Vestibular schwannoma is the most common benign neoplasm of the cerebellopontine angle. Its first symptoms include hearing loss, tinnitus, and vestibular symptoms, followed by cerebellar and brainstem symptoms, along with palsy of the adjacent cranial nerves. However, the clinical picture has unpredictable dynamics and currently, there are no reliable predictors of tumor behavior. Hence, it is desirable to have a fast routine method for analysis of vestibular schwannoma tissues at the molecular level. The major objective of this study was to verify whether a technique using in-sample specific protein digestion with trypsin would have the potential to provide a proteomic characterization of these pathological tissues. The achieved results showed that the use of this approach with subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of released peptides allowed a fast identification of a considerable number of proteins in two differential parts of vestibular schwannoma tissue as well as in tissues of control healthy samples. Furthermore, mathematical analysis of MS data was able to discriminate between pathological vestibular schwannoma tissues and healthy tissues. Thus, in-sample protein digestion combined with LC-MS/MS separation and identification of released specific peptides followed by mathematical analysis appears to have the potential for routine characterization of vestibular schwannomas at the molecular level. Data are available via ProteomeXchange with identifier PXD045261.
- MeSH
- chromatografie kapalinová metody MeSH
- lidé MeSH
- peptidové fragmenty * analýza chemie metabolismus MeSH
- peptidy metabolismus MeSH
- proteolýza MeSH
- proteomika metody MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- trypsin chemie MeSH
- vestibulární schwannom * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
For the understanding of pathological states of bone tissues in oral surgery, it would be desirable to have the possibility to simulate these processes on bone cell models in vitro. These cultures, similarly to bone tissues, contain numerous proteins entrapped in the insoluble matrix. The major goal of this study was to verify whether a method based on direct in-matrix protein digestion could be suitable for the discrimination between different induced pathological states of bone cell models cultivated in vitro. Using in-sample specific protein digestion with trypsin followed by liquid chromatography-tandem mass spectrometry analysis of released peptides, 446 proteins (in average per sample) were identified in a bone cell in vitro model with induced cancer, 440 proteins were found in a model with induced inflammation, 451 proteins were detected in control in vitro culture, and 491 proteins were distinguished in samples of vestibular laminas of maxillary bone tissues originating from six different patients. Subsequent partial least squares - discrimination analysis of obtained liquid chromatography-tandem mass spectrometry data was able to discriminate among in vitro cultures with induced cancer, with induced inflammation, and control cultivation. Thus, the direct in-sample protein digestion by trypsin followed by liquid chromatography-tandem mass spectrometry analysis of released specific peptide fragments from the insoluble matrix and mathematical analysis of the mass spectrometry data seems to be a promising tool for the routine proteomic characterization of in vitro human bone models with induced different pathological states.
- MeSH
- chromatografie kapalinová metody MeSH
- lidé MeSH
- peptidy analýza MeSH
- proteiny chemie MeSH
- proteolýza MeSH
- proteomika metody MeSH
- stomatochirurgické výkony * MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- trypsin chemie MeSH
- zánět MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The problem of a growing resistance of bacteria and other microorganisms to conventional antibiotics gave rise to a search for new potent antimicrobial agents. Insect antimicrobial peptides (AMPs) seem to be promising novel potential anti-infective therapeutics. The dipeptide β-alanyl-tyrosine (β-Ala-Tyr) is one of the endogenous insect toxins exhibiting antibacterial activity against both Gram-negative and Gram-positive bacteria. Prior to testing its other antimicrobial activities, it has to be prepared in a pure form. In this study, we have developed a capillary zone electrophoresis (CZE) method for analysis of β-Ala-Tyr isolated from the extract of the hemolymph of larvae of the fleshfly Neobellieria bullata by reversed-phase high-performance liquid chromatography (RP-HPLC). Based on our previously described correlation between CZE and free-flow zone electrophoresis (FFZE), analytical CZE separation of β-Ala-Tyr and its admixtures have been converted into preparative purification of β-Ala-Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the β-Ala-Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.
- MeSH
- antiinfekční látky chemie izolace a purifikace MeSH
- dipeptidy chemie izolace a purifikace MeSH
- elektroforéza metody MeSH
- hemolymfa chemie MeSH
- larva chemie MeSH
- Sarcophagidae chemie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Ameloblastin (Ambn) as an intrinsically disordered protein (IDP) stands for an important role in the formation of enamel-the hardest biomineralized tissue commonly formed in vertebrates. The human ameloblastin (AMBN) is expressed in two isoforms: full-length isoform I (AMBN ISO I) and isoform II (AMBN ISO II), which is about 15 amino acid residues shorter than AMBN ISO I. The significant feature of AMBN-its oligomerization ability-is enabled due to a specific sequence encoded by exon 5 present at the N-terminal part in both known isoforms. In this study, we characterized AMBN ISO I and AMBN ISO II by biochemical and biophysical methods to determine their common features and differences. We confirmed that both AMBN ISO I and AMBN ISO II form oligomers in in vitro conditions. Due to an important role of AMBN in biomineralization, we further addressed the calcium (Ca2+)-binding properties of AMBN ISO I and ISO II. The binding properties of AMBN to Ca2+ may explain the role of AMBN in biomineralization and more generally in Ca2+ homeostasis processes.
- MeSH
- biologické modely MeSH
- hydrodynamika MeSH
- lidé MeSH
- multimerizace proteinu MeSH
- protein - isoformy MeSH
- proteiny vázající vápník chemie metabolismus MeSH
- proteiny zubní skloviny chemie metabolismus MeSH
- spektrální analýza MeSH
- teplota MeSH
- vápník metabolismus MeSH
- vazba proteinů MeSH
- vnitřně neuspořádané proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In this study, the apparent binding constants and limiting mobilities of the multiply charged complexes of the Δ- and Λ-enantiomers of Ru(II)- and Fe(II)-polypyridyl associates ([Ru(2,2'-bipyridine)3 ]2+ , [Ru(1,10-phenanthroline)3 ]2+ , and [Fe(1,10-phenanthroline)3 ]2+ ) with single-isomer 2,3-diacetylated-6-sulfated-cyclodextrins (CDs) (12Ac-6S-α-CD, 14Ac-7S-β-CD, and 16Ac-8S-γ-CD) were determined by ACE using uncorrected and ionic strength corrected actual mobilities of the species involved. Two limiting models were tested for the ionic strength correction of the actual mobilities based on an empirical relation for the ionic strength correction of multivalent ionic species. In model 1, the nominal values of the charge numbers (zS,nom ) and analytical concentrations (cS,nom ) of the above CD selectors in the BGEs were applied for calculation of the BGE ionic strength, as usual. In model 2, the CD selectors were considered as singly charged species (zS = -1) with |zS,nom |-times higher concentrations in the BGE than their analytical concentrations (cS = |zS,nom | × cS,nom ) in the calculation of the BGE ionic strength. In all three cases-with uncorrected actual mobilities as well as with actual mobilities corrected according to the two limiting models-the measured effective mobilities of the above enantiomers fit well the theoretical curves of their mobility dependences on the CD selectors concentrations in the BGE, with high average coefficients of determination (R2 = 0.9890-0.9995). Nevertheless, the best physico-chemically meaningful values of the apparent binding constants and the limiting mobilities of the enantiomer-CDs complexes with low RSDs were obtained using the actual mobilities of the species involved corrected according to model 2.
The commonly used histological assessment of pathological states of alveolar bone tissues in oral surgery needs laborious and time-consuming processing by an experienced histologist. Therefore, a simpler and faster methodology is required in this field. Following this demand, this paper reports a straightforward approach using the tryptic cleavage of proteins directly in bone without its demineralization, followed by the capillary electrophoresis-ultraviolet detection profiling of the yielded protein digest. Cleavage-derived peptides were separated by capillary electrophoresis in acidic background electrolytes, pH 2.01-2.54. The best resolution of peptide fragments with the highest peak capacity was achieved in the background electrolyte composed of 55 mM H3 PO4 , 14 mM tris(hydroxymethyl)aminomethan, pH 2.01. The differences in the obtained capillary electrophoresis-ultraviolet detection profiles with characteristic patterns for particular bone samples were subsequently discriminated by linear discriminant analysis over principal components. This approach was first verified on porcine bone tissues as model samples; jawbone and calf bone tissues could be discriminated with an accuracy of 100%. Subsequently, the method was capable of differentiating unequivocally between human healthy and inflammatory alveolar bone tissues obtained from oral surgery. This procedure seems to be promising as complement or even an alternative to the traditional histological discrimination between healthy and inflammatory bone tissues in oral surgery.
CE using randomly highly sulfated α-, β-, and γ-CDs (S-α-CD, S-β-CD, S-γ-CD), sulfobutylether-β-CD (SBE-β-CD), single isomer (6-O-sulfo) α-, β-, and γ-CDs, and their derivatives as stereoselectors was applied to chiral analysis of polypyridyl complexes of [Ru(bpy)3 ](2+) , [Ru(phen)3 ](2+) , and [Fe(phen)3 ](2+) (bpy = 2,2'-bipyridine; phen = 1,10 phenanthroline). The best separations of Δ- and Λ-enantiomers of the these complexes with high resolution (up to R1,2 = 7.0) and short analysis times (10-20 min) were achieved in the BGE composed of 22 mM NaOH/35 mM H3 PO4 , pH 2.4, containing 1.5-6.0 mM S-α-CD or S-β-CD, or SBE-β-CD as chiral selectors. The developed method was applied to the assessment of enantiomeric purity of several samples of [Ru(bpy)3 ](2+) catalyst. CE experiments were performed in a homemade analyzer equipped with bare or hydroxypropylcellulose-coated fused-silica capillaries (total/effective length 40/29 cm, id/od 50/375 μm) and an UV absorption detector operating at 206 nm. In addition to chiral analysis, apparent binding constants of the complexes of [Ru(bpy)3 ](2+) , [Ru(phen)3 ](2+) , and [Fe(phen)3 ](2+) enantiomers with five sulfated CDs (S-α-CD, S-β-CD, S-γ-CD, SBE-β-CD, and 16Me-8S-γ-CD) were determined from the dependence of their effective electrophoretic mobilities on the concentration of the CDs in the BGE by nonlinear regression analysis. Calculated apparent binding constants of these complexes were found to be in the (1.10-4.66) × 10(3) L/mol range. Moreover, it was shown that at selected concentrations of some S-CDs and suppressed or very low electroosmotic flow, the exceptional enantioseparations with infinite resolution could be achieved.
Thirteen mono-N-acyl derivatives of 2,6-diaminopimelic acid (DAP)-new potential inhibitors of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18)-were analyzed and characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies and two capillary electromigration methods: capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Structural features of DAP derivatives were characterized by IR and NMR spectroscopies, whereas CZE and MEKC were applied to evaluate their purity and to investigate their electromigration properties. Effective electrophoretic mobilities of these compounds were determined by CZE in acidic and alkaline background electrolytes (BGEs) and by MEKC in acidic and alkaline BGEs containing a pseudostationary phase of anionic detergent sodium dodecyl sulfate (SDS) or cationic detergent cetyltrimethylammonium bromide (CTAB). The best separation of DAP derivatives, including diastereomers of some of them, was achieved by MEKC in an acidic BGE (500 mM acetic acid [pH 2.54] and 60mM SDS). All DAP derivatives were examined for their ability to inhibit catalytic activity of DapE from Haemophilus influenzae (HiDapE) and ArgE from Escherichia coli (EcArgE). None of these DAP derivatives worked as an effective inhibitor of HiDapE, but one derivative-N-fumaryl, Me-ester-DAP-was found to be a moderate inhibitor of EcArgE, thereby providing a promising lead structure for further studies on ArgE inhibitors.
- MeSH
- amidohydrolasy antagonisté a inhibitory MeSH
- elektroforéza kapilární metody MeSH
- Escherichia coli enzymologie MeSH
- Haemophilus influenzae enzymologie MeSH
- inhibitory enzymů chemie farmakologie MeSH
- kyselina diaminopimelová chemie MeSH
- magnetická rezonanční spektroskopie metody MeSH
- spektrofotometrie infračervená metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
New large-scale synthetic approach to antiretroviral agent 9-[2-(R)-(phosphonomethoxy)propyl]-2,6-diaminopurine, (R)-PMPDAP, was developed. Reaction of (R)-propanediol carbonate with 2,6-diaminopurine afforded exclusively (R)-9-(2-hydroxypropyl)-2,6-diaminopurine which was subsequently used for introduction of a phosphonomethyl residue using TsOCH(2)P(O)(OiPr)(2) or BrCH(2)P(O)(OiPr)(2) followed by deprotection of ester groups. All minor ingredients and by-products formed during the process were identified and further studied. The final product was obtained in high yield and its high enantiomeric purity (>99%) was confirmed by chiral capillary electrophoretic analysis using β-cyclodextrin as a chiral selector. Antiretroviral activity data of (R)-PMPDAP and its diverse prodrugs against HIV and FIV were investigated. Akin to (R)-PMPDAP, both prodrugs inhibit FIV replication in a selective manner. Compared to the parent molecule, the amidate prodrug was 10-fold less active against FIV in cell culture, whereas the alkoxyalkyl ester prodrug was 200-fold more potent in inhibiting FIV replication in vitro.
- MeSH
- adenin analogy a deriváty chemie farmakologie MeSH
- antivirové látky chemie farmakologie MeSH
- HIV-1 účinky léků MeSH
- organofosforové sloučeniny chemie farmakologie MeSH
- prekurzory léčiv chemie farmakologie MeSH
- stereoizomerie MeSH
- virus kočičí imunodeficience účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A new CZE method has been developed for chiral analysis of an important anti-acquired immunodeficiency syndrome drug, 9-(R)-[2-(phosphonomethoxy)propyl]adenine ((R)-PMPA, tenofovir), and six related antiviral acyclic nucleoside phosphonates using beta-CD as a chiral selector. The influence of the composition, concentration and pH of the BGE and the type and concentration of chiral selector on enantiomer resolution was investigated. Complete separations of (R,S)-enantiomers of PMPA with very good resolution (R(s)=1.50-3.64) were achieved within a short time (4-15 min) in 20-50 mM sodium borate or sodium tetraborate BGEs, pH 10.0, at 20 mg/mL concentration of beta-CD. (R,S)-enantiomers of five similar PMPA analogs containing purine bases (adenine, diaminopurine or guanine) and hydroxyl or fluor substituents at C3 carbon atom of propyl chain were baseline separated within 10-17 min in 35 mM sodium tetraborate BGE, pH 10.0, at 20 mg/mL beta-CD concentration. Another important antiviral used by acquired immunodeficiency syndrome patients, derived from pyrimidine base cytosine, 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (cidofovir), and the (R)-enantiomer of this drug were successfully separated in 50 mM sodium tetraborate BGE, pH 10.5, at 20 mg/mL beta-CD concentration within 45 min. Using the UV-absorption detection at 206 nm, the concentration detection limits of the analyzed acyclic nucleoside phosphonates were determined in the submicromolar to micromolar range (0.15-2.51 microg/mL level).
- MeSH
- adenin analogy a deriváty chemie MeSH
- beta-cyklodextriny chemie MeSH
- cytosin analogy a deriváty chemie MeSH
- elektroforéza kapilární metody MeSH
- elektrolyty MeSH
- koncentrace vodíkových iontů MeSH
- látky proti HIV chemie MeSH
- lineární modely MeSH
- organofosfonáty chemie MeSH
- puriny chemie MeSH
- pyrimidiny chemie MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- stereoizomerie MeSH
- Publikační typ
- práce podpořená grantem MeSH
