János Szöllosi

Dynamic, yet structured: The cell membrane three decades after the Singer–Nicolson model

The fluid mosaic membrane model proved to be a very useful hypothesis in explaining many, but certainly not all, phenomena taking place in biological membranes. New experimental data show that the compartmentalization of membrane components can be as important for effective signal transduction as is the fluidity of the membrane. In this work, we pay tribute to the Singer–Nicolson model, which is near its 30th anniversary, honoring its basic features, “mosaicism” and “diffusion,” which predict the interspersion of proteins and lipids and their ability to undergo dynamic rearrangement via Brownian motion. At the same time, modifications based on quantitative data are proposed, highlighting the often genetically predestined, yet flexible, multilevel structure implementing a vast complexity of cellular functions. This new “dynamically structured mosaic model” bears the following characteristics: emphasis is shifted from fluidity to mosaicism, which, in our interpretation, means nonrandom codistribution patterns of specific kinds of membrane proteins forming small-scale clusters at the molecular level and large-scale clusters (groups of clusters, islands) at the submicrometer level. The cohesive forces, which maintain these assemblies as principal elements of the membranes, originate from within a microdomain structure, where lipid–lipid, protein–protein, and protein–lipid interactions, as well as sub- and supramembrane (cytoskeletal, extracellular matrix, other cell) effectors, many of them genetically predestined, play equally important roles. The concept of fluidity in the original model now is interpreted as permissiveness of the architecture to continuous, dynamic restructuring of the molecular- and higher-level clusters according to the needs of the cell and as evoked by the environment.

Lipopolysaccharide and ceramide docking to CD14 provokes ligand‐specific receptor clustering in rafts

The glycosylphosphatidylinositol‐anchored receptor CD14 plays a major role in the inflammatory response of monocytes to lipopolysaccharide. Here, we describe that ceramide, a constituent of atherogenic lipoproteins, binds to CD14 and induces clustering of CD14 to co‐receptors in rafts. In resting cells, CD14 was associated with CD55, the Fcγ‐receptors CD32 and CD64 and the pentaspan CD47. Ceramide further recruited the complement receptor 3 (CD11b/CD18) and CD36 into proximity of CD14. Lipopolysaccharide, in addition, induced co‐clustering with Toll‐like receptor 4, Fcγ‐RIIIa (CD16a) and the tetraspanin CD81 while CD47 was dissociated. The different receptor complexes may be linked to ligand‐specific cellular responses initiated by CD14.

Application of fluorescence resonance energy transfer in the clinical laboratory: Routine and research

Fluorescence resonance energy transfer (FRET) phenomenon has been applied to a variety of scientific challenges in the past. The potential utility of this biophysical tool will be revisited in the 21st century. The rapid digital signal processing in conjunction with personal computers and the wide use of multicolor laser technology in clinical flow cytometry opened an opportunity for multiplexed assay systems. The concept is very simple. Color-coded microspheres are used as solid-phase matrix for the detection of fluorescent labeled molecules. It is the homogeneous assay methodology in which solid-phase particles behave similarly to the dynamics of a liquid environment. This approach offers a rapid cost-effective technology that harnesses a wide variety of fluorochromes and lasers. With this microsphere technology, the potential applications for clinical flow cytometry in the future are enormous. This new approach of well-established clinically proven methods sets the stage to briefly review the theoretical and practical aspects of FRET technology. The review shows various applications of FRET in research and clinical laboratories. Combination of FRET with monoclonal antibodies resulted in a boom of structural analysis of proteins in solutions and also in biological membranes. Cell surface mapping of cluster of differentiation molecules on immunocompetent cells has gained more and more interest in the last decade. Several examples for biological applications are discussed in detail. FRET can also be used to improve the spectral characteristics of fluorescent dyes and dye combinations, such as the tandem dyes in flow and image cytometry and the FRET primers in DNA sequencing and polymerase chain reactions. The advantages and disadvantages of donor-acceptor dye combinations are evaluated. In addition, the sensitivity of FRET provides the basis for establishing fast, robust, and accurate enzyme assays and immunoassays. Benefits and limitations of FRET-based assays are thoroughly scrutinized. At the end of the paper we review the future of FRET methodology.

IS THERE A RELATIONSHIP BETWEEN CELL PHONE USE AND SEMEN QUALITY

This study was conducted to determine a possible relationship between regular cell phone use and different human semen attributes. The history-taking of men in our university clinic was supplemented with questions concerning cell phone use habits, including possession, daily standby position and daily transmission times. Semen analyses were performed by conventional methods. Statistics were calculated with SPSS statistical software. A total of 371 were included in the study. The duration of possession and the daily transmission time correlated negatively with the proportion of rapid progressive motile sperm (r = − 0.12 and r = − 0.19, respectively), and positively with the proportion of slow progressive motile sperm (r = 0.12 and r = 0.28, respectively). The low and high transmitter groups also differed in the proportion of rapid progressive motile sperm (48.7% vs. 40.6%). The prolonged use of cell phones may have negative effects on the sperm motility characteristics.

Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces. Quantitative evaluation of the transfer efficiency on a cell-by-cell basis.

A method has been developed for the determination of the efficiency (E) of the fluorescence resonance energy transfer between moieties on cell surfaces by use of a computer-controlled flow cytometer capable of dual wavelength excitation. The absolute value of E may be calculated on a single-cell basis. The analysis requires the measurement of samples stained with donor and acceptor conjugated ligands alone as well as together. In model experiments HK 22 murine lymphoma cells labeled with fluorescein-conjugated concanavalin A (Con A) and/or rhodamine conjugated Con A were used to determine energy transfer histograms. Using the analytic solution to energy transfer in two dimensions, a high surface density of Con A binding sites was found that suggests that the Con A receptor sites on the cell surface are to a degree preclustered . We call this technique flow cytometric energy transfer ( FCET ).

Hsp90 restrains ErbB-2/HER2 signalling by limiting heterodimer formation

ErbB‐2/HER2 is an oncogenic tyrosine kinase that regulates a signalling network by forming ligand‐induced heterodimers with several growth factor receptors of the ErbB family. Hsp90 and co‐chaperones regulate degradation of ErbB‐2 but not other ErbB members. Here, we report that the role of Hsp90 in modulating the ErbB network extends beyond regulation of protein stability. The capacity of ErbB‐2 to recruit ligand‐bound receptors into active heterodimers is limited by Hsp90, which is dissociated from ErbB‐2 following ligand‐induced heterodimerization. We show that Hsp90 binds a specific loop within the kinase domain of ErbB‐2, thereby restraining heterodimer formation and catalytic function. These results define a role for Hsp90 as a molecular switch regulating the ErbB signalling network by limiting formation of ErbB‐2‐centred receptor complexes.

Is semen quality affected by male body fat distribution

The aim of this study was to examine the relationship of semen parameters, sexual function‐related hormones and waist/hip ratio. Eighty‐one selected patients presenting with infertility were examined. Weight, height, waist circumference and hip circumference were measured, and reproduction‐related hormone levels were determined. Semen was analysed by conventional methods. Semen volume, sperm concentration, motility, total sperm count, total motile sperm cell number, rapid progressive motile sperm count and reproduction‐related hormone levels [follicle‐stimulating hormone, luteinizing hormone, prolactin, testosterone, 17β‐oestradiol and sexual hormone‐binding globulin (SHBG)]. Significant correlations were found: (i) weight, waist circumference and hip circumference versus testosterone level, SHBG level, and testosterone/17β‐oestradiol ratio; (ii) hip circumference versus sperm concentration; (iii) waist circumference and hip circumference versus sperm count, total motile sperm cell number and rapid progressive motile sperm count; (iv) weight versus total sperm count and total motile sperm cell number; (v) waist circumference and hip circumference versus prolactin level (positively) and SHBG (negatively); (vi) waist circumference and waist/hip ratio versus semen volume. It can be concluded that the waist/hip ratio is correlated with the reproductive hormone levels. Although both the waist circumference and hip circumference correlated with the semen characteristics, the waist/hip ratio did not.

Unraveling the Biologic and Clinical Complexities of HER2

It has been over 20 years since the discovery of the human epidermal growth factor receptor 2 (HER2), a tyrosine kinase receptor that is a potent oncoprotein in breast and other cancers and has become an opportune target for therapy. HER2 plays a critical role in normal development, forming homodimers or heterodimers with other HER family members and triggering downstream signaling cascades controlling proliferation, cell survival, and apoptosis. However, amplification of the HER2 gene in cancer cells results in overexpression of HER2 receptors on the cell surface, leading to excessive and dysregulated signaling. HER2-driven signaling also upregulates transcription factors that act on the HER2 promoter, increasing its expression. In breast cancer, HER2 is gene amplified in 20%-25% of primary tumors and is associated with a more aggressive phenotype and poorer prognosis. The key role HER2 plays in tumorigenesis makes it an ideal target for therapy. Trastuzumab, a monoclonal antibody against HER2, inhibits downstream signaling and has proven to be effective against HER2-overexpressing metastatic breast cancer both as a single agent and in combination with chemotherapy. Seminal clinical trial data also show that the use of adjuvant trastuzumab in combination with chemotherapy or as a single agent after chemotherapy significantly increases disease-free and overall survival. Lapatinib, a dual tyrosine kinase inhibitor against HER1 and HER2, has been approved in combination with capecitabine for HER2-overexpressing advanced or metastatic breast cancer, which has progressed following previous anthracycline, taxane, and trastuzumab therapy. Other HER2-targeting strategies are also under active investigation.

Die Hard: Are Cancer Stem Cells the Bruce Willises of Tumor Biology?

In recent years, an exponentially growing number of studies have focused on identifying cancer stem cells (CSC) in human malignancies. The rare CSCs could be crucial in controlling and curing cancer: through asymmetric division CSCs supposedly drive tumor growth and evade therapy with the help of traits shared with normal stem cells such as quiescence, self‐renewal ability, and multidrug resistance pump activity. Here, we give a brief overview of techniques used to confirm the stem cell‐like behavior of putative CSCs and discuss markers and methods for identifying, isolating, and culturing them. We touch on the limitations of each marker and why the combined use of CSC markers, in vitro and in vivo assays may still fail to identify all relevant CSC populations. Finally, the various experimental findings supporting and contradicting the CSC hypothesis are summarized. The large number of tumor types thus far with a subpopulation of uniquely tumorigenic and therapy resistant cells suggests that despite the unanswered questions and inconsistencies, the CSC hypothesis has a legitimate role to play in tumor biology. At the same time, experimental evidence supporting the established alternative theory of clonal evolution can be found as well. Therefore, a model that describes cancer initiation and progression should combine elements of clonal evolution and CSC theory.

Effect of body weight on sperm concentration in normozoospermic males

A total of 274 men (aged: 26 ± 4.9 years) with normozoospermia were enrolled into this study. Their body mass index (BMI: kg/m2) varied between 17 and 39. According to BMI, the patients were divided into four groups: Group 1: 17–20, Group 2: 20.1–25, Group 3: 25.1–30 and Group 4: 30.1–39. Twenty-nine subjects were found in the first, 96 in the second, 91 in the third and 58 men in the fourth group. Sperm concentration was significantly lower in the obese group (29 × 106/ml, p < 0.05) than in the group of BMI 17–20, 20–25 and 25–30. In advance, in the obese group, sperm count continuously decreased with aging. We conclude that obesity is associated with a lower sperm count in case of normozoospermia.