaginellins. Inhibition of acetylcholinesterase (AChE) was mostly discerned in DCM extracts and was only exhibited in S. myosuroides, S. cupressina, Selaginella biformis, and S. apoda extracts with the half maximal inhibitory concentration (IC50) in the range of 19 ± 3 to 62 ± 1 mg/L. Substantial cytotoxicity against cancer cell lines was demonstrated by the MeOH extract of S. apoda, where the ratio of the IC50 HEK (human embryonic kidney) to IC50 HepG2 (hepatocellular carcinoma) was 7.9 ± 0.2. MeOH extracts inhibited the production of nitrate oxide and cytokines in a dose-dependent manner. Notably, S. biformis halved the production of NO, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 at the following concentrations: 105 ± 9, 11 ± 1, and 10 ± 1 mg/L, respectively. Our data confirmed that extracts from Selaginella species exhibited cytotoxicity against cancer cell lines and AChE inhibition. The activity observed in S. apoda was the most promising and is worth further exploration.

15.
Viruses ; 10(10)2018 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-30347798

RESUMO

The envelope glycoprotein (Env) plays a crucial role in the retroviral life cycle by mediating primary interactions with the host cell. As described previously and expanded on in this paper, Env mediates the trafficking of immature Mason-Pfizer monkey virus (M-PMV) particles to the plasma membrane (PM). Using a panel of labeled RabGTPases as endosomal markers, we identified Env mostly in Rab7a- and Rab9a-positive endosomes. Based on an analysis of the transport of recombinant fluorescently labeled M-PMV Gag and Env proteins, we propose a putative mechanism of the intracellular trafficking of M-PMV Env and immature particles. According to this model, a portion of Env is targeted from the trans-Golgi network (TGN) to Rab7a-positive endosomes. It is then transported to Rab9a-positive endosomes and back to the TGN. It is at the Rab9a vesicles where the immature particles may anchor to the membranes of the Env-containing vesicles, preventing Env recycling to the TGN. These Gag-associated vesicles are then transported to the plasma membrane.


Assuntos
Produtos do Gene env/metabolismo , Vírus dos Macacos de Mason-Pfizer/fisiologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vesículas Transportadoras/virologia , Animais , Membrana Celular/metabolismo , Membrana Celular/virologia , Endossomos/metabolismo , Endossomos/virologia , Produtos do Gene env/genética , Vírus dos Macacos de Mason-Pfizer/genética , Transporte Proteico , Vesículas Transportadoras/metabolismo , Montagem de Vírus
16.
Curr Pharm Biotechnol ; 18(14): 1167-1174, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29484986

RESUMO

BACKGROUND: The aim of this work was to compare water and organic extracts, infusions and tinctures from flowers and leaves of Calendula officinalis in terms of their biological activity and composition. The purpose of work was investigation whether the leaves and stems are really the waste or they contain interesting substances which could be utilized. Antimicrobial, antifungal, antioxidant and anti-inflammatory activities were studied. Then, the ability to inhibit collagenase was studied as well. Cytotoxicity was tested for all the samples on mammalian cell lines. METHODS: To determine the composition of extracts, infusions and tinctures phytochemical analysis (the set of colour reactions for the detection of groups of biologically active compounds) was carried out and showed that samples from flowers and leaves contain the same groups of biologically active substances (proteins and amino acids, reducing sugars, flavonoids, saponins, phenolics, terpenoids, steroids, glycosides). The antimicrobial activity of tested samples was proved, where the most sensitive bacterium was Micrococcus luteus and the most sensitive yeast was Geotrichum candidum. RESULTS: The study of anti-collagenase activity has shown that the enzymatic reaction of collagenase was affected by all tested samples and their effect was concentration dependent. Cytotoxicity of water and methanol extracts at cell lines HEK 293T and HepG2 was observed. CONCLUSION: Cells HepG2 were more sensitive than cells HEK 293T. Using cell line RAW 264.7, antiinflammatory activity of all samples was observed. Tincture of leaves was the most effective.


Assuntos
Calendula/química , Flores/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Animais , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/toxicidade , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/toxicidade , Antioxidantes/isolamento & purificação , Antioxidantes/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Extratos Vegetais/toxicidade
17.
Materials (Basel) ; 10(9)2017 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-28837101

RESUMO

Thermal plasma spray is a common, well-established technology used in various application fields. Nevertheless, in our work, this technology was employed in a completely new way; for the preparation of bulk titanium. The aim was to produce titanium with properties similar to human bone to be used for bone augmentations. Titanium rods sprayed on a thin substrate wire exerted a porosity of about 15%, which yielded a significant decrease of Young's modulus to the bone range and provided rugged topography for enhanced biological fixation. For the first verification of the suitability of the selected approach, tests of the mechanical properties in terms of compression, bending, and impact were carried out, the surface was characterized, and its compatibility with bone cells was studied. While preserving a high enough compressive strength of 628 MPa, the elastic modulus reached 11.6 GPa, thus preventing a stress-shielding effect, a generally known problem of implantable metals. U-2 OS and Saos-2 cells derived from bone osteosarcoma grown on the plasma-sprayed surface showed good viability.

18.
Mater Sci Eng C Mater Biol Appl ; 79: 550-562, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28629053

RESUMO

Recently, iron-based materials have been considered as candidates for the fabrication of biodegradable load-bearing implants. Alloying with palladium has been found to be a suitable approach to enhance the insufficient corrosion rate of iron-based alloys. In this work, we have extensively compared the microstructure, the mechanical and corrosion properties, and the cytotoxicity of an FePd2 (wt%) alloy prepared by three different routes - casting, mechanical alloying and spark plasma sintering (SPS), and mechanical alloying and the space holder technique (SHT). The properties of the FePd2 (wt%) were compared with pure Fe prepared in the same processes. The preparation route significantly influenced the material properties. Materials prepared by SPS possessed the highest values of mechanical properties (CYS~750-850MPa) and higher corrosion rates than the casted materials. Materials prepared by SHT contained approximately 60% porosity; therefore, their mechanical properties reached the lowest values, and they had the highest corrosion rates, approximately 0.7-1.2mm/a. Highly porous FePd2 was tested in vitro according to the ISO 10993-5 standard using L929 cells, and two-fold diluted extracts showed acceptable cytocompatibility. In general, alloying with Pd enhanced both mechanical properties and corrosion rates and did not decrease the cytocompatibility of the studied materials.


Assuntos
Ligas/química , Materiais Biocompatíveis , Corrosão , Ferro , Chumbo , Teste de Materiais , Suporte de Carga
19.
Mater Sci Eng C Mater Biol Appl ; 73: 736-742, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28183668

RESUMO

Effect of processing by equal channel angular pressing (ECAP) on the degradation behaviour of extruded LAE442 magnesium alloy was investigated in a 0.1M NaCl solution, Kirkland's biocorrosion medium (KBM) and Minimum Essential Medium (MEM), both with and without 10% of foetal bovine serum (FBS). Uniform degradation of as extruded and ECAP processed samples in NaCl solution was observed, nevertheless higher corrosion resistance was found in the latter material. The increase of corrosion resistance due to ECAP was observed also after 14-days immersion in all media used. Higher compactness of the corrosion layer formed on the samples after ECAP was responsible for the observed decrease of corrosion resistance, which was proven by scanning electron microscope investigation. Lower corrosion rate in media with FBS was observed and was explained by additional effect of protein incorporation on the corrosion layer stability. A cytotoxicity test using L929 cells was carried out to investigate possible effect of processing on the cell viability. Sufficient cytocompatibility of the extruded samples was observed with no adverse effects of the subsequent ECAP processing. In conclusion, this in vitro study proved that the degradation behaviour of the LAE442 alloy could be improved by subsequent ECAP processing and this material is a good candidate for future in vivo investigation.


Assuntos
Ligas/química , Magnésio/química , Teste de Materiais/métodos , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Corrosão , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Hidrogênio/análise , Íons , Camundongos , Cloreto de Sódio/farmacologia , Soluções
20.
Mater Sci Eng C Mater Biol Appl ; 76: 25-30, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28482525

RESUMO

New materials with appropriate mechanical properties and an antibacterial effect are constantly being sought for orthopedic and dental applications. The aim of this study was to investigate newly developed TiSi alloys coated with titania sol-gel containing silver. Titanium alloys with 5 or 10wt% of silicon were prepared by vacuum arc remelting and dip-coated with titania sol containing either AgNO3 or Ag3PO4 in two concentrations. The size and distribution of the particles in the layer were evaluated, as well as layer compactness (SEM). The antibacterial effect (against E. coli and S. epidermidis) and cytotoxicity (towards L929 and U-2 OS cell lines) of these materials were then tested. Despite cracking of the coatings after firing, the coatings demonstrated very good antibacterial effects against both E. coli and S. epidermidis after 24h of interaction. None of the tested materials were toxic to both cell lines. Collectively, our results suggest that these materials are promising candidates for orthopedic applications.


Assuntos
Antibacterianos/química , Ligas , Materiais Revestidos Biocompatíveis , Escherichia coli , Transição de Fase , Silicatos , Prata , Titânio
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