If Cell Mechanics Can Be Described by Elastic Modulus: Study of Different Models and Probes Used in Indentation Experiments

Here we investigated the question whether cells, being highly heterogeneous objects, could be described with the elastic modulus (effective Young’s modulus) in a self-consistent way. We performed a comparative analysis of the elastic modulus derived from the indentation data obtained with atomic force microscopy (AFM) on human cervical epithelial cells (both normal and cancerous). Both sharp (cone) and dull (2500-nm radius sphere) AFM probes were used. The indentation data were processed through different elastic models. The cell was approximated as a homogeneous elastic medium that had either 1), smooth hemispherical boundary (Hertz/Sneddon models) or 2), the boundary covered with a layer of glycocalyx and membrane protrusions (“brush” models). Consistency of these approximations was investigated. Specifically, we tested the independence of the elastic modulus of the indentation depth, which is assumed in these models. We demonstrated that only one model showed consistency in treating cells as a homogeneous elastic medium, namely, the brush model, when processing the indentation data collected with the dull AFM probe. The elastic modulus demonstrated strong depth dependence in all models: Hertz/Sneddon models (no brush taken into account), and when the brush model was applied to the data collected with sharp conical probes. We conclude that it is possible to describe the elastic properties of the cell body by means of an effective elastic modulus, used in a self-consistent way, when using the brush model to analyze data collected with a dull AFM probe. The nature of these results is discussed.     

Adult human retinal pigment epithelial cells - a potential source of cells for regeneration retina

Retinal pigment epithelium (RPE) arises from neuroectoderm and plays a key role in support of photoreceptor functions. Several degenerative eye diseases, such as macular degeneration or retinitis pigmentosa, are associated with impaired RPE function that may lead to photoreceptor loss and blindness. RPE cell culture derived from adult human eyes autopsy could be an important source for transplantation to cure such retinal degenerative diseases. RPE cells subsequent isolation and maintenance in culture are described. Besides the results of immunocytochemical analysis that characterizes dedifferentiated state of cultured adult human RPE cells are given. Our findings demonstrate that mature human RPE cells have the capacity to express neural markers in response to conditions that promote dedifferentiation.     

Ca/Mg-dependent endonuclease as a probe for detecting chromatin changes in lymphocytes in chronic lymphoid leukemia

Chromatin fragmentation of bovine peripheral blood lymphocytes from normal animals and the ones suffering from chronic lympholeucosis (CLL) by DNase I, micrococcal nuclease and purified Ca/Mg-dependent endonuclease from nuclei of human splenocytes was studied. The lymphocytes chromatin from CLL animals was shown to be more resistant to nucleases, than the one from normal animals. It was found that difference between fragmentation of chromatin samples from normal and CLL bovines was more dramatic when Ca/Mg- dependent endonuclease was used versus traditionally exploited DNase I and micrococcal nuclease. The data suggest that purified Ca/Mg-dependent endonuclease can be a useful enzymatic probe for detection of lymphocytes chromatin changes during CLL.     

The role of lung afferent system in respiratory control during head-down tilt

The role of lung receptors in respiratory control during acute head-down tilt (AHDT, -30 degrees) was investigated in anesthetized, tracheostomized rats. The results show that AHDT increased the mechanical respiratory load, slowed inspiratory flow, reduced the end expiratory lung volume, tidal volume and minute ventilation. On the other hand, during AHDT a significant rise in inspiratory swings of oesophageal pressure was recorded indicated a compensatory increase in inspiratory muscle contraction force. These effects were reduced after transaction of the vagus nerve. It was also shown that respiratory response on added mechanical load was reduced during AHDT as compared with the value in horizontal position. This deference disappeared after vagotomy. The data obtained suggested that afferent information from lung receptors take part in compensation of respiratory effects of AHDT. The cause of reduction in respiratory response to loading during AHDT involves weakness of lung reflexes evoked by volume changes.     

Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly ¹³C/¹⁵N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive ¹³C/¹⁵N-labeled amino acids. The most cost-effective production of ¹³C/¹⁵N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% ¹³C-glycerol and 0.5% ¹⁵N-ammonium sulfate, supplemented with only 0.025% of ¹³C/¹⁵N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state.     

Optimization of function of breath by means of training with additional resistive resistance

Research on studying influence of the rate of muscular training in conditions of additional resistive resistance to breath on a level of physical working capacity, aerobic productivity and a functional condition of system of breath was performed. It is shown, that regular muscular loadings in a combination to resistive resistance to breath in a mode of an interval exposition provide an increase of profitability and efficiency of functioning of breathing system as expressed in increase of expiratory force respiratory muscles, rationalization of parity of volumetric-time parameters of breath pattern that promotes more effective gas exchange and decrease in power cost of respiratory movements and as a result leads to increase in aerobic productivity and growth of the general and special working capacity. At continuous exposition of resistive resistance against a background of muscular training, the increase of inspiratory force of respiratory muscles, increase of functional capacity of system of breath were revealed at physical activity as well as a significant gain of the general physical working capacity.     

Germinal granules in interstitial cells of the colonial hydroids <Emphasis Type="Italic">Obelia longissima</Emphasis> pallas, 1766 and <Emphasis Type="Italic">Ectopleura crocea</Emphasis> Agassiz, 1862

Electron-dense germinal granules, which are usually regarded as markers and key organelles of germline cells, were revealed in the interstitial (stem) cells of the colonial hydroids Obelia longissima and Ectopleura crocea. The interstitial cells of O. longissima displayed intense alkaline phosphatase activity, a histochemical marker for vertebrate embryonic stem and primary germ cells, as well as positive reaction to proliferating cell nuclear antigen (PCNA), which is an immunochemical marker for cell reproduction. Our findings and the literature data suggest the evolutionary conservation and similarity of the morphological and functional organization of potentially gametogenic stem cells in asexually reproducing invertebrates and germ cells in all studied Metazoa. The self-renewing pool of such stem cells provides the cellular source for blastogenesis and gametogenesis and the cellular basis for life functions, including both asexual and sexual reproduction.     

Transmembrane adaptor protein WBP1L regulates CXCR4 signalling and murine haematopoiesis

WW domain binding protein 1‐like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6‐RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non‐haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4‐family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l‐deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis.     

The synthesis of imidodiphosphoric acid derivatives as potential substrates in the pyrophosphorolysis reaction

The preparation conditions for dichlorophosphinylphosphorimidic trichloride were optimized. It was used in the synthesis of esters of imidodiphosphoric acid. The interaction of the trichloride with amines resulted in the corresponding amidodiphosphates rather than in the expected amides of imidodiphosphoric acid.