crystallization protocol
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Galectin-4 is thought to play a role in the process of tumour conversion of cells of the alimentary tract and the breast tissue; however, its exact function remains unknown. With the aim of elucidating the structural basis of mouse galectin-4 (mGal-4) binding specificity, we have undertaken X-ray analysis of the N-terminal domain, CRD1, of mGal-4 in complex with lactose (the basic building block of known galectin-4 carbohydrate ligands). Crystals of CRD1 in complex with lactose were obtained using vapour-diffusion techniques. The crystals belong to tetragonal space group P42(1)2 with unit-cell parameters a = 91.1, b = 91.16, c = 57.10 A and preliminary X-ray diffraction data were collected to 3.2 A resolution. An optimized crystallization procedure and cryocooling protocol allowed us to extend resolution to 2.1 A. Structure refinement is currently under way; the initial electron-density maps clearly show non-protein electron density in the vicinity of the carbohydrate binding site, indicating the presence of one lactose molecule. The structure will help to improve understanding of the binding specificity and function of the potential colon cancer marker galectin-4.
- MeSH
- aminokyselinové motivy MeSH
- difrakce rentgenového záření MeSH
- financování organizované MeSH
- galektin 4 chemie metabolismus MeSH
- krystalizace MeSH
- laktosa chemie metabolismus MeSH
- ligandy MeSH
- myši MeSH
- nádorové biomarkery chemie metabolismus MeSH
- nádory tračníku chemie metabolismus MeSH
- peptidové fragmenty chemie metabolismus MeSH
- terciární struktura proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
Knowledge of the active pharmaceutical ingredient (API) solubility in a polymer is imperative for successful amorphous solid dispersion design and formulation but acquiring this information at storage temperature is challenging. Various solubility determination methods have been established, which utilize differential scanning calorimetry (DSC). In this work, three commonly used DSC-based protocols [i.e., melting point depression (MPD), recrystallization, and zero-enthalpy extrapolation (Z-EE)] and a method that we have developed called "step-wise dissolution" (S-WD) were analyzed. For temperature-composition phase diagram construction, two glass-transition temperature equations (i.e., those of Gordon-Taylor and Kwei) and three solid-liquid equilibrium curve modeling approaches [i.e., the Flory-Huggins model, an empirical equation, and the perturbed-chain statistical associating fluid theory (PC-SAFT) equation of state (EOS)] were considered. Indomethacin (IND) and Kollidon 12 PF (PVP K12) were selected as the API and polymer, respectively. An annealing time investigation revealed that the IND-PVP K12 dissolution process was remarkably faster than demixing, which contradicted previously published statements. Thus, the recrystallization method overestimated the solubility of IND in PVP K12 when a 2-h time of annealing was set as the benchmark. Likewise, the MPD and Z-EE methods overestimated the API solubility because of unreliable IND melting endotherm evaluation at lower API loadings and a relatively slow heating rate, respectively. When the experimental results obtained using the S-WD method (in conjunction with the Kwei equation) were applied to the PC-SAFT EOS, which was regarded as the most reliable combination, the predicted IND solubility in PVP K12 at T = 25 °C was approximately 40 wt %. When applicable, the S-WD method offers the advantage of using a limited number of DSC sample pans and API-polymer physical mixture compositions, which is both cost- and time-effective.
Nanocrystalline cerium dioxide is able to protect living cells from oxidative stress under the influence of various stress factors, in particular under the one of low temperatures. This study investigates the phase-structural transformations in aqueous solutions containing CeO2 nanoparticles (NPs) and their impact on the cryopreservation process. Differential scanning calorimetry and thermomechanical analysis were used to analyse the phase transitions in aqueous suspensions of CeO2 NPs and aqueous solutions of the cryoprotectant dimethyl sulfoxide (Me2SO) with CeO2 NPs. Various concentrations of CeO2 NPs were tested to observe their effects on the crystallization and melting behaviours. The addition of CeO2 NPs significantly altered the temperatures and enthalpies of melting and crystallization in water. Low concentrations of CeO2 NPs promoted crystallization, while higher concentrations inhibited it, reducing supercooling and recrystallization during thawing. In Me2SO solutions, CeO2 NPs raised the glass transition temperature and affected the recrystallization process, with higher concentrations leading to more pronounced vitrification and reduced recrystallization. We also investigated the regularities of the effect of CeO2 NPs on phase transitions in combined cryoprotective media with Ham's F12, fetal bovine serum and Me2SO, which can be used in future to design the cryopreservation protocols. In the complex media, CeO2 NPs decreased the metastability and altered eutectic crystallization patterns, indicating potential cryoprotective effects. In conclusion, CeO2 NPs modulate the thermophysical properties of cryoprotective solutions, enhancing vitrification and reducing recrystallization, which could improve cryopreservation efficiency. Optimizing NP concentrations is crucial for practical applications in cryopreservation.
- MeSH
- cer * chemie farmakologie MeSH
- diferenciální skenovací kalorimetrie * MeSH
- dimethylsulfoxid * chemie MeSH
- kryoprezervace * metody MeSH
- kryoprotektivní látky * chemie farmakologie MeSH
- krystalizace * MeSH
- nanočástice * chemie MeSH
- tranzitní teplota MeSH
- vitrifikace * účinky léků MeSH
- změna skupenství * MeSH
- Publikační typ
- časopisecké články MeSH
Schistosomiasis caused by parasitic blood flukes of the genus Schistosoma is a global health problem with over 200 million people infected. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated protease critical for digestion of host blood proteins as a source of nutrients. SmCB1 is a validated drug target, and inhibitors of SmCB1 represent promising anti-schistosomals. A comprehensive structural and functional characterization of SmCB1 provides a starting point for the rational design of selective and potent SmCB1 inhibitors. Here, we report optimized protocols for (1) the production of recombinant SmCB1 in the Pichia pastoris expression system and its purification, (2) the measurement of SmCB1 activity and inhibition in a kinetic fluorescence assay, and (3) the preparation and crystallization of SmCB1 in complex with a model vinyl sulfone inhibitor, and the determination of its crystal structure.
- MeSH
- aktivace enzymů MeSH
- elektroporace MeSH
- exprese genu MeSH
- genetické vektory metabolismus MeSH
- glykosylace MeSH
- kathepsin B antagonisté a inhibitory chemie izolace a purifikace metabolismus MeSH
- kinetika MeSH
- krystalizace MeSH
- mutace genetika MeSH
- rekombinantní proteiny izolace a purifikace metabolismus MeSH
- Saccharomycetales genetika MeSH
- Schistosoma mansoni enzymologie MeSH
- transformace genetická MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The advances in electron cryo-microscopy have enabled high-resolution structural studies of vitrified macromolecular complexes in situ by cryo-electron tomography (cryo-ET). Since utilization of cryo-ET is generally limited to the specimens with thickness < 500 nm, a complex sample preparation protocol to study larger samples such as single eukaryotic cells by cryo-ET was developed and optimized over the last decade. The workflow is based on the preparation of a thin cellular lamella by cryo-focused ion beam milling (cryo-FIBM) from the vitrified cells. The sample preparation protocol is a multi-step process which includes utilization of several high-end instruments and comprises sample manipulation prone to sample deterioration. Here, we present a workflow for preparation of three different model specimens that was optimized to provide high-quality lamellae for cryo-ET or electron diffraction tomography with high reproducibility. Preparation of lamellae from large adherent mammalian cells, small suspension eukaryotic cell line, and protein crystals of intermediate size is described which represents examples of the most frequently studied samples used for cryo-FIBM in life sciences.
- MeSH
- buňky ultrastruktura MeSH
- elektronová kryomikroskopie metody MeSH
- ionty MeSH
- makromolekulární látky ultrastruktura MeSH
- molekulární biologie metody MeSH
- odběr biologického vzorku metody MeSH
- proteiny ultrastruktura MeSH
- průběh práce MeSH
- reprodukovatelnost výsledků MeSH
- Saccharomyces cerevisiae ultrastruktura MeSH
- tomografie elektronová metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lectin-like transcript 1 (LLT1, gene clec2d) was identified to be a ligand for the single human NKR-P1 receptor present on NK and NK-T lymphocytes. Naturally, LLT1 is expressed on the surface of NK cells, stimulating IFN-γ production, and is up-regulated upon activation of other immune cells, e.g. TLR-stimulated dendritic cells and B cells or T cell receptor-activated T cells. While in normal tissues LLT1:NKR-P1 interaction (representing an alternative "missing-self" recognition system) play an immunomodulatory role in regulation of crosstalk between NK and antigen presenting cells, LLT1 is upregulated in glioblastoma cells, one of the most lethal tumors, where it acts as a mediator of immune escape of glioma cells. Here we report transient expression and characterization of soluble His176Cys mutant of LLT1 ectodomain in an eukaryotic expression system of human suspension-adapted HEK293S GnTI(-) cell line with uniform N-glycans. The His176Cys mutation is critical for C-type lectin-like domain stability, leading to the reconstruction of third canonical disulfide bridge in LLT1, as shown by mass spectrometry. Purified soluble LLT1 is homogeneous, deglycosylatable and forms a non-covalent homodimer whose dimerization is not dependent on presence of its N-glycans. As a part of production of soluble LLT1, we have adapted HEK293S GnTI(-) cell line to growth in suspension in media facilitating transient transfection and optimized novel high cell density transfection protocol, greatly enhancing protein yields. This transfection protocol is generally applicable for protein production within this cell line, especially for protein crystallography.
- MeSH
- buňky NK metabolismus MeSH
- disulfidy metabolismus MeSH
- DNA metabolismus MeSH
- glykosylace MeSH
- HEK293 buňky MeSH
- krystalizace MeSH
- lektiny typu C chemie izolace a purifikace metabolismus MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- multimerizace proteinu MeSH
- N-acetylglukosaminyltransferasy metabolismus MeSH
- polyethylenimin chemie MeSH
- polysacharidy metabolismus MeSH
- rozpustnost MeSH
- roztoky MeSH
- sbalování proteinů MeSH
- sekvence aminokyselin MeSH
- stabilita proteinů MeSH
- terciární struktura proteinů MeSH
- transfekce metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Some biologically active substances are unstable and poorly soluble in aqueous media, at the same time exhibiting low bioavailability. The incorporation of these biologically active compounds into the structure of a lipid-based lyotropic liquid crystalline phase or nanoparticles can increase or improve their stability and transport properties, subsequent bioavailability, and applicability in general. The aim of this short overview is (1) to clarify the principle of self-assembly of lipidic amphiphilic molecules in an aqueous environment and (2) to present lipidic bicontinuous cubic and hexagonal phases and their current biosensing (with a focus on electrochemical protocols) and biomedical applications.
- MeSH
- kapalné krystaly * chemie MeSH
- lipidy chemie MeSH
- nanočástice * chemie MeSH
- technologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The objectives of this study were to evaluate the relationships among urea and acetone content in cows' cervical mucus (CM), its crystallization type (CT) and sperm survival (SS) after timed AI. Samples of CM were collected from 192 Holstein cows treated by Ovsynch(®) protocol. Analysis of the urea and acetone content for identification of the metabolic status, the arborization test for evaluation of insemination timing and the short-term heat test of SS for assessment of its suitability as a biological matrix were performed. The data set was analyzed by the GLM procedure using SAS(®). The results documented the existence of substantial differences in individual response to the Ovsynch(®) protocol causing insemination of 55.2% cows at an inappropriate time. The urea content was found as a possible indicator of a cow's metabolism and/or of insemination timing, concentrations of less than 500 mg/L corresponded (P<0.05-0.01) to the cows' expected response to timed AI. The greater the urea content, the greater the proportion of cows inseminated at an inappropriate time. Effects (P<0.05-0.01) of CT, urea and acetone content on SS were determined. The greatest values of SS were detected in cows with an expected response to precisely timed oestrus documented by the corresponding CT. Greater values of urea (>260 mg/L) and acetone (>5mg/L) negatively affected SS as well (P<0.05-0.01). The results confirmed that the accuracy of insemination timing can be affected by the metabolism intensity, just as CM quality directly influences sperm survival.
- MeSH
- aceton chemie MeSH
- časové faktory MeSH
- cervikální hlen chemie MeSH
- cervix uteri fyziologie MeSH
- močovina chemie MeSH
- skot MeSH
- spermie fyziologie MeSH
- umělá inseminace veterinární MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mouse NKR-P1C(B6) receptor corresponding to NK1.1 alloantigen is one of the most widespread surface markers of mouse NK and NKT cells in C57BL/6 mice detected by monoclonal antibody PK136. Although functional studies revealed the ability of this receptor to activate both natural killing and production of cytokines upon antibody crosslinking, the ligand for NKR-P1C(B6) remains unknown. In order to initiate ligand identification, structural studies, and epitope mapping experiments, we developed a simple and efficient expression and purification protocol allowing to produce large amounts of pure soluble monomeric mouse NKR-P1C(B6). Our protein encompassed approximately half of the stalk region and the entire C-terminal globular ligand binding domain. The identity of protein that was devoid of N-terminal initiation methionine and had all three expected disulfides closed was confirmed using high resolution ion cyclotron resonance mass spectrometry. Protein produced into inclusion bodies in Escherichia coli was efficiently refolded into a unique three dimensional structure as confirmed by NMR using (1)H-(15)N-HSQC spectra of uniformly labeled protein. The exceptional purity of the protein should allow its crystallization and detailed structural investigations, and is a prerequisite for its use as a probe in ligand identification and antibody epitope mapping experiments.
- MeSH
- antigeny povrchové genetika imunologie izolace a purifikace metabolismus MeSH
- buněčná inkluze genetika metabolismus MeSH
- buňky NK imunologie metabolismus MeSH
- Escherichia coli MeSH
- exprese genu MeSH
- hmotnostní spektrometrie MeSH
- klonování DNA MeSH
- lektinové receptory NK-buněk - podrodina B genetika imunologie izolace a purifikace metabolismus MeSH
- ligandy MeSH
- molekulární sekvence - údaje MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- refolding proteinů MeSH
- rekombinantní proteiny genetika imunologie izolace a purifikace metabolismus MeSH
- rozpustnost MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Kolorektální karcinom patří ve vyspělých zemích mezi nejčastější nádorová onemocnění a Česká republika je celosvětově na špici v jeho výskytu. Roční incidence u nás přesahuje 7 000 osob ročně. Primárně je 20 % pacientů diagnostikováno ve stadiu IV a téměř u poloviny pacientů se v průběhu onemocnění vyskytnou metastázy. V posledních 15 letech onkologická léčba metastického onemocnění prodělala významný vývoj. Standardem se stala terapie fluorouracilem, jehož modulace leucovorinem vedla k prodloužení mediánu přežití u infúzních režimů až k 15 měsícům (symptomatická terapie 6–8 měsíců). Kombinace fluorouracilu s oxaliplatinou nebo irinotecanem vede k dalšímu prodloužení k 20 měsícům. Následné přidání biologické léčby (bevacizumab, cetuximab, panitumumab) posouvá medián nad 20 měsíců. Cetuximab je monoklonální chimérická IgG1 protilátka, která cíleně blokuje extracelulární část EGFR. V České republice je zaregistrován pro léčbu pacientů s generalizovaným kolorektálním karcinomem s EGFR pozitivitou a K-ras wild type v monoterapii nebo v kombinaci s chemoterapií po selhání terapie irinotecanem a oxaliplatinou. Ve 2 registračních studiích (EMR 62 202–007-BOND 1, IMCL CP02–9 923) vedl ke klinickému benefitu u více jak poloviny nemocných. Ve studii publikované Cunninghamem a spolupracovníky byli pacienti zařazeni do ramene s cetuximabem a irinotecanem anebo do ramene s cetuximabem samotným (PR byla dokumentována u 23 % pacientů, SD u 33 % léčených pacientů). Medián přežití byl prodloužen z 6,9 měsíců na 8,6 měsíců. Evropská léková agentura (EMEA) doporučila cetuximab k léčbě metastického kolorektálního karcinomu (s expresí EGFR, K-ras wild type) v kombinaci s chemoterapií do I. linie léčby na základě studie CRYSTAL. Letos v dubnu byly publikovány v JCO výsledky této studie, která potvrdila efekt cetuximabu v I. linii léčby K-ras wild type karcinomů kolorekta s efektem prodloužení přežití (medián OS = 23,5 versus 20,0 měsíců; hazard ratio (HR) = 0,796; P = 0,0093), PFS (medián PFS = 9,9 versus 8,4 měsíců; HR = 0,696; P = 0,0012) a četnost léčebných odpovědí (RR) (RR = 57,3 % versus 39,7 %; odds ratio = 2,069; P < 0,001) ve srovnání se samotným FOLFIRI.
Colorectal cancer in third-line treatment Colorectal cancer is among the most common malignancies in the developed countries and the Czech Republic is the leading country in terms of its incidence worldwide. The annual incidence in the Czech Republic exceeds 7 000 persons. Primarily, 20 % of patients are diagnosed with stage IV and nearly half of the patients develop metastases during the course of the disease. In the past 15 years, oncological treatment for metastatic disease has made significant progress. Treatment with fluorouracil has become a standard; its modulation with leucovorin has resulted in a prolonged median survival in infusion regimens of up to 15 months (6–8 months in symptomatic treatment). A combination of fluorouracil with oxaliplatin or irinotecan leads to a further extension up to 20 months. A subsequent addition of biological therapy (bevacizumab, cetuximab, panitumumab) pushes the median beyond 20 months. Cetuximab is a chimeric IgG1 monoclonal antibody which targets the extracellular domain of the EGFR. In the Czech Republic, it is registered for the treatment of patients with generalized colorectal cancer with EGFR positivity and K-ras wild type after a failed treatment with irinotecan. In two registration studies, it resulted in a clinical benefit in more than half of the patients (PR was documented in 23 % patients; SD in 33 % of the patients treated). The median survival increased from 6.9 months to 8.6 months. The European Medicines Agency (EMEA) has recommended cetuximab for the treatment of metastatic colorectal cancer (with EGFR expression, K-ras wild type) in combination with chemotherapy as the first-line treatment based on the CRYSTAL study. This year in April, the results of this study were published in the JCO and confirmed the effect of cetuximab in the first-line treatment of K-ras wild type colorectal cancer with an improved survival (median OS = 23.5 versus 20.0 months; hazard ratio (HR) = 0.796; P = 0.0093), PFS (median PFS = 9.9 versus 8.4 months; HR = 0.696; P = 0.0012) and response rate (RR) (RR = 57.3 % versus 39.7 %; odds ratio = 2.069; P < 0.001) compared to FOLFIRI alone.
- Klíčová slova
- XELOX, XELIRI, FOLFIRI, kanavitový krém,
- MeSH
- adenokarcinom farmakoterapie chirurgie radioterapie MeSH
- adjuvantní chemoterapie metody MeSH
- adjuvantní radioterapie metody MeSH
- chemoembolizace MeSH
- exantém chemicky indukované MeSH
- kamptothecin terapeutické užití MeSH
- kolorektální nádory farmakoterapie chirurgie radioterapie MeSH
- lidé středního věku MeSH
- lidé MeSH
- metastázy nádorů MeSH
- monoklonální protilátky aplikace a dávkování terapeutické užití toxicita MeSH
- nádory jater farmakoterapie sekundární MeSH
- přežití bez známek nemoci MeSH
- progrese nemoci MeSH
- protinádorové látky škodlivé účinky terapeutické užití MeSH
- protokoly protinádorové kombinované chemoterapie škodlivé účinky terapeutické užití MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
