Atypical applications of transverse diffusion of laminar flow profiles methodology for in-capillary reactions in capillary electrophoresis.

Capillary electrophoresis (CE) is a powerful separation technique offering quick and efficient analyses in various fields of bioanalytical chemistry. It is characterized by many well-known advantages, but one, which is perhaps the most important for this application field, is somewhat overlooked. It is the possibility to perform chemical and biochemical reactions at the nL scale inside the separation capillary. There are two basic formats applicable for this purpose, heterogeneous and homogeneous. In the former, one reactant is immobilized onto a particle or monolithic support or directly on the capillary wall, and the other is injected. In the latter, the reactant mixing inside a capillary is based on electromigration or diffusion. One of the diffusion-based methodologies, termed Transverse Diffusion of Laminar Flow Profiles, is the subject of this review. Since most studies utilizing in-capillary reactions in CE focus on enzymes, which are being continuously and exhaustively reviewed, this review covers the atypical applications of this methodology, but still in the bioanalytical field. As can be seen from the demonstrated applications, they are not limited to reactions, but can also be utilized for other biochemical systems.

Polyphosphate and tyrosine phosphorylation in the N-terminal domain of the human mitochondrial Lon protease disrupts its functions.

Phosphorylation plays a crucial role in the regulation of many fundamental cellular processes. Phosphorylation levels are increased in many cancer cells where they may promote changes in mitochondrial homeostasis. Proteomic studies on various types of cancer identified 17 phosphorylation sites within the human ATP-dependent protease Lon, which degrades misfolded, unassembled and oxidatively damaged proteins in mitochondria. Most of these sites were found in Lon's N-terminal (NTD) and ATPase domains, though little is known about the effects on their function. By combining the biochemical and cryo-electron microscopy studies, we show the effect of Tyr186 and Tyr394 phosphorylations in Lon's NTD, which greatly reduce all Lon activities without affecting its ability to bind substrates or perturbing its tertiary structure. A substantial reduction in Lon's activities is also observed in the presence of polyphosphate, whose amount significantly increases in cancer cells. Our study thus provides an insight into the possible fine-tuning of Lon activities in human diseases, which highlights Lon's importance in maintaining proteostasis in mitochondria.

Contactless conductivity detector as a tool for improving universality and sensitivity of capillary electrophoresis-frontal analysis: Proof of concept.

Drug binding to plasma proteins influences processes such as liberation, adsorption, disposition, metabolism, and elimination of drugs, which are thus one of the key steps of a new drug development. As a result, the characterization of drug-protein interactions is an essential part of these time- and money-consuming processes. It is important to determine not only the binding strength and the stoichiometry of interaction, but also the binding site of a drug on a protein molecule, because two drugs with the same binding site can mutually affect free drug concentration. Capillary electrophoresis-frontal analysis with mobility shift affinity capillary electrophoresis is one of the most used affinity capillary electrophoresis methods for the characterization of these interactions. In this study, a well-known sensitivity problem of most capillary electrophoresis-frontal analyses using ultraviolet detection is solved by its combination with contactless conductivity detection, which provided sixfold lower limits of quantitation and detection. Binding parameters of the human serum albumin-salicylic acid model affinity pair were evaluated by this newly developed approach and by the classical approach with ultraviolet detection primarily used for their mutual comparison. The results of both approaches agreed well and are also in agreement with literature data obtained using different techniques.

Comparison of Fungal Thermophilic and Mesophilic Catalase-Peroxidases for Their Antioxidative Properties.

Catalase-peroxidases (KatGs) are unique bifunctional oxidoreductases that contain heme in their active centers allowing both the peroxidatic and catalatic reaction modes. These originally bacterial enzymes are broadly distributed among various fungi allowing them to cope with reactive oxygen species present in the environment or inside the cells. We used various biophysical, biochemical, and bioinformatics methods to investigate differences between catalase-peroxidases originating in thermophilic and mesophilic fungi from different habitats. Our results indicate that the architecture of the active center with a specific post-translational modification is highly similar in mesophilic and thermophilic KatG and also the peroxidatic acitivity with ABTS, guaiacol, and L-DOPA. However, only the thermophilic variant CthedisKatG reveals increased manganese peroxidase activity at elevated temperatures. The catalatic activity releasing molecular oxygen is comparable between CthedisKatG and mesophilic MagKatG1 over a broad temperature range. Two constructed point mutations in the active center were performed selectively blocking the formation of described post-translational modification in the active center. They exhibited a total loss of catalatic activity and changes in the peroxidatic activity. Our results indicate the capacity of bifunctional heme enzymes in the variable reactivity for potential biotech applications.

Sensitivity enhancement of capillary electrophoresis-frontal analysis-based method for characterization of drug-protein interactions using on-line sample preconcentration.

Capillary electrophoresis-frontal analysis is one of the most frequently used approaches for the study of plasma protein-drug interactions as a substantial part of new drug development. However, the capillary electrophoresis-frontal analysis typically combined with ultraviolet-visible detection suffers from insufficient concentration sensitivity, particularly for substances with limited solubility and low molar absorption coefficient. The sensitivity problem has been solved in this work by its combination with an on-line sample preconcentration. According to the knowledge of the authors this combination has never been used to characterize plasma protein-drug binding. It resulted in a fully automated and versatile methodology for the characterization of binding interactions. Further, the validated method minimalizes the experimental errors due to a reduction in the manipulation of samples. Moreover, employing an on-line preconcentration strategy with capillary electrophoresis-frontal analysis using human serum albumin-salicylic acid as a model system improves the drug concentration sensitivity 17-fold compared to the conventional method. The value of binding constant (1.51 ± 0.63) · 104 L/mol obtained by this new capillary electrophoresis-frontal analysis modification is in agreement with the value (1.13 ± 0.28) ·104 L/mol estimated by a conventional variant of capillary electrophoresis-frontal analysis without the preconcentration step, as well as with literature data obtained using different techniques.

Defining the Functional Interactome of Spliceosome-Associated G-Patch Protein Gpl1 in the Fission Yeast Schizosaccharomyces pombe.

Pre-mRNA splicing plays a fundamental role in securing protein diversity by generating multiple transcript isoforms from a single gene. Recently, it has been shown that specific G-patch domain-containing proteins are critical cofactors involved in the regulation of splicing processes. In this study, using the knock-out strategy, affinity purification and the yeast-two-hybrid assay, we demonstrated that the spliceosome-associated G-patch protein Gpl1 of the fission yeast S. pombe mediates interactions between putative RNA helicase Gih35 (SPAC20H4.09) and WD repeat protein Wdr83, and ensures their binding to the spliceosome. Furthermore, RT-qPCR analysis of the splicing efficiency of deletion mutants indicated that the absence of any of the components of the Gpl1-Gih35-Wdr83 complex leads to defective splicing of fet5 and pwi1, the reference genes whose unspliced isoforms harboring premature stop codons are targeted for degradation by the nonsense-mediated decay (NMD) pathway. Together, our results shed more light on the functional interactome of G-patch protein Gpl1 and revealed that the Gpl1-Gih35-Wdr83 complex plays an important role in the regulation of pre-mRNA splicing in S. pombe.

Highly flexible metabolism of the marine euglenozoan protist Diplonema papillatum.

BACKGROUND: The phylum Euglenozoa is a group of flagellated protists comprising the diplonemids, euglenids, symbiontids, and kinetoplastids. The diplonemids are highly abundant and speciose, and recent tools have rendered the best studied representative, Diplonema papillatum, genetically tractable. However, despite the high diversity of diplonemids, their lifestyles, ecological functions, and even primary energy source are mostly unknown. RESULTS: We designed a metabolic map of D. papillatum cellular bioenergetic pathways based on the alterations of transcriptomic, proteomic, and metabolomic profiles obtained from cells grown under different conditions. Comparative analysis in the nutrient-rich and nutrient-poor media, as well as the absence and presence of oxygen, revealed its capacity for extensive metabolic reprogramming that occurs predominantly on the proteomic rather than the transcriptomic level. D. papillatum is equipped with fundamental metabolic routes such as glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, respiratory complexes, ß-oxidation, and synthesis of fatty acids. Gluconeogenesis is uniquely dominant over glycolysis under all surveyed conditions, while the TCA cycle represents an eclectic combination of standard and unusual enzymes. CONCLUSIONS: The identification of conventional anaerobic enzymes reflects the ability of this protist to survive in low-oxygen environments. Furthermore, its metabolism quickly reacts to restricted carbon availability, suggesting a high metabolic flexibility of diplonemids, which is further reflected in cell morphology and motility, correlating well with their extreme ecological valence.

10-HDA, A Major Fatty Acid of Royal Jelly, Exhibits pH Dependent Growth-Inhibitory Activity Against Different Strains of Paenibacillus larvae.

Paenibacillus larvae (P. larvae) is a bacterial pathogen causing American foulbrood (AFB), the most serious disease of honeybee larvae. The food of young larvae could play an important role in the resistance of larvae against AFB. It contains antibacterial substances produced by honeybees that may inhibit the propagation of the pathogen in larval midguts. In this study, we identified and investigated the antibacterial effects of one of these substances, trans-10-hydroxy-2-decenoic acid (10-HDA), against P. larvae strains including all Enterobacterial Repetitive Intergenic Consensus (ERIC) genotypes. Its inhibitory activities were studied by determining the minimum inhibitory concentrations (MICs). It was found that 10-HDA efficacy increases substantially with decreasing pH; up to 12-fold differences in efficacy were observed between pH = 5.5 and pH = 7.2. P. larvae strains showed different susceptibility to 10-HDA; up to 2.97-fold differences existed among various strains with environmentally important ERIC I and ERIC II genotypes. Germinating spores of the pathogen were generally more susceptible to 10-HDA than vegetative cells. Our findings suggest that 10-HDA could play significant role in conferring antipathogenic activity to larval food in the midguts of young larvae and contribute to the resistance of individual larvae to P. larvae.

A novel photopolymerizable derivative of hyaluronan for designed hydrogel formation.

A new photopolymerizable derivative of hyaluronan (methacrylhydrazide-HA, MAHA) was prepared by carbodiimide chemistry. The reaction conditions were optimized for molecular weight (Mw), reaction time and amount of reagents with a degree of methacrylation (DM) ranging from 2% to 58%. Methacrylhydrazide-HA was hydrolytically stable (PBS, 7days, 37°C) in contrast to commonly used methacrylester analoque (23% hydrolyzed). MAHA readily photopolymerized into densely crosslinked hydrogels under physiological conditions. The varied DM, Mw, irradiation time (texp) and macromer concentration in photocrosslinking afforded hydrogels with different physical (swelling ratio, degradation rate) and mechanical properties (stiffness, toughness). Three-dimensional fabrication and surface patterning of MAHA hydrogels were demonstrated by photolithography and light mediated micromolding. A live-dead assay with skin fibroblasts showed convenient biocompatibility of MAHA (16%, 116kDa) for potential scaffolding applications in tissue engineering and regenerative medicine.

Honeybee glucose oxidase--its expression in honeybee workers and comparative analyses of its content and H2O2-mediated antibacterial activity in natural honeys.

Antibacterial properties of honey largely depend on the accumulation of hydrogen peroxide (H2O2), which is generated by glucose oxidase (GOX)-mediated conversion of glucose in diluted honey. However, honeys exhibit considerable variation in their antibacterial activity. Therefore, the aim of the study was to identify the mechanism behind the variation in this activity and in the H2O2 content in honeys associated with the role of GOX in this process. Immunoblots and in situ hybridization analyses demonstrated that gox is solely expressed in the hypopharyngeal glands of worker bees performing various tasks and not in other glands or tissues. Real-time PCR with reference genes selected for worker heads shows that the gox expression progressively increases with ageing of the youngest bees and nurses and reached the highest values in processor bees. Immunoblot analysis of honey samples revealed that GOX is a regular honey component but its content significantly varied among honeys. Neither botanical source nor geographical origin of honeys affected the level of GOX suggesting that some other factors such as honeybee nutrition and/or genetic/epigenetic factors may take part in the observed variation. A strong correlation was found between the content of GOX and the level of generated H2O2 in honeys except honeydew honeys. Total antibacterial activity of most honey samples against Pseudomonas aeruginosa isolate significantly correlated with the H2O2 content. These results demonstrate that the level of GOX can significantly affect the total antibacterial activity of honey. They also support an idea that breeding of novel honeybee lines expressing higher amounts of GOX could help to increase the antibacterial efficacy of the hypopharyngeal gland secretion that could have positive influence on a resistance of colonies against bacterial pathogens.

Methylglyoxal-induced modifications of significant honeybee proteinous components in manuka honey: Possible therapeutic implications.

Methylglyoxal (MGO) is a major antibacterial component of manuka honey. Another antibacterial component found in Revamil honey, peptide defensin1, was not identified in manuka honey. The primary aim of the study was to evaluate the content of defensin1 in honeys of different botanical origins and to investigate a presumed effect of reactive MGO on defensin1 and a dominant protein of honey MRJP1 in manuka honey. Immunoblotting of honey samples showed that defensin1 was a regular but quantitatively variable component of honeys. One of the reasons of varying contents of defensin1 in different honeys seems to be constitutive but varying defensin1 expression in individual honeybees in bee populations that we documented on samples of nurse and forager bees by RT-PCR. Comparative analyses of honeys revealed a size modification of defensin1, MRJP1 and probably also α-glucosidase in manuka honey. We further showed that (i) the treatment of purified defensin1 in solution containing high amount of MGO caused a time-dependent loss of its antibacterial activity and (ii) increasing MGO concentrations in a non-manuka honey were connected with a gradual increase in the molecular weight of MRJP1. Obtained results demonstrate that MGO abrogates the antibacterial activity of defensin1 and modifies MRJP1 in manuka honey. We assume that MGO could also have negative effects on the structure and function of other proteins/peptides in manuka honey, including glucose oxidase, generating hydrogen peroxide.