The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligand binding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards and selected urinary proteins from real samples. It has been shown that the affinity purification of trypsin decreases significantly the number of unmatched peptides in peptide mass fingerprints. The presence of arginine in the digestion buffer was found to reduce intensity of autolytic peptides. As a result, the described purification procedure is applicable in a common proteomic routine.
- MeSH
- chromatografie afinitní MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- mikrochemie metody MeSH
- peptidové mapování metody MeSH
- proteinové hydrolyzáty chemie MeSH
- proteinurie moč MeSH
- skot MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- trypsin izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nanomaterials are now produced in large quantities due to their useful properties different from other materials. Possible toxicity of nanomaterials is currently addressed and discussed. Using a modified Tollens process, an aqueous dispersion of Ag nanoparticles was prepared, on which their unique properties and changes of material characteristics when approaching nano dimensions can be demonstrated. Also, experiments regarding toxicity of nanoparticles were proposed. Toxicity of the nanoparticles was tested on Paramecium caudatum. Following the instruction, the students discover that the prepared Ag nanoparticles are much less toxic than ionic Ag. The proposed experiments bring interdisciplinary chemical, physical, and biological findings and knowledge.
- MeSH
- antiinfekční látky toxicita MeSH
- chemické techniky analytické metody trendy výchova MeSH
- financování organizované MeSH
- koloidy chemie toxicita MeSH
- lidé MeSH
- mikrochemie metody výchova MeSH
- nanočástice mikrobiologie toxicita využití MeSH
- nanotechnologie metody trendy výchova MeSH
- Paramecium caudatum účinky léků MeSH
- přírodní vědy metody trendy výchova MeSH
- stříbro toxicita MeSH
- testy toxicity metody využití MeSH
- výchova a vzdělávání metody MeSH
- Check Tag
- lidé MeSH
The lipid components of pathogen cell membranes have been considered as a poor pharmacological target, due to their universal distribution and apparent homogeneity throughout living organisms. Among the rare exceptions to this view one could mention polyene antibiotics such as amphotericin, or peptide antibiotics such as the polymyxins and the gramicidins. In the last two decades, however, the above notion has been challenged by two main lines of discovery; first, natural antimicrobial peptides (AMPs) that kill pathogens by interaction with phospholipids and membrane permeabilization, and secondly, cell-penetrating peptides (CPPs), capable of introducing into cells a variety of cargoes in the absence of specific receptors, again by interaction at some point with membrane phospholipids. For both AMPs and CPPs, the pharmacological proof-of-concept has been successfully demonstrated, and promising applications as nanobiotechnological tools have been envisaged though not hitherto materialized in clinical settings. In this review we briefly examine the pros and cons of these two classes of therapeutic agents, as well as strategies aimed at rationalizing and expanding their potentiality.
- MeSH
- antibiotická rezistence genetika imunologie MeSH
- financování organizované MeSH
- infekční nemoci farmakoterapie MeSH
- kationické antimikrobiální peptidy genetika účinky léků MeSH
- lidé MeSH
- lipopolysacharidy biosyntéza MeSH
- mikrochemie metody trendy MeSH
- peptidy genetika metabolismus účinky léků MeSH
- permeabilita buněčné membrány genetika imunologie účinky léků MeSH
- rostliny MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
Microparticles are an intensively studied field of pharmaceutical technology. Reduced side effects and new drug delivery methods are some of the advantages they offer. The aim of this study was to develop and optimize the preparation of Eudragit® RS 100 microparticles containing diclofenac sodium. The emulsification - solvent evaporation method is one of the most common and widely used microencapsulation techniques. The influence of few selected processes and formulation variables on the microparticle characteristics was investigated for the emulsion of methanol in liquid paraffin. The following conditions were selected: polymer-to-drug ratio 2:1, emulsification with 1% Span® 80 at 19000 rpm and 40 °C. The dissolution of the drug from microparticles was controlled by diffusion.
- MeSH
- aplikace orální MeSH
- diklofenak aplikace a dávkování farmakokinetika farmakologie MeSH
- elektronová mikroskopie metody využití MeSH
- emulze MeSH
- farmaceutický průmysl metody trendy MeSH
- gastrointestinální trakt účinky léků MeSH
- klinické laboratorní techniky přístrojové vybavení využití MeSH
- lékové formy MeSH
- léky s prodlouženým účinkem farmakokinetika farmakologie MeSH
- lidé MeSH
- methanol MeSH
- methylmetakryláty farmakokinetika farmakologie MeSH
- mikrochemie dějiny metody MeSH
- nosiče léků farmakokinetika farmakologie MeSH
- polymery terapeutické užití MeSH
- rozpustnost MeSH
- statistika jako téma MeSH
- Check Tag
- lidé MeSH
OBJECTIVES: Some individually-housed male mice behave aggressively during encounters with strange males, while others are timid or sociable in the same situation. The objective of the present study was to examine concentrations of glutamate, aspartate, and GABA in the brain of aggressive, timid, and sociable mice. METHODS: Random-bred albino mice were housed individually for three weeks and then classified in three groups (aggressive, timid, and sociable mice) according to their behavior during social interaction with non-aggressive group-housed male mice in a neutral cage. One week after categorization, by means of the social conflict test, levels of glutamate, aspartate, and GABA were measured by in vivo microdialysis of the medial prefrontal cortex (mPFC) of the isolated and group-housed mice. RESULTS: Sociable mice had almost triple the levels of GABA in their mPFC than aggressive or timid mice. No significant differences in aspartate and glutamate levels were found in these three types of individually-housed mice. Forebrain chemistry of group-housed mice did not differ from that of individually-housed mice with the exception of levels of glutamate and GABA which were significantly lower in group-housed mice than in sociable individually-housed mice. CONCLUSION: The present results suggest that GABA might play a role in sociable behavior. Results also corroborate other findings indicating that the GABAergic system represents an important molecular and neuronal substrate for the selective attenuation of anxiety and aggression.
- MeSH
- agrese fyziologie MeSH
- biologické modely MeSH
- chování zvířat fyziologie MeSH
- excitační aminokyseliny analýza metabolismus MeSH
- GABA analýza metabolismus MeSH
- kyselina asparagová analýza metabolismus MeSH
- kyselina glutamová analýza metabolismus MeSH
- mikrochemie metody MeSH
- mikrodialýza metody MeSH
- mozek metabolismus MeSH
- myši inbrední ICR MeSH
- myši MeSH
- plachost MeSH
- sociální chování MeSH
- sociální žádoucnost MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this setup, the part of capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. In the case of haloalkane dehalogenase, a model enzyme selected for this study, the enzymatic reaction was performed in 20 mM glycine buffer (pH 8.6) whereas 20 mM beta-alanine-hydrochloric acid buffer (pH 3.5) was used as a background electrolyte in combination with direct detection at 200 nm. The whole study was performed on poorly soluble brominated substrate--1,2-dibromoethane. As a result it was first necessary to find the compromise between the concentrations of the enzyme and the substrate preserving both the adequate sensitivity of the assay and at the same time the attainable substrate solubility. By means of the developed EMMA methodology we were able to determine the Michaelis constant (K(M)) as well as the substrate inhibition constant (K(SI)). The value of K(M) and K(SI) obtained were 7.7+/-2.5 mM and 1.1+/-0.4 mM, respectively. Observation of the substrate inhibition of haloalkane dehalogenase by 1,2-dibromoethane is in accordance with previous literature data.
elektronický časopis
- Konspekt
- Chemie. Mineralogické vědy
- NLK Obory
- chemie, klinická chemie
- chemie, klinická chemie
- NLK Publikační typ
- elektronické časopisy
