Hepatocellular carcinoma (HCC) is one of the most frequent causes of cancer-related deaths worldwide. We recently showed that pharmacologically induced lipotoxicity represents a promising therapeutic strategy for the treatment of HCC. Synthetic LXRα agonists induce the production of toxic saturated fatty acids in tumor cells. When combined with DFG-out Raf inhibitors, which block fatty acid desaturation by inducing proteasomal degradation of stearoyl-CoA desaturase (SCD1), LXRα activation can trigger lipotoxicity-induced cancer cell death. However, the clinical translation of this therapeutic strategy is limited by the lack of specific LXRα agonists for clinical use. Here, we have developed a series of promising maleimide LXR agonists with increased potency for LXRα and enhanced specificity. Our agonist frontrunner 40 shows high selectivity for LXRα and strong therapeutic efficacy in HCC organoids, therefore illustrating a strong potential for advancing this lipotoxic treatment strategy to clinical application.
- MeSH
- hepatocelulární karcinom * farmakoterapie metabolismus patologie MeSH
- jaterní receptor X * agonisté metabolismus MeSH
- lidé MeSH
- maleimidy * farmakologie chemie terapeutické užití MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory jater * farmakoterapie metabolismus patologie MeSH
- protinádorové látky * farmakologie chemie terapeutické užití chemická syntéza MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane-disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, that is, (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid, and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.
- MeSH
- buněčná membrána metabolismus chemie MeSH
- buněčné linie MeSH
- lidé MeSH
- maleáty chemie MeSH
- membránové mikrodomény metabolismus chemie MeSH
- membránové proteiny * chemie metabolismus MeSH
- peptidy * chemie MeSH
- polystyreny chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An advantageous alternative to the use of detergents in biochemical studies on membrane proteins are the recently developed styrene-maleic acid (SMA) amphipathic copolymers. In our recent study [1] we demonstrated that using this approach, most T cell membrane proteins were fully solubilized (presumably in small nanodiscs), while two types of raft proteins, GPI-anchored proteins and Src family kinases, were mostly present in much larger (>250 nm) membrane fragments markedly enriched in typical raft lipids, cholesterol and lipids containing saturated fatty acid residues. In the present study we demonstrate that disintegration of membranes of several other cell types by means of SMA copolymer follows a similar pattern and we provide a detailed proteomic and lipidomic characterization of these SMA-resistant membrane fragments (SRMs).
BACKGROUND: CD16 was previously suggested to be a new marker of basophils that is subject to downregulation by FcεRI crosslinking. Certain compounds, including supraoptimal concentrations of the PKC inhibitors, bisindolylmaleimides, decouple the release of granules containing CD203c, CD63 and histamine, and may thus help to identify the mechanisms related to the CD16 externalization. OBJECTIVE: We hypothesized that CD16 is differentially expressed on the surface of basophils in patients with birch pollen or insect venom allergy and is subject to a regulation in response to allergens. We also employed CD203c and CD63 externalization decoupling by bisindolylmaleimides. METHODS: We performed a basophil activation test coupled with CD16 and histamine detection using cells isolated from patients with allergy to birch pollen or insect venom and negative controls. We employed two PKC inhibitors, bisindolylmaleimide II and Ro 31-8220 at their supraoptimal concentrations and, after difficulties reproducing previously published data, we analyzed the fluorescence of these inhibitors alone. We identified the CD16 isoforms by sequencing nested RT-PCR amplicons from flow cytometry sorted basophils and by cleaving the CD16b GPI anchor using a phospholipase C. RESULTS: We provide the first evidence that CD16a is expressed as a surface antigen on a small subpopulation of human basophils in patients with respiratory and insect venom allergy, and this antigen shows increased surface expression following allergen challenge or FcεRI crosslinking. We rejected the apparent decoupling of the surface expression of basophil activation markers following the administration of bisindolylmaleimides. CONCLUSIONS & CLINICAL RELEVANCE: The inclusion of αCD16 in negative selection cocktails selects against a subset of basophils that are CD16+ or CD16dim . Using CD16dim basophils and unstained leucocytes, we show that previous studies with supraoptimal concentrations of bisindolylmaleimides are likely flawed and are not associated with the differential expression of CD203c and CD63.
- MeSH
- alergie imunologie patologie MeSH
- antigeny CD63 imunologie MeSH
- bazofily imunologie patologie MeSH
- dospělí MeSH
- fosfodiesterasy imunologie MeSH
- GPI-vázané proteiny imunologie MeSH
- indoly chemie MeSH
- jedy členovců toxicita MeSH
- kousnutí a bodnutí hmyzem imunologie patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- maleimidy chemie MeSH
- pyrofosfatasy imunologie MeSH
- receptory IgG imunologie MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid Lo phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different - the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.
- MeSH
- anizotropie MeSH
- buněčná membrána chemie MeSH
- cholesterol chemie MeSH
- detergenty chemie MeSH
- gelová chromatografie MeSH
- Jurkat buňky MeSH
- lidé MeSH
- lipidové dvojvrstvy chemie MeSH
- lipidy chemie MeSH
- maleáty chemie MeSH
- mastné kyseliny chemie MeSH
- membránové mikrodomény chemie MeSH
- membránové proteiny chemie MeSH
- membrány umělé MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- polymery chemie MeSH
- radiační rozptyl MeSH
- rozpustnost MeSH
- styren chemie MeSH
- světlo MeSH
- T-lymfocyty cytologie MeSH
- ultracentrifugace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Over the last decades, multidrug-resistant bacteria have emerged and spread, increasing the number of bacteria, against which commonly used antibiotics are no longer effective. It has become a serious public health problem whose solution requires medical research in order to explore novel effective antimicrobial molecules. On the one hand, antimicrobial peptides (AMPs) are regarded as good alternatives because of their generally broad-spectrum activities, but sometimes they can be easily degraded by the organism or be toxic to animal cells. On the other hand, cationic carbosilane dendrons, whose focal point can be functionalized in many different ways, have also shown good antimicrobial activity. In this work, we synthetized first- and second-generation cationic carbosilane dendrons with a maleimide molecule on their focal point, enabling their functionalization with three different AMPs. After different microbiology studies, we found an additive effect between first-generation dendron and AMP3 whose study reveals three interesting effects: (i) bacteria aggregation due to AMP3, which could facilitate bacteria detection or even contribute to antibacterial activity by preventing host cell attack, (ii) bacteria disaggregation capability of second-generation cationic dendrons, and (iii) a higher AMP3 aggregation ability when dendrons were added previously to peptide treatment. These compounds and their different effects observed over bacteria constitute an interesting system for further mechanism studies.
Directing the organization of cells into a tissue with defined architectures is one use of biomaterials for regenerative medicine. To this end, hydrogels are widely investigated as they have mechanical properties similar to native soft tissues and can be formed in situ to conform to a defect. Herein, we describe the development of porous hydrogel tubes fabricated through a two-step polymerization process with an intermediate microsphere phase that provides macroscale porosity (66.5%) for cell infiltration. These tubes were investigated in a spinal cord injury model, with the tubes assembled to conform to the injury and to provide an orientation that guides axons through the injury. Implanted tubes had good apposition and were integrated with the host tissue due to cell infiltration, with a transient increase in immune cell infiltration at 1 week that resolved by 2 weeks post injury compared to a gelfoam control. The glial scar was significantly reduced relative to control, which enabled robust axon growth along the inner and outer surface of the tubes. Axon density within the hydrogel tubes (1744 axons/mm2) was significantly increased more than 3-fold compared to the control (456 axons/mm2), with approximately 30% of axons within the tube myelinated. Furthermore, implantation of hydrogel tubes enhanced functional recovery relative to control. This modular assembly of porous tubes to fill a defect and directionally orient tissue growth could be extended beyond spinal cord injury to other tissues, such as vascular or musculoskeletal tissue. STATEMENT OF SIGNIFICANCE: Tissue engineering approaches that mimic the native architecture of healthy tissue are needed following injury. Traditionally, pre-molded scaffolds have been implemented but require a priori knowledge of wound geometries. Conversely, hydrogels can conform to any injury, but do not guide bi-directional regeneration. In this work, we investigate the feasibility of a system of modular hydrogel tubes to promote bi-directional regeneration after spinal cord injury. This system allows for tubes to be cut to size during surgery and implanted one-by-one to fill any injury, while providing bi-directional guidance. Moreover, this system of tubes can be broadly applied to tissue engineering approaches that require a modular guidance system, such as repair to vascular or musculoskeletal tissues.
- MeSH
- axony účinky léků patologie MeSH
- hydrogely farmakologie MeSH
- jizva patologie MeSH
- lokomoce účinky léků MeSH
- maleimidy chemie MeSH
- mikrosféry MeSH
- myelinová pochva účinky léků metabolismus MeSH
- myši inbrední C57BL MeSH
- neuroglie patologie MeSH
- polyethylenglykoly chemie MeSH
- polymerizace MeSH
- poranění míchy patologie patofyziologie MeSH
- poréznost MeSH
- reagencia zkříženě vázaná chemie MeSH
- regenerace nervu účinky léků MeSH
- tkáňové podpůrné struktury chemie MeSH
- zadní končetina účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
Electrophoretic focusing on inverse electromigration dispersion (EMD) gradient is a new analytical technique based on a unique separation principle where weak non-amphoteric ionogenic species are focused, separated and transported to the detector by an EMD profile of suitable properties. The present work extends the theoretical description of this method by introducing the concept of resolution and deriving the fundamental equation expressing resolution as function of basic system parameters. The results indicate that at constant current operation, resolution is proportional to the square root of time. For variable current regimes (e.g. constant voltage), the time variable is replaced by the product of electric current and passed electric charge. Computer simulations for a model pair of substances support the validity of the presented theory and confirm the theoretical conclusion that resolution can be increased by allowing longer electromigration of the gradient in terms of time or passed charge. The experimental example shown comprises an anionic electrolyte system based on maleic acid and 2,6-lutidine, combined with ESI-MS detection and operated in the reverse mode due to strong electroosmotic flow and ESI suction. The practical implementation of the proposed methodology is done by application of negative pressure at the inlet vial, resulting in very substantial resolution enhancement and baseline separation of otherwise unresolved substances. The performance and high sensitivity of the developed technique is demonstrated on the example of simultaneous analysis of four sulfonamides and three dichlorophenols in waters with limits of detection on the 1 nM level.
- MeSH
- chlorfenoly analýza MeSH
- elektroforéza kapilární MeSH
- elektrolyty chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací * MeSH
- limita detekce MeSH
- maleáty chemie MeSH
- pitná voda analýza MeSH
- pyridiny chemie MeSH
- sulfonamidy analýza MeSH
- tlak MeSH
- Publikační typ
- časopisecké články MeSH
The paper is focused on pilot study of effects of novel synthetic protein kinase inhibitors-maleimide derivatives in different concentrations on normal, transformed and multipotent cell lines. Influence on cell proliferation and morphological characteristics has been demonstrated. The chosen agents cause antiproliferative effect on transformed cells and are not cytotoxic to normal cell lines. Moreover, different maleimide derivatives' effects on multipotent cells in attached and floating states has been shown. Described results can be used for further research of the maleimide derivatives as antitumor agents.
- MeSH
- buňky cytologie účinky léků MeSH
- endoteliální buňky cytologie účinky léků MeSH
- fibroblasty cytologie účinky léků MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- inhibiční koncentrace 50 MeSH
- kultivované buňky MeSH
- lidé MeSH
- maleimidy chemie farmakologie MeSH
- mezenchymální kmenové buňky cytologie účinky léků MeSH
- MFC-7 buňky MeSH
- multipotentní kmenové buňky cytologie účinky léků MeSH
- pupečník cytologie MeSH
- transformované buněčné linie MeSH
- tvar buňky účinky léků MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
INTRODUCTION AND OBJECTIVE: In Italy, rabbits are frequently reared for meat production. The aim of the study was to find the seroprevalence of T. gondii and N. caninum parasites, and risk factors of infection in rabbit farms. MATERIAL AND METHODS: Blood samples from 260 apparently healthy breeding rabbits were collected on 13 commercial farms in Northern Italy. Rabbits were divided into categories according to age, number of births, breed, province and size of farm. Samples were tested for antibodies to T. gondii and N. caninum using the indirect fluorescence antibody test (IFAT); samples with a titre ≥ 50 were considered positive. RESULTS: Antibodies to T. gondii and N. caninum were found in 38 (14.6 %) and 3 (1.2 %) rabbits, respectively. A statistically significant difference (p-value ≤ 0.05) was found only in T. gondii prevalence among different rabbit breeds and provinces. CONCLUSION: Rabbits from Northern Italy are at risk of T. gondii and N. caninum infection; however, it is lower compared to seroprevalence noted in other animal species or in humans.
- MeSH
- kokcidióza epidemiologie parazitologie přenos veterinární MeSH
- králíci * MeSH
- maleimidy MeSH
- Neospora fyziologie MeSH
- prevalence MeSH
- rizikové faktory MeSH
- séroepidemiologické studie MeSH
- Toxoplasma fyziologie MeSH
- toxoplazmóza zvířat epidemiologie parazitologie přenos MeSH
- železnaté sloučeniny MeSH
- zvířata MeSH
- Check Tag
- králíci * MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Itálie MeSH
