BACKGROUND: The main goal of this research was to study the interactions of a fully characterized set of silver nanomaterials (Ag ENMs) with cells in vitro, according to the standards of Good Laboratory Practices (GLP), to assure the quality of nanotoxicology research. We were interested in whether Ag ENMs synthesized by the same method, with the same size distribution, shape and specific surface area, but with different charges and surface compositions could give different biological responses. METHODS: A range of methods and toxicity endpoints were applied to study the impacts of interaction of the Ag ENMs with TK6 cells. As tests of viability, relative growth activity and trypan blue exclusion were applied. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. The mutagenic potential of Ag ENMs was investigated with the in vitro HPRT gene mutation test on V79-4 cells according to the OECD protocol. Ag ENM agglomeration, dissolution as well as uptake and distribution within the cells were investigated as crucial aspects of Ag ENM toxicity. Ag ENM stabilizers were included in addition to positive and negative controls. RESULTS: Different cytotoxic effects were observed including membrane damage, cell cycle arrest and cell death. Ag ENMs also induced various kinds of DNA damage including strand breaks and DNA oxidation, and caused gene mutation. We found that positive Ag ENMs had greater impact on cyto- and genotoxicity than did Ag ENMs with neutral or negative charge, assumed to be related to their greater uptake into cells and to their presence in the nucleus and mitochondria, implying that Ag ENMs might induce toxicity by both direct and indirect mechanisms. CONCLUSION: We showed that Ag ENMs could be cytotoxic, genotoxic and mutagenic. Our experiments with the HPRT gene mutation assay demonstrated that surface chemical composition plays a significant role in Ag ENM toxicity.

• MeSH
• biologický transport MeSH
• buněčná membrána účinky léků   patologie   MeSH
• Cricetulus MeSH
• hodnocení rizik MeSH
• hypoxanthinfosforibosyltransferasa genetika   MeSH
• kometový test MeSH
• kontrolní body buněčného cyklu účinky léků   MeSH
• kovové nanočástice * MeSH
• lidé MeSH
• mutace * MeSH
• mutační analýza DNA MeSH
• nádorové buněčné linie MeSH
• oxidační stres účinky léků   MeSH
• poškození DNA * MeSH
• povrchové vlastnosti MeSH
• proliferace buněk účinky léků   MeSH
• sloučeniny stříbra chemická syntéza   metabolismus   toxicita   MeSH
• tvar buňky účinky léků   MeSH
• velikost částic MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

• MeSH
• biofyzika MeSH
• biologický transport MeSH
• buněčná membrána MeSH
• membránové potenciály MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• fyzika, biofyzika
• cytologie, klinická cytologie
• NLK Publikační typ
• kolektivní monografie

• MeSH
• fyzikální chemie MeSH
• Publikační typ
• monografie MeSH

• Konspekt
• Fyzikální chemie
• NLK Obory
• chemie, klinická chemie

An investigation was made of the adhesion, growth and differentiation of osteoblast-like MG-63 and Saos-2 cells on titanium (Ti) and niobium (Nb) supports and on TiNb alloy with surfaces oxidized at 165°C under hydrothermal conditions and at 600°C in a stream of air. The oxidation mode and the chemical composition of the samples tuned the morphology, topography and distribution of the charge on their surfaces, which enabled us to evaluate the importance of these material characteristics in the interaction of the cells with the sample surface. Numbers of adhered MG-63 and Saos-2 cells correlated with the number of positively-charged (related with the Nb2O5 phase) and negatively-charged sites (related with the TiO2 phase) on the alloy surface. Proliferation of these cells is correlated with the presence of positively-charged (i.e. basic) sites of the Nb2O5 alloy phase, while cell differentiation is correlated with negatively-charged (acidic) sites of the TiO2 alloy phase. The number of charged sites and adhered cells was substantially higher on the alloy sample oxidized at 600°C than on the hydrothermally treated sample at 165°C. The expression values of osteoblast differentiation markers (collagen type I and osteocalcin) were higher for cells grown on the Ti samples than for those grown on the TiNb samples. This was more particularly apparent in the samples treated at 165°C. No considerable immune activation of murine macrophage-like RAW 264.7 cells on the tested samples was found. The secretion of TNF-α by these cells into the cell culture media was much lower than for either cells grown in the presence of bacterial lipopolysaccharide, or untreated control samples. Thus, oxidized Ti and TiNb are both promising materials for bone implantation; TiNb for applications where bone cell proliferation is desirable, and Ti for induction of osteogenic cell differentiation.

• MeSH
• biologické markery metabolismus   MeSH
• buněčná adheze účinky léků   MeSH
• buněčná diferenciace účinky léků   MeSH
• buněčné linie MeSH
• kolagen typu I metabolismus   MeSH
• lidé MeSH
• lipopolysacharidy farmakologie   MeSH
• makrofágy cytologie   účinky léků   metabolismus   MeSH
• myši MeSH
• osteoblasty cytologie   účinky léků   metabolismus   MeSH
• osteokalcin metabolismus   MeSH
• oxidace-redukce MeSH
• povrchové vlastnosti MeSH
• proliferace buněk účinky léků   MeSH
• slitiny chemie   farmakologie   MeSH
• statická elektřina MeSH
• tkáňové podpůrné struktury * MeSH
• TNF-alfa farmakologie   MeSH
• vysoká teplota MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Glutamate carboxypeptidase III (GCPIII) is a metalloenzyme that belongs to the transferrin receptor/glutamate carboxypeptidase II (GCPII; EC 3.4.17.21) superfamily. GCPIII has been studied mainly because of its evolutionary relationship to GCPII, an enzyme involved in a variety of neuropathologies and malignancies, such as glutamatergic neurotoxicity and prostate cancer. Given the potential functional and pharmacological overlap between GCPIII and GCPII, studies addressing the structural and physiological properties of GCPIII are crucial for obtaining a deeper understanding of the GCPII/GCPIII system. In the present study, we report high-resolution crystal structures of the human GCPIII ectodomain in a 'pseudo-unliganded' state and in a complex with: (a) L-glutamate (a product of hydrolysis); (b) a phosphapeptide transition state mimetic, namely (2S,3'S)-{[(3'-amino-3'-carboxy-propyl)-hydroxyphosphinoyl]methyl}-pentanedioic acid; and (c) quisqualic acid, a glutamate biostere. Our data reveal the overall fold and quaternary arrangement of the GCPIII molecule, define the architecture of the GCPIII substrate-binding cavity, and offer an experimental evidence for the presence of Zn(2+) ions in the bimetallic active site. Furthermore, the structures allow us to detail interactions between the enzyme and its ligands and to characterize the functional flexibility of GCPIII, which is essential for substrate recognition. A comparison of these GCPIII structures with the equivalent GCPII complexes reveals differences in the organization of specificity pockets, in surface charge distribution, and in the occupancy of the co-catalytic zinc sites. The data presented here provide information that should prove to be essential for the structurally-aided design of GCPIII-specific inhibitors and might comprise guidelines for future comparative GCPII/GCPIII studies.

• MeSH
• antigeny povrchové chemie   MeSH
• fosfopeptidy chemie   MeSH
• glutamátkarboxypeptidasa II chemie   MeSH
• karboxypeptidasy chemie   MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• kvarterní struktura proteinů MeSH
• kyselina chischalová chemie   MeSH
• kyselina glutamová chemie   MeSH
• lidé MeSH
• ligandy MeSH
• molekulární mimikry MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Positive selection acting on Toll-like receptors (TLRs) has been recently investigated to reveal evolutionary mechanisms of host-pathogen molecular co-adaptation. Much of this research, however, has focused mainly on the identification of sites predicted to be under positive selection, bringing little insight into the functional differences and similarities among species and a limited understanding of convergent evolution in the innate immune molecules. In this study, we provide evidence of phenotypic variability in the avian TLR4 ligand-binding region (LBR), the direct interface between host and pathogen molecular structures. We show that 55 passerine species vary substantially in the distribution of electrostatic potential on the surface of the receptor, and based on these distinct patterns, we identified four species clusters. Seven of the 34 evolutionarily nonconservative and positively selected residues correspond topologically to sites previously identified as being important for lipopolysaccharide, lipid IVa or MD-2 binding. Five of these positions codetermine the identity of the charge clusters. Groups of species that host-related communities of pathogens were predicted to cluster based on their TLR4 LBR charge. Despite some evidence for convergence among taxa, there were no clear associations between the TLR4 LBR charge distribution and any of the general ecological characteristics compared (migration, latitudinal distribution and diet). Closely related species, however, mostly belonged to the same surface charge cluster indicating that phylogenetic constraints are key determinants shaping TLR4 adaptive evolution. Our results suggest that host innate immune evolution is consistent with Fahrenholz's rule on the cospeciation of hosts and their parasites.

• MeSH
• glykolipidy chemie   genetika   MeSH
• interakce hostitele a patogenu genetika   MeSH
• konformace proteinů MeSH
• ligandy MeSH
• lipid A analogy a deriváty   chemie   genetika   MeSH
• lipopolysacharidy chemie   genetika   MeSH
• lymfocytární antigen 96 chemie   genetika   MeSH
• mikrobiota genetika   MeSH
• molekulární evoluce * MeSH
• molekulární modely MeSH
• přirozená imunita genetika   MeSH
• ptáci genetika   parazitologie   MeSH
• sekvenční analýza DNA MeSH
• selekce (genetika) * genetika   MeSH
• statická elektřina MeSH
• toll-like receptor 4 chemie   genetika   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We analyze the conditions of the adsorption of a flexible peptide onto a charged substrate in the 'wrong side' of the isoelectric point (WSIP), i.e. when surface and peptide charges have the same sign. As a model system, we focus on the casein macropeptide (CMP), both in the aglycosylated (aCMP) and fully glycosydated (gCMP) forms. We model the substrate as a uniformly charged plane while CMP is treated as a bead-and-spring model including electrostatic interactions, excluded volume effects and acid/base equilibria. Adsorption coverage, aminoacid charges and concentration profiles are computed by means of Monte Carlo simulations at fixed pH and salt concentration. We conclude that for different reasons the CMP can be adsorbed to both positively and negatively charged surfaces in the WSIP. For negatively charged surfaces, WSIP adsorption is due to the patchy distribution of charges: the peptide is attached to the surface by the positively charged end of the chain, while the repulsion of the surface for the negatively charged tail is screened by the small ions of the added salt. This effect increases with salt concentration. Conversely, a positively charged substrate induces strong charge regulation of the peptide: the acidic groups are deprotonated, and the peptide becomes negatively charged. This effect is stronger at low salt concentrations and it is more intense for gCMP than for aCMP, due to the presence of the additional sialic groups in gCMP.

• MeSH
• adsorpce MeSH
• cytidinmonofosfát MeSH
• izoelektrický bod MeSH
• kaseiny * MeSH
• peptidy * MeSH
• povrchové vlastnosti MeSH
• Publikační typ
• časopisecké články MeSH

Ultra-low fouling and functionalizable coatings represent emerging surface platforms for various analytical and biomedical applications such as those involving examination of cellular interactions in their native environments. Ultra-low fouling surface platforms as advanced interfaces enabling modulation of behavior of living cells via tuning surface physicochemical properties are presented and studied. The state-of-art ultra-low fouling surface-grafted polymer brushes of zwitterionic poly(carboxybetaine acrylamide), nonionic poly(N-(2-hydroxypropyl)methacrylamide), and random copolymers of carboxybetaine methacrylamide (CBMAA) and HPMAA [p(CBMAA-co-HPMAA)] with tunable molar contents of CBMAA and HPMAA are employed. Using a model Huh7 cell line, a systematic study of surface wettability, swelling, and charge effects on the cell growth, shape, and cytoskeleton distribution is performed. This study reveals that ultra-low fouling interfaces with a high content of zwitterionic moieties (>65 mol%) modulate cell behavior in a distinctly different way compared to coatings with a high content of nonionic HPMAA. These differences are attributed mostly to the surface hydration capabilities. The results demonstrate a high potential of carboxybetaine-rich ultra-low fouling surfaces with high hydration capabilities and minimum background signal interferences to create next-generation bioresponsive interfaces for advanced studies of living objects.

• MeSH
• biokompatibilní potahované materiály * chemie   farmakologie   MeSH
• cytoskelet metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• polymery * chemie   farmakologie   MeSH
• smáčivost MeSH
• testování materiálů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

N-(2-Hydroxypropyl)methacrylamide (HPMA) co-polymers containing disulfide and carbonyl thiazolidine-2-thione (TT) reactive groups in their side-chains (pHPMA-TT) were used as reductively removable chemical modification of the surface of cowpea mosaic viruses (CPMV). CPMV was used in this study as a model particle for viral gene delivery. The co-polymer reaction with CPMV surfaces carried out in aqueous solution was evaluated by monitoring the changes in the weight-average molecular weight and hydrodynamic size of the viruses using light scattering methods. The reaction conditions were optimized. The surface modification of CPMV with pHPMA-TT under selected conditions (concentrations of a coating polymer (c(p)) and NaCl) has not influenced the size distribution of the viral particles. The uncharged polymers bound to the viral surface via biodegradable S-S bonds can be fully removed by the exchange reaction with reductive dithiothreitol. To achieve optimal covering of viral surfaces, the positively charged reactive polymers (with or without biodegradable S-S bonds) should be applied at low concentrations (c(p)=0.1-0.5 mg/ml) and in presence of NaCl (0.15 M).

• MeSH
• biokompatibilní materiály chemická syntéza   chemie   MeSH
• biomimetika MeSH
• Comovirus chemie   MeSH
• disulfidy chemie   MeSH
• hydrofobní a hydrofilní interakce MeSH
• intracelulární prostor metabolismus   MeSH
• methakryláty chemie   MeSH
• polymery chemická syntéza   chemie   MeSH
• povrchové vlastnosti MeSH
• thiazolidiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Pancreatic ductal adenocarcinoma (PDAC) is a growing medical problem associated with extensive metastasis and high mortality. Intraperitoneal (IP) administration of therapeutics promises to help the treatment of cancers originated from organs in the peritoneal cavity. In this study, we evaluated how physicochemical properties of self-assembled polycation/siRNA nanoparticles affect their IP delivery efficacy in an orthotopic PDAC model. We have examined the effect of covalent polycation modification with lipophobic and hydrophobic tetrafluoro-p-toluic acid (TFTA), hydrophobic cholesterol, and hydrophilic poly(ethylene glycol) respectively. The surface charge of the three different nanoparticles was also modulated by coating the surface with serum albumin. We found that positively charged fluorine-containing particles with lipophobic properties based on a mixture of positively charged polymeric AMD3100 CXCR4 antagonist (PAMD) and PAMD modified with TFTA (mPAMD-TFTA)/siRNA displayed the best cell uptake and transfection efficacy in vitro. Biodistribution evaluation of the nanoparticles in a syngeneic orthotopic PDAC model revealed that the fluorine-containing formulation also achieved the highest PDAC tumor accumulation after IP administration. With a combination of CXCR4 inhibition by PAMD and PLK1 downregulation by siRNA, the treatment with mPAMD-TFTA/siPLK1 showed significant inhibition of both primary and metastatic PDAC tumors. Overall, our study provides insights into and guides the design of the nanoparticles for improved IP delivery of siRNA in PDAC.

• MeSH
• halogenace * MeSH
• lidé MeSH
• malá interferující RNA MeSH
• nádorové buněčné linie MeSH
• nádory slinivky břišní * farmakoterapie   MeSH
• polyelektrolyty MeSH
• tkáňová distribuce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH