The paper deals with the characterisation of the bioactive phenomena of glass-ceramic scaffold derived from Bioglass® (containing 77 wt.% of crystalline phases Na(2)O·2CaO·3SiO(2) and CaO·SiO(2) and 23 wt.% of residual glass phase) using simulated body fluid (SBF) buffered with tris-(hydroxymethyl) aminomethane (TRIS). A significant effect of the TRIS buffer on glass-ceramic scaffold dissolution in SBF was detected. To better understand the influence of the buffer, the glass-ceramic scaffold was exposed to a series of in vitro tests using different media as follows: (i) a fresh liquid flow of SBF containing tris (hydroxymethyl) aminomethane; (ii) SBF solution without TRIS buffer; (iii) TRIS buffer alone; and (iv) demineralised water. The in vitro tests were provided under static and dynamic arrangements. SBF buffered with TRIS dissolved both the crystalline and residual glass phases of the scaffold and a crystalline form of hydroxyapatite (HAp) developed on the scaffold surface. In contrast, when TRIS buffer was not present in the solutions only the residual glassy phase dissolved and an amorphous calcium phosphate (Ca-P) phase formed on the scaffold surface. It was confirmed that the TRIS buffer primarily dissolved the crystalline phase of the glass-ceramic, doubled the dissolving rate of the scaffold and moreover supported the formation of crystalline HAp. This significant effect of the buffer TRIS on bioactive glass-ceramic scaffold degradation in SBF has not been demonstrated previously and should be considered when analysing the results of SBF immersion bioactivity tests of such systems.
An international standard (ISO: 23317:2014) exists for the in vitro testing of inorganic biomaterials in simulated body fluid (SBF). This standard uses TRIS buffer to maintain neutral pH in SBF, but in our previous paper, we showed that the interaction of a tested glass-ceramic material with TRIS can produce false-positive results. In this study, we evaluated whether the HEPES buffer, which also belongs to the group of Good´s buffers, would be more suitable for SBF. We compared its suitability in two media: SBF with HEPES and demineralized water with HEPES. The tested scaffold (45S5 bioactive glass-based) was exposed to the media under a static-dynamic arrangement (solutions were replaced on a daily basis) for 15 days. Leachate samples were collected daily for the analysis of Ca2+ ions and Si (AAS), (PO4 )3- ions (UV-VIS), and to measure pH. The glass-ceramic scaffold was analyzed by SEM/EDS, XRD, and WD-XRF before and after 0.3, 1, 3, 7, 11, and 15 days of exposure. Our results confirmed the rapid selective dissolution of the glass-ceramic crystalline phase (Combeite) containing Ca2+ ions due to the presence of HEPES, hydroxyapatite supersaturation being reached within 24 h in both solutions. These new results suggest that, like TRIS, HEPES buffer is not suitable for the in vitro testing of highly reactive inorganic biomaterials (glass, glass-ceramics). The ISO standard for such tests requires revision, but HEPES is not a viable alternative to TRIS buffer. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 143-152, 2018.
The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris-sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.
- MeSH
- Biotechnology methods MeSH
- Time Factors MeSH
- Cryptococcus enzymology metabolism MeSH
- Cells, Immobilized enzymology metabolism MeSH
- beta-Fructofuranosidase isolation & purification MeSH
- Hydrogen-Ion Concentration MeSH
- Culture Media chemistry MeSH
- Buffers MeSH
- Sucrose MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- MeSH
- Conjunctivitis, Allergic * diagnosis drug therapy MeSH
- Anti-Allergic Agents * therapeutic use MeSH
- Histamine Antagonists adverse effects therapeutic use MeSH
- Adrenal Cortex Hormones adverse effects therapeutic use MeSH
- Clinical Trials as Topic MeSH
- Oxamic Acid analogs & derivatives administration & dosage adverse effects MeSH
- Humans MeSH
- Drug-Related Side Effects and Adverse Reactions MeSH
- Nitriles administration & dosage therapeutic use MeSH
- Ophthalmic Solutions * administration & dosage adverse effects therapeutic use MeSH
- Tromethamine analogs & derivatives administration & dosage adverse effects MeSH
- Check Tag
- Humans MeSH
Procedures for the extraction-spectrophotometric determination of tris(2-chloroethyl)amine, an alkylating agent known as a drug as well as a chemical warfare agent (nitrogen mustard HN-3), with 7 acid-base indicators of a triphenylmethane lactone type, phthaleins, were developed. Representatives of phthaleins without an oxygen bridge (thymolphthalein, o-cresolphthalein, naphtholphthalein) and with an oxygen bridge (fluorescein, 2',7'-dichlorofluorescein, eosin B and eosin Y) were used. The methods were based on the formation of ion pair complexes. Chloroform was used as a non-polar solvent for an extraction. The conditions to determine were optimized for the optimal pH of the buffer and the concentration of a phthalein as a reagent. The dependence on the reaction time in a water phase and the stoichiometry of extraction products were studied. The detection limits and the limits of the determination of separate procedures and conditional extraction constants were determined. Comparison with the spectrophotometric method of the group determination of alkyl halides and acyl halides using alkaline ethanol-water solution of thymolphthalein, the so-called T-135 agent, was conducted. While studying the selectivity, the possible interference of bis(2-chloroethyl)sulphide and 3 nitrogen mustards in the proposed procedures were verified. Copyright © 2016 John Wiley & Sons, Ltd.
- MeSH
- Alkylating Agents analysis isolation & purification MeSH
- Chemical Warfare Agents analysis isolation & purification MeSH
- Phenolphthaleins chemistry MeSH
- Hydrogen-Ion Concentration MeSH
- Limit of Detection MeSH
- Buffers MeSH
- Nitrogen Mustard Compounds analysis isolation & purification MeSH
- Spectrophotometry methods MeSH
- Water analysis MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Keywords
- lodoxamid tromethamin,
- MeSH
- Allergens adverse effects MeSH
- Conjunctivitis, Allergic * diagnosis drug therapy MeSH
- Anti-Allergic Agents * therapeutic use MeSH
- Histamine Antagonists adverse effects therapeutic use MeSH
- Adrenal Cortex Hormones adverse effects therapeutic use MeSH
- Clinical Trials as Topic MeSH
- Oxamic Acid analogs & derivatives therapeutic use MeSH
- Humans MeSH
- Drug-Related Side Effects and Adverse Reactions MeSH
- Nitriles therapeutic use MeSH
- Ophthalmic Solutions * MeSH
- Statistics as Topic MeSH
- Tromethamine analogs & derivatives therapeutic use MeSH
- Check Tag
- Humans MeSH
The proper course and reproducibility of diagnostic techniques depend on narrowly defined reaction conditions, including the reaction pH. Nevertheless, numerous assays are affected by an inaccurately defined reaction pH. Buffers are sometimes suggested for use outside their useful pH ranges, which complicates the reproducibility of results because the buffering capacity is insufficient to retain the disclosed pH. Here, we focus on the comet assay lysis buffer. Comet assay is broadly used for quantifying DNA breaks in eukaryotic cells. The most widespread comet assay protocols employ lysis of the cells before electrophoresis in a buffer containing Triton X-100, a high concentration of NaCl, sodium sarcosinate, EDTA, and Tris, with some modifications. However, nearly all researchers report that they use Tris buffer at pH 10, and some report the pH of the Tris additive alone. Alternatively, others report the pH of the final lysis buffer. However, the lysis solution used in the comet assay is buffered at a pH outside the useful range of Tris. Tris-based buffers have a useful pH range of 7.0 - 9.0. The buffer composed of 10 mM Tris has pKa 8.10 at 25°C and 8.69 at 4°C. The cell lysis conditions used in nearly all modifications of comet assay protocols remain imprecise and uncritically employed. Despite the pH of the lysis buffer likely has negligible effect on the detection of DNA breaks, precise lysis conditions are highly important for the use of comet assay in the detection of base modifications, which are often unstable and sensitive to pH.
- MeSH
- DNA * MeSH
- Comet Assay methods MeSH
- Hydrogen-Ion Concentration MeSH
- Humans MeSH
- DNA Damage * MeSH
- Reproducibility of Results MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Humanin (HN), Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-IIe-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala, recently discovered in the human brain, is an important neuroprotective peptide. Some derivatives of HN show even higher biological activity, for example [G-14]-HN, where Ser at position 14 is replaced with Gly. As structurally related HN peptide derivatives have similar chemical properties, their separation by CE is difficult. In this work, the electrophoretic behaviour of HN derivatives including [G-14]-HN, a tryptophan HN derivative [W-14]-HN, several other HN derivatives and HN fragments was studied. While phosphate buffer was used as the general BGE, the effects of the buffer concentration and various additives were examined, including sulphate, heptane sulphonate, 2-morpholinoethanesulphonic acid N-[tris(hydroxymethyl)methyl]-2-aminoethane sulphonic acid (TES), sulphated-beta-CD and beta-CD. Separation efficiency of 200,000 theoretical plates was achieved in a BGE of 80 mM phosphate at pH 2.5 where seven out of nine major peaks were partially separated. By investigating the influence of concentration of the interrogated ions on peptides migration, the association between positively charged protonated sites of peptides and various anions was proved. Especially a strong interaction with phosphate, sulphate and sulphonate groups was established. Conditional stability constant of the [Pep(z+), (H(2)PO(4)(-))(n)](z - n) ion associate (n = 1) for [G-14]-HN equals to log K approximately 1.78.
- MeSH
- Alkanesulfonates chemistry MeSH
- beta-Cyclodextrins chemistry MeSH
- Electrophoresis, Capillary methods MeSH
- Financing, Organized MeSH
- Phosphates chemistry MeSH
- Intracellular Signaling Peptides and Proteins chemical synthesis chemistry isolation & purification MeSH
- Hydrogen-Ion Concentration MeSH
- Humans MeSH
- Molecular Sequence Data MeSH
- Neuropeptides chemical synthesis chemistry isolation & purification MeSH
- Peptide Fragments chemical synthesis chemistry isolation & purification MeSH
- Buffers MeSH
- Solutions MeSH
- Amino Acid Sequence MeSH
- Sulfates chemistry MeSH
- Amino Acid Substitution MeSH
- Check Tag
- Humans MeSH
Water-soluble CdTe quantum dots (QDs) and their conjugates with antibodies and antigenes were prepared by optimized procedures for applications in CE immunoassays. The QD size of 3.5 nm, excitation spectrum in the range of 300-500 nm, the maximum wavelength of the emission spectrum at 610 nm, quantum yield of 0.25 and luminescence lifetimes in the range of 3.6-43 ns were determined. The 0.1 M solution of TRIS/TAPS (pH 8.3) was found to be the optimum buffer for the separation of the antiovalbumin-ovalbumin immunocomplex from the free conjugates of QDs.
In this work, electrophoretically mediated microanalysis (EMMA) was applied to the in-capillary tryptic digestion of proteins for proteomic purposes. Compared with classical in-solution tryptic digestion or the trypsin reactor commonly used for this purpose, the EMMA-based method is rapid, can be automated and requires only a small amount of trypsin preparation. Moreover, the protein digestion and the analysis of the resulting peptides are integrated into one procedure. A combination of the EMMA methodology with a partial filling technique was used in this study, since the pH optimum of the trypsin reaction differs strongly from the best pH for the CZE separation of peptides. In this set-up, a part of the capillary is filled with the best buffer for the tryptic digestion (50 mM Tris-HCl buffer, pH 8.5) whereas the rest of the capillary is filled with the BGE optimal for peptide separation (0.1 M phosphate buffer, pH 2.5). As the proteins differ in their isoelectric points, a sandwich type of injection was used. The analysed protein is thus injected between two trypsin zones, which ensures their mixing and digestion. The analysis of one protein comprising both the digestion and the peptide separation is then completed in 1 h using a commercial instrument for CE with no modifications.
- MeSH
- Cytochromes c chemistry MeSH
- Electrophoresis, Capillary methods MeSH
- Insulin chemistry MeSH
- Caseins chemistry MeSH
- Peptide Fragments chemistry MeSH
- Proteins chemistry MeSH
- Proteomics methods MeSH
- Trypsin chemistry MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
