Expression of Kv1.2 in microglia and its putative roles in modulating production of proinflammatory cytokines and reactive oxygen species

Microglial cells are endowed with different potassium ion channels but their expression and specific functions have remained to be fully clarified. This study has shown Kv1.2 expression in the amoeboid microglia in the rat brain between 1 (P1) and 10 (P10) days of age. Kv1.2 expression was localized in the ramified microglia at P14 and was hardly detected at P21. In postnatal rats exposed to hypoxia, Kv1.2 immunoreactivity in microglia was markedly enhanced. Quantitative RT-PCR analysis confirmed Kv1.2 mRNA expression in microglial cells in vitro . It was further shown that Kv1.2 and protein expression coupled with that of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) was significantly increased when the cells were subjected to hypoxia. The same increase was observed in cells exposed to adenosine 5'-triphosphate (ATP) and lipopolysaccharide (LPS). Concomitantly, the intracellular potassium concentration decreased significantly. Blockade of Kv1.2 channel with rTityustoxin-Kα (TsTx) resulted in partial recovery of intracellular potassium concentration accompanied by a reduced expression of IL-1β and TNF-α mRNA and protein expression and intracellular reactive oxygen species (ROS) production. We conclude that Kv1.2 in microglia modulates IL-1β and TNF-α expression and ROS production probably by regulating the intracellular potassium concentration.     

Calpain-2 regulates hypoxia/HIF-induced plasticity toward amoeboid cancer cell migration and metastasis

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The tapetum: Its form,function, and possible phylogeny inEmbryophyta

It appears that the tapetum is universally present in land plants, even though it is sometimes difficult to recognize, because it serves mostly as a tissue for meiocyte/spore nutrition. In addition to this main function, the tapetum has other functions, namely the production of the locular fluid, the production and release of callase, the conveying of P.A.S. positive material towards the loculus, the formation of exine precursors, viscin threads and orbicules (= Ubisch bodies), the production of sporophytic proteins and enzymes, and of pollenkitt/tryphine. Not all these functions are present in all land plants:Embryophyta. Two main tapetal types are usually distinguished in theSpermatophyta: the secretory or parietal type and the amoeboid or periplasmodial type; in lower groups, however, other types may be recognized, with greater or lesser differences. A hypothetical phylogenesis of the tapetum is proposed on the basis of its morphological appearance and of the nutritional relations with meiocytes/spores. The evolutionary trends of the tapeta tend towards a more and more intimate and increasingly greater contact with the spores/pollen grains. Three evolutionary trends can be recognized: 1) an intrusion of the tapetal cells between the spores, 2) a loss of tapetal cell walls, and 3) increasing nutrition through direct contact in narrow anthers.     

Synchroma grande spec. nov. (Synchromophyceae class. nov., Heterokontophyta): an amoeboid marine alga with unique plastid complexes

Chromist algae including the Heterokontophyta are supposed to have evolved monophyletically by secondary endosymbiosis from a eukaryotic host cell that engulfed a eukaryotic red alga. The red algal endosymbiont was then reduced to a secondary plastid surrounded by four enveloping membranes. On the basis of the amoeboid marine alga Synchroma grande gen. et spec. nov., the Synchromophyceae are described here as a new class of Heterokontophyta. Their taxonomic position is characterized by 18S rRNA and rbcL gene phylogenies, morphology, and pigment composition. The so far unique feature of the Synchromophyceae is the occurrence of conspicuous chloroplast complexes representing multiplastidic red secondary endosymbionts. In these remarkable secondary endosymbionts, several primary chloroplasts are aggregated in a common periplastidial compartment and are collectively enveloped by an additional outer membrane pair. The discovery of this novel plastid morphology is highly relevant for research on algal evolution and is discussed in terms of the postulated monophyletic origin of Chromista.     

RASSF1C oncogene elicits amoeboid invasion,cancer stemness,and extracellular vesicle release via a SRC/Rho axis

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal‐to‐amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC‐mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C‐induced amoeboid cells display increased expression of cancer stem‐like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C‐induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C‐driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early‐stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.     

A novel approach to studying the migratory morphology of embryonic mesenchymal cells

A polarized morphology, defined by extension of an anterior pseudopod, is essential for the amoeboid migration of embryonic mesenchymal cells. Leukocytes adopt a similar morphology immediately following suspension in simple buffers containing chemotactic factors. Polarization in suspension therefore provides a rapid and sensitive screening assay for putative regulators of leukocyte migration. The aim of the present study was to investigate whether this assay might also be used to study the motile behaviour of embryonic mesenchymal cells. Primary cultures of mesenchymal cells were established from explants of stage 28 chick embryo corneal-limbal stroma. Serum-starved, subconfluent cultures were harvested using ethylene-diamine tetra-acetic acid and resuspended in Hanks' solution for up to 15 min at 37 degrees C. A variety of cell shapes, including spherical cells, blebbed cells, and cells with either non-polarized or polarized pseudopodia were observed. The proportions of cells with pseudopodia increased significantly over time. Treatment of cells with the chemotactic mitogen platelet derived growth factor-BB (PDGF-BB, homodimer isoform) suppressed blebbing and increased both pseudopod formation and polarization. Optimal polarization occurred in concentrations of PDGF-BB that are similar to those required for optimal chemotaxis (10 ng x mL(-1)). The polarization observed in the absence of PDGF-BB suggests that the migration of cells examined in this study might be controlled at least in part by some intrinsic mechanism. In addition, the strong polarization response to PDGF-BB confirms the role for this growth factor during corneal development. Observations of mesenchymal cell morphology in suspension, therefore provide novel data regarding the motile behaviour of embryonic cells.     

Fast-crawling cell types migrate to avoid the direction of periodic substratum stretching

To investigate the relationship between mechanical stimuli from substrata and related cell functions, one of the most useful techniques is the application of mechanical stimuli via periodic stretching of elastic substrata. In response to this stimulus, Dictyostelium discoideum cells migrate in a direction perpendicular to the stretching direction. The origins of directional migration, higher migration velocity in the direction perpendicular to the stretching direction or the higher probability of a switch of migration direction to perpendicular to the stretching direction, however, remain unknown. In this study, we applied periodic stretching stimuli to neutrophil-like differentiated HL-60 cells, which migrate perpendicular to the direction of stretch. Detailed analysis of the trajectories of HL-60 cells and Dictyostelium cells obtained in a previous study revealed that the higher probability of a switch of migration direction to that perpendicular to the direction of stretching was the main cause of such directional migration. This directional migration appears to be a strategy adopted by fast-crawling cells in which they do not migrate faster in the direction they want to go, but migrate to avoid a direction they do not want to go.     

Evaluation of cancer stem cell migration using compartmentalizing microfluidic devices and live cell imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized. Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment. We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.     

盐胁迫预处理对大麦根尖分生区细胞超微结构的影响

盐胁迫吸胀后的大麦种子转入正常条件下萌发,其发芽势严重降低,发芽率则和对照差异不大。电镜观察表明,萌发３天的大麦根尖分生区细胞膜系统已修复完善,内质网和高尔基体丰富,变形质体和变形线粒体大量出现,表明短时盐胁迫对种萌发的抑制作用是可逆的,而且一旦抑制作用被解除,植物细胞内的大生代谢活动会更加活跃,这一现象称为“抑制效应的补偿作用”。由于盐胁迫吸胀后代谢加强总伴随着变形质体和变形线粒体的大量出现。因