Effect of X-Irradiation at Different Stages in the Cell Cycle on Individual Cell–Based Kinetics in an Asynchronous Cell Population

Using an asynchronously growing cell population, we investigated how X-irradiation at different stages of the cell cycle influences individual cell–based kinetics. To visualize the cell-cycle phase, we employed the fluorescent ubiquitination-based cell cycle indicator (Fucci). After 5 Gy irradiation, HeLa cells no longer entered M phase in an order determined by their previous stage of the cell cycle, primarily because green phase (S and G2) was less prolonged in cells irradiated during the red phase (G1) than in those irradiated during the green phase. Furthermore, prolongation of the green phase in cells irradiated during the red phase gradually increased as the irradiation timing approached late G1 phase. The results revealed that endoreduplication rarely occurs in this cell line under the conditions we studied. We next established a method for classifying the green phase into early S, mid S, late S, and G2 phases at the time of irradiation, and then attempted to estimate the duration of G2 arrest based on certain assumptions. The value was the largest when cells were irradiated in mid or late S phase and the smallest when they were irradiated in G1 phase. In this study, by closely following individual cells irradiated at different cell-cycle phases, we revealed for the first time the unique cell-cycle kinetics in HeLa cells that follow irradiation.     

Influence of Gangliosides on the IL-2– and IL-4–Dependent Cell Proliferation

Ganglioside-induced apoptosis in the cells of IL-2–dependent cytotoxic murine cell line CTLL-2 was shown to be caspase dependent: GM1-, GM2-, and GD3-induced suppression of cell proliferation was cancelled by a general caspase inhibitor Z-VAD-FMK. Ganglioside-induced apoptosis pathways are different for different individual glycolipids; the differences exist both at the initiation and effector stages of the caspase cascade. Only for GM1-induced process, molecular mechanisms of signal transduction coincide with the ones for CD95 and TNF: the participation of both the main initiation caspases 8, 1, and 4, and caspases 3 and 9 as well, has been shown. Caspase 3 participates in the pathway induced by GM3, GD1a, GD1b, and GT1b, but not by GM2. As morphological features show, tumor-associated ganglioside GM2 is also a stimulus of programmed cell death (PCD) for CTLL-2 cell line: addition of GM2 into cell culture has resulted in appearance of annexin V-positive cells and in accumulation of DNA breaks (shown by the TUNEL direct dyeing of the open ends). But a caspase 3 inhibitor Z-DEVD-FMK did not restore the cell proliferation suppressed by GM2, and addition of a fluorescent substrate of caspase 3 Ac-DEVD-AFC did not result in the fluorescence development. So caspase 3 does not participate in downstream pathways of GM2-induced cell apoptosis, and a PCD-effector system other than the apoptosome-mediated one is involved here.     

Photodynamic Therapy in Pythium insidiosum – An In Vitro Study of the Correlation of Sensitizer Localization and Cell Death

Pythiosis is an infectious disease caused by Pythium insidiosum, a fungus-like organism. Due to the lack of ergosterol on its cell membrane, antibiotic therapy is ineffective. The conventional treatment is surgery, but lesion recurrence is frequent, requiring several resections or limb amputation. Photodynamic therapy uses photo-activation of drugs and has the potential to be an attractive alternative option. The in vitro PDT response on the growing of Pythium insidiosum culture was investigated using three distinct photosensitizers: methylene blue, Photogem, and Photodithazine. The photosensitizer distribution in cell structures and the PDT response for incubation times of 30, 60, and 120 minutes were evaluated. Methylene blue did not penetrate in the pathogen''s cell and consequently there was no PDT inactivation. Photogem showed heterogenous distribution in the hyphal structure with small concentration inside the cells. Porphyrin-PDT response was heterogenous, death and live cells were observed in the treated culture. After 48 hours, hyphae regrowth was observed. Photodithazine showed more homogenous distribution inside the cell and with the specific intracellular localization dependent on incubation time. Photodithazine first accumulates in intracellular vacuoles, and at incubation times of one hour, it is located at all cell membranes. Higher inhibition of the growing rates was achieved with Photodithazine -PDT, over 98%. Our results showed that the photosensitizers that cross more efficiently the Pythium insidiosum membranes are able to cause extensive damage to the organism under illumination and therefore, are the best options for clinical treatment.     

Glutamate Receptor Requirement for Neuronal Death from Anoxia–Reoxygenation: An in Vitro Model for Assessment of the Neuroprotective Effects of Estrogens

1.Previous studies demonstrated that estrogens, specifically 17-estradiol, the potent, naturally occurring estrogen, are neuroprotective in a variety of models including glutamate toxicity. The aim of the present study is twofold: (1) to assess the requirement for glutamate receptors in neuronal cell death associated with anoxia–reoxygenation in three cell types, SK-N-SH and HT-22 neuronal cell lines and primary rat cortical neuronal cultures, and (2) to evaluate the neuroprotective activity of both 17-estradiol and its weaker isomer, 17-estradiol, in both anoxia-reoxygenation and glutamate toxicity.2.SK-N-SH and HT-22 cell lines, both of which lack NMDA receptors as assessed by MK-801 binding assays, were resistant to both anoxia–reoxygenation and glutamate-induced cell death. In contrast, primary rat cortical neurons, which exhibit both NMDA and AMPA receptors, were sensitive to brief periods of exposure to anoxia–reoxygenation or glutamate. As such, there appears to be an obligatory requirement for NMDA and/or AMPA receptors in neuronal cell death resulting from brief periods of anoxia followed by reoxygenation.3.Using primary rat cortical neuronal cultures, we evaluated the neuroprotective activity of 17-estradiol (1.3 or 133 nM) and 17-estradiol (133 nM) in both anoxia–reoxygenation and excitotoxicity models of cell death. We found that the 133 nM but not the 1.3 nM dose of the potent estrogen, 17-estradiol, protected 58.0, 57.5, and 85.3% of the primary rat cortical neurons from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively, and the 133 nM dose of the weak estrogen, 17-estradiol, protected 74.6, 81.7, and 85.8% of cells from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively. These data demonstrate that pretreatment with estrogens can attenuate glutamate excitotoxicity and that this protection is independent of the ability of the steroid to bind the estrogen receptor.     

Geographic Divisions and Modeling of Virological Data on Seasonal Influenza in the Chinese Mainland during the 2006–2009 Monitoring Years

Background

Seasonal influenza epidemics occur annually with bimodality in southern China and unimodality in northern China. Regional differences exist in surveillance data collected by the National Influenza Surveillance Network of the Chinese mainland. Qualitative and quantitative analyses on the spatiotemporal rules of the influenza virus''s activities are needed to lay the foundation for the surveillance, prevention and control of seasonal influenza.

Methods

The peak performance analysis and Fourier harmonic extraction methods were used to explore the spatiotemporal characteristics of the seasonal influenza virus activity and to obtain geographic divisions. In the first method, the concept of quality control was introduced and robust estimators were chosen to make the results more convincing. The dominant Fourier harmonics of the provincial time series were extracted in the second method, and the VARiable CLUSter (VARCLUS) procedure was used to variably cluster the extracted results. On the basis of the above geographic division results, three typical districts were selected and corresponding sinusoidal models were applied to fit the time series of the virological data.

Results

The predominant virus during every peak is visible from the bar charts of the virological data. The results of the two methods that were used to obtain the geographic divisions have some consistencies with each other and with the virus activity mechanism. Quantitative models were established for three typical districts: the south1 district, including Guangdong, Guangxi, Jiangxi and Fujian; the south2 district, including Hunan, Hubei, Shanghai, Jiangsu and Zhejiang; and the north district, including the 14 northern provinces except Qinghai. The sinusoidal fitting models showed that the south1 district had strong annual periodicity with strong winter peaks and weak summer peaks. The south2 district had strong semi-annual periodicity with similarly strong summer and winter peaks, and the north district had strong annual periodicity with only winter peaks.     

Cell Cycle–Dependent Differentiation Dynamics Balances Growth and Endocrine Differentiation in the Pancreas

Organogenesis relies on the spatiotemporal balancing of differentiation and proliferation driven by an expanding pool of progenitor cells. In the mouse pancreas, lineage tracing at the population level has shown that the expanding pancreas progenitors can initially give rise to all endocrine, ductal, and acinar cells but become bipotent by embryonic day 13.5, giving rise to endocrine cells and ductal cells. However, the dynamics of individual progenitors balancing self-renewal and lineage-specific differentiation has never been described. Using three-dimensional live imaging and in vivo clonal analysis, we reveal the contribution of individual cells to the global behaviour and demonstrate three modes of progenitor divisions: symmetric renewing, symmetric endocrinogenic, and asymmetric generating a progenitor and an endocrine progenitor. Quantitative analysis shows that the endocrine differentiation process is consistent with a simple model of cell cycle–dependent stochastic priming of progenitors to endocrine fate. The findings provide insights to define control parameters to optimize the generation of β-cells in vitro.     

Effects of miR-19b Overexpression on Proliferation, Differentiation, Apoptosis and Wnt/β-Catenin Signaling Pathway in P19 Cell Model of Cardiac Differentiation In Vitro

MicroRNA (miR)-19b is part of the miR-17–92 cluster associated with cardiac development. Here, we investigated the effects of overexpressing miR-19b on proliferation, differentiation, apoptosis, and regulation of the Wnt/β-catenin signaling pathway in the multipotent murine P19 cell line that can be induced to undergo cardiogenesis. P19 cells were transfected with the miR-19b plasmid or empty vector, and miR-19b overexpression was verified by Quantitative Real-Time PCR (qPCR). The miR-19b or vector control stable cell lines were selected using Blasticidin S HCl, and their proliferation, cell cycle, and apoptosis levels were analyzed using the Cell Counting Kit-8 and flow cytometry. P19 cell differentiation markers, apoptosis-related genes (bax, bcl-2), and Wnt/β-catenin signaling pathway-related genes were detected by qPCR, the corresponding proteins by Western blot. Expression of the Wnt pathway and differentiation marker proteins was also verified by immunofluorescence. Morphological changes associated with apoptosis were observed by electron microscopy and Hoechst staining. On the basis of these results, we demonstrated that miR-19b overexpression promoted proliferation and differentiation but inhibited apoptosis in P19 cells; Wnt and β-catenin expressions were decreased, while that of GSK3β was increased with miR-19b overexpression. Overexpression of miR-19b inhibited activation of the Wnt/β-catenin signaling pathway in P19 cells, which may regulate cardiomyocyte differentiation. Our findings may bring new insights into the mechanisms underlying cardiac diseases and suggest that miR-19b is a potential new therapeutic target for cardiovascular diseases.     

Cell Cycle–related Changes in the Conducting Properties of r-eag K+ Channels

Release from arrest in G2 phase of the cell cycle causes profound changes in rat ether-à-go-go (r-eag) K⁺ channels heterologously expressed in Xenopus oocytes. The most evident consequence of the onset of maturation is the appearance of rectification in the r-eag current. The trigger for these changes is located downstream of the activation of mitosis-promoting factor (MPF). We demonstrate here that the rectification is due to a voltage-dependent block by intracellular Na⁺ ions. Manipulation of the intracellular Na⁺ concentration indicates that the site of Na⁺ block is located ∼45% into the electrical distance of the pore and is only present in oocytes undergoing maturation. Since the currents through excised patches from immature oocytes exhibited a fast rundown, we studied CHO-K1 cells permanently transfected with r-eag. These cells displayed currents with a variable degree of block by Na⁺ and variable permeability to Cs⁺. Partial synchronization of the cultures in G0/G1 or M phases of the cell cycle greatly reduced the variability. The combined data obtained from mammalian cells and oocytes strongly suggest that the permeability properties of r-eag K⁺ channels are modulated during cell cycle–related processes.     

Effect of Glucose on the Lactoferrin’s Conformation and its Effect on MC 3T3-E1 Cell Proliferation

The objective of this research was to investigate the influence of glucose on bovine lactoferrin’s (LF) conformation, thermodynamic stability and osteoblastic cell proliferation. The conformation and thermodynamic stability of LF was detected by spectroscopic and differential scanning calorimetry (DSC). The osteoblastic cell proliferation of LF at physiological concentrations (100 μg/ml) was measured by BrdU incorporation. The binding constant between glucose and LF is KSV = 5 × 10⁻³, and Tyr residues of LF were located in a more hydrophobic environment, while Trp residues were located in a more hydrophilic environment. LF with glucose had increased α-helix and β-sheet contents by 6 and 14 %, respectively. It showed a two-step denaturation of LF. There was a gradual changs in the denaturation temperature and the calorimetric enthalpies (ΔHcal) with a growing concentration of glucose. It has also revealed that glucose dose dependently reduced the ability of LF to increase MC 3T3-E1 cell proliferation. Increasing the binding with glucose, LF might cause to change its native state, which reduced the stimulation activity of osteoblasts cell proliferation.     

Effects of Oxygen Concentration on the Proliferation and Differentiation of Mouse Neural Stem Cells In Vitro

Background and purpose Cerebral ischemia is known to elicit the activation of neural stem cells (NSCs); however its mechanism is not fully determined. Although oxygen concentration is known to mediate many ischemic actions, there has been little attention given to the role of pathological oxygen changes under cerebral ischemia on the activation of NSCs. We investigated the effects of various oxygen concentrations on mouse neural stem cells in vitro. Methods NSCs were cultured from the ganglionic eminence of fetal ICR mice on embryonic day 15.5 using a neurosphere method. The effects of oxygen concentrations on proliferation, differentiation, and cell death of NSCs were evaluated by bromodeoxyuridine (BrdU) incorporation, immunocytochemistry, and TUNEL assay, respectively. Results The highest proliferation and the neuronal differentiation of the NSCs were observed in 2% oxygen, which yielded significantly higher proportions of both BrdU-labeled cells and Tuj1-positive cells when compared with 20% and 4% oxygen. On the other hand, the differentiation to the astrocytes was not affected by oxygen concentrations, except in the case of anoxia (0% oxygen). The cell death of the NSCs increased in lower oxygen conditions and peaked at anoxia. Furthermore, the switching of the neuronal subtype differentiation from GABA-positive to glutamate-positive neurons was observed in lower oxygen conditions. Conclusions These findings raise the possibility that reduced oxygen levels occurring with cerebral ischemia enhance NSC proliferation and neural differentiation, and that mild hypoxia (2% oxygen), which is known to occur in the ischemic penumbra, is suitable for abundant neuronal differentiation.     

Mathematical Modeling of the Impact of Actin and Keratin Filaments on?Keratinocyte Cell Spreading

Keratin intermediate filaments (IFs) form cross-linked arrays to fulfill their structural support function in epithelial cells and tissues subjected to external stress. How the cross-linking of keratin IFs impacts the morphology and differentiation of keratinocytes in the epidermis and related surface epithelia remains an open question. Experimental measurements have established that keratinocyte spreading area is inversely correlated to the extent of keratin IF bundling in two-dimensional culture. In an effort to quantitatively explain this relationship, we developed a mathematical model in which isotropic cell spreading is considered as a first approximation. Relevant physical properties such as actin protrusion, adhesion events, and the corresponding response of lamellum formation at the cell periphery are included in this model. Through optimization with experimental data that relate time-dependent changes in keratinocyte surface area during spreading, our simulation results confirm the notion that the organization and mechanical properties of cross-linked keratin filaments affect cell spreading; in addition, our results provide details of the kinetics of this effect. These in silico findings provide further support for the notion that differentiation-related changes in the density and intracellular organization of keratin IFs affect tissue architecture in epidermis and related stratified epithelia.     

Effects of Huangqi （Hex） on Inducing Cell Differentiation and Cell Death in K562 and HEL Cells

InterestintraditionalChineseherbalremedieshasboomedin the western countries. It is very important to study theirmolecular mechanisms and purify effective compoundswith new knowledge and new techniques to meet a greatneed for human health. Ephedrine, the f…     

FGF23 Suppresses Chondrocyte Proliferation in the Presence of Soluble α-Klotho both in Vitro and in Vivo

Fibroblast growth factor-23 (FGF23) is well established to play crucial roles in the regulation of phosphate homeostasis. X-linked hypophosphatemic rickets (XLH) is characterized by impaired mineralization and growth retardation associated with elevated circulating FGF23 levels. Administration of phosphate and calcitriol is effective in improving growth retardation, but is not sufficient to fully reverse impaired growth, suggesting the existence of a disease-specific mechanism in the development of growth retardation in addition to dysregulated phosphate metabolism. However, the precise mechanisms of growth retardation in XLH remain elusive. Here, we postulated that FGF23 suppressed chondrocyte proliferation in the presence of soluble α-Klotho (sKL). In vitro and ex vivo studies revealed that FGF23 formed a protein complex with sKL through KL1 internal repeat and suppressed the linear growth of metatarsals in the presence of sKL, which was antagonized by co-incubation with neutralizing antibodies against FGF23 or by knocking-down FGFR3 expression. Additionally, FGF23 binding to FGFR3 was enhanced in the presence of sKL. Histologically, the length of the proliferating zone was diminished and was associated with decreased chondrocyte proliferation. FGF23/sKL suppressed Indian hedgehog (Ihh) expression and administration of Ihh protein partially rescued the suppressive effect of FGF23/sKL on metatarsal growth. Intraperitoneal administration of sKL in Hyp mice, a murine model for XLH, caused a decrease in the length of the proliferating zone associated with decreased chondrocyte proliferation without altering circulating phosphate levels. These findings suggest that suppression of chondrocyte proliferation by FGF23 could have a causative role in the development of growth retardation in XLH.     

In Vitro Measurement and Modeling of Platelet Adhesion on VWF-Coated Surfaces in Channel Flow

The process of platelet adhesion is initiated by glycoprotein (GP)Ib and GPIIbIIIa receptors on the platelet surface binding with von Willebrand factor on the vascular walls. This initial adhesion and detachment of a single platelet is a complex process that involves multiple bonds forming and breaking and is strongly influenced by the surrounding blood-flow environment. In addition to bond-level kinetics, external factors such as shear rate, hematocrit, and GPIb and GPIIbIIIa receptor densities have also been identified as influencing the platelet-level rate constants in separate studies, but this still leaves a gap in understanding between these two length scales. In this study, we investigate the fundamental relationship of the dynamics of platelet adhesion, including these interrelating factors, using a coherent strategy. We build a, to our knowledge, novel and computationally efficient multiscale model accounting for multibond kinetics and hydrodynamic effects due to the flow of a cellular suspension. The model predictions of platelet-level kinetics are verified by our microfluidic experiments, which systematically investigate the role of each external factor on platelet adhesion in an in vitro setting. We derive quantitative formulas describing how the rates of platelet adhesion, translocation, and detachment are defined by the molecular-level kinetic constants, the local platelet concentration near the reactive surface determined by red-blood-cell migration, the platelet effective reactive area due to its tumbling motion, and the platelet surface receptor density. Furthermore, if any of these aspects involved have abnormalities, e.g., in a disease condition, our findings also have clinical relevance in predicting the resulting change in the adhesion dynamics, which is essential to hemostasis and thrombosis.     

Targeting of β-Arrestin2 to the Centrosome and Primary Cilium: Role in Cell Proliferation Control

Background

The primary cilium is a sensory organelle generated from the centrosome in quiescent cells and found at the surface of most cell types, from where it controls important physiological processes. Specific sets of membrane proteins involved in sensing the extracellular milieu are concentrated within cilia, including G protein coupled receptors (GPCRs). Most GPCRs are regulated by β-arrestins, βarr1 and βarr2, which control both their signalling and endocytosis, suggesting that βarrs may also function at primary cilium.

Methodology/Principal Findings

In cycling cells, βarr2 was observed at the centrosome, at the proximal region of the centrioles, in a microtubule independent manner. However, βarr2 did not appear to be involved in classical centrosome-associated functions. In quiescent cells, both in vitro and in vivo, βarr2 was found at the basal body and axoneme of primary cilia. Interestingly, βarr2 was found to interact and colocalize with 14-3-3 proteins and Kif3A, two proteins known to be involved in ciliogenesis and intraciliary transport. In addition, as suggested for other centrosome or cilia-associated proteins, βarrs appear to control cell cycle progression. Indeed, cells lacking βarr2 were unable to properly respond to serum starvation and formed less primary cilia in these conditions.

Conclusions/Significance

Our results show that βarr2 is localized to the centrosome in cycling cells and to the primary cilium in quiescent cells, a feature shared with other proteins known to be involved in ciliogenesis or primary cilium function. Within cilia, βarr2 may participate in the signaling of cilia-associated GPCRs and, therefore, in the sensory functions of this cell “antenna”.