The combination of chemical cross-linking and mass spectrometry is currently a progressive technology for deriving structural information of proteins and protein complexes. In addition, chemical cross-linking is a powerful tool for stabilizing macromolecular complexes for single particle cryo-electron microscopy. Broad pallets of cross-linking chemistry, currently available for the majority of cross-linking experiments, still rely on the amine-reactive N-hydroxysuccinimide esters targeting mainly N-termini and lysine side chains. These cross-linkers are divided into two groups: water soluble and water insoluble; and research teams prefer one or another speculating on the benefits of their choice. However, the effect of cross-linker polarity on the outcome of cross-linking reaction has never been studied. Herein, we use both polar (bis(sulfosuccinimidyl) glutarate) and non-polar (disuccinimidyl glutarate) cross-linkers and systematically investigated the impact of cross-linker hydrophobicity on resulting distance constraints, using bovine serum albumin as a model protein. SIGNIFICANCE: Even though the amine reactive BS2G and DSG cross-linkers have the same length of spacer and are based on N-hydroxysuccinimidic group, our data showed that each of them formed preferentially different cross-links. We demonstrated that the choice of cross-linker can have a significant impact on the output data for structural characterization of biomolecules. Using equimolar mixtures of DSG with d6-BS2G, and BS2G with d6-DSG, we established that the polar BS2G preferentially bound to polar regions of modified molecule, whereas non-polar DSG bound to hydrophobic regions. This phenomenon established that the mixture of polar and non-polar cross-linkers acted as an efficient tool for the determination of distance constraints in proteins.
BACKGROUND: A modified high performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of methotrexate (MTX) and its main metabolite 7-hydroxymethotrexate (7-OHMTX) and compared to the immunochemical fluorescence polarization immunoassay (FPIA2) determination of methotrexate. METHODS: Methotrexate was determined by HPLC with UV detection at 303 nm after precipitation of proteins with trichloroacetic acid. Fluorescence polarization immunoassays (FPIA2) of methotrexate were performed on the TDx FLx Immunoassay Analyzer. RESULTS: Our data indicate good correlation between methotrexate levels > 1 micromol/L determined by HPLC and FPIA2. (r = 0.94, Spearman correlation coefficient). However, concentrations of methotrexate < 1 micromol/L measured by fluorescence polarization immunoassay were overestimated. CONCLUSIONS: The concentration of MTX < 1 micromol/L are overestimated due to the cross reactivity with metabolites 7-OHMTX and 2,4-diamino-N10-methylpteroic acid (DAMPA). The cross reaction may affect the therapy and lead to relapse in children with acute lymphoblastic leukemia.
- MeSH
- Precursor Cell Lymphoblastic Leukemia-Lymphoma blood drug therapy MeSH
- Biotransformation MeSH
- Child MeSH
- Fluorescence Polarization Immunoassay MeSH
- Humans MeSH
- Methotrexate analogs & derivatives analysis blood pharmacokinetics MeSH
- Adolescent MeSH
- Young Adult MeSH
- Drug Monitoring methods MeSH
- Bone Neoplasms blood drug therapy MeSH
- Osteosarcoma blood drug therapy MeSH
- Antimetabolites, Antineoplastic blood pharmacokinetics MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
- Validation Study MeSH
Berberine (BBR), a small molecule protoberberine isoquinoline alkaloid, is easy to cross the blood-brain barrier and is a potential drug for neurodegenerative diseases. Here, we explored the role and molecular mechanism of BBR in Alzheimer's disease (AD) progression. Weighted gene co-expression network analysis (WGCNA) was conducted to determine AD pathology-associated gene modules and differentially expressed genes (DEGs) were also identified. GO and KEGG analyses were performed for gene function and signaling pathway annotation. Cell counting kit-8 (CCK8) assay was applied to analyze cell viability. Immunofluorescence (IF) staining assay was conducted to measure the levels of polarization markers. The production of inflammatory cytokines was analyzed by enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) level and mitochondrial membrane potential (MMP) were detected using a ROS detection kit and a MMP Detection Kit (JC-1), respectively. AD pathology-associated DEGs were applied for GO function annotation and KEGG enrichment analysis, and the results uncovered that AD pathology was related to immune and inflammation. Lipopolysaccharide (LPS) exposure induced the M1 phenotype of microglia, and BBR suppressed LPS-induced M1 polarization and induced microglia toward M2 polarization. Through co-culture of microglia and neuronal cells, we found that BBR exerted a neuro-protective role by attenuating the injury of LPS-induced HMC3 on SH-SY5Y cells. Mechanically, BBR switched the M1/M2 phenotypes of microglia by activating PI3K-AKT signaling. In summary, BBR protected neuronal cells from activated microglia-mediated neuro-inflammation by switching the M1/M2 polarization in LPS-induced microglia via activating PI3K-AKT signaling. Key words Alzheimer's Disease, Berberine, Microglia polarization, Neuroinflammation, PI3K-AKT signaling.
- MeSH
- Alzheimer Disease * metabolism drug therapy pathology MeSH
- Berberine * pharmacology therapeutic use MeSH
- Phosphatidylinositol 3-Kinases * metabolism MeSH
- Humans MeSH
- Microglia * drug effects metabolism MeSH
- Mice MeSH
- Neuroprotective Agents * pharmacology MeSH
- Cell Polarity drug effects MeSH
- Proto-Oncogene Proteins c-akt * metabolism MeSH
- Signal Transduction * drug effects MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Inflammation of adipose tissue in relation to atherosclerosis is currently widely studied in patients with advanced disease. However, data regarding polarization of adipose tissue and arterial wall macrophages and their mutual link in the early stages of atherosclerosis are scarce. The main aim of this cross-sectional study was to characterize macrophage subpopulations in arterial wall and adjacent adipose tissue; and to determine links between different subpopulations in a relatively healthy population living kidney donors. METHODS: The presence of cardiovascular risk factors was established in 68 living kidney donors. Macrophage polarization was analyzed by flow cytometry and confirmed by RT-PCR in samples of visceral adipose tissue, renal artery and adjacent perivascular adipose tissue collected during hand-assisted retroperitoneoscopic nephrectomy. RESULTS: CD14+CD16+CD36high macrophages were found only in adipose tissues and were strongly positively associated with several cardiovascular risk factors. The CD14+CD16+CD36low subpopulation was positively associated with the presence of several cardiovascular risk factors to a lesser extent in all studied tissues. In contrast, the proportion of CD14+CD16-CD36low macrophages was negatively linked to several cardiovascular risk factors and increased in subjects on statin therapy. The proportion of CD14+CD16+CD36low macrophages in perivascular, not visceral adipose tissue was associated with that of both macrophage subtypes in the arterial wall, suggesting a direct link between perivascular adipose tissue and the arterial wall. CONCLUSIONS: We confirmed the association of three macrophage subtypes in adipose tissue and arterial wall to the studied cardiovascular risk factors. Macrophage polarization in perivascular, but not visceral adipose tissue was linked to macrophage polarization in the arterial wall.
- MeSH
- Atherosclerosis * MeSH
- Humans MeSH
- Macrophages MeSH
- Cross-Sectional Studies MeSH
- Hydroxymethylglutaryl-CoA Reductase Inhibitors * MeSH
- Adipose Tissue MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
... Mann Speech accommodation theory 33 Social Consequences of Accommodating One\'s Style of Speech: A Cross-National ... ... Esser Group polarization 53 Group Polarization: A Critical Review and Meta-Analysis Daniel J. ... ... Isenberg 54 The Group as a Polarizer of Attitudes Serge Moscovici & Marisa Zavalloni VOLUME IV Intergroup ... ... Steele &Joshua Aronson Prejudice 64 Personality and Sociocultural Factors in Intergroup Attitudes: A Cross-National ... ... Stephan & Cookie White Stephan Contact hypothesis 68 The Extended Contact Effect: Knowledge of Cross-Group ...
Sage Benchmarks in psychology
1st ed. 4 sv.
Simultaneous application of polarization microscopy and dark field techniques has been used to study the internal structure of microbial cells. The dark field technique displays subtle cell structures like glowing objects on a dark background. In the polarizing microscope, cross polarizing filters along with the first-order quartz compensator and a rotary table show the maximum birefringence of the individual structures. The material containing microorganisms was collected in the villages of Sýkořice and Zbečno (Křivoklátsko Protected Landscape Area). The objects were studied in a laboratory microscope Carl Zeiss Jena type NfpK equipped with In Ph 160 basic body with variable dark field, special condenser with interchangeable diaphragm apertures, a rotary table, Meopta Praha polarizer, analyzer, first-order quartz compensator from LOMO Sankt Petersburg, and a digital Nikon D 70 DSLR camera. Three orders of microorganisms were studied: Siphonocladales, Chlorococcales, and Peritricha. Anisotropic structures in different amounts and sizes (e.g., granules and microfibrils) or in different configurations (e.g., cell walls or pellicle) have been found in all Protista organisms under study. Filamentous algae of the genus Cladophora (Cladophoraceae, Siphonocladales, Ulvophyceae) featured a strongly birefringent cell wall (shape birefringence) surrounded by less birefringent periphyton (microbial biofilm), at the edges of which cyanobacterial fibers could be recognized-a very important finding. The coccal algae of the genus Scenedesmus (Scenedesmataceae, Chlorococcales, Chlorophyceae) exhibited not only strongly birefringent granules, but also strongly birefringent microfibrils in the cytoplasm outside the strongly birefringent cell walls-very important finding. Of all the studied microorganisms, the weakest birefringence was shown in the surface membrane (pellicle) of the Vorticella (Vorticellidae, Peritricha, Ciliata). On the other hand, the ring of cilia on the top of the body had a somewhat stronger birefringence-an important finding. In conclusion, the dark field technique provides a high contrast image in the microscope and, if supplemented simultaneously by polarization microscopy, will allow us to partially infer the composition of the examined structures.
- MeSH
- Anisotropy MeSH
- Chlorophyta * MeSH
- Cytoplasm MeSH
- Birefringence MeSH
- Microscopy, Polarization MeSH
- Publication type
- Journal Article MeSH
Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface.
- MeSH
- Calorimetry, Differential Scanning MeSH
- Phosphorylcholine analogs & derivatives metabolism MeSH
- Humans MeSH
- Carbon-13 Magnetic Resonance Spectroscopy MeSH
- Mitochondrial Membranes metabolism MeSH
- Oxidation-Reduction MeSH
- Permeability MeSH
- bcl-2-Associated X Protein metabolism MeSH
- Proton Magnetic Resonance Spectroscopy MeSH
- Unilamellar Liposomes MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
A simultaneous application of negative phase contrast and polarization microscopy was used to study the internal structure of microbial cells. Negative phase contrast allowed us to display the fine cell structures with a refractive index of light approaching that of the environment, e.g., the cytoplasm, and converted an invisible phase image to a visible amplitude one. In the polarizing microscope, cross-polarizing filters, together with first-order quartz compensator and a turntable, showed maximum birefringence of individual structures. Material containing algae was collected in ponds in the villages Sýkořice and Zbečno (Protected Landscape Area Křivoklátsko). Objects were studied in a laboratory microscope (Carl Zeiss Jena, type NfpK), equipped with a basic body In Ph 160 with an exchangeable module Ph, LOMO St. Petersburg turntable mounted on a centering holder of our own construction and a Nikon D 70 digital SLR camera. Anisotropic granules were found only in the members of two orders of algae (Euglenales, Euglenophyceae and Chlorococcales, Chlorophyceae). They always showed strong birefringence and differed in both number and size. An important finding concerned thin pellicles in genus Euglena (Euglenales, Euglenophyceae) which exhibited weak birefringence. In genus Pediastrum (Chlorococcales, Chlorophyceae), these granules were found only in living coenobium cells. In contrast, dead coenobium cells contained many granules without birefringence-an important finding. Another important finding included birefringent lamellar structure of the transverse cell wall and weak birefringence of pyrenoids in filamentous algae of genus Spirogyra (Zygnematales, Conjugatophyceae). It was clearly displayed by the negative phase contrast and has not been documented by other methods. This method can also record the very weak birefringence of the frustule of a diatom of genus Pinnularia (Naviculales, Bacillariophyceae), which was further reinforced by the use of quartz compensator-an important finding. Simultaneous use of negative phase contrast and polarization microscopy allowed us to study not only birefringent granules of storage substances in microorganisms, but also the individual lamellae of the cell walls of filamentous algae and very thin frustule walls in diatoms. These can be visualized only by this contrast method, which provides a higher resolution (subjective opinion only) than other methods such as positive phase contrast or relief contrast.
- MeSH
- Anisotropy MeSH
- Cell Biology instrumentation MeSH
- Cell Wall chemistry MeSH
- Chlorophyta chemistry cytology MeSH
- Cytological Techniques methods MeSH
- Cytoplasm chemistry MeSH
- Birefringence MeSH
- Euglenida chemistry cytology MeSH
- Microscopy, Phase-Contrast * MeSH
- Microscopy, Polarization * MeSH
- Diatoms chemistry cytology MeSH
- Zygnematales chemistry cytology MeSH
- Publication type
- Journal Article MeSH
Mitochondria are crucial compartments of eukaryotic cells because they function as the cellular power plant and play a central role in the early stages of programmed cell death (apoptosis). To avoid undesired cell death, this apoptotic pathway is tightly regulated by members of the Bcl-2 protein family, which interact on the external surface of the mitochondria, i.e., the mitochondrial outer membrane (MOM), and modulate its permeability to apoptotic factors, controlling their release into the cytosol. A growing body of evidence suggests that the MOM lipids play active roles in this permeabilization process. In particular, oxidized phospholipids (OxPls) formed under intracellular stress seem to directly induce apoptotic activity at the MOM. Here we show that the process of MOM pore formation is sensitive to the type of OxPls species that are generated. We created MOM-mimicking liposome systems, which resemble the cellular situation before apoptosis and upon triggering of oxidative stress conditions. These vesicles were studied using (31)P solid-state magic-angle-spinning nuclear magnetic resonance spectroscopy and differential scanning calorimetry, together with dye leakage assays. Direct polarization and cross-polarization nuclear magnetic resonance experiments enabled us to probe the heterogeneity of these membranes and their associated molecular dynamics. The addition of apoptotic Bax protein to OxPls-containing vesicles drastically changed the membranes' dynamic behavior, almost completely negating the previously observed effect of temperature on the lipids' molecular dynamics and inducing an ordering effect that led to more cooperative membrane melting. Our results support the hypothesis that the mitochondrion-specific lipid cardiolipin functions as a first contact site for Bax during its translocation to the MOM in the onset of apoptosis. In addition, dye leakage assays revealed that different OxPls species in the MOM-mimicking vesicles can have opposing effects on Bax pore formation.
- MeSH
- Apoptosis physiology MeSH
- Calorimetry, Differential Scanning MeSH
- Escherichia coli MeSH
- Fluorescent Dyes MeSH
- Phospholipids metabolism MeSH
- Cardiolipins metabolism MeSH
- Humans MeSH
- Lipid Bilayers chemistry MeSH
- Mitochondrial Membranes metabolism MeSH
- Mitochondria metabolism MeSH
- Nuclear Magnetic Resonance, Biomolecular MeSH
- Oxidation-Reduction MeSH
- Oxidative Stress physiology MeSH
- Cell Membrane Permeability MeSH
- bcl-2-Associated X Protein metabolism MeSH
- Temperature MeSH
- Unilamellar Liposomes chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
