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EN
In this paper, a new method is described for the horizontal electrophoresis of cells on a density cushion under near-isopycnic conditions. When cell sedimentation is minimized, the electrophoresis of red blood cells (RBC) used as model cells within an anti-convective porous matrix (with pores over 300 μm in diameter) was capable of separating a mixture of human and chicken RBC according to their electrophoretic mobilities. Samples taken from the separated RBC bands show over 90% purity for each species. The simultaneous electrophoresis of several RBC samples carried out under identical conditions permitted the use of comparative data based on the electrophoretic mobility of cells which differ in their surface properties. We believe that this relatively simple system, in which cell sedimentation and convection are minimized, has the potential to be modified and adapted for the separation of other cell types/organelles.
EN
The lacrimal gland (LG) is an exocrine gland important for secretion of the tear film. The kinase p38 has important signal transduction functions, e.g. in gene transcription, but has previously not been known to modulate exocrine secretion. The aim of the current study was to investigate the role of p38 in carbachol (Cch)-induced LG secretion in LG acinar cells in vitro. Western blotting was used to determine the phosphorylation status of p38 and p42/44 and determine expression of p38 isoforms. To determine the effect of p38 inhibition on LG secretion, PD 169316, a general p38 inhibitor, and SB 239063, an inhibitor of p38α and β, were added to the cells prior to secretion measurements. The results revealed activation of p38 mediated by Cch stimulation and inhibition of Cch-induced secretion as a result of p38 inhibition. The inhibition was observed with PD 169316 isoforms, but not with SB 239063. The p38δ isoform was shown to have robust expression both by Western blotting of acinar cells and immunofluorescence of the whole gland. In conclusion, p38 activation mediates secretion in cholinergic stimulation of rabbit LG cells.
EN
The number of different cell types (NCT) characterizing an organism is often used to quantify organismic complexity. This method results in the tautology that more complex organisms have a larger number of different kinds of cells, and that organisms with more different kinds of cells are more complex. This circular reasoning can be avoided (and simultaneously tested) when NCT is plotted against different measures of organismic information content (e.g., genome or proteome size). This approach is illustrated by plotting the NCT of representative diatoms, green and brown algae, land plants, invertebrates, and vertebrates against data for genome size (number of base-pairs), proteome size (number of amino acids), and proteome functional versatility (number of intrinsically disordered protein domains or residues). Statistical analyses of these data indicate that increases in NCT fail to keep pace with increases in genome size, but exceed a one-to-one scaling relationship with increasing proteome size and with increasing numbers of intrinsically disordered protein residues. We interpret these trends to indicate that comparatively small increases in proteome (and not genome size) are associated with disproportionate increases in NCT, and that proteins with intrinsically disordered domains enhance cell type diversity and thus contribute to the evolution of complex multicellularity.
EN
Mitochondrial dysfunction plays a crucial role in cell types that exhibit necrosislike death after activation of their death program. Tumour necrosis factor (TNF) induces abnormal, perinuclear clustering of mitochondria from an evenly spread distribution throughout the cytoplasm. The mitochondria withdraw from the cell periphery and aggregate in a unipolar perinuclear cluster. TNF-induced mitochondrial clustering is caused by impaired kinesin-mediated transportation of mitochondria. In this report, we describe a novel activity of menadione (MEN), namely the induction of an altered spatial distribution of mitochondria in the choriocarcinoma JAR cells. Strikingly, 2 hours of cell exposition to menadione did not disrupt the integrity of the plasma membrane, while the intracellular ATP level significantly decreased. Control (untreated) cells displayed a typically scattered distribution of filamentary mitochondria inside the cell. After 2 hours of MEN treatment the spatial distribution of the mitochondria was markedly altered to an asymmetric perinuclear clustered distribution. Menadione-stressed cells displayed a highly asymmetrical perinuclear clustered distribution of the mitochondria. The exposure of cells to MEN also results in a change in shape of the mitochondria into a population of enlarged granular structures. The results of our study demonstrate that in JAR cells menadione causes mitochondria to translocate from the cell periphery into the perinuclear region several hours before disruption of cell membrane integrity and cell death.
EN
Whereas several reports describing the ultrastructure of the intact pancreatic islets have been recorded, published experience with the ultrastructural integrity of the cultured pancreatic islets is limited. The present study was, therefore, undertaken to provide an ultrastructure identification of the different cells in the cultured islets of the adult rat pancreas, after marking their secretory granules with gold particles. Pancreatic islets were isolated from adult male Wistar rats by the intraductal perfusion of collagenase technique. The islets were cultured in RPMI-1640 medium for 3 days and processed for preparation of ultrathin sections. The sections were stained with the indirect immunogold technique for insulin, glucagon, somatostatin, and pancreatic polypeptide. Ultrastructural examination of the cultured islets clearly identified the presence of B, A, D and PP-cells, as indicated by the numerous gold particles concentrated predominantly over the secretory granules. The secretory granules of the various cell types of the cultured islets demonstrated several similarities as well as differences from the recorded results of the corresponding secretory granules of the intact islets. The differences probably reflect a deviation in the underlying mechanisms of synthesis, maturation and secretion of the different secretory products of the cells in the cultured islets as they adapt to the in vitro environment.
EN
Stomatal guard cells are highly differentiated cell types within the epidermis of higher plant leaves. These cells are intimately involved in regulating gas exchange, i.e. the release of water and the uptake of CO2, through the leaf surface. Guard cells represent an interesting cell type since they respond to various plant internal (e.g. hormones) and external (e.g. humidity, light, CO2) signals in a relatively simple manner. Stomatal pore size is changed by modulating the level of osmotically active compounds within the guard cells. In the past, guard cells have mainly been studied using electrophysiological, biochemical and whole-plant techniques. Only recently molecular techniques have been applied to address questions regarding control mechanisms of stomatal functioning. In the following a short overview is given on these molecular approaches.
EN
CC chemokine receptor 5 (CCR5) is a pro-inflammatory chemokine receptor that is expressed on cells of the immune system, and specializes in cell migration in response to inflammation and tissue damage. Due to its key role in cell communication and migration, this receptor is involved in various inflammatory and autoimmune diseases, in addition to HIV infection. Met-RANTES is a modified CCR5 ligand that has previously been shown to antagonize CCR5 activation and function in response to its natural ligands in vitro. In vivo, Met-RANTES is able to reduce inflammation in models of induced inflammatory and autoimmune diseases. However, due to the fact that Met-RANTES is also capable of partial agonist activity regarding receptor signaling and internalization, it is clear that Met-RANTES does not function as a conventional receptor antagonist. To further elucidate the effect of Met-RANTES on CCR5, receptor trafficking was investigated in a CHO-CCR5-GFP cell line using the Opera confocal plate reader. The internalization response of CCR5 was quantified, and showed that Met-RANTES internalized CCR5 in a slower, less potent manner than the agonists CCL3 and CCL5. Fluorescent organelle labeling and live cell imaging showed CCL3 and CCL5 caused CCR5 to traffic through sorting endosomes, recycling endosomes and the Golgi apparatus. In contrast, Met-RANTES caused CCR5 to traffic through sorting endosomes and the Golgi apparatus in a manner that was independent of recycling endosomes. As receptor trafficking impacts on cell surface expression and the ability of the receptor to respond to more ligand, this information may indicate an alternative regulation of CCR5 by Met-RANTES that allows the modified ligand to reduce inflammation through stimulation of a pro-inflammatory receptor.
EN
Since serum is a source of hormones, growth factors, cytokines and antioxidants being a survival factor for cell culture its withdrawal from the culture medium induces apoptosis in a variety of cell types. In the present study the effect of FCS - deprivation on Bcl-2 protein level, Bax transcript and apoptosis of L1210 leukaemic cells was examined. The possibility of the existence of cellular origin "death factors" was also investigated. A simultaneous flow cytometric analysis of Bcl-2 protein using FITC-conjugated Mo anti-Bcl-2 antibody and the percentage of apoptotic cells by measurement of sub-Gl region in the DNA (stained with DAPI) histogram was performed. Bax transcript was measured using the RT-PCR method with GAPDH as a reference gene. ROS generation was assessed by flow cytometry using the oxidation - sensitive fluorescent marker C-DCDHF-DA. FCS deprivation induced a massive apoptosis of leukaemic cells, reaching 40% of the cell population after 24h. Typical features of apoptosis such as cell shrinkage, condensation of chromatin, pyknosis and fragmentation of nuclei, followed by secondary necrosis (putrosis) were observed using fluorescence microscopy. An increase in intracellular ROS generation by 100% after 24h of serum deprivation was also observed. The apoptotic effect of FCS - deficiency was accompanied by a 32.7% decrease in the number of cells containing Bcl-2 protein (cells with specific anti-Bcl-2 fluorescence), thereby a negative relationship (r=0.98, P<0.01) between the extent of apoptosis and bcl-2 expression was calculated. Since serum deficiency eliminated primarily the cells with the lowest Bcl-2 level, the average Bcl-2 level in the population of surviving cells was even higher than in the control cultures, growing in optimal conditions (10% FCS/RPMI). The conditioned medium (CM), obtained from 24h FCS - deprived L1210 leukaemic cells, contained >10 kDa molecules, which were able to down- regulate Bcl-2, up-regulate Bax, and induce apoptosis. This effect was more visible when >10 kDa CM filtrate was administered to poorly-fed (2% FCS/RPMI) than better-fed (10% FCS/RPMI) cultures. It is concluded that: 1) the susceptibility of L1210 leukaemic cells to serum deprivation - induced apoptosis is dependent on Bcl-2 protein level, and 2) serum - deprived leukaemic cells release >10 kDa molecule/s being inductor/s of programmed cell death, acting through the regulation of Bax/Bcl-2 rheostat.
EN
The cellular organisation of the infective eggs of I. madagascariensis has been reconstructed at light (LM) and electron microscopy (EM) level from serial semithin (LM) and ultrathin sections (EM). The oncospheres are surrounded by parenchymatic capsules. The total number of oncospheral cells' is about 44 (50 nuclei). Among them, five major cell types have been distinguished. These consisted of: (1) a six-nucleate, U-shaped penetration gland; (2) a bi-nucleate medullary centre (= sunken perikaryon of oncospheral tegument); (3) two nerve cells of neurosecretory type; (4) about 30 somatic cells (= myocytons of hook and somatic musculature); and (5) about 12 germinative cells (two groups of 6 cells), localised in the posterior pole of the hexacanth. The functional ultrastructure of hook-muscle system and penetration gland and their role in host tissue penetration are discussed.
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