Proteomic Analysis Reveals that Topoisomerase 2A is Associated with Defective Sperm Head Morphology

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Highlights

• •Human spermatozoa possess cells of poor morphology that lack nuclear integrity.
• •These cells can be isolated by density separation.
• •Mass spectrometry reveals their nuclei contain excess protein.
• •TOP2A is a promising marker of this poor nuclear development.

     

Membrane electrical parameters in turtle bladder measured using impedance-analysis techniques

Summary Equivalent-circuit impedance analysis experiments were performed on the urinary bladders of freshwater turtles in order to quantify membrane ionic conductances and areas, and to investigate how changes in these parameters are associated with changes in the rate of proton secretion in this tissue. In all experiments, sodium reabsorption was inhibited thereby unmasking the electrogenic proton secretion process. We report the following: (1) transepithelial impedance is represented exceptionally well by a simple equivalent-circuit model, which results in estimates of the apical and basolateral membrane ionic conductances and capacitances; (2) when sodium transport is inhibited with mucosal amiloride and serosal ouabain, the apical and basolateral membrane conductances and capacitances exhibit a continual decline with time; (3) this decline in the membrane parameters is most likely caused by subtle time-dependent changes in cell volume, resulting in changes in the areas of the apical and basolateral membranes; (4) stable membrane parameters are obtained if the tissue is not treated with ouabain, and if the oncotic pressure of the serosal solution is increased by the addition of 2% albumin; (5) inhibition of proton secretion using acetazolamide in CO₂ and HCO ₃ ^– -free bathing solutions results in a decrease in the area of the apical membrane, with no significant change in its specific conductance; (6) stimulation of proton transport with CO₂ and HCO ₃ ^– -containing serosal solution results in an increase in the apical membrane area and specific conductance. These results show that our methods can be used to measure changes in the membrane electrophysiological parameters that are related to changes in the rate of proton transport. Notably, they can be used to quantify in the live tissue, changes in membrane area resulting from changes in the net rates of endocytosis and exocytosis which are postulated to be intimately involved in the regulation of proton transport.     

Depicting the Spatial Distribution of Proteins in Human Tumor Tissue Combining SELDI and MALDI Imaging and Immunohistochemistry

Carcinoma tissue consists of not only tumor cells but also fibroblasts, endothelial cells or vascular structures, and inflammatory cells forming the supportive tumor stroma. Therefore, the spatial distribution of proteins that promote growth and proliferation in these complex functional units is of high interest. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a newly developed technique that generates spatially resolved profiles of protein signals directly from thin tissue sections. Surface-enhanced laser desorption/ionization mass spectrometry (MS)combined with tissue microdissection allows analysis of defined parts of the tissue with a higher sensitivity and a broader mass range. Nevertheless, both MS-based techniques have a limited spatial resolution. IHC is a technique that allows a resolution down to the subcellular level. However, the detection and measurement of a specific protein expression level is possible only by semiquantitative methods. Moreover, prior knowledge about the identity of the proteins of interest is necessary. In this study, we combined all three techniques to gain highest spatial resolution, sensitivity, and quantitative information. We used frozen tissue from head and neck tumors and chose two exemplary proteins (HNP1–3 and S100A8) to highlight the advantages and disadvantages of each technique. It could be shown that the combination of these three techniques results in congruent but also synergetic data. (J Histochem Cytochem 58:929–937, 2010)     

The intracellular concentration of cyclic adenosine 3',5'-monophosphate is constant throughout the cell cycle of Escherichia coli

Abstract Both the intracellular and the extracellular concentration of cyclic AMP increases logarithmically in synchronously growing cultures of Escherichia coli . Thus, cyclic AMP by itself cannot regulate growth and division of the bacterium during the cell cycle.     

Multifunctional roles of the autoimmune disease-associated tyrosine phosphatase PTPN22 in regulating T cell homeostasis

The non-receptor tyrosine phosphatase PTPN22 has a vital function in inhibiting antigen-receptor signaling in T cells, while polymorphisms in the PTPN22 gene are important risk alleles in human autoimmune diseases. We recently reported that a key physiological function of PTPN22 was to prevent naïve T cell activation and effector cell responses in response to low affinity antigens. PTPN22 also has a more general role in limiting T cell receptor-induced proliferation. Here we present new data emphasizing this dual function for PTPN22 in T cells. Furthermore, we show that T cell activation modulates the expression of PTPN22 and additional inhibitory phosphatases. We discuss the implication of these findings for our understanding of the roles of PTPN22 in regulating T cell responses and in autoimmunity.     

Investigating the effect of cell substrate on cancer cell stiffness by optical tweezers

The mechanical properties of cells are influenced by their microenvironment. Here we report cell stiffness alteration by changing the cell substrate stiffness for isolated cells and cells in contact with other cells. Polydimethylsiloxane (PDMS) is used to prepare soft substrates with three different stiffness values (173, 88 and 17 kPa respectively). Breast cancer cells lines, namely HBL-100, MCF-7 and MDA-MB-231 with different level of aggressiveness are cultured on these substrates and their local elasticity is investigated by vertical indentation of the cell membrane. Our preliminary results show an unforeseen behavior of the MDA-MB-231 cells. When cultured on glass substrate as isolated cells, they are less stiff than the other two types of cells, in agreement with the general statement that more aggressive and metastatic cells are softer. However, when connected to other cells the stiffness of MDA-MB-231 cells becomes similar to the other two cell lines. Moreover, the stiffness of MDA-MB-231 cells cultured on soft PDMS substrates is significantly higher than the stiffness of the other cell types, demonstrating thus the strong influence of the environmental conditions on the mechanical properties of the cells.     

Binding of B‐cell maturation antigen to B‐cell activating factor induces survival of multiple myeloma cells by activating Akt and JNK signaling pathways

B‐cell maturation antigen (BCMA) is expressed on normal and malignant plasma cells and represents a potential target for therapeutic intervention. In this study, we characterized the mechanism underlying the protein kinase B (Akt) and c‐Jun N‐terminal kinase (JNK) pathways and BCMA interactions in regulating multiple myeloma (MM) cell survival. It was found that the expression levels of B cell‐activating factor (BAFF) and BCMA were increased in MM cells as compared with those in normal controls. The proliferation of U266 cells was induced by recombinant human BAFF (rhBAFF) and could also be decreased by BCMA siRNA. The expression of Bcl‐2 protein was up‐regulated, and Bax protein was down‐regulated after rhBAFF treatment, which could be reversed by BCMA siRNA. Similarly, the protein p‐JNK and p‐Akt were activated by rhBAFF and could be changed by BCMA siRNA. In addition, the BCMA mRNA and protein expression levels were decreased after treatment with Akt and JNK pathway inhibitors. These results suggest that Akt and JNK pathways are involved in the regulation of BCMA. A novel BAFF/BCMA signalling pathway in MM may be a new therapeutic target for MM. Copyright © 2016 John Wiley & Sons, Ltd.     

Mechanical forces: The missing link between idiopathic pulmonary fibrosis and lung cancer

Patients with idiopathic pulmonary fibrosis (IPF) have a high risk of developing lung cancer compared with the general population. The morbidity of lung cancer in IPF patient ranges from 3% to 22%, and in some cases exceeds 50%, and these patients have a reduced survival time. However, the mechanisms through which IPF increases the morbidity and mortality in lung cancer remain unclear.By carefully analyzing the pathological features of these two diseases, we uncovered that, first, similar to IPF, lung carcinomas are more frequently found in the peripheral area of the lungs and, second, lung cancers tend to develop from the honeycomb areas in IPF. In accordance with the above pathological features, due to the spatial location, the peripheral areas of the lung experience a high stretch force because the average distance between adjacent alveolar cells in this area tends to be larger than that at the central lung when inflated; furthermore, the honeycomb areas, comprised of condensed fibrous tissue, are characterized by increased stiffness. Both of these pathological features of lung cancer and IPF are coincidentally related to abnormal mechanical forces (stretch and tissue stiffness). Therefore, we believe that the aberrant mechanical forces that are generated in the lung with IPF may contribute to the onset and progression of lung cancer.In this review, we discuss the possible effects of mechanical forces that are generated in IPF on the initiation and progression of lung cancer from the perspective of the hallmarks of cancer, including proliferation, metastasis, angiogenesis, cancer stem cells, immunology, epigenetics, and metabolism, so as to advance our understanding of the pathogenesis of IPF-related lung cancer and to harness these concepts for lung cancer mechanotherapies.     

Ammonium photo-production by heterocytous cyanobacteria: potentials and constraints

Over the last decades, production of microalgae and cyanobacteria has been developed for several applications, including novel foods, cosmetic ingredients and more recently biofuel. The sustainability of these promising developments can be hindered by some constraints, such as water and nutrient footprints. This review surveys data on N₂-fixing cyanobacteria for biomass production and ways to induce and improve the excretion of ammonium within cultures under aerobic conditions. The nitrogenase complex is oxygen sensitive. Nevertheless, nitrogen fixation occurs under oxic conditions due to cyanobacteria-specific characteristics. For instance, in some cyanobacteria, the vegetative cell differentiation in heterocyts provides a well-adapted anaerobic microenvironment for nitrogenase protection. Therefore, cell cultures of oxygenic cyanobacteria have been grown in laboratory and pilot photobioreactors (Dasgupta et al., 2010; Fontes et al., 1987; Moreno et al., 2003; Nayak & Das, 2013). Biomass production under diazotrophic conditions has been shown to be controlled by environmental factors such as light intensity, temperature, aeration rate, and inorganic carbon concentration, also, more specifically, by the concentration of dissolved oxygen in the culture medium. Currently, there is little information regarding the production of extracellular ammonium by heterocytous cyanobacteria. This review compares the available data on maximum ammonium concentrations and analyses the specific rate production in cultures grown as free or immobilized filamentous cyanobacteria. Extracellular production of ammonium could be coupled, as suggested by recent research on non-diazotrophic cyanobacteria, to that of other high value metabolites. There is little information available regarding the possibility for using diazotrophic cyanobacteria as cellular factories may be in regard of the constraints due to nitrogen fixation.