Antitumor Cell-Complex Vaccines Employing Genetically Modified Tumor Cells and Fibroblasts

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Antonio Miguel

2014-02-01

Full Text Available The present study evaluates the immune response mediated by vaccination with cell complexes composed of irradiated B16 tumor cells and mouse fibroblasts genetically modified to produce GM-CSF. The animals were vaccinated with free B16 cells or cell complexes. We employed two gene plasmid constructions: one high producer (pMok and a low producer (p2F. Tumor transplant was performed by injection of B16 tumor cells. Plasma levels of total IgG and its subtypes were measured by ELISA. Tumor volumes were measured and survival curves were obtained. The study resulted in a cell complex vaccine able to stimulate the immune system to produce specific anti-tumor membrane proteins (TMP IgG. In the groups vaccinated with cells transfected with the low producer plasmid, IgG production was higher when we used free B16 cell rather than cell complexes. Nonspecific autoimmune response caused by cell complex was not greater than that induced by the tumor cells alone. Groups vaccinated with B16 transfected with low producer plasmid reached a tumor growth delay of 92% (p ≤ 0.01. When vaccinated with cell complex, the best group was that transfected with high producer plasmid, reaching a tumor growth inhibition of 56% (p ≤ 0.05. Significant survival (40% was only observed in the groups vaccinated with free transfected B16 cells.

Synaptic Basis for Differential Orientation Selectivity between Complex and Simple Cells in Mouse Visual Cortex.

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Li, Ya-tang; Liu, Bao-hua; Chou, Xiao-lin; Zhang, Li I; Tao, Huizhong W

2015-08-05

In the primary visual cortex (V1), orientation-selective neurons can be categorized into simple and complex cells primarily based on their receptive field (RF) structures. In mouse V1, although previous studies have examined the excitatory/inhibitory interplay underlying orientation selectivity (OS) of simple cells, the synaptic bases for that of complex cells have remained obscure. Here, by combining in vivo loose-patch and whole-cell recordings, we found that complex cells, identified by their overlapping on/off subfields, had significantly weaker OS than simple cells at both spiking and subthreshold membrane potential response levels. Voltage-clamp recordings further revealed that although excitatory inputs to complex and simple cells exhibited a similar degree of OS, inhibition in complex cells was more narrowly tuned than excitation, whereas in simple cells inhibition was more broadly tuned than excitation. The differential inhibitory tuning can primarily account for the difference in OS between complex and simple cells. Interestingly, the differential synaptic tuning correlated well with the spatial organization of synaptic input: the inhibitory visual RF in complex cells was more elongated in shape than its excitatory counterpart and also was more elongated than that in simple cells. Together, our results demonstrate that OS of complex and simple cells is differentially shaped by cortical inhibition based on its orientation tuning profile relative to excitation, which is contributed at least partially by the spatial organization of RFs of presynaptic inhibitory neurons. Simple and complex cells, two classes of principal neurons in the primary visual cortex (V1), are generally thought to be equally selective for orientation. In mouse V1, we report that complex cells, identified by their overlapping on/off subfields, has significantly weaker orientation selectivity (OS) than simple cells. This can be primarily attributed to the differential tuning selectivity

Terbutaline causes immobilization of single β2-adrenergic receptor-ligand complexes in the plasma membrane of living A549 cells as revealed by single-molecule microscopy

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Sieben, Anne; Kaminski, Tim; Kubitscheck, Ulrich; Häberlein, Hanns

2011-02-01

G-protein-coupled receptors are important targets for various drugs. After signal transduction, regulatory processes, such as receptor desensitization and internalization, change the lateral receptor mobility. In order to study the lateral diffusion of β2-adrenergic receptors (β2AR) complexed with fluorescently labeled noradrenaline (Alexa-NA) in plasma membranes of A549 cells, trajectories of single receptor-ligand complexes were monitored using single-particle tracking. We found that a fraction of 18% of all β2ARs are constitutively immobile. About 2/3 of the β2ARs moved with a diffusion constant of D2 = 0.03+/-0.001 μm2/s and about 17% were diffusing five-fold faster (D3 = 0.15+/-0.02 μm2/s). The mobile receptors moved within restricted domains and also showed a discontinuous diffusion behavior. Analysis of the trajectory lengths revealed two different binding durations with τ1 = 77+/-1 ms and τ2 = 388+/-11 ms. Agonistic stimulation of the β2AR-Alexa-NA complexes with 1 μM terbutaline caused immobilization of almost 50% of the receptors within 35 min. Simultaneously, the mean area covered by the mobile receptors decreased significantly. Thus, we demonstrated that agonistic stimulation followed by cell regulatory processes results in a change in β2AR mobility suggesting that different receptor dynamics characterize different receptor states.

High-resolution crystal structure of Streptococcus pyogenes β-NAD{sup +} glycohydrolase in complex with its endogenous inhibitor IFS reveals a highly water-rich interface

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Yoon, Ji Young; An, Doo Ri; Yoon, Hye-Jin [Seoul National University, Seoul 151-747 (Korea, Republic of); Kim, Hyoun Sook [Seoul National University, Seoul 151-747 (Korea, Republic of); Seoul National University, Seoul 151-742 (Korea, Republic of); Lee, Sang Jae [Seoul National University, Seoul 151-742 (Korea, Republic of); Im, Ha Na; Jang, Jun Young [Seoul National University, Seoul 151-747 (Korea, Republic of); Suh, Se Won, E-mail: sewonsuh@snu.ac.kr [Seoul National University, Seoul 151-747 (Korea, Republic of); Seoul National University, Seoul 151-747 (Korea, Republic of)

2013-11-01

The crystal structure of the complex between the C-terminal domain of Streptococcus pyogenes β-NAD{sup +} glycohydrolase and an endogenous inhibitor for SPN was determined at 1.70 Å. It reveals that the interface between the two proteins is highly rich in water molecules. One of the virulence factors produced by Streptococcus pyogenes is β-NAD{sup +} glycohydrolase (SPN). S. pyogenes injects SPN into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. As SPN is toxic to bacterial cells themselves, S. pyogenes possesses the ifs gene that encodes an endogenous inhibitor for SPN (IFS). IFS is localized intracellularly and forms a complex with SPN. This intracellular complex must be dissociated during export through the cell envelope. To provide a structural basis for understanding the interactions between SPN and IFS, the complex was overexpressed between the mature SPN (residues 38–451) and the full-length IFS (residues 1–161), but it could not be crystallized. Therefore, limited proteolysis was used to isolate a crystallizable SPN{sub ct}–IFS complex, which consists of the SPN C-terminal domain (SPN{sub ct}; residues 193–451) and the full-length IFS. Its crystal structure has been determined by single anomalous diffraction and the model refined at 1.70 Å resolution. Interestingly, our high-resolution structure of the complex reveals that the interface between SPN{sub ct} and IFS is highly rich in water molecules and many of the interactions are water-mediated. The wet interface may facilitate the dissociation of the complex for translocation across the cell envelope.

X-ray-enhanced cancer cell migration requires the linker of nucleoskeleton and cytoskeleton complex.

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Imaizumi, Hiromasa; Sato, Katsutoshi; Nishihara, Asuka; Minami, Kazumasa; Koizumi, Masahiko; Matsuura, Nariaki; Hieda, Miki

2018-04-01

The linker of nucleoskeleton and cytoskeleton (LINC) complex is a multifunctional protein complex that is involved in various processes at the nuclear envelope, including nuclear migration, mechanotransduction, chromatin tethering and DNA damage response. We recently showed that a nuclear envelope protein, Sad1 and UNC84 domain protein 1 (SUN1), a component of the LINC complex, has a critical function in cell migration. Although ionizing radiation activates cell migration and invasion in vivo and in vitro, the underlying molecular mechanism remains unknown. Here, we examined the involvement of the LINC complex in radiation-enhanced cell migration and invasion. A sublethal dose of X-ray radiation promoted human breast cancer MDA-MB-231 cell migration and invasion, whereas carbon ion beam radiation suppressed these processes in a dose-dependent manner. Depletion of SUN1 and SUN2 significantly suppressed X-ray-enhanced cell migration and invasion. Moreover, depletion or overexpression of each SUN1 splicing variant revealed that SUN1_888 containing 888 amino acids of SUN1 but not SUN1_916 was required for X-ray-enhanced migration and invasion. In addition, the results suggested that X-ray irradiation affected the expression level of SUN1 splicing variants and a SUN protein binding partner, nesprins. Taken together, our findings supported that the LINC complex contributed to photon-enhanced cell migration and invasion. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Mechanics of Cellulose Synthase Complexes in Living Plant Cells

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Zehfroosh, Nina; Liu, Derui; Ramos, Kieran P.; Yang, Xiaoli; Goldner, Lori S.; Baskin, Tobias I.

The polymer cellulose is one of the major components of the world's biomass with unique and fascinating characteristics such as its high tensile strength, renewability, biodegradability, and biocompatibility. Because of these distinctive aspects, cellulose has been the subject of enormous scientific and industrial interest, yet there are still fundamental open questions about cellulose biosynthesis. Cellulose is synthesized by a complex of transmembrane proteins called ``Cellulose Synthase A'' (CESA) in the plasma membrane. Studying the dynamics and kinematics of the CESA complex will help reveal the mechanism of cellulose synthesis and permit the development and validation of models of CESA motility. To understand what drives these complexes through the cell membrane, we used total internal reflection fluorescence microscopy (TIRFM) and variable angle epi-fluorescence microscopy to track individual, fluorescently-labeled CESA complexes as they move in the hypocotyl and root of living plants. A mean square displacement analysis will be applied to distinguish ballistic, diffusional, and other forms of motion. We report on the results of these tracking experiments. This work was funded by NSF/PHY-1205989.

Structurally related hydrazone-based metal complexes with different antitumor activities variably induce apoptotic cell death.

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Megger, Dominik A; Rosowski, Kristin; Radunsky, Christian; Kösters, Jutta; Sitek, Barbara; Müller, Jens

2017-04-05

Three new complexes bearing the tridentate hydrazone-based ligand 2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)pyridine (L) were synthesized and structurally characterized. Biological tests indicate that the Zn(ii) complex [ZnCl 2 (L)] is of low cytotoxicity against the hepatocellular carcinoma cell line HepG2. In contrast, the Cu(ii) and Mn(ii) complexes [CuCl 2 (L)] and [MnCl 2 (L)] are highly cytotoxic with EC 50 values of 1.25 ± 0.01 μM and 20 ± 1 μM, respectively. A quantitative proteome analysis reveals that treatment of the cells with the Cu(ii) complex leads to a significantly altered abundance of 102 apoptosis-related proteins, whereas 38 proteins were up- or down-regulated by the Mn(ii) complex. A closer inspection of those proteins regulated only by the Cu(ii) complex suggests that the superior cytotoxic activity of this complex is likely to be related to an initiation of the caspase-independent cell death (CICD). In addition, an increased generation of reactive oxygen species (ROS) and a strong up-regulation of proteins responsive to oxidative stress suggest that alterations of the cellular redox metabolism likely contribute to the cytotoxicity of the Cu(ii) complex.

Conjunctival lymphangioma in a 4-year-old girl revealed tuberous sclerosis complex

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Freiberg, Florentina Joyce

2016-09-01

Full Text Available Background: To present a case of conjunctival lymphangioma in a girl with tuberous sclerosis complex.Methods/results: A 4-year-old girl presented with a relapsing cystic lesion of the bulbar conjunctiva in the right eye with string-of-pearl-like dilation of lymphatic vessels and right-sided facial swelling with mild pain. Best-corrected vision was not impaired. Examination of the skin revealed three hypomelanotic macules and a lumbal Shagreen patch. Magnetic resonance imaging (MRI findings displayed minimal enhancement of buccal fat on the right side. Cranial and orbital MRI showed signal enhancement in the right cortical and subcortical areas. Genetic analysis revealed a heterozygous deletion encompassing exon 1 and 2 of the gene (tuberous sclerosis complex 1 gene, confirming the diagnosis of tuberous sclerosis complex.Conclusion: In conjunctival lymphangioma, tuberous sclerosis complex should be considered as the primary disease.

Quantitative proteomics reveals middle infrared radiation-interfered networks in breast cancer cells.

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Chang, Hsin-Yi; Li, Ming-Hua; Huang, Tsui-Chin; Hsu, Chia-Lang; Tsai, Shang-Ru; Lee, Si-Chen; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2015-02-06

Breast cancer is one of the leading cancer-related causes of death worldwide. Treatment of triple-negative breast cancer (TNBC) is complex and challenging, especially when metastasis has developed. In this study, we applied infrared radiation as an alternative approach for the treatment of TNBC. We used middle infrared (MIR) with a wavelength range of 3-5 μm to irradiate breast cancer cells. MIR significantly inhibited cell proliferation in several breast cancer cells but did not affect the growth of normal breast epithelial cells. We performed iTRAQ-coupled LC-MS/MS analysis to investigate the MIR-triggered molecular mechanisms in breast cancer cells. A total of 1749 proteins were identified, quantified, and subjected to functional enrichment analysis. From the constructed functionally enriched network, we confirmed that MIR caused G2/M cell cycle arrest, remodeled the microtubule network to an astral pole arrangement, altered the actin filament formation and focal adhesion molecule localization, and reduced cell migration activity and invasion ability. Our results reveal the coordinative effects of MIR-regulated physiological responses in concentrated networks, demonstrating the potential implementation of infrared radiation in breast cancer therapy.

Selective ligand activity at Nur/retinoid X receptor complexes revealed by dimer-specific bioluminescence resonance energy transfer-based sensors

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Giner, Xavier C; Cotnoir-White, David; Mader, Sylvie; Lévesque, Daniel

2017-01-01

Retinoid X receptors (RXR) play a role as master regulators due to their capacity to form heterodimers with other nuclear receptors. Accordingly, retinoid signaling is involved in multiple biological processes, including development, cell differentiation, metabolism and cell death. However, the role and functions of RXR in different heterodimer complexes remain unsolved, mainly because most RXR drugs (called rexinoids) are not selective to specific heterodimer complexes. This also strongly limits the use of rexinoids for specific therapeutic approaches. In order to better characterize rexinoids at specific nuclear receptor complexes, we have developed and optimized luciferase protein complementation-based Bioluminescence Resonance Energy Transfer (BRET) assays, which can directly measure recruitment of a co-activator motif fused to yellow fluorescent protein (YFP) by specific nuclear receptor dimers. To validate the assays, we compared rexinoid modulation of co-activator recruitment by RXR homodimer, and heterodimers Nur77/RXR and Nurr1/RXR. Results reveal that some rexinoids display selective co-activator recruitment activities with homo- or hetero-dimer complexes. In particular, SR11237 (BMS649) has increased potency for recruitment of co-activator motif and transcriptional activity with the Nur77/RXR heterodimer compared to other complexes. This technology should prove useful to identify new compounds with specificity for individual dimeric species formed by nuclear receptors. PMID:26148973

Chansporter complexes in cell signaling.

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Abbott, Geoffrey W

2017-09-01

Ion channels facilitate diffusion of ions across cell membranes for such diverse purposes as neuronal signaling, muscular contraction, and fluid homeostasis. Solute transporters often utilize ionic gradients to move aqueous solutes up their concentration gradient, also fulfilling a wide variety of tasks. Recently, an increasing number of ion channel-transporter ('chansporter') complexes have been discovered. Chansporter complex formation may overcome what could otherwise be considerable spatial barriers to rapid signal integration and feedback between channels and transporters, the ions and other substrates they transport, and environmental factors to which they must respond. Here, current knowledge in this field is summarized, covering both heterologous expression structure/function findings and potential mechanisms by which chansporter complexes fulfill contrasting roles in cell signaling in vivo. © 2017 Federation of European Biochemical Societies.

Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

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Mukerji Joya

2012-06-01

Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

Natural Product Screening Reveals Naphthoquinone Complex I Bypass Factors.

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Scott B Vafai

Full Text Available Deficiency of mitochondrial complex I is encountered in both rare and common diseases, but we have limited therapeutic options to treat this lesion to the oxidative phosphorylation system (OXPHOS. Idebenone and menadione are redox-active molecules capable of rescuing OXPHOS activity by engaging complex I-independent pathways of entry, often referred to as "complex I bypass." In the present study, we created a cellular model of complex I deficiency by using CRISPR genome editing to knock out Ndufa9 in mouse myoblasts, and utilized this cell line to develop a high-throughput screening platform for novel complex I bypass factors. We screened a library of ~40,000 natural product extracts and performed bioassay-guided fractionation on a subset of the top scoring hits. We isolated four plant-derived 1,4-naphthoquinone complex I bypass factors with structural similarity to menadione: chimaphilin and 3-chloro-chimaphilin from Chimaphila umbellata and dehydro-α-lapachone and dehydroiso-α-lapachone from Stereospermum euphoroides. We also tested a small number of structurally related naphthoquinones from commercial sources and identified two additional compounds with complex I bypass activity: 2-methoxy-1,4-naphthoquinone and 2-methoxy-3-methyl-1,4,-naphthoquinone. The six novel complex I bypass factors reported here expand this class of molecules and will be useful as tool compounds for investigating complex I disease biology.

Complexes of γ-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells

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Kukharskyy, Vitaliy; Sulimenko, Vadym; Macurek, Libor; Sulimenko, Tetyana; Draberova, Eduarda; Draber, Pavel

2004-01-01

Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that γ-tubulin (γ-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, γ-tubulin, and with anti-phosphotyrosine antibody revealed that γ-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in γ-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated γ-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing γ-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of γ-tubulin interaction with tubulin dimers or other proteins during neurogenesis

The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy

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Arnauld eSergé

2016-05-01

Full Text Available The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation and metastasis.

DNA entropy reveals a significant difference in complexity between housekeeping and tissue specific gene promoters.

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Thomas, David; Finan, Chris; Newport, Melanie J; Jones, Susan

2015-10-01

The complexity of DNA can be quantified using estimates of entropy. Variation in DNA complexity is expected between the promoters of genes with different transcriptional mechanisms; namely housekeeping (HK) and tissue specific (TS). The former are transcribed constitutively to maintain general cellular functions, and the latter are transcribed in restricted tissue and cells types for specific molecular events. It is known that promoter features in the human genome are related to tissue specificity, but this has been difficult to quantify on a genomic scale. If entropy effectively quantifies DNA complexity, calculating the entropies of HK and TS gene promoters as profiles may reveal significant differences. Entropy profiles were calculated for a total dataset of 12,003 human gene promoters and for 501 housekeeping (HK) and 587 tissue specific (TS) human gene promoters. The mean profiles show the TS promoters have a significantly lower entropy (pentropy distributions for the 3 datasets show that promoter entropies could be used to identify novel HK genes. Functional features comprise DNA sequence patterns that are non-random and hence they have lower entropies. The lower entropy of TS gene promoters can be explained by a higher density of positive and negative regulatory elements, required for genes with complex spatial and temporary expression. Copyright © 2015 Elsevier Ltd. All rights reserved.

RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization.

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Julia F Pielage

2008-03-01

Full Text Available Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of approximately 80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa-induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.

Evidence that the respiratory syncytial virus polymerase complex associates with lipid rafts in virus-infected cells: a proteomic analysis

International Nuclear Information System (INIS)

McDonald, Terence P.; Pitt, Andrew R.; Brown, Gaie; Rixon, Helen W. McL.; Sugrue, Richard J.

2004-01-01

The interaction between the respiratory syncytial virus (RSV) polymerase complex and lipid rafts was examined in HEp2 cells. Lipid-raft membranes were prepared from virus-infected cells and their protein content was analysed by Western blotting and mass spectrometry. This analysis revealed the presence of the N, P, L, M2-1 and M proteins. However, these proteins appeared to differ from one another in their association with these structures, with the M2-1 protein showing a greater partitioning into raft membranes compared to that of the N, P or M proteins. Determination of the polymerase activity profile of the gradient fractions revealed that 95% of the detectable viral enzyme activity was associated with lipid-raft membranes. Furthermore, analysis of virus-infected cells by confocal microscopy suggested an association between these proteins and the raft-lipid, GM1. Together, these results provide evidence that the RSV polymerase complex is able to associate with lipid rafts in virus-infected cells

Spindle cell carcinoma of the breast as complex cystic lesion: a case report

International Nuclear Information System (INIS)

Kitada, Masahiro; Hayashi, Satoshi; Matsuda, Yoshinari; Ishibashi, Kei; Oikawa, Keisuke; Miyokawa, Naoyuki

2014-01-01

Spindle cell carcinoma of the breast is a rare tumor. This tumor can proliferate rapidly and cause cystic changes because of internal tissue necrosis. We evaluated a 54-year-old woman with right breast lump. Mammography showed a category four mass with a diameter of 2.5 cm. Ultrasonography (US) revealed a complex cystic lesion, and fine-needle aspiration (FNA) cytology demonstrated bloody fluid and malignant cells. Partial breast resection and sentinel lymph node biopsy were performed. Immunohistology revealed spindle cells with positive results for cytokeratin (AE1/AE3) and vimentin, partially positive results for s-100, and negative results for desmin and α-actin. The pathological stage was IIA, and biochemical characterization showed that the tumor was triple negative. Six courses of FEC-100 chemotherapy (5-fluorouracil 500 mg/m 2 , epirubicin 100 mg/m 2 , and cyclophosphamide 500 mg/m 2 ) were administered. Radiotherapy was performed. This case is discussed with reference to the literature

Combining complexity measures of EEG data: multiplying measures reveal previously hidden information.

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Burns, Thomas; Rajan, Ramesh

2015-01-01

Many studies have noted significant differences among human electroencephalograph (EEG) results when participants or patients are exposed to different stimuli, undertaking different tasks, or being affected by conditions such as epilepsy or Alzheimer's disease. Such studies often use only one or two measures of complexity and do not regularly justify their choice of measure beyond the fact that it has been used in previous studies. If more measures were added to such studies, however, more complete information might be found about these reported differences. Such information might be useful in confirming the existence or extent of such differences, or in understanding their physiological bases. In this study we analysed publically-available EEG data using a range of complexity measures to determine how well the measures correlated with one another. The complexity measures did not all significantly correlate, suggesting that different measures were measuring unique features of the EEG signals and thus revealing information which other measures were unable to detect. Therefore, the results from this analysis suggests that combinations of complexity measures reveal unique information which is in addition to the information captured by other measures of complexity in EEG data. For this reason, researchers using individual complexity measures for EEG data should consider using combinations of measures to more completely account for any differences they observe and to ensure the robustness of any relationships identified.

Comparison of two binuclear vanadium-catecholate complexes: Synthesis, X-ray structure and effects in cancer cells

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Chi, Zixiang; Zhu, Linli; Lu, Xiaoming

2011-08-01

Two binuclear vanadium-catecholate complexes [Et 3NH] 2[V VO 2(μ-cat)] 2( 1) and [Et 3NH] 2[V VO 2(μ-N-2,3-D)] 2( 2) (cat = catechol, N-2,3-D = naphthalene-2,3-diol) have been synthesized and characterized by X-ray diffraction, IR, UV-vis spectroscopy and cyclic voltammetry (CV). X-ray analysis reveals that the structures of complexes 1 and 2 are both in the anion form of V. Et 3N works as counter-ions and connects the main frame by hydrogen bonding. The electrochemical behavior of the two complexes is studied in comparison to that of the free ligands and the two complexes display different redox potentials. Pharmaceutical screenings of complexes 1 and 2 have been made against two representative cancer cell-lines A-549 (lung cancer) and Bel-7402 (liver cancer) by MTT assay. The inhibition of cell proliferation was determined 72 h after cells were exposed to the tested compounds at a concentration of 5 μg/mL. Complex 1 exhibits well inhibition ratio against both two cell-lines (76.28% and 75.94%), while 2 displays positive and negative effect (65.36% and -68.82%) respectively. In association with X-ray and electrochemistry, a preliminary analysis about the possible inhibitory mechanism is provided.

The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes.

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Divakaruni, Arun V; Baida, Cyril; White, Courtney L; Gober, James W

2007-10-01

MreB, the bacterial actin homologue, is thought to function in spatially co-ordinating cell morphogenesis in conjunction with MreC, a protein that wraps around the outside of the cell within the periplasmic space. In Caulobacter crescentus, MreC physically associates with penicillin-binding proteins (PBPs) which catalyse the insertion of intracellularly synthesized precursors into the peptidoglycan cell wall. Here we show that MreC is required for the spatial organization of components of the peptidoglycan-synthesizing holoenzyme in the periplasm and MreB directs the localization of a peptidoglycan precursor synthesis protein in the cytosol. Additionally, fluorescent vancomycin (Van-FL) labelling revealed that the bacterial cytoskeletal proteins MreB and FtsZ, as well as MreC and RodA, were required for peptidoglycan synthetic activity. MreB and FtsZ were found to be required for morphogenesis of the polar stalk. FtsZ was required for a cell cycle-regulated burst of peptidoglycan synthesis early in the cell cycle resulting in the synthesis of cross-band structures, whereas MreB was required for lengthening of the stalk. Thus, the bacterial cytoskeleton and cell shape-determining proteins such as MreC, function in concert to orchestrate the localization of cell wall synthetic complexes resulting in spatially co-ordinated and efficient peptidoglycan synthetic activity.

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

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Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

2011-09-27

The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

Non-SMC condensin I complex proteins control chromosome segregation and survival of proliferating cells in the zebrafish neural retina

Directory of Open Access Journals (Sweden)

Harris William A

2009-07-01

Full Text Available Abstract Background The condensation of chromosomes and correct sister chromatid segregation during cell division is an essential feature of all proliferative cells. Structural maintenance of chromosomes (SMC and non-SMC proteins form the condensin I complex and regulate chromosome condensation and segregation during mitosis. However, due to the lack of appropriate mutants, the function of the condensin I complex during vertebrate development has not been described. Results Here, we report the positional cloning and detailed characterization of retinal phenotypes of a zebrafish mutation at the cap-g locus. High resolution live imaging reveals that the progression of mitosis between prometa- to telophase is delayed and that sister chromatid segregation is impaired upon loss of CAP-G. CAP-G associates with chromosomes between prometa- and telophase of the cell cycle. Loss of the interaction partners CAP-H and CAP-D2 causes cytoplasmic mislocalization of CAP-G throughout mitosis. DNA content analysis reveals increased genomic imbalances upon loss of non-SMC condensin I subunits. Within the retina, loss of condensin I function causes increased rates of apoptosis among cells within the proliferative ciliary marginal zone (CMZ whereas postmitotic retinal cells are viable. Inhibition of p53-mediated apoptosis partially rescues cell numbers in cap-g mutant retinae and allows normal layering of retinal cell types without alleviating their aberrant nuclear sizes. Conclusion Our findings indicate that the condensin I complex is particularly important within rapidly amplifying progenitor cell populations to ensure faithful chromosome segregation. In contrast, differentiation of postmitotic retinal cells is not impaired upon polyploidization.

Cdt1 revisited: complex and tight regulation during the cell cycle and consequences of deregulation in mammalian cells

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Fujita Masatoshi

2006-10-01

Full Text Available Abstract In eukaryotic cells, replication of genomic DNA initiates from multiple replication origins distributed on multiple chromosomes. To ensure that each origin is activated precisely only once during each S phase, a system has evolved which features periodic assembly and disassembly of essential pre-replication complexes (pre-RCs at replication origins. The pre-RC assembly reaction involves the loading of a presumptive replicative helicase, the MCM2-7 complexes, onto chromatin by the origin recognition complex (ORC and two essential factors, CDC6 and Cdt1. The eukaryotic cell cycle is driven by the periodic activation and inactivation of cyclin-dependent kinases (Cdks and assembly of pre-RCs can only occur during the low Cdk activity period from late mitosis through G1 phase, with inappropriate re-assembly suppressed during S, G2, and M phases. It was originally suggested that inhibition of Cdt1 function after S phase in vertebrate cells is due to geminin binding and that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance induces re-replication. However, recent progress has revealed that Cdt1 activity is more strictly regulated by two other mechanisms in addition to geminin: (1 functional and SCFSkp2-mediated proteolytic regulation through phosphorylation by Cdks; and (2 replication-coupled proteolysis mediated by the Cullin4-DDB1Cdt2 ubiquitin ligase and PCNA, an eukaryotic sliding clamp stimulating replicative DNA polymerases. The tight regulation implies that Cdt1 control is especially critical for the regulation of DNA replication in mammalian cells. Indeed, Cdt1 overexpression evokes chromosomal damage even without re-replication. Furthermore, deregulated Cdt1 induces chromosomal instability in normal human cells. Since Cdt1 is overexpressed in cancer cells, this could be a new molecular mechanism leading to carcinogenesis. In this review, recent insights into Cdt1 function and regulation in mammalian cells are discussed.

Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding

NARCIS (Netherlands)

Goeij, de J.M.; Kluijver, de A.; Duyl, van F.C.; Vacelet, J.; Wijffels, R.H.; Goeij, de A.F.P.M.; Cleutjens, J.P.M.; Schutte, B.

2009-01-01

This study reveals the peculiar in vivo cell kinetics and cell turnover of the marine sponge Halisarca caerulea under steady-state conditions. The tropical coral reef sponge shows an extremely high proliferation activity, a short cell cycle duration and massive cell shedding. Cell turnover is

Novel ruthenium methylcyclopentadienyl complex bearing a bipyridine perfluorinated ligand shows strong activity towards colorectal cancer cells.

Science.gov (United States)

Teixeira, Ricardo G; Brás, Ana Rita; Côrte-Real, Leonor; Tatikonda, Rajendhraprasad; Sanches, Anabela; Robalo, M Paula; Avecilla, Fernando; Moreira, Tiago; Garcia, M Helena; Haukka, Matti; Preto, Ana; Valente, Andreia

2018-01-01

Three new compounds have been synthesized and completely characterized by analytical and spectroscopic techniques. The new bipyridine-perfluorinated ligand L1 and the new organometallic complex [Ru(η 5 -MeCp)(PPh 3 ) 2 Cl] (Ru1) crystalize in the centrosymmetric triclinic space group P1¯. Analysis of the phenotypic effects induced by both organometallic complexes Ru1 and [Ru(η 5 -MeCp)(PPh 3 )(L1)][CF 3 SO 3 ] (Ru2), on human colorectal cancer cells (SW480 and RKO) survival, showed that Ru2 has a potent anti-proliferative activity, 4-6 times higher than cisplatin, and induce apoptosis in these cells. Data obtained in a noncancerous cell line derived from normal colon epithelial cells (NCM460) revealed an intrinsic selectivity of Ru2 for malignant cells at low concentrations, showing the high potential of this compound as a selective anticancer agent. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Quantum coherence spectroscopy reveals complex dynamics in bacterial light-harvesting complex 2 (LH2).

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Harel, Elad; Engel, Gregory S

2012-01-17

Light-harvesting antenna complexes transfer energy from sunlight to photosynthetic reaction centers where charge separation drives cellular metabolism. The process through which pigments transfer excitation energy involves a complex choreography of coherent and incoherent processes mediated by the surrounding protein and solvent environment. The recent discovery of coherent dynamics in photosynthetic light-harvesting antennae has motivated many theoretical models exploring effects of interference in energy transfer phenomena. In this work, we provide experimental evidence of long-lived quantum coherence between the spectrally separated B800 and B850 rings of the light-harvesting complex 2 (LH2) of purple bacteria. Spectrally resolved maps of the detuning, dephasing, and the amplitude of electronic coupling between excitons reveal that different relaxation pathways act in concert for optimal transfer efficiency. Furthermore, maps of the phase of the signal suggest that quantum mechanical interference between different energy transfer pathways may be important even at ambient temperature. Such interference at a product state has already been shown to enhance the quantum efficiency of transfer in theoretical models of closed loop systems such as LH2.

Crystal structure of Vδ1 T cell receptor in complex with CD1d-sulfatide shows MHC-like recognition of a self-lipid by human γδ T cells.

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Luoma, Adrienne M; Castro, Caitlin D; Mayassi, Toufic; Bembinster, Leslie A; Bai, Li; Picard, Damien; Anderson, Brian; Scharf, Louise; Kung, Jennifer E; Sibener, Leah V; Savage, Paul B; Jabri, Bana; Bendelac, Albert; Adams, Erin J

2013-12-12

The nature of the antigens recognized by γδ T cells and their potential recognition of major histocompatibility complex (MHC)-like molecules has remained unclear. Members of the CD1 family of lipid-presenting molecules are suggested ligands for Vδ1 TCR-expressing γδ T cells, the major γδ lymphocyte population in epithelial tissues. We crystallized a Vδ1 TCR in complex with CD1d and the self-lipid sulfatide, revealing the unusual recognition of CD1d by germline Vδ1 residues spanning all complementarity-determining region (CDR) loops, as well as sulfatide recognition separately encoded by nongermline CDR3δ residues. Binding and functional analysis showed that CD1d presenting self-lipids, including sulfatide, was widely recognized by gut Vδ1+ γδ T cells. These findings provide structural demonstration of MHC-like recognition of a self-lipid by γδ T cells and reveal the prevalence of lipid recognition by innate-like T cell populations. Copyright © 2013 Elsevier Inc. All rights reserved.

Isolation and characterization of antigen-Ia complexes involved in T cell recognition

DEFF Research Database (Denmark)

Buus, S; Sette, A; Colon, S M

1986-01-01

Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes, it is he......Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes...... with glutaraldehyde revealed that the ovalbumin peptide was cross-linked solely to the alpha chain of I-Ad. Planar membranes containing I-Ad-OVA complexes stimulated a T cell response with 2 X 10(4) less antigen than required when uncomplexed antigen was used, thus demonstrating the biologic importance...

Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

International Nuclear Information System (INIS)

Economou, Nicoleta J.; Zentner, Isaac J.; Lazo, Edwin; Jakoncic, Jean; Stojanoff, Vivian; Weeks, Stephen D.; Grasty, Kimberly C.; Cocklin, Simon; Loll, Patrick J.

2013-01-01

Using a carrier-protein strategy, the structure of teicoplanin bound to its bacterial cell-wall target has been determined. The structure reveals the molecular determinants of target recognition, flexibility in the antibiotic backbone and intrinsic radiation sensitivity of teicoplanin. Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex. The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance

CRIM1 complexes with ß-catenin and cadherins, stabilizes cell-cell junctions and is critical for neural morphogenesis.

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Virgilio G Ponferrada

Full Text Available In multicellular organisms, morphogenesis is a highly coordinated process that requires dynamically regulated adhesion between cells. An excellent example of cellular morphogenesis is the formation of the neural tube from the flattened epithelium of the neural plate. Cysteine-rich motor neuron protein 1 (CRIM1 is a single-pass (type 1 transmembrane protein that is expressed in neural structures beginning at the neural plate stage. In the frog Xenopus laevis, loss of function studies using CRIM1 antisense morpholino oligonucleotides resulted in a failure of neural development. The CRIM1 knockdown phenotype was, in some cases, mild and resulted in perturbed neural fold morphogenesis. In severely affected embryos there was a dramatic failure of cell adhesion in the neural plate and complete absence of neural structures subsequently. Investigation of the mechanism of CRIM1 function revealed that it can form complexes with ß-catenin and cadherins, albeit indirectly, via the cytosolic domain. Consistent with this, CRIM1 knockdown resulted in diminished levels of cadherins and ß-catenin in junctional complexes in the neural plate. We conclude that CRIM1 is critical for cell-cell adhesion during neural development because it is required for the function of cadherin-dependent junctions.

Gold nanoparticle-mediated laser stimulation induces a complex stress response in neuronal cells.

Science.gov (United States)

Johannsmeier, Sonja; Heeger, Patrick; Terakawa, Mitsuhiro; Kalies, Stefan; Heisterkamp, Alexander; Ripken, Tammo; Heinemann, Dag

2018-04-25

Stimulation of neuronal cells generally resorts to electric signals. Recent advances in laser-based stimulation methods could present an alternative with superior spatiotemporal resolution. The avoidance of electronic crosstalk makes these methods attractive for in vivo therapeutic application. In particular, nano-mediators, such as gold nanoparticles, can be used to transfer the energy from a laser pulse to the cell membrane and subsequently activate excitable cells. Although the underlying mechanisms of neuronal activation have been widely unraveled, the overall effect on the targeted cell is not understood. Little is known about the physiological and pathophysiological impact of a laser pulse targeted onto nanoabsorbers on the cell membrane. Here, we analyzed the reaction of the neuronal murine cell line Neuro-2A and murine primary cortical neurons to gold nanoparticle mediated laser stimulation. Our study reveals a severe, complex and cell-type independent stress response after laser irradiation, emphasizing the need for a thorough assessment of this approach's efficacy and safety.

'Big bang' of B-cell development revealed.

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Murre, Cornelis

2018-01-15

Earlier studies have identified transcription factors that specify B-cell fate, but the underlying mechanisms remain to be revealed. Two new studies by Miyai and colleagues (pp. 112-126) and Li and colleagues (pp. 96-111) in this issue of Genes & Development provide new and unprecedented insights into the genetic and epigenetic mechanisms that establish B-cell identity. © 2018 Murre; Published by Cold Spring Harbor Laboratory Press.

Lipid clustering correlates with membrane curvature as revealed by molecular simulations of complex lipid bilayers.

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Heidi Koldsø

2014-10-01

Full Text Available Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP2, in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP2, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.

Whole-transcriptome analysis of endothelial to hematopoietic stem cell transition reveals a requirement for Gpr56 in HSC generation.

Science.gov (United States)

Solaimani Kartalaei, Parham; Yamada-Inagawa, Tomoko; Vink, Chris S; de Pater, Emma; van der Linden, Reinier; Marks-Bluth, Jonathon; van der Sloot, Anthon; van den Hout, Mirjam; Yokomizo, Tomomasa; van Schaick-Solernó, M Lucila; Delwel, Ruud; Pimanda, John E; van IJcken, Wilfred F J; Dzierzak, Elaine

2015-01-12

Hematopoietic stem cells (HSCs) are generated via a natural transdifferentiation process known as endothelial to hematopoietic cell transition (EHT). Because of small numbers of embryonal arterial cells undergoing EHT and the paucity of markers to enrich for hemogenic endothelial cells (ECs [HECs]), the genetic program driving HSC emergence is largely unknown. Here, we use a highly sensitive RNAseq method to examine the whole transcriptome of small numbers of enriched aortic HSCs, HECs, and ECs. Gpr56, a G-coupled protein receptor, is one of the most highly up-regulated of the 530 differentially expressed genes. Also, highly up-regulated are hematopoietic transcription factors, including the "heptad" complex of factors. We show that Gpr56 (mouse and human) is a target of the heptad complex and is required for hematopoietic cluster formation during EHT. Our results identify the processes and regulators involved in EHT and reveal the surprising requirement for Gpr56 in generating the first HSCs. © 2015 Solaimani Kartalaei et al.

Genomewide effects of peroxisome proliferator-activated receptor gamma in macrophages and dendritic cells--revealing complexity through systems biology.

Science.gov (United States)

Cuaranta-Monroy, Ixchelt; Kiss, Mate; Simandi, Zoltan; Nagy, Laszlo

2015-09-01

Systems biology approaches have become indispensable tools in biomedical and basic research. These data integrating bioinformatic methods gained prominence after high-throughput technologies became available to investigate complex cellular processes, such as transcriptional regulation and protein-protein interactions, on a scale that had not been studied before. Immunology is one of the medical fields that systems biology impacted profoundly due to the plasticity of cell types involved and the accessibility of a wide range of experimental models. In this review, we summarize the most important recent genomewide studies exploring the function of peroxisome proliferator-activated receptor γ in macrophages and dendritic cells. PPARγ ChIP-seq experiments were performed in adipocytes derived from embryonic stem cells to complement the existing data sets and to provide comparators to macrophage data. Finally, lists of regulated genes generated from such experiments were analysed with bioinformatics and system biology approaches. We show that genomewide studies utilizing high-throughput data acquisition methods made it possible to gain deeper insights into the role of PPARγ in these immune cell types. We also demonstrate that analysis and visualization of data using network-based approaches can be used to identify novel genes and functions regulated by the receptor. The example of PPARγ in macrophages and dendritic cells highlights the crucial importance of systems biology approaches in establishing novel cellular functions for long-known signaling pathways. © 2015 Stichting European Society for Clinical Investigation Journal Foundation.

Cell complexes of transition metals in biochemistry and medicine

International Nuclear Information System (INIS)

Voloshin, Ya.Z.; Varzatskij, O.A.; Bubnov, Yu.N.

2007-01-01

Basic directions and prospects of use of cell complexes of transition metals in medicine and biochemistry are considered: incapsulation of radioactive metal ions for radiotherapy and diagnostics; preparation of contrast compounds for magnetic resonance tomography, antidotes and pharmaceutical preparation of prolonged effect, preparations for boron-neutron-capture therapy of neoplasms, antioxidants; membrane transport of metal ions; study of interaction of cell metal complexes with nucleic acids; possibility of use of self-assembly of cell complexes for imitation of ligases and use of clathrochelates as linkers; design of inhibitors of viruses for AIDS therapy [ru

B lymphocytes confer immune tolerance via cell surface GARP-TGF-β complex.

Science.gov (United States)

Wallace, Caroline H; Wu, Bill X; Salem, Mohammad; Ansa-Addo, Ephraim A; Metelli, Alessandra; Sun, Shaoli; Gilkeson, Gary; Shlomchik, Mark J; Liu, Bei; Li, Zihai

2018-04-05

GARP, a cell surface docking receptor for binding and activating latent TGF-β, is highly expressed by platelets and activated Tregs. While GARP is implicated in immune invasion in cancer, the roles of the GARP-TGF-β axis in systemic autoimmune diseases are unknown. Although B cells do not express GARP at baseline, we found that the GARP-TGF-β complex is induced on activated human and mouse B cells by ligands for multiple TLRs, including TLR4, TLR7, and TLR9. GARP overexpression on B cells inhibited their proliferation, induced IgA class-switching, and dampened T cell-independent antibody production. In contrast, B cell-specific deletion of GARP-encoding gene Lrrc32 in mice led to development of systemic autoimmune diseases spontaneously as well as worsening of pristane-induced lupus-like disease. Canonical TGF-β signaling more readily upregulates GARP in Peyer patch B cells than in splenic B cells. Furthermore, we demonstrated that B cells are required for the induction of oral tolerance of T cell-dependent antigens via GARP. Our studies reveal for the first time to our knowledge that cell surface GARP-TGF-β is an important checkpoint for regulating B cell peripheral tolerance, highlighting a mechanism of autoimmune disease pathogenesis.

Dynamic Regulation of a Cell Adhesion Protein Complex Including CADM1 by Combinatorial Analysis of FRAP with Exponential Curve-Fitting

Science.gov (United States)

Sakurai-Yageta, Mika; Maruyama, Tomoko; Suzuki, Takashi; Ichikawa, Kazuhisa; Murakami, Yoshinori

2015-01-01

Protein components of cell adhesion machinery show continuous renewal even in the static state of epithelial cells and participate in the formation and maintenance of normal epithelial architecture and tumor suppression. CADM1 is a tumor suppressor belonging to the immunoglobulin superfamily of cell adhesion molecule and forms a cell adhesion complex with an actin-binding protein, 4.1B, and a scaffold protein, MPP3, in the cytoplasm. Here, we investigate dynamic regulation of the CADM1-4.1B-MPP3 complex in mature cell adhesion by fluorescence recovery after photobleaching (FRAP) analysis. Traditional FRAP analysis were performed for relatively short period of around 10min. Here, thanks to recent advances in the sensitive laser detector systems, we examine FRAP of CADM1 complex for longer period of 60 min and analyze the recovery with exponential curve-fitting to distinguish the fractions with different diffusion constants. This approach reveals that the fluorescence recovery of CADM1 is fitted to a single exponential function with a time constant (τ) of approximately 16 min, whereas 4.1B and MPP3 are fitted to a double exponential function with two τs of approximately 40-60 sec and 16 min. The longer τ is similar to that of CADM1, suggesting that 4.1B and MPP3 have two distinct fractions, one forming a complex with CADM1 and the other present as a free pool. Fluorescence loss in photobleaching analysis supports the presence of a free pool of these proteins near the plasma membrane. Furthermore, double exponential fitting makes it possible to estimate the ratio of 4.1B and MPP3 present as a free pool and as a complex with CADM1 as approximately 3:2 and 3:1, respectively. Our analyses reveal a central role of CADM1 in stabilizing the complex with 4.1B and MPP3 and provide insight in the dynamics of adhesion complex formation. PMID:25780926

Polylox barcoding reveals haematopoietic stem cell fates realized in vivo

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Rössler, Jens; Wang, Xi; Postrach, Daniel; Busch, Katrin; Rode, Immanuel; Klapproth, Kay; Dietlein, Nikolaus; Quedenau, Claudia; Chen, Wei; Sauer, Sascha; Wolf, Stephan; Höfer, Thomas; Rodewald, Hans-Reimer

2017-01-01

Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping1 has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites2, viral barcodes3, and strategies based on transposons4 and CRISPR/Cas9 genome editing5; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system6,7. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs8–10. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure. PMID:28813413

Polylox barcoding reveals haematopoietic stem cell fates realized in vivo.

Science.gov (United States)

Pei, Weike; Feyerabend, Thorsten B; Rössler, Jens; Wang, Xi; Postrach, Daniel; Busch, Katrin; Rode, Immanuel; Klapproth, Kay; Dietlein, Nikolaus; Quedenau, Claudia; Chen, Wei; Sauer, Sascha; Wolf, Stephan; Höfer, Thomas; Rodewald, Hans-Reimer

2017-08-24

Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.

Chemoproteomics Reveals Chemical Diversity and Dynamics of 4-Oxo-2-nonenal Modifications in Cells.

Science.gov (United States)

Sun, Rui; Fu, Ling; Liu, Keke; Tian, Caiping; Yang, Yong; Tallman, Keri A; Porter, Ned A; Liebler, Daniel C; Yang, Jing

2017-10-01

4-Oxo-2-nonenal (ONE) derived from lipid peroxidation modifies nucleophiles and transduces redox signaling by its reactions with proteins. However, the molecular interactions between ONE and complex proteomes and their dynamics in situ remain largely unknown. Here we describe a quantitative chemoproteomic analysis of protein adduction by ONE in cells, in which the cellular target profile of ONE is mimicked by its alkynyl surrogate. The analyses reveal four types of ONE-derived modifications in cells, including ketoamide and Schiff-base adducts to lysine, Michael adducts to cysteine, and a novel pyrrole adduct to cysteine. ONE-derived adducts co-localize and exhibit crosstalk with many histone marks and redox sensitive sites. All four types of modifications derived from ONE can be reversed site-specifically in cells. Taken together, our study provides much-needed mechanistic insights into the cellular signaling and potential toxicities associated with this important lipid derived electrophile. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Multi-region and single-cell sequencing reveal variable genomic heterogeneity in rectal cancer.

Science.gov (United States)

Liu, Mingshan; Liu, Yang; Di, Jiabo; Su, Zhe; Yang, Hong; Jiang, Beihai; Wang, Zaozao; Zhuang, Meng; Bai, Fan; Su, Xiangqian

2017-11-23

Colorectal cancer is a heterogeneous group of malignancies with complex molecular subtypes. While colon cancer has been widely investigated, studies on rectal cancer are very limited. Here, we performed multi-region whole-exome sequencing and single-cell whole-genome sequencing to examine the genomic intratumor heterogeneity (ITH) of rectal tumors. We sequenced nine tumor regions and 88 single cells from two rectal cancer patients with tumors of the same molecular classification and characterized their mutation profiles and somatic copy number alterations (SCNAs) at the multi-region and the single-cell levels. A variable extent of genomic heterogeneity was observed between the two patients, and the degree of ITH increased when analyzed on the single-cell level. We found that major SCNAs were early events in cancer development and inherited steadily. Single-cell sequencing revealed mutations and SCNAs which were hidden in bulk sequencing. In summary, we studied the ITH of rectal cancer at regional and single-cell resolution and demonstrated that variable heterogeneity existed in two patients. The mutational scenarios and SCNA profiles of two patients with treatment naïve from the same molecular subtype are quite different. Our results suggest each tumor possesses its own architecture, which may result in different diagnosis, prognosis, and drug responses. Remarkable ITH exists in the two patients we have studied, providing a preliminary impression of ITH in rectal cancer.

Cadherin complexes recruit mRNAs and RISC to regulate epithelial cell signaling.

Science.gov (United States)

Kourtidis, Antonis; Necela, Brian; Lin, Wan-Hsin; Lu, Ruifeng; Feathers, Ryan W; Asmann, Yan W; Thompson, E Aubrey; Anastasiadis, Panos Z

2017-10-02

Cumulative evidence demonstrates that most RNAs exhibit specific subcellular distribution. However, the mechanisms regulating this phenomenon and its functional consequences are still under investigation. Here, we reveal that cadherin complexes at the apical zonula adherens (ZA) of epithelial adherens junctions recruit the core components of the RNA-induced silencing complex (RISC) Ago2, GW182, and PABPC1, as well as a set of 522 messenger RNAs (mRNAs) and 28 mature microRNAs (miRNAs or miRs), via PLEKHA7. Top canonical pathways represented by these mRNAs include Wnt/β-catenin, TGF-β, and stem cell signaling. We specifically demonstrate the presence and silencing of MYC, JUN, and SOX2 mRNAs by miR-24 and miR-200c at the ZA. PLEKHA7 knockdown dissociates RISC from the ZA, decreases loading of the ZA-associated mRNAs and miRNAs to Ago2, and results in a corresponding increase of MYC, JUN, and SOX2 protein expression. The present work reveals a mechanism that directly links junction integrity to the silencing of a set of mRNAs that critically affect epithelial homeostasis. © 2017 Kourtidis et al.

Information maximization explains the emergence of complex cell-like neurons

Directory of Open Access Journals (Sweden)

Takuma eTanaka

2013-11-01

Full Text Available We propose models and a method to qualitatively explain the receptive field properties of complex cells in the primary visual cortex. We apply a learning method based on the information maximization principle in a feedforward network, which comprises an input layer of image patches, simple cell-like first-output-layer neurons, and second-output-layer neurons (Model 1. The information maximization results in the emergence of the complex cell-like receptive field properties in the second-output-layer neurons. After learning, second-output-layer neurons receive connection weights having the same size from two first-output-layer neurons with sign-inverted receptive fields. The second-output-layer neurons replicate the phase invariance and iso-orientation suppression. Furthermore, on the basis of these results, we examine a simplified model showing the emergence of complex cell-like receptive fields (Model 2. We show that after learning, the output neurons of this model exhibit iso-orientation suppression, cross-orientation facilitation, and end stopping, which are similar to those found in complex cells. These properties of model neurons suggest that complex cells in the primary visual cortex become selective to features composed of edges to increase the variability of the output.

Phospho-eNOS Ser-1176 is associated with the nucleoli and the Golgi complex in C6 rat glioma cells.

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Klinz, Franz-Josef; Herberg, Natalie; Arnhold, Stefan; Addicks, Klaus; Bloch, Wilhelm

2007-06-29

Enzymatic activity of endothelial nitric oxide synthase (eNOS) is controlled by posttranslational modifications, protein-protein interactions, and subcellular localization. For example, N-terminal fatty acid modifications target eNOS to the Golgi complex where it becomes phosphorylated. We show here by immunofluorescence analysis that phospho-eNOS Ser-1176 is enriched in the perinuclear region of interphase C6 rat glioma cells. Confocal double immunofluorescence microscopy with the Golgi marker protein 58K revealed that phospho-eNOS Ser-1176 is associated with the Golgi complex. Surprisingly, we observed several spots in the nucleus of C6 cells that were positive for phospho-eNOS Ser-1176. Confocal double immunofluorescence analysis with the nucleolus marker protein fibrillarin revealed that within the nucleus phospho-eNOS Ser-1176 is exclusively associated with the nucleoli. It is known that in mitotic cells nucleoli are lost during prophase and rebuild during telophase. In agreement with this, we find no nucleoli-like distribution of phospho-eNOS Ser-1176 in metaphase and anaphase C6 glioma cells. Our finding that phospho-eNOS Ser-1176 is selectively associated with the nucleoli points to a so far unknown role for eNOS in interphase glioma cells.

Haplotyping the human T-cell receptor β-chain gene complex by use of restriction fragment length polymorphisms

International Nuclear Information System (INIS)

Charmley, P.; Chao, A.; Gatti, R.A.; Concannon, P.; Hood, L.

1990-01-01

The authors have studied the genetic segregation of human T-cell receptor β-chain (TCRβ) genes on chromosome 7q in 40 CEPH (Centre d'Etude du Polymorphisme Humain) families by using restriction fragment length polymorphisms (RFLPs). They constructed haplotypes from eight RFLPs by using variable- and constant-region cDNA probes, which detect polymorphisms that span more than 600 kilobases of the TCRβ gene complex. Analysis of allele distributions between TCRβ genes revealed significant linkage disequilibrium between only 6 of the 28 different pairs of RFLPs. This linkage disequilibrium strongly influences the most efficient order to proceed for typing of these RFLPs in order to achieve maximum genetic informativeness, which in this study revealed a 97.3% level of heterozygosity within the TCRβ gene complex. The results should provide new insight into recent reports of disease associations with the TCRβ gene complex and should assist in designing future experiments to detect or confirm the existence of disease-susceptibility loci in this region of the human genome

Survey of large protein complexes D. vulgaris reveals great structural diversity

Energy Technology Data Exchange (ETDEWEB)

Han, B.-G.; Dong, M.; Liu, H.; Camp, L.; Geller, J.; Singer, M.; Hazen, T. C.; Choi, M.; Witkowska, H. E.; Ball, D. A.; Typke, D.; Downing, K. H.; Shatsky, M.; Brenner, S. E.; Chandonia, J.-M.; Biggin, M. D.; Glaeser, R. M.

2009-08-15

An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr {approx} 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate {approx}10 times greater than that of previous 'proteomic' screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.

A New Method to Develop Human Dental Pulp Cells and Platelet-rich Fibrin Complex.

Science.gov (United States)

He, Xuan; Chen, Wen-Xia; Ban, Guifei; Wei, Wei; Zhou, Jun; Chen, Wen-Jin; Li, Xian-Yu

2016-11-01

Platelet-rich fibrin (PRF) has been used as a scaffold material in various tissue regeneration studies. In the previous methods to combine seed cells with PRF, the structure of PRF was damaged, and the manipulation time in vitro was also increased. The objective of this in vitro study was to explore an appropriate method to develop a PRF-human dental pulp cell (hDPC) complex to maintain PRF structure integrity and to find out the most efficient part of PRF. The PRF-hDPC complex was developed at 3 different time points during PRF preparation: (1) the before centrifugation (BC) group, the hDPC suspension was added to the venous blood before blood centrifugation; (2) the immediately after centrifugation (IAC) group, the hDPC suspension was added immediately after blood centrifugation; (3) the after centrifugation (AC) group, the hDPC suspension was added 10 minutes after blood centrifugation; and (4) the control group, PRF without hDPC suspension. The prepared PRF-hDPC complexes were cultured for 7 days. The samples were fixed for histologic, immunohistochemistry, and scanning electron microscopic evaluation. Real-time polymerase chain reaction was performed to evaluate messenger RNA expression of alkaline phosphatase and dentin sialophosphoprotein. Enzyme-linked immunosorbent assay quantification for growth factors was performed within the different parts of the PRF. Histologic, immunohistochemistry, and scanning electron microscopic results revealed that hDPCs were only found in the BC group and exhibited favorable proliferation. Real-time polymerase chain reaction revealed that alkaline phosphatase and dentin sialophosphoprotein expression increased in the cultured PRF-hDPC complex. The lower part of the PRF released the maximum quantity of growth factors. Our new method to develop a PRF-hDPCs complex maintained PRF structure integrity. The hDPCs were distributed in the buffy coat, which might be the most efficient part of PRF. Copyright © 2016 American

Complex N-Glycans Influence the Spatial Arrangement of Voltage Gated Potassium Channels in Membranes of Neuronal-Derived Cells.

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M Kristen Hall

Full Text Available The intrinsic electrical properties of a neuron depend on expression of voltage gated potassium (Kv channel isoforms, as well as their distribution and density in the plasma membrane. Recently, we showed that N-glycosylation site occupancy of Kv3.1b modulated its placement in the cell body and neurites of a neuronal-derived cell line, B35 neuroblastoma cells. To extrapolate this mechanism to other N-glycosylated Kv channels, we evaluated the impact of N-glycosylation occupancy of Kv3.1a and Kv1.1 channels. Western blots revealed that wild type Kv3.1a and Kv1.1 α-subunits had complex and oligomannose N-glycans, respectively, and that abolishment of the N-glycosylation site(s generated Kv proteins without N-glycans. Total internal reflection fluorescence microscopy images revealed that N-glycans of Kv3.1a contributed to its placement in the cell membrane while N-glycans had no effect on the distribution of Kv1.1. Based on particle analysis of EGFP-Kv proteins in the adhered membrane, glycosylated forms of Kv3.1a, Kv1.1, and Kv3.1b had differences in the number, size or density of Kv protein clusters in the cell membrane of neurites and cell body of B35 cells. Differences were also observed between the unglycosylated forms of the Kv proteins. Cell dissociation assays revealed that cell-cell adhesion was increased by the presence of complex N-glycans of Kv3.1a, like Kv3.1b, whereas cell adhesion was similar in the oligomannose and unglycosylated Kv1.1 subunit containing B35 cells. Our findings provide direct evidence that N-glycans of Kv3.1 splice variants contribute to the placement of these glycoproteins in the plasma membrane of neuronal-derived cells while those of Kv1.1 were absent. Further when the cell membrane distribution of the Kv channel was modified by N-glycans then the cell-cell adhesion properties were altered. Our study demonstrates that N-glycosylation of Kv3.1a, like Kv3.1b, provides a mechanism for the distribution of these

Substrate recognition by complement convertases revealed in the C5-cobra venom factor complex

DEFF Research Database (Denmark)

Laursen, Nick Stub; Andersen, Kasper Røjkjær; Braren, Ingke

2011-01-01

with a protease subunit (Bb or C2a). We determined the crystal structures of the C3b homologue cobra venom factor (CVF) in complex with C5, and in complex with C5 and the inhibitor SSL7 at 4.3 Å resolution. The structures reveal a parallel two-point attachment between C5 and CVF, where the presence of SSL7 only...

Single-cell analysis reveals a link between CD3- and CD59-mediated signaling pathways in Jurkat T cells

International Nuclear Information System (INIS)

Lipp, A. M.

2012-01-01

Elevation of intracellular free calcium concentration ([Ca2+]i) is a key signal during T cell activation and is commonly used as a read-out parameter for stimulation of T cell signaling. Upon T cell stimulation a variety of calcium signals is produced by individual cells of the T cell population and the type of calcium signal strongly influences cell fate decisions. The heterogeneous nature of T cells is masked in ensemble measurements, which highlights the need for single-cell measurements. In this study we used single-cell calcium measurements in Jurkat cells to investigate signaling pathways, which are triggered by different proteins, namely CD3 and CD59. By application of an automated cluster algorithm the presented assay provides unbiased analysis of a large data set of individual calcium time traces generated by the whole cell population. By using this method we could demonstrate that the Jurkat population generates heterogeneous calcium signals in a stimulus-dependent manner. Furthermore, our data revealed the existence of a link between CD3- and CD59-mediated signaling pathways. Single-cell calcium measurements in Jurkat cells expressing different levels of the T cell receptor (TCR) complex indicated that CD59-mediated calcium signaling is critically dependent on TCR surface expression levels. In addition, triggering CD59-mediated calcium signaling resulted in down-regulation of TCR surface expression levels, which is known to happen upon direct TCR triggering too. Moreover, by using siRNA-mediated protein knock-downs and protein knock-out Jurkat mutants we could show that CD3- and CD59-mediated calcium signaling require identical key proteins. We therefore explored by which mechanism CD59-mediated signaling couples into TCR-mediated signaling. Fluorescence recovery after photobleaching (FRAP) experiments and live-cell protein-protein interaction assays provided no evidence of a direct physical interaction between CD3- and CD59-mediated signaling pathways

Presynaptic dystroglycan-pikachurin complex regulates the proper synaptic connection between retinal photoreceptor and bipolar cells.

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Omori, Yoshihiro; Araki, Fumiyuki; Chaya, Taro; Kajimura, Naoko; Irie, Shoichi; Terada, Koji; Muranishi, Yuki; Tsujii, Toshinori; Ueno, Shinji; Koyasu, Toshiyuki; Tamaki, Yasuhiro; Kondo, Mineo; Amano, Shiro; Furukawa, Takahisa

2012-05-02

Dystroglycan (DG) is a key component of the dystrophin-glycoprotein complex (DGC) at the neuromuscular junction postsynapse. In the mouse retina, the DGC is localized at the presynapse of photoreceptor cells, however, the function of presynaptic DGC is poorly understood. Here, we developed and analyzed retinal photoreceptor-specific DG conditional knock-out (DG CKO) mice. We found that the DG CKO retina showed a reduced amplitude and a prolonged implicit time of the ERG b-wave. Electron microscopic analysis revealed that bipolar dendrite invagination into the photoreceptor terminus is perturbed in the DG CKO retina. In the DG CKO retina, pikachurin, a DG ligand in the retina, is markedly decreased at photoreceptor synapses. Interestingly, in the Pikachurin(-/-) retina, the DG signal at the ribbon synaptic terminus was severely reduced, suggesting that pikachurin is required for the presynaptic accumulation of DG at the photoreceptor synaptic terminus, and conversely DG is required for pikachurin accumulation. Furthermore, we found that overexpression of pikachurin induces formation and clustering of a DG-pikachurin complex on the cell surface. The Laminin G repeats of pikachurin, which are critical for its oligomerization and interaction with DG, were essential for the clustering of the DG-pikachurin complex as well. These results suggest that oligomerization of pikachurin and its interaction with DG causes DG assembly on the synapse surface of the photoreceptor synaptic terminals. Our results reveal that the presynaptic interaction of pikachurin with DG at photoreceptor terminals is essential for both the formation of proper photoreceptor ribbon synaptic structures and normal retinal electrophysiology.

Regulatory complexity revealed by integrated cytological and RNA-seq analyses of meiotic substages in mouse spermatocytes.

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Ball, Robyn L; Fujiwara, Yasuhiro; Sun, Fengyun; Hu, Jianjun; Hibbs, Matthew A; Handel, Mary Ann; Carter, Gregory W

2016-08-12

The continuous and non-synchronous nature of postnatal male germ-cell development has impeded stage-specific resolution of molecular events of mammalian meiotic prophase in the testis. Here the juvenile onset of spermatogenesis in mice is analyzed by combining cytological and transcriptomic data in a novel computational analysis that allows decomposition of the transcriptional programs of spermatogonia and meiotic prophase substages. Germ cells from testes of individual mice were obtained at two-day intervals from 8 to 18 days post-partum (dpp), prepared as surface-spread chromatin and immunolabeled for meiotic stage-specific protein markers (STRA8, SYCP3, phosphorylated H2AFX, and HISTH1T). Eight stages were discriminated cytologically by combinatorial antibody labeling, and RNA-seq was performed on the same samples. Independent principal component analyses of cytological and transcriptomic data yielded similar patterns for both data types, providing strong evidence for substage-specific gene expression signatures. A novel permutation-based maximum covariance analysis (PMCA) was developed to map co-expressed transcripts to one or more of the eight meiotic prophase substages, thereby linking distinct molecular programs to cytologically defined cell states. Expression of meiosis-specific genes is not substage-limited, suggesting regulation of substage transitions at other levels. This integrated analysis provides a general method for resolving complex cell populations. Here it revealed not only features of meiotic substage-specific gene expression, but also a network of substage-specific transcription factors and relationships to potential target genes.

89Zr-Oxine Complex PET Cell Imaging in Monitoring Cell-based Therapies

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Wu, Haitao; Asiedu, Kingsley O.; Szajek, Lawrence P.; Griffiths, Gary L.; Choyke, Peter L.

2015-01-01

Purpose To develop a clinically translatable method of cell labeling with zirconium 89 (89Zr) and oxine to track cells with positron emission tomography (PET) in mouse models of cell-based therapy. Materials and Methods This study was approved by the institutional animal care committee. 89Zr-oxine complex was synthesized in an aqueous solution. Cell labeling conditions were optimized by using EL4 mouse lymphoma cells, and labeling efficiency was examined by using dendritic cells (DCs) (n = 4), naïve (n = 3) and activated (n = 3) cytotoxic T cells (CTLs), and natural killer (NK) (n = 4), bone marrow (n = 4), and EL4 (n = 4) cells. The effect of 89Zr labeling on cell survival, proliferation, and function were evaluated by using DCs (n = 3) and CTLs (n = 3). Labeled DCs (444–555 kBq/[5 × 106] cells, n = 5) and CTLs (185 kBq/[5 × 106] cells, n = 3) transferred to mice were tracked with microPET/CT. In a melanoma immunotherapy model, tumor targeting and cytotoxic function of labeled CTLs were evaluated with imaging (248.5 kBq/[7.7 × 106] cells, n = 4) and by measuring the tumor size (n = 6). Two-way analysis of variance was used to compare labeling conditions, the Wilcoxon test was used to assess cell survival and proliferation, and Holm-Sidak multiple tests were used to assess tumor growth and perform biodistribution analyses. Results 89Zr-oxine complex was synthesized at a mean yield of 97.3% ± 2.8 (standard deviation). It readily labeled cells at room temperature or 4°C in phosphate-buffered saline (labeling efficiency range, 13.0%–43.9%) and was stably retained (83.5% ± 1.8 retention on day 5 in DCs). Labeling did not affect the viability of DCs and CTLs when compared with nonlabeled control mice (P > .05), nor did it affect functionality. 89Zr-oxine complex enabled extended cell tracking for 7 days. Labeled tumor-specific CTLs accumulated in the tumor (4.6% on day 7) and induced tumor regression (P cell tracking technique for use with PET that is

(89)Zr-Oxine Complex PET Cell Imaging in Monitoring Cell-based Therapies.

Science.gov (United States)

Sato, Noriko; Wu, Haitao; Asiedu, Kingsley O; Szajek, Lawrence P; Griffiths, Gary L; Choyke, Peter L

2015-05-01

To develop a clinically translatable method of cell labeling with zirconium 89 ((89)Zr) and oxine to track cells with positron emission tomography (PET) in mouse models of cell-based therapy. This study was approved by the institutional animal care committee. (89)Zr-oxine complex was synthesized in an aqueous solution. Cell labeling conditions were optimized by using EL4 mouse lymphoma cells, and labeling efficiency was examined by using dendritic cells (DCs) (n = 4), naïve (n = 3) and activated (n = 3) cytotoxic T cells (CTLs), and natural killer (NK) (n = 4), bone marrow (n = 4), and EL4 (n = 4) cells. The effect of (89)Zr labeling on cell survival, proliferation, and function were evaluated by using DCs (n = 3) and CTLs (n = 3). Labeled DCs (444-555 kBq/[5 × 10(6)] cells, n = 5) and CTLs (185 kBq/[5 × 10(6)] cells, n = 3) transferred to mice were tracked with microPET/CT. In a melanoma immunotherapy model, tumor targeting and cytotoxic function of labeled CTLs were evaluated with imaging (248.5 kBq/[7.7 × 10(6)] cells, n = 4) and by measuring the tumor size (n = 6). Two-way analysis of variance was used to compare labeling conditions, the Wilcoxon test was used to assess cell survival and proliferation, and Holm-Sidak multiple tests were used to assess tumor growth and perform biodistribution analyses. (89)Zr-oxine complex was synthesized at a mean yield of 97.3% ± 2.8 (standard deviation). It readily labeled cells at room temperature or 4°C in phosphate-buffered saline (labeling efficiency range, 13.0%-43.9%) and was stably retained (83.5% ± 1.8 retention on day 5 in DCs). Labeling did not affect the viability of DCs and CTLs when compared with nonlabeled control mice (P > .05), nor did it affect functionality. (89)Zr-oxine complex enabled extended cell tracking for 7 days. Labeled tumor-specific CTLs accumulated in the tumor (4.6% on day 7) and induced tumor regression (P cell tracking technique for use with PET that is applicable to a broad range of

Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba δ-endotoxin complex revealed by electron crystallography: Implications for toxin-pore formation

International Nuclear Information System (INIS)

Ounjai, Puey; Unger, Vinzenz M.; Sigworth, Fred J.; Angsuthanasombat, Chanan

2007-01-01

The insecticidal nature of Cry δ-endotoxins produced by Bacillus thuringiensis is generally believed to be caused by their ability to form lytic pores in the midgut cell membrane of susceptible insect larvae. Here we have analyzed membrane-associated structures of the 65-kDa dipteran-active Cry4Ba toxin by electron crystallography. The membrane-associated toxin complex was crystallized in the presence of DMPC via detergent dialysis. Depending upon the charge of the adsorbed surface, 2D crystals of the oligomeric toxin complex have been captured in two distinct conformations. The projection maps of those crystals have been generated at 17 A resolution. Both complexes appeared to be trimeric; as in one crystal form, its projection structure revealed a symmetrical pinwheel-like shape with virtually no depression in the middle of the complex. The other form revealed a propeller-like conformation displaying an obvious hole in the center region, presumably representing the toxin-induced pore. These crystallographic data thus demonstrate for the first time that the 65-kDa activated Cry4Ba toxin in association with lipid membranes could exist in at least two different trimeric conformations, conceivably implying the closed and open states of the pore

Cationic Amphiphilic Tris-Cyclometalated Iridium(III) Complexes Induce Cancer Cell Death via Interaction with Ca2+-Calmodulin Complex.

Science.gov (United States)

Hisamatsu, Yosuke; Suzuki, Nozomi; Masum, Abdullah-Al; Shibuya, Ai; Abe, Ryo; Sato, Akira; Tanuma, Sei-Ichi; Aoki, Shin

2017-02-15

In our previous paper, we reported on the preparation of some cationic amphiphilic Ir complexes (2c, 2d) containing KKGG peptides that induce and detect cell death of Jurkat cells. Mechanistic studies suggest that 2c interacts with anionic molecules and/or membrane receptors on the cell surface to trigger an intracellular Ca 2+ response, resulting in the induction of cell death, accompanied by membrane disruption. We have continued the studies of cell death of Jurkat cells induced by 2c and found that xestospongin C, a selective inhibitor of an inositol 1,4,5-trisphosphate receptor located on the endoplasmic reticulum (ER), reduces the cytotoxicity of 2c, suggesting that 2c triggers the release of Ca 2+ from the ER, leading to an increase in the concentration of cytosolic Ca 2+ , thus inducing cell death. Moreover, we synthesized a series of new amphiphilic cationic Ir complexes 5a-c containing photoreactive 3-trifluoromethyl-3-phenyldiazirine (TFPD) groups, in an attempt to identify the target molecules of 2c. Interestingly, it was discovered that a TFPD group functions as a triplet quencher of Ir complexes. It was also found that 5b is useful as a turn-on phosphorescent probe of acidic proteins such as bovine serum albumin (BSA) (pI = 4.7) and their complexation was confirmed by luminescence titrations and SDS-PAGE of photochemical products between them. These successful results allowed us to carry out photoaffinity labeling of the target biomolecules of 5b (2c and analogues thereof) in Jurkat cells. A proteomic analysis of the products obtained by the photoirradiation of 5b with Jurkat cells suggests that the Ca 2+ -binding protein "calmodulin (CaM)" is one of target proteins of the Ir complexes. Indeed, 5b was found to interact with the Ca 2+ -CaM complex, as evidenced by luminescence titrations and the results of photochemical reactions of 5b with CaM in the presence of Ca 2+ (SDS-PAGE). A plausible mechanism for cell death induced by a cationic amphiphilic Ir

Spatio-temporal diversification of the cell wall matrix materials in the developing stomatal complexes of Zea mays.

Science.gov (United States)

Giannoutsou, E; Apostolakos, P; Galatis, B

2016-11-01

The matrix cell wall materials, in developing Zea mays stomatal complexes are asymmetrically distributed, a phenomenon appearing related to the local cell wall expansion and deformation, the establishment of cell polarity, and determination of the cell division plane. In cells of developing Zea mays stomatal complexes, definite cell wall regions expand determinately and become locally deformed. This differential cell wall behavior is obvious in the guard cell mother cells (GMCs) and the subsidiary cell mother cells (SMCs) that locally protrude towards the adjacent GMCs. The latter, emitting a morphogenetic stimulus, induce polarization/asymmetrical division in SMCs. Examination of immunolabeled specimens revealed that homogalacturonans (HGAs) with a high degree of de-esterification (2F4- and JIM5-HGA epitopes) and arabinogalactan proteins are selectively distributed in the extending and deformed cell wall regions, while their margins are enriched with rhamnogalacturonans (RGAs) containing highly branched arabinans (LM6-RGA epitope). In SMCs, the local cell wall matrix differentiation constitutes the first structural event, indicating the establishment of cell polarity. Moreover, in the premitotic GMCs and SMCs, non-esterified HGAs (2F4-HGA epitope) are preferentially localized in the cell wall areas outlining the cytoplasm where the preprophase band is formed. In these areas, the forthcoming cell plate fuses with the parent cell walls. These data suggest that the described heterogeneity in matrix cell wall materials is probably involved in: (a) local cell wall expansion and deformation, (b) the transduction of the inductive GMC stimulus, and (c) the determination of the division plane in GMCs and SMCs.

Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells

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Iain C. Macaulay

2016-02-01

Full Text Available The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment.

Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes.

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Xiaoyun Wu

Full Text Available The retinoblastoma (Rb tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.

High-frequency microrheology reveals cytoskeleton dynamics in living cells

Science.gov (United States)

Rigato, Annafrancesca; Miyagi, Atsushi; Scheuring, Simon; Rico, Felix

2017-08-01

Living cells are viscoelastic materials, dominated by an elastic response on timescales longer than a millisecond. On shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce. Here, we develop high-frequency microrheology experiments to probe the viscoelastic response of living cells from 1 Hz to 100 kHz. We report the viscoelasticity of different cell types under cytoskeletal drug treatments. On previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequencies, providing a unique mechanical fingerprint. Microrheology over a wide dynamic range--up to the frequency characterizing the molecular components--provides a mechanistic understanding of cell mechanics.

Modification and uptake of a cisplatin carbonato complex by Jurkat cells.

Science.gov (United States)

Centerwall, Corey R; Tacka, Kirk A; Kerwood, Deborah J; Goodisman, Jerry; Toms, Bonnie B; Dubowy, Ronald L; Dabrowiak, James C

2006-07-01

The interactions of Jurkat cells with cisplatin, cis-[Pt(15NH3)2Cl2]1, are studied using 1H-15N heteronuclear single quantum coherence (HSQC) NMR and inductively coupled plasma mass spectrometry. We show that Jurkat cells in culture rapidly modify the monocarbonato complex cis-[Pt(15NH3)2(CO3)Cl]- (4), a cisplatin species that forms in culture media and probably also in blood. Analysis of the HSQC NMR peak intensity for 4 in the presence of different numbers of Jurkat cells reveals that each cell is capable of modifying 0.0028 pmol of 4 within approximately 0.6 h. The amounts of platinum taken up by the cell, weakly bound to the cell surface, remaining in the culture medium, and bound to genomic DNA were measured as functions of time of exposure to different concentrations of drug. The results show that most of the 4 that has been modified by the cells remains in the culture medium as a substance of molecular mass <3 kDa, which is HSQC NMR silent, and is not taken up by the cell. These results are consistent with a hitherto undocumented extracellular detoxification mechanism in which the cells rapidly modify 4, which is present in the culture medium, so it cannot bind to the cell. Because there is only a slow decrease in the amount of unmodified 4 remaining in the culture medium after 1 h, -1.1 +/- 0.4 microM h(-1), the cells subsequently lose their ability to modify 4. These observations have important implications for the mechanism of action of cisplatin.

Telomere dysfunction and cell survival: Roles for distinct TIN2-containing complexes

Energy Technology Data Exchange (ETDEWEB)

Kim, Sahn-ho; Davalos, Albert R.; Heo, Seok-Jin; Rodier, Francis; Zou, Ying; Beausejour, Christian; Kaminker, Patrick; Yannone, Steven M.; Campisi, Judith

2007-10-02

Telomeres are maintained by three DNA binding proteins (TRF1, TRF2 and POT1), and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. Along with two other proteins, TPP1 and hRap1, these form a soluble complex that may be the core telomere maintenance complex. It is not clear whether sub-complexes also exist in vivo. We provide evidence for two TIN2 sub-complexes with distinct functions in human cells. We isolated these two TIN2 sub-complexes from nuclear lysates of unperturbed cells and cells expressing TIN2 mutants TIN2-13, TIN2-15C, which cannot bind TRF2 or TRF1, respectively. In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere dysfunction and cell death. Our findings suggest that distinct TIN2 complexes exist, and that TIN2-15C-sensitive subcomplexes are particularly important for cell survival in the absence of functional p53.

Arp2/3 complex activity in filopodia of spreading cells

Directory of Open Access Journals (Sweden)

Mendes Paula M

2008-12-01

Full Text Available Abstract Background Cells use filopodia to explore their environment and to form new adhesion contacts for motility and spreading. The Arp2/3 complex has been implicated in lamellipodial actin assembly as a major nucleator of new actin filaments in branched networks. The interplay between filopodial and lamellipodial protrusions is an area of much interest as it is thought to be a key determinant of how cells make motility choices. Results We find that Arp2/3 complex localises to dynamic puncta in filopodia as well as lamellipodia of spreading cells. Arp2/3 complex spots do not appear to depend on local adhesion or on microtubules for their localisation but their inclusion in filopodia or lamellipodia depends on the activity of the small GTPase Rac1. Arp2/3 complex spots in filopodia are capable of incorporating monomeric actin, suggesting the presence of available filament barbed ends for polymerisation. Arp2/3 complex in filopodia co-localises with lamellipodial proteins such as capping protein and cortactin. The dynamics of Arp2/3 complex puncta suggests that they are moving bi-directionally along the length of filopodia and that they may be regions of lamellipodial activity within the filopodia. Conclusion We suggest that filopodia of spreading cells have regions of lamellipodial activity and that this activity affects the morphology and movement of filopodia. Our work has implications for how we understand the interplay between lamellipodia and filopodia and for how actin networks are generated spatially in cells.

GlcNAc-1-P-transferase–tunicamycin complex structure reveals basis for inhibition of N-glycosylation

Energy Technology Data Exchange (ETDEWEB)

Yoo, Jiho; Mashalidis, Ellene H.; Kuk, Alvin C. Y.; Yamamoto, Kazuki; Kaeser, Benjamin; Ichikawa, Satoshi; Lee, Seok-Yong

2018-02-19

N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase or GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity, inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structural information. Here we present crystal structures of human GPT in complex with tunicamycin. In conclusion, structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We demonstrate that this difference could be exploited to design MraY-specific inhibitors as potential antibiotics.

Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

Science.gov (United States)

Hsu, Li-Jin; Hong, Qunying; Chen, Shur-Tzu; Kuo, Hsiang-Lin; Schultz, Lori; Heath, John; Lin, Sing-Ru; Lee, Ming-Hui; Li, Dong-Zhang; Li, Zih-Ling; Cheng, Hui-Ching; Armand, Gerard; Chang, Nan-Shan

2017-03-21

Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

Long-Term Live Cell Imaging Reveals New Roles For Salmonella Effector Proteins SseG and SteA

Science.gov (United States)

McQuate, Sarah E.; Young, Alexandra M.; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S.; Palmer, Amy E.

2016-01-01

Summary Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here we establish a pipeline for long-term (16 hours) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages, and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyperreplication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. PMID:27376507

Carbohydrate linked organotin(IV) complexes as human topoisomerase Iα inhibitor and their antiproliferative effects against the human carcinoma cell line.

Science.gov (United States)

Khan, Rais Ahmad; Yadav, Shipra; Hussain, Zahid; Arjmand, Farukh; Tabassum, Sartaj

2014-02-14

Dimethyltin(IV) complexes with ethanolamine (1) and biologically significant N-glycosides (2 and 3) were designed and synthesized. The structural elucidation of complexes 1-3 was done using elemental and spectroscopic methods; in addition, complex 1 was studied by single crystal X-ray diffraction studies. The in vitro DNA binding profile of complexes 2 and 3 was carried out by employing different biophysical methods to ascertain the feasibility of glycosylated complexes. Further, the cleaving ability of 2 and 3 was investigated by the agarose gel electrophoretic mobility assay with supercoiled pBR322 DNA, and demonstrated significantly good nuclease activity. Furthermore, both the complexes exhibited significant inhibitory effects on the catalytic activity of human Topo I at lower concentration than standard drugs. Computer-aided molecular docking techniques were used to ascertain the mode and mechanism of action towards the molecular target DNA and Topo I. The cytotoxicity of 2 and 3 against human hepatoma cancer cells (Huh7) was evaluated, which revealed significant regression in cancerous cells as compared with the standard drug. The antiproliferative activities of 2 and 3 were tested against human hepatoma cancer cells (Huh7), and results showed significantly good activity. Additionally, to validate the remarkable antiproliferative activity of complexes 2 and 3, specific regulatory gene expression (MMP-2 and TGF-β) was obtained by real time PCR.

Diverse phosphorylation patterns of B cell receptor-associated signaling in naïve and memory human B cells revealed by phosphoflow, a powerful technique to study signaling at the single cell level

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Franklin R Toapanta

2012-10-01

Full Text Available Following interaction with cognate antigens, B cells undergo cell activation, proliferation and differentiation. Ligation of the B cell receptor (BCR leads to the phosphorylation of BCR-associated signaling proteins within minutes of antigen binding, a process with profound consequences for the fate of the cells and development of effector immunity. Phosphoflow allows a rapid evaluation of various signaling pathways in complex heterogenous cell subsets. This novel technique was used in combination with multi-chromatic flow cytometry and fluorescent-cell barcoding to study phosphorylation of BCR-associated signaling pathways in naïve and memory human B cell subsets. Proteins of the initiation (Syk, propagation (Btk, Akt and integration (p38MAPK and Erk1/2 signaling units were studied. Switched memory (Sm CD27+ and Sm CD27- phosphorylation patterns were similar when stimulated with anti-IgA or -IgG. In contrast, naïve and unswitched memory (Um cells showed significant differences following IgM stimulation. Enhanced phosphorylation of Syk was observed in Um cells, suggesting a lower activation threshold. This is likely the result of higher amounts of IgM on the cell surface, higher pan-Syk levels and enhanced susceptibility to phosphatase inhibition. All other signaling proteins evaluated also showed some degree of enhanced phosphorylation in Um cells. Furthermore, both the PLC-γ2 and PI3K pathways were activated in Um cells, while only the PI3K pathway was activated on naïve cells. Um cells were the only ones that activated signaling pathways when stimulated with fluorescently-labeled S. Typhi and S. pneumoniae. Finally, simultaneous evaluation of signaling proteins at the single cell level (multi-phosphorylated cells revealed that interaction with gram positive and negative bacteria resulted in complex and diverse signaling patterns. Phosphoflow holds great potential to accelerate vaccine development by identifying signaling profiles in good

Novel piplartine-containing ruthenium complexes: synthesis, cell growth inhibition, apoptosis induction and ROS production on HCT116 cells.

Science.gov (United States)

D'Sousa Costa, Cinara O; Araujo Neto, João H; Baliza, Ingrid R S; Dias, Rosane B; Valverde, Ludmila de F; Vidal, Manuela T A; Sales, Caroline B S; Rocha, Clarissa A G; Moreira, Diogo R M; Soares, Milena B P; Batista, Alzir A; Bezerra, Daniel P

2017-11-28

Piplartine (piperlongumine) is a plant-derived molecule that has been receiving intense interest due to its anticancer characteristics that target the oxidative stress. In the present paper, two novel piplartine-containing ruthenium complexes [Ru(piplartine)(dppf)(bipy)](PF 6 ) 2 (1) and [Ru(piplartine)(dppb)(bipy)](PF 6 ) 2 (2) were synthesized and investigated for their cellular and molecular responses on cancer cell lines. We found that both complexes are more potent than metal-free piplartine in a panel of cancer cell lines on monolayer cultures, as well in 3D model of cancer multicellular spheroids formed from human colon carcinoma HCT116 cells. Mechanistic studies uncovered that the complexes reduced the cell growth and caused phosphatidylserine externalization, internucleosomal DNA fragmentation, caspase-3 activation and loss of the mitochondrial transmembrane potential on HCT116 cells. Moreover, the pre-treatment with Z-VAD(OMe)-FMK, a pan-caspase inhibitor, reduced the complexes-induced apoptosis, indicating cell death by apoptosis through caspase-dependent and mitochondrial intrinsic pathways. Treatment with the complexes also caused a marked increase in the production of reactive oxygen species (ROS), including hydrogen peroxide, superoxide anion and nitric oxide, and decreased reduced glutathione levels. Application of N-acetyl-cysteine, an antioxidant, reduced the ROS levels and apoptosis induced by the complexes, indicating activation of ROS-mediated apoptosis pathway. RNA transcripts of several genes, including gene related to the cell cycle, apoptosis and oxidative stress, were regulated under treatment. However, the complexes failed to induce DNA intercalation. In conclusion, the complexes are more potent than piplartine against different cancer cell lines and are able to induce caspase-dependent and mitochondrial intrinsic apoptosis on HCT116 cells by ROS-mediated pathway.

Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.

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Amatsu, Sho; Sugawara, Yo; Matsumura, Takuhiro; Kitadokoro, Kengo; Fujinaga, Yukako

2013-12-06

Clostridium botulinum HA is a component of the large botulinum neurotoxin complex and is critical for its oral toxicity. HA plays multiple roles in toxin penetration in the gastrointestinal tract, including protection from the digestive environment, binding to the intestinal mucosal surface, and disruption of the epithelial barrier. At least two properties of HA contribute to these roles: the sugar-binding activity and the barrier-disrupting activity that depends on E-cadherin binding of HA. HA consists of three different proteins, HA1, HA2, and HA3, whose structures have been partially solved and are made up mainly of β-strands. Here, we demonstrate structural and functional reconstitution of whole HA and present the complete structure of HA of serotype B determined by x-ray crystallography at 3.5 Å resolution. This structure reveals whole HA to be a huge triskelion-shaped molecule. Our results suggest that whole HA is functionally and structurally separable into two parts: HA1, involved in recognition of cell-surface carbohydrates, and HA2-HA3, involved in paracellular barrier disruption by E-cadherin binding.

Viral Transmission Dynamics at Single-Cell Resolution Reveal Transiently Immune Subpopulations Caused by a Carrier State Association.

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William Cenens

2015-12-01

Full Text Available Monitoring the complex transmission dynamics of a bacterial virus (temperate phage P22 throughout a population of its host (Salmonella Typhimurium at single cell resolution revealed the unexpected existence of a transiently immune subpopulation of host cells that emerged from peculiarities preceding the process of lysogenization. More specifically, an infection event ultimately leading to a lysogen first yielded a phage carrier cell harboring a polarly tethered P22 episome. Upon subsequent division, the daughter cell inheriting this episome became lysogenized by an integration event yielding a prophage, while the other daughter cell became P22-free. However, since the phage carrier cell was shown to overproduce immunity factors that are cytoplasmically inherited by the P22-free daughter cell and further passed down to its siblings, a transiently resistant subpopulation was generated that upon dilution of these immunity factors again became susceptible to P22 infection. The iterative emergence and infection of transiently resistant subpopulations suggests a new bet-hedging strategy by which viruses could manage to sustain both vertical and horizontal transmission routes throughout an infected population without compromising a stable co-existence with their host.

Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival.

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Christian Frezza

Full Text Available Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS, to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.

Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans

OpenAIRE

Fraune, Johanna; Alsheimer, Manfred; Volff, Jean-Nicolas; Busch, Karoline; Fraune, Sebastian; Bosch, Thomas C. G.; Benavente, Ricardo

2012-01-01

The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed...

B-Cell-Specific Diversion of Glucose Carbon Utilization Reveals a Unique Vulnerability in B Cell Malignancies.

Science.gov (United States)

Xiao, Gang; Chan, Lai N; Klemm, Lars; Braas, Daniel; Chen, Zhengshan; Geng, Huimin; Zhang, Qiuyi Chen; Aghajanirefah, Ali; Cosgun, Kadriye Nehir; Sadras, Teresa; Lee, Jaewoong; Mirzapoiazova, Tamara; Salgia, Ravi; Ernst, Thomas; Hochhaus, Andreas; Jumaa, Hassan; Jiang, Xiaoyan; Weinstock, David M; Graeber, Thomas G; Müschen, Markus

2018-04-05

B cell activation during normal immune responses and oncogenic transformation impose increased metabolic demands on B cells and their ability to retain redox homeostasis. While the serine/threonine-protein phosphatase 2A (PP2A) was identified as a tumor suppressor in multiple types of cancer, our genetic studies revealed an essential role of PP2A in B cell tumors. Thereby, PP2A redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway (PPP) to salvage oxidative stress. This unique vulnerability reflects constitutively low PPP activity in B cells and transcriptional repression of G6PD and other key PPP enzymes by the B cell transcription factors PAX5 and IKZF1. Reflecting B-cell-specific transcriptional PPP-repression, glucose carbon utilization in B cells is heavily skewed in favor of glycolysis resulting in lack of PPP-dependent antioxidant protection. These findings reveal a gatekeeper function of the PPP in a broad range of B cell malignancies that can be efficiently targeted by small molecule inhibition of PP2A and G6PD. Copyright © 2018 Elsevier Inc. All rights reserved.

Characterising the cellulose synthase complexes of cell walls

NARCIS (Netherlands)

Mansoori Zangir, N.

2012-01-01

One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as

SCAR/WAVE: A complex issue.

Science.gov (United States)

Davidson, Andrew J; Insall, Robert H

2013-11-01

The SCAR/WAVE complex drives the actin polymerisation that underlies protrusion of the front of the cell and thus drives migration. However, it is not understood how the activity of SCAR/WAVE is regulated to generate the infinite range of cellular shape changes observed during cell motility. What are the relative roles of the subunits of the SCAR/WAVE complex? What signaling molecules do they interact with? And how does the complex integrate all this information in order to control the temporal and spatial polymerisation of actin during protrusion formation? Unfortunately, the interdependence of SCAR complex members has made genetic dissection hard. In our recent paper,(1) we describe stabilization of the Dictyostelium SCAR complex by a small fragment of Abi. Here we summarize the main findings and discuss how this approach can help reveal the inner workings of this impenetrable complex.

The immune complex CTA1-DD/IgG adjuvant specifically targets connective tissue mast cells through FcγRIIIA and augments anti-HPV immunity after nasal immunization.

Science.gov (United States)

Fang, Y; Zhang, T; Lidell, L; Xu, X; Lycke, N; Xiang, Z

2013-11-01

We have previously reported that CTA1-DD/IgG immune complexes augment antibody responses in a mast cell-dependent manner following intranasal (IN) immunizations. However, from a safety perspective, mast cell activation could preclude clinical use. Therefore, we have extended these studies and demonstrate that CTA1-DD/IgG immune complexes administered IN did not trigger an anaphylactic reaction. Importantly, CTA1-DD/IgE immune complexes did not activate mast cells. Interestingly, only connective tissue, but not mucosal, mast cells could be activated by CTA1-DD/IgG immune complexes. This effect was mediated by FcγRIIIA, only expressed on connective tissue mast cells, and found in the nasal submucosa. FcγRIIIA-deficient mice had compromised responses to immunization adjuvanted by CTA1-DD/IgG. Proof-of-concept studies revealed that IN immunized mice with human papillomavirus (HPV) type 16 L1 virus-like particles (VLP) and CTA1-DD/IgG immune complexes demonstrated strong and sustained specific antibody titers in serum and vaginal secretions. From a mast cell perspective, CTA1-DD/IgG immune complexes appear to be safe and effective mucosal adjuvants.

Single-Cell Analyses of ESCs Reveal Alternative Pluripotent Cell States and Molecular Mechanisms that Control Self-Renewal

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Dmitri Papatsenko

2015-08-01

Full Text Available Analyses of gene expression in single mouse embryonic stem cells (mESCs cultured in serum and LIF revealed the presence of two distinct cell subpopulations with individual gene expression signatures. Comparisons with published data revealed that cells in the first subpopulation are phenotypically similar to cells isolated from the inner cell mass (ICM. In contrast, cells in the second subpopulation appear to be more mature. Pluripotency Gene Regulatory Network (PGRN reconstruction based on single-cell data and published data suggested antagonistic roles for Oct4 and Nanog in the maintenance of pluripotency states. Integrated analyses of published genomic binding (ChIP data strongly supported this observation. Certain target genes alternatively regulated by OCT4 and NANOG, such as Sall4 and Zscan10, feed back into the top hierarchical regulator Oct4. Analyses of such incoherent feedforward loops with feedback (iFFL-FB suggest a dynamic model for the maintenance of mESC pluripotency and self-renewal.

Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells

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Andrea-Anneliese Keller

2013-02-01

Full Text Available Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.

Trichomonas vaginalis perturbs the junctional complex in epithelial cells

Institute of Scientific and Technical Information of China (English)

无

2005-01-01

Trichomonas vaginalis, a protist parasite of the urogenital tract in humans, is the causative agent of trichomonosis,which in recent years have been associated with the cervical cancer development. In the present study we analyzed the modifications at the junctional complex level of Caco-2 cells after interaction with two isolates of T. vaginalis and the influence of the iron concentration present in the parasite's culture medium on the interaction effects. Our results show that T. vaginalis adheres to the epithelial cell causing alterations in the junctional complex, such as: (a) a decrease in transepithelial electrical resistance; (b) alteration in the pattern of junctional complex proteins distribution as obseryed for E-cadherin, occludin and ZO-1; and (c) enlargement of the spaces between epithelial cells. These effects were dependent on (a) the degree of the parasite virulence isolate, (b) the iron concentration in the culture medium, and (c) the expression of adhesin proteins on the parasite surface.

Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed

Science.gov (United States)

Hsu, Li-Jin; Hong, Qunying; Chen, Shur-Tzu; Kuo, Hsiang-Lin; Schultz, Lori; Heath, John; Lin, Sing-Ru; Lee, Ming-Hui; Li, Dong-Zhang; Li, Zih-Ling; Cheng, Hui-Ching; Armand, Gerard; Chang, Nan-Shan

2017-01-01

Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed. PMID:27845895

Crystal structures of the CPAP/STIL complex reveal its role in centriole assembly and human microcephaly

Science.gov (United States)

Cottee, Matthew A; Muschalik, Nadine; Wong, Yao Liang; Johnson, Christopher M; Johnson, Steven; Andreeva, Antonina; Oegema, Karen; Lea, Susan M; Raff, Jordan W; van Breugel, Mark

2013-01-01

Centrioles organise centrosomes and template cilia and flagella. Several centriole and centrosome proteins have been linked to microcephaly (MCPH), a neuro-developmental disease associated with small brain size. CPAP (MCPH6) and STIL (MCPH7) are required for centriole assembly, but it is unclear how mutations in them lead to microcephaly. We show that the TCP domain of CPAP constitutes a novel proline recognition domain that forms a 1:1 complex with a short, highly conserved target motif in STIL. Crystal structures of this complex reveal an unusual, all-β structure adopted by the TCP domain and explain how a microcephaly mutation in CPAP compromises complex formation. Through point mutations, we demonstrate that complex formation is essential for centriole duplication in vivo. Our studies provide the first structural insight into how the malfunction of centriole proteins results in human disease and also reveal that the CPAP–STIL interaction constitutes a conserved key step in centriole biogenesis. DOI: http://dx.doi.org/10.7554/eLife.01071.001 PMID:24052813

Pea border cell maturation and release involve complex cell wall structural dynamics

DEFF Research Database (Denmark)

Mravec, Jozef; Guo, Xiaoyuan; Hansen, Aleksander Riise

2017-01-01

The adhesion of plant cells is vital for support and protection of the plant body and is maintained by a variety of molecular associations between cell wall components. In some specialized cases though, plant cells are programmed to detach and root cap-derived border cells are examples of this....... Border cells (in some species known as border-like cells) provide an expendable barrier between roots and the environment. Their maturation and release is an important but poorly characterized cell separation event. To gain a deeper insight into the complex cellular dynamics underlying this process, we...... undertook a systematic, detailed analysis of pea (Pisum sativum) root tip cell walls. Our study included immuno-carbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy (FT-IR), quantitative RT-PCR of cell wall biosynthetic genes, analysis...

Live cell imaging reveals marked variability in myoblast proliferation and fate

Science.gov (United States)

2013-01-01

Background During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. PMID:23638706

Assembly and architecture of the EBV B cell entry triggering complex.

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Karthik Sathiyamoorthy

2014-08-01

Full Text Available Epstein-Barr Virus (EBV is an enveloped double-stranded DNA virus of the gammaherpesvirinae sub-family that predominantly infects humans through epithelial cells and B cells. Three EBV glycoproteins, gH, gL and gp42, form a complex that targets EBV infection of B cells. Human leukocyte antigen (HLA class II molecules expressed on B cells serve as the receptor for gp42, triggering membrane fusion and virus entry. The mechanistic role of gHgL in herpesvirus entry has been largely unresolved, but it is thought to regulate the activation of the virally-encoded gB protein, which acts as the primary fusogen. Here we study the assembly and function of the reconstituted B cell entry complex comprised of gHgL, gp42 and HLA class II. The structure from negative-stain electron microscopy provides a detailed snapshot of an intermediate state in EBV entry and highlights the potential for the triggering complex to bring the two membrane bilayers into proximity. Furthermore, gHgL interacts with a previously identified, functionally important hydrophobic pocket on gp42, defining the overall architecture of the complex and playing a critical role in membrane fusion activation. We propose a macroscopic model of the initiating events in EBV B cell fusion centered on the formation of the triggering complex in the context of both viral and host membranes. This model suggests how the triggering complex may bridge the two membrane bilayers, orienting critical regions of the N- and C- terminal ends of gHgL to promote the activation of gB and efficient membrane fusion.

Doubly end-on azido bridged mixed-valence cobalt trinuclear complex: Spectral study, VTM, inhibitory effect and antimycobacterial activity on human carcinoma and tuberculosis cells

Science.gov (United States)

Datta, Amitabha; Das, Kuheli; Sen, Chandana; Karan, Nirmal Kumar; Huang, Jui-Hsien; Lin, Chia-Her; Garribba, Eugenio; Sinha, Chittaranjan; Askun, Tulin; Celikboyun, Pinar; Mane, Sandeep B.

2015-09-01

Doubly end-on azido-bridged mixed-valence trinuclear cobalt complex, [Co3(L)2(N3)6(CH3OH)2] (1) is afforded by employing a potential monoanionic tetradentate-N2O2 Schiff base precursor (2-[{[2-(dimethylamino)ethyl]imino}methyl]-6-methoxyphenol; HL). Single crystal X-ray structure reveals that in 1, the adjacent CoII and CoIII ions are linked by double end-on azido bridges and thus the full molecule is generated by the site symmetry of a crystallographic twofold rotation axis. Complex 1 is subjected on different spectral analysis such as IR, UV-vis, emission and EPR spectroscopy. On variable temperature magnetic study, we observe that during cooling, the χMT values decrease smoothly until 15 K and then reaches to the value 1.56 cm3 K mol-1 at 2 K. Complex 1 inhibits the cell growth on human lung carcinoma (A549 cells), human colorectal (COLO 205 and HT-29 cells), and human heptacellular (PLC5 cells) carcinoma cells. Complex 1 exhibits anti-mycobacterial activity and considerable efficacy on Mycobacterium tuberculosis H37Rv ATCC 27294 and H37Ra ATCC 25177 strains.

Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function.

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Shah, Prajay T; Stratton, Jo A; Stykel, Morgan Gail; Abbasi, Sepideh; Sharma, Sandeep; Mayr, Kyle A; Koblinger, Kathrin; Whelan, Patrick J; Biernaskie, Jeff

2018-05-03

Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche. Copyright © 2018 Elsevier Inc. All rights reserved.

Generation of tooth-periodontium complex structures using high-odontogenic potential dental epithelium derived from mouse embryonic stem cells.

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Zhang, Yancong; Li, Yongliang; Shi, Ruirui; Zhang, Siqi; Liu, Hao; Zheng, Yunfei; Li, Yan; Cai, Jinglei; Pei, Duanqing; Wei, Shicheng

2017-06-08

A number of studies have shown that tooth-like structures can be regenerated using induced pluripotent stem cells and mouse embryonic stem (mES) cells. However, few studies have reported the regeneration of tooth-periodontium complex structures, which are more suitable for clinical tooth transplantation. We established an optimized approach to induce high-odontogenic potential dental epithelium derived from mES cells by temporally controlling bone morphogenic protein 4 (BMP4) function and regenerated tooth-periodontium complex structures in vivo. First, immunofluorescence and quantitative reverse transcription-polymerase chain reaction were used to identify the watershed of skin and the oral ectoderm. LDN193189 was then used to inhibit the BMP4 receptor around the watershed, followed by the addition of exogenous BMP4 to promote BMP4 function. The generated dental epithelium was confirmed by western blot analysis and immunofluorescence. The generated epithelium was ultimately combined with embryonic day 14.5 mouse mesenchyme and transplanted into the renal capsules of nude mice. After 4 weeks, the tooth-periodontium complex structure was examined by micro-computed tomography (CT) and hematoxylin and eosin (H&E) staining. Our study found that the turning point of oral ectoderm differentiation occurred around day 3 after the embryoid body was transferred to a common culture plate. Ameloblastin-positive dental epithelial cells were detected following the temporal regulation of BMP4. Tooth-periodontium complex structures, which included teeth, a periodontal membrane, and alveolar bone, were formed when this epithelium was combined with mouse dental mesenchyme and transplanted into the renal capsules of nude mice. Micro-CT and H&E staining revealed that the generated tooth-periodontium complex structures shared a similar histological structure with normal mouse teeth. An optimized induction method was established to promote the differentiation of mES cells into dental

Aquation Is a Crucial Activation Step for Anticancer Action of Ruthenium(II) Polypyridyl Complexes to Trigger Cancer Cell Apoptosis.

Science.gov (United States)

Li, Meng; Lai, Lanhai; Zhao, Zhennan; Chen, Tianfeng

2016-01-01

Aquation has been proposed as crucial chemical action step for ruthenium (Ru) complexes, but its effects on the action mechanisms remain elusive. Herein, we have demonstrated the aquation process of a potent Ru polypyridyl complex (RuBmp=[Ru(II) (bmbp)(phen)Cl]ClO4 , bmbp=2,6-bis(6-methylbenzimidazol-2-yl) pyridine, phen=phenanthroline) with a chloride ligand, and revealed that aquation of RuBmp effectively enhanced its hydrophilicity and cellular uptake, thus significantly increasing its anticancer efficacy. The aquation products (H-RuBmp=[Ru(II) (bmbp)(phen)Cl]ClO4 , [Ru(II) (bmbp)(phen)(H2 O)]ClO4 , bmbp) exhibited a much higher apoptosis-inducing ability than the intact complex, with involvement of caspase activation, mitochondria dysfunction, and interaction with cell membrane death receptors. H-RuBmp demonstrated a higher interaction potency with the cell membrane and induced higher levels of ROS overproduction in cancer cells to regulate the AKT, MAPK, and p53 signaling pathways. Taken together, this study could provide useful information for fine-tuning the rational design of next-generation metal medicines. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bacterial actin MreB assembles in complex with cell shape protein RodZ.

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van den Ent, Fusinita; Johnson, Christopher M; Persons, Logan; de Boer, Piet; Löwe, Jan

2010-03-17

Bacterial actin homologue MreB is required for cell shape maintenance in most non-spherical bacteria, where it assembles into helical structures just underneath the cytoplasmic membrane. Proper assembly of the actin cytoskeleton requires RodZ, a conserved, bitopic membrane protein that colocalises to MreB and is essential for cell shape determination. Here, we present the first crystal structure of bacterial actin engaged with a natural partner and provide a clear functional significance of the interaction. We show that the cytoplasmic helix-turn-helix motif of Thermotoga maritima RodZ directly interacts with monomeric as well as filamentous MreB and present the crystal structure of the complex. In vitro and in vivo analyses of mutant T. maritima and Escherichia coli RodZ validate the structure and reveal the importance of the MreB-RodZ interaction in the ability of cells to propagate as rods. Furthermore, the results elucidate how the bacterial actin cytoskeleton might be anchored to the membrane to help constrain peptidoglycan synthesis in the periplasm.

Transforming growth factor beta 1 modulates extracellular matrix organization and cell-cell junctional complex formation during in vitro angiogenesis.

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Merwin, J R; Anderson, J M; Kocher, O; Van Itallie, C M; Madri, J A

1990-01-01

Transforming growth factor-beta 1 (TGF-beta 1) is angiogenic in vivo. In two-dimensional (2-D) culture systems microvascular endothelial cell proliferation is inhibited up to 80% by TGF-beta 1; however, in three-dimensional (3-D) collagen gels TGF-beta 1 is found to have no effect on proliferation while eliciting the formation of calcium and magnesium dependent tube-like structures mimicking angiogenesis. DNA analyses performed on 3-D cell cultures reveal no significant difference in the amount of DNA or cell number in control versus TGF-beta 1 treated cultures. In 2-D cultures TGF-beta 1 is known to increase cellular fibronectin accumulation; however, in 3-D cultures no difference is seen between control and TGF-beta 1 treated cells as established by ELISA testing for type IV collagen, fibronectin, and laminin. In 3-D cultures there is increased synthesis and secretion of type V collagen in both control and TGF-beta 1 treated cultures over 2-D cultures. Even though an equal amount of type V collagen is seen in both 3-D conditions, there is a reorganization of the protein with concentration along an organizing basal lamina in TGF-beta 1 treated cultures. EM morphological analyses on 3-D cultures illustrate quiescent, control cells lacking cell contacts. In contrast, TGF-beta 1 treated cells show increased pseudopod formation, cell-cell contact, and organized basal lamina-like material closely apposed to the "abluminal" plasma membranes. TGF-beta 1 treated cells also appear to form junctional complexes between adjoining cells. Immunofluorescence using specific antibodies to the tight junction protein ZO-1 results in staining at apparent cell-cell junctions in the 3-D cultures. Northern blots of freshly isolated microvascular endothelium, 2-D and 3-D cultures, using cDNA and cRNA probes specific for the ZO-1 tight junction protein, reveal the presence of the 7.8 kb mRNA. Western blots of rat epididymal fat pad endothelial cells (RFC) monolayer lysates probed with

Comprehensive prediction in 78 human cell lines reveals rigidity and compactness of transcription factor dimers

Science.gov (United States)

Jankowski, Aleksander; Szczurek, Ewa; Jauch, Ralf; Tiuryn, Jerzy; Prabhakar, Shyam

2013-01-01

The binding of transcription factors (TFs) to their specific motifs in genomic regulatory regions is commonly studied in isolation. However, in order to elucidate the mechanisms of transcriptional regulation, it is essential to determine which TFs bind DNA cooperatively as dimers and to infer the precise nature of these interactions. So far, only a small number of such dimeric complexes are known. Here, we present an algorithm for predicting cell-type–specific TF–TF dimerization on DNA on a large scale, using DNase I hypersensitivity data from 78 human cell lines. We represented the universe of possible TF complexes by their corresponding motif complexes, and analyzed their occurrence at cell-type–specific DNase I hypersensitive sites. Based on ∼1.4 billion tests for motif complex enrichment, we predicted 603 highly significant cell-type–specific TF dimers, the vast majority of which are novel. Our predictions included 76% (19/25) of the known dimeric complexes and showed significant overlap with an experimental database of protein–protein interactions. They were also independently supported by evolutionary conservation, as well as quantitative variation in DNase I digestion patterns. Notably, the known and predicted TF dimers were almost always highly compact and rigidly spaced, suggesting that TFs dimerize in close proximity to their partners, which results in strict constraints on the structure of the DNA-bound complex. Overall, our results indicate that chromatin openness profiles are highly predictive of cell-type–specific TF–TF interactions. Moreover, cooperative TF dimerization seems to be a widespread phenomenon, with multiple TF complexes predicted in most cell types. PMID:23554463

Nuclear DAMP complex-mediated RAGE-dependent macrophage cell death

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Chen, Ruochan [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Fu, Sha; Fan, Xue-Gong [Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Lotze, Michael T.; Zeh, Herbert J. [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Tang, Daolin, E-mail: tangd2@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Kang, Rui, E-mail: kangr@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States)

2015-03-13

High mobility group box 1 (HMGB1), histone, and DNA are essential nuclear components involved in the regulation of chromosome structure and function. In addition to their nuclear function, these molecules act as damage-associated molecular patterns (DAMPs) alone or together when released extracellularly. The synergistic effect of these nuclear DNA-HMGB1-histone complexes as DAMP complexes (nDCs) on immune cells remains largely unexplored. Here, we demonstrate that nDCs limit survival of macrophages (e.g., RAW264.7 and peritoneal macrophages) but not cancer cells (e.g., HCT116, HepG2 and Hepa1-6). nDCs promote production of inflammatory tumor necrosis factor α (TNFα) release, triggering reactive oxygen species-dependent apoptosis and necrosis. Moreover, the receptor for advanced glycation end products (RAGE), but not toll-like receptor (TLR)-4 and TLR-2, was required for Akt-dependent TNFα release and subsequent cell death following treatment with nDCs. Genetic depletion of RAGE by RNAi, antioxidant N-Acetyl-L-cysteine, and TNFα neutralizing antibody significantly attenuated nDC-induced cell death. These findings provide evidence supporting novel signaling mechanisms linking nDCs and inflammation in macrophage cell death. - Highlights: • Nuclear DAMP complexes (nDCs) selectively induce cell death in macrophages, but not cancer cells. • TNFα-mediated oxidative stress is required for nDC-induced death. • RAGE-mediated Akt activation is required for nDC-induced TNFα release. • Blocking RAGE and TNFα inhibits nDC-induced macrophage cell death.

Multilayer Stochastic Block Models Reveal the Multilayer Structure of Complex Networks

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Toni Vallès-Català

2016-03-01

Full Text Available In complex systems, the network of interactions we observe between systems components is the aggregate of the interactions that occur through different mechanisms or layers. Recent studies reveal that the existence of multiple interaction layers can have a dramatic impact in the dynamical processes occurring on these systems. However, these studies assume that the interactions between systems components in each one of the layers are known, while typically for real-world systems we do not have that information. Here, we address the issue of uncovering the different interaction layers from aggregate data by introducing multilayer stochastic block models (SBMs, a generalization of single-layer SBMs that considers different mechanisms of layer aggregation. First, we find the complete probabilistic solution to the problem of finding the optimal multilayer SBM for a given aggregate-observed network. Because this solution is computationally intractable, we propose an approximation that enables us to verify that multilayer SBMs are more predictive of network structure in real-world complex systems.

In silico and cell-based analyses reveal strong divergence between prediction and observation of T-cell-recognized tumor antigen T-cell epitopes.

Science.gov (United States)

Schmidt, Julien; Guillaume, Philippe; Dojcinovic, Danijel; Karbach, Julia; Coukos, George; Luescher, Immanuel

2017-07-14

Tumor exomes provide comprehensive information on mutated, overexpressed genes and aberrant splicing, which can be exploited for personalized cancer immunotherapy. Of particular interest are mutated tumor antigen T-cell epitopes, because neoepitope-specific T cells often are tumoricidal. However, identifying tumor-specific T-cell epitopes is a major challenge. A widely used strategy relies on initial prediction of human leukocyte antigen-binding peptides by in silico algorithms, but the predictive power of this approach is unclear. Here, we used the human tumor antigen NY-ESO-1 (ESO) and the human leukocyte antigen variant HLA-A*0201 (A2) as a model and predicted in silico the 41 highest-affinity, A2-binding 8-11-mer peptides and assessed their binding, kinetic complex stability, and immunogenicity in A2-transgenic mice and on peripheral blood mononuclear cells from ESO-vaccinated melanoma patients. We found that 19 of the peptides strongly bound to A2, 10 of which formed stable A2-peptide complexes and induced CD8 + T cells in A2-transgenic mice. However, only 5 of the peptides induced cognate T cells in humans; these peptides exhibited strong binding and complex stability and contained multiple large hydrophobic and aromatic amino acids. These results were not predicted by in silico algorithms and provide new clues to improving T-cell epitope identification. In conclusion, our findings indicate that only a small fraction of in silico -predicted A2-binding ESO peptides are immunogenic in humans, namely those that have high peptide-binding strength and complex stability. This observation highlights the need for improving in silico predictions of peptide immunogenicity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells

DEFF Research Database (Denmark)

Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas

2016-01-01

to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially...... kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified...... as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human...

Differential effects of a complex organochlorine mixture on the proliferation of breast cancer cell lines

Energy Technology Data Exchange (ETDEWEB)

Aube, Michel, E-mail: 4aubem@videotron.ca [Axe de recherche en sante des populations et environnementale, Centre de recherche du Centre hospitalier universitaire de Quebec and Universite Laval, 2875 Boulevard Laurier, Edifice Delta 2, bureau 600, Quebec, QC, Canada G1V 2M2 (Canada); Larochelle, Christian, E-mail: christian.larochelle@inspq.qc.ca [Axe de recherche en sante des populations et environnementale, Centre de recherche du Centre hospitalier universitaire de Quebec and Universite Laval, 2875 Boulevard Laurier, Edifice Delta 2, bureau 600, Quebec, QC, Canada G1V 2M2 (Canada); Ayotte, Pierre, E-mail: pierre.ayotte@inspq.qc.ca [Axe de recherche en sante des populations et environnementale, Centre de recherche du Centre hospitalier universitaire de Quebec and Universite Laval, 2875 Boulevard Laurier, Edifice Delta 2, bureau 600, Quebec, QC, Canada G1V 2M2 (Canada); Laboratoire de Toxicologie, Institut national de sante publique du Quebec, 945 avenue Wolfe, Quebec, QC, Canada G1V 5B3 (Canada)

2011-04-15

Organochlorine compounds (OCs) are a group of persistent chemicals that accumulate in fatty tissues with age. Although OCs has been tested individually for their capacity to induce breast cancer cell proliferation, few studies examined the effect of complex mixtures that comprise compounds frequently detected in the serum of women. We constituted such an OC mixture containing 15 different components in environmentally relevant proportions and assessed its proliferative effects in four breast cancer cell lines (MCF-7, T47D, CAMA-1, MDAMB231) and in non-cancerous CV-1 cells. We also determined the capacity of the mixture to modulate cell cycle stage of breast cancer cells and to induce estrogenic and antiandrogenic effects using gene reporter assays. We observed that low concentrations of the mixture (100x10{sup 3} and 50x10{sup 3} dilutions) stimulated the proliferation of MCF-7 cells while higher concentrations (10x10{sup 3} and 5x10{sup 3} dilutions) had the opposite effect. In contrast, the mixture inhibited the proliferation of non-hormone-dependent cell lines. The mixture significantly increased the number of MCF-7 cells entering the S phase, an effect that was blocked by the antiestrogen ICI 182,780. Low concentrations of the mixture also caused an increase in CAMA-1 cell proliferation but only in the presence estradiol and dihydrotestosterone (p<0.05 at the 50x10{sup 3} dilution). DDT analogs and polychlorinated biphenyls all had the capacity to stimulate the proliferation of CAMA-1 cells in the presence of sex steroids. Reporter gene assays further revealed that the mixture and several of its constituents (DDT analogs, aldrin, dieldrin, {beta}-hexachlorocyclohexane, toxaphene) induced estrogenic effects, whereas the mixture and several components (DDT analogs, aldrin, dieldrin and PCBs) inhibited the androgen signaling pathway. Our results indicate that the complex OC mixture increases the proliferation of MCF-7 cells due to its estrogenic potential. The

Taurine-modified Ru(ii)-complex targets cancerous brain cells for photodynamic therapy.

Science.gov (United States)

Du, Enming; Hu, Xunwu; Roy, Sona; Wang, Peng; Deasy, Kieran; Mochizuki, Toshiaki; Zhang, Ye

2017-05-30

The precision and efficacy of photodynamic therapy (PDT) is essential for the treatment of brain tumors because the cancer cells are within or adjacent to the delicate nervous system. Taurine is an abundant amino acid in the brain that serves the central nervous system (CNS). A taurine-modified polypyridyl Ru-complex was shown to have optimized intracellular affinity in cancer cells through accumulation in lysosomes. Symmetrical modification of this Ru-complex by multiple taurine molecules enhanced the efficiency of molecular emission with boosted generation of reactive oxygen species. These characteristic features make the taurine-modified Ru-complex a potentially effective photosensitizer for PDT of target cancer cells, with outstanding efficacy in cancerous brain cells.

Mistletoe lectin I in complex with galactose and lactose reveals distinct sugar-binding properties

International Nuclear Information System (INIS)

Mikeska, Ruth; Wacker, Roland; Arni, Raghuvir; Singh, Tej P.; Mikhailov, Albert; Gabdoulkhakov, Azat; Voelter, Wolfgang; Betzel, Christian

2004-01-01

The structures of mistletoe lectin I in complex with lactose and galactose reveal differences in binding by the two known sites in subdomains α1 and γ2 and suggest the presence of a third low-affinity site in subdomain β1. The structures of mistletoe lectin I (ML-I) from Viscum album complexed with lactose and galactose have been determined at 2.3 Å resolution and refined to R factors of 20.9% (R free = 23.6%) and 20.9 (R free = 24.6%), respectively. ML-I is a heterodimer and belongs to the class of ribosome-inactivating proteins of type II, which consist of two chains. The A-chain has rRNA N-glycosidase activity and irreversibly inhibits eukaryotic ribosomes. The B-chain is a lectin and preferentially binds to galactose-terminated glycolipids and glycoproteins on cell membranes. Saccharide binding is performed by two binding sites in subdomains α1 and γ2 of the ML-I B-chain separated by ∼62 Å from each other. The favoured binding of galactose in subdomain α1 is achieved via hydrogen bonds connecting the 4-hydroxyl and 3-hydroxyl groups of the sugar moiety with the side chains of Asp23B, Gln36B and Lys41B and the main chain of 26B. The aromatic ring of Trp38B on top of the preferred binding pocket supports van der Waals packing of the apolar face of galactose and stabilizes the sugar–lectin complex. In the galactose-binding site II of subdomain γ2, Tyr249B provides the hydrophobic stacking and the side chains of Asp235B, Gln238B and Asn256B are hydrogen-bonding partners for galactose. In the case of the galactose-binding site I, the 2-hydroxyl group also stabilizes the sugar–protein complex, an interaction thus far rarely detected in galactose-specific lectins. Finally, a potential third low-affinity galactose-binding site in subunit β1 was identified in the present ML-I structures, in which a glycerol molecule from the cryoprotectant buffer has bound, mimicking the sugar compound

Stochasticity in the enterococcal sex pheromone response revealed by quantitative analysis of transcription in single cells.

Science.gov (United States)

Breuer, Rebecca J; Bandyopadhyay, Arpan; O'Brien, Sofie A; Barnes, Aaron M T; Hunter, Ryan C; Hu, Wei-Shou; Dunny, Gary M

2017-07-01

In Enterococcus faecalis, sex pheromone-mediated transfer of antibiotic resistance plasmids can occur under unfavorable conditions, for example, when inducing pheromone concentrations are low and inhibiting pheromone concentrations are high. To better understand this paradox, we adapted fluorescence in situ hybridization chain reaction (HCR) methodology for simultaneous quantification of multiple E. faecalis transcripts at the single cell level. We present direct evidence for variability in the minimum period, maximum response level, and duration of response of individual cells to a specific inducing condition. Tracking of induction patterns of single cells temporally using a fluorescent reporter supported HCR findings. It also revealed subpopulations of rapid responders, even under low inducing pheromone concentrations where the overall response of the entire population was slow. The strong, rapid induction of small numbers of cells in cultures exposed to low pheromone concentrations is in agreement with predictions of a stochastic model of the enterococcal pheromone response. The previously documented complex regulatory circuitry controlling the pheromone response likely contributes to stochastic variation in this system. In addition to increasing our basic understanding of the biology of a horizontal gene transfer system regulated by cell-cell signaling, demonstration of the stochastic nature of the pheromone response also impacts any future efforts to develop therapeutic agents targeting the system. Quantitative single cell analysis using HCR also has great potential to elucidate important bacterial regulatory mechanisms not previously amenable to study at the single cell level, and to accelerate the pace of functional genomic studies.

Stage-specific damage to synaptonemal complexes and metaphase chromosomes induced by X rays in male mouse germ cells

International Nuclear Information System (INIS)

Backer, L.C.; Sontag, M.R.; Allen, J.W.

1991-01-01

Synaptonemal complexes (SCs) reveal mutagen-induced effects in germ cell meiotic chromosomes. The study was aimed at characterizing relationships between SC and metaphase I chromosome damage following radiation exposure at various stages of spermatogenesis. Male mice were irradiated with doses of 0, 2, or 4 Gy, and spermatocytes were harvested at times consistent with earlier exposures as spermatogonial stem cells, preleptotene cells (premeiotic DNA synthesis), or meiotic prophase cells. After stem-cell exposure, twice as many rearrangements were observed in SCs as in metaphase I chromosomes. Irradiation during premeiotic DNA synthesis resulted in dose-related increases in SC breakage and rearrangements (including novel forms) and in metaphase chromosomal aberrations. Following prophase exposure, various types and levels of SC and metaphase damage were observed. Irradiation of zygotene cells led to high frequencies of chromosome multivalents in metaphase I without a correspondingly high level of damage in preceding prophase SCs. Thus, irradiation of premeiotic and meiotic cells results in variable relationships between SC and metaphase chromosome damage

Modeling of Complex Life Cycle Prediction Based on Cell Division

Directory of Open Access Journals (Sweden)

Fucheng Zhang

2017-01-01

Full Text Available Effective fault diagnosis and reasonable life expectancy are of great significance and practical engineering value for the safety, reliability, and maintenance cost of equipment and working environment. At present, the life prediction methods of the equipment are equipment life prediction based on condition monitoring, combined forecasting model, and driven data. Most of them need to be based on a large amount of data to achieve the problem. For this issue, we propose learning from the mechanism of cell division in the organism. We have established a moderate complexity of life prediction model across studying the complex multifactor correlation life model. In this paper, we model the life prediction of cell division. Experiments show that our model can effectively simulate the state of cell division. Through the model of reference, we will use it for the equipment of the complex life prediction.

A kinome-wide RNAi screen in Drosophila Glia reveals that the RIO kinases mediate cell proliferation and survival through TORC2-Akt signaling in glioblastoma.

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Renee D Read

Full Text Available Glioblastoma, the most common primary malignant brain tumor, is incurable with current therapies. Genetic and molecular analyses demonstrate that glioblastomas frequently display mutations that activate receptor tyrosine kinase (RTK and Pi-3 kinase (PI3K signaling pathways. In Drosophila melanogaster, activation of RTK and PI3K pathways in glial progenitor cells creates malignant neoplastic glial tumors that display many features of human glioblastoma. In both human and Drosophila, activation of the RTK and PI3K pathways stimulates Akt signaling along with other as-yet-unknown changes that drive oncogenesis. We used this Drosophila glioblastoma model to perform a kinome-wide genetic screen for new genes required for RTK- and PI3K-dependent neoplastic transformation. Human orthologs of novel kinases uncovered by these screens were functionally assessed in mammalian glioblastoma models and human tumors. Our results revealed that the atypical kinases RIOK1 and RIOK2 are overexpressed in glioblastoma cells in an Akt-dependent manner. Moreover, we found that overexpressed RIOK2 formed a complex with RIOK1, mTor, and mTor-complex-2 components, and that overexpressed RIOK2 upregulated Akt signaling and promoted tumorigenesis in murine astrocytes. Conversely, reduced expression of RIOK1 or RIOK2 disrupted Akt signaling and caused cell cycle exit, apoptosis, and chemosensitivity in glioblastoma cells by inducing p53 activity through the RpL11-dependent ribosomal stress checkpoint. These results imply that, in glioblastoma cells, constitutive Akt signaling drives RIO kinase overexpression, which creates a feedforward loop that promotes and maintains oncogenic Akt activity through stimulation of mTor signaling. Further study of the RIO kinases as well as other kinases identified in our Drosophila screen may reveal new insights into defects underlying glioblastoma and related cancers and may reveal new therapeutic opportunities for these cancers.

Surface complexation of neptunium (V) onto whole cells and cell componets of Shewanella alga

Energy Technology Data Exchange (ETDEWEB)

Reed, Donald Timothy [Los Alamos National Laboratory; Deo, Randhir P [ASU; Rittmann, Bruce E [ASU; Songkasiri, Warinthorn [UNAFFILIATED

2008-01-01

We systematically quantified surface complexation of neptunium(V) onto whole cells of Shewanella alga strain BrY and onto cell wall and extracellular polymeric substances (EPS) of S. alga. We first performed acid and base titrations and used the mathematical model FITEQL with constant-capacitance surface-complexation to determine the concentrations and deprotonation constants of specific surface functional groups. Deprotonation constants most likely corresponded to a carboxyl site associated with amino acids (pK{sub a} {approx} 2.4), a carboxyl group not associated with amino acids (pK{sub a} {approx} 5), a phosphoryl site (pK{sub a} {approx} 7.2), and an amine site (pK{sub a} > 10). We then carried out batch sorption experiments with Np(V) and each of the S. alga components at different pHs. Results show that solution pH influenced the speciation of Np(V) and each of the surface functional groups. We used the speciation sub-model of the biogeochemical model CCBATCH to compute the stability constants for Np(V) complexation to each surface functional group. The stability constants were similar for each functional group on S. alga bacterial whole cells, cell walls, and EPS, and they explain the complicated sorption patterns when they are combined with the aqueous-phase speciation of Np(V). For pH < 8, NpO{sub 2}{sup +} was the dominant form of Np(V), and its log K values for the low-pK{sub a} carboxyl, other carboxyl, and phosphoryl groups were 1.75, 1.75, and 2.5 to 3.1, respectively. For pH greater than 8, the key surface ligand was amine >XNH3+, which complexed with NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-}. The log K for NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-} complexed onto the amine groups was 3.1 to 3.6. All of the log K values are similar to those of Np(V) complexes with aqueous carboxyl and N-containing carboxyl ligands. These results point towards the important role of surface complexation in defining key actinide-microbiological interactions in the subsurface.

Radio metal (169Yb) uptake in normal and tumour cells in vitro. Influence of metabolic cell activity and complex structure

International Nuclear Information System (INIS)

Franke, W.G.; Kampf, G.

1996-01-01

Trivalent radio metal tracers have been used for tumour imaging and metastatic pain palliation. For better understanding their tumour accumulation, basic model studies of uptake of different 169 Yb complexes into cultured normal and tumour cells were performed. Whereas the uptake of 169 Yb citrate is strongly dependent on the metabolic activity and is not tumour-cell pacific, the uptake of 169 Yb complexed with amino carbonic acid (NTA, EDTA, DTPA) does not correlate to the metabolic activities. These complexes are taken up to a greater amount by the tumour cells (by a factor of about 2). Uptake of both complex types leads to a stable association to cellular compounds, 169 Yb is not releasable by the strong complexing agent DTPA. Protein binding of the 169 Yb complexes shows great influence on their cellular uptake. The bound proportion is no more available,for cellular uptake. The results indicate that i 0 uptake of 169 Yb citrate is an active cellular transport process which i not tumor-specific, ii) the 169 Yb amino carbonic acid complexes show a weak favouring by the tumour cells, iii) different from earlier acceptions the Yb complexes studied are not taken up by the cells in protein-bound form. The structure of the Yb complex is decisive for its protein binding and cellular uptake. (author). 13 refs., 6 figs

Proteomic analysis reveals the diversity and complexity of membrane proteins in chickpea (Cicer arietinum L.

Directory of Open Access Journals (Sweden)

Jaiswal Dinesh Kumar

2012-10-01

Full Text Available Abstract Background Compartmentalization is a unique feature of eukaryotes that helps in maintaining cellular homeostasis not only in intra- and inter-organellar context, but also between the cells and the external environment. Plant cells are highly compartmentalized with a complex metabolic network governing various cellular events. The membranes are the most important constituents in such compartmentalization, and membrane-associated proteins play diverse roles in many cellular processes besides being part of integral component of many signaling cascades. Results To obtain valuable insight into the dynamic repertoire of membrane proteins, we have developed a proteome reference map of a grain legume, chickpea, using two-dimensional gel electrophoresis. MALDI-TOF/TOF and LC-ESI-MS/MS analysis led to the identification of 91 proteins involved in a variety of cellular functions viz., bioenergy, stress-responsive and signal transduction, metabolism, protein synthesis and degradation, among others. Significantly, 70% of the identified proteins are putative integral membrane proteins, possessing transmembrane domains. Conclusions The proteomic analysis revealed many resident integral membrane proteins as well as membrane-associated proteins including those not reported earlier. To our knowledge, this is the first report of membrane proteome from aerial tissues of a crop plant. The findings may provide a better understanding of the biochemical machinery of the plant membranes at the molecular level that might help in functional genomics studies of different developmental pathways and stress-responses.

Involvement of F-Actin in Chaperonin-Containing t-Complex 1 Beta Regulating Mouse Mesangial Cell Functions in a Glucose-Induction Cell Model

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Jin-Shuen Chen

2011-01-01

Full Text Available The aim of this study is to investigate the role of chaperonin-containing t-complex polypeptide 1 beta (CCT2 in the regulation of mouse mesangial cell (mMC contraction, proliferation, and migration with filamentous/globular-(F/G- actin ratio under high glucose induction. A low CCT2 mMC model induced by treatment of small interference RNA was established. Groups with and without low CCT2 induction examined in normal and high (H glucose conditions revealed the following major results: (1 low CCT2 or H glucose showed the ability to attenuate F/G-actin ratio; (2 groups with low F/G-actin ratio all showed less cell contraction; (3 suppression of CCT2 may reduce the proliferation and migration which were originally induced by H glucose. In conclusion, CCT2 can be used as a specific regulator for mMC contraction, proliferation, and migration affected by glucose, which mechanism may involve the alteration of F-actin, particularly for cell contraction.

454 sequencing reveals extreme complexity of the class II Major Histocompatibility Complex in the collared flycatcher

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Gustafsson Lars

2010-12-01

Full Text Available Abstract Background Because of their functional significance, the Major Histocompatibility Complex (MHC class I and II genes have been the subject of continuous interest in the fields of ecology, evolution and conservation. In some vertebrate groups MHC consists of multiple loci with similar alleles; therefore, the multiple loci must be genotyped simultaneously. In such complex systems, understanding of the evolutionary patterns and their causes has been limited due to challenges posed by genotyping. Results Here we used 454 amplicon sequencing to characterize MHC class IIB exon 2 variation in the collared flycatcher, an important organism in evolutionary and immuno-ecological studies. On the basis of over 152,000 sequencing reads we identified 194 putative alleles in 237 individuals. We found an extreme complexity of the MHC class IIB in the collared flycatchers, with our estimates pointing to the presence of at least nine expressed loci and a large, though difficult to estimate precisely, number of pseudogene loci. Many similar alleles occurred in the pseudogenes indicating either a series of recent duplications or extensive concerted evolution. The expressed alleles showed unambiguous signals of historical selection and the occurrence of apparent interlocus exchange of alleles. Placing the collared flycatcher's MHC sequences in the context of passerine diversity revealed transspecific MHC class II evolution within the Muscicapidae family. Conclusions 454 amplicon sequencing is an effective tool for advancing our understanding of the MHC class II structure and evolutionary patterns in Passeriformes. We found a highly dynamic pattern of evolution of MHC class IIB genes with strong signals of selection and pronounced sequence divergence in expressed genes, in contrast to the apparent sequence homogenization in pseudogenes. We show that next generation sequencing offers a universal, affordable method for the characterization and, in perspective

VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells

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Ayumi Yoshida

2015-09-01

Full Text Available Neuropilin-1 (NRP1 has been identified as a VEGF-A receptor. DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1. In the present study, the RNA interference of VEGF-A or NRP1 suppressed DJM-1 cell proliferation. Furthermore, the overexpression of the NRP1 wild type restored shNRP1-treated DJM-1 cell proliferation, whereas NRP1 cytoplasmic deletion mutants did not. A co-immunoprecipitation analysis revealed that VEGF-A induced interactions between NRP1 and GIPC1, a scaffold protein, and complex formation between GIPC1 and Syx, a RhoGEF. The knockdown of GIPC1 or Syx reduced active RhoA and DJM-1 cell proliferation without affecting the MAPK or Akt pathway. C3 exoenzyme or Y27632 inhibited the VEGF-A-induced proliferation of DJM-1 cells. Conversely, the overexpression of the constitutively active form of RhoA restored the proliferation of siVEGF-A-treated DJM-1 cells. Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor. A cell-penetrating oligopeptide that targeted GIPC1/Syx complex formation inhibited the VEGF-A-induced activation of RhoA and suppressed DJM-1 cell proliferation. In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.

Neurosecretory cells of the amygdaloid complex during estrous cycle.

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Akhmadeev, A V; Kalimullina, L B

2005-02-01

Ultrastructure of neurosecretory cells of the dorsomedial nucleus of the cerebral amygdaloid complex (one of the main zones of sexual dimorphism) was studied in different phases of the estrous cycle. The characteristics of the "light" and "dark" cells change depending on the concentrations of sex steroids during estrus and metestrus.

Cyclometalated Ruthenium(II) Anthraquinone Complexes Exhibit Strong Anticancer Activity in Hypoxic Tumor Cells.

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Zeng, Leli; Chen, Yu; Huang, Huaiyi; Wang, Jinquan; Zhao, Donglei; Ji, Liangnian; Chao, Hui

2015-10-19

Hypoxia is the critical feature of the tumor microenvironment that is known to lead to resistance to many chemotherapeutic drugs. Six novel ruthenium(II) anthraquinone complexes were designed and synthesized; they exhibit similar or superior cytotoxicity compared to cisplatin in hypoxic HeLa, A549, and multidrug-resistant (A549R) tumor cell lines. Their anticancer activities are related to their lipophilicity and cellular uptake; therefore, these physicochemical properties of the complexes can be changed by modifying the ligands to obtain better anticancer candidates. Complex 1, the most potent member of the series, is highly active against hypoxic HeLa cancer cells (IC50 =0.53 μM). This complex likely has 46-fold better activity than cisplatin (IC50 =24.62 μM) in HeLa cells. This complex tends to accumulate in the mitochondria and the nucleus of hypoxic HeLa cells. Further mechanistic studies show that complex 1 induced cell apoptosis during hypoxia through multiple pathways, including those of DNA damage, mitochondrial dysfunction, and the inhibition of DNA replication and HIF-1α expression, making it an outstanding candidate for further in vivo studies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Toroidal surface complexes of bacteriophage φ12 are responsible for host-cell attachment

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Leo-Macias, Alejandra; Katz, Garrett; Wei Hui; Alimova, Alexandra; Katz, A.; Rice, William J.; Diaz-Avalos, Ruben; Hu Guobin; Stokes, David L.; Gottlieb, Paul

2011-01-01

Cryo-electron tomography and subtomogram averaging are utilized to determine that the bacteriophage φ12, a member of the Cystoviridae family, contains surface complexes that are toroidal in shape, are composed of six globular domains with six-fold symmetry, and have a discrete density connecting them to the virus membrane-envelope surface. The lack of this kind of spike in a reassortant of φ12 demonstrates that the gene for the hexameric spike is located in φ12's medium length genome segment, likely to the P3 open reading frames which are the proteins involved in viral-host cell attachment. Based on this and on protein mass estimates derived from the obtained averaged structure, it is suggested that each of the globular domains is most likely composed of a total of four copies of P3a and/or P3c proteins. Our findings may have implications in the study of the evolution of the cystovirus species in regard to their host specificity. - Research Highlights: → Subtomogram averaging reveals enhanced detail of a φ12 cystovirus surface protein complex. → The surface protein complex has a toroidal shape and six-fold symmetry. → It is encoded by the medium-size genome segment. → The proteins of the surface complex most likely are one copy of P3a and three copies of P3c.

Single-cell transcriptomic reconstruction reveals cell cycle and multi-lineage differentiation defects in Bcl11a-deficient hematopoietic stem cells.

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Tsang, Jason C H; Yu, Yong; Burke, Shannon; Buettner, Florian; Wang, Cui; Kolodziejczyk, Aleksandra A; Teichmann, Sarah A; Lu, Liming; Liu, Pentao

2015-09-21

Hematopoietic stem cells (HSCs) are a rare cell type with the ability of long-term self-renewal and multipotency to reconstitute all blood lineages. HSCs are typically purified from the bone marrow using cell surface markers. Recent studies have identified significant cellular heterogeneities in the HSC compartment with subsets of HSCs displaying lineage bias. We previously discovered that the transcription factor Bcl11a has critical functions in the lymphoid development of the HSC compartment. In this report, we employ single-cell transcriptomic analysis to dissect the molecular heterogeneities in HSCs. We profile the transcriptomes of 180 highly purified HSCs (Bcl11a (+/+) and Bcl11a (-/-)). Detailed analysis of the RNA-seq data identifies cell cycle activity as the major source of transcriptomic variation in the HSC compartment, which allows reconstruction of HSC cell cycle progression in silico. Single-cell RNA-seq profiling of Bcl11a (-/-) HSCs reveals abnormal proliferative phenotypes. Analysis of lineage gene expression suggests that the Bcl11a (-/-) HSCs are constituted of two distinct myeloerythroid-restricted subpopulations. Remarkably, similar myeloid-restricted cells could also be detected in the wild-type HSC compartment, suggesting selective elimination of lymphoid-competent HSCs after Bcl11a deletion. These defects are experimentally validated in serial transplantation experiments where Bcl11a (-/-) HSCs are myeloerythroid-restricted and defective in self-renewal. Our study demonstrates the power of single-cell transcriptomics in dissecting cellular process and lineage heterogeneities in stem cell compartments, and further reveals the molecular and cellular defects in the Bcl11a-deficient HSC compartment.

Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum.

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Yeoman, Jeffrey A; Hanssen, Eric; Maier, Alexander G; Klonis, Nectarios; Maco, Bohumil; Baum, Jake; Turnbull, Lynne; Whitchurch, Cynthia B; Dixon, Matthew W A; Tilley, Leann

2011-04-01

The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.

Telomere dysfunction and cell survival: roles for distinctTIN2-containing complexes

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Kim, Sahn-Ho; Davalos, Albert R.; Heo, Seok-Jin; Rodier, Francis; Beausejour, Christian; Kaminker, Patrick; Campisi, Judith

2006-11-07

Telomeres are maintained by three DNA binding proteins, TRF1, TRF2 and POT1, and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. These and two other proteins form a soluble complex that may be the core telomere-maintenance complex. It is not clear whether subcomplexes exist or function in vivo. Here, we provide evidence for two TIN2 subcomplexes with distinct functions in human cells. TIN2 ablation by RNA interference caused telomere uncapping and p53-independent cell death in all cells tested. However, we isolated two TIN2 complexes from cell lysates, each selectively sensitive to a TIN2 mutant (TIN2-13, TIN2-15C). In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN215C more than TIN2-13 caused genomic instability and cell death. Thus, TIN2 subcomplexes likely have distinct functions in telomere maintenance, and may provide selective targets for eliminating cells with mutant p53.

Identifying off-target effects of etomoxir reveals that carnitine palmitoyltransferase I is essential for cancer cell proliferation independent of β-oxidation.

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Cong-Hui Yao

2018-03-01

Full Text Available It has been suggested that some cancer cells rely upon fatty acid oxidation (FAO for energy. Here we show that when FAO was reduced approximately 90% by pharmacological inhibition of carnitine palmitoyltransferase I (CPT1 with low concentrations of etomoxir, the proliferation rate of various cancer cells was unaffected. Efforts to pharmacologically inhibit FAO more than 90% revealed that high concentrations of etomoxir (200 μM have an off-target effect of inhibiting complex I of the electron transport chain. Surprisingly, however, when FAO was reduced further by genetic knockdown of CPT1, the proliferation rate of these same cells decreased nearly 2-fold and could not be restored by acetate or octanoic acid supplementation. Moreover, CPT1 knockdowns had altered mitochondrial morphology and impaired mitochondrial coupling, whereas cells in which CPT1 had been approximately 90% inhibited by etomoxir did not. Lipidomic profiling of mitochondria isolated from CPT1 knockdowns showed depleted concentrations of complex structural and signaling lipids. Additionally, expression of a catalytically dead CPT1 in CPT1 knockdowns did not restore mitochondrial coupling. Taken together, these results suggest that transport of at least some long-chain fatty acids into the mitochondria by CPT1 may be required for anabolic processes that support healthy mitochondrial function and cancer cell proliferation independent of FAO.

Cytotoxicity of Titanate-Calcium Complexes to MC3T3 Osteoblast-Like Cells

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Drury, Jeanie L.; Moussi, Joelle; Taylor-Pashow, Kathryn M. L.

2016-01-01

Monosodium titanates (MST) are a relatively novel form of particulate titanium dioxide that have been proposed for biological use as metal sorbents or delivery agents, most recently calcium (II). In these roles, the toxicity of the titanate or its metal complex is crucial to its biological utility. The aim of this study was to determine the cytotoxicity of MST and MST-calcium complexes with MC3T3 osteoblast-like cells; MST-Ca(II) complexes could be useful to promote bone formation in various hard tissue applications. MC3T3 cells were exposed to native MST or MST-Ca(II) complexes for 24–72 h. A CellTiter-Blue® assay was employed to assess the metabolic activity of the cells. The results showed that MST and MST-Ca(II) suppressed MC3T3 metabolic activity significantly in a dose-, time-, and cell-density-dependent fashion. MST-Ca(II) suppressed MC3T3 metabolism in a statistically identical manner as native MST at all concentrations. We concluded that MST and MST-Ca(II) are significantly cytotoxic to MC3T3 cells through a mechanism yet unknown; this is a potential problem to the biological utility of these complexes. PMID:28044136

Cytotoxicity of Titanate-Calcium Complexes to MC3T3 Osteoblast-Like Cells

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Yen-Wei Chen

2016-01-01

Full Text Available Monosodium titanates (MST are a relatively novel form of particulate titanium dioxide that have been proposed for biological use as metal sorbents or delivery agents, most recently calcium (II. In these roles, the toxicity of the titanate or its metal complex is crucial to its biological utility. The aim of this study was to determine the cytotoxicity of MST and MST-calcium complexes with MC3T3 osteoblast-like cells; MST-Ca(II complexes could be useful to promote bone formation in various hard tissue applications. MC3T3 cells were exposed to native MST or MST-Ca(II complexes for 24–72 h. A CellTiter-Blue® assay was employed to assess the metabolic activity of the cells. The results showed that MST and MST-Ca(II suppressed MC3T3 metabolic activity significantly in a dose-, time-, and cell-density-dependent fashion. MST-Ca(II suppressed MC3T3 metabolism in a statistically identical manner as native MST at all concentrations. We concluded that MST and MST-Ca(II are significantly cytotoxic to MC3T3 cells through a mechanism yet unknown; this is a potential problem to the biological utility of these complexes.

Beyond Contagion: Reality Mining Reveals Complex Patterns of Social Influence.

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Aamena Alshamsi

Full Text Available Contagion, a concept from epidemiology, has long been used to characterize social influence on people's behavior and affective (emotional states. While it has revealed many useful insights, it is not clear whether the contagion metaphor is sufficient to fully characterize the complex dynamics of psychological states in a social context. Using wearable sensors that capture daily face-to-face interaction, combined with three daily experience sampling surveys, we collected the most comprehensive data set of personality and emotion dynamics of an entire community of work. From this high-resolution data about actual (rather than self-reported face-to-face interaction, a complex picture emerges where contagion (that can be seen as adaptation of behavioral responses to the behavior of other people cannot fully capture the dynamics of transitory states. We found that social influence has two opposing effects on states: adaptation effects that go beyond mere contagion, and complementarity effects whereby individuals' behaviors tend to complement the behaviors of others. Surprisingly, these effects can exhibit completely different directions depending on the stable personality or emotional dispositions (stable traits of target individuals. Our findings provide a foundation for richer models of social dynamics, and have implications on organizational engineering and workplace well-being.

Beyond Contagion: Reality Mining Reveals Complex Patterns of Social Influence.

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Alshamsi, Aamena; Pianesi, Fabio; Lepri, Bruno; Pentland, Alex; Rahwan, Iyad

2015-01-01

Contagion, a concept from epidemiology, has long been used to characterize social influence on people's behavior and affective (emotional) states. While it has revealed many useful insights, it is not clear whether the contagion metaphor is sufficient to fully characterize the complex dynamics of psychological states in a social context. Using wearable sensors that capture daily face-to-face interaction, combined with three daily experience sampling surveys, we collected the most comprehensive data set of personality and emotion dynamics of an entire community of work. From this high-resolution data about actual (rather than self-reported) face-to-face interaction, a complex picture emerges where contagion (that can be seen as adaptation of behavioral responses to the behavior of other people) cannot fully capture the dynamics of transitory states. We found that social influence has two opposing effects on states: adaptation effects that go beyond mere contagion, and complementarity effects whereby individuals' behaviors tend to complement the behaviors of others. Surprisingly, these effects can exhibit completely different directions depending on the stable personality or emotional dispositions (stable traits) of target individuals. Our findings provide a foundation for richer models of social dynamics, and have implications on organizational engineering and workplace well-being.

Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans.

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Fraune, Johanna; Alsheimer, Manfred; Volff, Jean-Nicolas; Busch, Karoline; Fraune, Sebastian; Bosch, Thomas C G; Benavente, Ricardo

2012-10-09

The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed no sequence homology. This discrepancy challenged the hypothesis that the SC arose only once in evolution. To pursue this matter we focused on the evolution of SYCP1 and SYCP3, the two major structural SC proteins of mammals. Remarkably, our comparative bioinformatic and expression studies revealed that SYCP1 and SYCP3 are also components of the SC in the basal metazoan Hydra. In contrast to previous assumptions, we therefore conclude that SYCP1 and SYCP3 form monophyletic groups of orthologous proteins across metazoans.

Geometric Mechanics Reveals Optimal Complex Terrestrial Undulation Patterns

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Gong, Chaohui; Astley, Henry; Schiebel, Perrin; Dai, Jin; Travers, Matthew; Goldman, Daniel; Choset, Howie; CMU Team; GT Team

Geometric mechanics offers useful tools for intuitively analyzing biological and robotic locomotion. However, utility of these tools were previously restricted to systems that have only two internal degrees of freedom and in uniform media. We show kinematics of complex locomotors that make intermittent contacts with substrates can be approximated as a linear combination of two shape bases, and can be represented using two variables. Therefore, the tools of geometric mechanics can be used to analyze motions of locomotors with many degrees of freedom. To demonstrate the proposed technique, we present studies on two different types of snake gaits which utilize combinations of waves in the horizontal and vertical planes: sidewinding (in the sidewinder rattlesnake C. cerastes) and lateral undulation (in the desert specialist snake C. occipitalis). C. cerastes moves by generating posteriorly traveling body waves in the horizontal and vertical directions, with a relative phase offset equal to +/-π/2 while C. occipitalismaintains a π/2 offset of a frequency doubled vertical wave. Geometric analysis reveals these coordination patterns enable optimal movement in the two different styles of undulatory terrestrial locomotion. More broadly, these examples demonstrate the utility of geometric mechanics in analyzing realistic biological and robotic locomotion.

Internalisation of gonadotrophin-receptor complex in ovarian luteal cells

International Nuclear Information System (INIS)

Conn, P.M.; Conti, M.; Harwood, J.P.; Dufau, M.L.; Catt, K.J.

1978-01-01

Following evidence that certain protein hormones can enter target cells the present investigation was undertaken which shows that gonadotrophin-induced receptor loss may occur by a process of internalisation of the hormone-receptor complex following the initial interaction of gonadotrophin with the cell surface. Localisation studies were carried out in 33-d old female rats previously treated with pregnant mare serum gonadotrophin and human chorionic gonadotrophin (hCG) to induce ovarian luteinisation. Animals were injected with 125 I-hCG to label the ovarian receptors for luteinising hormone in vivo. Microscope autoradiographs demonstrating distribution of 125 I-hCG in ovaries at various times following injection are shown. The combined results from the autoradiographs and from solubilisation experiments were used to determine the location and nature of the hCG-receptor complex following occupancy and loss of receptors from the plasma membrane of luteinised ovarian cells. (U.K.)

Organometallic tris(8-hydroxyquinoline)aluminum complexes as buffer layers and dopants in inverted organic solar cells

International Nuclear Information System (INIS)

Tolkki, Antti; Kaunisto, Kimmo; Heiskanen, Juha P.; Omar, Walaa A.E.; Huttunen, Kirsi; Lehtimäki, Suvi; Hormi, Osmo E.O.; Lemmetyinen, Helge

2012-01-01

Tris(8-hydroxyquinoline)aluminum (Alq 3 ) is a frequently used material for organic light emitting diodes. The electronic properties and solubility can be tuned by chemical tailoring of the quinoline part, which makes it an interesting candidate for organic solar cells. Steady-state absorption and fluorescence, as well as time-resolved fluorescence properties of the parent Alq 3 and a series of complexes consisting of derivatives, such as 4-substituted pyrazol, methylpyrazol, arylvinyl, and pyridinoanthrene moieties, of the quinoline ligand, were studied in solutions and in thin films. Suitability of the complexes as anodic buffer layers or dopants in inverted organic solar cells based on the well known bulk heterojunction of poly(3-hexylthiophene) (P3HT) and phenyl-C 61 -butyric acid methyl ester (PCBM) was tested. The devices equipped with the derivatives showed higher power conversion efficiency (η) compared to the photocells containing the parent Alq 3 . Open circuit voltage (V oc ) was increased when the derivatives were utilized as the anodic buffer layer. Doping of the P3HT:PCBM with a small amount of Alq 3 or its derivative improved short circuit current density, V oc , fill factor, and η, while the series resistance decreased. In addition, the devices were stable in air over several weeks without encapsulation. Possible mechanisms leading to the improvements in the photovoltaic performance by using the parent Alq 3 or its derivative as buffer layer or dopant are discussed. - Highlights: ► Tris(8-hydroxyquinoline)aluminum (Alq 3 ) complexes in inverted organic solar cells. ► The Alq 3 complexes were used as an anodic buffer layer and as a dopant. ► Efficiency increased and the derivatives revealed varying open circuit voltage. ► Photovoltaic performance was stable after storage in a dark ambient atmosphere.

Surface-enhanced Raman scattering reveals adsorption of mitoxantrone on plasma membrane of living cells

International Nuclear Information System (INIS)

Breuzard, G.; Angiboust, J.-F.; Jeannesson, P.; Manfait, M.; Millot, J.-M.

2004-01-01

Surface-enhanced Raman scattering (SERS) spectroscopy was applied to analyze mitoxantrone (MTX) adsorption on the plasma membrane microenvironment of sensitive (HCT-116 S) or BCRP/MXR-type resistant (HCT-116 R) cells. The addition of silver colloid to MTX-treated cells revealed an enhanced Raman scattering of MTX. Addition of extracellular DNA induced a total extinction of MTX Raman intensity for both cell lines, which revealed an adsorption of MTX on plasma membrane. A threefold higher MTX Raman intensity was observed for HCT-116 R, suggesting a tight MTX adsorption in the plasma membrane microenvironment. Fluorescence confocal microscopy confirmed a relative MTX emission around plasma membrane for HCT-116 R. After 30 min at 4 deg. C, a threefold decrease of the MTX Raman scattering was observed for HCT-116 R, contrary to HCT-116 S. Permeation with benzyl alcohol revealed a threefold decrease of membrane MTX adsorption on HCT-116 R, exclusively. This additional MTX adsorption should correspond to the drug bound to an unstable site on the HCT-116 R membrane. This study showed that SERS spectroscopy could be a direct method to reveal drug adsorption to the membrane environment of living cells

Design of magnetic gene complexes as effective and serum resistant gene delivery systems for mesenchymal stem cells.

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Zhang, Tian-Yuan; Wu, Jia-He; Xu, Qian-Hao; Wang, Xia-Rong; Lu, Jingxiong; Hu, Ying; Jo, Jun-Ichiro; Yamamoto, Masaya; Ling, Daishun; Tabata, Yasuhiko; Gao, Jian-Qing

2017-03-30

Gene engineered mesenchymal stem cells (MSCs) have been proposed as promising tools for their various applications in biomedicine. Nevertheless, the lack of an effective and safe way to genetically modify these stem cells is still a major obstacle in the current studies. Herein, we designed novel magnetic complexes by assembling cationized pullulan derivatives with magnetic iron oxide nanoparticles for delivering target genes to MSCs. Results showed that this complexes achieved effective gene expression with the assistance of external magnetic field, and resisted the adverse effect induced by serum proteins on the gene delivery. Moreover, neither significant cytotoxicity nor the interference on the osteogenic differentiation to MSCs were observed after magnetofection. Further studies revealed that this effective and serum resistant gene transfection was partly due to the accelerated and enhanced intracellular uptake process driven by external magnetic field. To conclude, the current study presented a novel option for genetic modification of MSCs in an effective, relatively safe and serum compatible way. Copyright © 2017 Elsevier B.V. All rights reserved.

Tutorial: Electroporation of cells in complex materials and tissue

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Rems, L.; Miklavčič, D.

2016-05-01

Electroporation is being successfully used in biology, medicine, food processing, and biotechnology, and in some environmental applications. Recent applications also include in addition to classical electroporation, where cells are exposed to micro- or milliseconds long pulses, exposures to extremely short nanosecond pulses, i.e., high-frequency electroporation. Electric pulses are applied to cells in different structural configurations ranging from suspended cells to cells in tissues. Understanding electroporation of cells in tissues and other complex environments is a key to its successful use and optimization in various applications. Thus, explanation will be provided theoretically/numerically with relation to experimental observations by scaling our understanding of electroporation from the molecular level of the cell membrane up to the tissue level.

Cytotoxicity and anti-tumor effects of new ruthenium complexes on triple negative breast cancer cells.

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Cecília P Popolin

Full Text Available Triple-negative breast cancer (TNBC is a highly aggressive breast cancer subtype. The high rate of metastasis associated to the fact that these cells frequently display multidrug resistance, make the treatment of metastatic disease difficult. Development of antitumor metal-based drugs was started with the discovery of cisplatin, however, the severe side effects represent a limitation for its clinical use. Ruthenium (Ru complexes with different ligands have been successfully studied as prospective antitumor drugs. In this work, we demonstrated the activity of a series of biphosphine bipyridine Ru complexes (1 [Ru(SO4(dppb(bipy], (2 [Ru(CO3(dppb(bipy], (3 [Ru(C2O4(dppb(bipy] and (4 [Ru(CH3CO2(dppb(bipy]PF6 [where dppb = 1,4-bis(diphenylphosphinobutane and bipy = 2,2'-bipyridine], on proliferation of TNBC (MDA-MB-231, estrogen-dependent breast tumor cells (MCF-7 and a non-tumor breast cell line (MCF-10A. Complex (4 was most effective among the complexes and was selected to be further investigated on effects on tumor cell adhesion, migration, invasion and in apoptosis. Moreover, DNA and HSA binding properties of this complex were also investigated. Results show that complex (4 was more efficient inhibiting proliferation of MDA-MB-231 cells over non-tumor cells. In addition, complex (4 was able to inhibit MDA-MB231 cells adhesion, migration and invasion and to induce apoptosis and inhibit MMP-9 secretion in TNBC cells. Complex (4 should be further investigated in vivo in order to stablish its potential to improve breast cancer treatment.

Genetic recombination within the human T-cell receptor α-chain gene complex

International Nuclear Information System (INIS)

Robinson, M.A.; Kindt, T.J.

1987-01-01

Genetic analyses of the human T-cell receptor (TCR) α-chain genes indicate that recombination events may occur frequently within this gene complex. Examination of the inheritance of restriction fragment length polymorphisms (RFLP) detected by using probes for constant or variable region gene segments made it possible to assign TCRα haplotypes to the 16 parents and 43 offspring of eight families studied. A total of six RFLP, three for the constant region and three for variable region segments, were examined in the present studies. Most enzyme and probe combinations tested revealed no polymorphism and those finally selected for the study showed limited polymorphism in that only two or, in one case, three allelic forms of the gene were seen. In spite of limited variability at this level, extensive heterogeneity was observed for the combinations of markers present in haplotypes, suggesting that frequent recombination events have occurred. Most strikingly, multiple combinations of RFLP occurring in close proximity of the TCRα constant region gene were observed in this study. A high recombination frequency for the TCRα gene complex is further supported by the observation that two children, one in each of two families, inherited recombinant TCRα haplotypes

Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices

International Nuclear Information System (INIS)

Lee, P.-F.; Yeh, Alvin T.; Bayless, Kayla J.

2009-01-01

The interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are fundamental in mediating various steps of angiogenesis, including cell adhesion, migration and sprout formation. Here, we used a noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique to optically image endothelial sprouting morphogenesis in three-dimensional (3D) collagen matrices. We simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Dynamic 3D imaging revealed EC interactions with collagen fibers along with quantifiable alterations in collagen matrix density elicited by EC movement through and morphogenesis within the matrix. Specifically, we observed increased collagen density in the area between bifurcation points of sprouting structures and anisotropic increases in collagen density around the perimeter of lumenal structures, but not advancing sprout tips. Proteinase inhibition studies revealed membrane-associated matrix metalloproteinase were utilized for sprout advancement and lumen expansion. Rho-associated kinase (p160ROCK) inhibition demonstrated that the generation of cell tension increased collagen matrix alterations. This study followed sprouting ECs within a 3D matrix and revealed that the advancing structures recognize and significantly alter their extracellular environment at the periphery of lumens as they progress

Biotin-tagged platinum(iv) complexes as targeted cytostatic agents against breast cancer cells.

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Muhammad, Nafees; Sadia, Nasreen; Zhu, Chengcheng; Luo, Cheng; Guo, Zijian; Wang, Xiaoyong

2017-09-05

A biotin-guided platinum IV complex is highly cytotoxic against breast cancer cells but hypotoxic against mammary epithelial cells. The mono-biotinylated Pt IV complex is superior to the di-biotinylated one and hence a promising drug candidate for the targeted therapy of breast cancer.

Substrate recognition by complement convertases revealed in the C5-cobra venom factor complex

DEFF Research Database (Denmark)

Laursen, Nick Stub; Andersen, Kasper Røjkjær; Braren, Ingke

2011-01-01

with a protease subunit (Bb or C2a). We determined the crystal structures of the C3b homologue cobra venom factor (CVF) in complex with C5, and in complex with C5 and the inhibitor SSL7 at 4.3 Å resolution. The structures reveal a parallel two-point attachment between C5 and CVF, where the presence of SSL7 only...... slightly affects the C5-CVF interface, explaining the IgA dependence for SSL7-mediated inhibition of C5 cleavage. CVF functions as a relatively rigid binding scaffold inducing a conformational change in C5, which positions its cleavage site in proximity to the serine protease Bb. A general model...

Naturally death-resistant precursor cells revealed as the origin of retinoblastoma

DEFF Research Database (Denmark)

Trinh, Emmanuelle; Lazzerini Denchi, Eros; Helin, Kristian

2004-01-01

The molecular mechanisms and the cell-of-origin leading to retinoblastoma are not well defined. In this issue of Cancer Cell, Bremner and colleagues describe the first inheritable model of retinoblastoma, revealing that loss of the pocket proteins pRb and p107 deregulates cell cycle exit in retinal...... precursors. The authors show that a subset of these precursors contain an inherent resistance to apoptosis, and that while most terminally differentiate, some are likely to acquire additional mutations, leading to tumor formation. Thus, this work defines the cell-of-origin of retinoblastoma and suggests...... that mutations giving increased proliferative capacity are required for retinoblastoma development....

Different types of nsP3-containing protein complexes in Sindbis virus-infected cells.

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Gorchakov, Rodion; Garmashova, Natalia; Frolova, Elena; Frolov, Ilya

2008-10-01

Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.

Mapping the HLA ligandome of Colorectal Cancer Reveals an Imprint of Malignant Cell Transformation.

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Löffler, Markus W; Kowalewski, Daniel J; Backert, Linus; Bernhardt, Jörg; Adam, Patrick; Schuster, Heiko; Dengler, Florian; Backes, Daniel; Kopp, Hans-Georg; Beckert, Stefan; Wagner, Silvia; Königsrainer, Ingmar; Kohlbacher, Oliver; Kanz, Lothar; Königsrainer, Alfred; Rammensee, Hans-Georg; Stevanovic, Stefan; Haen, Sebastian P

2018-05-22

Immune cell infiltrates have proven highly relevant for colorectal carcinoma (CRC) prognosis, making CRC a promising candidate for immunotherapy. Since tumors interact with the immune system via HLA-presented peptide ligands, exact knowledge of the peptidome constitution is fundamental for understanding this relationship. Here we comprehensively describe the naturally presented HLA-ligandome of CRC and corresponding non-malignant colon (NMC) tissue. Mass spectrometry identified 35,367 and 28,132 HLA-class I ligands on CRC and NMC, attributable to 7,684 and 6,312 distinct source proteins, respectively. Cancer-exclusive peptides were assessed on source protein level using Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein analysis through evolutionary relationships (PANTHER), revealing pathognomonic CRC-associated pathways including Wnt, TGF-β, PI3K, p53, and RTK-RAS. Relative quantitation of peptide presentation on paired CRC and NMC tissue further identified source proteins from cancer- and infection-associated pathways to be over-represented merely within the CRC ligandome. From the pool of tumor-exclusive peptides, a selected HLA-ligand subset was assessed for immunogenicity, with the majority exhibiting an existing T cell repertoire. Overall, these data show that the HLA-ligandome reflects cancer-associated pathways implicated in CRC oncogenesis, suggesting that alterations in tumor cell metabolism could result in cancer-specific, albeit not mutation-derived tumor-antigens. Hence, a defined pool of unique tumor peptides, attributable to complex cellular alterations that are exclusive to malignant cells might comprise promising candidates for immunotherapeutic applications. Copyright ©2018, American Association for Cancer Research.

Comprehensive transcriptome analyses correlated with untargeted metabolome reveal differentially expressed pathways in response to cell wall alterations.

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Reem, Nathan T; Chen, Han-Yi; Hur, Manhoi; Zhao, Xuefeng; Wurtele, Eve Syrkin; Li, Xu; Li, Ling; Zabotina, Olga

2018-03-01

This research provides new insights into plant response to cell wall perturbations through correlation of transcriptome and metabolome datasets obtained from transgenic plants expressing cell wall-modifying enzymes. Plants respond to changes in their cell walls in order to protect themselves from pathogens and other stresses. Cell wall modifications in Arabidopsis thaliana have profound effects on gene expression and defense response, but the cell signaling mechanisms underlying these responses are not well understood. Three transgenic Arabidopsis lines, two with reduced cell wall acetylation (AnAXE and AnRAE) and one with reduced feruloylation (AnFAE), were used in this study to investigate the plant responses to cell wall modifications. RNA-Seq in combination with untargeted metabolome was employed to assess differential gene expression and metabolite abundance. RNA-Seq results were correlated with metabolite abundances to determine the pathways involved in response to cell wall modifications introduced in each line. The resulting pathway enrichments revealed the deacetylation events in AnAXE and AnRAE plants induced similar responses, notably, upregulation of aromatic amino acid biosynthesis and changes in regulation of primary metabolic pathways that supply substrates to specialized metabolism, particularly those related to defense responses. In contrast, genes and metabolites of lipid biosynthetic pathways and peroxidases involved in lignin polymerization were downregulated in AnFAE plants. These results elucidate how primary metabolism responds to extracellular stimuli. Combining the transcriptomics and metabolomics datasets increased the power of pathway prediction, and demonstrated the complexity of pathways involved in cell wall-mediated signaling.

An Integrated Cell Purification and Genomics Strategy Reveals Multiple Regulators of Pancreas Development

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Benitez, Cecil M.; Qu, Kun; Sugiyama, Takuya; Pauerstein, Philip T.; Liu, Yinghua; Tsai, Jennifer; Gu, Xueying; Ghodasara, Amar; Arda, H. Efsun; Zhang, Jiajing; Dekker, Joseph D.; Tucker, Haley O.; Chang, Howard Y.; Kim, Seung K.

2014-01-01

The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus. PMID:25330008

An integrated cell purification and genomics strategy reveals multiple regulators of pancreas development.

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Cecil M Benitez

2014-10-01

Full Text Available The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus.

Cellular responses induced by Cu(II quinolinonato complexes in human tumor and hepatic cells

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Trávníček Zdeněk

2012-12-01

Full Text Available Abstract Background Inspired by the unprecedented historical success of cisplatin, one of the most important research directions in bioinorganic and medicinal chemistry is dedicated to the development of new anticancer compounds with the potential to surpass it in antitumor activity, while having lower unwanted side-effects. Therefore, a series of copper(II mixed-ligand complexes of the type [Cu(qui(L]Y · xH2O (1–6, where Hqui = 2-phenyl-3-hydroxy-4(1H-quinolinone, Y = NO3 (1, 3, 5 or BF4 (2, 4, 6, and L = 1,10-phenanthroline (phen (1, 2, 5-methyl-1,10-phenanthroline (mphen (3, 4 and bathophenanthroline (bphen (5, 6, was studied for their in vitro cytotoxicity against several human cancer cell lines (A549 lung carcinoma, HeLa cervix epitheloid carcinoma, G361 melanoma cells, A2780 ovarian carcinoma, A2780cis cisplatin-resistant ovarian carcinoma, LNCaP androgen-sensitive prostate adenocarcinoma and THP-1 monocytic leukemia. Results The tested complexes displayed a stronger cytotoxic effect against all the cancer cells as compared to cisplatin. The highest cytotoxicity was found for the complexes 4 (IC50 = 0.36 ± 0.05 μM and 0.56 ± 0.15 μM, 5 (IC50 = 0.66 ± 0.07 μM and 0.73 ± 0.08 μM and 6 (IC50 = 0.57 ± 0.11 μM and 0.70 ± 0.20 μM against A2780, and A2780cis respectively, as compared with the values of 12.0 ± 0.8 μM and 27.0 ± 4.6 μM determined for cisplatin. Moreover, the tested complexes were much less cytotoxic to primary human hepatocytes than to the cancer cells. The complexes 5 and 6 exhibited significantly high ability to modulate secretion of the pro-inflammatory cytokines TNF-α (2873 ± 238 pg/mL and 3284 ± 139 pg/mL for 5, and 6 respectively and IL-1β (1177 ± 128 pg/mL and 1087 ± 101 pg/mL for 5, and 6 respectively tested on the lipopolysaccharide (LPS-stimulated THP-1 cells as compared with the values of 1173�

Opto-acoustic microscopy reveals adhesion mechanics of single cells

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Abi Ghanem, Maroun; Dehoux, Thomas; Liu, Liwang; Le Saux, Guillaume; Plawinski, Laurent; Durrieu, Marie-Christine; Audoin, Bertrand

2018-01-01

Laser-generated GHz-ultrasonic-based technologies have shown the ability to image single cell adhesion and stiffness simultaneously. Using this new modality, we here demonstrate quantitative indicators to investigate contact mechanics and adhesion processes of the cell. We cultured human cells on a rigid substrate, and we used an inverted pulsed opto-acoustic microscope to generate acoustic pulses containing frequencies up to 100 GHz in the substrate. We map the reflection of the acoustic pulses at the cell-substrate interface to obtain images of the acoustic impedance of the cell, Zc, as well as of the stiffness of the interface, K, with 1 μm lateral resolution. Our results show that the standard deviation ΔZc reveals differences between different cell types arising from the multiplicity of local conformations within the nucleus. From the distribution of K-values within the nuclear region, we extract a mean interfacial stiffness, Km, that quantifies the average contact force in areas of the cell displaying weak bonding. By analogy with classical contact mechanics, we also define the ratio of the real to nominal contact areas, Sr/St. We show that Km can be interpreted as a quantitative indicator of passive contact at metal-cell interfaces, while Sr/St is sensitive to active adhesive processes in the nuclear region. The ability to separate the contributions of passive and active adhesion processes should allow gaining insight into cell-substrate interactions, with important applications in tissue engineering.

Role of Conserved Oligomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast Cancer Cells

Science.gov (United States)

2006-05-01

of COG complex function we utilized RNA interference assay to knockdown COG3p subunit of COG complex in normal and breast cancer cells and other tumor...protein trafficking, but the role of the COG complex in the abnormal glycosylation and secretion of tumor markers in breast cancer cells remains... COG complex in breast cancer cells MCF7 had been elevated 2-4 times in comparison to HB2 cells (Figure 5 A). The expression of HeLa COG3 CD44 ab

Induction of cell-cell fusion by ectromelia virus is not inhibited by its fusion inhibitory complex

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Fuchs Pinhas

2009-09-01

Full Text Available Abstract Background Ectromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes. This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium. Results We show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within" or by infection with a high amount of virus particles per cell (fusion "from without". Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection. Conclusion Ectromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.

Gaussian graphical modeling reveals specific lipid correlations in glioblastoma cells

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Mueller, Nikola S.; Krumsiek, Jan; Theis, Fabian J.; Böhm, Christian; Meyer-Bäse, Anke

2011-06-01

Advances in high-throughput measurements of biological specimens necessitate the development of biologically driven computational techniques. To understand the molecular level of many human diseases, such as cancer, lipid quantifications have been shown to offer an excellent opportunity to reveal disease-specific regulations. The data analysis of the cell lipidome, however, remains a challenging task and cannot be accomplished solely based on intuitive reasoning. We have developed a method to identify a lipid correlation network which is entirely disease-specific. A powerful method to correlate experimentally measured lipid levels across the various samples is a Gaussian Graphical Model (GGM), which is based on partial correlation coefficients. In contrast to regular Pearson correlations, partial correlations aim to identify only direct correlations while eliminating indirect associations. Conventional GGM calculations on the entire dataset can, however, not provide information on whether a correlation is truly disease-specific with respect to the disease samples and not a correlation of control samples. Thus, we implemented a novel differential GGM approach unraveling only the disease-specific correlations, and applied it to the lipidome of immortal Glioblastoma tumor cells. A large set of lipid species were measured by mass spectrometry in order to evaluate lipid remodeling as a result to a combination of perturbation of cells inducing programmed cell death, while the other perturbations served solely as biological controls. With the differential GGM, we were able to reveal Glioblastoma-specific lipid correlations to advance biomedical research on novel gene therapies.

A protocadherin-cadherin-FLRT3 complex controls cell adhesion and morphogenesis.

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Xuejun Chen

2009-12-01

Full Text Available Paraxial protocadherin (PAPC and fibronectin leucine-rich domain transmembrane protein-3 (FLRT3 are induced by TGFbeta signaling in Xenopus embryos and both regulate morphogenesis by inhibiting C-cadherin mediated cell adhesion.We have investigated the functional and physical relationships between PAPC, FLRT3, and C-cadherin. Although neither PAPC nor FLRT3 are required for each other to regulate C-cadherin adhesion, they do interact functionally and physically, and they form a complex with cadherins. By itself PAPC reduces cell adhesion physiologically to induce cell sorting, while FLRT3 disrupts adhesion excessively to cause cell dissociation. However, when expressed together PAPC limits the cell dissociating and tissue disrupting activity of FLRT3 to make it effective in physiological cell sorting. PAPC counteracts FLRT3 function by inhibiting the recruitment of the GTPase RND1 to the FLRT3 cytoplasmic domain.PAPC and FLRT3 form a functional complex with cadherins and PAPC functions as a molecular "governor" to maintain FLRT3 activity at the optimal level for physiological regulation of C-cadherin adhesion, cell sorting, and morphogenesis.

Single-cell RNA-Seq reveals cell heterogeneity and hierarchy within mouse mammary epithelia.

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Sun, Heng; Miao, Zhengqiang; Zhang, Xin; Chan, Un In; Su, Sek Man; Guo, Sen; Wong, Chris Koon Ho; Xu, Xiaoling; Deng, Chu-Xia

2018-04-17

The mammary gland is very intricately and well organized into distinct tissues, including epithelia, endothelia, adipocytes, and stromal and immune cells. Many mammary gland diseases, such as breast cancer arise from abnormalities in the mammary epithelium, which is mainly composed of two distinct lineages, the basal and luminal cells. Because of the limitation of traditional transcriptome analysis of bulk mammary cells, the hierarchy and heterogeneity of mammary cells within these two lineages remain unclear. To this end, using single-cell RNA-Seq coupled with FACS analysis and principal component analysis, we determined gene expression profiles of mammary epithelial cells of virgin and pregnant mice. These analyses revealed a much higher heterogeneity among the mammary cells than has been previously reported and enabled cell classification into distinct subgroups according to signature gene markers present in each group. We also identified and verified a rare CDH5+ cell subpopulation within a basal cell lineage as quiescent mammary stem cells (MaSCs). Moreover, using pseudo-temporal analysis, we reconstructed the developmental trajectory of mammary epithelia and uncovered distinct changes in gene expression and in biological functions of mammary cells along the developmental process. In conclusion, our work greatly refines the resolution of the cellular hierarchy in developing mammary tissues. The discovery of CDH5+ cells as MaSCs in these tissues may have implications for our understanding of the initiation, development, and pathogenesis of mammary tumors. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Phenotypic diversity of diploid and haploid Emiliania huxleyi cells and of cells in different growth phases revealed by comparative metabolomics.

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Mausz, Michaela A; Pohnert, Georg

2015-01-01

In phytoplankton a high species diversity of microalgae co-exists at a given time. But diversity is not only reflected by the species composition. Within these species different life phases as well as different metabolic states can cause additional diversity. One important example is the coccolithophore Emiliania huxleyi. Diploid cells play an important role in marine ecosystems since they can form massively abundant algal blooms but in addition the less abundant haploid life phase of E. huxleyi occurs in lower quantities. Both life phases may fulfill different functions in the plankton. We hypothesize that in addition to the functional diversity caused by this life phase transition the growth stage of cells can also influence the metabolic composition and thus the ecological impact of E. huxleyi. Here we introduce a metabolomic survey in dependence of life phases as well as different growth phases to reveal such changes. The comparative metabolomic approach is based on the extraction of intracellular metabolites from intact microalgae, derivatization and analysis by gas chromatography coupled to mass spectrometry (GC-MS). Automated data processing and statistical analysis using canonical analysis of principal coordinates (CAP) revealed unique metabolic profiles for each life phase. Concerning the correlations of metabolites to growth phases, complex patterns were observed. As for example the saccharide mannitol showed its highest concentration in the exponential phase, whereas fatty acids were correlated to stationary and sterols to declining phase. These results are indicative for specific ecological roles of these stages of E. huxleyi and are discussed in the context of previous physiological and ecological studies. Copyright © 2014 Elsevier GmbH. All rights reserved.

Synergy analysis reveals association between insulin signaling and desmoplakin expression in palmitate treated HepG2 cells.

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Xuewei Wang

Full Text Available The regulation of complex cellular activities in palmitate treated HepG2 cells, and the ensuing cytotoxic phenotype, involves cooperative interactions between genes. While previous approaches have largely focused on identifying individual target genes, elucidating interacting genes has thus far remained elusive. We applied the concept of information synergy to reconstruct a "gene-cooperativity" network for palmititate-induced cytotoxicity in liver cells. Our approach integrated gene expression data with metabolic profiles to select a subset of genes for network reconstruction. Subsequent analysis of the network revealed insulin signaling as the most significantly enriched pathway, and desmoplakin (DSP as its top neighbor. We determined that palmitate significantly reduces DSP expression, and treatment with insulin restores the lost expression of DSP. Insulin resistance is a common pathological feature of fatty liver and related ailments, whereas loss of DSP has been noted in liver carcinoma. Reduced DSP expression can lead to loss of cell-cell adhesion via desmosomes, and disrupt the keratin intermediate filament network. Our findings suggest that DSP expression may be perturbed by palmitate and, along with insulin resistance, may play a role in palmitate induced cytotoxicity, and serve as potential targets for further studies on non-alcoholic fatty liver disease (NAFLD.

The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells.

Science.gov (United States)

Hares, Michelle C; Hinchliffe, Stewart J; Strong, Philippa C R; Eleftherianos, Ioannis; Dowling, Andrea J; ffrench-Constant, Richard H; Waterfield, Nick

2008-11-01

The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode approximately 1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalian-pathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 degrees C and 37 degrees C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multi-nucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further

Live cell CRISPR-imaging in plants reveals dynamic telomere movements

KAUST Repository

Dreissig, Steven

2017-05-16

Elucidating the spatio-temporal organization of the genome inside the nucleus is imperative to understand the regulation of genes and non-coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long-standing gap between sequencing studies which reveal genomic information and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial CRISPR-Cas9 system. By fusing eGFP/mRuby2 to the catalytically inactive version of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR-dCas9, we reveal dynamic telomere movements of up to 2 μm within 30 minutes during interphase. Furthermore, we show that CRISPR-dCas9 can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for imaging of multiple genomic loci in live plants cells. CRISPR-imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells.

Effects of reactive Mn(III)-oxalate complexes on structurally intact plant cell walls

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Summering, J. A.; Keiluweit, M.; Goni, M. A.; Nico, P. S.; Kleber, M.

2011-12-01

Lignin components in the in plant litter are commonly assumed to have longer residence times in soil than many other compounds, which are supposedly, more easily degradable. The supposed resistance of lignin compounds to decomposition is generally attributed to the complex chain of biochemical steps required to create footholds in the non-porous structure of ligno-cellulose in cell walls. Interestingly, Mn(III) complexes have shown the ability to degrade ligno-cellulose. Mn(III) chelated by ligands such as oxalate are soluble oxidizers with a high affinity for lignin structures. Here we determined (i) the formation and decay kinetics of the Mn(III)-oxalate complexes in aqueous solution and (ii) the effects that these complexes have on intact ligno-cellulose. UV/vis spectroscopy and iodometric titrations confirmed the transient nature of Mn(III)-oxalate complexes with decay rates being in the order of hours. Zinnia elegans tracheary elements - a model ligno-cellulose substrate - were treated with Mn(III)-oxalate complexes in a newly developed flow-through reactor. Soluble decomposition products released during the treatment were analyzed by GC/MS and the degree of cell integrity was measured by cell counts, pre- and post-treatment counts indicate a decrease in intact Zinnia elegans as a result of Mn(III)-treatment. GC/MS results showed the release of a multitude of solubilized lignin breakdown products from plant cell walls. We conclude that Mn(III)-oxalate complexes have the ability to lyse intact plant cells and solubilize lignin. Lignin decomposition may thus be seen as resource dependent, with Mn(III) a powerful resource that should be abundant in terrestrial characterized by frequent redox fluctuations.

Peripheral Light-Harvesting LH2 Complex Can Be Assembled in Cells of Nonsulfur Purple Bacterium Rhodoblastus acidophilus without Carotenoids.

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Bol'shakov, M A; Ashikhmin, A A; Makhneva, Z K; Moskalenko, A A

2015-09-01

The effect of carotenoids on the assembly of LH2 complex in cells of the purple nonsulfur bacterium Rhodoblastus acidophilus was investigated. For this purpose, the bacterial culture was cultivated with an inhibitor of carotenoid biosynthesis - 71 µM diphenylamine (DPA). The inhibitor decreased the level of biosynthesis of the colored carotenoids in membranes by ~58%. It was found that a large amount of phytoene was accumulated in them. This carotenoid precursor was bound nonspecifically to LH2 complex and did not stabilize its structure. Thermostability testing of the isolated LH2 complex together with analysis of carotenoid composition revealed that the population of this complex was heterogeneous with respect to carotenoid composition. One fraction of the LH2 complex with carotenoid content around 90% remains stable and was not destroyed under heating for 15 min at 50°C. The other fraction of LH2 complex containing on average less than one molecule of carotenoid per complex was destroyed under heating, forming a zone of free pigments (and polypeptides). The data suggest that a certain part of the LH2 complexes is assembled without carotenoids in cells of the nonsulfur bacterium Rbl. acidophilus grown with DPA. These data contradict the fact that the LH2 complex from nonsulfur bacteria cannot be assembled without carotenoids, but on the other hand, they are in good agreement with the results demonstrated in our earlier studies of the sulfur bacteria Allochromatium minutissimum and Ectothiorhodospira haloalkaliphila. Carotenoidless LH2 complex was obtained from these bacteria with the use of DPA (Moskalenko, A. A., and Makhneva, Z. K. (2012) J. Photochem. Photobiol., 108, 1-7; Ashikhmin, A., et al. (2014) Photosynth. Res., 119, 291-303).

A recombinant antibody with the antigen-specific, major histocompatibility complex-restricted specificity of T cells

DEFF Research Database (Denmark)

Andersen, P S; Stryhn, A; Hansen, B E

1996-01-01

Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might ...

A Recombinant Antibody with the Antigen-Specific, Major Histocompatibility Complex-Restricted Specificity of T Cells

Science.gov (United States)

Andersen, Peter S.; Stryhn, Anette; Hansen, Bjarke E.; Fugger, Lars; Engberg, Jan; Buus, Soren

1996-03-01

Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes.

Single-Cell Gene Expression Analysis of a Human ESC Model of Pancreatic Endocrine Development Reveals Different Paths to β-Cell Differentiation.

Science.gov (United States)

Petersen, Maja Borup Kjær; Azad, Ajuna; Ingvorsen, Camilla; Hess, Katja; Hansson, Mattias; Grapin-Botton, Anne; Honoré, Christian

2017-10-10

The production of insulin-producing β cells from human embryonic stem cells (hESCs) in vitro represents a promising strategy for a cell-based therapy for type 1 diabetes mellitus. To explore the cellular heterogeneity and temporal progression of endocrine progenitors and their progeny, we performed single-cell qPCR on more than 500 cells across several stages of in vitro differentiation of hESCs and compared them with human islets. We reveal distinct subpopulations along the endocrine differentiation path and an early lineage bifurcation toward either polyhormonal cells or β-like cells. We uncover several similarities and differences with mouse development and reveal that cells can take multiple paths to the same differentiation state, a principle that could be relevant to other systems. Notably, activation of the key β-cell transcription factor NKX6.1 can be initiated before or after endocrine commitment. The single-cell temporal resolution we provide can be used to improve the production of functional β cells. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer

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Chiara Francavilla

2017-03-01

Full Text Available Our understanding of the molecular determinants of cancer is still inadequate because of cancer heterogeneity. Here, using epithelial ovarian cancer (EOC as a model system, we analyzed a minute amount of patient-derived epithelial cells from either healthy or cancerous tissues by single-shot mass-spectrometry-based phosphoproteomics. Using a multi-disciplinary approach, we demonstrated that primary cells recapitulate tissue complexity and represent a valuable source of differentially expressed proteins and phosphorylation sites that discriminate cancer from healthy cells. Furthermore, we uncovered kinase signatures associated with EOC. In particular, CDK7 targets were characterized in both EOC primary cells and ovarian cancer cell lines. We showed that CDK7 controls cell proliferation and that pharmacological inhibition of CDK7 selectively represses EOC cell proliferation. Our approach defines the molecular landscape of EOC, paving the way for efficient therapeutic approaches for patients. Finally, we highlight the potential of phosphoproteomics to identify clinically relevant and druggable pathways in cancer.

Opto-acoustic microscopy reveals adhesion mechanics of single cells.

Science.gov (United States)

Abi Ghanem, Maroun; Dehoux, Thomas; Liu, Liwang; Le Saux, Guillaume; Plawinski, Laurent; Durrieu, Marie-Christine; Audoin, Bertrand

2018-01-01

Laser-generated GHz-ultrasonic-based technologies have shown the ability to image single cell adhesion and stiffness simultaneously. Using this new modality, we here demonstrate quantitative indicators to investigate contact mechanics and adhesion processes of the cell. We cultured human cells on a rigid substrate, and we used an inverted pulsed opto-acoustic microscope to generate acoustic pulses containing frequencies up to 100 GHz in the substrate. We map the reflection of the acoustic pulses at the cell-substrate interface to obtain images of the acoustic impedance of the cell, Z c , as well as of the stiffness of the interface, K, with 1 μm lateral resolution. Our results show that the standard deviation ΔZ c reveals differences between different cell types arising from the multiplicity of local conformations within the nucleus. From the distribution of K-values within the nuclear region, we extract a mean interfacial stiffness, K m , that quantifies the average contact force in areas of the cell displaying weak bonding. By analogy with classical contact mechanics, we also define the ratio of the real to nominal contact areas, S r /S t . We show that K m can be interpreted as a quantitative indicator of passive contact at metal-cell interfaces, while S r /S t is sensitive to active adhesive processes in the nuclear region. The ability to separate the contributions of passive and active adhesion processes should allow gaining insight into cell-substrate interactions, with important applications in tissue engineering.

Iodinated chlorin p6 copper complex induces anti-proliferative effect in oral cancer cells through elevation of intracellular reactive oxygen species.

Science.gov (United States)

Sarbadhikary, Paromita; Dube, Alok

2017-11-01

We investigated the anticancer chemotoxicity of previously reported iodinated chlorin p 6 copper complex (ICp 6 -Cu), a novel chlorophyll derivative in which copper is attached to the side chain carboxylate groups via coordination. Human oral carcinoma cells NT8e, 4451 and the non-cancerous keratinocyte HaCaT cells were treated with ICp 6 -Cu for 48 h in dark and cell viability, proliferation and morphological alterations were examined. ICp 6 -Cu showed pronounced cytotoxicity in cancer cells with IC 50 ∼40 μM, whereas, the viability of HaCaT cells was not affected. Cell proliferation assay revealed that ICp 6 -Cu at IC 50 concentration led to complete inhibition of cell proliferation in both the cell lines. Cell morphology studied by confocal microscopy showed absence of cell death via necrosis or apoptosis. Instead, the treated cells displayed distinct features of non-apoptotic death such as highly vacuolated cytoplasm, lysosomal membrane permeabilization and damage to cytoskeleton F-actin filaments. In addition, ICp 6 -Cu treatment led to time dependent increase in the intracellular level of reactive oxygen species (ROS) and the cytotoxicity of ICp 6 -Cu was significantly inhibited by pre-treatment of cells with antioxidants (glutathione and trolox). These findings revealed that ICp 6 -Cu is a potent chemotoxic agent which can induce cytotoxic effect in cancer cells through elevation of intracellular ROS. It is suggested that ICp 6 -Cu may provide tumor selective chemotoxicity by exploiting difference of redox environment in normal and cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states.

Science.gov (United States)

Schlau-Cohen, Gabriela S; Wang, Quan; Southall, June; Cogdell, Richard J; Moerner, W E

2013-07-02

Photosynthetic organisms flourish under low light intensities by converting photoenergy to chemical energy with near unity quantum efficiency and under high light intensities by safely dissipating excess photoenergy and deleterious photoproducts. The molecular mechanisms balancing these two functions remain incompletely described. One critical barrier to characterizing the mechanisms responsible for these processes is that they occur within proteins whose excited-state properties vary drastically among individual proteins and even within a single protein over time. In ensemble measurements, these excited-state properties appear only as the average value. To overcome this averaging, we investigate the purple bacterial antenna protein light harvesting complex 2 (LH2) from Rhodopseudomonas acidophila at the single-protein level. We use a room-temperature, single-molecule technique, the anti-Brownian electrokinetic trap, to study LH2 in a solution-phase (nonperturbative) environment. By performing simultaneous measurements of fluorescence intensity, lifetime, and spectra of single LH2 complexes, we identify three distinct states and observe transitions occurring among them on a timescale of seconds. Our results reveal that LH2 complexes undergo photoactivated switching to a quenched state, likely by a conformational change, and thermally revert to the ground state. This is a previously unobserved, reversible quenching pathway, and is one mechanism through which photosynthetic organisms can adapt to changes in light intensities.

Pea Border Cell Maturation and Release Involve Complex Cell Wall Structural Dynamics.

Science.gov (United States)

Mravec, Jozef; Guo, Xiaoyuan; Hansen, Aleksander Riise; Schückel, Julia; Kračun, Stjepan Krešimir; Mikkelsen, Maria Dalgaard; Mouille, Grégory; Johansen, Ida Elisabeth; Ulvskov, Peter; Domozych, David S; Willats, William George Tycho

2017-06-01

The adhesion of plant cells is vital for support and protection of the plant body and is maintained by a variety of molecular associations between cell wall components. In some specialized cases, though, plant cells are programmed to detach, and root cap-derived border cells are examples of this. Border cells (in some species known as border-like cells) provide an expendable barrier between roots and the environment. Their maturation and release is an important but poorly characterized cell separation event. To gain a deeper insight into the complex cellular dynamics underlying this process, we undertook a systematic, detailed analysis of pea ( Pisum sativum ) root tip cell walls. Our study included immunocarbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy, quantitative reverse transcription-PCR of cell wall biosynthetic genes, analysis of hydrolytic activities, transmission electron microscopy, and immunolocalization of cell wall components. Using this integrated glycobiology approach, we identified multiple novel modes of cell wall structural and compositional rearrangement during root cap growth and the release of border cells. Our findings provide a new level of detail about border cell maturation and enable us to develop a model of the separation process. We propose that loss of adhesion by the dissolution of homogalacturonan in the middle lamellae is augmented by an active biophysical process of cell curvature driven by the polarized distribution of xyloglucan and extensin epitopes. © 2017 American Society of Plant Biologists. All Rights Reserved.

Towards comprehensive cell lineage reconstructions in complex organisms using light-sheet microscopy.

Science.gov (United States)

Amat, Fernando; Keller, Philipp J

2013-05-01

Understanding the development of complex multicellular organisms as a function of the underlying cell behavior is one of the most fundamental goals of developmental biology. The ability to quantitatively follow cell dynamics in entire developing embryos is an indispensable step towards such a system-level understanding. In recent years, light-sheet fluorescence microscopy has emerged as a particularly promising strategy for recording the in vivo data required to realize this goal. Using light-sheet fluorescence microscopy, entire complex organisms can be rapidly imaged in three dimensions at sub-cellular resolution, achieving high temporal sampling and excellent signal-to-noise ratio without damaging the living specimen or bleaching fluorescent markers. The resulting datasets allow following individual cells in vertebrate and higher invertebrate embryos over up to several days of development. However, the complexity and size of these multi-terabyte recordings typically preclude comprehensive manual analyses. Thus, new computational approaches are required to automatically segment cell morphologies, accurately track cell identities and systematically analyze cell behavior throughout embryonic development. We review current efforts in light-sheet microscopy and bioimage informatics towards this goal, and argue that comprehensive cell lineage reconstructions are finally within reach for many key model organisms, including fruit fly, zebrafish and mouse. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

Science.gov (United States)

Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

2018-01-01

DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1 ) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P correlation (R T4  > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

Colocalization and Disposition of Cellulosomes in Clostridium clariflavum as Revealed by Correlative Superresolution Imaging

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Lior Artzi

2018-02-01

Full Text Available Cellulosomes are multienzyme complexes produced by anaerobic, cellulolytic bacteria for highly efficient breakdown of plant cell wall polysaccharides. Clostridium clariflavum is an anaerobic, thermophilic bacterium that produces the largest assembled cellulosome complex in nature to date, comprising three types of scaffoldins: a primary scaffoldin, ScaA; an adaptor scaffoldin, ScaB; and a cell surface anchoring scaffoldin, ScaC. This complex can contain 160 polysaccharide-degrading enzymes. In previous studies, we proposed potential types of cellulosome assemblies in C. clariflavum and demonstrated that these complexes are released into the extracellular medium. In the present study, we explored the disposition of the highly structured, four-tiered cell-anchored cellulosome complex of this bacterium. Four separate, integral cellulosome components were subjected to immunolabeling: ScaA, ScaB, ScaC, and the cellulosome’s most prominent enzyme, GH48. Imaging of the cells by correlating scanning electron microscopy and three-dimensional (3D superresolution fluorescence microscopy revealed that some of the protuberance-like structures on the cell surface represent cellulosomes and that the components are highly colocalized and organized by a defined hierarchy on the cell surface. The display of the cellulosome on the cell surface was found to differ between cells grown on soluble or insoluble substrates. Cell growth on microcrystalline cellulose and wheat straw exhibited dramatic enhancement in the amount of cellulosomes displayed on the bacterial cell surface.

Fluoxetine induces autophagic cell death via eEF2K-AMPK-mTOR-ULK complex axis in triple negative breast cancer.

Science.gov (United States)

Sun, Dejuan; Zhu, Lingjuan; Zhao, Yuqian; Jiang, Yingnan; Chen, Lixia; Yu, Yang; Ouyang, Liang

2018-04-01

Triple negative breast cancer (TNBC) is a complex and intrinsically aggressive tumour with poor prognosis, and the discovery of targeted small-molecule drugs for TNBC treatment still remains in its infancy. In this study, we aimed to discover a small-molecule agent for TNBC treatment and illuminate its potential mechanisms. Cell viability was detected by using methylthiazoltetrazolium (MTT) assay. Electron microscopy, GFP-LC3 transfection, monodansylcadaverine staining and apoptosis assay were performed to determine Fluoxetine-induced autophagy and apoptosis. Western blotting and siRNA transfection were carried out to investigate the mechanisms of Fluoxetine-induced autophagy. iTRAQ-based proteomics analysis was used to explore the underlying mechanisms. We have demonstrated that Fluoxetine had remarkable anti-proliferative activities and induced autophagic cell death in MDA-MB-231 and MDA-MB-436 cells. The mechanism for Fluoxetine-induced autophagic cell death was associated with inhibition of eEF2K and activation of AMPK-mTOR-ULK complex axis. Further iTRAQ-based proteomics and network analyses revealed that Fluoxetine-induced mechanism was involved in BIRC6, BNIP1, SNAP29 and Bif-1. These results demonstrate that Fluoxetine induces apoptosis and autophagic cell death in TNBC, which will hold a promise for the future TNBC therapy. © 2017 John Wiley & Sons Ltd.

Three-dimensional bioprinting of complex cell laden alginate hydrogel structures.

Science.gov (United States)

Tabriz, Atabak Ghanizadeh; Hermida, Miguel A; Leslie, Nicholas R; Shu, Wenmiao

2015-12-21

Different bioprinting techniques have been used to produce cell-laden alginate hydrogel structures, however these approaches have been limited to 2D or simple three-dimension (3D) structures. In this study, a new extrusion based bioprinting technique was developed to produce more complex alginate hydrogel structures. This was achieved by dividing the alginate hydrogel cross-linking process into three stages: primary calcium ion cross-linking for printability of the gel, secondary calcium cross-linking for rigidity of the alginate hydrogel immediately after printing and tertiary barium ion cross-linking for long-term stability of the alginate hydrogel in culture medium. Simple 3D structures including tubes were first printed to ensure the feasibility of the bioprinting technique and then complex 3D structures such as branched vascular structures were successfully printed. The static stiffness of the alginate hydrogel after printing was 20.18 ± 1.62 KPa which was rigid enough to sustain the integrity of the complex 3D alginate hydrogel structure during the printing. The addition of 60 mM barium chloride was found to significantly extend the stability of the cross-linked alginate hydrogel from 3 d to beyond 11 d without compromising the cellular viability. The results based on cell bioprinting suggested that viability of U87-MG cells was 93 ± 0.9% immediately after bioprinting and cell viability maintained above 88% ± 4.3% in the alginate hydrogel over the period of 11 d.

5C analysis of the Epidermal Differentiation Complex locus reveals distinct chromatin interaction networks between gene-rich and gene-poor TADs in skin epithelial cells.

Directory of Open Access Journals (Sweden)

Krzysztof Poterlowicz

2017-09-01

Full Text Available Mammalian genomes contain several dozens of large (>0.5 Mbp lineage-specific gene loci harbouring functionally related genes. However, spatial chromatin folding, organization of the enhancer-promoter networks and their relevance to Topologically Associating Domains (TADs in these loci remain poorly understood. TADs are principle units of the genome folding and represents the DNA regions within which DNA interacts more frequently and less frequently across the TAD boundary. Here, we used Chromatin Conformation Capture Carbon Copy (5C technology to characterize spatial chromatin interaction network in the 3.1 Mb Epidermal Differentiation Complex (EDC locus harbouring 61 functionally related genes that show lineage-specific activation during terminal keratinocyte differentiation in the epidermis. 5C data validated by 3D-FISH demonstrate that the EDC locus is organized into several TADs showing distinct lineage-specific chromatin interaction networks based on their transcription activity and the gene-rich or gene-poor status. Correlation of the 5C results with genome-wide studies for enhancer-specific histone modifications (H3K4me1 and H3K27ac revealed that the majority of spatial chromatin interactions that involves the gene-rich TADs at the EDC locus in keratinocytes include both intra- and inter-TAD interaction networks, connecting gene promoters and enhancers. Compared to thymocytes in which the EDC locus is mostly transcriptionally inactive, these interactions were found to be keratinocyte-specific. In keratinocytes, the promoter-enhancer anchoring regions in the gene-rich transcriptionally active TADs are enriched for the binding of chromatin architectural proteins CTCF, Rad21 and chromatin remodeler Brg1. In contrast to gene-rich TADs, gene-poor TADs show preferential spatial contacts with each other, do not contain active enhancers and show decreased binding of CTCF, Rad21 and Brg1 in keratinocytes. Thus, spatial interactions between gene

Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes in Jurkat T-cells treated with HIV-1 Tat protein.

Science.gov (United States)

Liao, Wenting; Tan, Guangguo; Zhu, Zhenyu; Chen, Qiuli; Lou, Ziyang; Dong, Xin; Zhang, Wei; Pan, Wei; Chai, Yifeng

2012-11-02

HIV-1 Tat protein is released by infected cells and can affect bystander uninfected T cells and induce numerous biological responses which contribute to its pathogenesis. To elucidate the complex pathogenic mechanism, we conducted a comprehensive investigation on Tat protein-related extracellular and intracellular metabolic changes in Jurkat T-cells using combined gas chromatography-mass spectrometry (GC-MS), reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS)-based metabonomics approach. Quantitative real-time PCR (qRT-PCR) analyses were further employed to measure expressions of several relevant enzymes together with perturbed metabolic pathways. Combined metabonomic and qRT-PCR analyses revealed that HIV-1 Tat caused significant and comprehensive metabolic changes, as represented by significant changes of 37 metabolites and 10 relevant enzymes in HIV-1 Tat-treated cells. Using MetaboAnalyst 2.0, it was found that 11 pathways (Impact-value >0.10) among the regulated pathways were acutely perturbed, including sphingolipid metabolism, glycine, serine and threonine metabolism, pyruvate metabolism, inositol phosphate metabolism, arginine and proline metabolism, citrate cycle, phenylalanine metabolism, tryptophan metabolism, pentose phosphate pathway, glycerophospholipid metabolism, glycolysis or gluconeogenesis. These results provide metabolic evidence of the complex pathogenic mechanism of HIV-1 Tat protein as a "viral toxin", and would help obligate Tat protein as "an important target" for therapeutic intervention and vaccine development.

Dye-sensitized solar cells and complexes between pyridines and iodines

DEFF Research Database (Denmark)

Hansen, Poul Erik; Phuong, Nguyen Tuyet; Krake, Jacob

2012-01-01

Interactions between triiodide (I3–) and 4-tert-butylpyridine (4TBP) as postulated in dye-sensitized solar cells (DSC) are investigated by means of 13C NMR and IR spectroscopy supported by DFT calculations. The charge transfer (CT) complex 4TBP∙I2 and potential salts such as (4TBP)2I+, I3– were...... synthesized and characterized by IR and 13C NMR spectroscopy. However, mixing (butyl)4N+, I3– and 4TBP at concentrations comparable to those of the DSC solar cell did not lead to any reaction. Neither CT complexes nor cationic species like (4TBP)2I+ were observed, judging from the 13C NMR spectroscopic...

Complexities in human herpesvirus-6A and -6B binding to host cells

International Nuclear Information System (INIS)

Pedersen, Simon Metz; Hoellsberg, Per

2006-01-01

Human herpesvirus-6A and -6B uses the cellular receptor CD46 for fusion and infection of the host cell. The viral glycoprotein complex gH-gL from HHV-6A binds to the short consensus repeat 2 and 3 in CD46. Although all the major isoforms of CD46 bind the virus, certain isoforms may have higher affinity than others for the virus. Within recent years, elucidation of the viral complex has identified additional HHV-6A and -6B specific glycoproteins. Thus, gH-gL associates with a gQ1-gQ2 dimer to form a heterotetrameric complex. In addition, a novel complex consisting of gH-gL-gO has been described that does not bind CD46. Accumulating evidence suggests that an additional HHV-6A and -6B receptor exists. The previous simple picture of HHV-6A/B-host cell contact therefore includes more layers of complexities on both the viral and the host cell side of the interaction

Complex heterogeneous tissue constructs containing multiple cell types prepared by inkjet printing technology.

Science.gov (United States)

Xu, Tao; Zhao, Weixin; Zhu, Jian-Ming; Albanna, Mohammad Z; Yoo, James J; Atala, Anthony

2013-01-01

This study was designed to develop a versatile method for fabricating complex and heterogeneous three-dimensional (3D) tissue constructs using simultaneous ink-jetting of multiple cell types. Human amniotic fluid-derived stem cells (hAFSCs), canine smooth muscle cells (dSMCs), and bovine aortic endothelial cells (bECs), were separately mixed with ionic cross-linker calcium chloride (CaCl(2)), loaded into separate ink cartridges and printed using a modified thermal inkjet printer. The three cell types were delivered layer-by-layer to pre-determined locations in a sodium alginate-collagen composite located in a chamber under the printer. The reaction between CaCl(2) and sodium alginate resulted in a rapid formation of a solid composite gel and the printed cells were anchored in designated areas within the gel. The printing process was repeated for several cycles leading to a complex 3D multi-cell hybrid construct. The biological functions of the 3D printed constructs were evaluated in vitro and in vivo. Each of the printed cell types maintained their viability and normal proliferation rates, phenotypic expression, and physiological functions within the heterogeneous constructs. The bioprinted constructs were able to survive and mature into functional tissues with adequate vascularization in vivo. These findings demonstrate the feasibility of fabricating complex heterogeneous tissue constructs containing multiple cell types using inkjet printing technology. Copyright © 2012 Elsevier Ltd. All rights reserved.

Organometallic tris(8-hydroxyquinoline)aluminum complexes as buffer layers and dopants in inverted organic solar cells

Energy Technology Data Exchange (ETDEWEB)

Tolkki, Antti, E-mail: antti.tolkki@tut.fi [Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, FI-33101, Tampere (Finland); Kaunisto, Kimmo [Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, FI-33101, Tampere (Finland); Heiskanen, Juha P. [Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, FI-33101, Tampere (Finland); Department of Chemistry, University of Oulu, P.O. Box 3000, FI-90014, Oulu (Finland); Omar, Walaa A.E. [Department of Chemistry, University of Oulu, P.O. Box 3000, FI-90014, Oulu (Finland); Chemistry Branch, Department of Science and Mathematics, Suez Canal University, Suez 43721 (Egypt); Huttunen, Kirsi; Lehtimaeki, Suvi [Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, FI-33101, Tampere (Finland); Hormi, Osmo E.O. [Department of Chemistry, University of Oulu, P.O. Box 3000, FI-90014, Oulu (Finland); Lemmetyinen, Helge [Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, FI-33101, Tampere (Finland)

2012-04-30

Tris(8-hydroxyquinoline)aluminum (Alq{sub 3}) is a frequently used material for organic light emitting diodes. The electronic properties and solubility can be tuned by chemical tailoring of the quinoline part, which makes it an interesting candidate for organic solar cells. Steady-state absorption and fluorescence, as well as time-resolved fluorescence properties of the parent Alq{sub 3} and a series of complexes consisting of derivatives, such as 4-substituted pyrazol, methylpyrazol, arylvinyl, and pyridinoanthrene moieties, of the quinoline ligand, were studied in solutions and in thin films. Suitability of the complexes as anodic buffer layers or dopants in inverted organic solar cells based on the well known bulk heterojunction of poly(3-hexylthiophene) (P3HT) and phenyl-C{sub 61}-butyric acid methyl ester (PCBM) was tested. The devices equipped with the derivatives showed higher power conversion efficiency ({eta}) compared to the photocells containing the parent Alq{sub 3}. Open circuit voltage (V{sub oc}) was increased when the derivatives were utilized as the anodic buffer layer. Doping of the P3HT:PCBM with a small amount of Alq{sub 3} or its derivative improved short circuit current density, V{sub oc}, fill factor, and {eta}, while the series resistance decreased. In addition, the devices were stable in air over several weeks without encapsulation. Possible mechanisms leading to the improvements in the photovoltaic performance by using the parent Alq{sub 3} or its derivative as buffer layer or dopant are discussed. - Highlights: Black-Right-Pointing-Pointer Tris(8-hydroxyquinoline)aluminum (Alq{sub 3}) complexes in inverted organic solar cells. Black-Right-Pointing-Pointer The Alq{sub 3} complexes were used as an anodic buffer layer and as a dopant. Black-Right-Pointing-Pointer Efficiency increased and the derivatives revealed varying open circuit voltage. Black-Right-Pointing-Pointer Photovoltaic performance was stable after storage in a dark ambient

Mass transport and chloride ion complexes in occluded cell

International Nuclear Information System (INIS)

Tsuru, T.; Hashimoto, K.; Nishikata, A.; Haruyama, S.

1989-01-01

Changes in the transport and the concentration of ions in a model occluded cell are traced during galvanostatic anodic polarization of a mild steel and a stainless steel. Apparent transport numbers of anions and cations, which were estimated from chemical analysis of solution, were different from those calculated from known mobility data. At the initial stage of the polarization, the transport number of chloride ion was almost unity, and then decreased gradually. For the mild steel, the concentration of total chloride ion accumulated in the occluded compartment increased with the anodic charge passed, and the amount of chloride ion complexed with cations also increased. The chloride complex was estimated as FeCl + . For SUS304 stainless steel, the total chloride ion increased, however, the free chloride ion, which responded to an Ag/AgCl electrode remained approximately 2 mol/dm 3 . Therefore, most of the chloride ions transferred into the occluded cell formed complex ions, such as CrCl n 3-n . The number of chloride ion coordinated to ferrous and chromic ions was estimated from the data fo mass transport for the case of the mild steel and the stainless steel. (author) 9 refs., 14 figs

Neurog1 Genetic Inducible Fate Mapping (GIFM) Reveals the Existence of Complex Spatiotemporal Cyto-Architectures in the Developing Cerebellum.

Science.gov (United States)

Obana, Edwin A; Lundell, Travis G; Yi, Kevin J; Radomski, Kryslaine L; Zhou, Qiong; Doughty, Martin L

2015-06-01

Neurog1 is a pro-neural basic helix-loop-helix (bHLH) transcription factor expressed in progenitor cells located in the ventricular zone and subsequently the presumptive white matter tracts of the developing mouse cerebellum. We used genetic inducible fate mapping (GIFM) with a transgenic Neurog1-CreER allele to characterize the contributions of Neurog1 lineages to cerebellar circuit formation in mice. GIFM reveals Neurog1-expressing progenitors are fate-mapped to become Purkinje cells and all GABAergic interneuron cell types of the cerebellar cortex but not glia. The spatiotemporal sequence of GIFM is unique to each neuronal cell type. GIFM on embryonic days (E) 10.5 to E12.5 labels Purkinje cells with different medial-lateral settling patterns depending on the day of tamoxifen delivery. GIFM on E11.5 to P7 labels interneurons and the timing of tamoxifen administration correlates with the final inside-to-outside resting position of GABAergic interneurons in the cerebellar cortex. Proliferative status and long-term BrdU retention of GIFM lineages reveals Purkinje cells express Neurog1 around the time they become post-mitotic. In contrast, GIFM labels mitotic and post-mitotic interneurons. Neurog1-CreER GIFM reveals a correlation between the timing of Neurog1 expression and the spatial organization of GABAergic neurons in the cerebellar cortex with possible implications for cerebellar circuit assembly.

Epigenetic landscapes reveal transcription factors regulating CD8+ T cell differentiation

Science.gov (United States)

Yu, Bingfei; Zhang, Kai; Milner, J. Justin; Toma, Clara; Chen, Runqiang; Scott-Browne, James P.; Pereira, Renata M.; Crotty, Shane; Chang, John T.; Pipkin, Matthew E.; Wang, Wei; Goldrath, Ananda W.

2017-01-01

Dynamic changes in the expression of transcription factors (TFs) can influence specification of distinct CD8+ T cell fates, but the observation of equivalent expression of TF among differentially-fated precursor cells suggests additional underlying mechanisms. Here, we profiled genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8+ T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that TF expression and binding contributed to establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal novel TFs influencing the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8+ T cell differentiation, regulated the formation of terminal-effector and memory-precursor cell-fates, respectively. Our data define the epigenetic landscape of differentiation intermediates, facilitating identification of TFs with previously unappreciated roles in CD8+ T cell differentiation. PMID:28288100

Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

Directory of Open Access Journals (Sweden)

Pitter F Huesgen

Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer.

Science.gov (United States)

Francavilla, Chiara; Lupia, Michela; Tsafou, Kalliopi; Villa, Alessandra; Kowalczyk, Katarzyna; Rakownikow Jersie-Christensen, Rosa; Bertalot, Giovanni; Confalonieri, Stefano; Brunak, Søren; Jensen, Lars J; Cavallaro, Ugo; Olsen, Jesper V

2017-03-28

Our understanding of the molecular determinants of cancer is still inadequate because of cancer heterogeneity. Here, using epithelial ovarian cancer (EOC) as a model system, we analyzed a minute amount of patient-derived epithelial cells from either healthy or cancerous tissues by single-shot mass-spectrometry-based phosphoproteomics. Using a multi-disciplinary approach, we demonstrated that primary cells recapitulate tissue complexity and represent a valuable source of differentially expressed proteins and phosphorylation sites that discriminate cancer from healthy cells. Furthermore, we uncovered kinase signatures associated with EOC. In particular, CDK7 targets were characterized in both EOC primary cells and ovarian cancer cell lines. We showed that CDK7 controls cell proliferation and that pharmacological inhibition of CDK7 selectively represses EOC cell proliferation. Our approach defines the molecular landscape of EOC, paving the way for efficient therapeutic approaches for patients. Finally, we highlight the potential of phosphoproteomics to identify clinically relevant and druggable pathways in cancer. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

Energy Technology Data Exchange (ETDEWEB)

Rockel, Beate [Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried (Germany); Schmaler, Tilo; Huang, Xiaohua [Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin (Germany); Dubiel, Wolfgang, E-mail: Wolfgang.dubiel@charite.de [Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin (Germany)

2014-07-25

Highlights: • Deneddylation rates of human erythrocyte and mouse fibroblast CSN are very similar. • 3D models of native human and mouse CSN reveal common architectures. • The cryo-structure of native mammalian CSN shows a horseshoe subunit arrangement. - Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14 years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts

Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

International Nuclear Information System (INIS)

Rockel, Beate; Schmaler, Tilo; Huang, Xiaohua; Dubiel, Wolfgang

2014-01-01

Highlights: • Deneddylation rates of human erythrocyte and mouse fibroblast CSN are very similar. • 3D models of native human and mouse CSN reveal common architectures. • The cryo-structure of native mammalian CSN shows a horseshoe subunit arrangement. - Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14 years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts

Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells.

Science.gov (United States)

Fletcher, Anne L; Malhotra, Deepali; Acton, Sophie E; Lukacs-Kornek, Veronika; Bellemare-Pelletier, Angelique; Curry, Mark; Armant, Myriam; Turley, Shannon J

2011-01-01

Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

REST-mediated recruitment of polycomb repressor complexes in mammalian cells

DEFF Research Database (Denmark)

Dietrich, Nikolaj; Lerdrup, Mads; Landt, Eskild

2012-01-01

Polycomb Repressive Complex (PRC) 1 and PRC2 regulate genes involved in differentiation and development. However, the mechanism for how PRC1 and PRC2 are recruited to genes in mammalian cells is unclear. Here we present evidence for an interaction between the transcription factor REST, PRC1......, and increased gene expression. Genome-wide analysis of Polycomb binding in Rest¿/¿ and Eed¿/¿ mouse embryonic stem (mES) cells showed that Rest was required for PRC1 recruitment to a subset of Polycomb regulated neuronal genes. Furthermore, we found that PRC1 can be recruited to Rest binding sites independently...... of CpG islands and the H3K27Me3 mark. Surprisingly, PRC2 was frequently increased around Rest binding sites located in CpG-rich regions in the Rest¿/¿ mES cells, indicating a more complex interplay where Rest also can limit PRC2 recruitment. Therefore, we propose that Rest has context...

Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis.

Directory of Open Access Journals (Sweden)

Jingwei Cheng

2017-10-01

Full Text Available Merkel cell carcinoma (MCC frequently contains integrated copies of Merkel cell polyomavirus DNA that express a truncated form of Large T antigen (LT and an intact Small T antigen (ST. While LT binds RB and inactivates its tumor suppressor function, it is less clear how ST contributes to MCC tumorigenesis. Here we show that ST binds specifically to the MYC homolog MYCL (L-MYC and recruits it to the 15-component EP400 histone acetyltransferase and chromatin remodeling complex. We performed a large-scale immunoprecipitation for ST and identified co-precipitating proteins by mass spectrometry. In addition to protein phosphatase 2A (PP2A subunits, we identified MYCL and its heterodimeric partner MAX plus the EP400 complex. Immunoprecipitation for MAX and EP400 complex components confirmed their association with ST. We determined that the ST-MYCL-EP400 complex binds together to specific gene promoters and activates their expression by integrating chromatin immunoprecipitation with sequencing (ChIP-seq and RNA-seq. MYCL and EP400 were required for maintenance of cell viability and cooperated with ST to promote gene expression in MCC cell lines. A genome-wide CRISPR-Cas9 screen confirmed the requirement for MYCL and EP400 in MCPyV-positive MCC cell lines. We demonstrate that ST can activate gene expression in a EP400 and MYCL dependent manner and this activity contributes to cellular transformation and generation of induced pluripotent stem cells.

Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis.

Science.gov (United States)

Cheng, Jingwei; Park, Donglim Esther; Berrios, Christian; White, Elizabeth A; Arora, Reety; Yoon, Rosa; Branigan, Timothy; Xiao, Tengfei; Westerling, Thomas; Federation, Alexander; Zeid, Rhamy; Strober, Benjamin; Swanson, Selene K; Florens, Laurence; Bradner, James E; Brown, Myles; Howley, Peter M; Padi, Megha; Washburn, Michael P; DeCaprio, James A

2017-10-01

Merkel cell carcinoma (MCC) frequently contains integrated copies of Merkel cell polyomavirus DNA that express a truncated form of Large T antigen (LT) and an intact Small T antigen (ST). While LT binds RB and inactivates its tumor suppressor function, it is less clear how ST contributes to MCC tumorigenesis. Here we show that ST binds specifically to the MYC homolog MYCL (L-MYC) and recruits it to the 15-component EP400 histone acetyltransferase and chromatin remodeling complex. We performed a large-scale immunoprecipitation for ST and identified co-precipitating proteins by mass spectrometry. In addition to protein phosphatase 2A (PP2A) subunits, we identified MYCL and its heterodimeric partner MAX plus the EP400 complex. Immunoprecipitation for MAX and EP400 complex components confirmed their association with ST. We determined that the ST-MYCL-EP400 complex binds together to specific gene promoters and activates their expression by integrating chromatin immunoprecipitation with sequencing (ChIP-seq) and RNA-seq. MYCL and EP400 were required for maintenance of cell viability and cooperated with ST to promote gene expression in MCC cell lines. A genome-wide CRISPR-Cas9 screen confirmed the requirement for MYCL and EP400 in MCPyV-positive MCC cell lines. We demonstrate that ST can activate gene expression in a EP400 and MYCL dependent manner and this activity contributes to cellular transformation and generation of induced pluripotent stem cells.

Photodynamic killing of cancer cells by a Platinum(II) complex with cyclometallating ligand

Science.gov (United States)

Doherty, Rachel E.; Sazanovich, Igor V.; McKenzie, Luke K.; Stasheuski, Alexander S.; Coyle, Rachel; Baggaley, Elizabeth; Bottomley, Sarah; Weinstein, Julia A.; Bryant, Helen E.

2016-03-01

Photodynamic therapy that uses photosensitizers which only become toxic upon light-irradiation provides a strong alternative to conventional cancer treatment due to its ability to selectively target tumour material without affecting healthy tissue. Transition metal complexes are highly promising PDT agents due to intense visible light absorption, yet the majority are toxic even without light. This study introduces a small, photostable, charge-neutral platinum-based compound, Pt(II) 2,6-dipyrido-4-methyl-benzenechloride, complex 1, as a photosensitizer, which works under visible light. Activation of the new photosensitizer at low concentrations (0.1-1 μM) by comparatively low dose of 405 nm light (3.6 J cm-2) causes significant cell death of cervical, colorectal and bladder cancer cell lines, and, importantly, a cisplatin resistant cell line EJ-R. The photo-index of the complex is 8. We demonstrate that complex 1 induces irreversible DNA single strand breaks following irradiation, and that oxygen is essential for the photoinduced action. Neither light, nor compound alone led to cell death. The key advantages of the new drug include a remarkably fast accumulation time (diffusion-controlled, minutes), and photostability. This study demonstrates a highly promising new agent for photodynamic therapy, and attracts attention to photostable metal complexes as viable alternatives to conventional chemotherapeutics, such as cisplatin.

Interaction of Cosup(III) complexes with radiation on 'in vitro' mammalian cells and on molecular systems

International Nuclear Information System (INIS)

Alvarez, M.V.; Cabildo, P.; Lopez Zumel, M.C.

1978-01-01

Cosup(III) complexes could be considered as potential radiosensitizers in dependence of the electronic configuration of the metal. Due to the high redox state that causes its powerful electrophilia they possibly act through electron-affinic mechanisms. In this paper the effect of [Co(NH 3 ) 5 Cl]Cl 2 on TC.SV-40 hamster cells is reported, in aerobic and hypoxic conditions, and its action, at a non-toxic concentration, in a combined treatment with X-rays. Results show the small protection induced by the product in the survival curves. As a contribution to the knowledge of its possible mechanism of action some molecular proofs and the interaction test with -SHNP cellular groups are made. A high degree of reaction is observed as is the case with good radiosensitizers but at molecular level the behaviour of the complex formed between DNA and the product reveals mutual protection due to their corresponding radiodegradation. (orig.) [de

Gamma-irradiation and neutron effect on DNA-membrane complexes of mammalian cells

International Nuclear Information System (INIS)

Lapidus, I.L.; Nazarov, V.M.; Ehrtsgreber, G.

1984-01-01

The first results of radiobiological investigations in the biophysical channel of the JINR reactor IBR-2 are presented. Sedimentation behaviour of DNA-membrane complexes has been studied at irradiation of the Chinese hamster cells (VT9-4) in a wide dose range of 137 Cs γ-irradiation and neutrons. An earlier assumption of the authors on the role of DNA double-strand breaks in changing the relative sedimentation velocity of complexes at irradiation of cells with doses over 50 Gy has been confirmed

Interactions of trivalent and tetravalent heavy metal-siderophore complexes with pseudomonas fluorescens

International Nuclear Information System (INIS)

Yoshida, T.; Ozaki, T.; Ohnuki, T.; Francis, A.J.

2004-01-01

We investigated the interactions of the Fe(III)-, Eu(III)-, and Hf(IV)-desferrioxamine B (DFO) complexes with the Gram-negative aerobic bacterium Pseudomonas fluorescens. Potentiometric titration of 1:1 Fe(III)-, Eu(III)-, and Hf(IV)-DFO complexes showed that Hf(IV) formed a strong complex with DFO whose stability was comparable to that of the Fe(III)-DFO complex, while Eu(III) formed a weaker one. DFO in a growth medium was not degraded by P. fluorescens. Contact of P. fluorescens cells with the Fe(III)-, Eu(III)-, and Hf(IV)-DFO complexes at pH 4-9 revealed that there was negligible adsorption of Hf(IV) and Fe(III), whereas Eu(III) was dissociated from DFO and was readily adsorbed by the cells. These results suggest that Fe(III) and Hf(IV) form stable complexes with DFO and are not adsorbed by P. fluorescens cells. Europium(III) forms a weaker complex with DFO than Fe(III) and Hf(IV) do and its DFO complex is readily dissociated in the presence of the cells. (orig.)

SUMO modification through rapamycin-mediated heterodimerization reveals a dual role for Ubc9 in targeting RanGAP1 to nuclear pore complexes

International Nuclear Information System (INIS)

Zhu Shanshan; Zhang Hong; Matunis, Michael J.

2006-01-01

SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMO substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification

Feeding cells induced by phytoparasitic nematodes require γ-tubulin ring complex for microtubule reorganization.

Directory of Open Access Journals (Sweden)

Mohamed Youssef Banora

2011-12-01

Full Text Available Reorganization of the microtubule network is important for the fast isodiametric expansion of giant-feeding cells induced by root-knot nematodes. The efficiency of microtubule reorganization depends on the nucleation of new microtubules, their elongation rate and activity of microtubule severing factors. New microtubules in plants are nucleated by cytoplasmic or microtubule-bound γ-tubulin ring complexes. Here we investigate the requirement of γ-tubulin complexes for giant feeding cells development using the interaction between Arabidopsis and Meloidogyne spp. as a model system. Immunocytochemical analyses demonstrate that γ-tubulin localizes to both cortical cytoplasm and mitotic microtubule arrays of the giant cells where it can associate with microtubules. The transcripts of two Arabidopsis γ-tubulin (TUBG1 and TUBG2 and two γ-tubulin complex proteins genes (GCP3 and GCP4 are upregulated in galls. Electron microscopy demonstrates association of GCP3 and γ-tubulin as part of a complex in the cytoplasm of giant cells. Knockout of either or both γ-tubulin genes results in the gene dose-dependent alteration of the morphology of feeding site and failure of nematode life cycle completion. We conclude that the γ-tubulin complex is essential for the control of microtubular network remodelling in the course of initiation and development of giant-feeding cells, and for the successful reproduction of nematodes in their plant hosts.

Control of cell fate by the formation of an architecturally complex bacterial community.

Science.gov (United States)

Vlamakis, Hera; Aguilar, Claudio; Losick, Richard; Kolter, Roberto

2008-04-01

Bacteria form architecturally complex communities known as biofilms in which cells are held together by an extracellular matrix. Biofilms harbor multiple cell types, and it has been proposed that within biofilms individual cells follow different developmental pathways, resulting in heterogeneous populations. Here we demonstrate cellular differentiation within biofilms of the spore-forming bacterium Bacillus subtilis, and present evidence that formation of the biofilm governs differentiation. We show that motile, matrix-producing, and sporulating cells localize to distinct regions within the biofilm, and that the localization and percentage of each cell type is dynamic throughout development of the community. Importantly, mutants that do not produce extracellular matrix form unstructured biofilms that are deficient in sporulation. We propose that sporulation is a culminating feature of biofilm formation, and that spore formation is coupled to the formation of an architecturally complex community of cells.

Contribution of Human Oral Cells to Astringency by Binding Salivary Protein/Tannin Complexes.

Science.gov (United States)

Soares, Susana; Ferrer-Galego, Raúl; Brandão, Elsa; Silva, Mafalda; Mateus, Nuno; Freitas, Victor de

2016-10-10

The most widely accepted mechanism to explain astringency is the interaction and precipitation of salivary proteins by food tannins, in particular proline-rich proteins. However, other mechanisms have been arising to explain astringency, such as binding of tannins to oral cells. In this work, an experimental method was adapted to study the possible contribution of both salivary proteins and oral cells to astringency induced by grape seed procyanidin fractions. Overall, in the absence of salivary proteins, the extent of procyanidin complexation with oral cells increased with increasing procyanidin degree of polymerization (mDP). Procyanidin fractions rich in monomers were the ones with the lowest ability to bind to oral cells. In the presence of salivary proteins and for procyanidins with mDP 2 the highest concentrations (1.5 and 2.0 mM) resulted in an increased binding of procyanidins to oral cells. This was even more evident for fractions III and IV at 1.0 mM and upper concentrations. Regarding the salivary proteins affected, it was possible to observe a decrease of P-B peptide and aPRP proteins for fractions II and III. This decrease is greater as the procyanidins' mDP increases. In fact, for fraction IV an almost total depletion of all salivary proteins was observed. This decrease is due to the formation of insoluble salivary protein/procyanidin complexes. Altogether, these data suggest that some procyanidins are able to bind to oral cells and that the salivary proteins interact with procyanidins forming salivary protein/procyanidin complexes that are also able to link to oral cells. The procyanidins that remain unbound to oral cells are able to bind to salivary proteins forming a large network of salivary protein/procyanidin complexes. Overall, the results presented herein provide one more step to understand food oral astringency onset.

Stem cell heterogeneity revealed

DEFF Research Database (Denmark)

Andersen, Marianne S; Jensen, Kim B

2016-01-01

The skin forms a protective, water-impermeable barrier consisting of heavily crosslinked epithelial cells. However, the specific role of stem cells in sustaining this barrier remains a contentious issue. A detailed analysis of the interfollicular epidermis now proposes a model for how a composite...... of cells with different properties are involved in its maintenance....

Metabolomics reveals mycoplasma contamination interferes with the metabolism of PANC-1 cells.

Science.gov (United States)

Yu, Tao; Wang, Yongtao; Zhang, Huizhen; Johnson, Caroline H; Jiang, Yiming; Li, Xiangjun; Wu, Zeming; Liu, Tian; Krausz, Kristopher W; Yu, Aiming; Gonzalez, Frank J; Huang, Min; Bi, Huichang

2016-06-01

Mycoplasma contamination is a common problem in cell culture and can alter cellular functions. Since cell metabolism is either directly or indirectly involved in every aspect of cell function, it is important to detect changes to the cellular metabolome after mycoplasma infection. In this study, liquid chromatography mass spectrometry (LC/MS)-based metabolomics was used to investigate the effect of mycoplasma contamination on the cellular metabolism of human pancreatic carcinoma cells (PANC-1). Multivariate analysis demonstrated that mycoplasma contamination induced significant metabolic changes in PANC-1 cells. Twenty-three metabolites were identified and found to be involved in arginine and purine metabolism and energy supply. This study demonstrates that mycoplasma contamination significantly alters cellular metabolite levels, confirming the compelling need for routine checking of cell cultures for mycoplasma contamination, particularly when used for metabolomics studies. Graphical abstract Metabolomics reveals mycoplasma contamination changes the metabolome of PANC-1 cells.

Transmitter receptors reveal segregation of the arcopallium/amygdala complex in pigeons (Columba livia).

Science.gov (United States)

Herold, Christina; Paulitschek, Christina; Palomero-Gallagher, Nicola; Güntürkün, Onur; Zilles, Karl

2018-02-15

At the beginning of the 20th century it was suggested that a complex group of nuclei in the avian posterior ventral telencephalon is comparable to the mammalian amygdala. Subsequent findings, however, revealed that most of these structures share premotor characteristics, while some indeed constitute the avian amygdala. These developments resulted in 2004 in a change of nomenclature of these nuclei, which from then on were named arcopallial or amygdala nuclei and referred to as the arcopallium/amygdala complex. The structural basis for the similarities between avian and mammalian arcopallial and amygdala subregions is poorly understood. Therefore, we analyzed binding site densities for glutamatergic AMPA, NMDA and kainate, GABAergic GABA A , muscarinic M 1 , M 2 and nicotinic acetylcholine (nACh; α 4 β 2 subtype), noradrenergic α 1 and α 2 , serotonergic 5-HT 1A and dopaminergic D 1/5 receptors using quantitative in vitro receptor autoradiography combined with a detailed analysis of the cyto- and myelo-architecture. Our approach supports a segregation of the pigeon's arcopallium/amygdala complex into the following subregions: the arcopallium anterius (AA), the arcopallium ventrale (AV), the arcopallium dorsale (AD), the arcopallium intermedium (AI), the arcopallium mediale (AM), the arcopallium posterius (AP), the nucleus posterioris amygdalopallii pars basalis (PoAb) and pars compacta (PoAc), the nucleus taeniae amgygdalae (TnA) and the area subpallialis amygdalae (SpA). Some of these subregions showed further subnuclei and each region of the arcopallium/amygdala complex are characterized by a distinct multi-receptor density expression. Here we provide a new detailed map of the pigeon's arcopallium/amygdala complex and compare the receptor architecture of the subregions to their possible mammalian counterparts. © 2017 Wiley Periodicals, Inc.

Endometrial natural killer (NK) cells reveal a tissue-specific receptor repertoire.

Science.gov (United States)

Feyaerts, D; Kuret, T; van Cranenbroek, B; van der Zeeuw-Hingrez, S; van der Heijden, O W H; van der Meer, A; Joosten, I; van der Molen, R G

2018-02-13

Is the natural killer (NK) cell receptor repertoire of endometrial NK (eNK) cells tissue-specific? The NK cell receptor (NKR) expression profile in pre-pregnancy endometrium appears to have a unique tissue-specific phenotype, different from that found in NK cells in peripheral blood, suggesting that these cells are finely tuned towards the reception of an allogeneic fetus. NK cells are important for successful pregnancy. After implantation, NK cells encounter extravillous trophoblast cells and regulate trophoblast invasion. NK cell activity is amongst others regulated by C-type lectin heterodimer (CD94/NKG2) and killer cell immunoglobulin-like (KIR) receptors. KIR expression on decidual NK cells is affected by the presence of maternal HLA-C and biased towards KIR2D expression. However, little is known about NKR expression on eNK cells prior to pregnancy. In this study, matched peripheral and menstrual blood (a source of endometrial cells) was obtained from 25 healthy females with regular menstrual cycles. Menstrual blood was collected during the first 36 h of menstruation using a menstrual cup, a non-invasive technique to obtain endometrial cells. KIR and NKG2 receptor expression on eNK cells was characterized by 10-color flow cytometry, and compared to matched pbNK cells of the same female. KIR and HLA-C genotypes were determined by PCR-SSOP techniques. Anti-CMV IgG antibodies in plasma were measured by chemiluminescence immunoassay. KIR expression patterns of eNK cells collected from the same female do not differ over consecutive menstrual cycles. The percentage of NK cells expressing KIR2DL2/L3/S2, KIR2DL3, KIR2DL1, LILRB1 and/or NKG2A was significantly higher in eNK cells compared to pbNK cells, while no significant difference was observed for NKG2C, KIR2DL1/S1, and KIR3DL1. The NKR repertoire of eNK cells was clearly different from pbNK cells, with eNK cells co-expressing more than three NKR simultaneously. In addition, outlier analysis revealed 8 and 15 NKR

Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells

Directory of Open Access Journals (Sweden)

Anne L Fletcher

2011-09-01

Full Text Available Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

Composite cell sheet for periodontal regeneration: crosstalk between different types of MSCs in cell sheet facilitates complex periodontal-like tissue regeneration.

Science.gov (United States)

Zhang, Hao; Liu, Shiyu; Zhu, Bin; Xu, Qiu; Ding, Yin; Jin, Yan

2016-11-14

Tissue-engineering strategies based on mesenchymal stem cells (MSCs) and cell sheets have been widely used for periodontal tissue regeneration. However, given the complexity in periodontal structure, the regeneration methods using a single species of MSC could not fulfill the requirement for periodontal regeneration. We researched the interaction between the periodontal ligament stem cells (PDLSCs) and jaw bone marrow-derived mesenchymal stem cells (JBMMSCs), and constructed a composite cell sheet comprising both of the above MSCs to regenerate complex periodontium-like structures in nude mice. Our results show that by co-culturing PDLSCs and JBMMSCs, the expressions of bone and extracellular matrix (ECM)-related genes and proteins were significantly improved in both MSCs. Further investigations showed that, compared to the cell sheet using PDLSCs or JBMMSCs, the composite stem cell sheet (CSCS), which comprises these two MSCs, expressed higher levels of bone- and ECM-related genes and proteins, and generated a composite structure more similar to the native periodontal tissue physiologically in vivo. In conclusion, our results demonstrate that the crosstalk between PDLSCs and JBMMSCs in cell sheets facilitate regeneration of complex periodontium-like structures, providing a promising new strategy for physiological and functional regeneration of periodontal tissue.

Cell array-based intracellular localization screening reveals novel functional features of human chromosome 21 proteins

Directory of Open Access Journals (Sweden)

Kahlem Pascal

2006-06-01

Full Text Available Abstract Background Trisomy of human chromosome 21 (Chr21 results in Down's syndrome, a complex developmental and neurodegenerative disease. Molecular analysis of Down's syndrome, however, poses a particular challenge, because the aneuploid region of Chr21 contains many genes of unknown function. Subcellular localization of human Chr21 proteins may contribute to further understanding of the functions and regulatory mechanisms of the genes that code for these proteins. Following this idea, we used a transfected-cell array technique to perform a rapid and cost-effective analysis of the intracellular distribution of Chr 21 proteins. Results We chose 89 genes that were distributed over the majority of 21q, ranging from RBM11 (14.5 Mb to MCM3AP (46.6 Mb, with part of them expressed aberrantly in the Down's syndrome mouse model. Open reading frames of these genes were cloned into a mammalian expression vector with an amino-terminal His6 tag. All of the constructs were arrayed on glass slides and reverse transfected into HEK293T cells for protein expression. Co-localization detection using a set of organelle markers was carried out for each Chr21 protein. Here, we report the subcellular localization properties of 52 proteins. For 34 of these proteins, their localization is described for the first time. Furthermore, the alteration in cell morphology and growth as a result of protein over-expression for claudin-8 and claudin-14 genes has been characterized. Conclusion The cell array-based protein expression and detection approach is a cost-effective platform for large-scale functional analyses, including protein subcellular localization and cell phenotype screening. The results from this study reveal novel functional features of human Chr21 proteins, which should contribute to further understanding of the molecular pathology of Down's syndrome.

Cell surface clustering of Cadherin adhesion complex induced by antibody coated beads

Institute of Scientific and Technical Information of China (English)

无

2000-01-01

Cadherin receptors mediate cell-cell adhesion, signal transduction and assembly of cytoskeletons. How a single transmembrane molecule Cadherin can be involved in multiple functions through modulating its binding activities with many membrane adhesion molecules and cytoskeletal components is an unanswered question which can be elucidated by clues from bead experiments. Human lung cells expressing N-Cadherin were examined. After co-incubation with anti-N-Cadherin monoclonal antibody coated beads, cell surface clustering of N-Cadherin was induced. Immunofluorescent detection demonstrated that in addition to Cadherin, β-Catenin, α-Catenin, α-Actinin and Actin fluorescence also aggregated respectively at the membrane site of bead attachment. Myosin heavy chain (MHC), another major component of Actin cytoskeleton, did not aggregate at the membrane site of bead attachment. Adhesion unrelated protein Con A and polylysine conjugated beads did not induce the clustering of adhesion molecules. It is indicated that the Cadherin/Catenins/α-Actinin/Actin complex is formed at Cadherin mediated cell adherens junction; occupancy and cell surface clustering of Cadherin is crucial for the formation of Cadherin adhesion protein complexes.

A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies.

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Masakazu Kohda

2016-01-01

Full Text Available Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4 as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3 and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21 as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.

High-Resolution Imaging Reveals New Features of Nuclear Export of mRNA through the Nuclear Pore Complexes

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Joseph M. Kelich

2014-08-01

Full Text Available The nuclear envelope (NE of eukaryotic cells provides a physical barrier for messenger RNA (mRNA and the associated proteins (mRNPs traveling from sites of transcription in the nucleus to locations of translation processing in the cytoplasm. Nuclear pore complexes (NPCs embedded in the NE serve as a dominant gateway for nuclear export of mRNA. However, the fundamental characterization of export dynamics of mRNPs through the NPC has been hindered by several technical limits. First, the size of NPC that is barely below the diffraction limit of conventional light microscopy requires a super-resolution microscopy imaging approach. Next, the fast transit of mRNPs through the NPC further demands a high temporal resolution by the imaging approach. Finally, the inherent three-dimensional (3D movements of mRNPs through the NPC demand the method to provide a 3D mapping of both transport kinetics and transport pathways of mRNPs. This review will highlight the recently developed super-resolution imaging techniques advanced from 1D to 3D for nuclear export of mRNPs and summarize the new features in the dynamic nuclear export process of mRNPs revealed from these technical advances.

The number of cell types, information content, and the evolution of complex multicellularity

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Karl J. Niklas

2014-12-01

Full Text Available 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.

The Capsaspora genome reveals a complex unicellular prehistory of animals.

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Suga, Hiroshi; Chen, Zehua; de Mendoza, Alex; Sebé-Pedrós, Arnau; Brown, Matthew W; Kramer, Eric; Carr, Martin; Kerner, Pierre; Vervoort, Michel; Sánchez-Pons, Núria; Torruella, Guifré; Derelle, Romain; Manning, Gerard; Lang, B Franz; Russ, Carsten; Haas, Brian J; Roger, Andrew J; Nusbaum, Chad; Ruiz-Trillo, Iñaki

2013-01-01

To reconstruct the evolutionary origin of multicellular animals from their unicellular ancestors, the genome sequences of diverse unicellular relatives are essential. However, only the genome of the choanoflagellate Monosiga brevicollis has been reported to date. Here we completely sequence the genome of the filasterean Capsaspora owczarzaki, the closest known unicellular relative of metazoans besides choanoflagellates. Analyses of this genome alter our understanding of the molecular complexity of metazoans' unicellular ancestors showing that they had a richer repertoire of proteins involved in cell adhesion and transcriptional regulation than previously inferred only with the choanoflagellate genome. Some of these proteins were secondarily lost in choanoflagellates. In contrast, most intercellular signalling systems controlling development evolved later concomitant with the emergence of the first metazoans. We propose that the acquisition of these metazoan-specific developmental systems and the co-option of pre-existing genes drove the evolutionary transition from unicellular protists to metazoans.

Polycomb repressive complex 2 (PRC2 restricts hematopoietic stem cell activity.

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Ian J Majewski

2008-04-01

Full Text Available Polycomb group proteins are transcriptional repressors that play a central role in the establishment and maintenance of gene expression patterns during development. Using mice with an N-ethyl-N-nitrosourea (ENU-induced mutation in Suppressor of Zeste 12 (Suz12, a core component of Polycomb Repressive Complex 2 (PRC2, we show here that loss of Suz12 function enhances hematopoietic stem cell (HSC activity. In addition to these effects on a wild-type genetic background, mutations in Suz12 are sufficient to ameliorate the stem cell defect and thrombocytopenia present in mice that lack the thrombopoietin receptor (c-Mpl. To investigate the molecular targets of the PRC2 complex in the HSC compartment, we examined changes in global patterns of gene expression in cells deficient in Suz12. We identified a distinct set of genes that are regulated by Suz12 in hematopoietic cells, including eight genes that appear to be highly responsive to PRC2 function within this compartment. These data suggest that PRC2 is required to maintain a specific gene expression pattern in hematopoiesis that is indispensable to normal stem cell function.

Chemical genetics analysis of an aniline mustard anticancer agent reveals complex I of the electron transport chain as a target.

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Fedeles, Bogdan I; Zhu, Angela Y; Young, Kellie S; Hillier, Shawn M; Proffitt, Kyle D; Essigmann, John M; Croy, Robert G

2011-09-30

The antitumor agent 11β (CAS 865070-37-7), consisting of a DNA-damaging aniline mustard linked to an androgen receptor (AR) ligand, is known to form covalent DNA adducts and to induce apoptosis potently in AR-positive prostate cancer cells in vitro; it also strongly prevents growth of LNCaP xenografts in mice. The present study describes the unexpectedly strong activity of 11β against the AR-negative HeLa cells, both in cell culture and tumor xenografts, and uncovers a new mechanism of action that likely explains this activity. Cellular fractionation experiments indicated that mitochondria are the major intracellular sink for 11β; flow cytometry studies showed that 11β exposure rapidly induced oxidative stress, mitochondria being an important source of reactive oxygen species (ROS). Additionally, 11β inhibited oxygen consumption both in intact HeLa cells and in isolated mitochondria. Specifically, 11β blocked uncoupled oxygen consumption when mitochondria were incubated with complex I substrates, but it had no effect on oxygen consumption driven by substrates acting downstream of complex I in the mitochondrial electron transport chain. Moreover, 11β enhanced ROS generation in isolated mitochondria, suggesting that complex I inhibition is responsible for ROS production. At the cellular level, the presence of antioxidants (N-acetylcysteine or vitamin E) significantly reduced the toxicity of 11β, implicating ROS production as an important contributor to cytotoxicity. Collectively, our findings establish complex I inhibition and ROS generation as a new mechanism of action for 11β, which supplements conventional DNA adduct formation to promote cancer cell death.

Revealing mechanisms of selective, concentration-dependent potentials of 4-hydroxy-2-nonenal to induce apoptosis in cancer cells through inactivation of membrane-associated catalase.

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Bauer, Georg; Zarkovic, Neven

2015-04-01

Tumor cells generate extracellular superoxide anions and are protected against superoxide anion-mediated intercellular apoptosis-inducing signaling by the expression of membrane-associated catalase. 4-Hydroxy-2-nonenal (4-HNE), a versatile second messenger generated during lipid peroxidation, has been shown to induce apoptosis selectively in malignant cells. The findings described in this paper reveal the strong, concentration-dependent potential of 4-HNE to specifically inactivate extracellular catalase of tumor cells both indirectly and directly and to consequently trigger apoptosis in malignant cells through superoxide anion-mediated intercellular apoptosis-inducing signaling. Namely, 4-HNE caused apoptosis selectively in NOX1-expressing tumor cells through inactivation of their membrane-associated catalase, thus reactivating subsequent intercellular signaling through the NO/peroxynitrite and HOCl pathways, followed by the mitochondrial pathway of apoptosis. Concentrations of 4-HNE of 1.2 µM and higher directly inactivated membrane-associated catalase of tumor cells, whereas at lower concentrations, 4-HNE triggered a complex amplificatory pathway based on initial singlet oxygen formation through H2O2 and peroxynitrite interaction. Singlet-oxygen-dependent activation of the FAS receptor and caspase-8 increased superoxide anion generation by NOX1 and amplification of singlet oxygen generation, which allowed singlet-oxygen-dependent inactivation of catalase. 4-HNE and singlet oxygen cooperate in complex autoamplificatory loops during this process. The finding of these novel anticancer pathways may be useful for understanding the role of 4-HNE in the control of malignant cells and for the optimization of ROS-dependent therapeutic approaches including antioxidant treatments. Copyright © 2015 Elsevier Inc. All rights reserved.

In vitro formation of the Merkel cell-neurite complex in embryonic mouse whiskers using organotypic co-cultures.

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Ishida, Kentaro; Saito, Tetsuichiro; Mitsui, Toshiyuki

2018-06-01

A Merkel cell-neurite complex is a touch receptor composed of specialized epithelial cells named Merkel cells and peripheral sensory nerves in the skin. Merkel cells are found in touch-sensitive skin components including whisker follicles. The nerve fibers that innervate Merkel cells of a whisker follicle extend from the maxillary branch of the trigeminal ganglion. Whiskers as a sensory organ attribute to the complicated architecture of the Merkel cell-neurite complex, and therefore it is intriguing how the structure is formed. However, observing the dynamic process of the formation of a Merkel cell-neurite complex in whiskers during embryonic development is still difficult. In this study, we tried to develop an organotypic co-culture method of a whisker pad and a trigeminal ganglion explant to form the Merkel cell-neurite complex in vitro. We initially developed two distinct culture methods of a single whisker row and a trigeminal ganglion explant, and then combined them. By dissecting and cultivating a single row from a whisker pad, the morphogenesis of whisker follicles could be observed under a microscope. After the co-cultivation of the whisker row with a trigeminal ganglion explant, a Merkel cell-neurite complex composed of Merkel cells, which were positive for both cytokeratin 8 and SOX2, Neurofilament-H-positive trigeminal nerve fibers and Schwann cells expressing Nestin, SOX2 and SOX10 was observed via immunohistochemical analyses. These results suggest that the process for the formation of a Merkel cell-neurite complex can be observed under a microscope using our organotypic co-culture method. © 2018 Japanese Society of Developmental Biologists.

Unraveling the Complexities of Androgen Receptor Signaling in Prostate Cancer Cells

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Heemers, Hannelore V.; Tindall, Donald J.

2009-01-01

Androgen signaling is critical for proliferation of prostate cancer cells but cannot be fully inhibited by current androgen deprivation therapies. A study by Xu et al. in this issue of Cancer Cell provides insights into the complexities of androgen signaling in prostate cancer and suggests avenues to target a subset of androgen-sensitive genes.

Dynamic differentiation of GABAA-sensitive influences on orientation selectivity of complex cells in the cat striate cortex.

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Pfleger, B; Bonds, A B

1995-01-01

The influence of GABAA receptors on orientation selectivity of cat complex cells was tested by iontophoresis of the GABAA receptor blockers bicuculline and N-methyl-bicuculline while stimulating with drifting sinusoidal gratings. Reduction of orientation tuning was markedly less than reported in previous studies that used drifting bars as visual stimuli. Only 3/31 cells lost orientation selectivity, with an average increase in bandwidth of 33%, as opposed to half the cells losing selectivity and a bandwidth increase for the remainder of 47% as reported previously. Infusion of GABAA blockers revealed a prominent stimulus onset transient response, lasting about 120 ms, that showed a broadening of orientation selectivity comparable to that found using drifting bars under similar circumstances. We believe that drifting gratings emphasize a steady-state response component that retains, in the presence of GABAA blockers, significant orientation selectivity. Because the onset transient is initially unselective for orientation, we suggest that the steady-state, orientation-selective response component develops from an alternate inhibitory mechanism, possibly mediated by GABAB receptors.

Cell type-specific characterization of nuclear DNA contents within complex tissues and organs

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Lambert Georgina M

2005-10-01

Full Text Available Abstract Background Eukaryotic organisms are defined by the presence of a nucleus, which encloses the chromosomal DNA, and is characterized by its DNA content (C-value. Complex eukaryotic organisms contain organs and tissues that comprise interspersions of different cell types, within which polysomaty, endoreduplication, and cell cycle arrest is frequently observed. Little is known about the distribution of C-values across different cell types within these organs and tissues. Results We have developed, and describe here, a method to precisely define the C-value status within any specific cell type within complex organs and tissues of plants. We illustrate the application of this method to Arabidopsis thaliana, specifically focusing on the different cell types found within the root. Conclusion The method accurately and conveniently charts C-value within specific cell types, and provides novel insight into developmental processes. The method is, in principle, applicable to any transformable organism, including mammals, within which cell type specificity of regulation of endoreduplication, of polysomaty, and of cell cycle arrest is suspected.

The dynamic relationship between cerebellar Purkinje cell simple spikes and the spikelet number of complex spikes.

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Burroughs, Amelia; Wise, Andrew K; Xiao, Jianqiang; Houghton, Conor; Tang, Tianyu; Suh, Colleen Y; Lang, Eric J; Apps, Richard; Cerminara, Nadia L

2017-01-01

Purkinje cells are the sole output of the cerebellar cortex and fire two distinct types of action potential: simple spikes and complex spikes. Previous studies have mainly considered complex spikes as unitary events, even though the waveform is composed of varying numbers of spikelets. The extent to which differences in spikelet number affect simple spike activity (and vice versa) remains unclear. We found that complex spikes with greater numbers of spikelets are preceded by higher simple spike firing rates but, following the complex spike, simple spikes are reduced in a manner that is graded with spikelet number. This dynamic interaction has important implications for cerebellar information processing, and suggests that complex spike spikelet number may maintain Purkinje cells within their operational range. Purkinje cells are central to cerebellar function because they form the sole output of the cerebellar cortex. They exhibit two distinct types of action potential: simple spikes and complex spikes. It is widely accepted that interaction between these two types of impulse is central to cerebellar cortical information processing. Previous investigations of the interactions between simple spikes and complex spikes have mainly considered complex spikes as unitary events. However, complex spikes are composed of an initial large spike followed by a number of secondary components, termed spikelets. The number of spikelets within individual complex spikes is highly variable and the extent to which differences in complex spike spikelet number affects simple spike activity (and vice versa) remains poorly understood. In anaesthetized adult rats, we have found that Purkinje cells recorded from the posterior lobe vermis and hemisphere have high simple spike firing frequencies that precede complex spikes with greater numbers of spikelets. This finding was also evident in a small sample of Purkinje cells recorded from the posterior lobe hemisphere in awake cats. In addition

Enlightening intracellular complexity of living cells with quantitative phase microscopy

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Martinez Torres, C.; Laperrousaz, B.; Berguiga, L.; Boyer Provera, E.; Elezgaray, J.; Nicolini, F. E.; Maguer-Satta, V.; Arneodo, A.; Argoul, F.

2016-03-01

The internal distribution of refractive indices (RIs) of a living cell is much more complex than usually admitted in multi-shell models. The reconstruction of RI maps from single phase images has rarely been achieved for several reasons: (i) we still have very little knowledge of the impact of internal macromolecular complexes on the local RI and (ii) phase changes produced by light propagation through the sample are mixed with diffraction effects by internal cell bodies. We propose the implementation a 2D wavelet-based contour chain detection method to distinguish internal boundaries thanks to their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are morphological indicators for distinguishing cells of different origins and to follow their transformation in pathologic situations. We use this method to compare non adherent blood cells from primary and laboratory culture origins, in healthy and pathological situations (chronic myelogenous leukaemia). In a second part of this presentation, we concentrate on the temporal dynamics of the phase contour chains and we discuss the spectral decomposition of their dynamics in both health and disease.

Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL) Reveals the Sequential Differentiation of Sieve Element-Like Cells.

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Kondo, Yuki; Nurani, Alif Meem; Saito, Chieko; Ichihashi, Yasunori; Saito, Masato; Yamazaki, Kyoko; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Fukuda, Hiroo

2016-06-01

Cell differentiation is a complex process involving multiple steps, from initial cell fate specification to final differentiation. Procambial/cambial cells, which act as vascular stem cells, differentiate into both xylem and phloem cells during vascular development. Recent studies have identified regulatory cascades for xylem differentiation. However, the molecular mechanism underlying phloem differentiation is largely unexplored due to technical challenges. Here, we established an ectopic induction system for phloem differentiation named Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL). Our results verified similarities between VISUAL-induced Arabidopsis thaliana phloem cells and in vivo sieve elements. We performed network analysis using transcriptome data with VISUAL to dissect the processes underlying phloem differentiation, eventually identifying a factor involved in the regulation of the master transcription factor gene APL Thus, our culture system opens up new avenues not only for genetic studies of phloem differentiation, but also for future investigations of multidirectional differentiation from vascular stem cells. © 2016 American Society of Plant Biologists. All rights reserved.

Redox reactions of the α-synuclein-Cu(2+) complex and their effects on neuronal cell viability.

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Wang, Chengshan; Liu, Lin; Zhang, Lin; Peng, Yong; Zhou, Feimeng

2010-09-21

α-Synuclein (α-syn), a presynaptic protein believed to play an important role in neuropathology in Parkinson's disease (PD), is known to bind Cu(2+). Cu(2+) has been shown to accelerate the aggregation of α-syn to form various toxic aggregates in vitro. Copper is also a redox-active metal whose complexes with amyloidogenic proteins/peptides have been linked to oxidative stress in major neurodegenerative diseases. In this work, the formation of the Cu(2+) complex with α-syn or with an N-terminal peptide, α-syn(1-19), was confirmed with electrospray-mass spectrometry (ES-MS). The redox potentials of the Cu(2+) complex with α-syn (α-syn-Cu(2+)) and α-syn(1-19) were determined to be 0.018 and 0.053 V, respectively. Furthermore, the Cu(2+) center(s) can be readily reduced to Cu(+), and possible reactions of α-syn-Cu(2+) with cellular species (e.g., O(2), ascorbic acid, and dopamine) were investigated. The occurrence of a redox reaction can be rationalized by comparing the redox potential of the α-syn-Cu(2+) complex to that of the specific cellular species. For example, ascorbic acid can directly reduce α-syn-Cu(2+) to α-syn-Cu(+), setting up a redox cycle in which O(2) is reduced to H(2)O(2) and cellular redox species is continuously exhausted. In addition, the H(2)O(2) generated was demonstrated to reduce viability of the neuroblastoma SY-HY5Y cells. Although our results ruled out the direct oxidation of dopamine by α-syn-Cu(2+), the H(2)O(2) generated in the presence of α-syn-Cu(2+) can oxidize dopamine. Our results suggest that oxidative stress is at least partially responsible for the loss of dopaminergic cells in PD brain and reveal the multifaceted role of the α-syn-Cu(2+) complex in oxidative stress associated with PD symptoms.

Biofilm growth program and architecture revealed by single-cell live imaging

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Yan, Jing; Sabass, Benedikt; Stone, Howard; Wingreen, Ned; Bassler, Bonnie

Biofilms are surface-associated bacterial communities. Little is known about biofilm structure at the level of individual cells. We image living, growing Vibrio cholerae biofilms from founder cells to ten thousand cells at single-cell resolution, and discover the forces underpinning the architectural evolution of the biofilm. Mutagenesis, matrix labeling, and simulations demonstrate that surface-adhesion-mediated compression causes V. cholerae biofilms to transition from a two-dimensional branched morphology to a dense, ordered three-dimensional cluster. We discover that directional proliferation of rod-shaped bacteria plays a dominant role in shaping the biofilm architecture, and this growth pattern is controlled by a single gene. Competition analyses reveal the advantages of the dense growth mode in providing the biofilm with superior mechanical properties. We will further present continuum theory to model the three-dimensional growth of biofilms at the solid-liquid interface as well as solid-air interface.

Functional cooperation between FACT and MCM is coordinated with cell cycle and differential complex formation

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Lin Chih-Li

2010-02-01

Full Text Available Abstract Background Functional cooperation between FACT and the MCM helicase complex constitutes an integral step during DNA replication initiation. However, mode of regulation that underlies the proper functional interaction of FACT and MCM is poorly understood. Methods & Results Here we present evidence indicating that such interaction is coordinated with cell cycle progression and differential complex formation. We first demonstrate the existence of two distinct FACT-MCM subassemblies, FACT-MCM2/4/6/7 and FACT-MCM2/3/4/5. Both complexes possess DNA unwinding activity and are subject to cell cycle-dependent enzymatic regulation. Interestingly, analysis of functional attributes further suggests that they act at distinct, and possibly sequential, steps during origin establishment and replication initiation. Moreover, we show that the phosphorylation profile of the FACT-associated MCM4 undergoes a cell cycle-dependent change, which is directly correlated with the catalytic activity of the FACT-MCM helicase complexes. Finally, at the quaternary structure level, physical interaction between FACT and MCM complexes is generally dependent on persistent cell cycle and further stabilized upon S phase entry. Cessation of mitotic cycle destabilizes the complex formation and likely leads to compromised coordination and activities. Conclusions Together, our results correlate FACT-MCM functionally and temporally with S phase and DNA replication. They further demonstrate that enzymatic activities intrinsically important for DNA replication are tightly controlled at various levels, thereby ensuring proper progression of, as well as exit from, the cell cycle and ultimately euploid gene balance.

Relationship between macular ganglion cell complex thickness and macular outer retinal thickness: a spectral-domain optical coherence tomography study.

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Kita, Yoshiyuki; Kita, Ritsuko; Takeyama, Asuka; Anraku, Ayako; Tomita, Goji; Goldberg, Ivan

2013-01-01

To assess the relationship between macular ganglion cell complex and macular outer retinal thicknesses. Case-control study. Forty-two normal eyes and 91 eyes with primary open-angle glaucoma were studied. Spectral-domain optical coherence tomography (RTVue-100) was used to measure the macular ganglion cell complex and macular outer retinal thickness. Ganglion cell complex to outer retinal thickness ratio was also calculated. The relationships between the ganglion cell complex and outer retinal thicknesses and between the ganglion cell complex to outer retinal thickness ratio and outer retinal thickness were evaluated. There was a positive correlation between ganglion cell complex and outer retinal thicknesses in the normal group and the glaucoma group (r = 0.53, P variation in the outer retinal thickness. Therefore, when determining the ganglion cell complex, it seems necessary to consider the outer retinal thickness as well. We propose the ratio as a suitable parameter to account for individual variations in outer retinal thickness. © 2013 The Authors. Clinical and Experimental Ophthalmology © 2013 Royal Australian and New Zealand College of Ophthalmologists.

Radiation-induced dissociation of stable DNA-protein complexes in Erlich ascites carcinoma cells

International Nuclear Information System (INIS)

Juhasz, P.P.; Sirota, N.P.; Gaziev, A.I.

1982-01-01

DNA of Ehrlich ascites carcinoma cells prepared under conditions that were highly denaturing for proteins but not for DNA, contained a group of nonhistone residual proteins. The amount of these proteins increased during DNA replication. The DNA-protein complex observed was sensitive to proteolytic enzymes and/or SH-reagents. γ-irradiation cells with moderate doses leads to a decrease in the amount of DNA-protein complexes. High-dose gamma-irradiation produces enhanced linking of chromosomal proteins with DNA. (author)

Role of Conserved Oligomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast Cancer Cells

National Research Council Canada - National Science Library

Zolov, Sergey N

2006-01-01

...: protein glycosylation and its sorting. For analysis of COG complex function we utilized RNA interference assay to knockdown COG3p subunit of COG complex in normal and breast cancer cells and other tumor cell lines...

Protein C inhibits endocytosis of thrombin-thrombomodulin complexes in A549 lung cancer cells and human umbilical vein endothelial cells

International Nuclear Information System (INIS)

Maruyama, I.; Majerus, P.W.

1987-01-01

We investigated the effect of protein C on the endocytosis of thrombin-thrombomodulin complexes. We previously showed that exposure of umbilical vein endothelial cells to thrombin stimulated the internalization and degradation of thrombin. A similar internalization was stimulated by a monoclonal antithrombomodulin antibody. We have repeated these studies in the presence of protein C and found that endocytosis of 125 I-thrombin-thrombomodulin complexes, but not 125 I-antithrombomodulin-thrombomodulin complexes, is inhibited. Activated protein C did not inhibit endocytosis of thrombin-thrombomodulin complexes. Protein C inhibited both internalization and degradation of 125 I-thrombin and diisopropylphosphoryl (DIP) 125 I-thrombin in human lung cancer cells (A549). These effects were observed at protein C concentrations found in human plasma. Protein S had no effect on the inhibition of endocytosis of thrombin-thrombomodulin complexes by protein C. We propose that protein C may regulate the rate of endocytosis of thrombin-thrombomodulin complexes in vivo and thereby control the capacity for endothelium to activate protein C

Pea Border Cell Maturation and Release Involve Complex Cell Wall Structural Dynamics1[OPEN

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2017-01-01

The adhesion of plant cells is vital for support and protection of the plant body and is maintained by a variety of molecular associations between cell wall components. In some specialized cases, though, plant cells are programmed to detach, and root cap-derived border cells are examples of this. Border cells (in some species known as border-like cells) provide an expendable barrier between roots and the environment. Their maturation and release is an important but poorly characterized cell separation event. To gain a deeper insight into the complex cellular dynamics underlying this process, we undertook a systematic, detailed analysis of pea (Pisum sativum) root tip cell walls. Our study included immunocarbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy, quantitative reverse transcription-PCR of cell wall biosynthetic genes, analysis of hydrolytic activities, transmission electron microscopy, and immunolocalization of cell wall components. Using this integrated glycobiology approach, we identified multiple novel modes of cell wall structural and compositional rearrangement during root cap growth and the release of border cells. Our findings provide a new level of detail about border cell maturation and enable us to develop a model of the separation process. We propose that loss of adhesion by the dissolution of homogalacturonan in the middle lamellae is augmented by an active biophysical process of cell curvature driven by the polarized distribution of xyloglucan and extensin epitopes. PMID:28400496

Cell division control by the Chromosomal Passenger Complex

Energy Technology Data Exchange (ETDEWEB)

Waal, Maike S. van der; Hengeveld, Rutger C.C.; Horst, Armando van der; Lens, Susanne M.A., E-mail: s.m.a.lens@umcutrecht.nl

2012-07-15

The Chromosomal Passenger Complex (CPC) consisting of Aurora B kinase, INCENP, Survivin and Borealin, is essential for genomic stability by controlling multiple processes during both nuclear and cytoplasmic division. In mitosis it ensures accurate segregation of the duplicated chromosomes by regulating the mitotic checkpoint, destabilizing incorrectly attached spindle microtubules and by promoting the axial shortening of chromosomal arms in anaphase. During cytokinesis the CPC most likely prevents chromosome damage by imposing an abscission delay when a chromosome bridge connects the two daughter cells. Moreover, by controlling proper cytoplasmic division, the CPC averts tetraploidization. This review describes recent insights on how the CPC is capable of conducting its various functions in the dividing cell to ensure chromosomal stability.

Comparative analysis of fungal genomes reveals different plant cell wall degrading capacity in fungi

Science.gov (United States)

2013-01-01

Background Fungi produce a variety of carbohydrate activity enzymes (CAZymes) for the degradation of plant polysaccharide materials to facilitate infection and/or gain nutrition. Identifying and comparing CAZymes from fungi with different nutritional modes or infection mechanisms may provide information for better understanding of their life styles and infection models. To date, over hundreds of fungal genomes are publicly available. However, a systematic comparative analysis of fungal CAZymes across the entire fungal kingdom has not been reported. Results In this study, we systemically identified glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and glycosyltransferases (GTs) as well as carbohydrate-binding modules (CBMs) in the predicted proteomes of 103 representative fungi from Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. Comparative analysis of these CAZymes that play major roles in plant polysaccharide degradation revealed that fungi exhibit tremendous diversity in the number and variety of CAZymes. Among them, some families of GHs and CEs are the most prevalent CAZymes that are distributed in all of the fungi analyzed. Importantly, cellulases of some GH families are present in fungi that are not known to have cellulose-degrading ability. In addition, our results also showed that in general, plant pathogenic fungi have the highest number of CAZymes. Biotrophic fungi tend to have fewer CAZymes than necrotrophic and hemibiotrophic fungi. Pathogens of dicots often contain more pectinases than fungi infecting monocots. Interestingly, besides yeasts, many saprophytic fungi that are highly active in degrading plant biomass contain fewer CAZymes than plant pathogenic fungi. Furthermore, analysis of the gene expression profile of the wheat scab fungus Fusarium graminearum revealed that most of the CAZyme genes related to cell wall degradation were up-regulated during plant infection. Phylogenetic analysis also

The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components.

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Lukas Aeberhard

2015-06-01

Full Text Available Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.

The GARP/Latent TGF-β1 complex on Treg cells modulates the induction of peripherally derived Treg cells during oral tolerance.

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Edwards, Justin P; Hand, Timothy W; Morais da Fonseca, Denise; Glass, Deborah D; Belkaid, Yasmine; Shevach, Ethan M

2016-06-01

Treg cells can secrete latent TGF-β1 (LTGF-β1), but can also utilize an alternative pathway for transport and expression of LTGF-β1 on the cell surface in which LTGF-β1 is coupled to a distinct LTGF-β binding protein termed glycoprotein A repetitions predominant (GARP)/LRRC32. The function of the GARP/LTGF-β1 complex has remained elusive. Here, we examine in vivo the roles of GARP and TGF-β1 in the induction of oral tolerance. When Foxp3(-) OT-II T cells were transferred to wild-type recipient mice followed by OVA feeding, the conversion of Foxp3(-) to Foxp3(+) OT-II cells was dependent on recipient Treg cells. Neutralization of IL-2 in the recipient mice also abrogated this conversion. The GARP/LTGF-β1 complex on recipient Treg cells, but not dendritic cell-derived TGF-β1, was required for efficient induction of Foxp3(+) T cells and for the suppression of delayed hypersensitivity. Expression of the integrin αvβ8 by Treg cells (or T cells) in the recipients was dispensable for induction of Foxp3 expression. Transient depletion of the bacterial flora enhanced the development of oral tolerance by expanding Treg cells with enhanced expression of the GARP/LTGF-β1 complex. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Live cell linear dichroism imaging reveals extensive membrane ruffling within the docking structure of natural killer cell immune synapses

DEFF Research Database (Denmark)

Benninger, Richard K P; Vanherberghen, Bruno; Young, Stephen

2009-01-01

We have applied fluorescence imaging of two-photon linear dichroism to measure the subresolution organization of the cell membrane during formation of the activating (cytolytic) natural killer (NK) cell immune synapse (IS). This approach revealed that the NK cell plasma membrane is convoluted...... into ruffles at the periphery, but not in the center of a mature cytolytic NK cell IS. Time-lapse imaging showed that the membrane ruffles formed at the initial point of contact between NK cells and target cells and then spread radialy across the intercellular contact as the size of the IS increased, becoming...... absent from the center of the mature synapse. Understanding the role of such extensive membrane ruffling in the assembly of cytolytic synapses is an intriguing new goal....

Predictive ethoinformatics reveals the complex migratory behaviour of a pelagic seabird, the Manx Shearwater

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Freeman, Robin; Dean, Ben; Kirk, Holly; Leonard, Kerry; Phillips, Richard A.; Perrins, Chris M.; Guilford, Tim

2013-01-01

Understanding the behaviour of animals in the wild is fundamental to conservation efforts. Advances in bio-logging technologies have offered insights into the behaviour of animals during foraging, migration and social interaction. However, broader application of these systems has been limited by device mass, cost and longevity. Here, we use information from multiple logger types to predict individual behaviour in a highly pelagic, migratory seabird, the Manx Shearwater (Puffinus puffinus). Using behavioural states resolved from GPS tracking of foraging during the breeding season, we demonstrate that individual behaviours can be accurately predicted during multi-year migrations from low cost, lightweight, salt-water immersion devices. This reveals a complex pattern of migratory stopovers: some involving high proportions of foraging, and others of rest behaviour. We use this technique to examine three consecutive years of global migrations, revealing the prominence of foraging behaviour during migration and the importance of highly productive waters during migratory stopover. PMID:23635496

Llgl1 Connects Cell Polarity with Cell-Cell Adhesion in Embryonic Neural Stem Cells.

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Jossin, Yves; Lee, Minhui; Klezovitch, Olga; Kon, Elif; Cossard, Alexia; Lien, Wen-Hui; Fernandez, Tania E; Cooper, Jonathan A; Vasioukhin, Valera

2017-06-05

Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. We report here that mice with embryonic neural stem-cell-specific deletion of Llgl1 (Nestin-Cre/Llgl1 fl/fl ), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1 fl/fl brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. We show that LLGL1 directly binds to and promotes internalization of N-cadherin, and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1, restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Detection of autoreactive CD4 T cells using major histocompatibility complex class II dextramers

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Kuszynski Charles

2011-07-01

Full Text Available Abstract Background Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells. However, unlike CD8 T cells, CD4 T cells - especially self-reactive cells - are challenging to detect with major histocompatibility complex (MHC class II tetramers because of low frequencies and low affinities of their T cell receptors to MHC-peptide complexes. Here, we report the use of fluorescent multimers, designated MHC dextramers that contain a large number of peptide-MHC complexes per reagent. Results The utility of MHC dextramers was evaluated in three autoimmune disease models: 1 proteolipid protein (PLP 139-151-induced experimental autoimmune encephalomyelitis in SJL/J (H-2s mice; 2 myelin oligodendrocyte glycoprotein (MOG 35-55-induced experimental autoimmune encephalomyelitis in C57Bl/6 (H-2b mice; and 3 cardiac myosin heavy chain (Myhc-α 334-352-induced experimental autoimmune myocarditis in A/J (H-2a mice. Flow cytometrically, we demonstrate that IAs/PLP 139-151, IAb/MOG 35-55 and IAk/Myhc-α 334-352 dextramers detect the antigen-sensitized cells with specificity, and with a detection sensitivity significantly higher than that achieved with conventional tetramers. Furthermore, we show that binding of dextramers, but not tetramers, is less dependent on the activation status of cells, permitting enumeration of antigen-specific cells ex vivo. Conclusions The data suggest that MHC dextramers are useful tools to track the generation and functionalities of self-reactive CD4 cells in various experimental systems.

Deep Neural Networks Reveal a Gradient in the Complexity of Neural Representations across the Ventral Stream.

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Güçlü, Umut; van Gerven, Marcel A J

2015-07-08

Converging evidence suggests that the primate ventral visual pathway encodes increasingly complex stimulus features in downstream areas. We quantitatively show that there indeed exists an explicit gradient for feature complexity in the ventral pathway of the human brain. This was achieved by mapping thousands of stimulus features of increasing complexity across the cortical sheet using a deep neural network. Our approach also revealed a fine-grained functional specialization of downstream areas of the ventral stream. Furthermore, it allowed decoding of representations from human brain activity at an unsurpassed degree of accuracy, confirming the quality of the developed approach. Stimulus features that successfully explained neural responses indicate that population receptive fields were explicitly tuned for object categorization. This provides strong support for the hypothesis that object categorization is a guiding principle in the functional organization of the primate ventral stream. Copyright © 2015 the authors 0270-6474/15/3510005-10$15.00/0.

Metabolic profiling reveals potential metabolic markers associated with Hypoxia Inducible Factor-mediated signalling in hypoxic cancer cells.

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Armitage, Emily G; Kotze, Helen L; Allwood, J William; Dunn, Warwick B; Goodacre, Royston; Williams, Kaye J

2015-10-28

Hypoxia inducible factors (HIFs) plays an important role in oxygen compromised environments and therefore in tumour survival. In this research, metabolomics has been applied to study HIFs metabolic function in two cell models: mouse hepatocellular carcinoma and human colon carcinoma, whereby the metabolism has been profiled for a range of oxygen potentials. Wild type cells have been compared to cells deficient in HIF signalling to reveal its effect on cellular metabolism under normal oxygen conditions as well as low oxygen, hypoxic and anoxic environments. Characteristic responses to hypoxia that were conserved across both cell models involved the anti-correlation between 2-hydroxyglutarate, 2-oxoglutarate, fructose, hexadecanoic acid, hypotaurine, pyruvate and octadecenoic acid with 4-hydroxyproline, aspartate, cysteine, glutamine, lysine, malate and pyroglutamate. Further to this, network-based correlation analysis revealed HIF specific pathway responses to each oxygen condition that were also conserved between cell models. From this, 4-hydroxyproline was revealed as a regulating hub in low oxygen survival of WT cells while fructose appeared to be in HIF deficient cells. Pathways surrounding these hubs were built from the direct connections of correlated metabolites that look beyond traditional pathways in order to understand the mechanism of HIF response to low oxygen environments.

DNA fragmentation and cell death mediated by T cell antigen receptor/CD3 complex on a leukemia T cell line.

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Takahashi, S; Maecker, H T; Levy, R

1989-10-01

An anti-T cell receptor (TcR) monoclonal antibody (mAb), LC4, directed against a human leukemic T cell line, SUP-T13, caused DNA fragmentation ("apoptosis") and cell death upon binding to this cell line. Cross-linking of receptor molecules was necessary for this effect since F(ab')2, but not Fab', fragments of LC4 could induce cell death. Five anti-CD3 mAb tested also caused apoptosis, but only when they were presented on a solid phase. Interestingly, soluble anti-CD3 mAb induced calcium flux and had an additive effect on the calcium flux and interleukin 2 receptor expression induced by LC4, but these anti-CD3 mAb reversed the growth inhibition and apoptosis caused by LC4. The calcium ionophore A23187, but not the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), also induced apoptosis, suggesting that protein kinase C activation alone does not cause apoptosis, although PMA is growth inhibitory. These results suggest that two distinct biological phenomena can accompany stimulation of the TcR/CD3 complex. In both cases, calcium flux and interleukin 2 receptor expression is induced, but only in one case is apoptosis and cell death seen. The signal initiating apoptosis can be selectively prevented by binding CD3 portion of the receptor in this cell line. This difference in signals mediated by the TcR/CD3 complex may be important in explaining the process of thymic selection, as well as in choosing anti-TcR mAb for therapeutic use.

Epigenetic landscapes reveal transcription factors that regulate CD8+ T cell differentiation.

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Yu, Bingfei; Zhang, Kai; Milner, J Justin; Toma, Clara; Chen, Runqiang; Scott-Browne, James P; Pereira, Renata M; Crotty, Shane; Chang, John T; Pipkin, Matthew E; Wang, Wei; Goldrath, Ananda W

2017-05-01

Dynamic changes in the expression of transcription factors (TFs) can influence the specification of distinct CD8 + T cell fates, but the observation of equivalent expression of TFs among differentially fated precursor cells suggests additional underlying mechanisms. Here we profiled the genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8 + T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that the expression and binding of TFs contributed to the establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal key TFs that influence the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8 + T cell differentiation, regulated the formation of terminal-effector cell fates and memory-precursor cell fates, respectively. Our data define the epigenetic landscape of differentiation intermediates and facilitate the identification of TFs with previously unappreciated roles in CD8 + T cell differentiation.

Organizational changes of the daughter basal complex during the parasite replication of Toxoplasma gondii.

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Ke Hu

2008-01-01

Full Text Available The apicomplexans are a large group of parasitic protozoa, many of which are important human and animal pathogens, including Plasmodium falciparum and Toxoplasma gondii. These parasites cause disease only when they replicate, and their replication is critically dependent on the proper assembly of the parasite cytoskeletons during cell division. In addition to their importance in pathogenesis, the apicomplexan parasite cytoskeletons are spectacular structures. Therefore, understanding the cytoskeletal biogenesis of these parasites is important not only for parasitology but also of general interest to broader cell biology. Previously, we found that the basal end of T. gondii contains a novel cytoskeletal assembly, the basal complex, a cytoskeletal compartment constructed in concert with the daughter cortical cytoskeleton during cell division. This study focuses on key events during the biogenesis of the basal complex using high resolution light microscopy, and reveals that daughter basal complexes are established around the duplicated centrioles independently of the structural integrity of the daughter cortical cytoskeleton, and that they are dynamic "caps" at the growing ends of the daughters. Compartmentation and polarization of the basal complex is first revealed at a late stage of cell division upon the recruitment of an EF-hand containing calcium binding protein, TgCentrin2. This correlates with the constriction of the basal complex, a process that can be artificially induced by increasing cellular calcium concentration. The basal complex is therefore likely to be a new kind of centrin-based contractile apparatus.

Comparative live-cell imaging analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa reveal novel features of the filamentous fungal polarisome.

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Alexander Lichius

Full Text Available A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk, whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture.

Role for a Novel Usher Protein Complex in Hair Cell Synaptic Maturation

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Zallocchi, Marisa; Meehan, Daniel T.; Delimont, Duane; Rutledge, Joseph; Gratton, Michael Anne; Flannery, John; Cosgrove, Dominic

2012-01-01

The molecular mechanisms underlying hair cell synaptic maturation are not well understood. Cadherin-23 (CDH23), protocadherin-15 (PCDH15) and the very large G-protein coupled receptor 1 (VLGR1) have been implicated in the development of cochlear hair cell stereocilia, while clarin-1 has been suggested to also play a role in synaptogenesis. Mutations in CDH23, PCDH15, VLGR1 and clarin-1 cause Usher syndrome, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa. Here we show developmental expression of these Usher proteins in afferent spiral ganglion neurons and hair cell synapses. We identify a novel synaptic Usher complex comprised of clarin-1 and specific isoforms of CDH23, PCDH15 and VLGR1. To establish the in vivo relevance of this complex, we performed morphological and quantitative analysis of the neuronal fibers and their synapses in the Clrn1−/− mouse, which was generated by incomplete deletion of the gene. These mice showed a delay in neuronal/synaptic maturation by both immunostaining and electron microscopy. Analysis of the ribbon synapses in Ames waltzerav3J mice also suggests a delay in hair cell synaptogenesis. Collectively, these results show that, in addition to the well documented role for Usher proteins in stereocilia development, Usher protein complexes comprised of specific protein isoforms likely function in synaptic maturation as well. PMID:22363448

Role for a novel Usher protein complex in hair cell synaptic maturation.

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Marisa Zallocchi

Full Text Available The molecular mechanisms underlying hair cell synaptic maturation are not well understood. Cadherin-23 (CDH23, protocadherin-15 (PCDH15 and the very large G-protein coupled receptor 1 (VLGR1 have been implicated in the development of cochlear hair cell stereocilia, while clarin-1 has been suggested to also play a role in synaptogenesis. Mutations in CDH23, PCDH15, VLGR1 and clarin-1 cause Usher syndrome, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa. Here we show developmental expression of these Usher proteins in afferent spiral ganglion neurons and hair cell synapses. We identify a novel synaptic Usher complex comprised of clarin-1 and specific isoforms of CDH23, PCDH15 and VLGR1. To establish the in vivo relevance of this complex, we performed morphological and quantitative analysis of the neuronal fibers and their synapses in the Clrn1-/- mouse, which was generated by incomplete deletion of the gene. These mice showed a delay in neuronal/synaptic maturation by both immunostaining and electron microscopy. Analysis of the ribbon synapses in Ames waltzer(av3J mice also suggests a delay in hair cell synaptogenesis. Collectively, these results show that, in addition to the well documented role for Usher proteins in stereocilia development, Usher protein complexes comprised of specific protein isoforms likely function in synaptic maturation as well.

Small-angle X-ray Solution Scattering Study of the Multi-aminoacyl-tRNA Synthetase Complex Reveals an Elongated and Multi-armed particle*

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Dias, José; Renault, Louis; Pérez, Javier; Mirande, Marc

2013-01-01

In animal cells, nine aminoacyl-tRNA synthetases are associated with the three auxiliary proteins p18, p38, and p43 to form a stable and conserved large multi-aminoacyl-tRNA synthetase complex (MARS), whose molecular mass has been proposed to be between 1.0 and 1.5 MDa. The complex acts as a molecular hub for coordinating protein synthesis and diverse regulatory signal pathways. Electron microscopy studies defined its low resolution molecular envelope as an overall rather compact, asymmetric triangular shape. Here, we have analyzed the composition and homogeneity of the native mammalian MARS isolated from rabbit liver and characterized its overall internal structure, size, and shape at low resolution by hydrodynamic methods and small-angle x-ray scattering in solution. Our data reveal that the MARS exhibits a much more elongated and multi-armed shape than expected from previous reports. The hydrodynamic and structural features of the MARS are large compared with other supramolecular assemblies involved in translation, including ribosome. The large dimensions and non-compact structural organization of MARS favor a large protein surface accessibility for all its components. This may be essential to allow structural rearrangements between the catalytic and cis-acting tRNA binding domains of the synthetases required for binding the bulky tRNA substrates. This non-compact architecture may also contribute to the spatiotemporal controlled release of some of its components, which participate in non-canonical functions after dissociation from the complex. PMID:23836901

Structure insight of GSDMD reveals the basis of GSDMD autoinhibition in cell pyroptosis.

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Kuang, Siyun; Zheng, Jun; Yang, Hui; Li, Suhua; Duan, Shuyan; Shen, Yanfang; Ji, Chaoneng; Gan, Jianhua; Xu, Xue-Wei; Li, Jixi

2017-10-03

Recent findings have revealed that the protein gasdermin D (GSDMD) plays key roles in cell pyroptosis. GSDMD binds lipids and forms pore structures to induce pyroptosis upon microbial infection and associated danger signals. However, detailed structural information for GSDMD remains unknown. Here, we report the crystal structure of the C-terminal domain of human GSDMD (GSDMD-C) at 2.64-Å resolution. The first loop on GSDMD-C inserts into the N-terminal domain (GSDMD-N), which helps stabilize the conformation of the full-length GSDMD. Substitution of this region by a short linker sequence increased levels of cell death. Mutants F283A and F283R can increase protein heterogeneity in vitro and are capable of undergoing cell pyroptosis in 293T cells. The small-angle X-ray-scattering envelope of human GSDMD is consistent with the modeled GSDMD structure and mouse GSDMA3 structure, which suggests that GSDMD adopts an autoinhibited conformation in solution. The positive potential surface of GSDMD-N covered by GSDMD-C is exposed after being released from the autoinhibition state and can form high-order oligomers via a charge-charge interaction. Furthermore, by mapping different regions of GSDMD, we determined that one short segment is sufficient to kill bacteria in vitro and can efficiently inhibit cell growth in Escherichia coli and Mycobacterium Smegmatis These findings reveal that GSDMD-C acts as an auto-inhibition executor and GSDMD-N could form pore structures via a charge-charge interaction upon cleavage by caspases during cell pyroptosis.

Calcium Imaging Reveals Coordinated Simple Spike Pauses in Populations of Cerebellar Purkinje Cells

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Jorge E. Ramirez

2016-12-01

Full Text Available The brain’s control of movement is thought to involve coordinated activity between cerebellar Purkinje cells. The results reported here demonstrate that somatic Ca2+ imaging is a faithful reporter of Na+-dependent “simple spike” pauses and enables us to optically record changes in firing rates in populations of Purkinje cells in brain slices and in vivo. This simultaneous calcium imaging of populations of Purkinje cells reveals a striking spatial organization of pauses in Purkinje cell activity between neighboring cells. The source of this organization is shown to be the presynaptic gamma-Aminobutyric acid producing (GABAergic network, and blocking ionotropic gamma-Aminobutyric acid receptor (GABAARs abolishes the synchrony. These data suggest that presynaptic interneurons synchronize (inactivity between neighboring Purkinje cells, and thereby maximize their effect on downstream targets in the deep cerebellar nuclei.

Non-canonical TAF complexes regulate active promoters in human embryonic stem cells.

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Maston, Glenn A; Zhu, Lihua Julie; Chamberlain, Lynn; Lin, Ling; Fang, Minggang; Green, Michael R

2012-11-13

The general transcription factor TFIID comprises the TATA-box-binding protein (TBP) and approximately 14 TBP-associated factors (TAFs). Here we find, unexpectedly, that undifferentiated human embryonic stem cells (hESCs) contain only six TAFs (TAFs 2, 3, 5, 6, 7 and 11), whereas following differentiation all TAFs are expressed. Directed and global chromatin immunoprecipitation analyses reveal an unprecedented promoter occupancy pattern: most active genes are bound by only TAFs 3 and 5 along with TBP, whereas the remaining active genes are bound by TBP and all six hESC TAFs. Consistent with these results, hESCs contain a previously undescribed complex comprising TAFs 2, 6, 7, 11 and TBP. Altering the composition of hESC TAFs, either by depleting TAFs that are present or ectopically expressing TAFs that are absent, results in misregulated expression of pluripotency genes and induction of differentiation. Thus, the selective expression and use of TAFs underlies the ability of hESCs to self-renew.DOI:http://dx.doi.org/10.7554/eLife.00068.001.

Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes

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Bestembayeva, Aizhan; Kramer, Armin; Labokha, Aksana A.; Osmanović, Dino; Liashkovich, Ivan; Orlova, Elena V.; Ford, Ian J.; Charras, Guillaume; Fassati, Ariberto; Hoogenboom, Bart W.

2015-01-01

The nuclear pore complex (NPC) is the gate for transport between the cell nucleus and the cytoplasm. Small molecules cross the NPC by passive diffusion, but molecules larger than ∼5 nm must bind to nuclear transport receptors to overcome a selective barrier within the NPC. Although the structure and shape of the cytoplasmic ring of the NPC are relatively well characterized, the selective barrier is situated deep within the central channel of the NPC and depends critically on unstructured nuclear pore proteins, and is therefore not well understood. Here, we show that stiffness topography with sharp atomic force microscopy tips can generate nanoscale cross-sections of the NPC. The cross-sections reveal two distinct structures, a cytoplasmic ring and a central plug structure, which are consistent with the three-dimensional NPC structure derived from electron microscopy. The central plug persists after reactivation of the transport cycle and resultant cargo release, indicating that the plug is an intrinsic part of the NPC barrier. Added nuclear transport receptors accumulate on the intact transport barrier and lead to a homogenization of the barrier stiffness. The observed nanomechanical properties in the NPC indicate the presence of a cohesive barrier to transport and are quantitatively consistent with the presence of a central condensate of nuclear pore proteins in the NPC channel.

Breast-axillary complex in HIV/AIDS patients.

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Eni, U E; Naaya, H U; Yawe, K D T; Lawan, M A; Bakari, A A

2010-01-01

HIV/AIDS have not only increased the health care burden especially in developing countries, it equally complicates the presentation of many diseases. Some well known disease entities now occur in fulminant complexities not previously described or known as such. The objective of this article is to report an unusual presentation of HIV/AIDS patients to the surgeon with Axillary and ipsilateral breast swelling. This is a report of three cases seen and managed by the authors. Three adult female patients presented with progressively increasing axillary and ipsilateral breast swellings. They also had associated fevers and weight loss. Their main concern had been development of breast cancer. One of the patients was a known retroviral positive on Highly Active Anti-Retroviral Therapy (HAART). Examination revealed axillary abscess and ipsilateral breast oedema in two cases. The patient on HAART had a hard breast-axillary mass complex. Biopsy (FNAB) revealed inflammatory cells and no malignancy in all three cases. HIV screening was positive in all cases. One of the patients had excision of breast-axillary mass complex, and the histology showed features of chronic inflammation, with no malignant cells. The other two had incision and drainage of their axillary abscess. This shows the ubiquitous presentation of HIV/AIDS in our environment and surgeons should be aware of the breast axillary complex in HIV/AIDS. Medical practitioners should be careful to obtain accurate diagnosis before embarking on treatment especially mutilating surgical procedures.

Investigation of the complex structure, comparative DNA-binding and DNA cleavage of two water-soluble mono-nuclear lanthanum(III) complexes and cytotoxic activity of chitosan-coated magnetic nanoparticles as drug delivery for the complexes

Science.gov (United States)

Asadi, Zahra; Nasrollahi, Neda; Karbalaei-Heidari, Hamidreza; Eigner, Vaclav; Dusek, Michal; Mobaraki, Nabiallah; Pournejati, Roya

2017-05-01

Two water-soluble mono-nuclear macrocyclic lanthanum(III) complexes of 2,6-diformyl-4-methylphenol with 1,3-diamino-2-propanol (C1) or 1,3-propylenediamine (C2) were synthesized and characterized by UV-Vis, FT-IR, 13C and 1H NMR spectroscopy and elemental analysis. C1 complex was structurally characterized by single-crystal X-ray diffraction, which revealed that the complex was mononuclear and ten-coordinated. The coordination sites around lanthanum(III) were occupied with a five-dentate ligand, two bidentate nitrates, and one water molecule. The interaction of complexes with DNA was studied in buffered aqueous solution at pH 7.4. UV-Vis absorption spectroscopy, emission spectroscopy, circular dichroism (CD) and viscometric measurements provided clear evidence of the intercalation mechanism of binding. The obtained intrinsic binding constants (Kb) 9.3 × 103 and 1.2 × 103 M- 1 for C1 and C2, respectively confirmed that C1 is better intercalator than C2. The DNA docking studies suggested that the complexes bind with DNA in a groove binding mode with the binding affinity of C1 > C2. Moreover, agarose gel electrophoresis study of the DNA-complex for both compounds revealed that the C1 intercalation cause ethidium bromide replacement in a competitive manner which confirms the suggested mechanism of binding. Finally, the anticancer experiments for the treated cancerous cell lines with both synthesized compounds show that these hydrophilic molecules need a suitable carrier to pass through the hydrophobic nature of cell membrane efficiently.

Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement.

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Carmen Herranz, Ma; Sanchez-Navarro, Jesús-Angel; Saurí, Ana; Mingarro, Ismael; Pallás, Vicente

2005-08-15

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed.

Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

International Nuclear Information System (INIS)

Carmen Herranz, Ma; Sanchez-Navarro, Jesus-Angel; Sauri, Ana; Mingarro, Ismael; Pallas, Vicente

2005-01-01

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed

Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility

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Seumois, Grégory; Chavez, Lukas; Gerasimova, Anna; Lienhard, Matthias; Omran, Nada; Kalinke, Lukas; Vedanayagam, Maria; Ganesan, Asha Purnima V; Chawla, Ashu; Djukanović, Ratko; Ansel, K Mark; Peters, Bjoern; Rao, Anjana; Vijayanand, Pandurangan

2014-01-01

A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4+ T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis. PMID:24997565

Biochemical characterization of native Usher protein complexes from a vesicular subfraction of tracheal epithelial cells.

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Zallocchi, Marisa; Sisson, Joseph H; Cosgrove, Dominic

2010-02-16

Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in nondetergent buffer and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15, and VLGR-1 and a different one at the top of the gradient that included all of the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100-200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins cosediment into the gradient at a sedimentation coefficient of approximately 50 S, correlating with a predicted molecular mass of 2 x 10(6) Da. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher protein complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors.

Granzyme A Cleaves a Mitochondrial Complex I Protein to Initiate Caspase-Independent Cell Death

Science.gov (United States)

Martinvalet, Denis; Dykxhoorn, Derek M.; Ferrini, Roger; Lieberman, Judy

2010-01-01

SUMMARY The killer lymphocyte protease granzyme A (GzmA) triggers caspase-independent target cell death with morphological features of apoptosis. We previously showed that GzmA acts directly on mitochondria to generate reactive oxygen species (ROS) and disrupt the transmembrane potential (ΔΨm) but does not permeabilize the mitochondrial outer membrane. Mitochondrial damage is critical to GzmA-induced cell death since cells treated with superoxide scavengers are resistant to GzmA. Here we find that GzmA accesses the mitochondrial matrix to cleave the complex I protein NDUFS3, an iron-sulfur subunit of the NADH:ubiquinone oxidoreductase complex I, after Lys56 to interfere with NADH oxidation and generate superoxide anions. Target cells expressing a cleavage site mutant of NDUFS3 are resistant to GzmA-mediated cell death but remain sensitive to GzmB. PMID:18485875

System-wide analysis reveals a complex network of tumor-fibroblast interactions involved in tumorigenicity.

Directory of Open Access Journals (Sweden)

Megha Rajaram

Full Text Available Many fibroblast-secreted proteins promote tumorigenicity, and several factors secreted by cancer cells have in turn been proposed to induce these proteins. It is not clear whether there are single dominant pathways underlying these interactions or whether they involve multiple pathways acting in parallel. Here, we identified 42 fibroblast-secreted factors induced by breast cancer cells using comparative genomic analysis. To determine what fraction was active in promoting tumorigenicity, we chose five representative fibroblast-secreted factors for in vivo analysis. We found that the majority (three out of five played equally major roles in promoting tumorigenicity, and intriguingly, each one had distinct effects on the tumor microenvironment. Specifically, fibroblast-secreted amphiregulin promoted breast cancer cell survival, whereas the chemokine CCL7 stimulated tumor cell proliferation while CCL2 promoted innate immune cell infiltration and angiogenesis. The other two factors tested had minor (CCL8 or minimally (STC1 significant effects on the ability of fibroblasts to promote tumor growth. The importance of parallel interactions between fibroblasts and cancer cells was tested by simultaneously targeting fibroblast-secreted amphiregulin and the CCL7 receptor on cancer cells, and this was significantly more efficacious than blocking either pathway alone. We further explored the concept of parallel interactions by testing the extent to which induction of critical fibroblast-secreted proteins could be achieved by single, previously identified, factors produced by breast cancer cells. We found that although single factors could induce a subset of genes, even combinations of factors failed to induce the full repertoire of functionally important fibroblast-secreted proteins. Together, these results delineate a complex network of tumor-fibroblast interactions that act in parallel to promote tumorigenicity and suggest that effective anti

The human CTC1/STN1/TEN1 complex regulates telomere maintenance in ALT cancer cells

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Huang, Chenhui; Jia, Pingping; Chastain, Megan; Shiva, Olga; Chai, Weihang, E-mail: wchai@wsu.edu

2017-06-15

Maintaining functional telomeres is important for long-term proliferation of cells. About 15% of cancer cells are telomerase-negative and activate the alternative-lengthening of telomeres (ALT) pathway to maintain their telomeres. Recent studies have shown that the human CTC1/STN1/TEN1 complex (CST) plays a multi-faceted role in telomere maintenance in telomerase-expressing cancer cells. However, the role of CST in telomere maintenance in ALT cells is unclear. Here, we report that human CST forms a functional complex localizing in the ALT-associated PML bodies (APBs) in ALT cells throughout the cell cycle. Suppression of CST induces telomere instabilities including telomere fragility and elevates telomeric DNA recombination, leading to telomere dysfunction. In addition, CST deficiency significantly diminishes the abundance of extrachromosomal circular telomere DNA known as C-circles and t-circles. Suppression of CST also results in multinucleation in ALT cells and impairs cell proliferation. Our findings imply that the CST complex plays an important role in regulating telomere maintenance in ALT cells. - Highlights: • CST localizes at telomeres and ALT-associated PML bodies in ALT cells throughout the cell cycle. • CST is important for promoting telomeric DNA replication in ALT cells. • CST deficiency decreases ECTR formation and increases T-SCE. • CST deficiency impairs ALT cell proliferation and results in multinucleation.

Characterization of tissue plasminogen activator binding proteins isolated from endothelial cells and other cell types

International Nuclear Information System (INIS)

Beebe, D.P.; Wood, L.L.; Moos, M.

1990-01-01

Human tissue plasminogen activator (t-PA) was shown to bind specifically to human osteosarcoma cells (HOS), and human epidermoid carcinoma cells (A-431 cells). Crosslinking studies with DTSSP demonstrated high molecular weight complexes (130,000) between 125 I-t-PA and cell membrane protein on human umbilical vein endothelial cells (HUVEC), HOS, and A-431 cells. A 48-65,000 molecular weight complex was demonstrated after crosslinking t-PA peptide (res. 7-20) to cells. Ligand blotting of cell lysates which had been passed over a t-PA affinity column revealed binding of t-PA to 54,000 and 95,000 molecular weight proteins. Several t-PA binding proteins were identified in immunopurified cell lysates, including tubulin beta chain, plasminogen activator inhibitor type 1 and single chain urokinase

CAFET algorithm reveals Wnt/PCP signature in lung squamous cell carcinoma.

Directory of Open Access Journals (Sweden)

Yue Hu

Full Text Available We analyzed the gene expression patterns of 138 Non-Small Cell Lung Cancer (NSCLC samples and developed a new algorithm called Coverage Analysis with Fisher's Exact Test (CAFET to identify molecular pathways that are differentially activated in squamous cell carcinoma (SCC and adenocarcinoma (AC subtypes. Analysis of the lung cancer samples demonstrated hierarchical clustering according to the histological subtype and revealed a strong enrichment for the Wnt signaling pathway components in the cluster consisting predominantly of SCC samples. The specific gene expression pattern observed correlated with enhanced activation of the Wnt Planar Cell Polarity (PCP pathway and inhibition of the canonical Wnt signaling branch. Further real time RT-PCR follow-up with additional primary tumor samples and lung cancer cell lines confirmed enrichment of Wnt/PCP pathway associated genes in the SCC subtype. Dysregulation of the canonical Wnt pathway, characterized by increased levels of β-catenin and epigenetic silencing of negative regulators, has been reported in adenocarcinoma of the lung. Our results suggest that SCC and AC utilize different branches of the Wnt pathway during oncogenesis.

Proteomic analysis of human bladder epithelial cells by 2D blue native SDS-PAGE reveals TCDD-induced alterations of calcium and iron homeostasis possibly mediated by nitric oxide.

Science.gov (United States)

Verma, Nisha; Pink, Mario; Petrat, Frank; Rettenmeier, Albert W; Schmitz-Spanke, Simone

2015-01-02

A proteomic analysis of the interaction among multiprotein complexes involved in 2,3,7,8-dibenzo-p-dioxin (TCDD)-mediated toxicity in urinary bladder epithelial RT4 cells was performed using two-dimensional blue native SDS-PAGE (2D BN/SDS-PAGE). To enrich the protein complexes, unexposed and TCDD-exposed cells were fractionated. BN/SDS-PAGE of the resulting fractions led to an effective separation of proteins and protein complexes of various origins, including cell membrane, mitochondria, and other intracellular compartments. Major differences between the proteome of control and exposed cells involved the alteration of many calcium-regulated proteins (calmodulin, protein S100-A2, annexin A5, annexin A10, gelsolin isoform b) and iron-regulated proteins (ferritin, heme-binding protein 2, transferrin). On the basis of these findings, the intracellular calcium concentration was determined, revealing a significant increase after 24 h of exposure to TCDD. Moreover, the concentration of the labile iron pool (LIP) was also significantly elevated in TCDD-exposed cells. This increase was strongly inhibited by the calmodulin (CaM) antagonist W-7, which pointed toward a possible interaction between iron and calcium signaling. Because nitric oxide (NO) production was significantly enhanced in TCDD-exposed cells and was also inhibited by W-7, we hypothesize that alterations in calcium and iron homeostasis upon exposure to TCDD may be linked through NO generated by CaM-activated nitric oxide synthase. In our model, we propose that NO produced upon TCDD exposure interacts with the iron centers of iron-regulatory proteins (IRPs) that modulate the alteration of ferritin and transferrin, resulting in an augmented cellular LIP and, hence, increased toxicity.

Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

Energy Technology Data Exchange (ETDEWEB)

Vorhagen, Susanne; Niessen, Carien M., E-mail: carien.niessen@uni-koeln.de

2014-11-01

Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.

The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells.

Science.gov (United States)

Lucato, Christina M; Halls, Michelle L; Ooms, Lisa M; Liu, Heng-Jia; Mitchell, Christina A; Whisstock, James C; Ellisdon, Andrew M

2015-08-21

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP3 and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP3/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP3 and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity

Science.gov (United States)

Chiu, Isaac M; Barrett, Lee B; Williams, Erika K; Strochlic, David E; Lee, Seungkyu; Weyer, Andy D; Lou, Shan; Bryman, Gregory S; Roberson, David P; Ghasemlou, Nader; Piccoli, Cara; Ahat, Ezgi; Wang, Victor; Cobos, Enrique J; Stucky, Cheryl L; Ma, Qiufu; Liberles, Stephen D; Woolf, Clifford J

2014-01-01

The somatosensory nervous system is critical for the organism's ability to respond to mechanical, thermal, and nociceptive stimuli. Somatosensory neurons are functionally and anatomically diverse but their molecular profiles are not well-defined. Here, we used transcriptional profiling to analyze the detailed molecular signatures of dorsal root ganglion (DRG) sensory neurons. We used two mouse reporter lines and surface IB4 labeling to purify three major non-overlapping classes of neurons: 1) IB4+SNS-Cre/TdTomato+, 2) IB4−SNS-Cre/TdTomato+, and 3) Parv-Cre/TdTomato+ cells, encompassing the majority of nociceptive, pruriceptive, and proprioceptive neurons. These neurons displayed distinct expression patterns of ion channels, transcription factors, and GPCRs. Highly parallel qRT-PCR analysis of 334 single neurons selected by membership of the three populations demonstrated further diversity, with unbiased clustering analysis identifying six distinct subgroups. These data significantly increase our knowledge of the molecular identities of known DRG populations and uncover potentially novel subsets, revealing the complexity and diversity of those neurons underlying somatosensation. DOI: http://dx.doi.org/10.7554/eLife.04660.001 PMID:25525749

Arabidopsis thaliana plants lacking the ARP2/3 complex show defects in cell wall assembly and auxin distribution.

Science.gov (United States)

Pratap Sahi, Vaidurya; Cifrová, Petra; García-González, Judith; Kotannal Baby, Innu; Mouillé, Gregory; Gineau, Emilie; Müller, Karel; Baluška, František; Soukup, Aleš; Petrášek, Jan; Schwarzerová, Katerina

2017-12-25

The cytoskeleton plays an important role in the synthesis of plant cell walls. Both microtubules and actin cytoskeleton are known to be involved in the morphogenesis of plant cells through their role in cell wall building. The role of ARP2/3-nucleated actin cytoskeleton in the morphogenesis of cotyledon pavement cells has been described before. Seedlings of Arabidopsis mutants lacking a functional ARP2/3 complex display specific cell wall-associated defects. In three independent Arabidopsis mutant lines lacking subunits of the ARP2/3 complex, phenotypes associated with the loss of the complex were analysed throughout plant development. Organ size and anatomy, cell wall composition, and auxin distribution were investigated. ARP2/3-related phenotype is associated with changes in cell wall composition, and the phenotype is manifested especially in mature tissues. Cell walls of mature plants contain less cellulose and a higher amount of homogalacturonan, and display changes in cell wall lignification. Vascular bundles of mutant inflorescence stems show a changed pattern of AUX1-YFP expression. Plants lacking a functional ARP2/3 complex have decreased basipetal auxin transport. The results suggest that the ARP2/3 complex has a morphogenetic function related to cell wall synthesis and auxin transport. © The Author(s) 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cancer initiation and progression: an unsimplifiable complexity

Directory of Open Access Journals (Sweden)

Frezza Eldo E

2006-10-01

Full Text Available Abstract Background Cancer remains one of the most complex diseases affecting humans and, despite the impressive advances that have been made in molecular and cell biology, how cancer cells progress through carcinogenesis and acquire their metastatic ability is still widely debated. Conclusion There is no doubt that human carcinogenesis is a dynamic process that depends on a large number of variables and is regulated at multiple spatial and temporal scales. Viewing cancer as a system that is dynamically complex in time and space will, however, probably reveal more about its underlying behavioural characteristics. It is encouraging that mathematicians, biologists and clinicians continue to contribute together towards a common quantitative understanding of cancer complexity. This way of thinking may further help to clarify concepts, interpret new and old experimental data, indicate alternative experiments and categorize the acquired knowledge on the basis of the similarities and/or shared behaviours of very different tumours.

Cytosolic calcium mediates RIP1/RIP3 complex-dependent necroptosis through JNK activation and mitochondrial ROS production in human colon cancer cells.

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Sun, Wen; Wu, Xiaxia; Gao, Hongwei; Yu, Jie; Zhao, Wenwen; Lu, Jin-Jian; Wang, Jinhua; Du, Guanhua; Chen, Xiuping

2017-07-01

Necroptosis is a form of programmed necrosis mediated by signaling complexes with receptor-interacting protein 1 (RIP1) and RIP3 kinases as the main mediators. However, the underlying execution pathways of this phenomenon have yet to be elucidated in detail. In this study, a RIP1/RIP3 complex was formed in 2-methoxy-6-acetyl-7-methyljuglone (MAM)-treated HCT116 and HT29 colon cancer cells. With this formation, mitochondrial reactive oxygen species (ROS) levels increased, mitochondrial depolarization occurred, and ATP concentrations decreased. This process was identified as necroptosis. This finding was confirmed by experiments showing that MAM-induced cell death was attenuated by the pharmacological or genetic blockage of necroptosis signaling, including RIP1 inhibitor necrostatin-1s (Nec-1s) and siRNA-mediated gene silencing of RIP1 and RIP3, but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM54). Transmission electron microscopy (TEM) analysis further revealed the ultrastructural features of MAM-induced necroptosis. MAM-induced RIP1/RIP3 complex triggered necroptosis through cytosolic calcium (Ca 2+ ) accumulation and sustained c-Jun N-terminal kinase (JNK) activation. Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate necroptotic features, including mitochondrial ROS elevation, mitochondrial depolarization, and ATP depletion. 2-thenoyltrifluoroacetone (TTFA), which is a mitochondrial complex II inhibitor, was found to effectively reverse both MAM induced mitochondrial ROS generation and cell death, indicating the complex II was the ROS-producing site. The essential role of mitochondrial ROS was confirmed by the protective effect of overexpression of manganese superoxide dismutase (MnSOD). MAM-induced necroptosis was independent of TNFα, p53, MLKL, and lysosomal membrane permeabilization. In summary, our study demonstrated that RIP1/RIP3 complex-triggered cytosolic calcium

Raman spectrum reveals Mesenchymal stem cells inhibiting HL60 cells growth

Science.gov (United States)

Su, Xin; Fang, Shaoyin; Zhang, Daosen; Zhang, Qinnan; Lu, Xiaoxu; Tian, Jindong; Fan, Jinping; Zhong, Liyun

2017-04-01

Though some research results reveals that Mesenchymal stem cells (MSCs) have the ability of inhibiting tumor cells proliferation, it remains controversial about the precise interaction mechanism during MSCs and tumor cells co-culture. In this study, combing Raman spectroscopic data and principle component analysis (PCA), the biochemical changes of MSCs or Human promyelocytic leukemia (HL60) cells during their co-culture were presented. The obtained results showed that some main Raman peaks of HL60 assigned to nucleic acids or proteins were greatly higher in intensity in the late stage of co-culture than those in the early stage of co-culture while they were still lower relative to the control group, implicating that the effect of MSCs inhibiting HL60 proliferation appeared in the early stage but gradually lost the inhibiting ability in the late stage of co-culture. Moreover, some other peaks of HL60 assigned to proteins were decreased in intensity in the early stage of co-culture relative to the control group but rebounded to the level similar to the control group in the late stage, showing that the content and structure changes of these proteins might be generated in the early stage but returned to the original state in the late stage of co-culture. As a result, in the early stage of MSCs-HL60 co-culture, along with the level of Akt phosphorylation of HL60 was lowered relative to its control group, the proliferation rate of HL60 cells was decreased. And in the late stage of co-culture, along with the level of Akt phosphorylation was rebounded, the reverse transfer of Raman peaks within 875-880 cm- 1 appeared, thus MSCs lost the ability to inhibit HL60 growth and HL60 proliferation was increased. In addition, it was observed that the peak at 811 cm- 1, which is a marker of RNA, was higher in intensity in the late stage than that in the control group, indicating that MSCs might be differentiated into myofibroblast-like MSCs. In addition, PCA results also exhibited

Atomic force microscopy stiffness tomography on living Arabidopsis thaliana cells reveals the mechanical properties of surface and deep cell-wall layers during growth.

Science.gov (United States)

Radotić, Ksenija; Roduit, Charles; Simonović, Jasna; Hornitschek, Patricia; Fankhauser, Christian; Mutavdžić, Dragosav; Steinbach, Gabor; Dietler, Giovanni; Kasas, Sandor

2012-08-08

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Complex (Nonstandard) Six-Layer Polytypes of Lizardite Revealed from Oblique-Texture Electron Diffraction Patterns

International Nuclear Information System (INIS)

Zhukhlistov, A.P.; Zinchuk, N.N.; Kotel'nikov, D.D.

2004-01-01

Association of simple (1T and 3R) and two complex (nonstandard) orthogonal polytypes of the serpentine mineral lizardite from the Catoca kimberlite pipe (West Africa) association is revealed from oblique-texture electron diffraction patterns. A six-layer polytype with an ordered superposition of equally oriented layers (notation 3 2 3 2 3 4 3 4 3 6 3 6 or ++ - -00) belonging to the structural group A and a three-layer (336 or I,I,II) or a six-layer (336366 or I,I,II,I,II,II) polytype with alternating oppositely oriented layers and semi-disordered structure are identified using polytype analysis

Generation of a haptoglobin-hemoglobin complex-specific Fab antibody blocking the binding of the complex to CD163

DEFF Research Database (Denmark)

Horn, Ivo R; Nielsen, Marianne Jensby; Madsen, Mette

2003-01-01

During intravascular hemolysis hemoglobin (Hb) binds to haptoglobin (Hp) leading to endocytosis of the complex by the macrophage receptor, CD163. In the present study, we used a phage-display Fab antibody strategy to explore if the complex formation between Hp and Hb leads to exposure of antigenic...... epitopes specific for the complex. By Hp-Hb-affinity screening of a phage-Fab library, we isolated a phage clone against the ligand complex. Surface plasmon resonance analyses of the Fab part expressed as a recombinant protein revealed a high affinity binding (KD = 3.9 nm) to Hp-Hb, whereas no binding...... was measured for non-complexed Hp or Hb. The Fab antibody completely inhibited the binding of 125I-labeled Hp-Hb complexes to CD163 and blocked their uptake in CD163-transfected cells. In conclusion, we have raised a receptor-blocking antibody specifically recognizing the Hp-Hb complex. In addition to provide...

Mutagenicity of complex mixtures

International Nuclear Information System (INIS)

Pelroy, R.A.

1985-01-01

The effect of coal-derived complex chemical mixtures on the mutagenicity of 6-aminochrysene (6-AC) was determined with Salmonella typhimurium TA98. Previous results suggested that the mutagenic potency of 6-AC for TA98 in the standard microsomal activation (Ames) assay increased if it was presented to the cells mixed with high-boiling coal liquids (CL) from the solvent refined coal (SRC) process. In this year's work, the apparent mutational synergism of CL and 6-AC was independently verified in a fluctuation bioassay which allowed quantitation of mutational frequencies and cell viability. The results of this assay system were similar to those in the Ames assay. Moreover, the fluctation assay revealed that mutagenesis and cellular toxicity induced by 6-AC were both strongly enhanced if 6-AC was presented to the cells mixed in a high-boiling CL. 4 figures

Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle signatures in joint diseases.

Directory of Open Access Journals (Sweden)

Bence György

Full Text Available INTRODUCTION: Microvesicles (MVs, earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF samples of patients with osteoarthritis (OA, rheumatoid arthritis (RA and juvenile idiopathic arthritis (JIA. To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM, Nanoparticle Tracking Analysis (NTA and mass spectrometry (MS. For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+ and CD8(+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections. In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction. CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

Nuclear dynamics of influenza A virus ribonucleoproteins revealed by live-cell imaging studies

International Nuclear Information System (INIS)

Loucaides, Eva M.; Kirchbach, Johann C. von; Foeglein, Agnes; Sharps, Jane; Fodor, Ervin; Digard, Paul

2009-01-01

The negative sense RNA genome of influenza A virus is transcribed and replicated in the nuclei of infected cells by the viral RNA polymerase. Only four viral polypeptides are required but multiple cellular components are potentially involved. We used fluorescence recovery after photobleaching (FRAP) to characterise the dynamics of GFP-tagged viral ribonucleoprotein (RNP) components in living cells. The nucleoprotein (NP) displayed very slow mobility that significantly increased on formation of transcriptionally active RNPs. Conversely, single or dimeric polymerase subunits showed fast nuclear dynamics that decreased upon formation of heterotrimers, suggesting increased interaction of the full polymerase complex with a relatively immobile cellular component(s). Treatment with inhibitors of cellular transcription indicated that in part, this reflected an interaction with cellular RNA polymerase II. Analysis of mutated influenza virus polymerase complexes further suggested that this was through an interaction between PB2 and RNA Pol II separate from PB2 cap-binding activity.

Lymphocyte depletion in thymic nurse cells: a tool to identify in situ lympho-epithelial complexes having thymic nurse cell characteristics

NARCIS (Netherlands)

Leene, W.; de Waal Malefijt, R.; Roholl, P. J.; Hoeben, K. A.

1988-01-01

In situ pre-existing complexes of epithelial cells and thymocytes having thymic nurse cell characteristics were visualized in the murine thymus cortex using dexamethasone as a potent killer of cortisone-sensitive thymocytes. The degradation and subsequent depletion of cortisone-sensitive thymocytes

Synthetic Strigolactone Analogues Reveal Anti-Cancer Activities on Hepatocellular Carcinoma Cells

KAUST Repository

Hasan, Mohammed Nihal

2018-02-09

Hepatocellular carcinoma (HCC) remains one of the leading causes of death worldwide. The complex etiology is attributed to many factors like heredity, cirrhosis, hepatitis infections or the dysregulation of the different molecular pathways. Nevertheless, the current treatment regimens have either severe side effects or tumors gradually acquire resistance upon prolonged use. Thus, developing a new selective treatment for HCC is the need of the hour. Many anticancer agents derived from plants have been evaluated for their cytotoxicity towards many human cancer cell lines. Strigolactones (SLs)-a newly discovered class of phytohormones, play a crucial role in the development of plant-root and shoot. Recently, many synthetic analogues of SLs have demonstrated pro-apoptotic effects on different cancer cell lines like prostate, breast, colon and lung. In this study, we tested synthetic SLs analogues on HCC cell line-HepG2 and evaluated their capability to induce cell proliferation inhibition and apoptosis. Primary WST-1 assays, followed by annexin-V/7AAD staining, demonstrated the anti-proliferative effects. The SLs analogues TIT3 and TIT7 were found to significantly reduce HepG2 cell viability in a dose- and time-dependent manner and induce apoptosis. Interestingly, though TIT3 and TIT7 strongly affected cancer cell proliferation, both compounds showed moderate anti-proliferative effect on normal cells. Further, migration of cancer cells was suppressed upon treatment with TIT3 and TIT7 in a wound healing assay. In summary, these findings suggest that two SLs analogues TIT3 and TIT7 exert selective inhibitory effects on cancer cells most likely through targeting microtubules. SLs analogues could be used in future as potential anti-cancer candidates in chemotherapy.

Synthetic Strigolactone Analogues Reveal Anti-Cancer Activities on Hepatocellular Carcinoma Cells

KAUST Repository

Hasan, Mohammed Nihal; Choudhry, Hani; Razvi, Syed Shoeb; Moselhy, Said Salama; Kumosani, Taha Abduallah; Zamzami, Mazin A.; Omran, Ziad; Halwani, Majed A.; Al-Babili, Salim; Abualnaja, Khalid Omer; Al-Malki, Abdulrahman Labeed; Alhosin, Mahmoud; Asami, Tadao

2018-01-01

Hepatocellular carcinoma (HCC) remains one of the leading causes of death worldwide. The complex etiology is attributed to many factors like heredity, cirrhosis, hepatitis infections or the dysregulation of the different molecular pathways. Nevertheless, the current treatment regimens have either severe side effects or tumors gradually acquire resistance upon prolonged use. Thus, developing a new selective treatment for HCC is the need of the hour. Many anticancer agents derived from plants have been evaluated for their cytotoxicity towards many human cancer cell lines. Strigolactones (SLs)-a newly discovered class of phytohormones, play a crucial role in the development of plant-root and shoot. Recently, many synthetic analogues of SLs have demonstrated pro-apoptotic effects on different cancer cell lines like prostate, breast, colon and lung. In this study, we tested synthetic SLs analogues on HCC cell line-HepG2 and evaluated their capability to induce cell proliferation inhibition and apoptosis. Primary WST-1 assays, followed by annexin-V/7AAD staining, demonstrated the anti-proliferative effects. The SLs analogues TIT3 and TIT7 were found to significantly reduce HepG2 cell viability in a dose- and time-dependent manner and induce apoptosis. Interestingly, though TIT3 and TIT7 strongly affected cancer cell proliferation, both compounds showed moderate anti-proliferative effect on normal cells. Further, migration of cancer cells was suppressed upon treatment with TIT3 and TIT7 in a wound healing assay. In summary, these findings suggest that two SLs analogues TIT3 and TIT7 exert selective inhibitory effects on cancer cells most likely through targeting microtubules. SLs analogues could be used in future as potential anti-cancer candidates in chemotherapy.

The subclonal complexity of STIL-TAL1+ T-cell acute lymphoblastic leukaemia.

Science.gov (United States)

Furness, Caroline L; Mansur, Marcela B; Weston, Victoria J; Ermini, Luca; van Delft, Frederik W; Jenkinson, Sarah; Gale, Rosemary; Harrison, Christine J; Pombo-de-Oliveira, Maria S; Sanchez-Martin, Marta; Ferrando, Adolfo A; Kearns, Pamela; Titley, Ian; Ford, Anthony M; Potter, Nicola E; Greaves, Mel

2018-03-20

Single-cell genetics were used to interrogate clonal complexity and the sequence of mutational events in STIL-TAL1+ T-ALL. Single-cell multicolour FISH was used to demonstrate that the earliest detectable leukaemia subclone contained the STIL-TAL1 fusion and copy number loss of 9p21.3 (CDKN2A/CDKN2B locus), with other copy number alterations including loss of PTEN occurring as secondary subclonal events. In three cases, multiplex qPCR and phylogenetic analysis were used to produce branching evolutionary trees recapitulating the snapshot history of T-ALL evolution in this leukaemia subtype, which confirmed that mutations in key T-ALL drivers, including NOTCH1 and PTEN, were subclonal and reiterative in distinct subclones. Xenografting confirmed that self-renewing or propagating cells were genetically diverse. These data suggest that the STIL-TAL1 fusion is a likely founder or truncal event. Therapies targeting the TAL1 auto-regulatory complex are worthy of further investigation in T-ALL.

Apoptotic effect of novel Schiff based CdCl₂(C₁₄H₂₁N₃O₂) complex is mediated via activation of the mitochondrial pathway in colon cancer cells.

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Hajrezaie, Maryam; Paydar, Mohammadjavad; Looi, Chung Yeng; Moghadamtousi, Soheil Zorofchian; Hassandarvish, Pouya; Salga, Muhammad Saleh; Karimian, Hamed; Shams, Keivan; Zahedifard, Maryam; Majid, Nazia Abdul; Ali, Hapipah Mohd; Abdulla, Mahmood Ameen

2015-03-13

The development of metal-based agents has had a tremendous role in the present progress in cancer chemotherapy. One well-known example of metal-based agents is Schiff based metal complexes, which hold great promise for cancer therapy. Based on the potential of Schiff based complexes for the induction of apoptosis, this study aimed to examine the cytotoxic and apoptotic activity of a CdCl2(C14H21N3O2) complex on HT-29 cells. The complex exerted a potent suppressive effect on HT-29 cells with an IC50 value of 2.57 ± 0.39 after 72 h of treatment. The collapse of the mitochondrial membrane potential and the elevated release of cytochrome c from the mitochondria to the cytosol indicate the involvement of the intrinsic pathway in the induction of apoptosis. The role of the mitochondria-dependent apoptotic pathway was further proved by the significant activation of the initiator caspase-9 and the executioner caspases-3 and -7. In addition, the activation of caspase-8, which is associated with the suppression of NF-κB translocation to the nucleus, also revealed the involvement of the extrinsic pathway in the induced apoptosis. The results suggest that the CdCl2(C14H21N3O2) complex is able to induce the apoptosis of colon cancer cells and is a potential candidate for future cancer studies.

A curcumin-based TPA four-branched copper(II) complex probe for in vivo early tumor detection

Energy Technology Data Exchange (ETDEWEB)

Pi, Zongxin [Department of Chemical and Chemical Engineering, Hefei Normal University, Hefei 230001 (China); Wang, Jiafeng; Jiang, Bo [Department of Pharmacy, Anhui University of Chinese Medicine, Hefei 230038 (China); Cheng, Gang [Department of Chemical and Chemical Engineering, Hefei Normal University, Hefei 230001 (China); Zhou, Shuangsheng, E-mail: zshuangsheng@126.com [Department of Pharmacy, Anhui University of Chinese Medicine, Hefei 230038 (China); Center of Modern Experimental Technology, Anhui University, Hefei 230038 (China)

2015-01-01

A multibranched Cu(II) complex CuL{sub 2} curcumin-based was synthesized and characterized by single-crystal X-ray diffraction analysis. The photophysical properties of the complex have been investigated both experimentally and theoretically. The results show that the target complex exhibits higher quantum yield and larger two-photon absorption (TPA) cross-section in the near infrared (NIR) region compared with its free ligand. The cell imaging studies in vitro and in vivo reveal that the complex shows good photostability and excellent tumor targeting capability to tested cancerous cells, which can be potentially used for early tumor detection. - Graphical abstract: A multibranched Cu(II) complex was prepared from curcumin. The photophysical properties of the obtained complex have been investigated. The results exhibit that the complex has high capability to test cancerous cells and can distinguish between the cancerous and noncancerous cells, which should be potentially used for early tumor detection. - Highlights: • A novel multi-branched copper complex was synthesized. • The obtained compounds exhibited obvious TPA in high polar solvents. • The complex is a low toxicity at low-micromolar concentrations. • The complex exhibits larger TPA cross-section and brighter TPF imaging. • The complex has excellent targeting capability to tested cancerous cells.

3D complex: a structural classification of protein complexes.

Directory of Open Access Journals (Sweden)

Emmanuel D Levy

2006-11-01

Full Text Available Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes.

Genomic binding profiles of functionally distinct RNA polymerase III transcription complexes in human cells.

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Moqtaderi, Zarmik; Wang, Jie; Raha, Debasish; White, Robert J; Snyder, Michael; Weng, Zhiping; Struhl, Kevin

2010-05-01

Genome-wide occupancy profiles of five components of the RNA polymerase III (Pol III) machinery in human cells identified the expected tRNA and noncoding RNA targets and revealed many additional Pol III-associated loci, mostly near short interspersed elements (SINEs). Several genes are targets of an alternative transcription factor IIIB (TFIIIB) containing Brf2 instead of Brf1 and have extremely low levels of TFIIIC. Strikingly, expressed Pol III genes, unlike nonexpressed Pol III genes, are situated in regions with a pattern of histone modifications associated with functional Pol II promoters. TFIIIC alone associates with numerous ETC loci, via the B box or a novel motif. ETCs are often near CTCF binding sites, suggesting a potential role in chromosome organization. Our results suggest that human Pol III complexes associate preferentially with regions near functional Pol II promoters and that TFIIIC-mediated recruitment of TFIIIB is regulated in a locus-specific manner.

A heteromeric molecular complex regulates the migration of lung alveolar epithelial cells during wound healing.

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Ghosh, Manik C; Makena, Patrudu S; Kennedy, Joseph; Teng, Bin; Luellen, Charlean; Sinclair, Scott E; Waters, Christopher M

2017-05-19

Alveolar type II epithelial cells (ATII) are instrumental in early wound healing in response to lung injury, restoring epithelial integrity through spreading and migration. We previously reported in separate studies that focal adhesion kinase-1 (FAK) and the chemokine receptor CXCR4 promote epithelial repair mechanisms. However, potential interactions between these two pathways were not previously considered. In the present study, we found that wounding of rat ATII cells promoted increased association between FAK and CXCR4. In addition, protein phosphatase-5 (PP5) increased its association with this heteromeric complex, while apoptosis signal regulating kinase-1 (ASK1) dissociated from the complex. Cell migration following wounding was decreased when PP5 expression was decreased using shRNA, but migration was increased in ATII cells isolated from ASK1 knockout mice. Interactions between FAK and CXCR4 were increased upon depletion of ASK1 using shRNA in MLE-12 cells, but unaffected when PP5 was depleted. Furthermore, we found that wounded rat ATII cells exhibited decreased ASK1 phosphorylation at Serine-966, decreased serine phosphorylation of FAK, and decreased association of phosphorylated ASK1 with FAK. These changes in phosphorylation were dependent upon expression of PP5. These results demonstrate a unique molecular complex comprising CXCR4, FAK, ASK1, and PP5 in ATII cells during wound healing.

Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types.

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Kilian, A; Bowtell, D D; Abud, H E; Hime, G R; Venter, D J; Keese, P K; Duncan, E L; Reddel, R R; Jefferson, R A

1997-11-01

Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

Single-particle fusion of influenza viruses reveals complex interactions with target membranes

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van der Borg, Guus; Braddock, Scarlett; Blijleven, Jelle S.; van Oijen, Antoine M.; Roos, Wouter H.

2018-05-01

The first step in infection of influenza A virus is contact with the host cell membrane, with which it later fuses. The composition of the target bilayer exerts a complex influence on both fusion efficiency and time. Here, an in vitro, single-particle approach is used to study this effect. Using total internal reflection fluorescence (TIRF) microscopy and a microfluidic flow cell, the hemifusion of single virions is visualized. Hemifusion efficiency and kinetics are studied while altering target bilayer cholesterol content and sialic-acid donor. Cholesterol ratios tested were 0%, 10%, 20%, and 40%. Sialic-acid donors GD1a and GYPA were used. Both cholesterol ratio and sialic-acid donors proved to have a significant effect on hemifusion efficiency. Furthermore, comparison between GD1a and GYPA conditions shows that the cholesterol dependence of the hemifusion time is severely affected by the sialic-acid donor. Only GD1a shows a clear increasing trend in hemifusion efficiency and time with increasing cholesterol concentration of the target bilayer with maximum rates for GD1A and 40% cholesterol. Overall our results show that sialic acid donor and target bilayer composition should be carefully chosen, depending on the desired hemifusion time and efficiency in the experiment.

The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

International Nuclear Information System (INIS)

Loublier, Sandrine; Bayot, Aurelien; Rak, Malgorzata; El-Khoury, Riyad; Benit, Paule; Rustin, Pierre

2011-01-01

Highlights: → NDUFB6 is required for activity of mitochondrial complex I in human cell lines. → Lentivirus based RNA interference results in frequent off target insertions. → Flp-In recombinase mediated miRNA insertion allows gene-specific extinction. -- Abstract: Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.

The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

Energy Technology Data Exchange (ETDEWEB)

Loublier, Sandrine; Bayot, Aurelien; Rak, Malgorzata; El-Khoury, Riyad; Benit, Paule [Inserm U676, Hopital Robert Debre, F-75019 Paris (France); Universite Paris 7, Faculte de medecine Denis Diderot, IFR02 Paris (France); Rustin, Pierre, E-mail: pierre.rustin@inserm.fr [Inserm U676, Hopital Robert Debre, F-75019 Paris (France); Universite Paris 7, Faculte de medecine Denis Diderot, IFR02 Paris (France)

2011-10-22

Highlights: {yields} NDUFB6 is required for activity of mitochondrial complex I in human cell lines. {yields} Lentivirus based RNA interference results in frequent off target insertions. {yields} Flp-In recombinase mediated miRNA insertion allows gene-specific extinction. -- Abstract: Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.

COMBINED DELAY AND GRAPH EMBEDDING OF EPILEPTIC DISCHARGES IN EEG REVEALS COMPLEX AND RECURRENT NONLINEAR DYNAMICS.

Science.gov (United States)

Erem, B; Hyde, D E; Peters, J M; Duffy, F H; Brooks, D H; Warfield, S K

2015-04-01

The dynamical structure of the brain's electrical signals contains valuable information about its physiology. Here we combine techniques for nonlinear dynamical analysis and manifold identification to reveal complex and recurrent dynamics in interictal epileptiform discharges (IEDs). Our results suggest that recurrent IEDs exhibit some consistent dynamics, which may only last briefly, and so individual IED dynamics may need to be considered in order to understand their genesis. This could potentially serve to constrain the dynamics of the inverse source localization problem.

Basal cell carcinoma of the nipple-areola complex: a case report.

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Huang, Ching-Wen; Pan, Ching-Kuen; Shih, Teng-Fu; Tsai, Cheng-Chien; Juan, Chung-Chou; Ker, Chen-Guo

2005-10-01

Basal cell carcinoma (BCC) of the nipple-areola complex is very rare. Only 24 cases were reported in the literature and 17 (70.8%) of these cases arose in men. Most of the cases were treated with simple excision. We report on a case of BCC of the nipple-areola complex in a 46-year-old woman, treated with partial mastectomy. Metastasis to the axillary lymph nodes had been noted in 3 (12.5%) of the 24 reported cases of BCC of the nipple-areola complex. Thus, we applied the concept of the sentinel lymph node to detect possible metastases of axillary lymph nodes, letting us avoid the unnecessary axillary lymph node dissection and possible future morbidity.

Complex dynamics in the development of the first tarsal segment of Drosophila melanogaster

Directory of Open Access Journals (Sweden)

Juan Nicolas Malagon

2016-09-01

Full Text Available Gene, protein and cell interactions are vital for the development of a multicellular organism. As a result, complexity theory can be a fundamental tool to understand how diverse developmental and evolutionary processes occur. However, in most scientific programs these two fields are separated. In an effort to create a connection between the Evo-devo and complexity science, this article shows how the cell dynamics of epithelia can display behaviours with similar features to complex systems. Here, I propose that these cell dynamics, in addition to control cell density in epithelia, can provide high evolvability to this type of tissue. To achieve this goal, I used a as a systems the development of Drosophila melanogaster front legs. First, I provide an example in which order at the tissue level emerge from apparently random cell dynamics. Then, I show that small modifications in epithelial cellular components can produce highly organized or the opposite random cell dynamics. Therefore, this work shows that a developing epithelium displays signs of complex behaviours and I propose that the feedback between tension and cellular processes are key for understanding how multicellular organisms development and evolve. Such studies may reveal the mechanistic basis of complex processes that bridge several levels of organization.

Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria.

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Domínguez-Escobar, Julia; Chastanet, Arnaud; Crevenna, Alvaro H; Fromion, Vincent; Wedlich-Söldner, Roland; Carballido-López, Rut

2011-07-08

The peptidoglycan cell wall and the actin-like MreB cytoskeleton are major determinants of cell shape in rod-shaped bacteria. The prevailing model postulates that helical, membrane-associated MreB filaments organize elongation-specific peptidoglycan-synthesizing complexes along sidewalls. We used total internal reflection fluorescence microscopy to visualize the dynamic relation between MreB isoforms and cell wall synthesis in live Bacillus subtilis cells. During exponential growth, MreB proteins did not form helical structures. Instead, together with other morphogenetic factors, they assembled into discrete patches that moved processively along peripheral tracks perpendicular to the cell axis. Patch motility was largely powered by cell wall synthesis, and MreB polymers restricted diffusion of patch components in the membrane and oriented patch motion.

A new synthesis route for Os-complex modified redox polymers for potential biofuel cell applications.

Science.gov (United States)

Pöller, Sascha; Beyl, Yvonne; Vivekananthan, Jeevanthi; Guschin, Dmitrii A; Schuhmann, Wolfgang

2012-10-01

A new synthesis route for Os-complex modified redox polymers was developed. Instead of ligand exchange reactions for coordinative binding of suitable precursor Os-complexes at the polymer, Os-complexes already exhibiting the final ligand shell containing a suitable functional group were bound to the polymer via an epoxide opening reaction. By separation of the polymer synthesis from the ligand exchange reaction at the Os-complex, the modification of the same polymer backbone with different Os-complexes or the binding of the same Os-complex to a number of different polymer backbones becomes feasible. In addition, the Os-complex can be purified and characterized prior to its binding to the polymer. In order to further understand and optimize suitable enzyme/redox polymer systems concerning their potential application in biosensors or biofuel cells, a series of redox polymers was synthesized and used as immobilization matrix for Trametes hirsuta laccase. The properties of the obtained biofuel cell cathodes were compared with similar biocatalytic interfaces derived from redox polymers obtained via ligand exchange reaction of the parent Os-complex with a ligand integrated into the polymer backbone during the polymer synthesis. Copyright © 2011 Elsevier B.V. All rights reserved.

Age-dependent transition from cell-level to population-level control in murine intestinal homeostasis revealed by coalescence analysis.

Directory of Open Access Journals (Sweden)

Zheng Hu

Full Text Available In multi-cellular organisms, tissue homeostasis is maintained by an exquisite balance between stem cell proliferation and differentiation. This equilibrium can be achieved either at the single cell level (a.k.a. cell asymmetry, where stem cells follow strict asymmetric divisions, or the population level (a.k.a. population asymmetry, where gains and losses in individual stem cell lineages are randomly distributed, but the net effect is homeostasis. In the mature mouse intestinal crypt, previous evidence has revealed a pattern of population asymmetry through predominantly symmetric divisions of stem cells. In this work, using population genetic theory together with previously published crypt single-cell data obtained at different mouse life stages, we reveal a strikingly dynamic pattern of stem cell homeostatic control. We find that single-cell asymmetric divisions are gradually replaced by stochastic population-level asymmetry as the mouse matures to adulthood. This lifelong process has important developmental and evolutionary implications in understanding how adult tissues maintain their homeostasis integrating the trade-off between intrinsic and extrinsic regulations.

Trans- and cis-2-phenylindole platinum(II) complexes as cytotoxic agents against human breast adenocarcinoma cell lines

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Tomé, Maria; López, Concepción; González, Asensio; Ozay, Bahadir; Quirante, Josefina; Font-Bardía, Mercè; Calvet, Teresa; Calvis, Carme; Messeguer, Ramon; Baldomá, Laura; Badía, Josefa

2013-09-01

The synthesis and characterization of the new 2-phenylindole derivative: C8H3N-2-C6H5-3NOMe-5OMe (3c) and the trans- and cis-isomers of [Pt(3c)Cl2(DMSO)] complexes (4c and 5c, respectively) are described. The crystal structures of 4c·CH2Cl2 and 5c confirm: (a) the existence of a Pt-Nindole bond, (b) the relative arrangement of the Cl- ligands [trans- (in 4c) or cis- (in 5c)] and (c) the anti-(E) configuration of the oxime. The cytotoxic assessment of C8H3N-2-(C6H4-4‧R1)-3NOMe-5R2 [with R1 = R2 = H (3a); R1 = Cl, R2 = H (3b) and R1 = H, R2 = OMe (3c)] and the geometrical isomers of [Pt(L)Cl2(DMSO)] with L = 3a-3c [trans- (4a-4c) and cis- (5a-5c), respectively] against human breast adenocarcinoma cell lines (MDA-MB231 and MCF-7) is also reported and reveals that all the platinum(II) complexes (except 4a) are more cytotoxic than cisplatin in front of the MCF7 cell line. Electrophoretic DNA migration studies of the synthesized compounds in the absence and in the presence of topoisomerase-I have been performed, in order to get further insights into their mechanism of action.

A bioaccumulative cyclometalated platinum(II) complex with two-photon-induced emission for live cell imaging.

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Koo, Chi-Kin; Wong, Ka-Leung; Man, Cornelia Wing-Yin; Lam, Yun-Wah; So, Leo King-Yan; Tam, Hoi-Lam; Tsao, Sai-Wah; Cheah, Kok-Wai; Lau, Kai-Chung; Yang, Yang-Yi; Chen, Jin-Can; Lam, Michael Hon-Wah

2009-02-02

The cyclometalated platinum(II) complex [Pt(L)Cl], where HL is a new cyclometalating ligand 2-phenyl-6-(1H-pyrazol-3-yl)pyridine containing C(phenyl), N(pyridyl), and N(pyrazolyl) donor moieties, was found to possess two-photon-induced luminescent properties. The two-photon-absorption cross section of the complex in N,N-dimethylformamide at room temperature was measured to be 20.8 GM. Upon two-photon excitation at 730 nm from a Ti:sapphire laser, bright-green emission was observed. Besides its two-photon-induced luminescent properties, [Pt(L)Cl] was able to be rapidly accumulated in live HeLa and NIH3T3 cells. The two-photon-induced luminescence of the complex was retained after live cell internalization and can be observed by two-photon confocal microscopy. Its bioaccumulation properties enabled time-lapse imaging of the internalization process of the dye into living cells. Cytotoxicity of [Pt(L)Cl] to both tested cell lines was low, according to MTT assays, even at loadings as high as 20 times the dose concentration for imaging for 6 h.

Poly(amido)amine (PAMAM) dendrimer-cisplatin complexes for chemotherapy of cisplatin-resistant ovarian cancer cells

Science.gov (United States)

Yellepeddi, Venkata Kashyap; Vangara, Kiran Kumar; Palakurthi, Srinath

2013-09-01

Dendrimer-cisplatin complexes were prepared using PAMAM dendrimers with terminal -NH2 and -COOH groups as well as biotin-conjugated dendrimers. Preformulation parameters of dendrimer-cisplatin complexes were studied using differential scanning calorimetry (DSC) and inductively coupled plasma-mass spectrometry (ICP-MS). Cytotoxicity and mechanism of cytotoxicity of dendrimer-cisplatin complexes was investigated in OVCAR-3, SKOV, A2780 and cisplatin-resistant CP70 human ovarian cancer cell lines. The loading of cisplatin in dendrimers was 11 % (w/w). PAMAM G4 dendrimers with amine surface groups (biotinylated and native) have shown 2.5- to 3.0-fold reduction in IC50 values in ovarian cancer cells when compared with carboxylate surface dendrimers ( p cisplatin complexes resulted in a 7.0-fold increase ( p cisplatin chemotherapy of ovarian cancer.

Global terrestrial water storage connectivity revealed using complex climate network analyses

Science.gov (United States)

Sun, A. Y.; Chen, J.; Donges, J.

2015-07-01

Terrestrial water storage (TWS) exerts a key control in global water, energy, and biogeochemical cycles. Although certain causal relationship exists between precipitation and TWS, the latter quantity also reflects impacts of anthropogenic activities. Thus, quantification of the spatial patterns of TWS will not only help to understand feedbacks between climate dynamics and the hydrologic cycle, but also provide new insights and model calibration constraints for improving the current land surface models. This work is the first attempt to quantify the spatial connectivity of TWS using the complex network theory, which has received broad attention in the climate modeling community in recent years. Complex networks of TWS anomalies are built using two global TWS data sets, a remote sensing product that is obtained from the Gravity Recovery and Climate Experiment (GRACE) satellite mission, and a model-generated data set from the global land data assimilation system's NOAH model (GLDAS-NOAH). Both data sets have 1° × 1° grid resolutions and cover most global land areas except for permafrost regions. TWS networks are built by first quantifying pairwise correlation among all valid TWS anomaly time series, and then applying a cutoff threshold derived from the edge-density function to retain only the most important features in the network. Basinwise network connectivity maps are used to illuminate connectivity of individual river basins with other regions. The constructed network degree centrality maps show the TWS anomaly hotspots around the globe and the patterns are consistent with recent GRACE studies. Parallel analyses of networks constructed using the two data sets reveal that the GLDAS-NOAH model captures many of the spatial patterns shown by GRACE, although significant discrepancies exist in some regions. Thus, our results provide further measures for constraining the current land surface models, especially in data sparse regions.

Basal cell carcinoma of the nipple-areola complex.

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Ferguson, Mark S; Nouraei, S A Reza; Davies, Ben J H; McLean, N R

2009-11-01

Basal cell carcinoma (BCC) of the nipple-areola complex is uncommon. It has been suggested that BCCs in this region behave more aggressively, with a higher potential for distant spread, than in other anatomical sites. To address questions about etiology, behavior, optimal treatment, and prognosis of this entity. A literature search identifying all cases of BCC of the nipple and nipple-areola complex in the English literature from 1893 to 2008. Thirty-four cases of BCC of the nipple, areola, or both were identified, mostly affecting middle-aged men. The majority of patients were treated with tissue-sparing surgery. There was a metastatic rate of 9.1%, and one patient died from the disease (3.0%). The optimal treatment of this condition should be local excision, but patients with this condition should be followed up for primary site recurrence and axillary metastasis, because there is greater incidence than with BCC at other anatomical sites. Furthermore, proven axillary metastasis should be surgically treated.

Cell-specific STORM superresolution imaging reveals nanoscale organization of cannabinoid signaling

Science.gov (United States)

Szabó, Szilárd I.; Szabadits, Eszter; Pintér, Balázs; Woodhams, Stephen G.; Henstridge, Christopher M.; Balla, Gyula Y.; Nyilas, Rita; Varga, Csaba; Lee, Sang-Hun; Matolcsi, Máté; Cervenak, Judit; Kacskovics, Imre; Watanabe, Masahiko; Sagheddu, Claudia; Melis, Miriam; Pistis, Marco; Soltesz, Ivan; Katona, István

2014-01-01

A major challenge in neuroscience is to determine the nanoscale position and quantity of signaling molecules in a cell-type-, and subcellular compartment-specific manner. We therefore developed a novel approach combining cell-specific physiological and anatomical characterization with superresolution imaging, and studied the molecular and structural parameters shaping the physiological properties of synaptic endocannabinoid signaling in the mouse hippocampus. We found that axon terminals of perisomatically-projecting GABAergic interneurons possess increased CB1 receptor number, active-zone complexity, and receptor/effector ratio compared to dendritically-projecting interneurons, in agreement with higher efficiency of cannabinoid signaling at somatic versus dendritic synapses. Furthermore, chronic Δ9-tetrahydrocannabinol administration, which reduces cannabinoid efficacy on GABA release, evoked dramatic CB1-downregulation in a dose-dependent manner. Full receptor recovery required several weeks after cessation of Δ9-tetrahydrocannabinol treatment. These findings demonstrate that cell-type-specific nanoscale analysis of endogenous protein distribution is possible in brain circuits, and identify novel molecular properties controlling endocannabinoid signaling and cannabis-induced cognitive dysfunction. PMID:25485758

Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

OpenAIRE

Herranz, M. Carmen; Sánchez Navarro, Jesús A.; Saurí Peris, Ana; Mingarro Muñoz, Ismael; Pallás Benet, Vicente

2005-01-01

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive c...

Octamer-binding protein 4 affects the cell biology and phenotypic transition of lung cancer cells involving β-catenin/E-cadherin complex degradation.

Science.gov (United States)

Chen, Zhong-Shu; Ling, Dong-Jin; Zhang, Yang-De; Feng, Jian-Xiong; Zhang, Xue-Yu; Shi, Tian-Sheng

2015-03-01

Clinical studies have reported evidence for the involvement of octamer‑binding protein 4 (Oct4) in the tumorigenicity and progression of lung cancer; however, the role of Oct4 in lung cancer cell biology in vitro and its mechanism of action remain to be elucidated. Mortality among lung cancer patients is more frequently due to metastasis rather than their primary tumors. Epithelial‑mesenchymal transition (EMT) is a prominent biological event for the induction of epithelial cancer metastasis. The aim of the present study was to investigate whether Oct4 had the capacity to induce lung cancer cell metastasis via the promoting the EMT in vitro. Moreover, the effect of Oct4 on the β‑catenin/E‑cadherin complex, associated with EMT, was examined using immunofluorescence and immunoprecipitation assays as well as western blot analysis. The results demonstrated that Oct4 enhanced cell invasion and adhesion accompanied by the downregulation of epithelial marker cytokeratin, and upregulation of the mesenchymal markers vimentin and N‑cadherin. Furthermore, Oct4 induced EMT of lung cancer cells by promoting β‑catenin/E‑cadherin complex degradation and regulating nuclear localization of β‑catenin. In conclusion, the present study indicated that Oct4 affected the cell biology of lung cancer cells in vitro through promoting lung cancer cell metastasis via EMT; in addition, the results suggested that the association and degradation of the β‑catenin/E‑cadherin complex was regulated by Oct4 during the process of EMT.

Mycobacterium tuberculosis complex enhances susceptibility of CD4 T cells to HIV through a TLR2-mediated pathway.

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Seema M Thayil

Full Text Available Among HIV-infected individuals, co-infection with Mycobacterium tuberculosis is associated with faster progression to AIDS. We investigated the hypothesis that M. bovis BCG and M. tuberculosis (Mtb complex could enhance susceptibility of CD4+ cells to HIV infection. Peripheral blood mononuclear cells (PBMCs collected from healthy donors were stimulated with M. bovis BCG, M. tuberculosis CDC1551 and M. smegmatis MC(2155, and stimulated CD4+ cells were infected with R5-and X4-tropic single replication-competent pseudovirus. CD4+ cells stimulated with Mtb complex showed enhanced infection with R5- and X4-tropic HIV, compared to unstimulated cells or cells stimulated with M. smegmatis (p<0.01. Treatment with TLR2 siRNA reversed the increased susceptibility of CD4+ cells with R5- and X4-tropic virus induced by Mtb complex. These findings suggest that TB infection and/or BCG vaccination may be a risk factor for HIV acquisition.

New iridium complex as additive to the spiro-OMeTAD in perovskite solar cells with enhanced stability

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Laura Badia

2014-08-01

Full Text Available A new iridium complex, IrCp*Cl(PyPyz[TFSI], has been synthesized and used as additive for the hole transporter material, spiro-OMeTAD, in perovskite solar cells. The cells prepared with this Ir additive present higher efficiency than reference cells, and similar to cells prepared with Co additive. We have determined that the presence of metal complexes as additives decreases the recombination rate, as it has been observed by impedance spectroscopy. Very interestingly, while the efficiency after 3 months decreases by 22% and 70% for reference cell and cell with Co additive, respectively, the efficiency of devices containing the Ir additive is only decreased by a 4%.

Universal lab-on-a-chip platform for complex, perfused 3D cell cultures

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Sonntag, F.; Schmieder, F.; Ströbel, J.; Grünzner, S.; Busek, M.; Günther, K.; Steege, T.; Polk, C.; Klotzbach, U.

2016-03-01

The miniaturization, rapid prototyping and automation of lab-on-a-chip technology play nowadays a very important role. Lab-on-a-chip technology is successfully implemented not only for environmental analysis and medical diagnostics, but also as replacement of animals used for the testing of substances in the pharmaceutical and cosmetics industries. For that purpose the Fraunhofer IWS and partners developed a lab-on-a-chip platform for perfused cell-based assays in the last years, which includes different micropumps, valves, channels, reservoirs and customized cell culture modules. This technology is already implemented for the characterization of different human cell cultures and organoids, like skin, liver, endothelium, hair follicle and nephron. The advanced universal lab-on-a-chip platform for complex, perfused 3D cell cultures is divided into a multilayer basic chip with integrated micropump and application-specific 3D printed cell culture modules. Moreover a technology for surface modification of the printed cell culture modules by laser micro structuring and a complex and flexibly programmable controlling device based on an embedded Linux system was developed. A universal lab-on-a-chip platform with an optional oxygenator and a cell culture module for cubic scaffolds as well as first cell culture experiments within the cell culture device will be presented. The module is designed for direct interaction with robotic dispenser systems. This offers the opportunity to combine direct organ printing of cells and scaffolds with the microfluidic cell culture module. The characterization of the developed system was done by means of Micro-Particle Image Velocimetry (μPIV) and an optical oxygen measuring system.

Werner complex deficiency in cells disrupts the Nuclear Pore Complex and the distribution of lamin B1.

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Li, Zhi; Zhu, Yizhou; Zhai, Yujia; R Castroagudin, Michelle; Bao, Yifei; White, Tommy E; Glavy, Joseph S

2013-12-01

From the surrounding shell to the inner machinery, nuclear proteins provide the functional plasticity of the nucleus. This study highlights the nuclear association of Pore membrane (POM) protein NDC1 and Werner protein (WRN), a RecQ helicase responsible for the DNA instability progeria disorder, Werner Syndrome. In our previous publication, we connected the DNA damage sensor Werner's Helicase Interacting Protein (WHIP), a binding partner of WRN, to the NPC. Here, we confirm the association of the WRN/WHIP complex and NDC1. In established WRN/WHIP knockout cell lines, we further demonstrate the interdependence of WRN/WHIP and Nucleoporins (Nups). These changes do not completely abrogate the barrier of the Nuclear Envelope (NE) but do affect the distribution of FG Nups and the RAN gradient, which are necessary for nuclear transport. Evidence from WRN/WHIP knockout cell lines demonstrates changes in the processing and nucleolar localization of lamin B1. The appearance of "RAN holes" void of RAN corresponds to regions within the nucleolus filled with condensed pools of lamin B1. From WRN/WHIP knockout cell line extracts, we found three forms of lamin B1 that correspond to mature holoprotein and two potential post-translationally modified forms of the protein. Upon treatment with topoisomerase inhibitors lamin B1 cleavage occurs only in WRN/WHIP knockout cells. Our data suggest the link of the NDC1 and WRN as one facet of the network between the nuclear periphery and genome stability. Loss of WRN complex leads to multiple alterations at the NPC and the nucleolus. © 2013. Published by Elsevier B.V. All rights reserved.

Structural Studies of Complex Carbohydrates of Plant Cell Walls

Energy Technology Data Exchange (ETDEWEB)

Darvill, Alan [Univ. of Georgia, Athens, GA (United States); Hahn, Michael G. [Univ. of Georgia, Athens, GA (United States); O' Neill, Malcolm A. [Univ. of Georgia, Athens, GA (United States); York, William S. [Univ. of Georgia, Athens, GA (United States)

2015-02-17

Most of the solar energy captured by land plants is converted into the polysaccharides (cellulose, hemicellulose, and pectin) that are the predominant components of the cell wall. These walls, which account for the bulk of plant biomass, have numerous roles in the growth and development of plants. Moreover, these walls have a major impact on human life as they are a renewable source of biomass, a source of diverse commercially useful polymers, a major component of wood, and a source of nutrition for humans and livestock. Thus, understanding the molecular mechanisms that lead to wall assembly and how cell walls and their component polysaccharides contribute to plant growth and development is essential to improve and extend the productivity and value of plant materials. The proposed research will develop and apply advanced analytical and immunological techniques to study specific changes in the structures and interactions of the hemicellulosic and pectic polysaccharides that occur during differentiation and in response to genetic modification and chemical treatments that affect wall biosynthesis. These new techniques will make it possible to accurately characterize minute amounts of cell wall polysaccharides so that subtle changes in structure that occur in individual cell types can be identified and correlated to the physiological or developmental state of the plant. Successful implementation of this research will reveal fundamental relationships between polysaccharide structure, cell wall architecture, and cell wall functions.

Cell Wall Remodeling by a Synthetic Analog Reveals Metabolic Adaptation in Vancomycin Resistant Enterococci.

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Pidgeon, Sean E; Pires, Marcos M

2017-07-21

Drug-resistant bacterial infections threaten to overburden our healthcare system and disrupt modern medicine. A large class of potent antibiotics, including vancomycin, operate by interfering with bacterial cell wall biosynthesis. Vancomycin-resistant enterococci (VRE) evade the blockage of cell wall biosynthesis by altering cell wall precursors, rendering them drug insensitive. Herein, we reveal the phenotypic plasticity and cell wall remodeling of VRE in response to vancomycin in live bacterial cells via a metabolic probe. A synthetic cell wall analog was designed and constructed to monitor cell wall structural alterations. Our results demonstrate that the biosynthetic pathway for vancomycin-resistant precursors can be hijacked by synthetic analogs to track the kinetics of phenotype induction. In addition, we leveraged this probe to interrogate the response of VRE cells to vancomycin analogs and a series of cell wall-targeted antibiotics. Finally, we describe a proof-of-principle strategy to visually inspect drug resistance induction. Based on our findings, we anticipate that our metabolic probe will play an important role in further elucidating the interplay among the enzymes involved in the VRE biosynthetic rewiring.

Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

International Nuclear Information System (INIS)

Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.

2005-01-01

The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH 2 -terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

Heterogeneity of Purkinje cell simple spike-complex spike interactions: zebrin- and non-zebrin-related variations.

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Tang, Tianyu; Xiao, Jianqiang; Suh, Colleen Y; Burroughs, Amelia; Cerminara, Nadia L; Jia, Linjia; Marshall, Sarah P; Wise, Andrew K; Apps, Richard; Sugihara, Izumi; Lang, Eric J

2017-08-01

Cerebellar Purkinje cells (PCs) generate two types of action potentials, simple and complex spikes. Although they are generated by distinct mechanisms, interactions between the two spike types exist. Zebrin staining produces alternating positive and negative stripes of PCs across most of the cerebellar cortex. Thus, here we compared simple spike-complex spike interactions both within and across zebrin populations. Simple spike activity undergoes a complex modulation preceding and following a complex spike. The amplitudes of the pre- and post-complex spike modulation phases were correlated across PCs. On average, the modulation was larger for PCs in zebrin positive regions. Correlations between aspects of the complex spike waveform and simple spike activity were found, some of which varied between zebrin positive and negative PCs. The implications of the results are discussed with regard to hypotheses that complex spikes are triggered by rises in simple spike activity for either motor learning or homeostatic functions. Purkinje cells (PCs) generate two types of action potentials, called simple and complex spikes (SSs and CSs). We first investigated the CS-associated modulation of SS activity and its relationship to the zebrin status of the PC. The modulation pattern consisted of a pre-CS rise in SS activity, and then, following the CS, a pause, a rebound, and finally a late inhibition of SS activity for both zebrin positive (Z+) and negative (Z-) cells, though the amplitudes of the phases were larger in Z+ cells. Moreover, the amplitudes of the pre-CS rise with the late inhibitory phase of the modulation were correlated across PCs. In contrast, correlations between modulation phases across CSs of individual PCs were generally weak. Next, the relationship between CS spikelets and SS activity was investigated. The number of spikelets/CS correlated with the average SS firing rate only for Z+ cells. In contrast, correlations across CSs between spikelet numbers and the

Principles of assembly reveal a periodic table of protein complexes.

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Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

2015-12-11

Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. Copyright © 2015, American Association for the Advancement of Science.

Structures of the APC–ARM domain in complexes with discrete Amer1/WTX fragments reveal that it uses a consensus mode to recognize its binding partners

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Zhang, Zhenyi; Akyildiz, Senem; Xiao, Yafei; Gai, Zhongchao; An, Ying; Behrens, Jürgen; Wu, Geng

2015-01-01

The tumor suppressor APC employs its conserved armadillo repeat (ARM) domain to recognize many of its binding partners, including Amer1/WTX, which is mutated in Wilms' tumor and bone overgrowth syndrome. The APC–Amer1 complex has important roles in regulating Wnt signaling and cell adhesion. Three sites A1, A2, and A3 of Amer1 have been reported to mediate its interaction with APC-ARM. In this study, crystal structures of APC–ARM in complexes with Amer1-A1, -A2, and -A4, which is newly identified in this work, were determined. Combined with our GST pull-down, yeast two-hybrid, and isothermal titration calorimetry (ITC) assay results using mutants of APC and Amer1 interface residues, our structures demonstrate that Amer1-A1, -A2, and -A4, as well as other APC-binding proteins such as Asef and Sam68, all employ a common recognition pattern to associate with APC–ARM. In contrast, Amer1-A3 binds to the C-terminal side of APC–ARM through a bipartite interaction mode. Composite mutations on either APC or Amer1 disrupting all four interfaces abrogated their association in cultured cells and impaired the membrane recruitment of APC by Amer1. Our study thus comprehensively elucidated the recognition mechanism between APC and Amer1, and revealed a consensus recognition sequence employed by various APC–ARM binding partners. PMID:27462415

Structures of the APC-ARM domain in complexes with discrete Amer1/WTX fragments reveal that it uses a consensus mode to recognize its binding partners.

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Zhang, Zhenyi; Akyildiz, Senem; Xiao, Yafei; Gai, Zhongchao; An, Ying; Behrens, Jürgen; Wu, Geng

2015-01-01

The tumor suppressor APC employs its conserved armadillo repeat (ARM) domain to recognize many of its binding partners, including Amer1/WTX, which is mutated in Wilms' tumor and bone overgrowth syndrome. The APC-Amer1 complex has important roles in regulating Wnt signaling and cell adhesion. Three sites A1, A2, and A3 of Amer1 have been reported to mediate its interaction with APC-ARM. In this study, crystal structures of APC-ARM in complexes with Amer1-A1, -A2, and -A4, which is newly identified in this work, were determined. Combined with our GST pull-down, yeast two-hybrid, and isothermal titration calorimetry (ITC) assay results using mutants of APC and Amer1 interface residues, our structures demonstrate that Amer1-A1, -A2, and -A4, as well as other APC-binding proteins such as Asef and Sam68, all employ a common recognition pattern to associate with APC-ARM. In contrast, Amer1-A3 binds to the C-terminal side of APC-ARM through a bipartite interaction mode. Composite mutations on either APC or Amer1 disrupting all four interfaces abrogated their association in cultured cells and impaired the membrane recruitment of APC by Amer1. Our study thus comprehensively elucidated the recognition mechanism between APC and Amer1, and revealed a consensus recognition sequence employed by various APC-ARM binding partners.

Microspectroscopic Study of Liposome-to-cell Interaction Revealed by Förster Resonance Energy Transfer.

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Yefimova, Svetlana L; Kurilchenko, Irina Yu; Tkacheva, Tatyana N; Kavok, Nataliya S; Todor, Igor N; Lukianova, Nataliya Yu; Chekhun, Vasyl F; Malyukin, Yuriy V

2014-03-01

We report the Förster resonance energy transfer (FRET)-labeling of liposomal vesicles as an effective approach to study in dynamics the interaction of liposomes with living cells of different types (rat hepatocytes, rat bone marrow, mouse fibroblast-like cells and human breast cancer cells) and cell organelles (hepatocyte nuclei). The in vitro experiments were performed using fluorescent microspectroscopic technique. Two fluorescent dyes (DiO as the energy donor and DiI as an acceptor) were preloaded in lipid bilayers of phosphatidylcholine liposomes that ensures the necessary distance between the dyes for effective FRET. The change in time of the donor and acceptor relative fluorescence intensities was used to visualize and trace the liposome-to-cell interaction. We show that FRET-labeling of liposome vesicles allows one to reveal the differences in efficiency and dynamics of these interactions, which are associated with composition, fluidity, and metabolic activity of cell plasma membranes.

EPR spectroscopy of complex biological iron-sulfur systems.

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Hagen, Wilfred R

2018-02-21

From the very first discovery of biological iron-sulfur clusters with EPR, the spectroscopy has been used to study not only purified proteins but also complex systems such as respiratory complexes, membrane particles and, later, whole cells. In recent times, the emphasis of iron-sulfur biochemistry has moved from characterization of individual proteins to the systems biology of iron-sulfur biosynthesis, regulation, degradation, and implications for human health. Although this move would suggest a blossoming of System-EPR as a specific, non-invasive monitor of Fe/S (dys)homeostasis in whole cells, a review of the literature reveals limited success possibly due to technical difficulties in adherence to EPR spectroscopic and biochemical standards. In an attempt to boost application of System-EPR the required boundary conditions and their practical applications are explicitly and comprehensively formulated.

Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells

KAUST Repository

Joshi, Rubin N.

2017-09-25

Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4CD25 T cells (Tcons) independently of IP levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer.

Toxic effect of C60 fullerene-doxorubicin complex towards tumor and normal cells in vitro

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Prylutska S. V.

2014-09-01

Full Text Available Creation of new nanostructures possessing high antitumor activity is an important problem of modern biotechnology. Aim. To evaluate cytotoxicity of created complex of pristine C60 fullerene with the anthracycline antibiotic doxorubicin (Dox, as well as of free C60 fullerene and Dox, towards different cell types – tumor, normal immunocompetent and hepatocytes. Methods. Measurement of size distribution for particles in C60 + Dox mixture was performed by a dynamic light scattering (DLS technique. Toxic effect of C60 + Dox complex in vitro towards tumor and normal cells was studied using the MTT assay. Results. DLS experiment demonstrated that the main fraction of the particles in C60 + Dox mixture had a diameter in the range of about 132 nm. The toxic effect of C60 + Dox complex towards normal (lymphocytes, macrophages, hepatocytes and tumor (Ehrlich ascites carcinoma, leukemia L1210, Lewis lung carcinoma cells was decreased by ~10–16 % and ~7–9 %, accordingly, compared with the same effect of free Dox. Conclusions. The created C60 + Dox composite may be considered as a new pharmacological agent that kills effectively tumor cells in vitro and simultaneously prevents a toxic effect of the free form of Dox on normal cells.

Complex evolutionary patterns revealed by mitochondrial genomes of the domestic horse.

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Ning, T; Li, J; Lin, K; Xiao, H; Wylie, S; Hua, S; Li, H; Zhang, Y-P

2014-01-01

The domestic horse is the most widely used and important stock and recreational animal, valued for its strength and endurance. The energy required by the domestic horse is mainly supplied by mitochondria via oxidative phosphorylation. Thus, selection may have played an essential role in the evolution of the horse mitochondria. Besides, demographic events also affect the DNA polymorphic pattern on mitochondria. To understand the evolutionary patterns of the mitochondria of the domestic horse, we used a deep sequencing approach to obtain the complete sequences of 15 mitochondrial genomes, and four mitochondrial gene sequences, ND6, ATP8, ATP6 and CYTB, collected from 509, 363, 363 and 409 domestic horses, respectively. Evidence of strong substitution rate heterogeneity was found at nonsynonymous sites across the genomes. Signatures of recent positive selection on mtDNA of domestic horse were detected. Specifically, five amino acids in the four mitochondrial genes were identified as the targets of positive selection. Coalescentbased simulations imply that recent population expansion is the most probable explanation for the matrilineal population history for domestic horse. Our findings reveal a complex pattern of non-neutral evolution of the mitochondrial genome in the domestic horses.

Cellular responses of BRCA1-defective and triple-negative breast cancer cells and in vitro BRCA1 interactions induced by metallo-intercalator ruthenium(II) complexes containing chloro-substituted phenylazopyridine

International Nuclear Information System (INIS)

Nhukeaw, Tidarat; Temboot, Pornvichai; Hansongnern, Kanidtha; Ratanaphan, Adisorn

2014-01-01

. This study has revealed the ability of ruthenium complexes to inhibit cell proliferation, induce cell cycle progression and apoptosis. Ruthenium treatment upregulated the marker genes involved in apoptosis and cell cycle progression while it downregulated BRCA1 mRNA and replication of HCC1937 cells. Our results could provide an alternative approach to finding effective therapeutic ruthenium-based agents with promising anticancer activity, and demonstrated that the BRCA1 RING domain protein was a promising therapeutic target for breast cancers

Boudin trafficking reveals the dynamic internalisation of specific septate junction components in Drosophila.

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Tempesta, Camille; Hijazi, Assia; Moussian, Bernard; Roch, Fernando

2017-01-01

The maintenance of paracellular barriers in invertebrate epithelia depends on the integrity of specific cell adhesion structures known as septate junctions (SJ). Multiple studies in Drosophila have revealed that these junctions have a stereotyped architecture resulting from the association in the lateral membrane of a large number of components. However, little is known about the dynamic organisation adopted by these multi-protein complexes in living tissues. We have used live imaging techniques to show that the Ly6 protein Boudin is a component of these adhesion junctions and can diffuse systemically to associate with the SJ of distant cells. We also observe that this protein and the claudin Kune-kune are endocytosed in epidermal cells during embryogenesis. Our data reveal that the SJ contain a set of components exhibiting a high membrane turnover, a feature that could contribute in a tissue-specific manner to the morphogenetic plasticity of these adhesion structures.

Chromosome Mis-segregation Generates Cell-Cycle-Arrested Cells with Complex Karyotypes that Are Eliminated by the Immune System.

Science.gov (United States)

Santaguida, Stefano; Richardson, Amelia; Iyer, Divya Ramalingam; M'Saad, Ons; Zasadil, Lauren; Knouse, Kristin A; Wong, Yao Liang; Rhind, Nicholas; Desai, Arshad; Amon, Angelika

2017-06-19

Aneuploidy, a state of karyotype imbalance, is a hallmark of cancer. Changes in chromosome copy number have been proposed to drive disease by modulating the dosage of cancer driver genes and by promoting cancer genome evolution. Given the potential of cells with abnormal karyotypes to become cancerous, do pathways that limit the prevalence of such cells exist? By investigating the immediate consequences of aneuploidy on cell physiology, we identified mechanisms that eliminate aneuploid cells. We find that chromosome mis-segregation leads to further genomic instability that ultimately causes cell-cycle arrest. We further show that cells with complex karyotypes exhibit features of senescence and produce pro-inflammatory signals that promote their clearance by the immune system. We propose that cells with abnormal karyotypes generate a signal for their own elimination that may serve as a means for cancer cell immunosurveillance. Copyright © 2017 Elsevier Inc. All rights reserved.

CD4/CD8/Dendritic cell complexes in the spleen: CD8+ T cells can directly bind CD4+ T cells and modulate their response

Science.gov (United States)

Barinov, Aleksandr; Galgano, Alessia; Krenn, Gerald; Tanchot, Corinne; Vasseur, Florence

2017-01-01

CD4+ T cell help to CD8+ T cell responses requires that CD4+ and CD8+ T cells interact with the same antigen presenting dendritic cell (Ag+DC), but it remains controversial whether helper signals are delivered indirectly through a licensed DC and/or involve direct CD4+/CD8+ T cell contacts and/or the formation of ternary complexes. We here describe the first in vivo imaging of the intact spleen, aiming to evaluate the first interactions between antigen-specific CD4+, CD8+ T cells and Ag+DCs. We show that in contrast to CD4+ T cells which form transient contacts with Ag+DC, CD8+ T cells form immediate stable contacts and activate the Ag+DC, acquire fragments of the DC membranes by trogocytosis, leading to their acquisition of some of the DC properties. They express MHC class II, and become able to present the specific Marilyn peptide to naïve Marilyn CD4+ T cells, inducing their extensive division. In vivo, these CD8+ T cells form direct stable contacts with motile naïve CD4+ T cells, recruiting them to Ag+DC binding and to the formation of ternary complexes, where CD4+ and CD8+ T cells interact with the DC and with one another. The presence of CD8+ T cells during in vivo immune responses leads to the early activation and up-regulation of multiple functions by CD4+ T lymphocytes. Thus, while CD4+ T cell help is important to CD8+ T cell responses, CD8+ T cells can interact directly with naïve CD4+ T cells impacting their recruitment and differentiation. PMID:28686740

Hard wiring of T cell receptor specificity for the major histocompatibility complex is underpinned by TCR adaptability

Energy Technology Data Exchange (ETDEWEB)

Burrows, Scott R.; Chen, Zhenjun; Archbold, Julia K.; Tynan, Fleur E.; Beddoe, Travis; Kjer-Nielsen, Lars; Miles, John J.; Khanna, Rajiv; Moss, Denis J.; Liu, Yu Chih; Gras, Stephanie; Kostenko, Lyudmila; Brennan, Rebekah M.; Clements, Craig S.; Brooks, Andrew G.; Purcell, Anthony W.; McCluskey, James; Rossjohn, Jamie (Monash); (Queensland Inst. of Med. Rsrch.); (Melbourne)

2010-07-07

{alpha}{beta} T cell receptors (TCRs) are genetically restricted to corecognize peptide antigens bound to self-major histocompatibility complex (pMHC) molecules; however, the basis for this MHC specificity remains unclear. Despite the current dogma, evaluation of the TCR-pMHC-I structural database shows that the nongermline-encoded complementarity-determining region (CDR)-3 loops often contact the MHC-I, and the germline-encoded CDR1 and -2 loops frequently participate in peptide-mediated interactions. Nevertheless, different TCRs adopt a roughly conserved docking mode over the pMHC-I, in which three MHC-I residues (65, 69, and 155) are invariably contacted by the TCR in one way or another. Nonetheless, the impact of mutations at these three positions, either individually or together, was not uniformly detrimental to TCR recognition of pHLA-B*0801 or pHLA-B*3508. Moreover, when TCR-pMHC-I recognition was impaired, this could be partially restored by expression of the CD8 coreceptor. The structure of a TCR-pMHC-I complex in which these three (65, 69, and 155) MHC-I positions were all mutated resulted in shifting of the TCR footprint relative to the cognate complex and formation of compensatory interactions. Collectively, our findings reveal the inherent adaptability of the TCR in maintaining peptide recognition while accommodating changes to the central docking site on the pMHC-I.

N-cadherin in adult rat cardiomyocytes in culture. II. Spatio-temporal appearance of proteins involved in cell-cell contact and communication. Formation of two distinct N-cadherin/catenin complexes.

Science.gov (United States)

Hertig, C M; Butz, S; Koch, S; Eppenberger-Eberhardt, M; Kemler, R; Eppenberger, H M

1996-01-01

The spatio-temporal appearance and distribution of proteins forming the intercalated disc were investigated in adult rat cardiomyocytes (ARC). The 'redifferentiation model' of ARC involves extensive remodelling of the plasma membrane and of the myofibrillar apparatus. It represents a valuable system to elucidate the formation of cell-cell contact between cardiomyocytes and to assess the mechanisms by which different proteins involved in the cell-cell adhesion process are sorted in a precise manner to the sites of function. Appearance of N-cadherin, the catenins and connexin43 within newly formed adherens and gap junctions was studied. Here first evidence is provided for a formation of two distinct and separable N-cadherin/catenin complexes in cardiomyocytes. Both complexes are composed of N-cadherin and alpha-catenin which bind to either beta-catenin or plakoglobin in a mutually exclusive manner. The two N-cadherin/catenin complexes are assumed to be functionally involved in the formation of cell-cell contacts in ARC; however, the differential appearance and localization of the two types of complexes may also point to a specific role during ARC differentiation. The newly synthesized beta-catenin containing complex is more abundant during the first stages in culture after ARC isolation, while the newly synthesized plakoglobin containing complex progressively accumulates during the morphological changes of ARC. ARC formed a tissue-like pattern in culture whereby the new cell-cell contacts could be dissolved through Ca2+ depletion. Presence of cAMP and replenishment of Ca2+ content in the culture medium not only allowed reformation of cell-cell contacts but also affected the relative protein ratio between the two N-cadherin/catenin complexes, increasing the relative amount of newly synthesized beta-catenin over plakoglobin at a particular stage of ARC differentiation. The clustered N-cadherin/catenin complexes at the plasma membrane appear to be a prerequisite for the

Comparison of MukB homodimer versus MukBEF complex molecular architectures by electron microscopy reveals a higher-order multimerization

International Nuclear Information System (INIS)

Matoba, Kyoko; Yamazoe, Mitsuyoshi; Mayanagi, Kouta; Morikawa, Kosuke; Hiraga, Sota

2005-01-01

The complex of MukF, MukE, and MukB proteins participates in organization of sister chromosomes and partitioning into both daughter cells in Escherichia coli. We purified the MukB homodimer and the MukBEF complex and analyzed them by electron microscopy to compare both structures. A MukB homodimer shows a long rod-hinge-rod v-shape with small globular domains at both ends. The MukBEF complex shows a similar structure having larger globular domains than those of the MukB homodimer. These results suggest that MukF and MukE bind to the globular domains of a MukB homodimer. The globular domains of the MukBEF complex frequently associate with each other in an intramolecular fashion, forming a ring. In addition, MukBEF complex molecules tend to form multimers by the end-to-end joining with other MukBEF molecules in an intermolecular fashion, resulting in fibers and rosette-form structures in the absence of ATP and DNA in vitro

Microencapsulated cells of Lactobacillus paracasei subsp. paracasei in biopolymer complex coacervates and their function in a yogurt matrix.

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Bosnea, L A; Moschakis, T; Biliaderis, C G

2017-02-22

L. paracasei subsp. paracasei E6 cells were encapsulated by complex coacervation using whey protein isolate (WPI) and gum arabic and introduced in stirred yogurts after fermentation. For comparison purposes, yogurts without addition of L. paracasei and yogurts with free cells of L. paracasei were produced. The survival of free and microencapsulated L. paracasei cells was evaluated during storage of the yogurts for 45 days at 4 °C. In addition, yogurts were exposed to simulated gastric juice and the reduction in viable numbers of L. paracasei cells was assessed. The effect of complex coacervates' addition on the rheological properties of yogurts was also evaluated. Yogurts containing encapsulated L. paracasei cells showed a slightly improved cell survival (≤0.22 log CFU g -1 reduction) during storage when compared to yogurts containing free cells (≤0.64 log CFU g -1 reduction). Moreover, the microencapsulated L. paracasei cells exhibited greater survival compared to free cells upon exposure of the yogurt samples to simulated gastric juice (pH 2.0) for 3 h. Finally, the incorporation of complex coacervates did not significantly affect the rheological properties of yogurts especially when added at concentrations less than 10% w/w. Consequently, the inclusion of microencapsulated bacteria by complex coacervation in yogurts, could become an effective vehicle for successful delivery of probiotics to the gut, and hence contributing to the improvement of the gastrointestinal tract health, without altering the texture of the product.

Stem cell-like differentiation potentials of endometrial side population cells as revealed by a newly developed in vivo endometrial stem cell assay.

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Kaoru Miyazaki

Full Text Available Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP, but not endometrial main population cells (EMP, exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay.ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom, a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells.We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo endometrial tissue reconstitution. Using this assay, we demonstrated that ESP

Proliferative, necrotizing and crescentic immune complex-mediated glomerulonephritis in a cat

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Carolyn Gross

2015-09-01

Full Text Available Case Summary A 5-year-old cat was examined for vomiting and anorexia of 2 days’ duration. Azotemia, hyperphosphatemia and hypoalbuminemia were the main biochemical findings. Serial analyses of the urine revealed isosthenuria, proteinuria and eventual glucosuria. Hyperechoic perirenal fat was detected surrounding the right kidney by ultrasonography. Histopathologic evaluation of ante-mortem ultrasound-guided needle biopsies of the right kidney was consistent with proliferative, necrotizing and crescentic glomerulonephritis with fibrin thrombi, proteinaceous and red blood cell casts, and moderate multifocal chronic-active interstitial nephritis. Owing to a lack of clinical improvement, the cat was eventually euthanized. Post-mortem renal biopsies were processed for light microscopy, transmission electron microscopy and immunofluorescence. This revealed severe focal proliferative and necrotizing glomerulonephritis with cellular crescent formation, podocyte injury and secondary segmental sclerosis. Ultrastructural analysis revealed scattered electron-dense deposits in the mesangium, and immunofluorescence demonstrated positive granular staining for λ light chains, consistent with immune complex-mediated glomerulonephritis. Severe diffuse acute tubular epithelial injury and numerous red blood cell casts were also seen. Relevance and novel information To our knowledge, this is the first report of naturally occurring proliferative, necrotizing and crescentic immune complex glomerulonephritis in a cat.

Digital sorting of complex tissues for cell type-specific gene expression profiles.

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Zhong, Yi; Wan, Ying-Wooi; Pang, Kaifang; Chow, Lionel M L; Liu, Zhandong

2013-03-07

Cellular heterogeneity is present in almost all gene expression profiles. However, transcriptome analysis of tissue specimens often ignores the cellular heterogeneity present in these samples. Standard deconvolution algorithms require prior knowledge of the cell type frequencies within a tissue or their in vitro expression profiles. Furthermore, these algorithms tend to report biased estimations. Here, we describe a Digital Sorting Algorithm (DSA) for extracting cell-type specific gene expression profiles from mixed tissue samples that is unbiased and does not require prior knowledge of cell type frequencies. The results suggest that DSA is a specific and sensitivity algorithm in gene expression profile deconvolution and will be useful in studying individual cell types of complex tissues.

Synthesis, Structural Characterization, Antimicrobial Activity, and In Vitro Biocompatibility of New Unsaturated Carboxylate Complexes with 2,2'-Bipyridine.

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Vasile Scăețeanu, Gina; Chifiriuc, Mariana Carmen; Bleotu, Coralia; Kamerzan, Crina; Măruţescu, Luminiţa; Daniliuc, Constantin G; Maxim, Cătălin; Calu, Larisa; Olar, Rodica; Badea, Mihaela

2018-01-12

The synthesis, structural characterization, cytotoxicity, and antimicrobial properties of four new complexes formed by employing acrylate anion and 2,2'-bipyridine are reported herein. X-ray crystallography revealed the trinuclear nature of [Mn₃(2,2'-bipy)₂(C₃H₃O₂)₆] ( 1 ), meanwhile complexes with general formula [M(2,2'-bipy)(C₃H₃O₂)₂(H₂O) x ]∙ y H₂O (( 2 ) M: Ni, x = 1, y = 0; ( 3 ) M: Cu, x = 1, y = 0; ( 4 ) M: Zn, x = 0, y = 1; 2,2'-bipy: 2,2'-bipyridine; C₃H₃O₂: acrylate anion) were shown to be mononuclear. The lowest minimum inhibitory concentration (MIC) of 128 μg mL -1 was recorded for all four tested complexes against Candida albicans , for complex ( 3 ) against Escherichia coli , and for complex ( 4 ) against Staphylocococcus aureus . Compounds ( 3 ) and ( 4 ) were also potent efflux pumps activity inhibitors (EPI), proving their potential for use in synergistic combinations with antibiotics. Complexes ( 1 )-( 4 ) revealed that they were not cytotoxic to HCT-8 cells. They also proved to interfere with the cellular cycle of tumour HCT-8 cells by increasing the number of cells found in the S and G2/M phases. Taken together, these results demonstrate the potential of zinc and copper complexes for use in the development of novel antimicrobial and anti-proliferative agents.

Mapping photothermally induced gene expression in living cells and tissues by nanorod-locked nucleic acid complexes.

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Riahi, Reza; Wang, Shue; Long, Min; Li, Na; Chiou, Pei-Yu; Zhang, Donna D; Wong, Pak Kin

2014-04-22

The photothermal effect of plasmonic nanostructures has numerous applications, such as cancer therapy, photonic gene circuit, large cargo delivery, and nanostructure-enhanced laser tweezers. The photothermal operation can also induce unwanted physical and biochemical effects, which potentially alter the cell behaviors. However, there is a lack of techniques for characterizing the dynamic cell responses near the site of photothermal operation with high spatiotemporal resolution. In this work, we show that the incorporation of locked nucleic acid probes with gold nanorods allows photothermal manipulation and real-time monitoring of gene expression near the area of irradiation in living cells and animal tissues. The multimodal gold nanorod serves as an endocytic delivery reagent to transport the probes into the cells, a fluorescence quencher and a binding competitor to detect intracellular mRNA, and a plasmonic photothermal transducer to induce cell ablation. We demonstrate the ability of the gold nanorod-locked nucleic acid complex for detecting the spatiotemporal gene expression in viable cells and tissues and inducing photothermal ablation of single cells. Using the gold nanorod-locked nucleic acid complex, we systematically characterize the dynamic cellular heat shock responses near the site of photothermal operation. The gold nanorod-locked nucleic acid complex enables mapping of intracellular gene expressions and analyzes the photothermal effects of nanostructures toward various biomedical applications.

Dynamic complexity: plant receptor complexes at the plasma membrane.

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Burkart, Rebecca C; Stahl, Yvonne

2017-12-01

Plant receptor complexes at the cell surface perceive many different external and internal signalling molecules and relay these signals into the cell to regulate development, growth and immunity. Recent progress in the analyses of receptor complexes using different live cell imaging approaches have shown that receptor complex formation and composition are dynamic and take place at specific microdomains at the plasma membrane. In this review we focus on three prominent examples of Arabidopsis thaliana receptor complexes and how their dynamic spatio-temporal distribution at the PM has been studied recently. We will elaborate on the newly emerging concept of plasma membrane microdomains as potential hubs for specific receptor complex assembly and signalling outputs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

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Liu, Betty R; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

2016-01-01

Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.

Survey reveals public open to ban on hand-held cell phone use and texting.

Science.gov (United States)

2013-01-01

A study performed by the Bureau of Transportation Statistics (BTS) reveals that the public is open to a ban on hand-held cell phone use while driving. The study is based on data from 2009s Omnibus Household Survey (OHS), which is administered by B...

The Complex Interaction of Matrix Metalloproteinases in the Migration of Cancer Cells through Breast Tissue Stroma

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Kerry J. Davies

2014-01-01

Full Text Available Breast cancer mortality is directly linked to metastatic spread. The metastatic cell must exhibit a complex phenotype that includes the capacity to escape from the primary tumour mass, invade the surrounding normal tissue, and penetrate into the circulation before proliferating in the parenchyma of distant organs to produce a metastasis. In the normal breast, cellular structures change cyclically in response to ovarian hormones leading to regulated cell proliferation and apoptosis. Matrix metalloproteinases (MMPs are a family of zinc dependent endopeptidases. Their primary function is degradation of proteins in the extracellular matrix to allow ductal progression through the basement membrane. A complex balance between matrix metalloproteinases and their inhibitors regulate these changes. These proteinases interact with cytokines, growth factors, and tumour necrosis factors to stimulate branching morphologies in normal breast tissues. In breast cancer this process is disrupted facilitating tumour progression and metastasis and inhibiting apoptosis increasing the life of the metastatic cells. This paper highlights the role of matrix metalloproteinases in cell progression through the breast stroma and reviews the complex relationships between the different proteinases and their inhibitors in relation to breast cancer cells as they metastasise.

Toxicity study of complex CNT-PEG(-NH2)-DOX synthesis on neuroblastoma cells

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Nurulhuda, I.; Mazatulikhma, M. Z.; Alrokayan, S.; Khan, H.; Rusop, M.

2018-05-01

The synthesized carbon nanotubes was functionalized with PEG and drug (doxorubicin) was tested on neuroblastoma cells. The treatment was done for 24 and 48 h. The concentration of CNT and doxorubicin were at 2.5, 5, 10 µg/ml and 0.5, 0.1, 0.05 µM, respectively. The result showed the longer time treatment do have effect on the cells viability and the complex functionalized CNT have high cells viability rather than the drug and CNT treatment alone.

A new approach for determining phase response curves reveals that Purkinje cells can act as perfect integrators.

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Elena Phoka

2010-04-01

Full Text Available Cerebellar Purkinje cells display complex intrinsic dynamics. They fire spontaneously, exhibit bistability, and via mutual network interactions are involved in the generation of high frequency oscillations and travelling waves of activity. To probe the dynamical properties of Purkinje cells we measured their phase response curves (PRCs. PRCs quantify the change in spike phase caused by a stimulus as a function of its temporal position within the interspike interval, and are widely used to predict neuronal responses to more complex stimulus patterns. Significant variability in the interspike interval during spontaneous firing can lead to PRCs with a low signal-to-noise ratio, requiring averaging over thousands of trials. We show using electrophysiological experiments and simulations that the PRC calculated in the traditional way by sampling the interspike interval with brief current pulses is biased. We introduce a corrected approach for calculating PRCs which eliminates this bias. Using our new approach, we show that Purkinje cell PRCs change qualitatively depending on the firing frequency of the cell. At high firing rates, Purkinje cells exhibit single-peaked, or monophasic PRCs. Surprisingly, at low firing rates, Purkinje cell PRCs are largely independent of phase, resembling PRCs of ideal non-leaky integrate-and-fire neurons. These results indicate that Purkinje cells can act as perfect integrators at low firing rates, and that the integration mode of Purkinje cells depends on their firing rate.

Biomarker screening of oral cancer cell lines revealed sub-populations of CD133-, CD44-, CD24- and ALDH1- positive cancer stem cells

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Kendall K

2013-05-01

Full Text Available Head and neck squamous cell carcinoma (HNSCC ranks sixth worldwide for cancer-related mortality. For the past several decades the mainstay of treatment for HNSCC has been surgery and external beam radiation, although more recent trials combining chemotherapy and radiation have demonstrated improvements. However, cancer recurrence and treatment failures continue to occur in a significant percentage of patients. Recent advances in tumor biology have led to the discovery that many cancers, including HNSCC, may contain subpopulations of cells with stem cell-like properties that may explain relapse and recurrence. The objective of this study was to screen existing oral cancer cell lines for biomarkers specific for cells with stem cell-like properties. RNA was isolated for RT-PCR screening using primers for specific mRNA of the biomarkers: CD44, CD24, CD133, NANOG, Nestin, ALDH1, and ABCG2 in CAL27, SCC25 and SCC15 cells. This analysis revealed that some oral cancer cell lines (CAL27 and SCC25 may contain small subpopulations of adhesion- and contact-independent cells (AiDC that also express tumor stem cell markers, including CD44, CD133, and CD24. In addition, CAL27 cells also expressed the intracellular tumor stem cell markers, ALDH1 and ABCG2. Isolation and culture of the adhesion- and contact-independent cells from CAL27 and SCC25 populations revealed differential proliferation rates and more robust inhibition by the MEK inhibitor PD98059, as well as the chemotherapeutic agents Cisplatin and Paclitaxel, within the AiDC CAL27 cells. At least one oral cancer cell line (CAL27 contained subpopulations of cells that express specific biomarkers associated with tumor stem cells which were morphologically and phenotypically distinct from other cells within this cell line.

The Allelic Landscape of Human Blood Cell Trait Variation and Links to Common Complex Disease.

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Astle, William J; Elding, Heather; Jiang, Tao; Allen, Dave; Ruklisa, Dace; Mann, Alice L; Mead, Daniel; Bouman, Heleen; Riveros-Mckay, Fernando; Kostadima, Myrto A; Lambourne, John J; Sivapalaratnam, Suthesh; Downes, Kate; Kundu, Kousik; Bomba, Lorenzo; Berentsen, Kim; Bradley, John R; Daugherty, Louise C; Delaneau, Olivier; Freson, Kathleen; Garner, Stephen F; Grassi, Luigi; Guerrero, Jose; Haimel, Matthias; Janssen-Megens, Eva M; Kaan, Anita; Kamat, Mihir; Kim, Bowon; Mandoli, Amit; Marchini, Jonathan; Martens, Joost H A; Meacham, Stuart; Megy, Karyn; O'Connell, Jared; Petersen, Romina; Sharifi, Nilofar; Sheard, Simon M; Staley, James R; Tuna, Salih; van der Ent, Martijn; Walter, Klaudia; Wang, Shuang-Yin; Wheeler, Eleanor; Wilder, Steven P; Iotchkova, Valentina; Moore, Carmel; Sambrook, Jennifer; Stunnenberg, Hendrik G; Di Angelantonio, Emanuele; Kaptoge, Stephen; Kuijpers, Taco W; Carrillo-de-Santa-Pau, Enrique; Juan, David; Rico, Daniel; Valencia, Alfonso; Chen, Lu; Ge, Bing; Vasquez, Louella; Kwan, Tony; Garrido-Martín, Diego; Watt, Stephen; Yang, Ying; Guigo, Roderic; Beck, Stephan; Paul, Dirk S; Pastinen, Tomi; Bujold, David; Bourque, Guillaume; Frontini, Mattia; Danesh, John; Roberts, David J; Ouwehand, Willem H; Butterworth, Adam S; Soranzo, Nicole

2016-11-17

Many common variants have been associated with hematological traits, but identification of causal genes and pathways has proven challenging. We performed a genome-wide association analysis in the UK Biobank and INTERVAL studies, testing 29.5 million genetic variants for association with 36 red cell, white cell, and platelet properties in 173,480 European-ancestry participants. This effort yielded hundreds of low frequency (<5%) and rare (<1%) variants with a strong impact on blood cell phenotypes. Our data highlight general properties of the allelic architecture of complex traits, including the proportion of the heritable component of each blood trait explained by the polygenic signal across different genome regulatory domains. Finally, through Mendelian randomization, we provide evidence of shared genetic pathways linking blood cell indices with complex pathologies, including autoimmune diseases, schizophrenia, and coronary heart disease and evidence suggesting previously reported population associations between blood cell indices and cardiovascular disease may be non-causal. Copyright © 2016 Elsevier Inc. All rights reserved.

Structural and functional characterizations of SsgB, a conserved activator of developmental cell division in morphologically complex actinomycetes.

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Xu, Qingping; Traag, Bjørn A; Willemse, Joost; McMullan, Daniel; Miller, Mitchell D; Elsliger, Marc-André; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Bakolitsa, Constantina; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chruszcz, Maksymilian; Clayton, Thomas; Das, Debanu; Deller, Marc C; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; Farr, Carol L; Feuerhelm, Julie; Grant, Joanna C; Grzechnik, Anna; Grzechnik, Slawomir K; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Krishna, S Sri; Kumar, Abhinav; Marciano, David; Minor, Wladek; Mommaas, A Mieke; Morse, Andrew T; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L; Sefcovic, Natasha; Tien, Henry J; Trame, Christine B; van den Bedem, Henry; Wang, Shuren; Weekes, Dana; Hodgson, Keith O; Wooley, John; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A; van Wezel, Gilles P

2009-09-11

SsgA-like proteins (SALPs) are a family of homologous cell division-related proteins that occur exclusively in morphologically complex actinomycetes. We show that SsgB, a subfamily of SALPs, is the archetypal SALP that is functionally conserved in all sporulating actinomycetes. Sporulation-specific cell division of Streptomyces coelicolor ssgB mutants is restored by introduction of distant ssgB orthologues from other actinomycetes. Interestingly, the number of septa (and spores) of the complemented null mutants is dictated by the specific ssgB orthologue that is expressed. The crystal structure of the SsgB from Thermobifida fusca was determined at 2.6 A resolution and represents the first structure for this family. The structure revealed similarities to a class of eukaryotic "whirly" single-stranded DNA/RNA-binding proteins. However, the electro-negative surface of the SALPs suggests that neither SsgB nor any of the other SALPs are likely to interact with nucleotide substrates. Instead, we show that a conserved hydrophobic surface is likely to be important for SALP function and suggest that proteins are the likely binding partners.

Structural and Functional Characterizations of SsgB, a Conserved Activator of Developmental Cell Division in Morphologically Complex Actinomycetes*

Science.gov (United States)

Xu, Qingping; Traag, Bjørn A.; Willemse, Joost; McMullan, Daniel; Miller, Mitchell D.; Elsliger, Marc-André; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Bakolitsa, Constantina; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chruszcz, Maksymilian; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; Farr, Carol L.; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Grzechnik, Slawomir K.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; Minor, Wladek; Mommaas, A. Mieke; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; van den Bedem, Henry; Wang, Shuren; Weekes, Dana; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.; van Wezel, Gilles P.

2009-01-01

SsgA-like proteins (SALPs) are a family of homologous cell division-related proteins that occur exclusively in morphologically complex actinomycetes. We show that SsgB, a subfamily of SALPs, is the archetypal SALP that is functionally conserved in all sporulating actinomycetes. Sporulation-specific cell division of Streptomyces coelicolor ssgB mutants is restored by introduction of distant ssgB orthologues from other actinomycetes. Interestingly, the number of septa (and spores) of the complemented null mutants is dictated by the specific ssgB orthologue that is expressed. The crystal structure of the SsgB from Thermobifida fusca was determined at 2.6 Å resolution and represents the first structure for this family. The structure revealed similarities to a class of eukaryotic “whirly” single-stranded DNA/RNA-binding proteins. However, the electro-negative surface of the SALPs suggests that neither SsgB nor any of the other SALPs are likely to interact with nucleotide substrates. Instead, we show that a conserved hydrophobic surface is likely to be important for SALP function and suggest that proteins are the likely binding partners. PMID:19567872

Cell–cell signaling drives the evolution of complex traits: introduction—lung evo-devo

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Torday, John S.; Rehan, V. K.

2009-01-01

Physiology integrates biology with the environment through cell–cell interactions at multiple levels. The evolution of the respiratory system has been “deconvoluted” (Torday and Rehan in Am J Respir Cell Mol Biol 31:8–12, 2004) through Gene Regulatory Networks (GRNs) applied to cell–cell communication for all aspects of lung biology development, homeostasis, regeneration, and aging. Using this approach, we have predicted the phenotypic consequences of failed signaling for lung development, homeostasis, and regeneration based on evolutionary principles. This cell–cell communication model predicts other aspects of vertebrate physiology as adaptational responses. For example, the oxygen-induced differentiation of alveolar myocytes into alveolar adipocytes was critical for the evolution of the lung in land dwelling animals adapting to fluctuating Phanarezoic oxygen levels over the past 500 million years. Adipocytes prevent lung injury due to oxygen radicals and facilitate the rise of endothermy. In addition, they produce the class I cytokine leptin, which augments pulmonary surfactant activity and alveolar surface area, increasing selection pressure for both respiratory oxygenation and metabolic demand initially constrained by high-systemic vascular pressure, but subsequently compensated by the evolution of the adrenomedullary beta-adrenergic receptor mechanism. Conserted positive selection for the lung and adrenals created further selection pressure for the heart, which becomes progressively more complex phylogenetically in tandem with the lung. Developmentally, increasing heart complexity and size impinges precociously on the gut mesoderm to induce the liver. That evolutionary-developmental interaction is significant because the liver provides regulated sources of glucose and glycogen to the evolving physiologic system, which is necessary for the evolution of the neocortex. Evolution of neocortical control furthers integration of physiologic systems. Such

Deliberate ROS production and auxin synergistically trigger the asymmetrical division generating the subsidiary cells in Zea mays stomatal complexes.

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Livanos, Pantelis; Galatis, Basil; Apostolakos, Panagiotis

2016-07-01

Subsidiary cell generation in Poaceae is an outstanding example of local intercellular stimulation. An inductive stimulus emanates from the guard cell mother cells (GMCs) towards their laterally adjacent subsidiary cell mother cells (SMCs) and triggers the asymmetrical division of the latter. Indole-3-acetic acid (IAA) immunolocalization in Zea mays protoderm confirmed that the GMCs function as local sources of auxin and revealed that auxin is polarly accumulated between GMCs and SMCs in a timely-dependent manner. Besides, staining techniques showed that reactive oxygen species (ROS) exhibit a closely similar, also time-dependent, pattern of appearance suggesting ROS implication in subsidiary cell formation. This phenomenon was further investigated by using the specific NADPH-oxidase inhibitor diphenylene iodonium, the ROS scavenger N-acetyl-cysteine, menadione which leads to ROS overproduction, and H2O2. Treatments with diphenylene iodonium, N-acetyl-cysteine, and menadione specifically blocked SMC polarization and asymmetrical division. In contrast, H2O2 promoted the establishment of SMC polarity and subsequently subsidiary cell formation in "younger" protodermal areas. Surprisingly, H2O2 favored the asymmetrical division of the intervening cells of the stomatal rows leading to the creation of extra apical subsidiary cells. Moreover, H2O2 altered IAA localization, whereas synthetic auxin analogue 1-napthaleneacetic acid enhanced ROS accumulation. Combined treatments with ROS modulators along with 1-napthaleneacetic acid or 2,3,5-triiodobenzoic acid, an auxin efflux inhibitor, confirmed the crosstalk between ROS and auxin functioning during subsidiary cell generation. Collectively, our results demonstrate that ROS are critical partners of auxin during development of Z. mays stomatal complexes. The interplay between auxin and ROS seems to be spatially and temporarily regulated.

Single-cell tracking reveals antibiotic-induced changes in mycobacterial energy metabolism.

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Maglica, Željka; Özdemir, Emre; McKinney, John D

2015-02-17

ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in Mycobacterium smegmatis based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening. New antimicrobials are urgently needed to stem the rising tide of antibiotic-resistant bacteria. All antibiotics are expected to affect bacterial energy metabolism, directly or indirectly, yet tools to assess the impact of antibiotics on the ATP content of individual bacterial cells are lacking. The

Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells

DEFF Research Database (Denmark)

Hattori, Naoko; Niwa, Tohru; Kimura, Kana

2013-01-01

. Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell......Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which...... population, namely, ESCs at an early stage of differentiation, and this revealed that the bivalent modification disappeared in a highly concerted manner, whereas phenotypic differentiation proceeded with large variations among cells. Also, using this method, we were able to visualize a combination...

Human decidua-derived mesenchymal stem cells differentiate into functional alveolar type II-like cells that synthesize and secrete pulmonary surfactant complexes.

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Alejandro Cerrada

Full Text Available Lung alveolar type II (ATII cells are specialized in the synthesis and secretion of pulmonary surfactant, a lipid-protein complex that reduces surface tension to minimize the work of breathing. Surfactant synthesis, assembly and secretion are closely regulated and its impairment is associated with severe respiratory disorders. At present, well-established ATII cell culture models are not available. In this work, Decidua-derived Mesenchymal Stem Cells (DMSCs have been differentiated into Alveolar Type II- Like Cells (ATII-LCs, which display membranous cytoplasmic organelles resembling lamellar bodies, the organelles involved in surfactant storage and secretion by native ATII cells, and accumulate disaturated phospholipid species, a surfactant hallmark. Expression of characteristic ATII cells markers was demonstrated in ATII-LCs at gene and protein level. Mimicking the response of ATII cells to secretagogues, ATII-LCs were able to exocytose lipid-rich assemblies, which displayed highly surface active capabilities, including faster interfacial adsorption kinetics than standard native surfactant, even in the presence of inhibitory agents. ATII-LCs could constitute a highly useful ex vivo model for the study of surfactant biogenesis and the mechanisms involved in protein processing and lipid trafficking, as well as the packing and storage of surfactant complexes.

Quantitative genome-wide genetic interaction screens reveal global epistatic relationships of protein complexes in Escherichia coli.

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Mohan Babu

2014-02-01

Full Text Available Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI screens can provide insights into the biological role(s of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.

Basal cell carcinomas of the areola-nipple complex: case reports and review of the literature.

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Betti, Roberto; Martino, Patrizia; Moneghini, Laura; Vergani, Raffaelle; Tolomio, Elena; Crosti, Carlo

2003-11-01

Two white men 57 and 39 years old, and a 47-year-old white woman were seen with slowly developing papulo-nodular lesions of the areola-nipple complex. None of the patients presented with regional lymphadenopathy, history of trauma, or relevant sun-exposure. After excison of the mass, the histologic diagnosis of basal cell carcinoma was made. At two years of follow-up, no recurrence was evident. The low incidence of basal cell carcinoma in this particular site allows us to consider the areola-nipple complex location as unusual. Moreover, literature reports do not suggest that these BCCs have an increased potential for malignancy. The treatment options depend on the extension of the tumor and on the possible involvement of the areola-nipple complex and mammary tissue.

Global phosphoproteome profiling reveals unanticipated networks responsive to cisplatin treatment of embryonic stem cells

DEFF Research Database (Denmark)

Pines, Alex; Kelstrup, Christian D; Vrouwe, Mischa G

2011-01-01

(stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia...... rearrangements. Integration of transcriptomic and proteomic data revealed a poor correlation between changes in the relative levels of transcripts and their corresponding proteins, but a large overlap in affected pathways at the levels of mRNA, protein, and phosphoprotein. This study provides an integrated view...

Single-Cell Analysis of SMN Reveals Its Broader Role in Neuromuscular Disease

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Natalia Rodriguez-Muela

2017-02-01

Full Text Available The mechanism underlying selective motor neuron (MN death remains an essential question in the MN disease field. The MN disease spinal muscular atrophy (SMA is attributable to reduced levels of the ubiquitous protein SMN. Here, we report that SMN levels are widely variable in MNs within a single genetic background and that this heterogeneity is seen not only in SMA MNs but also in MNs derived from controls and amyotrophic lateral sclerosis (ALS patients. Furthermore, cells with low SMN are more susceptible to cell death. These findings raise the important clinical implication that some SMN-elevating therapeutics might be effective in MN diseases besides SMA. Supporting this, we found that increasing SMN across all MN populations using an Nedd8-activating enzyme inhibitor promotes survival in both SMA and ALS-derived MNs. Altogether, our work demonstrates that examination of human neurons at the single-cell level can reveal alternative strategies to be explored in the treatment of degenerative diseases.

Design of hypoxia-targeting radiopharmaceuticals: selective uptake of copper-64 complexes in hypoxic cells in vitro

International Nuclear Information System (INIS)

Dearling, J.L.J.; Lewis, J.S.; Mullen, G.E.D.; Rae, M.T.; Zweit, J.; Blower, P.J.

1998-01-01

The well-known perfusion tracer CuPTSM, labelled with 62 Cu or 64 Cu, is believed to be trapped in cells non-selectively by a bioreductive mechanism. It is proposed that by modifying the ligand to increase its electron donor strength (for example by adding alkyl functionality or replacing sulphur ligands with oxygen ligands), the copper complexes will become less easily reduced and tracers with selectivity for hypoxic tissues could thus be developed. The aim of this work was to prepare 64 Cu-labelled complexes of two series of ligands, based on the bis(thiosemicarbazone) (13 ligands) and bis(salicylaldimine) (3 ligands) skeletons, and to evaluate the hypoxia dependence of their uptake in cells. The complexes were incubated with Chinese hamster ovary cells under normoxic and hypoxic conditions, and the cells isolated by centrifugation to determine radioactivity uptake at various time points up to 90 min. Several members of both series demonstrated significant (P 60 Cu, 61 Cu, 62 Cu, 64 Cu) and targeted radiotherapy ( 64 Cu, 67 Cu). (orig.)

Complex forms of mitochondrial DNA in human B cells transformed by Epstein-Barr virus (EBV)

DEFF Research Database (Denmark)

Christiansen, Gunna; Christiansen, C; Zeuthen, J

1983-01-01

Human lymphocytes and lymphoid cell lines were analyzed for the presence of complex forms of mitochondrial DNA (mtDNA) by electron microscopy. A high frequency (9%-14.5%) of catenated dimers, circular dimers, or oligomers were found in samples from Epstein-Barr-virus-(EBV) transformed lymphoblast......Human lymphocytes and lymphoid cell lines were analyzed for the presence of complex forms of mitochondrial DNA (mtDNA) by electron microscopy. A high frequency (9%-14.5%) of catenated dimers, circular dimers, or oligomers were found in samples from Epstein-Barr-virus-(EBV) transformed...

Ultrastructural characterization of effector-target interactions for human neonatal and adult NK cells reveals reduced intercellular surface contacts of neonatal cells

NARCIS (Netherlands)

Ribeiro-do-Couto, Laura M.; Poelen, Martien; Hooibrink, Berend; Dormans, Jan A. M. A.; Roholl, Paul J. M.; Boog, Claire J. P.

2003-01-01

Limitations in neonatal natural killer (NK) cell responses may be associated with the less efficient newborn capacity to solve viral infections. Although these limitations have been extensively reported they are poorly characterized. Making use of the major histocompatibility complex (MHC) class I

Breaking down pluripotency in the porcine embryo reveals both a premature and reticent stem cell state in the inner cell mass and unique expression profiles of the naive and primed stem cell states.

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Hall, Vanessa Jane; Hyttel, Poul

2014-09-01

To date, it has been difficult to establish bona fide porcine embryonic stem cells (pESC) and stable induced pluripotent stem cells. Reasons for this remain unclear, but they may depend on inappropriate culture conditions. This study reports the most insights to date on genes expressed in the pluripotent cells of the porcine embryo, namely the inner cell mass (ICM), the trophectoderm-covered epiblast (EPI), and the embryonic disc epiblast (ED). Specifically, we reveal that the early porcine ICM represents a premature state of pluripotency due to lack of translation of key pluripotent proteins, and the late ICM enters a transient, reticent pluripotent state which lacks expression of most genes associated with pluripotency. We describe a unique expression profile of the porcine EPI, reflecting the naive stem cell state, including expression of OCT4, NANOG, CRIPTO, and SSEA-1; weak expression of NrOB1 and REX1; but very limited expression of genes in classical pathways involved in regulating pluripotency. The porcine ED, reflecting the primed stem cell state, can be characterized by the expression of OCT4, NANOG, SOX2, KLF4, cMYC, REX1, CRIPTO, and KLF2. Further cell culture experiments using inhibitors against FGF, JAK/STAT, BMP, WNT, and NODAL pathways on cell cultures derived from day 5 and 10 embryos reveal the importance of FGF, JAK/STAT, and BMP signaling in maintaining cell proliferation of pESCs in vitro. Together, this article provides new insights into the regulation of pluripotency, revealing unique stem cell states in the different porcine stem cell populations derived from the early developing embryo.

[Prostatic granulomas revealing a peripheral T-cell lymphoma].

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Foguem, C; Curlier, E; Rouamba, M-M; Regent, A; Philippe, P

2009-02-01

The presence of granulomas on tissue biopsie has been reported in a wide range of disorders. The clinical presentation and the diagnostic work-up of granulomatosis can be difficult as it is illustrated in the following report. A 59-year-old patient was referred in 2002 for a granulomatous prostatitis. Physical examination was normal. Except for the increase of prostate-specific antigen (which motivated a biopsy), the laboratory results were normal. Thoracic CT-scan disclosed mediastinal lymph nodes. A minor salivary gland biopsy was consistent with the diagnosis of sarcoidosis. In 2004, the patient presented an epidermal necrolysis, and in 2005 the deterioration of general status raised suspicion of a lymphoproliferative disorder. Liver and bone marrow biopsies revealed a granulomatous process. Despite steroid therapy, the patient died. Autopsy discloses a anaplasic T cell lymphoma. This report illustrates the relationship between sarcoidosis and lymphoma as a mode of presentation, a complication, or an accidental but misleading association? The association between anaplastic lymphoma and sarcoidosis is exceptional.

Antiproliferative and apoptosis-inducing activity of an oxidovanadium(IV) complex with the flavonoid silibinin against osteosarcoma cells.

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Leon, I E; Porro, V; Di Virgilio, A L; Naso, L G; Williams, P A M; Bollati-Fogolín, M; Etcheverry, S B

2014-01-01

Flavonoids are a large family of polyphenolic compounds synthesized by plants. They display interesting biological effects mainly related to their antioxidant properties. On the other hand, vanadium compounds also exhibit different biological and pharmacological effects in cell culture and in animal models. Since coordination of ligands to metals can improve or change the pharmacological properties, we report herein, for the first time, a detailed study of the mechanisms of action of an oxidovanadium(IV) complex with the flavonoid silibinin, Na2[VO(silibinin)2]·6H2O (VOsil), in a model of the human osteosarcoma derived cell line MG-63. The complex inhibited the viability of osteosarcoma cells in a dose-dependent manner with a greater potency than that of silibinin and oxidovanadium(IV) (p cell cycle arrest and activated caspase 3, triggering apoptosis as determined by flow cytometry. As a whole, these results show the main mechanisms of the deleterious effects of VOsil in the osteosarcoma cell line, demonstrating that this complex is a promising compound for cancer treatments.

Quantitative FLIM-FRET Microscopy to Monitor Nanoscale Chromatin Compaction In Vivo Reveals Structural Roles of Condensin Complexes

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David Llères

2017-02-01

Full Text Available How metazoan genomes are structured at the nanoscale in living cells and tissues remains unknown. Here, we adapted a quantitative FRET (Förster resonance energy transfer-based fluorescence lifetime imaging microscopy (FLIM approach to assay nanoscale chromatin compaction in living organisms. Caenorhabditis elegans was chosen as a model system. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1 and SETDB1 H3-lysine-9 methyltransferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.

Modeling two-phase flow in three-dimensional complex flow-fields of proton exchange membrane fuel cells

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Kim, Jinyong; Luo, Gang; Wang, Chao-Yang

2017-10-01

3D fine-mesh flow-fields recently developed by Toyota Mirai improved water management and mass transport in proton exchange membrane (PEM) fuel cell stacks, suggesting their potential value for robust and high-power PEM fuel cell stack performance. In such complex flow-fields, Forchheimer's inertial effect is dominant at high current density. In this work, a two-phase flow model of 3D complex flow-fields of PEMFCs is developed by accounting for Forchheimer's inertial effect, for the first time, to elucidate the underlying mechanism of liquid water behavior and mass transport inside 3D complex flow-fields and their adjacent gas diffusion layers (GDL). It is found that Forchheimer's inertial effect enhances liquid water removal from flow-fields and adds additional flow resistance around baffles, which improves interfacial liquid water and mass transport. As a result, substantial improvements in high current density cell performance and operational stability are expected in PEMFCs with 3D complex flow-fields, compared to PEMFCs with conventional flow-fields. Higher current density operation required to further reduce PEMFC stack cost per kW in the future will necessitate optimizing complex flow-field designs using the present model, in order to efficiently remove a large amount of product water and hence minimize the mass transport voltage loss.

Conserved properties of dentate gyrus neurogenesis across postnatal development revealed by single-cell RNA sequencing.

Science.gov (United States)

Hochgerner, Hannah; Zeisel, Amit; Lönnerberg, Peter; Linnarsson, Sten