Noncovalent molecular interactions between helquats, a new class of dicationic helical extended diquats, and several chiral acidic aromatic drugs and catalysts have been investigated using partial-filling affinity capillary electrophoresis (PF-ACE). Helquats dissolved at 1mM concentration in the aqueous background electrolyte (40mM Tris, 20mM acetic acid, pH 8.1) were introduced as ligand zones of variable length (0-130mm) into the hydroxypropylcellulose coated fused silica capillary whereas 0.1mM solutions of negatively charged chiral drugs or catalysts (warfarin, ibuprofen, mandelic acid, etodolac, binaphthyl phosphate and 11 other acidic aromatic compounds) were applied as a short analyte zone at the injection capillary end. After application of electric field, analyte and ligand migrated against each other and in case of their interactions, migration time of the analyte was increasing with increasing length of the ligand zone. From the tested compounds, only isomers of those exhibiting helical chirality and/or possessing conjugated aromatic systems were enantioselectively separated through their differential interactions with helquats. Some compounds with conjugated aromatic groups interacted with helquats moderately strongly but non-enantiospecifically. Small compounds with single benzene ring exhibited no or very weak non-enantiospecific interactions. PF-ACE method allowed to determine binding constants of the analyte-helquat complexes from the changes of migration times of the analytes. Binding constants of the weakest complexes of the analytes with helquats were less than 50L/mol, whereas binding constants of the strongest complexes were in the range 1 000-1 400L/mol.

• MeSH
• Algorithms MeSH
• Hydrocarbons, Aromatic analysis   MeSH
• Coloring Agents MeSH
• Cellulose analogs & derivatives   MeSH
• Electrophoresis, Capillary methods   MeSH
• Indicators and Reagents MeSH
• Pharmaceutical Preparations analysis   MeSH
• Ligands MeSH
• Stereoisomerism MeSH
• Publication type
• Journal Article MeSH

Affinity capillary electrophoresis (ACE) is typically used for the determination of stability constant, Kst, of weak to moderately strong complexes. Sensitive detection such as mass spectrometry (MS) is required for extension of ACE methodology for estimation of Kst of stronger complexes. Consequently, an efficient interface for hyphenation of CE with MS detection is necessary. For evaluation of interfaces for electrospray ionization mass spectrometric (ESI/MS) detection in ACE conditions, potassium-crown ether complexation was used as model system. The effective mobilities of the crown ether ligands and the Kst of their potassium complexes were measured/determined by ACE-ESI/MS using two lab-made interfaces: (i) a sheathless porous tip CE-ESI/MS interface and (ii) a nano-sheath liquid flow CE-ESI/MS interface, and, in turn, compared with those obtained by ACE with UV spectrophotometric detection. Apparent stability constant of potassium-crown ether complexes in 60/40 (v/v) methanol/water mixed solvent, pH* 5.5, was about 1300 L/mol for dibenzo-18-crown-6, 1600 L/mol for benzo-18-crown-6 and 5200 L/mol for 18-crown-6 ligands, respectively. It was observed that electrophoretic mobilities from CE-MS experiments differ from reference values determined by UV detection by ∼7% depending on the CE-MS interface used. Good agreement of CE-MS and CE-UV data was achieved for nano-sheath liquid flow interface, in which the spray potential and the CE separation potential can be effectively decoupled. As for sheathless porous tip interface, a correction procedure involving a mobility marker has been proposed. It provides typically only ca. 1% difference of effective mobilities and Kst values obtained from CE-MS data as compared to those received by the reference ACE-UV method.

• MeSH
• Crown Ethers chemistry   MeSH
• Electricity MeSH
• Electrophoresis, Capillary methods   MeSH
• Spectrometry, Mass, Electrospray Ionization methods   MeSH
• Kinetics MeSH
• Porosity MeSH
• Rheology * MeSH
• Solvents MeSH
• Water MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH

Ornithine-delta-aminotransferase (OAT, EC 2.6.1.13) catalyzes the transamination of L-ornithine to L-glutamate-gamma-semialdehyde. The physiological role of OAT in plants is not yet well understood. It is probably related to arginine catabolism resulting in glutamate but the enzyme has also been associated with stress-induced proline biosynthesis. We investigated the enzyme from pea (PsOAT) to assess whether diamines and polyamines may serve as substrates or they show inhibitory properties. First, a cDNA coding for PsOAT was cloned and expressed in Escherichia coli to obtain a recombinant protein with a C-terminal 6xHis tag. Recombinant PsOAT was purified under native conditions by immobilized metal affinity chromatography and its molecular and kinetic properties were characterized. Protein identity was confirmed by peptide mass fingerprinting after proteolytic digestion. The purified PsOAT existed as a monomer of 50 kDa and showed typical spectral properties of enzymes containing pyridoxal-5'-phosphate as a prosthetic group. The cofactor content of PsOAT was estimated to be 0.9 mol per mol of the monomer by a spectrophotometric analysis with phenylhydrazine. L-Ornithine was the best substrate (K(m)=15 mM) but PsOAT also slowly converted N(alpha)-acetyl-L-ornithine. In these reactions, 2-oxoglutarate was the exclusive amino group acceptor (K(m)=2mM). The enzyme had a basic optimal pH of 8.8 and displayed relatively high temperature optimum. Diamines and polyamines were not accepted as substrates. On the other hand, putrescine, spermidine and others represented weak non-competitive inhibitors. A model of the molecular structure of PsOAT was obtained using the crystal structure of human OAT as a template.

• MeSH
• Biocatalysis MeSH
• Chromatography, Affinity MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Pisum sativum enzymology   MeSH
• Molecular Sequence Data MeSH
• Molecular Weight MeSH
• Polyamines pharmacology   MeSH
• Recombinant Proteins antagonists & inhibitors   chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
• Substrate Specificity MeSH
• Ornithine-Oxo-Acid Transaminase antagonists & inhibitors   chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The total solute retention by a chemically modified stationary phase (CMSP) has been shown several times to be a potential tool for studying the binding abilities of the bound compound. In this article, we present a methodology for the deconvolution of the total retention into structure-specific contributions. Three complementary silica-based CMSPs were prepared: 1) non-modified silica, 2) silica modified by syn-bis-Tröger's base (a molecular tweezer) and 3) silica modified by anti-bis-Tröger's base (a non-tweezer molecule). These were characterized by elemental analysis and Raman spectroscopy, and used to assemble liquid chromatography (LC) columns. The total retention factors were estimated for electron-deficient nitro- and cyano-derivatives of benzene in both normal and reverse elution modes. The total retention factor was considered to be the sum of structure-specific retention factors, each related to the affinity (the binding constant) of a specific structure (the binding site), and its content in the modified silica, as defined for weak-affinity chromatography (WAC). The obtained structure-specific contributions are in line with the binding studies of ligands in solution. They reveal details of the retention mechanism, suggesting a more suitable attachment of ligands, and expose the shortcomings of evaluations based solely on the total retentions.

• MeSH
• Chromatography, Liquid methods   MeSH
• Ligands MeSH
• Silicon Dioxide chemistry   MeSH
• Solutions MeSH
• Stereoisomerism MeSH
• Publication type
• Journal Article MeSH

• Keywords
• elektroforéza kapilární,

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• fyzika, biofyzika
• biomedicínské inženýrství

Interaction between a host cell and pathogen is a permanent event and can have either adverse outcome leading to disease or great benefit for their mutual co-existence. Understanding pathological host-pathogen interaction is a prerequisite for unveiling the strategies of pathogens virulence. A number of methods exist today for deciphering and characterizing host-pathogen interaction. To increase their sensitivity and accuracy, these methods are commonly used in combinations, such as affinity purification and liquid chromatography-mass spectrometry analysis, cross-linking together with liquid chromatography-mass spectrometry analysis, or stable isotope labeling with amino acids in cell culture with affinity purification. In this review, we focus on study of the early interaction time interval when the pathogen binds and invades the host cell and activates sophisticated mechanisms to overcome the host defense barrier. We briefly describe the methods applied in identifying bacterial-host cell protein interactions while emphasizing these methods' various strengths and weaknesses.

• MeSH
• Host-Pathogen Interactions * MeSH
• Humans MeSH
• Disease Transmission, Infectious MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH

Efficient selective sample enrichment is often a key procedure in protocols for analyses of complex samples. This applies not only to trace sample components but also to species with weak detection response. For example enrichment of phosphopeptides using selective affinity techniques prior to mass spectrometry analysis is necessary to increase detection sensitivity of phosphopeptides from highly complex peptide mixtures. In this work we have developed inorganic nanofibrous materials based on titanium or zirconium dioxides for selective and efficient enrichment of phosphopeptides for MALDI/MS detection. In comparison to the common bead based materials the presented nanofibrous materials exhibit much higher permeability allowing their use not only for batch mode or packed in the column operation, but also in the pipette tip format without the need for high pressure. Both the methods of preparation and characterization of the resulting materials are presented.

• MeSH
• Phosphopeptides isolation & purification   MeSH
• Nanofibers * MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Titanium chemistry   MeSH
• Zirconium chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

"This book presents a clear and precise discussion of the biochemistry of eukaryotic cells, particularly those of mammalian tissues, relates biochemical events at a cellular level to the subsequent physiological processes in the whole animal, and cites examples of abnormal biochemical processes in human disease. The organization and content are tied together to provide students with the complete picture of biochemistry and how it relates to human diseases"--Provided by publisher.

• MeSH
• Biochemical Phenomena MeSH
• Publication type
• Textbook MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• Učební osnovy. Vyučovací předměty. Učebnice
• NML Fields
• biochemie

• MeSH
• Cell Biology MeSH
• Molecular Biology MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• biologie
• cytologie, klinická cytologie

"As the amount of information in biology expands dramatically, it becomes increasingly important for textbooks to distill the vast amount of scientific knowledge into concise principles and enduring concepts. As with previous editions, Molecular Biology of the Cell, Sixth Edition accomplishes this goal with clear writing and beautiful illustrations. The Sixth Edition has been extensively revised and updated with the latest research in the field of cell biology, and it provides an exceptional framework for teaching and learning. The entire illustration program has been greatly enhanced. Protein structures better illustrate structure-function relationships, icons are simpler and more consistent within and between chapters, and micrographs have been refreshed and updated with newer, clearer, or better images. As a new feature, each chapter now contains intriguing open-ended questions highlighting "What We Don't Know," introducing students to challenging areas of future research. Updated end-of-chapter problems reflect new research discussed in the text. Thought-provoking end-of-chapter questions have been expanded to all chapters, including questions on developmental biology, tissues and stem cells, the immune system, and pathogens"--Provided by publisher.

• MeSH
• Cells * MeSH
• Molecular Biology MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• molekulární biologie, molekulární medicína
• NML Publication type
• učebnice vysokých škol