Novel Transmission Electron Microscopic Method to Quantify Protein Clustering on the Cell Surface.

The cell surface distribution patterns (clustering) of membrane proteins have been widely investigated in cell biology. Here we describe a novel transmission electron microscopic (TEM) protocol designed to improve the quality of information obtained about the protein distribution patterns detected. This novel method makes it possible to study the clustering of all transmembrane proteins on one half of the cytoplasmic membrane of a whole cell. To achieve better imaging, we combine various methods, including critical-point drying, fixation of gold beads with a carbon layer, and a newly developed chemical thinning method. In addition, in our image-processing algorithm, we implemented pair correlation and pair cross-correlation functions, providing more details and better quantitative accuracy in characterizing the size and numbers of possible protein clusters. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and transmission electron micrography Alternate Protocol: Direct cell labeling for transmission electron micrography Basic Protocol 2: Analysis of TEM images to detect immunogold-labeled proteins.

HLA DQ protein changes the cell surface distribution pattern of HLA proteins as monitored by Förster resonance energy transfer and high-resolution electron microscopy.

Peptide presentation by MHC class I and MHC class II molecules plays important roles in the regulation of the immune response. One factor in these displays is the density of antigen, which must exceed a critical threshold for the effective activation of T cells. Nonrandom distribution of MHC class I and class II has already been detected at the nanometer level and at higher hierarchical levels. It is not clear how the absence and reappearance of some protein molecules can influence the nonrandom distribution. Therefore, we performed experiments on HLA II-deficient bare lymphocyte syndrome (BLS1) cells: we created a stable transfected cell line, tDQ6-BLS-1, and were able to detect the effect of the appearance of HLA-DQ6 molecules on the homo and heteroassociation of different cell surface molecules by comparing Förster resonance energy transfer (FRET) efficiency on transfected cells to that on nontransfected BLS-1 and JY human B-cell lines. Our FRET results show a decrease in homoassociation FRET between HLA I chains in HLA-DQ6-transfected tDQ6-BLS-1 cells compared with the parent BLS-1 cell line and an increase in heteroassociation FRET between HLA I and HLA II (compared with JY cells), suggesting a similar pattern of antigen presentation by the HLA-DQ6 allele. Transmission electron microscopy (TEM) revealed that both HLA class I and class II molecules formed clusters at higher hierarchical levels on the tDQ6-BLS-1 cells, and the de novo synthesized HLA DQ molecules did not intersperse with HLA class I islands. These observations could be important in understanding the fine tuning of the immune response.

Diverse effect of BMP-2 homodimer on mesenchymal progenitors of different origin.

Bone morphogenetic protein-2 (BMP-2), is a potential factor to enhance osseointegration of dental implants. However, the appropriate cellular system to investigate the osteogenic effect of BMP-2 in vitro in a standardized manner still needs to be defined. The aim of this study was to examine the effect of BMP-2 on the cell proliferation and osteogenic differentiation of human osteogenic progenitors of various origins: dental pulp stem cells (DPSC), human osteosarcoma cell line (Saos-2) and human embryonic palatal mesenchymal cell line (HEPM). For induction of osteogenic differentiation, cell culture medium was supplemented with BMP-2 homodimer alone or in combination with conventionally used differentiation inducing agents. Differentiation was monitored for 6-18 days. To assess differentiation, proliferation rate, alkaline phosphatase activity, calcium deposition and the expression level of osteogenic differentiation marker genes (Runx2, BMP-2) were measured. BMP-2 inhibited cell proliferation in a concentration and time-dependent manner. In a concentration which caused maximal cell proliferation, BMP-2 did not induce osteogenic differentiation in any of the tested systems. However, it had a synergistic effect with the osteoinductive medium in both DPSC and Saos-2, but not in HEPM cells. We also found that the differentiation process was faster in Saos-2 than in DPSCs. Osteogenic differentiation could not be induced in the osteoblast progenitor HEPM cells. Our data suggest that in a concentration that inhibits proliferation the differentiation inducing effect of BMP-2 is evident only in the presence of permissive osteoinductive components. ß-glycerophosphate, was identified interacting with BMP-2 in a synergistic manner.

Understanding FRET as a research tool for cellular studies.

Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resolution are methods based on Förster Resonance Energy Transfer (FRET). FRET is effective at a distance of 1-10 nm which is equivalent to the size of macromolecules, thus providing an unprecedented level of detail on molecular interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a molecular complex in real-time making it possible to establish the functional significance of the studied molecules in a native environment. Now, FRET is widely used in biological sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochemical methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for determining the molecular heterogeneity of the plasma membrane in various cell types.

[Effect of BMP-2 treatment on the morphology and proliferation of human embryonic palatal derived mesenchymal preosteoblast cells]. / BMP-2 hatása a humán embrionális szájpadlásból származó mesenchymalis preosteoblast sejtek morfológiájára és proliferációjára.

In dental implantation missing tooth or teeth are replaced by artificial root. To reduce the time required for the integration newest trends are the enhancement of bone formation around the implant by bioactive molecules, growth factors. Such a molecule is bone morphogenetic protein-2 (BMP-2) accepted by US Food and Drug Administration (FDA). In these kind of applications effect of BMP-2 is tested in vitro on appropriate cell lines. One of these cell lines is the osteoblast like human embrionic palatal mesenchymal cell line (HEPM). In our experiments the effect of BMP-2 homodimer treatment was investigated on the differentiation of HEPM cells to osteoblasts reflected by changes in morphology, and proliferation after a short, 3 days BMP-2 treatment. Results showed that after three days BMP-2 treatment facilitates cell attachment on a concentration dependent manner however changes in cell morphology and proliferation could not be observed. Continuing the BMP-2 treatment inhibitory effect was measured in cell proliferation, which may refer to cell differentiation.

CD1d favors MHC neighborhood, GM1 ganglioside proximity and low detergent sensitive membrane regions on the surface of B lymphocytes.

BACKGROUND: Cluster of differentiation 1 (CD1) represents a family of proteins which is involved in lipid-based antigen presentation. Primarily, antigen presenting cells, like B cells, express CD1 proteins. Here, we examined the cell-surface distribution of CD1d, a subtype of CD1 receptors, on B lymphocytes. METHODS: Fluorescence labeling methods, including fluorescence resonance energy transfer (FRET),were employed to investigate plasma membrane features of CD1d receptors. RESULTS: High FRET efficiency was observed between CD1d and MHC I heavy chain (MHC I-HC), ß2-microglobulin(ß2m) and MHC II proteins in the plasma membrane. In addition, overexpression of CD1d reduced the expression of MHC II and increased the expression of MHC I-HC and ß2m proteins on the cell-surface. Surprisingly, ß2m dependent CD1d isoform constituted only ~15% of the total membrane CD1d proteins. Treatment of B cells with methyl-ß-cyclodextrin (MßCD) / simvastatin caused protein rearrangement; however, FRET demonstrated only minimal effect of these chemicals on the association between CD1d and GM1 ganglioside on cell-surface.Likewise, a modest effect was only observed in a co-culture assay between MßCD/simvastatin treated C1R­CD1d cells and invariant natural killer T cells on measuring secreted cytokines (IFNγ and IL4). Furthermore,CD1d rich regions were highly sensitive to low concentration of Triton X-100. Physical proximity between CD1d, MHC and GM1 molecules was also detected in the plasma membrane. CONCLUSIONS: An intricate relationship between CD1d, MHC, and lipid species was found on the membrane of human B cells. GENERAL SIGNIFICANCE: Organization of CD1d on the plasma membrane might be critical for its biological functions.

[Comperative study of implant surface characteristics]. / Implantátumok felületi sajátosságainak összehasonlító vizsgálata.

The osseointegration between the implant and its' bone environment is very important. The implants shall meet the following requirements: biocompatibility, rigidity, resistance against corrosion and technical producibility. In our present study surface morphology and material characteristics of different implants (Denti Bone Level, Denti Zirconium C, Bionika CorticaL, Straumann SLA, Straumann SLA Active, Dentsply Ankylos and Biotech Kontact implant) were investigated with scanning electron microscopy and energy-dispersive X-ray spectroscopy. The possible surface alterations caused by the manufacturing technology were also investigated. During grit-blasting the implants' surface is blasted with hard ceramic particles (titanium oxide, alumina, calcium phosphate). Properties of blasting material are critical because the osseointegration of dental implants should not be hampered. The physical and chemical features of blasting particles could importantly affect the produced surfaces of implants. Titanium surfaces with micro pits are created after immersion in mixtures of strong acids. On surfaces after dual acid-etching procedures the crosslinking between fibrin and osteogenetic cells could be enhanced therefore bone formation could be directly facilitated on the surface of the implant. Nowadays there are a number of surface modification techniques available. These can be used as a single method or in combination with each other. The effect of the two most commonly used surface modifications (acid-etching and grit-blasting) on different implants are demonstrated in our investigation.

Comparative study of the three different fluorophore antibody conjugation strategies.

The progression in bioconjugational chemistry has significantly contributed to the evolution and success of protein biology. Mainly, antibody chemistry has been a subject of intensive study owing to the expansion of research areas warranted by using various derivatives of conjugated antibodies. Three reactive moieties (amine, sulfhydryl and carbohydrate) in the antibodies are chiefly favored for the conjugational purpose. This feature is known for decades, nevertheless, amine based conjugation is still the most preferred strategy despite the appreciation the other two methods receive in conserving the antigen binding affinity (ABA). No single report has been published, according to our knowledge, where these three conjugation strategies were applied to the same fluorophore antibody systems. In this study, we evaluated conjugation yield, time demand and cost efficiency of these conjugation procedures. Our results showed that amine based conjugations was by far the best technique due to its simplicity, rapidity, ease of operation, higher conjugate yield, cheaper cost and potential for larger fluorophore/protein labeling ratio without having much effect in ABA. Furthermore, sulfhydryl labeling clearly excelled in terms of reduced non-specific binding and mild effect in ABA but was usually complicated by an asymmetric antibody reduction due to mercaptoethylamine while carbohydrate oxidation based strategy performed the worst during our experiment.

Angiotensin II increases the permeability and PV-1 expression of endothelial cells.

Angiotensin II (ANG II), the major effector molecule of the renin-angiotensin system (RAS), is a powerful vasoactive mediator associated with hypertension and renal failure. In this study the permeability changes and its morphological attributes in endothelial cells of human umbilical vein (HUVECs) were studied considering the potential regulatory role of ANG II. The effects of ANG II were compared with those of vascular endothelial growth factor (VEGF). Permeability was determined by 40 kDa FITC-Dextran and electrical impedance measurements. Plasmalemmal vesicle-1 (PV-1) mRNA levels were measured by PCR. Endothelial cell surface was studied by atomic force microscopy (AFM), and caveolae were visualized by transmission electron microscopy (TEM) in HUVEC monolayers. ANG II (10(-7) M), similarly to VEGF (100 ng/ml), increased the endothelial permeability parallel with an increase in the number of cell surface openings and caveolae. AT1 and VEGF-R2 receptor blockers (candesartan and ZM-323881, respectively) blunted these effects. ANG II and VEGF increased the expression of PV-1, which could be blocked by candesartan or ZM-323881 pretreatments and by the p38 mitogem-activated protein (MAP) kinase inhibitor SB-203580. Additionally, SB-203580 blocked the increase in endothelial permeability and the number of surface openings and caveolae. In conclusion, we have demonstrated that ANG II plays a role in regulation of permeability and formation of cell surface openings through AT1 receptor and PV-1 protein synthesis in a p38 MAP kinase-dependent manner in endothelial cells. The surface openings that increase in parallel with permeability may represent transcellular channels, caveolae, or both. These morphological and permeability changes may be involved in (patho-) physiological effects of ANG II.

Coating conditions matter to collagen matrix formation regarding von Willebrand factor and platelet binding.

INTRODUCTION: Von Willebrand factor (VWF) and platelet binding needs a uniform collagen matrix therefore we aimed to find an optimal condition for the preparation of human type-I and type-III collagen matrices. METHOD: The effects of pH, salt and ligand concentration and binding time were tested when collagen matrices were prepared by adsorption. Surface-bound collagen and collagen-bound VWF measured by specific antibodies. Platelet adhesion was tested under flow conditions at a shear rate of 1800s(-1) for 2 min. Matrices and platelets were visualized by atomic force and scanning electron microscope. RESULTS: The extent of human collagens type-I and III binding to the surface was 10 and 4 times greater and binding was maximal under 8-16 hours, when coated from physiological buffer solution versus acid solution. Collagen fibrils were more developed and platelet adhesion was higher, with more organized and denser aggregates. VWF binding was parallel to the surface bound collagen in both collagen types. CONCLUSION: Collagen coating of surfaces for VWF binding and platelet adhesion studies is very variable from acid solution. Our experiments provide evidences that neutralizing the acid and adding NaCl in physiological concentration, thereby facilitating formation of collagen fibril molecules in solution, results in efficient coating of human type-I and type III collagens, which then bind normal VWF equally well.

Bare lymphocyte syndrome: an opportunity to discover our immune system.

Bare lymphocyte syndrome (BLS) is a rare immunodeficiency disorder manifested by the partial or complete disappearance of major histocompatibility complex (MHC) proteins from the surface of the cells. Based on this specific feature, it is categorized into three different types depending on which type of MHC protein is affected. These proteins are mainly involved in generating the effective immune responses by differentiating 'self' from 'non-self' antigens through a process referred to as antigen presentation. Investigations on BLS have immensely contributed to our understanding of the transcriptional regulation of these molecules and have led to the discovery of several important proteins of the antigen presentation pathway. Reviews on this subject consistently project type II BLS, MHC II deficiency as BLS syndrome, although literatures' document cases of other types of BLS too. Therefore, in this article, we have assembled information on the BLS syndrome to produce a systematic narration while emphasizing the importance of BLS system in studying various aspects of immune biology.

Strength in numbers: effects of acceptor abundance on FRET efficiency.

Fluorescence resonance energy transfer (FRET) is a strongly distance-dependent process between a donor and an acceptor molecule, which can be used for sensitive distance measurements and characterization of molecular interactions at the nanometer level. The original mathematical description of this process, however, is only valid for the interaction of one donor with one acceptor. This criterion is not always met, especially in biological systems, where multiple structures can interact simultaneously, often making distance estimations based on transfer efficiency values error-prone. Herein we investigate how the interaction of multiple acceptors and donors influences the transfer efficiency value in an intramolecular cellular FRET system by manipulating the fluorophore/protein ratio of the fluorophore-conjugated antibodies. We show that the labeling ratio of the acceptor has the largest influence on measured transfer efficiency and decreasing or increasing the acceptor labeling ratio can be utilized to manipulate the FRET response of the acceptor-donor pair and therefore is a tool for optimizing sensitivity of FRET measurements.

Non-random distribution of interleukin receptors on the cell surface.

Spatial organization of cell surface proteins plays a key role in the process of transmembrane signalling. Receptor clustering and changes in their cell surface distribution are often determining factors in the final outcome of ligand-receptor interactions. There are several techniques for assessing the distribution of protein molecules. Fluorescence resonance energy transfer (FRET) is an excellent tool for determining distance relationships of cell surface molecules. However, it does not provide information on the distribution of molecular clusters. Different kinds of microscopies fill this gap. The evaluation of the images provided by the listed techniques is often questionable. Herein we show the applicability of Ripley's K(t) function as a tool for analyzing the cell surface receptor patterns (Y. Nakamura, et al., Nature 1994, 369, 330-333). We have implemented an effective image processing algorithm for fast localization of gold labels on biological samples. We investigated spatial organization of Interleukin-2R alpha and -15R alpha (IL-2R alpha and IL-15R alpha) on a human CD4+leukaemia T-cell line, Kit225 FT7.10 by using transmission electron microscopy (TEM). TEM analysis showed co-clustering of the two types of alpha-chains even on the few-hundred-nanometer scale. The analysis of our data may contribute to our understanding the action of the IL-2/IL-15 receptor system in T-cell function.

NMR spectroscopic elucidation of the structure and stereochemistry of tricyclic 3-styrylpyrazolines.

Diastereomeric mixtures of tricyclic 3-styrylpyrazolines have been prepared by the reaction of 3-cynnamylidenechroman-4-ones and their 1-thio analogs with hydrazine in hot acetic acid or propionic acid solutions. The diastereomeric mixtures were separated by column chromatography to obtain the pure diastereomers. The elucidation of their structure and stereochemistry and complete (1)H and (13)C assignments have been performed by a combination of various one- and two-dimensional NMR experiments.

DNA flow cytometry of human spermatozoa: consistent stoichiometric staining of sperm DNA using a novel decondensation protocol.

Rapid flow cytometric measurement of the frequency of aneuploid human sperms is in increasing demand but development of an exploitable method is hindered by difficulties of stoichiometric staining of sperm DNA. An aggressive decondensation protocol is needed after which cell integrity still remains intact. We used flow cytometry to examine the effect of lithium diiodosalicylate (LIS, chaotropic agent) on fluorescence intensity of propidium iodide-treated human spermatozoa from 10 normozoospermic men. When flow cytometric identification of diploid spermatozoa was achieved, validation was performed after sorting by three-color FISH. In contrast with the extremely variable histograms of nondecondensed sperms, consistent identification of haploid and diploid spermatozoa was possible if samples were decondensed with LIS prior to flow cytometry. A 76-fold enrichment of diploid sperms was observed in the sorted fractions by FISH. A significant correlation was found between the proportion of sorted cells and of diploid sperms by FISH. Application of LIS during the preparation of sperm for flow cytometry appears to ensure the stoichiometric staining of sperm DNA, making quantification of aneuploid sperm percentage possible. To our knowledge this is the first report in terms of separating spermatozoa with confirmedly abnormal chromosomal content. High correlation between the proportion of cells identified as having double DNA content by flow cytometry and diploid sperm by FISH allows rapid calculation of diploidy rate.

Nanometer-scale organization of the alpha subunits of the receptors for IL2 and IL15 in human T lymphoma cells.

Interleukin 2 and interleukin 15 (IL2 and IL15, respectively) provide quite distinct contributions to T-cell-mediated immunity, despite having similar receptor composition and signaling machinery. As most of the proposed mechanisms underlying this apparent paradox attribute key significance to the individual alpha-chains of IL2 and IL15 receptors, we investigated the spatial organization of the receptors IL2Ralpha and IL15Ralpha at the nanometer scale expressed on a human CD4+ leukemia T cell line using single-molecule-sensitive near-field scanning optical microscopy (NSOM). In agreement with previous findings, we here confirm clustering of IL2Ralpha and IL15Ralpha at the submicron scale. In addition to clustering, our single-molecule data reveal that a non-negligible percentage of the receptors are organized as monomers. Only a minor fraction of IL2Ralpha molecules reside outside the clustered domains, whereas approximately 30% of IL15Ralpha molecules organize as monomers or small clusters, excluded from the main domain regions. Interestingly, we also found that the packing densities per unit area of both IL2Ralpha and IL15Ralpha domains remained constant, suggesting a 'building block' type of assembly involving repeated structures and composition. Finally, dual-color NSOM demonstrated co-clustering of the two alpha-chains. Our results should aid understanding the action of the IL2R-IL15R system in T cell function and also might contribute to the more rationale design of IL2R- or IL15R-targeted immunotherapy agents for treating human leukemia.

Steady-state fluorescence quenching applications for studying protein structure and dynamics.

Fluorescence quenching methods are useful to obtain information about the conformational and/or dynamic changes of proteins in complex macromolecular systems. In this review steady-state methods are described and the data interpretation is thoroughly discussed. As a special case of fluorescence quenching mechanism, fluorescence resonance energy transfer (FRET) phenomenon is also presented. Application of a FRET based method to characterize the temperature dependence of the flexibility of protein matrix is clearly demonstrated.

Computer program for analyzing donor photobleaching FRET image series.

BACKGROUND: The photobleaching fluorescence resonance energy transfer (pbFRET) technique is a spectroscopic method to measure proximity relations between fluorescently labeled macromolecules using digital imaging microscopy. To calculate the energy transfer values one has to determine the bleaching time constants in pixel-by-pixel fashion from the image series recorded on the donor-only and donor and acceptor double-labeled samples. Because of the large number of pixels and the time-consuming calculations, this procedure should be assisted by powerful image data processing software. There is no commercially available software that is able to fulfill these requirements. METHODS: New evaluation software was developed to analyze pbFRET data for Windows platform in National Instrument LabVIEW 6.1. This development environment contains a mathematical virtual instrument package, in which the Levenberg-Marquardt routine is also included. As a reference experiment, FRET efficiency between the two chains (beta2-microglobulin and heavy chain) of major histocompatibility complex (MHC) class I glycoproteins and FRET between MHC I and MHC II molecules were determined in the plasma membrane of JY, human B lymphoma cells. RESULTS: The bleaching time constants calculated on pixel-by-pixel basis can be displayed as a color-coded map or as a histogram from raw image format. CONCLUSION: In this report we introduce a new version of pbFRET analysis and data processing software that is able to generate a full analysis pattern of donor photobleaching image series under various conditions. .

[In vitro study of the effects of 10-30 % hydrogen peroxide solutions on the surface of human tooth enamel]. / 10-30%-os hidrogén-peroxid humán fogzománcra gyakorolt hatásának in vitro vizsgálata.

The aim of the present study was to investigate the effects of hydrogen peroxide solutions (10-30%) on the enamel surface and the inner structure of human teeth. Prepared enamel samples were exposed to various concentrations of hydrogen peroxide solutions for one hour. Changes in the enamel were analyzed by two different methods. The inner structure was studied by the Ellman's reagent to detect possible changes in the amount of free thiol groups in the remaining organic phase of matured enamel. Surface alterations were imaged by atomic force microscope. Ellman's reaction revealed the presence of three groups of free thiols: one with easy, the second one with medium and the third one with difficult accessibility by hydrogen peroxide. Atomic force microscopy revealed severe alterations on the enamel surface after treatment with both low and high concentration of hydrogen peroxide. Our observations indicated that hydrogen peroxide induced alterations in the surface structure and in the inner phases of mature enamel in both low and high concentrations.

Computer program for determining fluorescence resonance energy transfer efficiency from flow cytometric data on a cell-by-cell basis.

The determination of fluorescence resonance energy transfer (FRET) with flow cytometry (FCET) is one of the most efficient tools to study the proximity relationships of cell membrane components in cell populations on a cell-by-cell basis. Because of the high amount of data and the relatively tedious calculations, this procedure should be assisted by powerful data processing software. The currently available programs are not able to fulfill this requirement. We developed a Windows-based program to calculate fluorescence resonance energy transfer efficiency values from list mode flow cytometry standard (FCS) files. This program displays the measured data in standard plots by generating one- and two-parameter histograms on linear or logarithmic scales. A graphical gating tool allows the user to select the desired cell population according to any combination of the parameter values. The program performs several statistical calculations, including mean, S.D., percent of the gated data. We have implemented two types of data sheet for FRET calculations to aid and guide the user during the analysis: one with population-mean-based autofluorescence correction and the other with spectrum-based cell-by-cell autofluorescence correction. In this paper, we describe the gating algorithms, the file opening procedure and the rules of gating. The structure of the program and a short description of the graphical user-interface (GUI) are also presented in this article.