Mobile Transcripts and Intercellular Communication in Plants.

Science.gov (United States)

Saplaoura, E; Kragler, F

2016-01-01

Phloem serves as a highway for mobile signals in plants. Apart from sugars and hormones, proteins and RNAs are transported via the phloem and contribute to the intercellular communication coordinating growth and development. Different classes of RNAs have been found mobile and in the phloem exudate such as viral RNAs, small interfering RNAs (siRNAs), microRNAs, transfer RNAs, and messenger RNAs (mRNAs). Their transport is considered to be mediated via ribonucleoprotein complexes formed between phloem RNA-binding proteins and mobile RNA molecules. Recent advances in the analysis of the mobile transcriptome indicate that thousands of transcripts move along the plant axis. Although potential RNA mobility motifs were identified, research is still in progress on the factors triggering siRNA and mRNA mobility. In this review, we discuss the approaches used to identify putative mobile mRNAs, the transport mechanism, and the significance of mRNA trafficking. © 2016 Elsevier Inc. All rights reserved.

Use of cultured cells with defects of citrulline metabolism in diagnosis and in the study of intercellular communication

Energy Technology Data Exchange (ETDEWEB)

Davidson, J S

1985-01-01

Citrullinemia and argininosuccinic aciduria are two disorders resulting from defects in two consecutive enzymes of the urea cycle, argininosuccinate synthetase and argininosuccinate lyase. Fibroblast cell lines were derived from patients with these disorders and the diagnoses, which had been made on the basis of amino acid levels in plasma and urine, were confirmed by demonstrating that the cell lines were unable to incorporate /sup 14/C-citrulline into protein. DNA from the argininosuccinate synthetase-deficient (ASS-) cells was analysed by restriction enzyme digestion and hybridisation to a cDNA probe which had been cloned from human argininosuccinate synthetase mRNA. No defect in the patient's DNA could be demonstrated, indicating that no major deletions in the argininosuccinate synthetase genes were present in this patient. Co-cultures of the ASS- and argininosuccinate lyase-deficient (ASL-) fibroblasts were able to incorporate /sup 14/C-citrulline into protein. Co-cultures of ASS- and ASL-cells were used as an assay system for measuring intercellular junctional communication. This allowed quantitation of the effects of pH and extra-cellular divalent cations on junctional communication. Tumor promoters such as phorbol esters and organochlorine pesticides have been reported to inhibit intercellular junctional communication in other systems, and this inhibitory activity may be related to the mechanism of tumor promotion. Retinoic acid and other retinoids also inhibited junctional communication, and the inhibitory effects of retinoic acid and TPA were additive. It is concluded that co-cultures of ASS- and ASL-cells constitute a useful system for providing quantitative measurements of intercellular junctional communication under a wide range of experimental conditions.

Intercellular communication via gap junctions affected by mechanical load in the bovine annulus fibrosus.

Science.gov (United States)

Desrochers, Jane; Duncan, Neil A

2014-01-01

Cells in the intervertebral disc, as in other connective tissues including tendon, ligament and bone, form interconnected cellular networks that are linked via functional gap junctions. These cellular networks may be necessary to affect a coordinated response to mechanical and environmental stimuli. Using confocal microscopy with fluorescence recovery after photobleaching methods, we explored the in situ strain environment of the outer annulus of an intact bovine disc and the effect of high-level flexion on gap junction signalling. The in situ strain environment in the extracellular matrix of the outer annulus under high flexion load was observed to be non-uniform with the extensive cellular processes remaining crimped sometimes at flexion angles greater than 25°. A significant transient disruption of intercellular communication via functional gap junctions was measured after 10 and 20 min under high flexion load. This study illustrates that in healthy annulus fibrosus tissue, high mechanical loads can impede the functioning of the gap junctions. Future studies will explore more complex loading conditions to determine whether losses in intercellular communication can be permanent and whether gap junctions in aged and degenerated tissues become more susceptible to load. The current research suggests that cellular structures such as gap junctions and intercellular networks, as well as other cell-cell and cell-matrix interconnections, need to be considered in computational models in order to fully understand how macroscale mechanical signals are transmitted across scales to the microscale and ultimately into a cellular biosynthetic response in collagenous tissues.

Methylmercury inhibits gap junctional intercellular communication in primary cultures of rat proximal tubular cells

Energy Technology Data Exchange (ETDEWEB)

Yoshida, Minoru; Sumi, Yawara [Department of Chemistry, St. Marianna University School of Medicine, Kawasagi (Japan); Kujiraoka, Toru [Department of Physiology, St. Marianna University School of Medicine, Kawasagi (Japan); Hara, Masayuki [Department of Anatomy, St. Marianna University School of Medicine, Kawasagi (Japan); Nakazawa, Hirokazu [Department of Chemistry, Faculty of Sciences, Meisei University (Japan)

1998-03-01

Methylmercury (MeHg) causes renal injury in addition to central and peripheral neuropathy. To clarify the mechanism of nephrotoxicity by MeHg, we investigated the effect of this compound on intercellular communication through gap junction channels in primary cultures of rat renal proximal tubular cells. Twenty minutes after exposure to 30 {mu}M MeHg, gap junctional intercellular communication (GJIC), which was assessed by dye coupling, was markedly inhibited before appearance of cytotoxicity. When the medium containing MeHg was exchanged with MeHg-free medium, dye coupling recovered abruptly. However, the dye-coupling was abolished again 30 min after replacement with control medium, and the cells were damaged. Intracellular calcium concentration, [Ca{sup 2+}]{sub i}, which modulates the function of gap junctions, significantly increased following exposure of the cells to 30 {mu}M MeHg and returned to control level following replacement with MeHg-free medium. These results suggest that the inhibiting effect of MeHg on GJIC is related to the change in [Ca{sup 2+}]{sub i}, and may be involved in the pathogenesis of renal dysfunction. (orig.) With 5 figs., 23 refs.

The use of cultured cells with defects of citrulline metabolism in diagnosis and in the study of intercellular communication

International Nuclear Information System (INIS)

Davidson, J.S.

1985-02-01

Citrullinemia and argininosuccinic aciduria are two disorders resulting from defects in two consecutive enzymes of the urea cycle, argininosuccinate synthetase and argininosuccinate lyase. Fibroblast cell lines were derived from patients with these disorders and the diagnoses, which had been made on the basis of amino acid levels in plasma and urine, were confirmed by demonstrating that the cell lines were unable to incorporate 14 C-citrulline into protein. DNA from the argininosuccinate synthetase-deficient (ASS-) cells was analysed by restriction enzyme digestion and hybridisation to a cDNA probe which had been cloned from human argininosuccinate synthetase mRNA. No defect in the patient's DNA could be demonstrated, indicating that no major deletions in the argininosuccinate synthetase genes were present in this patient. Co-cultures of the ASS- and argininosuccinate lyase-deficient (ASL-) fibroblasts were able to incorporate 14 C-citrulline into protein. Co-cultures of ASS- and ASL-cells were used as an assay system for measuring intercellular junctional communication. This allowed quantitation of the effects of pH and extra-cellular divalent cations on junctional communication. Tumor promoters such as phorbol esters and organochlorine pesticides have been reported to inhibit intercellular junctional communication in other systems, and this inhibitory activity may be related to the mechanism of tumor promotion. Retinoic acid and other retinoids also inhibited junctional communication, and the inhibitory effects of retinoic acid and TPA were additive. It is concluded that co-cultures of ASS- and ASL-cells constitute a useful system for providing quantitative measurements of intercellular junctional communication under a wide range of experimental conditions

Intercellular signal communication among odontoblasts and trigeminal ganglion neurons via glutamate.

Science.gov (United States)

Nishiyama, A; Sato, M; Kimura, M; Katakura, A; Tazaki, M; Shibukawa, Y

2016-11-01

Various stimuli to the exposed surface of dentin induce changes in the hydrodynamic force inside the dentinal tubules resulting in dentinal pain. Recent evidences indicate that mechano-sensor channels, such as the transient receptor potential channels, in odontoblasts receive these hydrodynamic forces and trigger the release of ATP to the pulpal neurons, to generate dentinal pain. A recent study, however, has shown that odontoblasts also express glutamate receptors (GluRs). This implies that cells in the dental pulp tissue have the ability to release glutamate, which acts as a functional intercellular mediator to establish inter-odontoblast and odontoblast-trigeminal ganglion (TG) neuron signal communication. To investigate the intercellular signal communication, we applied mechanical stimulation to odontoblasts and measured the intracellular free Ca 2+ concentration ([Ca 2+ ] i ). During mechanical stimulation in the presence of extracellular Ca 2+ , we observed a transient [Ca 2+ ] i increase not only in single stimulated odontoblasts, but also in adjacent odontoblasts. We could not observe these responses in the absence of extracellular Ca 2+ . [Ca 2+ ] i increases in the neighboring odontoblasts during mechanical stimulation of single odontoblasts were inhibited by antagonists of metabotropic glutamate receptors (mGluRs) as well as glutamate-permeable anion channels. In the odontoblast-TG neuron coculture, we observed an increase in [Ca 2+ ] i in the stimulated odontoblasts and TG neurons, in response to direct mechanical stimulation of single odontoblasts. These [Ca 2+ ] i increases in the neighboring TG neurons were inhibited by antagonists for mGluRs. The [Ca 2+ ] i increases in the stimulated odontoblasts were also inhibited by mGluRs antagonists. We further confirmed that the odontoblasts express group I, II, and III mGluRs. However, we could not record any currents evoked from odontoblasts near the mechanically stimulated odontoblast, with or without

Intercellular communication and cell proliferation in precision-cut rat liver slices : effect of medium composition and DDT

NARCIS (Netherlands)

Graaf, I.A.M.; Tajima, O.; Groten, J.P.; Wolterbeek, A.P.M.

2000-01-01

Gap junctional intercellular communication (GJIC) and cell proliferation were studied in control and 1,1'-bis(p-chlorophenyl)-2,2,2,-trichloroethane (DDT) treated precision-cut liver slices of rat by evaluating connexin 32 (Cx32) expression and 5-bromo-2'-deoxyuridine (BrdU) incorporation. In

INHIBITION OF GAP JUNCTIONAL INTERCELLULAR COMMUNICATION BY PERFLUORINATED COMPOUNDS IN RAT LIVER AND DOLPHIN KIDNEY EPITHELIAL CELL LINES IN VITRO AND SPRAGUE-DAWLEY RATS IN VIVO

Science.gov (United States)

Abstract Gap Junctional Intercellular Communication (GJIC) is the major pathway of intercellular signal transduction, and is, thus, important for normal cell growth and function. Recent studies have revealed a global distribution of some perfluorinated organic compounds e...

Intercellular bridges in vertebrate gastrulation.

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Luca Caneparo

Full Text Available The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape. To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries. We find that intercellular bridges join a significant fraction of epiblast cells in the zebrafish embryo, reaching several cell diameters in length and spanning across different regions of the developing embryos. These intercellular bridges are distinct from the cellular protrusions previously reported as extending from hypoblast cells (1-2 cellular diameters in length or epiblast cells (which were shorter. Most of the intercellular bridges were formed at pre-gastrula stages by the daughters of a dividing cell maintaining a membrane tether as they move apart after mitosis. These intercellular bridges persist during gastrulation and can mediate the transfer of proteins between distant cells. These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.

Inhibition of hepatocyte gap junctional intercellular communication by tumor promoters

International Nuclear Information System (INIS)

Ruch, R.J.

1988-01-01

The mechanisms by which tumor promoters enhance neoplasia are poorly understood. One effect common to most tumor promoters is their ability to inhibit the cell-to-cell exchange of small molecules and ions through gap junctions, i.e., gap junctional intercellular communication (IC). IC maybe necessary for normal growth control and the loss of IC may predispose cells to enhanced growth. In the present studies, the effects of liver tumor promoters and other agents on IC between rodent hepatocytes in primary culture has been studied. IC was detected between hepatocytes: (1) autoradiographically following the passage and incorporation of [5- 3 H]uridine nucleotides from pre-labeled donor hepatocytes to non-labeled, adjacent recipient hepatocytes and (2) by fluorescence microscopy after microinjection of fluorescent Lucifer Yellow CH dye into hepatocytes and visualizing dye spread into adjacent hepatocytes

Short-range intercellular calcium signaling in bone

DEFF Research Database (Denmark)

Jørgensen, Niklas Rye

2005-01-01

into biological effects in bone. Intercellular calcium waves are increases in intracellular calcium concentration in single cells, subsequently propagating to adjacent cells, and can be a possible mechanism for the coupling of bone formation to bone resorption. The aim of the present studies was to investigate...... whether bone cells are capable of communicating via intercellular calcium signals, and determine by which mechanisms the cells propagate the signals. First, we found that osteoblastic cells can propagate intercellular calcium transients upon mechanical stimulation, and that there are two principally...... different mechanisms for this propagation. One mechanism involves the secretion of a nucleotide, possibly ATP, acting in an autocrine action to purinergic P2Y2 receptors on the neighboring cells, leading to intracellular IP3 generation and subsequent release of calcium from intracellular stores. The other...

Short-range intercellular calcium signaling in bone

DEFF Research Database (Denmark)

Jørgensen, Niklas R

2005-01-01

The regulation of bone turnover is a complex and finely tuned process. Many factors regulate bone remodeling, including hormones, growth factors, cytokines etc. However, little is known about the signals coupling bone formation to bone resorption, and how mechanical forces are translated...... into biological effects in bone. Intercellular calcium waves are increases in intracellular calcium concentration in single cells, subsequently propagating to adjacent cells, and can be a possible mechanism for the coupling of bone formation to bone resorption. The aim of the present studies was to investigate...... whether bone cells are capable of communicating via intercellular calcium signals, and determine by which mechanisms the cells propagate the signals. First, we found that osteoblastic cells can propagate intercellular calcium transients upon mechanical stimulation, and that there are two principally...

Inter-cellular transport of ran GTPase.

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Deepak Khuperkar

Full Text Available Ran, a member of the Ras-GTPase superfamily, has a well-established role in regulating the transport of macromolecules across the nuclear envelope (NE. Ran has also been implicated in mitosis, cell cycle progression, and NE formation. Over-expression of Ran is associated with various cancers, although the molecular mechanism underlying this phenomenon is unclear. Serendipitously, we found that Ran possesses the ability to move from cell-to-cell when transiently expressed in mammalian cells. Moreover, we show that the inter-cellular transport of Ran is GTP-dependent. Importantly, Ran displays a similar distribution pattern in the recipient cells as that in the donor cell and co-localizes with the Ran binding protein Nup358 (also called RanBP2. Interestingly, leptomycin B, an inhibitor of CRM1-mediated export, or siRNA mediated depletion of CRM1, significantly impaired the inter-cellular transport of Ran, suggesting a function for CRM1 in this process. These novel findings indicate a possible role for Ran beyond nucleo-cytoplasmic transport, with potential implications in inter-cellular communication and cancers.

Intercellular odontoblast communication via ATP mediated by pannexin-1 channel and phospholipase C-coupled receptor activation.

Directory of Open Access Journals (Sweden)

Masaki eSato

2015-11-01

Full Text Available Extracellular ATP released via pannexin-1 channels, in response to the activation of mechanosensitive-TRP channels during odontoblast mechanical stimulation, mediates intercellular communication among odontoblasts in dental pulp slice preparation dissected form rat incisor. Recently, odontoblast cell lines, such as mouse odontoblast lineage cells, have been widely used to investigate physiological/pathological cellular functions. To clarify whether the odontoblast cell lines also communicate with each other by diffusible chemical substance(s, we investigated the chemical intercellular communication among cells from mouse odontoblast cell lines following mechanical stimulation. A single cell was stimulated using a glass pipette filled with standard extracellular solution. We measured intracellular free Ca2+ concentration ([Ca2+]i by fura-2 in stimulated cells, as well as in cells located nearby. Direct mechanical stimulation to a single odontoblast increased [Ca2+]i, which showed sensitivity to capsazepine. In addition, we observed increases in [Ca2+]i not only in the mechanically stimulated odontoblast, but also in nearby odontoblasts. We could observe mechanical stimulation-induced increase in [Ca2+]i in a stimulated human embryo kidney (HEK 293 cell, but not in nearby HEK293 cells. The increase in [Ca2+]i in nearby odontoblasts, but not in the stimulated odontoblast, was inhibited by adenosine triphosphate (ATP release channel (pannexin-1 inhibitor in a concentration- and spatial-dependent manner. Moreover, in the presence of phospholipase C (PLC inhibitor, the increase in [Ca2+]i in nearby odontoblasts, following mechanical stimulation of a single odontoblast, was abolished. We could record some inward currents evoked from odontoblasts near the stimulated odontoblast, but the currents were observed in only 4.8% of the recorded odontoblasts. The results of this study showed that ATP is released via pannexin-1, from a mechanically stimulated

Connexin 26-mediated gap junctional intercellular communication suppresses paracellular permeability of human intestinal epithelial cell monolayers

International Nuclear Information System (INIS)

Morita, Hidekazu; Katsuno, Tatsuro; Hoshimoto, Aihiro; Hirano, Noriaki; Saito, Yasushi; Suzuki, Yasuo

2004-01-01

In some cell types, gap junctional intercellular communication (GJIC) is associated with tight junctions. The present study was performed to determine the roles of GJIC in regulation of the barrier function of tight junctions. Caco-2 human colonic cells were used as a monolayer model, and barrier function was monitored by measuring mannitol permeability and transepithelial electrical resistance (TER). The monolayers were chemically disrupted by treatment with oleic acid and taurocholic acid. Western blotting analyses were performed to evaluate the protein levels of connexins, which are components of gap junctional intercellular channels. Cx26 expression was detected in preconfluent Caco-2 cells, and its level increased gradually after the monolayer reached confluency. These results prompted us to examine whether overexpression of Cx26 affects barrier function. Monolayers of Caco-2 cells stably expressing Cx26 showed significantly lower mannitol permeability and higher TER than mock transfectants when the monolayers were chemically disrupted. The levels of claudin-4, an important component of tight junctions, were significantly increased in the stable Cx26 transfectant. These results suggest that Cx26-mediated GJIC may play a crucial role in enhancing the barrier function of Caco-2 cell monolayers

Chlorpromazine reduces the intercellular communication via gap junctions in mammalian cells

International Nuclear Information System (INIS)

Orellana, Juan A.; Palacios-Prado, Nicolas; Saez, Juan C.

2006-01-01

In the work presented herein, we evaluated the effect of chlorpromazine (CPZ) on gap junctions expressed by two mammalian cell types; Gn-11 cells (cell line derived from mouse LHRH neurons) and rat cortical astrocytes maintained in culture. We also attempted to elucidate possible mechanisms of action of CPZ effects on gap junctions. CPZ, in concentrations comparable with doses used to treat human diseases, was found to reduce the intercellular communication via gap junctions as evaluated with measurements of dye coupling (Lucifer yellow). In both cell types, maximal inhibition of functional gap junctions was reached within about 1 h of treatment with CPZ, an recovery was almost complete at about 5 h after CPZ wash out. In both cell types, CPZ treatment increased the phosphorylation state of connexin43 (Cx43), a gap junction protein subunit. Moreover, CPZ reduced the reactivity of Cx43 (immunofluorescence) at cell interfaces and concomitantly increased its reactivity in intracellular vesicles, suggesting an increased retrieval from and/or reduced insertion into the plasma membrane. CPZ also caused cellular retraction reducing cell-cell contacts in a reversible manner. The reduction in contact area might destabilize existing gap junctions and abrogate formation of new ones. Moreover, the CPZ-induced reduction in gap junctional communication may depend on the connexins (Cxs) forming the junctions. If Cx43 were the only connexin expressed, MAPK-dependent phosphorylation of this connexin would induce closure of gap junction channels

Participation of intercellular communication and intracellular signal transduction in the radio-adaptive response of human fibroblastic cells

International Nuclear Information System (INIS)

Ishii, Keiichiro; Hoshi, Yuko; Iwasaki, Toshiyasu; Watanabe, Masami

1997-01-01

To investigate the radio-adaptive response of normal cells to low-dose radiation, we irradiated human embryonic cells with low-dose X-rays and examined the changes in sensitivity to subsequent high-dose X-irradiation. When the cells were irradiated by 200 cGy, the growth ratio of the viable cells five days after the irradiation decreased to 37% of that of the cells which received no X-irradiation. When the cells received a conditioning irradiation of 10 to 20 cGy four hours before the irradiation of 200 cGy, the growth ratio increased significantly to 45-53%, and a peak was reached at a conditioning dose of 13 cGy. Cells blocked off intercellular communication either in Ca 2+ ion-free medium or in TPA added medium during the conditioning irradiation of 13 cGy did not show the improvement of growth ratio. Addition of H-7, as an inhibitor of PKC, to the medium during the conditioning irradiation inhibited the induction of the radio-adaptive response. However, addition of either inhibitor of A kinase, H-89, or inhibitor of G kinase, H-8, failed to inhibit the induction of the radio-adaptive response. These results suggest that: (1) normal cells show an adaptive response to low-dose radiation, (2) intercellular communication may play a role in radio-adaptive responses, (3) the transduction of the signal induced in cells by low-dose X-irradiation via protein kinase C was involved in radio-adaptive responses, not via A kinase nor G kinase. (author)

Extracellular ultrathin fibers sensitive to intracellular reactive oxygen species: Formation of intercellular membrane bridges

Energy Technology Data Exchange (ETDEWEB)

Jung, Se-Hui; Park, Jin-Young; Joo, Jung-Hoon; Kim, Young-Myeong; Ha, Kwon-Soo, E-mail: ksha@kangwon.ac.kr

2011-07-15

Membrane bridges are key cellular structures involved in intercellular communication; however, dynamics for their formation are not well understood. We demonstrated the formation and regulation of novel extracellular ultrathin fibers in NIH3T3 cells using confocal and atomic force microscopy. At adjacent regions of neighboring cells, phorbol 12-myristate 13-acetate (PMA) and glucose oxidase induced ultrathin fiber formation, which was prevented by Trolox, a reactive oxygen species (ROS) scavenger. The height of ROS-sensitive ultrathin fibers ranged from 2 to 4 nm. PMA-induced formation of ultrathin fibers was inhibited by cytochalasin D, but not by Taxol or colchicine, indicating that ultrathin fibers mainly comprise microfilaments. PMA-induced ultrathin fibers underwent dynamic structural changes, resulting in formation of intercellular membrane bridges. Thus, these fibers are formed by a mechanism(s) involving ROS and involved in formation of intercellular membrane bridges. Furthermore, ultrastructural imaging of ultrathin fibers may contribute to understanding the diverse mechanisms of cell-to-cell communication and the intercellular transfer of biomolecules, including proteins and cell organelles.

Inhibition of Connexin 26/43 and Extracellular-Regulated Kinase Protein Plays a Critical Role in Melatonin Facilitated Gap Junctional Intercellular Communication in Hydrogen Peroxide-Treated HaCaT Keratinocyte Cells

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Hyo-Jung Lee

2012-01-01

Full Text Available Though melatonin was known to regulate gap junctional intercellular communication (GJIC in chick astrocytes and mouse hepatocytes, the underlying mechanism by melatonin was not elucidated in hydrogen peroxide- (H2O2- treated HaCaT keratinocyte cells until now. In the current study, though melatonin at 2 mM and hydrogen peroxide (H2O2 at 300 μM showed weak cytotoxicity in HaCaT keratinocyte cells, melatonin significantly suppressed the formation of reactive oxygen species (ROS in H2O2-treated HaCaT cells compared to untreated controls. Also, the scrape-loading dye-transfer assay revealed that melatonin enhances the intercellular communication by introducing Lucifer Yellow into H2O2-treated cells. Furthermore, melatonin significantly enhanced the expression of connexin 26 (Cx26 and connexin 43 (Cx43 at mRNA and protein levels, but not that of connexin 30 (Cx30 in H2O2-treated HaCaT cells. Of note, melatonin attenuated the phosphorylation of extracellular signal-regulated protein kinases (ERKs more than p38 MAPK or JNK in H2O2-treated HaCaT cells. Conversely, ERK inhibitor PD98059 promoted the intercellular communication in H2O2-treated HaCaT cells. Furthermore, combined treatment of melatonin (200 μM and vitamin C (10 μg/mL significantly reduced ROS production in H2O2-treated HaCaT cells. Overall, these findings support the scientific evidences that melatonin facilitates gap junctional intercellular communication in H2O2-treated HaCaT keratinocyte cells via inhibition of connexin 26/43 and ERK as a potent chemopreventive agent.

Studies of Bystander Effect and Intercellular Communication in Human Epithelial Cell Cultures Irradiated with X-rays

International Nuclear Information System (INIS)

Romppanen, E.; Trott, K. R.; Musatonen, R.; Leszcznski, D.; Belyakov, O.

2004-01-01

The bystander effect is a phenomenon whereby biological consequences of irradiation are expressed in nonexposed cells in the vicinity of exposed cells. Two main pathways have been proposed to mediate the bystander effect: Gap Junction Intercellular Communication (GJIC) and medium borne soluble factors dependent mechanisms. The present study was designed to evaluate the relative contributions of gap junction intercellular communication and of soluble extracellular factors on the bystander effects of low dose X-ray irradiation. HaCaT human epithelial cell monolayers were exposed to X-ray using specially constructed shield, which cover 95% or 56% or 0% of the cells from the radiation. To evaluate whether the GJIC is involved in transmission of the bystander signal from irradiated to nonirradiated cells, irradiations were performed in presence or absence of GJIC inhibitor lindane. The cytochalasin B block technique was used to quantify fractions of micronucleated cells 48 hours after the irradiation. Our results suggest that more micronucleated cells are induced in partially shielded monolayers than expected according to back extrapolation of the data from open field irradiation. Treatment with lindane considerably reduced amount of the bystander damage. We demonstrated that fraction of micronucleated cells after X-rays irradiation of 5% of cells with 1 Gy was 0.07±0.08 (without lindane) and 0.05±0.004 (in presence of lindane). Irradiation of 100% of cells with the same dose resulted in 0.023±0.04 /without lindane) and 0.013±0.02 (in presence of lindane) fractions of micronucleated cells. Comparison with open field data showed that the fraction of micronucleated cells after irradiation of 5% of the cell culture was 5-10 times greater than the estimated fraction assuming no bystander effect. Irradiation of 44% of cells ded not demonstrate a pronounced bystander effect. (Author) 20 refs

Intercellular protein-protein interactions at synapses.

Science.gov (United States)

Yang, Xiaofei; Hou, Dongmei; Jiang, Wei; Zhang, Chen

2014-06-01

Chemical synapses are asymmetric intercellular junctions through which neurons send nerve impulses to communicate with other neurons or excitable cells. The appropriate formation of synapses, both spatially and temporally, is essential for brain function and depends on the intercellular protein-protein interactions of cell adhesion molecules (CAMs) at synaptic clefts. The CAM proteins link pre- and post-synaptic sites, and play essential roles in promoting synapse formation and maturation, maintaining synapse number and type, accumulating neurotransmitter receptors and ion channels, controlling neuronal differentiation, and even regulating synaptic plasticity directly. Alteration of the interactions of CAMs leads to structural and functional impairments, which results in many neurological disorders, such as autism, Alzheimer's disease and schizophrenia. Therefore, it is crucial to understand the functions of CAMs during development and in the mature neural system, as well as in the pathogenesis of some neurological disorders. Here, we review the function of the major classes of CAMs, and how dysfunction of CAMs relates to several neurological disorders.

Modulatory effects of cAMP and PKC activation on gap junctional intercellular communication among thymic epithelial cells

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Neves-dos-Santos Sandra

2010-01-01

Full Text Available Abstract Background We investigated the effects of the signaling molecules, cyclic AMP (cAMP and protein-kinase C (PKC, on gap junctional intercellular communication (GJIC between thymic epithelial cells (TEC. Results Treatment with 8-Br-cAMP, a cAMP analog; or forskolin, which stimulates cAMP production, resulted in an increase in dye transfer between adjacent TEC, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. These treatments also increased Cx43 mRNA expression, and stimulated Cx43 protein accumulation in regions of intercellular contacts. VIP, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested. The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators. Stimulation of PKC by phorbol-myristate-acetate inhibited inter-TEC GJIC. Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations. Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC. Conclusions Overall, our data indicate that cAMP and PKC intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. This control is phylogenetically conserved in the thymus, since it was seen in both mouse and human TEC preparations. Lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.

Distinct moieties underlie biphasic H+ gating of connexin43 channels, producing a pH optimum for intercellular communication

Science.gov (United States)

Garciarena, Carolina D.; Malik, Akif; Swietach, Pawel; Moreno, Alonso P.; Vaughan-Jones, Richard D.

2018-01-01

Most mammalian cells can intercommunicate via connexin-assembled, gap-junctional channels. To regulate signal transmission, connexin (Cx) channel permeability must respond dynamically to physiological and pathophysiological stimuli. One key stimulus is intracellular pH (pHi), which is modulated by a tissue’s metabolic and perfusion status. Our understanding of the molecular mechanism of H+ gating of Cx43 channels—the major isoform in the heart and brain—is incomplete. To interrogate the effects of acidic and alkaline pHi on Cx43 channels, we combined voltage-clamp electrophysiology with pHi imaging and photolytic H+ uncaging, performed over a range of pHi values. We demonstrate that Cx43 channels expressed in HeLa or N2a cell pairs are gated biphasically by pHi via a process that consists of activation by H+ ions at alkaline pHi and inhibition at more acidic pHi. For Cx43 channel–mediated solute/ion transmission, the ensemble of these effects produces a pHi optimum, near resting pHi. By using Cx43 mutants, we demonstrate that alkaline gating involves cysteine residues of the C terminus and is independent of motifs previously implicated in acidic gating. Thus, we present a molecular mechanism by which cytoplasmic acid–base chemistry fine tunes intercellular communication and establishes conditions for the optimal transmission of solutes and signals in tissues, such as the heart and brain.—Garciarena, C. D., Malik, A., Swietach, P., Moreno, A. P., Vaughan-Jones, R. D. Distinct moieties underlie biphasic H+ gating of connexin43 channels, producing a pH optimum for intercellular communication. PMID:29183963

Silver nanoparticles increase connexin43-mediated gap junctional intercellular communication in HaCaT cells through activation of reactive oxygen species and mitogen-activated protein kinase signal pathway

DEFF Research Database (Denmark)

Qin, Yu; Han, Limin; Yang, Di

2018-01-01

Silver nanoparticles (AgNPs) are widely used in health and consumer products that routinely contact skin. However, the biological effects and possible mechanisms of AgNPs on skin remain unclear. Gap junctional intercellular communication (GJIC) plays a critical role in multicellular organisms to ...

Interaction between Na+/K+-pump and Na+/Ca2+-exchanger modulates intercellular communication.

Science.gov (United States)

Matchkov, Vladimir V; Gustafsson, Helena; Rahman, Awahan; Briggs Boedtkjer, Donna M; Gorintin, Sarah; Hansen, Anne Kirstine; Bouzinova, Elena V; Praetorius, Helle A; Aalkjaer, Christian; Nilsson, Holger

2007-04-13

Ouabain, a specific inhibitor of the Na(+)/K(+)-pump, has previously been shown to interfere with intercellular communication. Here we test the hypothesis that the communication between vascular smooth muscle cells is regulated through an interaction between the Na(+)/K(+)-pump and the Na(+)/Ca(2+)-exchanger leading to an increase in the intracellular calcium concentration ([Ca(2+)](i)) in discrete areas near the plasma membrane. [Ca(2+)](i) in smooth muscle cells was imaged in cultured rat aortic smooth muscle cell pairs (A7r5) and in rat mesenteric small artery segments simultaneously with force. In A7r5 coupling between cells was estimated by measuring membrane capacitance. Smooth muscle cells were uncoupled when the Na(+)/K(+)-pump was inhibited either by a low concentration of ouabain, which also caused a localized increase of [Ca(2+)](i) near the membrane, or by ATP depletion. Reduction of Na(+)/K(+)-pump activity by removal of extracellular potassium ([K(+)](o)) also uncoupled cells, but only after inhibition of K(ATP) channels. Inhibition of the Na(+)/Ca(2+)-exchange activity by SEA0400 or by a reduction of the equilibrium potential (making it more negative) also uncoupled the cells. Depletion of intracellular Na(+) and clamping of [Ca(2+)](i) at low concentrations prevented the uncoupling. The experiments suggest that the Na(+)/K(+)-pump may affect gap junction conductivity via localized changes in [Ca(2+)](i) through modulation of Na(+)/Ca(2+)-exchanger activity.

Heavy-ion-induced bystander killing of human lung cancer cells. Role of gap junctional intercellular communication

International Nuclear Information System (INIS)

Harada, Kosaku; Nonaka, Tetsuo; Hamada, Nobuyuki; Sakurai, Hideyuki; Hasegawa, Masatoshi; Kobayashi, Yasuhiko; Nakano, Takashi; Funayama, Tomoo; Kakizaki, Takehiko

2009-01-01

The aim of the present study was to clarify the mechanisms of cell death induced by heavy-ion irradiation focusing on the bystander effect in human lung cancer A549 cells. In microbeam irradiation, each of 1, 5, and 25 cells under confluent cell conditions was irradiated with 1, 5, or 10 particles of carbon ions (220 MeV), and then the surviving fraction of the population was measured by a clonogenic assay in order to investigate the bystander effect of heavy-ions. In this experiment, the limited number of cells (0.0001-0.002%, 5-25 cells) under confluent cell conditions irradiated with 5 or 10 carbon ions resulted in an exaggerated 8-14% increase in cell death by clonogenic assay. However, these overshooting responses were not observed under exponentially growing cell conditions. Furthermore, these responses were inhibited in cells treated with an inhibitor of gap junctional intercellular communication (GJIC), whereas they were markedly enhanced by the addition of a stimulator of GJIC. The present results suggest that bystander cell killing by heavy-ions was induced mainly by direct cell-to-cell communication, such as GJIC, which might play important roles in bystander responses. (author)

Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication.

Science.gov (United States)

Shender, Victoria O; Pavlyukov, Marat S; Ziganshin, Rustam H; Arapidi, Georgij P; Kovalchuk, Sergey I; Anikanov, Nikolay A; Altukhov, Ilya A; Alexeev, Dmitry G; Butenko, Ivan O; Shavarda, Alexey L; Khomyakova, Elena B; Evtushenko, Evgeniy; Ashrafyan, Lev A; Antonova, Irina B; Kuznetcov, Igor N; Gorbachev, Alexey Yu; Shakhparonov, Mikhail I; Govorun, Vadim M

2014-12-01

Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Transient suppression of gap junctional intercellular communication after exposure to 100-nanosecond pulsed electric fields.

Science.gov (United States)

Steuer, Anna; Schmidt, Anke; Labohá, Petra; Babica, Pavel; Kolb, Juergen F

2016-12-01

Gap junctional intercellular communication (GJIC) is an important mechanism that is involved and affected in many diseases and injuries. So far, the effect of nanosecond pulsed electric fields (nsPEFs) on the communication between cells was not investigated. An in vitro approach is presented with rat liver epithelial WB-F344 cells grown and exposed in a monolayer. In order to observe sub-lethal effects, cells were exposed to pulsed electric fields with a duration of 100ns and amplitudes between 10 and 20kV/cm. GJIC strongly decreased within 15min after treatment but recovered within 24h. Gene expression of Cx43 was significantly decreased and associated with a reduced total amount of Cx43 protein. In addition, MAP kinases p38 and Erk1/2, involved in Cx43 phosphorylation, were activated and Cx43 became hyperphosphorylated. Immunofluorescent staining of Cx43 displayed the disassembly of gap junctions. Further, a reorganization of the actin cytoskeleton was observed whereas tight junction protein ZO-1 was not significantly affected. All effects were field- and time-dependent and most pronounced within 30 to 60min after treatment. A better understanding of a possible manipulation of GJIC by nsPEFs might eventually offer a possibility to develop and improve treatments. Copyright © 2016 Elsevier B.V. All rights reserved.

Serum deprivation induces glucose response and intercellular coupling in human pancreatic adenocarcinoma PANC-1 cells.

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Hiram-Bab, Sahar; Shapira, Yuval; Gershengorn, Marvin C; Oron, Yoram

2012-03-01

This study aimed to investigate whether the previously described differentiating islet-like aggregates of human pancreatic adenocarcinoma cells (PANC-1) develop glucose response and exhibit intercellular communication. Fura 2-loaded PANC-1 cells in serum-free medium were assayed for changes in cytosolic free calcium ([Ca]i) induced by depolarization, tolbutamide inhibition of K(ATP) channels, or glucose. Dye transfer, assayed by confocal microscopy or by FACS, was used to detect intercellular communication. Changes in messenger RNA (mRNA) expression of genes of interest were assessed by quantitative real-time polymerase chain reaction. Proliferation was assayed by the MTT method. Serum-deprived PANC-1 cell aggregates developed [Ca]i response to KCl, tolbutamide, or glucose. These responses were accompanied by 5-fold increase in glucokinase mRNA level and, to a lesser extent, of mRNAs for K(ATP) and L-type calcium channels, as well as increase in mRNA levels of glucagon and somatostatin. Trypsin, a proteinase-activated receptor 2 agonist previously shown to enhance aggregation, modestly improved [Ca]i response to glucose. Glucose-induced coordinated [Ca]i oscillations and dye transfer demonstrated the emergence of intercellular communication. These findings suggest that PANC-1 cells, a pancreatic adenocarcinoma cell line, can be induced to express a differentiated phenotype in which cells exhibit response to glucose and form a functional syncytium similar to those observed in pancreatic islets.

1,4-Naphthoquinones: From Oxidative Damage to Cellular and Inter-Cellular Signaling

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Lars-Oliver Klotz

2014-09-01

Full Text Available Naphthoquinones may cause oxidative stress in exposed cells and, therefore, affect redox signaling. Here, contributions of redox cycling and alkylating properties of quinones (both natural and synthetic, such as plumbagin, juglone, lawsone, menadione, methoxy-naphthoquinones, and others to cellular and inter-cellular signaling processes are discussed: (i naphthoquinone-induced Nrf2-dependent modulation of gene expression and its potentially beneficial outcome; (ii the modulation of receptor tyrosine kinases, such as the epidermal growth factor receptor by naphthoquinones, resulting in altered gap junctional intercellular communication. Generation of reactive oxygen species and modulation of redox signaling are properties of naphthoquinones that render them interesting leads for the development of novel compounds of potential use in various therapeutic settings.

The intercellular cell adhesion molecule-1 (icam-1) in lung cancer: implications for disease progression and prognosis.

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Kotteas, Elias A; Boulas, Panagiotis; Gkiozos, Ioannis; Tsagkouli, Sofia; Tsoukalas, George; Syrigos, Konstantinos N

2014-09-01

The intercellular cell-adhesion molecule-1 (ICAM-1) is a transmembrane molecule and a distinguished member of the Immunoglobulin superfamily of proteins that participates in many important processes, including leukocyte endothelial transmigration, cell signaling, cell-cell interaction, cell polarity and tissue stability. ICAM-1and its soluble part are highly expressed in inflammatory conditions, chronic diseases and a number of malignancies. In the present article we present the implications of ICAM-1 in the progression and prognosis of one of the major global killers of our era: lung cancer. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

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Balda Maria S

2009-12-01

Full Text Available Abstract Background Tight junctions are an intercellular adhesion complex of epithelial and endothelial cells, and form a paracellular barrier that restricts the diffusion of solutes on the basis of size and charge. Tight junctions are formed by multiprotein complexes containing cytosolic and transmembrane proteins. How these components work together to form functional tight junctions is still not well understood and will require a complete understanding of the molecular composition of the junction. Results Here we identify a new transmembrane component of tight junctions: MarvelD3, a four-span transmembrane protein. Its predicted transmembrane helices form a Marvel (MAL and related proteins for vesicle traffic and membrane link domain, a structural motif originally discovered in proteins involved in membrane apposition and fusion events, such as the tight junction proteins occludin and tricellulin. In mammals, MarvelD3 is expressed as two alternatively spliced isoforms. Both isoforms exhibit a broad tissue distribution and are expressed by different types of epithelial as well as endothelial cells. MarvelD3 co-localises with occludin at tight junctions in intestinal and corneal epithelial cells. RNA interference experiments in Caco-2 cells indicate that normal MarvelD3 expression is not required for the formation of functional tight junctions but depletion results in monolayers with increased transepithelial electrical resistance. Conclusions Our data indicate that MarvelD3 is a third member of the tight junction-associated occludin family of transmembrane proteins. Similar to occludin, normal expression of MarvelD3 is not essential for the formation of functional tight junctions. However, MarvelD3 functions as a determinant of epithelial paracellular permeability properties.

Gap junction protein connexin-43 interacts directly with microtubules

NARCIS (Netherlands)

Giepmans, B N; Verlaan, I; Hengeveld, T; Janssen, H; Calafat, J; Falk, M M; Moolenaar, W H

2001-01-01

Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated

Mammalian Tissue Response to Low Dose Ionizing Radiation: The Role of Oxidative Metabolism and Intercellular Communication

Energy Technology Data Exchange (ETDEWEB)

Azzam, Edouard I

2013-01-16

The objective of the project was to elucidate the mechanisms underlying the biological effects of low dose/low dose rate ionizing radiation in organs/tissues of irradiated mice that differ in their susceptibility to ionizing radiation, and in human cells grown under conditions that mimic the natural in vivo environment. The focus was on the effects of sparsely ionizing cesium-137 gamma rays and the role of oxidative metabolism and intercellular communication in these effects. Four Specific Aims were proposed. The integrated outcome of the experiments performed to investigate these aims has been significant towards developing a scientific basis to more accurately estimate human health risks from exposures to low doses ionizing radiation. By understanding the biochemical and molecular changes induced by low dose radiation, several novel markers associated with mitochondrial functions were identified, which has opened new avenues to investigate metabolic processes that may be affected by such exposure. In particular, a sensitive biomarker that is differentially modulated by low and high dose gamma rays was discovered.

Laminin-332 alters connexin profile, dye coupling and intercellular Ca2+ waves in ciliated tracheal epithelial cells

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Olsen Colin E

2006-08-01

Full Text Available Abstract Background Tracheal epithelial cells are anchored to a dynamic basement membrane that contains a variety of extracellular matrix proteins including collagens and laminins. During development, wound repair and disease of the airway epithelium, significant changes in extracellular matrix proteins may directly affect cell migration, differentiation and events mediated by intercellular communication. We hypothesized that alterations in cell matrix, specifically type I collagen and laminin α3β3γ2 (LM-332 proteins within the matrix, directly affect intercellular communication in ciliated rabbit tracheal epithelial cells (RTEC. Methods Functional coupling of RTEC was monitored by microinjection of the negatively charged fluorescent dyes, Lucifer Yellow and Alexa 350, into ciliated RTEC grown on either a LM-332/collagen or collagen matrix. Coupling of physiologically significant molecules was evaluated by the mechanism and extent of propagated intercellular Ca2+ waves. Expression of connexin (Cx mRNA and proteins were assayed by reverse transcriptase – polymerase chain reaction and immunocytochemistry, respectively. Results When compared to RTEC grown on collagen alone, RTEC grown on LM-332/collagen displayed a significant increase in dye transfer. Although mechanical stimulation of RTEC grown on either LM-332/collagen or collagen alone resulted in intercellular Ca2+ waves, the mechanism of transfer was dependent on matrix: RTEC grown on LM-332/collagen propagated Ca2+waves via extracellular purinergic signaling whereas RTEC grown on collagen used gap junctions. Comparison of RTEC grown on collagen or LM-332/collagen matrices revealed a reorganization of Cx26, Cx43 and Cx46 proteins. Conclusion Alterations in airway basement membrane proteins such as LM-332 can induce connexin reorganizations and result in altered cellular communication mechanisms that could contribute to airway tissue function.

Methoxychlor and vinclozolin induce rapid changes in intercellular and intracellular signaling in liver progenitor cells

Czech Academy of Sciences Publication Activity Database

Babica, Pavel; Zurabian, R.; Kumar, E. R.; Chopra, R.; Mianecki, M. J.; Park, J.-S.; Jaša, Libor; Trosko, J. E.; Upham, B. L.

2016-01-01

Roč. 153, č. 1 (2016), s. 174-185 ISSN 1096-6080 R&D Projects: GA MŠk LH12034 Institutional support: RVO:67985939 Keywords : endocrine disrupters * gap junctional intercellular communication * resveratrol Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.081, year: 2016

The stability of the three transmembrane and the four transmembrane human vitamin K epoxide reductase models

Science.gov (United States)

Wu, Sangwook

2016-04-01

The three transmembrane and the four transmembrane helix models are suggested for human vitamin K epoxide reductase (VKOR). In this study, we investigate the stability of the human three transmembrane/four transmembrane VKOR models by employing a coarse-grained normal mode analysis and molecular dynamics simulation. Based on the analysis of the mobility of each transmembrane domain, we suggest that the three transmembrane human VKOR model is more stable than the four transmembrane human VKOR model.

TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication

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Li, Hanjun; Spagnol, Gaelle; Naslavsky, Naava; Caplan, Steve; Sorgen, Paul L.

2014-01-01

ABSTRACT Protein kinases have long been reported to regulate connexins; however, little is known about the involvement of phosphatases in the modulation of intercellular communication through gap junctions and the subsequent downstream effects on cellular processes. Here, we identify an interaction between the T-cell protein tyrosine phosphatase (TC-PTP, officially known as PTPN2) and the carboxyl terminus of connexin43 (Cx43, officially known as GJA1). Two cell lines, normal rat kidney (NRK) cells endogenously expressing Cx43 and an NRK-derived cell line expressing v-Src with temperature-sensitive activity, were used to demonstrate that EGF and v-Src stimulation, respectively, induced TC-PTP to colocalize with Cx43 at the plasma membrane. Cell biology experiments using phospho-specific antibodies and biophysical assays demonstrated that the interaction is direct and that TC-PTP dephosphorylates Cx43 residues Y247 and Y265, but does not affect v-Src. Transfection of TC-PTP also indirectly led to the dephosphorylation of Cx43 S368, by inactivating PKCα and PKCδ, with no effect on the phosphorylation of S279 and S282 (MAPK-dependent phosphorylation sites). Dephosphorylation maintained Cx43 gap junctions at the plaque and partially reversed the channel closure caused by v-Src-mediated phosphorylation of Cx43. Understanding dephosphorylation, along with the well-documented roles of Cx43 phosphorylation, might eventually lead to methods to modulate the regulation of gap junction channels, with potential benefits for human health. PMID:24849651

Gap junction intercellular communication mediated by connexin43 in astrocytes is essential for their resistance to oxidative stress.

Science.gov (United States)

Le, Hoa T; Sin, Wun Chey; Lozinsky, Shannon; Bechberger, John; Vega, José Luis; Guo, Xu Qiu; Sáez, Juan C; Naus, Christian C

2014-01-17

Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H2O2) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H2O2 transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H2O2-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H2O2 insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H2O2 treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.

Exploring the role of lipids in intercellular conduits: breakthroughs in the pipeline

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Elise eDelage

2013-12-01

Full Text Available It has been known for more than a century that most of the plant cells are connected to their neighbors through membranous pores perforating the cell wall, namely plasmodesmata (PDs. The recent discovery of tunneling nanotubes (TNTs, thin membrane bridges established between distant mammalian cells, suggests that intercellular communication mediated through cytoplasmic continuity could be a conserved feature of eukaryotic organisms. Although TNTs differ from PDs in their formation and architecture, both are characterized by a continuity of the plasma membrane between two cells, delimiting a nanotubular channel supported by actin-based cytoskeleton. Due to this unusual membrane organization, lipids are likely to play critical roles in the formation and stability of intercellular conduits like TNTs and PDs, but also in regulating the transfer through these structures. While it is crucial for a better understanding of those fascinating communication highways, the study of TNT lipid composition and dynamics turned out to be extremely challenging. The present review aims to give an overview of the recent findings in this context. We will also discuss some of the promising imaging approaches, which might be the key for future breakthroughs in the field and could also benefit the research on PDs.

The antiarrhythmic peptide analog rotigaptide (ZP123) stimulates gap junction intercellular communication in human osteoblasts and prevents decrease in femoral trabecular bone strength in ovariectomized rats

DEFF Research Database (Denmark)

Jørgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne

2005-01-01

Gap junctions play an important role in bone development and function, but the lack of pharmacological tools has hampered the gap junction research. The antiarrhythmic peptides stimulate gap junction communication between cardiomyocytes, but effects in noncardiac tissue are unknown. The purpose...... of this study was to examine whether antiarrhythmic peptides, which are small peptides increasing gap junctional conductivity, show specific binding to osteoblasts and investigate the effect of the stable analog rotigaptide (ZP123) on gap junctional intercellular communication in vitro and on bone mass...... and strength in vivo. Cell coupling and calcium signaling were assessed in vitro on human, primary, osteoblastic cells. In vivo effects of rotigaptide on bone strength and density were determined 4 wk after ovariectomy in rats treated with either vehicle, sc injection twice daily (300 nmol per kilogram body...

Stat3 is a positive regulator of gap junctional intercellular communication in cultured, human lung carcinoma cells

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Geletu Mulu

2012-12-01

Full Text Available Abstract Background Neoplastic transformation of cultured cells by a number of oncogenes such as src suppresses gap junctional, intercellular communication (GJIC; however, the role of Src and its effector Signal transducer and activator of transcription-3 (Stat3 upon GJIC in non small cell lung cancer (NSCLC has not been defined. Immunohistochemical analysis revealed high Src activity in NSCLC biopsy samples compared to normal tissues. Here we explored the potential effect of Src and Stat3 upon GJIC, by assessing the levels of tyr418-phosphorylated Src and tyr705-phosphorylated Stat3, respectively, in a panel of NSCLC cell lines. Methods Gap junctional communication was examined by electroporating the fluorescent dye Lucifer yellow into cells grown on a transparent electrode, followed by observation of the migration of the dye to the adjacent, non-electroporated cells under fluorescence illumination. Results An inverse relationship between Src activity levels and GJIC was noted; in five lines with high Src activity GJIC was absent, while two lines with extensive GJIC (QU-DB and SK-LuCi6 had low Src levels, similar to a non-transformed, immortalised lung epithelial cell line. Interestingly, examination of the mechanism indicated that Stat3 inhibition in any of the NSCLC lines expressing high endogenous Src activity levels, or in cells where Src was exogenously transduced, did not restore GJIC. On the contrary, Stat3 downregulation in immortalised lung epithelial cells or in the NSCLC lines displaying extensive GJIC actually suppressed junctional permeability. Conclusions Our findings demonstrate that although Stat3 is generally growth promoting and in an activated form it can act as an oncogene, it is actually required for gap junctional communication both in nontransformed lung epithelial cells and in certain lung cancer lines that retain extensive GJIC.

Cell-to-cell communication in plants, animals, and fungi: a comparative review.

Science.gov (United States)

Bloemendal, Sandra; Kück, Ulrich

2013-01-01

Cell-to-cell communication is a prerequisite for differentiation and development in multicellular organisms. This communication has to be tightly regulated to ensure that cellular components such as organelles, macromolecules, hormones, or viruses leave the cell in a precisely organized way. During evolution, plants, animals, and fungi have developed similar ways of responding to this biological challenge. For example, in higher plants, plasmodesmata connect adjacent cells and allow communication to regulate differentiation and development. In animals, two main general structures that enable short- and long-range intercellular communication are known, namely gap junctions and tunneling nanotubes, respectively. Finally, filamentous fungi have also developed specialized structures called septal pores that allow intercellular communication via cytoplasmic flow. This review summarizes the underlying mechanisms for intercellular communication in these three eukaryotic groups and discusses its consequences for the regulation of differentiation and developmental processes.

Intra- and Intercellular Quality Control Mechanisms of Mitochondria

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Yoshimitsu Kiriyama

2017-12-01

Full Text Available Mitochondria function to generate ATP and also play important roles in cellular homeostasis, signaling, apoptosis, autophagy, and metabolism. The loss of mitochondrial function results in cell death and various types of diseases. Therefore, quality control of mitochondria via intra- and intercellular pathways is crucial. Intracellular quality control consists of biogenesis, fusion and fission, and degradation of mitochondria in the cell, whereas intercellular quality control involves tunneling nanotubes and extracellular vesicles. In this review, we outline the current knowledge on the intra- and intercellular quality control mechanisms of mitochondria.

Role of protein dynamics in transmembrane receptor signalling

DEFF Research Database (Denmark)

Wang, Yong; Bugge, Katrine Østergaard; Kragelund, Birthe Brandt

2018-01-01

Cells are dependent on transmembrane receptors to communicate and transform chemical and physical signals into intracellular responses. Because receptors transport 'information', conformational changes and protein dynamics play a key mechanistic role. We here review examples where experiment...... to function. Because the receptors function in a heterogeneous environment and need to be able to switch between distinct functional states, they may be particularly sensitive to small perturbations that complicate studies linking dynamics to function....

Intercellular Communication between Keratinocytes and Fibroblasts Induces Local Osteoclast Differentiation: a Mechanism Underlying Cholesteatoma-Induced Bone Destruction.

Science.gov (United States)

Iwamoto, Yoriko; Nishikawa, Keizo; Imai, Ryusuke; Furuya, Masayuki; Uenaka, Maki; Ohta, Yumi; Morihana, Tetsuo; Itoi-Ochi, Saori; Penninger, Josef M; Katayama, Ichiro; Inohara, Hidenori; Ishii, Masaru

2016-06-01

Bone homeostasis is maintained by a balance in activity between bone-resorbing osteoclasts and bone-forming osteoblasts. Shifting the balance toward bone resorption causes osteolytic bone diseases such as rheumatoid arthritis and periodontitis. Osteoclast differentiation is regulated by receptor activator of nuclear factor κB ligand (RANKL), which, under some pathological conditions, is produced by T and B lymphocytes and synoviocytes. However, the mechanism underlying bone destruction in other diseases is little understood. Bone destruction caused by cholesteatoma, an epidermal cyst in the middle ear resulting from hyperproliferation of keratinizing squamous epithelium, can lead to lethal complications. In this study, we succeeded in generating a model for cholesteatoma, epidermal cyst-like tissue, which has the potential for inducing osteoclastogenesis in mice. Furthermore, an in vitro coculture system composed of keratinocytes, fibroblasts, and osteoclast precursors was used to demonstrate that keratinocytes stimulate osteoclast differentiation through the induction of RANKL in fibroblasts. Thus, this study demonstrates that intercellular communication between keratinocytes and fibroblasts is involved in the differentiation and function of osteoclasts, which may provide the molecular basis of a new therapeutic strategy for cholesteatoma-induced bone destruction. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The B[a]P-increased intercellular communication via translocation of connexin-43 into gap junctions reduces apoptosis

International Nuclear Information System (INIS)

Tekpli, X.; Rivedal, E.; Gorria, M.; Landvik, N.E.; Rissel, M.; Dimanche-Boitrel, M.-T.; Baffet, G.; Holme, J.A.; Lagadic-Gossmann, D.

2010-01-01

Gap junctions are channels in plasma membrane composed of proteins called connexins. These channels are organized in special domains between cells, and provide for direct gap junctional intercellular communication (GJIC), allowing diffusion of signalling molecules < 1 kD. GJIC regulates cell homeostasis and notably the balance between proliferation, cell cycle arrest, cell survival and apoptosis. Here, we have investigated benzo[a]pyrene (B[a]P) effects on GJIC and on the subcellular localization of the major protein of gap junction: connexin-43 (Cx43). Our results showed that B[a]P increased GJIC between mouse hepatoma Hepa1c1c7 cells via translocation of Cx43 from Golgi apparatus and lipid rafts into gap junction plaques. Interestingly, inhibition of GJIC by chlordane or small interference RNA directed against Cx43 enhanced B[a]P-induced apoptosis in Hepa1c1c7 cells. The increased apoptosis caused by inhibition of GJIC appeared to be mediated by ERK/MAPK pathway. It is suggested that B[a]P could induce transfer of cell survival signal or dilute cell death signal via regulation of ERK/MAPK through GJIC.

Extract from the Zooxanthellate Jellyfish Cotylorhiza tuberculata Modulates Gap Junction Intercellular Communication in Human Cell Cultures

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Stefano Piraino

2013-05-01

Full Text Available On a global scale, jellyfish populations in coastal marine ecosystems exhibit increasing trends of abundance. High-density outbreaks may directly or indirectly affect human economical and recreational activities, as well as public health. As the interest in biology of marine jellyfish grows, a number of jellyfish metabolites with healthy potential, such as anticancer or antioxidant activities, is increasingly reported. In this study, the Mediterranean “fried egg jellyfish” Cotylorhiza tuberculata (Macri, 1778 has been targeted in the search forputative valuable bioactive compounds. A medusa extract was obtained, fractionated, characterized by HPLC, GC-MS and SDS-PAGE and assayed for its biological activity on breast cancer cells (MCF-7 and human epidermal keratinocytes (HEKa. The composition of the jellyfish extract included photosynthetic pigments, valuable ω-3 and ω-6 fatty acids, and polypeptides derived either from jellyfish tissues and their algal symbionts. Extract fractions showed antioxidant activity and the ability to affect cell viability and intercellular communication mediated by gap junctions (GJIC differentially in MCF-7and HEKa cells. A significantly higher cytotoxicity and GJIC enhancement in MCF-7 compared to HEKa cells was recorded. A putative action mechanism for the anticancer bioactivity through the modulation of GJIC has been hypothesized and its nutraceutical and pharmaceutical potential was discussed.

Connexin-based intercellular communication and astrocyte heterogeneity.

Science.gov (United States)

Theis, Martin; Giaume, Christian

2012-12-03

This review gives an overview of the current knowledge on connexin-mediated communication in astrocytes, covering gap junction and hemichannel functions mediated by connexins. Astroglia is the main brain cell type that expresses the largest amount of connexin and exhibits high level of gap junctional communication compared to neurons and oligodendrocytes. However, in certain developmental and regional situations, astrocytes are also coupled with oligodendrocytes and neurons. This heterotypic coupling is infrequent and minor in terms of extent of the coupling area, which does not mean that it is not important in terms of cell interaction. Here, we present an update on heterogeneity of connexin expression and function at the molecular, subcellular, cellular and networking levels. Interestingly, while astrocytes were initially considered as a homogenous population, there is now increasing evidence for morphological, developmental, molecular and physiological heterogeneity of astrocytes. Consequently, the specificity of gap junction channel- and hemichannel-mediated communication, which tends to synchronize cell populations, is also a parameter to take into account when neuroglial interactions are investigated. This article is part of a Special Issue entitled Electrical Synapses. Copyright © 2012 Elsevier B.V. All rights reserved.

Transmembrane Signaling Proteoglycans

DEFF Research Database (Denmark)

Couchman, John R

2010-01-01

Virtually all metazoan cells contain at least one and usually several types of transmembrane proteoglycans. These are varied in protein structure and type of polysaccharide, but the total number of vertebrate genes encoding transmembrane proteoglycan core proteins is less than 10. Some core prote...... proteins, including those of the syndecans, always possess covalently coupled glycosaminoglycans; others do not. Syndecan has a long evolutionary history, as it is present in invertebrates, but many other transmembrane proteoglycans are vertebrate inventions. The variety of proteins...... proteins has been obtained in mouse knockout experiments. Here some of the latest developments in the field are examined in hopes of stimulating further interest in this fascinating group of molecules. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 26...

Possible Mechanisms of Mercury Toxicity and Cancer Promotion: Involvement of Gap Junction Intercellular Communications and Inflammatory Cytokines

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Roberto Zefferino

2017-01-01

Full Text Available A number of observations indicate that heavy metals are able to alter cellular metabolic pathways through induction of a prooxidative state. Nevertheless, the outcome of heavy metal-mediated effects in the development of human diseases is debated and needs further insights. Cancer is a well-established DNA mutation-linked disease; however, epigenetic events are perhaps more important and harmful than genetic alterations. Unfortunately, we do not have reliable screening methods to assess/validate the epigenetic (promoter effects of a physical or a chemical agent. We propose a mechanism of action whereby mercury acts as a possible promoter carcinogen. In the present contribution, we resume our previous studies on mercury tested at concentrations comparable with its occurrence as environmental pollutant. It is shown that Hg(II elicits a prooxidative state in keratinocytes linked to inhibition of gap junction-mediated intercellular communication and proinflammatory cytokine production. These combined effects may on one hand isolate cells from tissue-specific homeostasis promoting their proliferation and on the other hand tamper the immune system defense/surveillance checkmating the whole organism. Since Hg(II is not a mutagenic/genotoxic compound directly affecting gene expression, in a broader sense, mercury might be an example of an epigenetic tumor promoter or, further expanding this concept, a “metagenetic” effector.

Identification and characterization of RBM44 as a novel intercellular bridge protein.

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Tokuko Iwamori

2011-02-01

Full Text Available Intercellular bridges are evolutionarily conserved structures that connect differentiating germ cells. We previously reported the identification of TEX14 as the first essential intercellular bridge protein, the demonstration that intercellular bridges are required for male fertility, and the finding that intercellular bridges utilize components of the cytokinesis machinery to form. Herein, we report the identification of RNA binding motif protein 44 (RBM44 as a novel germ cell intercellular bridge protein. RBM44 was identified by proteomic analysis after intercellular bridge enrichment using TEX14 as a marker protein. RBM44 is highly conserved between mouse and human and contains an RNA recognition motif of unknown function. RBM44 mRNA is enriched in testis, and immunofluorescence confirms that RBM44 is an intercellular bridge component. However, RBM44 only partially localizes to TEX14-positive intercellular bridges. RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids. We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation. To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice. Rbm44 null male mice produce somewhat increased sperm, and show enhanced fertility of unknown etiology. Thus, although RBM44 localizes to intercellular bridges during meiosis, RBM44 is not required for fertility in contrast to TEX14.

Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity.

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Lai, Julian; Koh, Chuan Hock; Tjota, Monika; Pieuchot, Laurent; Raman, Vignesh; Chandrababu, Karthik Balakrishna; Yang, Daiwen; Wong, Limsoon; Jedd, Gregory

2012-09-25

Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity.

Phloroglucinol functions as an intracellular and intercellular chemical messenger influencing gene expression in Pseudomonas protegens.

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Clifford, Jennifer C; Buchanan, Alex; Vining, Oliver; Kidarsa, Teresa A; Chang, Jeff H; McPhail, Kerry L; Loper, Joyce E

2016-10-01

Bacteria can be both highly communicative and highly competitive in natural habitats and antibiotics are thought to play a role in both of these processes. The soil bacterium Pseudomonas protegens Pf-5 produces a spectrum of antibiotics, two of which, pyoluteorin and 2,4-diacetylphloroglucinol (DAPG), function in intracellular and intercellular communication, both as autoinducers of their own production. Here, we demonstrate that phloroglucinol, an intermediate in DAPG biosynthesis, can serve as an intercellular signal influencing the expression of pyoluteorin biosynthesis genes, the production of pyoluteorin, and inhibition of Pythium ultimum, a phytopathogenic oomycete sensitive to pyoluteorin. Through analysis of RNAseq data sets, we show that phloroglucinol had broad effects on the transcriptome of Pf-5, significantly altering the transcription of more than two hundred genes. The effects of nanomolar versus micromolar concentrations of phloroglucinol differed both quantitatively and qualitatively, influencing the expression of distinct sets of genes or having opposite effects on transcript abundance of certain genes. Therefore, our results support the concept of hormesis, a phenomenon associated with signalling molecules that elicit distinct responses at different concentrations. Phloroglucinol is the first example of an intermediate of antibiotic biosynthesis that functions as a chemical messenger influencing gene expression in P. protegens. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

The Impact of the ‘Austrian’ Mutation of the Amyloid Precursor Protein Transmembrane Helix is Communicated to the Hinge Region

DEFF Research Database (Denmark)

Stelzer, Walter; Scharnagl, Christina; Leurs, Ulrike

2016-01-01

The transmembrane helix of the amyloid precursor protein is subject to proteolytic cleavages by γ-secretase at different sites resulting in Aβ peptides of different length and toxicity. A number of point mutations within this transmembrane helix alter the cleavage pattern thus enhancing production...... destabilizes amide hydrogen bonds in the hinge which connects dimerization and cleavage regions. Weaker intrahelical hydrogen bonds at the hinge may enhance helix bending and thereby affect recognition of the transmembrane substrate by the enzyme and/or presentation of its cleavage sites to the catalytic cleft....

Intercellular Ca2+ Waves: Mechanisms and Function

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Sanderson, Michael J.

2012-01-01

Intercellular calcium (Ca2+) waves (ICWs) represent the propagation of increases in intracellular Ca2+ through a syncytium of cells and appear to be a fundamental mechanism for coordinating multicellular responses. ICWs occur in a wide diversity of cells and have been extensively studied in vitro. More recent studies focus on ICWs in vivo. ICWs are triggered by a variety of stimuli and involve the release of Ca2+ from internal stores. The propagation of ICWs predominately involves cell communication with internal messengers moving via gap junctions or extracellular messengers mediating paracrine signaling. ICWs appear to be important in both normal physiology as well as pathophysiological processes in a variety of organs and tissues including brain, liver, retina, cochlea, and vascular tissue. We review here the mechanisms of initiation and propagation of ICWs, the key intra- and extracellular messengers (inositol 1,4,5-trisphosphate and ATP) mediating ICWs, and the proposed physiological functions of ICWs. PMID:22811430

Modelling of intercellular synchronization in the Drosophila circadian clock

International Nuclear Information System (INIS)

Jun-Wei, Wang; Ai-Min, Chen; Jia-Jun, Zhang; Zhan-Jiang, Yuan; Tian-Shou, Zhou

2009-01-01

In circadian rhythm generation, intercellular signaling factors are shown to play a crucial role in both sustaining intrinsic cellular rhythmicity and acquiring collective behaviours across a population of circadian neurons. However, the physical mechanism behind their role remains to be fully understood. In this paper, we propose an indirectly coupled multicellular model for the synchronization of Drosophila circadian oscillators combining both intracellular and intercellular dynamics. By simulating different experimental conditions, we find that such an indirect coupling way can synchronize both heterogeneous self-sustained circadian neurons and heterogeneous mutational damped circadian neurons. Moreover, they can also be entrained to ambient light-dark (LD) cycles depending on intercellular signaling. (cross-disciplinary physics and related areas of science and technology)

Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

DEFF Research Database (Denmark)

Jørgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne

2003-01-01

The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting...... in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal...... of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx...

Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y12/13 receptors among BV-2 microglia.

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Pengchong Jiang

Full Text Available Nerve injury is accompanied by a liberation of diverse nucleotides, some of which act as 'find/eat-me' signals in mediating neuron-glial interplay. Intercellular Ca2+ wave (ICW communication is the main approach by which glial cells interact and coordinate with each other to execute immune defense. However, the detailed mechanisms on how these nucleotides participate in ICW communication remain largely unclear. In the present work, we employed a mechanical stimulus to an individual BV-2 microglia to simulate localized injury. Remarkable ICW propagation was observed no matter whether calcium was in the environment or not. Apyrase (ATP/ADP-hydrolyzing enzyme, suramin (broad-spectrum P2 receptor antagonist, 2-APB (IP3 receptor blocker and thapsigargin (endoplasmic reticulum calcium pump inhibitor potently inhibited these ICWs, respectively, indicating the dependence of nucleotide signals and P2Y receptors. Then, we detected the involvement of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose by desensitizing receptors. Results showed that desensitization with ATP and ADP could block ICW propagation in a dose-dependent manner, whereas other nucleotides had little effect. Meanwhile, the expression of P2Y receptors in BV-2 microglia was identified and their contributions were analyzed, from which we suggested P2Y12/13 receptors activation mostly contributed to ICWs. Besides, we estimated that extracellular ATP and ADP concentration sensed by BV-2 microglia was about 0.3 μM during ICWs by analyzing calcium dynamic characteristics. Taken together, these results demonstrated that the nucleotides ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICW communication through acting on P2Y12/13 receptors in BV-2 microglia.

Estrogenic compounds inhibit gap junctional intercellular communication in mouse Leydig TM3 cells

International Nuclear Information System (INIS)

Iwase, Yumiko; Fukata, Hideki; Mori, Chisato

2006-01-01

Some estrogenic compounds are reported to cause testicular disorders in humans and/or experimental animals by direct action on Leydig cells. In carcinogenesis and normal development, gap junctional intercellular communication (GJIC) plays an essential role in maintaining homeostasis. In this study, we examine the effects of diethylstilbestrol (DES, a synthetic estrogen), 17β-estradiol (E 2 , a natural estrogen), and genistein (GEN, a phytoestrogen) on GJIC between mouse Leydig TM3 cells using Lucifer yellow microinjection. The three compounds tested produced GJIC inhibition in the TM3 cells after 24 h. Gradually, 10 μM DES began to inhibit GJIC for 24 h and this effect was observed until 72 h. On the other hand, both 20 μM E 2 and 25 μM GEN rapidly inhibited GJIC in 6 h and 2 h, respectively. The effects continued until 24 h, but weakened by 72 h. Furthermore, a combined effect at μM level between DES and E 2 on GJIC inhibition was observed, but not between GEN and E 2 . DES and E 2 showed GJIC inhibition at low dose levels (nearly physiological estrogen levels) after 72 h, but GEN did not. DES-induced GJIC inhibition at 10 pM and 10 μM was completely counteracted by ICI 182,780 (ICl), an estrogen receptor antagonist. On the other hand, the inhibitory effects on GJIC with E 2 (10 pM and 20 μM) and GEN (25 μM) were partially blocked by ICI or calphostin C, a protein kinase C (PKC) inhibitor, and were completely blocked by the combination of ICI and calphostin C. These results demonstrate that DES inhibits GJIC between Leydig cells via the estrogen receptor (ER), and that E 2 and GEN inhibit GJIC via ER and PKC. These estrogenic compounds may have different individual nongenotoxic mechanism including PKC pathway on testicular carcinogenesis or development

Electrotonic potentials in Aloe vera L.: Effects of intercellular and external electrodes arrangement.

Science.gov (United States)

Volkov, Alexander G; Nyasani, Eunice K; Tuckett, Clayton; Scott, Jessenia M; Jackson, Mariah M Z; Greeman, Esther A; Greenidge, Ariane S; Cohen, Devin O; Volkova, Maia I; Shtessel, Yuri B

2017-02-01

Electrostimulation of plants can induce plant movements, activation of ion channels, ion transport, gene expression, enzymatic systems activation, electrical signaling, plant-cell damage, enhanced wound healing, and influence plant growth. Here we found that electrical networks in plant tissues have electrical differentiators. The amplitude of electrical responses decreases along a leaf and increases by decreasing the distance between polarizing Pt-electrodes. Intercellular Ag/AgCl electrodes inserted in a leaf and extracellular Ag/AgCl electrodes attached to the leaf surface were used to detect the electrotonic potential propagation along a leaf of Aloe vera. There is a difference in duration and amplitude of electrical potentials measured by electrodes inserted in a leaf and those attached to a leaf's surface. If the external reference electrode is located in the soil near the root, it changes the amplitude and duration of electrotonic potentials due to existence of additional resistance, capacitance, ion channels and ion pumps in the root. The information gained from this study can be used to elucidate extracellular and intercellular communication in the form of electrical signals within plants. Copyright © 2016 Elsevier B.V. All rights reserved.

Gap-junction-mediated communication in human periodontal ligament cells.

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Kato, R; Ishihara, Y; Kawanabe, N; Sumiyoshi, K; Yoshikawa, Y; Nakamura, M; Imai, Y; Yanagita, T; Fukushima, H; Kamioka, H; Takano-Yamamoto, T; Yamashiro, T

2013-07-01

Periodontal tissue homeostasis depends on a complex cellular network that conveys cell-cell communication. Gap junctions (GJs), one of the intercellular communication systems, are found between adjacent human periodontal ligament (hPDL) cells; however, the functional GJ coupling between hPDL cells has not yet been elucidated. In this study, we investigated functional gap-junction-mediated intercellular communication in isolated primary hPDL cells. SEM images indicated that the cells were in contact with each other via dendritic processes, and also showed high anti-connexin43 (Cx43) immunoreactivity on these processes. Gap-junctional intercellular communication (GJIC) among hPDL cells was assessed by fluorescence recovery after a photobleaching (FRAP) analysis, which exhibited dye coupling between hPDL cells, and was remarkably down-regulated when the cells were treated with a GJ blocker. Additionally, we examined GJs under hypoxic stress. The fluorescence recovery and expression levels of Cx43 decreased time-dependently under the hypoxic condition. Exposure to GJ inhibitor or hypoxia increased RANKL expression, and decreased OPG expression. This study shows that GJIC is responsible for hPDL cells and that its activity is reduced under hypoxia. This is consistent with the possible role of hPDL cells in regulating the biochemical reactions in response to changes in the hypoxic environment.

PI3K/Akt signaling is involved in the disruption of gap junctional communication caused by v-Src and TNF-α.

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Ito, Satoko; Hyodo, Toshinori; Hasegawa, Hitoki; Yuan, Hong; Hamaguchi, Michinari; Senga, Takeshi

2010-09-17

Gap junctional communication, which is mediated by the connexin protein family, is essential for the maintenance of normal tissue function and homeostasis. Loss of intercellular communication results in a failure to coordinately regulate cellular functions, and it can facilitate tumorigenesis. Expression of oncogenes and stimulation with cytokines has been shown to suppress intercellular communication; however, the exact mechanism by which intercellular communication is disrupted by these factors remains uncertain. In this report, we show that Akt is essential for the disruption of gap junctional communication in v-Src-transformed cells. In addition, inhibition of Akt restores gap junctional communication after it is suppressed by TNF-α signaling. Furthermore, we demonstrate that the expression of a constitutively active form of Akt1, but not of Akt2 or Akt3, is sufficient to suppress gap junctional communication. Our results clearly define Akt1 as one of the critical regulators of gap junctional communication. Copyright © 2010 Elsevier Inc. All rights reserved.

Evolution of altruism in spatial prisoner's dilemma: Intra- and inter-cellular interactions

Science.gov (United States)

Yokoi, Hiroki; Uehara, Takashi; Sakata, Tomoyuki; Naito, Hiromi; Morita, Satoru; Tainaka, Kei-ichi

2014-12-01

Iterated prisoner's dilemma game is carried out on lattice with “colony” structure. Each cell is regarded as a colony which contains plural players with an identical strategy. Both intra- and inter-cellular interactions are assumed. In the former a player plays with all other players in the same colony, while in the latter he plays with one player each from adjacent colonies. Spatial patterns among four typical strategies exhibit various dynamics and winners. Both theory and simulation reveal that All Cooperation (AC) wins, when the members of colony or the intensity of noise increases. This result explains the evolution of altruism in animal societies, even though errors easily occur in animal communications.

Plasmodesmata: channels for intercellular signaling during plant growth and development.

Science.gov (United States)

Sevilem, Iris; Yadav, Shri Ram; Helariutta, Ykä

2015-01-01

Plants have evolved strategies for short- and long-distance communication to coordinate plant development and to adapt to changing environmental conditions. Plasmodesmata (PD) are intercellular nanochannels that provide an effective pathway for both selective and nonselective movement of various molecules that function in diverse biological processes. Numerous non-cell-autonomous proteins (NCAP) and small RNAs have been identified that have crucial roles in cell fate determination and organ patterning during development. Both the density and aperture size of PD are developmentally regulated, allowing formation of spatial symplastic domains for establishment of tissue-specific developmental programs. The PD size exclusion limit (SEL) is controlled by reversible deposition of callose, as well as by some PD-associated proteins. Although a large number of PD-associated proteins have been identified, many of their functions remain unknown. Despite the fact that PD are primarily membranous structures, surprisingly very little is known about their lipid composition. Thus, future studies in PD biology will provide deeper insights into the high-resolution structure and tightly regulated functions of PD and the evolution of PD-mediated cell-to-cell communication in plants.

Control of phospholipid flip-flop by transmembrane peptides

International Nuclear Information System (INIS)

Kaihara, Masanori; Nakao, Hiroyuki; Yokoyama, Hirokazu; Endo, Hitoshi; Ishihama, Yasushi; Handa, Tetsurou; Nakano, Minoru

2013-01-01

Highlights: ► Phospholipid flip-flop in transmembrane peptide-containing vesicles was investigated. ► Peptides that contained polar residues in the center of the transmembrane region promoted phospholipid flip-flop. ► A bioinformatics approach revealed the presence of polar residues in the transmembrane region of ER membrane proteins. ► Polar residues in ER membrane proteins possibly provide flippase-like activity. - Abstract: We designed three types of transmembrane model peptides whose sequence originates from a frequently used model peptide KALP23, and we investigated their effects on phospholipid flip-flop. Time-resolved small-angle neutron scattering and a dithionite fluorescent quenching assay demonstrated that TMP-L, which has a fully hydrophobic transmembrane region, did not enhance phospholipid flip-flop, whereas TMP-K and TMP-E, which have Lys and Glu, respectively, in the center of their transmembrane regions, enhanced phospholipid flip-flop. Introduction of polar residues in the membrane-spanning helices is considered to produce a locally polar region and enable the lipid head group to interact with the polar side-chain inside the bilayers, thereby reducing the activation energy for the flip-flop. A bioinformatics approach revealed that acidic and basic residues account for 4.5% of the central region of the transmembrane domain in human ER membrane proteins. Therefore, polar residues in ER membrane proteins are considered to provide flippase-like activity

Proteins mediating intra- and intercellular transport of lipids and lipid-modified proteins

NARCIS (Netherlands)

Neumann, S.

2008-01-01

Proteins mediating intra- and intercellular transport of lipids and lipid-modified proteins In this thesis, I studied the intra- and intercellular transport of lipidic molecules, in particular glycosphingolipids and lipid-modified proteins. The first part focuses on the intracellular transport of

Drosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves

Science.gov (United States)

Restrepo, Simon; Basler, Konrad

2016-08-01

Calcium signalling is a highly versatile cellular communication system that modulates basic functions such as cell contractility, essential steps of animal development such as fertilization and higher-order processes such as memory. We probed the function of calcium signalling in Drosophila wing imaginal discs through a combination of ex vivo and in vivo imaging and genetic analysis. Here we discover that wing discs display slow, long-range intercellular calcium waves (ICWs) when mechanically stressed in vivo or cultured ex vivo. These slow imaginal disc intercellular calcium waves (SIDICs) are mediated by the inositol-3-phosphate receptor, the endoplasmic reticulum (ER) calcium pump SERCA and the key gap junction component Inx2. The knockdown of genes required for SIDIC formation and propagation negatively affects wing disc recovery after mechanical injury. Our results reveal a role for ICWs in wing disc homoeostasis and highlight the utility of the wing disc as a model for calcium signalling studies.

On the involvement of host proteins in Cowpea mosaic virus intercellular spread

NARCIS (Netherlands)

Hollander, den P.W.

2014-01-01

Abstract of thesis Paulus den Hollander entitled “On the involvement of host proteins in Cowpea mosaic virus intercellular spread”.

Defence: 18th of November 13.30 h

Abstract

Intercellular spread of Cowpea mosaic virus (CPMV) occurs via movement

ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells

DEFF Research Database (Denmark)

Jorgensen, N R; Geist, S T; Civitelli, R

1997-01-01

mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein......Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium...... stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied...

Intercellular signalling in Stigmatella aurantiaca.

Science.gov (United States)

Plaga, W; Ulrich, S H

1999-12-01

The myxobacterium Stigmatella aurantiaca is a prokaryotic model used to study intercellular signalling and the genetic determination of morphogenesis. Signalling factors and genes required for the generation of the elaborate multicellular fruiting body are to be identified. Recently, the structure of stigmolone, which is the pheromone necessary for fruiting body formation, was elucidated, and genes involved in development were characterised. Progress has also been made in the genetic accessibility of S. aurantiaca.

TMFoldWeb: a web server for predicting transmembrane protein fold class.

Science.gov (United States)

Kozma, Dániel; Tusnády, Gábor E

2015-09-17

Here we present TMFoldWeb, the web server implementation of TMFoldRec, a transmembrane protein fold recognition algorithm. TMFoldRec uses statistical potentials and utilizes topology filtering and a gapless threading algorithm. It ranks template structures and selects the most likely candidates and estimates the reliability of the obtained lowest energy model. The statistical potential was developed in a maximum likelihood framework on a representative set of the PDBTM database. According to the benchmark test the performance of TMFoldRec is about 77 % in correctly predicting fold class for a given transmembrane protein sequence. An intuitive web interface has been developed for the recently published TMFoldRec algorithm. The query sequence goes through a pipeline of topology prediction and a systematic sequence to structure alignment (threading). Resulting templates are ordered by energy and reliability values and are colored according to their significance level. Besides the graphical interface, a programmatic access is available as well, via a direct interface for developers or for submitting genome-wide data sets. The TMFoldWeb web server is unique and currently the only web server that is able to predict the fold class of transmembrane proteins while assigning reliability scores for the prediction. This method is prepared for genome-wide analysis with its easy-to-use interface, informative result page and programmatic access. Considering the info-communication evolution in the last few years, the developed web server, as well as the molecule viewer, is responsive and fully compatible with the prevalent tablets and mobile devices.

Evolution of Microbial Quorum Sensing to Human Global Quorum Sensing: An Insight into How Gap Junctional Intercellular Communication Might Be Linked to the Global Metabolic Disease Crisis

Directory of Open Access Journals (Sweden)

James E. Trosko

2016-06-01

Full Text Available The first anaerobic organism extracted energy for survival and reproduction from its source of nutrients, with the genetic means to ensure protection of its individual genome but also its species survival. While it had a means to communicate with its community via simple secreted molecules (“quorum sensing”, the eventual shift to an aerobic environment led to multi-cellular metazoan organisms, with evolutionary-selected genes to form extracellular matrices, stem cells, stem cell niches, and a family of gap junction or “connexin” genes. These germinal and somatic stem cells responded to extracellular signals that triggered intra-cellular signaling to regulate specific genes out of the total genome. These extra-cellular induced intra-cellular signals also modulated gap junctional intercellular communication (GJIC in order to regulate the new cellular functions of symmetrical and asymmetrical cell division, cell differentiation, modes of cell death, and senescence. Within the hierarchical and cybernetic concepts, differentiated by neurons organized in the brain of the Homo sapiens, the conscious mind led to language, abstract ideas, technology, myth-making, scientific reasoning, and moral decision–making, i.e., the creation of culture. Over thousands of years, this has created the current collision between biological and cultural evolution, leading to the global “metabolic disease” crisis.

Evolution of Microbial Quorum Sensing to Human Global Quorum Sensing: An Insight into How Gap Junctional Intercellular Communication Might Be Linked to the Global Metabolic Disease Crisis.

Science.gov (United States)

Trosko, James E

2016-06-15

The first anaerobic organism extracted energy for survival and reproduction from its source of nutrients, with the genetic means to ensure protection of its individual genome but also its species survival. While it had a means to communicate with its community via simple secreted molecules ("quorum sensing"), the eventual shift to an aerobic environment led to multi-cellular metazoan organisms, with evolutionary-selected genes to form extracellular matrices, stem cells, stem cell niches, and a family of gap junction or "connexin" genes. These germinal and somatic stem cells responded to extracellular signals that triggered intra-cellular signaling to regulate specific genes out of the total genome. These extra-cellular induced intra-cellular signals also modulated gap junctional intercellular communication (GJIC) in order to regulate the new cellular functions of symmetrical and asymmetrical cell division, cell differentiation, modes of cell death, and senescence. Within the hierarchical and cybernetic concepts, differentiated by neurons organized in the brain of the Homo sapiens, the conscious mind led to language, abstract ideas, technology, myth-making, scientific reasoning, and moral decision-making, i.e., the creation of culture. Over thousands of years, this has created the current collision between biological and cultural evolution, leading to the global "metabolic disease" crisis.

The association between soluble intercellular adhesion molecule-1 levels in drained dialysate and peritoneal injury in peritoneal dialysis.

Science.gov (United States)

Igarashi, Yusuke; Morishita, Yoshiyuki; Yoshizawa, Hiromichi; Imai, Reika; Imai, Toshimi; Hirahara, Ichiro; Akimoto, Tetsu; Ookawara, Susumu; Ishibashi, Kenichi; Muto, Shigeaki; Nagata, Daisuke

2017-11-01

Chronic inflammation of the peritoneum causes peritoneal injury in patients on peritoneal dialysis. Intercellular adhesion molecule-1 and its circulating form, soluble intercellular adhesion molecule-1, play pivotal roles in inflammation. However, their role in peritoneal injury is unclear. We measured changes in intercellular adhesion molecule-1 expression in the peritoneum of a peritoneal injury model in rats. The associations between soluble intercellular adhesion molecule-1 levels in drained dialysate and the solute transport rate (D/P-Cr and D/D0-glucose) determined by the peritoneal equilibration test, and matrix metalloproteinase-2 levels in drained dialysate were investigated in 94 peritoneal drained dialysate samples. Intercellular adhesion molecule-1 expression was increased in the peritoneum of rats with peritoneal injury. Soluble intercellular adhesion molecule-1 levels in drained dialysate were significantly positively correlated with D/P-Cr (r = .51, p molecule-1expression is increased in the peritoneum of a peritoneal injury model in the rat, and soluble intercellular adhesion molecule-1 levels in drained dialysate are associated with peritoneal injury in patients on peritoneal dialysis. These results suggest that soluble intercellular adhesion molecule-1 could be a novel biomarker of peritoneal injury in patients on peritoneal dialysis.

GAP junctional intercellular communication confers an enhanced bystander effect as well as a protective effect in HSV-TK/gancyclovir mediated cytotoxicity

International Nuclear Information System (INIS)

Wygoda, Marc R.; Rehemtulla, Alnawaz; Davis, Mary A.; Lawrence, Theodore S.

1996-01-01

Purpose/Objective: The 'Bystander Effect' is the phenomenon by which cells which are not transduced with the Herpes Simplex Virus - Thymidine Kinase (HSV-TK) gene are killed by the guanosine analog Gancyclovir (GCV) when they are nearby HSV-TK positive cells. Since currently available gene transfer technologies have a low efficiency (typically less than 10% of cells are transduced), a better understanding of the mechanism underlying the bystander effect is crucial for the success of the HSV-TK/GCV enzyme prodrug gene therapy approach. Gap junctions, which are specialized cell membrane channels allowing the passage of molecules < 1000 daltons in size directly from cell to cell, have been proposed as a possible mediator of the bystander effect. In particular, gap junctions could permit the transfer of toxic metabolites from the HSV-TK positive cells to the bystander cells. Our aim was to assess the role of gap junctional intercellular communication (GJIC) in the bystander effect. Materials and Methods: Co-culture of an HSV-TK transduced rat liver epithelial cell line (WB-TK), with either its communication competent parental cell line (WB), or an isogenic cell line differing by its communication incompetence (aB1). The ratios of effector cells (WB-TK) to bystander cells (WB or aB1), were (100(0)), (50(50)), (5(95)), or(0(100)) . These various mixed populations were exposed for 24 hours to 10μM GCV and thereafter placed at clonal density into selective media, allowing for growth of either the bystander (TK negative) cells or the effector (TK-positive) cells. Results: 1) When the effector/bystander ratio was (50(50)) the surviving fraction of the bystander cells was 3.5% and 37% for the WB and aB1, respectively. When the effector/bystander ratio was (5(95)), the surviving fraction was 51% and 65% for the WB and aB1, respectively. Either cell line (WB or aB1) cultured alone was unaffected by GCV. These results demonstrate that the bystander effect is much higher when

Autophagy and gap junctional intercellular communication inhibition are involved in cadmium-induced apoptosis in rat liver cells

Energy Technology Data Exchange (ETDEWEB)

Zou, Hui [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China); Zhuo, Liling [College of Life Science, Zaozhuang University, Zaozhuang, Shandong, 277160 (China); Han, Tao; Hu, Di; Yang, Xiaokang; Wang, Yi; Yuan, Yan; Gu, Jianhong; Bian, Jianchun; Liu, Xuezhong [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China); Liu, Zongping, E-mail: liuzongping@yzu.edu.cn [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China)

2015-04-17

Cadmium (Cd) is known to induce hepatotoxicity, yet the underlying mechanism of how this occurs is not fully understood. In this study, Cd-induced apoptosis was demonstrated in rat liver cells (BRL 3A) with apoptotic nuclear morphological changes and a decrease in cell index (CI) in a time- and concentration-dependent manner. The role of gap junctional intercellular communication (GJIC) and autophagy in Cd-induced apoptosis was investigated. Cd significantly induced GJIC inhibition as well as downregulation of connexin 43 (Cx43). The prototypical gap junction blocker carbenoxolone disodium (CBX) exacerbated the Cd-induced decrease in CI. Cd treatment was also found to cause autophagy, with an increase in mRNA expression of autophagy-related genes Atg-5, Atg-7, Beclin-1, and microtubule-associated protein light chain 3 (LC3) conversion from cytosolic LC3-I to membrane-bound LC3-II. The autophagic inducer rapamycin (RAP) prevented the Cd-induced CI decrease, while the autophagic inhibitor chloroquine (CQ) caused a further reduction in CI. In addition, CBX promoted Cd-induced autophagy, as well as changes in expression of Atg-5, Atg-7, Beclin-1 and LC3. CQ was found to block the Cd-induced decrease in Cx43 and GJIC inhibition, whereas RAP had opposite effect. These results demonstrate that autophagy plays a protective role during Cd-induced apoptosis in BRL 3A cells during 6 h of experiment, while autophagy exacerbates Cd-induced GJIC inhibition which has a negative effect on cellular fate. - Highlights: • GJIC and autophagy is crucial for biological processes. • Cd exposure causes GJIC inhibition and autophagy increase in BRL 3A cells. • Autophagy protects Cd induced BRL 3A cells apoptosis at an early stage. • Autophagy exacerbates Cd-induced GJIC inhibition. • GJIC plays an important role in autophagy induced cell death or survival.

Development of schizogenous intercellular spaces in plants

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Kimitsune eIshizaki

2015-07-01

Full Text Available Gas exchange is essential for multicellular organisms. In contrast to the circulatory systems of animals, land plants have tissues with intercellular spaces (ICSs, called aerenchyma, that are critical for efficient gas exchange. Plants form ICSs by two different mechanisms: schizogeny, where localized cell separation creates spaces; and lysogeny, where cells die to create intercellular spaces. In schizogenous ICS formation, specific molecular mechanisms regulate the sites of cell separation and coordinate extensive reorganization of cell walls. Emerging evidence suggests the involvement of extracellular signaling, mediated by peptide ligands and leucine-rich repeat receptor-like kinases, in the regulation of cell wall remodeling during cell separation. Recent work on the liverwort Marchantia polymorpha has demonstrated a critical role for a plasma membrane-associated plant U-box E3 ubiquitin ligase in ICS formation. In this review, I discuss the mechanism of schizogenous ICS formation, focusing on the potential role of extracellular signaling in the regulation of cell separation.

Hidden markov model for the prediction of transmembrane proteins using MATLAB.

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Chaturvedi, Navaneet; Shanker, Sudhanshu; Singh, Vinay Kumar; Sinha, Dhiraj; Pandey, Paras Nath

2011-01-01

Since membranous proteins play a key role in drug targeting therefore transmembrane proteins prediction is active and challenging area of biological sciences. Location based prediction of transmembrane proteins are significant for functional annotation of protein sequences. Hidden markov model based method was widely applied for transmembrane topology prediction. Here we have presented a revised and a better understanding model than an existing one for transmembrane protein prediction. Scripting on MATLAB was built and compiled for parameter estimation of model and applied this model on amino acid sequence to know the transmembrane and its adjacent locations. Estimated model of transmembrane topology was based on TMHMM model architecture. Only 7 super states are defined in the given dataset, which were converted to 96 states on the basis of their length in sequence. Accuracy of the prediction of model was observed about 74 %, is a good enough in the area of transmembrane topology prediction. Therefore we have concluded the hidden markov model plays crucial role in transmembrane helices prediction on MATLAB platform and it could also be useful for drug discovery strategy. The database is available for free at bioinfonavneet@gmail.comvinaysingh@bhu.ac.in.

Inhibition of gap-junctional intercellular communication and activation of mitogen-activated protein kinases by cyanobacterial extracts--indications of novel tumor-promoting cyanotoxins?

Science.gov (United States)

Bláha, Ludĕk; Babica, Pavel; Hilscherová, Klára; Upham, Brad L

2010-01-01

Toxicity and liver tumor promotion of cyanotoxins microcystins have been extensively studied. However, recent studies document that other metabolites present in the complex cyanobacterial water blooms may also have adverse health effects. In this study we used rat liver epithelial stem-like cells (WB-F344) to examine the effects of cyanobacterial extracts on two established markers of tumor promotion, inhibition of gap-junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) - ERK1/2. Extracts of cyanobacteria (laboratory cultures of Microcystis aeruginosa and Aphanizomenon flos-aquae and water blooms dominated by these species) inhibited GJIC and activated MAPKs in a dose-dependent manner (effective concentrations ranging 0.5-5mgd.w./mL). Effects were independent of the microcystin content and the strongest responses were elicited by the extracts of Aphanizomenon sp. Neither pure microcystin-LR nor cylindrospermopsin inhibited GJIC or activated MAPKs. Modulations of GJIC and MAPKs appeared to be specific to cyanobacterial extracts since extracts from green alga Chlamydomonas reinhardtii, heterotrophic bacterium Klebsiella terrigena, and isolated bacterial lipopolysaccharides had no comparable effects. Our study provides the first evidence on the existence of unknown cyanobacterial toxic metabolites that affect in vitro biomarkers of tumor promotion, i.e. inhibition of GJIC and activation of MAPKs.

Topology of transmembrane channel-like gene 1 protein.

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Labay, Valentina; Weichert, Rachel M; Makishima, Tomoko; Griffith, Andrew J

2010-10-05

Mutations of transmembrane channel-like gene 1 (TMC1) cause hearing loss in humans and mice. TMC1 is the founding member of a family of genes encoding proteins of unknown function that are predicted to contain multiple transmembrane domains. The goal of our study was to define the topology of mouse TMC1 expressed heterologously in tissue culture cells. TMC1 was retained in the endoplasmic reticulum (ER) membrane of five tissue culture cell lines that we tested. We used anti-TMC1 and anti-HA antibodies to probe the topologic orientation of three native epitopes and seven HA epitope tags along full-length TMC1 after selective or complete permeabilization of transfected cells with digitonin or Triton X-100, respectively. TMC1 was present within the ER as an integral membrane protein containing six transmembrane domains and cytosolic N- and C-termini. There is a large cytoplasmic loop, between the fourth and fifth transmembrane domains, with two highly conserved hydrophobic regions that might associate with or penetrate, but do not span, the plasma membrane. Our study is the first to demonstrate that TMC1 is a transmembrane protein. The topologic organization revealed by this study shares some features with that of the shaker-TRP superfamily of ion channels.

Tanshinone IIA increases the bystander effect of herpes simplex virus thymidine kinase/ganciclovir gene therapy via enhanced gap junctional intercellular communication.

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Jianyong Xiao

Full Text Available The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV. This effect is reported to be mediated by gap junctional intercellular communication (GJIC, and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA, a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy.

Cavitation of intercellular spaces is critical to establishment of hydraulic properties of compression wood of Chamaecyparis obtusa seedlings.

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Nakaba, Satoshi; Hirai, Asami; Kudo, Kayo; Yamagishi, Yusuke; Yamane, Kenichi; Kuroda, Katsushi; Nugroho, Widyanto Dwi; Kitin, Peter; Funada, Ryo

2016-03-01

When the orientation of the stems of conifers departs from the vertical as a result of environmental influences, conifers form compression wood that results in restoration of verticality. It is well known that intercellular spaces are formed between tracheids in compression wood, but the function of these spaces remains to be clarified. In the present study, we evaluated the impact of these spaces in artificially induced compression wood in Chamaecyparis obtusa seedlings. We monitored the presence or absence of liquid in the intercellular spaces of differentiating xylem by cryo-scanning electron microscopy. In addition, we analysed the relationship between intercellular spaces and the hydraulic properties of the compression wood. Initially, we detected small intercellular spaces with liquid in regions in which the profiles of tracheids were not rounded in transverse surfaces, indicating that the intercellular spaces had originally contained no gases. In the regions where tracheids had formed secondary walls, we found that some intercellular spaces had lost their liquid. Cavitation of intercellular spaces would affect hydraulic conductivity as a consequence of the induction of cavitation in neighbouring tracheids. Our observations suggest that cavitation of intercellular spaces is the critical event that affects not only the functions of intercellular spaces but also the hydraulic properties of compression wood. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Multicellular models of intercellular synchronization in circadian neural networks

International Nuclear Information System (INIS)

Henson, Michael A.

2013-01-01

The circadian clock generates 24 h rhythms that drive physiological and behavioral processes in a diverse range of organisms including microbes, plants, insects, and mammals. Recent experimental advances have produced improved understanding of the molecular mechanisms involved in circadian rhythm generation at the single cell level. However, the intercellular mechanisms that allow large populations of coupled pacemaker cells to synchronize and coordinate their rhythms remain poorly understood. The purpose of this article is to review recent progress in dynamic modeling of the circadian clock with a focus on multicellular models required to describe cell population synchronization. Mammalian systems are emphasized to illustrate the highly heterogeneous structure and rich dynamical behavior of multicellular circadian systems. Available multicellular models are characterized with respect to their single cell descriptions, intercellular coupling mechanisms, and network topologies. Examples drawn from our own research are used to demonstrate the advantages associated with integrating detailed single cell models within realistic multicellular networks for prediction of mammalian system dynamics. Mathematical modeling is shown to represent a powerful tool for understanding the intracellular and intercellular mechanisms utilized to robustly synchronize large populations of highly heterogeneous and sparsely coupled single cell oscillators. The article concludes with some possible directions for future research

In vivo relevance of intercellular calcium signaling in Drosophila wing development

OpenAIRE

Brodskiy, Pavel; Brito-Robinson, Teresa; Levis, Megan; Narciso, Cody; Jangula, Jamison; Huizar, Francisco; Wu, Qinfeng; Zartman, Jeremiah

2017-01-01

Recently, organ-scale intercellular Ca2+ transients (ICTs) were reported in the Drosophila wing disc. However, the functional in vivo significance of ICTs remains largely unknown. Here we demonstrate the in vivo relevance of intercellular Ca2+ signaling and its impact on wing development. We report that Ca2+ signaling in vivo decreases as wing discs mature. Ca2+ signaling ex vivo responds to fly extract in a dose-dependent manner. This suggests ICTs occur in vivo due to chemical stimulus that...

Phosphatidylinositol-bisphosphate regulates intercellular coupling in cardiac myocytes

DEFF Research Database (Denmark)

Hofgaard, Johannes P; Banach, Kathrin; Mollerup, Sarah

2008-01-01

that agonist-induced changes in PIP(2) can result in a reduction of the functional coupling of cardiomyocytes and, consequently, in changes in conduction velocity. Intercellular coupling was measured by Lucifer Yellow dye transfer in cultured neonatal rat cardiomyocytes. Conduction velocity was measured...

Multi-level communication of human retinal pigment epithelial cells via tunneling nanotubes.

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Dierk Wittig

Full Text Available BACKGROUND: Tunneling nanotubes (TNTs may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis. METHODOLOGY AND PRINCIPAL FINDINGS: Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca²⁺ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells. CONCLUSIONS AND SIGNIFICANCE: Our observations indicate that

Catalytic combustion of propane in a membrane reactor with separate feed of reactants—II. Operation in presence of trans-membrane pressure gradients

NARCIS (Netherlands)

Saracco, Guido; Veldsink, Jan Willem; Versteeg, Geert F.; Swaaij, Wim P.M. van

1995-01-01

This is the second communication of a series dealing with an experimental and modelling study on propane catalytic combustion in a membrane reactor with separate feed of reactants. In paper I the behaviour of the reactor in the absence of trans-membrane pressure gradients was presented and

Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

Energy Technology Data Exchange (ETDEWEB)

Ma, W.H.; Liu, Y.J.; Wang, W.; Zhang, Y.Z. [The Third Hospital of Hebei Medical University, The Provincial Key Laboratory for Orthopedic Biomechanics of Hebei, Shijiazhuang, Hebei Province (China)

2015-02-13

Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation.

Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

International Nuclear Information System (INIS)

Ma, W.H.; Liu, Y.J.; Wang, W.; Zhang, Y.Z.

2015-01-01

Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation

Low dose gamma irradiation enhances defined signaling components of intercellular reactive oxygen-mediated apoptosis induction

International Nuclear Information System (INIS)

Bauer, G

2011-01-01

Transformed cells are selectively removed by intercellular ROS-mediated induction of apoptosis. Signaling is based on the HOCl and the NO/peroxynitrite pathway (major pathways) and the nitryl chloride and the metal-catalyzed Haber-Weiss pathway (minor pathways). During tumor progression, resistance against intercellular induction of apoptosis is acquired through expression of membrane-associated catalase. Low dose radiation of nontransformed cells has been shown to enhance intercellular induction of apoptosis. The present study was performed to define the signaling components which are modulated by low dose gamma irradiation. Low dose radiation induced the release of peroxidase from nontransformed, transformed and tumor cells. Extracellular superoxide anion generation was strongly enhanced in the case of transformed cells and tumor cells, but not in nontransformed cells. Enhancement of peroxidase release and superoxide anion generation either increased intercellular induction of apoptosis of transformed cells, or caused a partial protection under specific signaling conditions. In tumor cells, low dose radiation enhanced the production of major signaling components, but this had no effect on apoptosis induction, due to the strong resistance mechanism of tumor cells. Our data specify the nature of low dose radiation-induced effects on specific signaling components of intercellular induction of apoptosis at defined stages of multistep carcinogenesis.

Effect of sound on gap-junction-based intercellular signaling: Calcium waves under acoustic irradiation.

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Deymier, P A; Swinteck, N; Runge, K; Deymier-Black, A; Hoying, J B

2015-01-01

We present a previously unrecognized effect of sound waves on gap-junction-based intercellular signaling such as in biological tissues composed of endothelial cells. We suggest that sound irradiation may, through temporal and spatial modulation of cell-to-cell conductance, create intercellular calcium waves with unidirectional signal propagation associated with nonconventional topologies. Nonreciprocity in calcium wave propagation induced by sound wave irradiation is demonstrated in the case of a linear and a nonlinear reaction-diffusion model. This demonstration should be applicable to other types of gap-junction-based intercellular signals, and it is thought that it should be of help in interpreting a broad range of biological phenomena associated with the beneficial therapeutic effects of sound irradiation and possibly the harmful effects of sound waves on health.

Inhibition of gap junctional Intercellular communication in WB-F344 rat liver epithelial cells by triphenyltin chloride through MAPK and PI3-kinase pathways

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Tsai Ming-Che

2010-06-01

Full Text Available Abstract Background Organotin compounds (OTCs have been widely used as stabilizers in the production of plastic, agricultural pesticides, antifoulant plaints and wood preservation. The toxicity of triphenyltin (TPT compounds was known for their embryotoxic, neurotoxic, genotoxic and immunotoxic effects in mammals. The carcinogenicity of TPT was not well understood and few studies had discussed the effects of OTCs on gap junctional intercellular communication (GJIC of cells. Method In the present study, the effects of triphenyltin chloride (TPTC on GJIC in WB-F344 rat liver epithelial cells were evaluated, using the scrape-loading dye transfer technique. Results TPTC inhibited GJIC after a 30-min exposure in a concentration- and time-dependent manner. Pre-incubation of cells with the protein kinase C (PKC inhibitor did not modify the response, but the specific MEK 1 inhibitor PD98059 and PI3K inhibitor LY294002 decreased substantially the inhibition of GJIC by TPTC. After WB-F344 cells were exposed to TPTC, phosphorylation of Cx43 increased as seen in Western blot analysis. Conclusions These results show that TPTC inhibits GJIC in WB-F344 rat liver epithelial cells by altering the Cx43 protein expression through both MAPK and PI3-kinase pathways.

Intercellular signaling pathways active during intervertebral disc growth, differentiation, and aging.

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Dahia, Chitra Lekha; Mahoney, Eric J; Durrani, Atiq A; Wylie, Christopher

2009-03-01

Intervertebral discs at different postnatal ages were assessed for active intercellular signaling pathways. To generate a spatial and temporal map of the signaling pathways active in the postnatal intervertebral disc (IVD). The postnatal IVD is a complex structure, consisting of 3 histologically distinct components, the nucleus pulposus, fibrous anulus fibrosus, and endplate. These differentiate and grow during the first 9 weeks of age in the mouse. Identification of the major signaling pathways active during and after the growth and differentiation period will allow functional analysis using mouse genetics and identify targets for therapy for individual components of the disc. Antibodies specific for individual cell signaling pathways were used on cryostat sections of IVD at different postnatal ages to identify which components of the IVD were responding to major classes of intercellular signal, including sonic hedgehog, Wnt, TGFbeta, FGF, and BMPs. We present a spatial/temporal map of these signaling pathways during growth, differentiation, and aging of the disc. During growth and differentiation of the disc, its different components respond at different times to different intercellular signaling ligands. Most of these are dramatically downregulated at the end of disc growth.

The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

Science.gov (United States)

Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

2012-01-01

The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

Tunneling Nanotubes: Intimate Communication between Myeloid Cells.

Science.gov (United States)

Dupont, Maeva; Souriant, Shanti; Lugo-Villarino, Geanncarlo; Maridonneau-Parini, Isabelle; Vérollet, Christel

2018-01-01

Tunneling nanotubes (TNT) are dynamic connections between cells, which represent a novel route for cell-to-cell communication. A growing body of evidence points TNT towards a role for intercellular exchanges of signals, molecules, organelles, and pathogens, involving them in a diverse array of functions. TNT form among several cell types, including neuronal cells, epithelial cells, and almost all immune cells. In myeloid cells (e.g., macrophages, dendritic cells, and osteoclasts), intercellular communication via TNT contributes to their differentiation and immune functions. Importantly, TNT enable myeloid cells to communicate with a targeted neighboring or distant cell, as well as with other cell types, therefore creating a complex variety of cellular exchanges. TNT also contribute to pathogen spread as they serve as "corridors" from a cell to another. Herein, we addressed the complexity of the definition and in vitro characterization of TNT in innate immune cells, the different processes involved in their formation, and their relevance in vivo . We also assess our current understanding of how TNT participate in immune surveillance and the spread of pathogens, with a particular interest for HIV-1. Overall, despite recent progress in this growing research field, we highlight that further investigation is needed to better unveil the role of TNT in both physiological and pathological conditions.

Tunneling Nanotubes: Intimate Communication between Myeloid Cells

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Maeva Dupont

2018-01-01

Full Text Available Tunneling nanotubes (TNT are dynamic connections between cells, which represent a novel route for cell-to-cell communication. A growing body of evidence points TNT towards a role for intercellular exchanges of signals, molecules, organelles, and pathogens, involving them in a diverse array of functions. TNT form among several cell types, including neuronal cells, epithelial cells, and almost all immune cells. In myeloid cells (e.g., macrophages, dendritic cells, and osteoclasts, intercellular communication via TNT contributes to their differentiation and immune functions. Importantly, TNT enable myeloid cells to communicate with a targeted neighboring or distant cell, as well as with other cell types, therefore creating a complex variety of cellular exchanges. TNT also contribute to pathogen spread as they serve as “corridors” from a cell to another. Herein, we addressed the complexity of the definition and in vitro characterization of TNT in innate immune cells, the different processes involved in their formation, and their relevance in vivo. We also assess our current understanding of how TNT participate in immune surveillance and the spread of pathogens, with a particular interest for HIV-1. Overall, despite recent progress in this growing research field, we highlight that further investigation is needed to better unveil the role of TNT in both physiological and pathological conditions.

Cell-cell communication in the kidney microcirculation

DEFF Research Database (Denmark)

Sørensen, Charlotte Mehlin; von Holstein-Rathlou, Niels-Henrik

2012-01-01

the postglomerular vasculature. Cxs form gap junctions between neighboring cells, and as in other organ systems, the major function of Cxs in the kidney appears to be mediation of intercellular communication. Cxs may also form hemichannels that allow cellular secretion of signaling molecules like ATP, and thereby...... mediate paracrine signaling. Renal Cxs facilitate vascular conduction, juxtaglomerlar apparatus calcium signaling, and enable ECs and VSMCs to communicate. Thus, current research suggests multiple roles for Cxs in important regulatory mechanisms within the kidney, including the renin-angiotensin system...

Cysteine-rich peptides (CRPs) mediate diverse aspects of cell-cell communication in plant reproduction and development.

Science.gov (United States)

Marshall, Eleanor; Costa, Liliana M; Gutierrez-Marcos, Jose

2011-03-01

Cell-cell communication in plants is essential for the correct co-ordination of reproduction, growth, and development. Studies to dissect this mode of communication have previously focussed primarily on the action of plant hormones as mediators of intercellular signalling. In animals, peptide signalling is a well-documented intercellular communication system, however, relatively little is known about this system in plants. In recent years, numerous reports have emerged about small, secreted peptides controlling different aspects of plant reproduction. Interestingly, most of these peptides are cysteine-rich, and there is convincing evidence suggesting multiple roles for related cysteine-rich peptides (CRPs) as signalling factors in developmental patterning as well as during plant pathogen responses and symbiosis. In this review, we discuss how CRPs are emerging as key signalling factors in regulating multiple aspects of vegetative growth and reproductive development in plants.

Maize plasma membrane aquaporin ZmPIP2;5, but not ZmPIP1;2, facilitates transmembrane diffusion of hydrogen peroxide.

Science.gov (United States)

Bienert, Gerd P; Heinen, Robert B; Berny, Marie C; Chaumont, François

2014-01-01

Plant aquaporins play important roles in transmembrane water transport processes, but some also facilitate the diffusion of other small uncharged solutes ranging from gases to metalloids. Recent evidence suggests that the transmembrane movement of hydrogen peroxide, an intra- and intercellular multifunctional signaling and defense compound, can be regulated by aquaporins. We addressed the question whether maize aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily facilitate hydrogen peroxide diffusion using heterologous expression in the yeast Saccharomyces cerevisiae. We showed that ZmPIP proteins belonging to the PIP1 and PIP2 groups were significantly expressed in yeast cells only after codon optimization of their cDNA. In accordance with previous localization studies in oocytes and plants, ZmPIP1;2 was mainly retained in intracellular membranes, while ZmPIP2;5 was localized to the plasma membrane. However, upon co-expression with ZmPIP2;5, ZmPIP1;2 was re-localized to the plasma membrane. Using a non-functional plasma membrane-localized ZmPIP2;5 mutant to deliver ZmPIP1;2 to the plasma membrane, we demonstrated that, in contrast to wild type ZmPIP2;5, ZmPIP1;2 was not permeable to hydrogen peroxide. Our study further highlighted the fact that, when using the yeast system, which is widely employed to study substrates for plant aquaporins and other transporters, although positive transport assay results allow direct conclusions to be drawn regarding solute permeability, negative results require additional control experiments to show that the protein is expressed and localized correctly before concluding on the lack of transport activity. © 2013.

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

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Koji Mizuhashi

Full Text Available Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119, encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice.First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS and osteoid maturation time (Omt, and significantly decreased mineral apposition rate (MAR and bone formation rate per bone surface (BFR/BS. In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant.Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

Science.gov (United States)

Mizuhashi, Koji; Chaya, Taro; Kanamoto, Takashi; Omori, Yoshihiro; Furukawa, Takahisa

2015-01-01

Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice. First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant. Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

Intercellular and intracellular signaling pathways mediating ionizing radiation-induced bystander effects

International Nuclear Information System (INIS)

Hamada, Nobuyuki; Hara, Takamitsu; Kobayashi, Yasuhiko; Matsumoto, Hideki

2007-01-01

A rapidly growing body of experimental evidence indicates that ionizing radiation induces biological effects in non-irradiated bystander cells that have received signals from adjacent or distant irradiated cells. This phenomenon, which has been termed the ionizing radiation-induced bystander effect, challenges the long-standing paradigm that radiation traversal through the nucleus of a cell is a prerequisite to elicit genetic damage or a biological response. Bystander effects have been observed in a number of experimental systems, and cells whose nucleus or cytoplasm is irradiated exert bystander responses. Bystander cells manifest a multitude of biological consequences, such as genetic and epigenetic changes, alterations in gene expression, activation of signal transduction pathways, and delayed effects in their progeny. Several mediating mechanisms have been proposed. These involve gap junction-mediated intercellular communication, secreted soluble factors, oxidative metabolism, plasma membrane-bound lipid rafts, and calcium fluxes. This paper reviews briefly the current knowledge of the bystander effect with a focus on proposed mechanisms. The potential benefit of bystander effects to cancer radiotherapy will also be discussed. (author)

PDBTM: Protein Data Bank of transmembrane proteins after 8 years.

Science.gov (United States)

Kozma, Dániel; Simon, István; Tusnády, Gábor E

2013-01-01

The PDBTM database (available at http://pdbtm.enzim.hu), the first comprehensive and up-to-date transmembrane protein selection of the Protein Data Bank, was launched in 2004. The database was created and has been continuously updated by the TMDET algorithm that is able to distinguish between transmembrane and non-transmembrane proteins using their 3D atomic coordinates only. The TMDET algorithm can locate the spatial positions of transmembrane proteins in lipid bilayer as well. During the last 8 years not only the size of the PDBTM database has been steadily growing from ∼400 to 1700 entries but also new structural elements have been identified, in addition to the well-known α-helical bundle and β-barrel structures. Numerous 'exotic' transmembrane protein structures have been solved since the first release, which has made it necessary to define these new structural elements, such as membrane loops or interfacial helices in the database. This article reports the new features of the PDBTM database that have been added since its first release, and our current efforts to keep the database up-to-date and easy to use so that it may continue to serve as a fundamental resource for the scientific community.

Intercellular Uptake of Technetium-99m Pertechnetate by Different Types of Cell Lines

International Nuclear Information System (INIS)

Safri Zainal Abidin; Raizulnasuha Abdul Rashid; Muhammad Afiq Khairil Anuar; Wan Nordiana A Abd Rahman

2015-01-01

The purpose of this study is to determine the technetium-99m pertechnetate ( 99m TcO 4 ) intercellular uptake by different types of cell lines. HeLa, human fetal osteoblast (hFOB), glial and glioma cell lines grown in 6-wells culture plates were incubated with 99m TcO 4 of activity of 200, 400, 600, 800 and 1000 μCi for 30 minutes at 37 degree Celsius and 5 % CO 2 humidified atmosphere. After incubation, the cells were washed 3 times with phosphate buffer saline to remove the extracellular traces of 99m TcO 4 . Measurement of the intercellular 99m TcO 4 into the cells was calculated. The intercellular uptake of 99m TcO 4 was found to be inversely correlate to the radioactivity. HHeLa cell shows the highest uptake followed by hFOB, glial and glioma cell lines. Comparison of uptake between normal and cancer cells present indistinguishable results. The findings of this study suggest that the intercellular uptake of 99m TcO 4 is highly dependent on the type of cells despite no significant different of uptake was found between normal and cancer cell lines. The level of radioactivity is also an important determinant factor that influence the uptake of 99m TcO 4 into the cell. The study will be the first precedent toward understanding the cellular characteristics and pharmacokinetic of non-invasive imaging tracer for future molecular imaging and therapy. (author)

Antiproliferative Action of Conjugated Linoleic Acid on Human MCF-7 Breast Cancer Cells Mediated by Enhancement of Gap Junctional Intercellular Communication through Inactivation of NF-κB

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Md. Abdur Rakib

2013-01-01

Full Text Available The major conjugated linoleic acid (CLA isomers, c9,t11-CLA and t10,c12-CLA, have anticancer effects; however, the exact mechanisms underlying these effects are unknown. Evidence suggests that reversal of reduced gap junctional intercellular communication (GJIC in cancer cells inhibits cell growth and induces cell death. Hence, we determined that CLA isomers enhance GJIC in human MCF-7 breast cancer cells and investigated the underlying molecular mechanisms. The CLA isomers significantly enhanced GJIC of MCF-7 cells at 40 μM concentration, whereas CLA inhibited cell growth and induced caspase-dependent apoptosis. CLA increased connexin43 (Cx43 expression both at the transcriptional and translational levels. CLA inhibited nuclear factor-κB (NF-κB activity and enhanced reactive oxygen species (ROS generation. No significant difference was observed in the efficacy of c9,t11-CLA and t10,c12-CLA. These results suggest that the anticancer effect of CLA is associated with upregulation of GJIC mediated by enhanced Cx43 expression through inactivation of NF-κB and generation of ROS in MCF-7 cells.

Intercellular Adhesion Molecule-1 Levels in Experimental Brain Injury and the Effects of Alpha-tocopherol

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Nilgun Senol

2014-06-01

Full Text Available Aim: The mechanisms, responsible for the secondary injuries occuring after acute injury of the brain are; release of nitrous oxide which is an inflammatory mediator, abnormal formation of free oxygen radicals and excessive stimulation of excitatory aminoacids. In this study, it is aimed to investigate changes in intercellular adhesion molecule levels in the brain, that occur subsequent to blunt head trauma, and after administration of an antioxidant agent, vitamin E. Material and Method: In this study, rats were divided into 4 groups. In group A; rats had only skin incision, group B; rats were traumatized after the skin incision, group C; isotonic (30mg/kg was given intraperitoneally after 30 minutes of the trauma, group D; alpha-tocopherol (30mg/kg was given intraperitoneally, after 30 minutes of the trauma. All the rats in these groups were sacrified after 24 hours. Biparietal and bifrontal lobs were taken about 3x5x1mm tickness and intercellular adhesion molecule-1 levels were studied by enzyme-linked immunosorbent assay kit. Results: As the result of the statistical analysis, it is detected that although there is an increase in intercellular adhesion molecule levels in brain parenchyma after trauma, it is statistically unsignificant. However, as the traumatized group and the group given alpha-tocopherol after trauma was compared, a statistically significant decrease in intercellular adhesion molecule-1 levels in the alpha-tocopherol given group was seen. Discussion: Alpha-tocopherol, an antioxidant agent, causes decrease in intercellular adhesion molecule levels, by decreasing inflammation.

Low dose/low fluence ionizing radiation-induced biological effects: The role of intercellular communication and oxidative metabolism

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Azzam, Edouard

Mechanistic investigations have been considered critical to understanding the health risks of exposure to ionizing radiation. To gain greater insight in the biological effects of exposure to low dose/low fluence space radiations with different linear energy transfer (LET) properties, we examined short and long-term biological responses to energetic protons and high charge (Z) and high energy (E) ions (HZE particles) in human cells maintained in culture and in targeted and non-targeted tissues of irradiated rodents. Particular focus of the studies has been on mod-ulation of gene expression, proliferative capacity, induction of DNA damage and perturbations in oxidative metabolism. Exposure to mean doses of 1000 MeV/nucleon iron ions, by which a small to moderate proportion of cells in an exposed population is targeted through the nucleus by an HZE particle, induced stressful effects in the irradiated and non-irradiated cells in the population. Direct intercellular communication via gap-junctions was a primary mediator of the propagation of stressful effects from irradiated to non-irradiated cells. Compromised prolif-erative capacity, elevated level of DNA damage and oxidative stress evaluated by measurements of protein carbonylation, lipid peroxidation and activity of metabolic enzymes persisted in the progeny of irradiated and non-irradiated cells. In contrast, progeny of cells exposed to high or low doses from 150-1000 MeV protons retained the ability to form colonies and harbored similar levels of micronuclei, a surrogate form of DNA damage, as control, which correlated with normal reactive oxygen species (ROS) levels. Importantly, a significant increase in the spontaneous neoplastic transformation frequency was observed in progeny of bystander mouse embryo fibroblasts (MEFs) co-cultured with MEFs irradiated with energetic iron ions but not protons. Of particular significance, stressful effects were detected in non-targeted tissues of rats that received partial

Glycine Perturbs Local and Global Conformational Flexibility of a Transmembrane Helix

DEFF Research Database (Denmark)

Högel, Philipp; Götz, Alexander; Kuhne, Felix

2018-01-01

Flexible transmembrane helices frequently support the conformational transitions between different functional states of membrane proteins. While proline is well known to distort and destabilize transmembrane helices, the role of glycine is still debated. Here, we systematically investigated the e...

Regulation of intercellular tight junctions by zonula occludens toxin and its eukaryotic analogue zonulin.

Science.gov (United States)

Fasano, A

2000-01-01

The intestinal epithelium represents the largest interface between the external environment and the internal host milieu and constitutes the major barrier through which molecules can either be absorbed or secreted. There is now substantial evidence that tight junctions (tj) play a major role in regulating epithelial permeability by influencing paracellular flow of fluid and solutes. Tj are one of the hallmarks of absorptive and secretory epithelia. Evidence now exists that tj are dynamic rather than static structures and readily adapt to a variety of developmental, physiological, and pathological circumstances. These adaptive mechanisms are still incompletely understood. Activation of PKC either by Zonula occludens toxin (Zot) or by phorbol esters increases paracellular permeability. Alteration of epithelial tj is a recently described property for infectious agents. Clostridium difficile toxin A and B and influenza and vesicular stomatitis viruses have been shown to loosen tj in tissue culture monolayers. Unlike what occurs after the Zot stimulus, these changes appear to be irreversible and are associated with destruction of the tj complex. On the basis of this observation, we postulated that Zot may mimic the effect of a functionally and immunologically related endogenous modulator of epithelial tj. We were able to identify an intestinal Zot analogue, which we named zonulin. It is conceivable that the zonulins participate in the physiological regulation of intercellular tj not only in the small intestine, but also throughout a wide range of extraintestinal epithelia as well as the ubiquitous vascular endothelium, including the blood-brain barrier. Disregulation of this hypothetical zonulin model may contribute to disease states that involve disordered intercellular communication, including developmental and intestinal disorders, tissue inflammation, malignant transformation, and metastasis.

Levels Of Serum Intercellular And Vascular Adhesion Molecules In ...

African Journals Online (AJOL)

The study evaluated the possible significant role of soluble intercellular and vascular adhesion molecule-1 (sICAM-1 and sVCAM-1), sE-selectin and interluekin-1β in development nephropathy in patients with insulin dependent diabetes mellitus (IDDM). This study included 60 patients with type 1 diabetes mellitus (IDDM) ...

Biological amine transport in chromaffin ghosts. Coupling to the transmembrane proton and potential gradients.

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Johnson, R G; Pfister, D; Carty, S E; Scarpa, A

1979-11-10

The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in chromaffin ghosts. The chromaffin ghosts were formed by hypo-osmotic lysis of isolated bovine chromaffin granules and extensive dialysis in order to remove intragranular binding components and dissipate the endogenous electrochemical gradients. Upon ATP addition to suspensions of chromaffin ghosts, a transmembrane proton gradient alone, a transmembrane gradient alone, or both, could be established, depending upon the compositions of the media in which the ghosts were formed and resuspended. When chloride was present in the medium, addition of ATP resulted in the generation of a transmembrane proton gradient, acidic inside of 1 pH unit (measured by [14C]methylamine distribution), and no transmembrane potential (measured by [14C]-thiocyanate distribution). When ATP was added to chromaffin ghosts suspended in a medium in which chloride was substituted by isethionate, a transmembrane potential, inside positive, of 45 mV and no transmembrane proton gradient, was measured. In each medium, the addition of agents known to affect proton or potential gradients, respectively, exerted a predictable mechanism of action. Accumulation of [14C]epinephrine or [14C]5-hydroxytryptamine was over 1 order of magnitude greater in the presence of the transmembrane proton gradient or the transmembrane potential than in the absence of any gradient and, moreover, was related to the magnitude of the proton or potential gradient in a dose-dependent manner. When ghosts were added to a medium containing chloride and isethionate, both a delta pH and delta psi could be generated upon addition of ATP. In this preparation, the maximal rate of amine accumulation was observed. The results indicate that amine accumulation into chromaffin ghosts can occur in the presence of either a transmembrane proton gradient, or a transmembrane potential

When a transmembrane channel isn't, or how biophysics and biochemistry (mis)communicate.

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Reviakine, Ilya

2018-02-12

Annexins are a family of soluble proteins that bind to acidic phospholipids such as phosphatidylserine in a calcium-dependent manner. The archetypical member of the annexin family is annexin A5. For many years, its function remained unknown despite the availability of a high-resolution structure. This, combined with the observations of specific ion conductance in annexin-bound membranes, fueled speculations about the possible membrane-spanning forms of annexins that functioned as ion channels. The channel hypothesis remained controversial and did not gather sufficient evidence to become accepted. Yet, it continues to draw attention as a framework for interpreting indirect (e.g., biochemical) data. The goal of the mini-review is to examine the data on annexin-lipid interactions from the last ~30 years from the point of view of the controversy between the two lines of inquiry: the well-characterized peripheral assembly of the annexins at membranes vs. their putative transmembrane insertion. In particular, the potential role of lipid rearrangements induced by annexin binding is highlighted. Copyright © 2018 Elsevier B.V. All rights reserved.

The intercellular synchronization of Ca2+ oscillations evaluates Cx36-dependent coupling.

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Sabine Bavamian

Full Text Available Connexin36 (Cx36 plays an important role in insulin secretion by controlling the intercellular synchronization of Ca(2+ transients induced during stimulation. The lack of drugs acting on Cx36 channels is a major limitation in further unraveling the molecular mechanism underlying this effect. To screen for such drugs, we have developed an assay allowing for a semi-automatic, fluorimetric quantification of Ca(2+ transients in large populations of MIN6 cells. Here, we show that (1 compared to control cells, MIN6 cells with reduced Cx36 expression or function showed decreased synchrony of glucose-induced Ca(2+ oscillations; (2 glibenclamide, a sulphonylurea which promotes Cx36 junctions and coupling, increased the number of synchronous MIN6 cells, whereas quinine, an antimalarial drug which inhibits Cx36-dependent coupling, decreased this proportion; (3 several drugs were identified that altered the intercellular Ca(2+ synchronization, cell coupling and distribution of Cx36; (4 some of them also affected insulin content. The data indicate that the intercellular synchronization of Ca(2+ oscillations provides a reliable and non-invasive measurement of Cx36-dependent coupling, which is useful to identify novel drugs affecting the function of β-cells, neurons, and neuron-related cells that express Cx36.

Transmembrane-sequence-dependent overexpression and secretion of glycoproteins in Saccharomyces cerevisiae.

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Schuster, M; Wasserbauer, E; Aversa, G; Jungbauer, A

2001-02-01

Protein expression using the secretory pathway in Saccharomyces cerevisiae can lead to high amounts of overexpressed and secreted proteins in culture supernatants in a short period of time. These post-translational modified expression products can be purified up to >90% in a single step. The overexpression and secretion of the transmembrane glycoprotein signaling lymphocytic activation molecule (SLAM) was studied. SLAM belongs to the immunoglobulin superfamily and its engagement results in T-cell expansion and INF-gamma production. The molecule is composed of an extracellular, a single-span transmembrane and a cytoplasmatic domain. The extracellular part may be relevant for stimulation studies in vitro since SLAM is a high-affinity self-ligand. Therefore several fragments of this region have been expressed as Flag-fusions in S. cerevisiae: a full-length fragment containing the transmembrane region and the autologous signal sequence, another without the transmembrane region, and two fragments without the autologous signal sequence with and without the transmembrane region. By molecular cloning, the different deletion mutants of the cDNA encoding the full-length construct have been inserted in a yeast episomal plasmid. Upstream of the cDNA, the alpha-leader sequence of a yeast mating pheromone has been cloned to direct the fusion proteins into the secretory protein maturation pathway. All four fragments were expressed but yield, location, and maturation were highly influenced by the transmembrane domain and the autologous signal sequence. Only the fragment without autologous signal sequence and transmembrane domain could be efficiently secreted. High-mannose glycosylation was analyzed by lectin mapping and digestion with specific glycosidases. After enzyme treatment, a single band product with the theoretical size could be detected and identified as SLAM by a specific monoclonal antibody. The fusion protein concentration in the supernatant was 30 microg/ml. The

Optimal cellular mobility for synchronization arising from the gradual recovery of intercellular interactions

International Nuclear Information System (INIS)

Uriu, Koichiro; Ares, Saúl; Oates, Andrew C; Morelli, Luis G

2012-01-01

Cell movement and intercellular signaling occur simultaneously during the development of tissues, but little is known about how movement affects signaling. Previous theoretical studies have shown that faster moving cells favor synchronization across a population of locally coupled genetic oscillators. An important assumption in these studies is that cells can immediately interact with their new neighbors after arriving at a new location. However, intercellular interactions in cellular systems may need some time to become fully established. How movement affects synchronization in this situation has not been examined. Here, we develop a coupled phase oscillator model in which we consider cell movement and the gradual recovery of intercellular coupling experienced by a cell after movement, characterized by a moving rate and a coupling recovery rate, respectively. We find (1) an optimal moving rate for synchronization and (2) a critical moving rate above which achieving synchronization is not possible. These results indicate that the extent to which movement enhances synchrony is limited by a gradual recovery of coupling. These findings suggest that the ratio of time scales of movement and signaling recovery is critical for information transfer between moving cells. (paper)

Expression of a defence-related intercellular barley peroxidase in transgenic tobacco

DEFF Research Database (Denmark)

Kristensen, B.K.; Brandt, J.; Bojsen, K.

1997-01-01

genetically, phenotypically and biochemically. The T-DNA was steadily inherited through three generations. The barley peroxidase is expressed and sorted to the intercellular space in the transgenic tobacco plants. The peroxidase can be extracted from the intercellular space in two molecular forms from both...... barley and transgenic tobacco. The tobacco expressed forms are indistinguishable from the barley expressed forms as determined by analytical isoelectric focusing (pI 8.5) and Western-blotting. Staining for N-glycosylation showed that one form only was glycosylated. The N-terminus of purified Prx8 from...... transgenic tobacco was blocked by pyroglutamate, after the removal of which, N-terminal sequencing verified the transit signal-peptide cleavage site deduced from the cDNA sequence. Phenotype comparisons show that the constitutive expression of Prx8 lead to growth retardation. However, an infection assay...

Extracellular vesicle communication pathways as regulatory targets of oncogenic transformation.

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Choi, Dongsic; Lee, Tae Hoon; Spinelli, Cristiana; Chennakrishnaiah, Shilpa; D'Asti, Esterina; Rak, Janusz

2017-07-01

Pathogenesis of human cancers bridges intracellular oncogenic driver events and their impact on intercellular communication. Among multiple mediators of this 'pathological connectivity' the role of extracellular vesicles (EVs) and their subsets (exosomes, ectosomes, oncosomes) is of particular interest for several reasons. The release of EVs from cancer cells represents a unique mechanism of regulated expulsion of bioactive molecules, a process that also mediates cell-to-cell transfer of lipids, proteins, and nucleic acids. Biological effects of these processes have been implicated in several aspects of cancer-related pathology, including tumour growth, invasion, angiogenesis, metastasis, immunity and thrombosis. Notably, the emerging evidence suggests that oncogenic mutations may impact several aspects of EV-mediated cell-cell communication including: (i) EV release rate and protein content; (ii) molecular composition of cancer EVs; (iii) the inclusion of oncogenic and mutant macromolecules in the EV cargo; (iv) EV-mediated release of genomic DNA; (v) deregulation of mechanisms responsible for EV biogenesis (vesiculome) and (vi) mechanisms of EV uptake by cancer cells. Intriguingly, EV-mediated intercellular transfer of mutant and oncogenic molecules between subpopulations of cancer cells, their indolent counterparts and stroma may exert profound biological effects that often resemble (but are not tantamount to) oncogenic transformation, including changes in cell growth, clonogenicity and angiogenic phenotype, or cause cell stress and death. However, several biological barriers likely curtail a permanent horizontal transformation of normal cells through EV-mediated mechanisms. The ongoing analysis and targeting of EV-mediated intercellular communication pathways can be viewed as a new therapeutic paradigm in cancer, while the analysis of oncogenic cargo contained in EVs released from cancer cells into biofluids is being developed for clinical use as a biomarker

Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins

KAUST Repository

Chan, Yuk-kit

2015-04-01

Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient, and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1, and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future. This article is protected by copyright. All rights reserved.

Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins

KAUST Repository

Chan, Yuk-kit; Zhang, Huoming; Liu, Pei; Tsao, George Sai-wah; Li Lung, Maria; Mak, Nai-ki; Ngok-shun Wong, Ricky; Ying-kit Yue, Patrick

2015-01-01

Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient, and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1, and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future. This article is protected by copyright. All rights reserved.

Dysfunction in gap junction intercellular communication induces aberrant behavior of the inner cell mass and frequent collapses of expanded blastocysts in mouse embryos.

Science.gov (United States)

Togashi, Kazue; Kumagai, Jin; Sato, Emiko; Shirasawa, Hiromitsu; Shimoda, Yuki; Makino, Kenichi; Sato, Wataru; Kumazawa, Yukiyo; Omori, Yasufumi; Terada, Yukihiro

2015-06-01

We investigated the role of gap junctions (GJs) in embryological differentiation, and observed the morphological behavior of the inner cell mass (ICM) by time-lapse movie observation (TLM) with gap junction inhibitors (GJis). ICR mouse embryos were exposed to two types of GJis in CZB medium: oleamide (0 to 50 μM) and 1-heptanol (0 to 10 mM). We compared the rate of blastocyst formation at embryonic day 4.5 (E4.5) with E5.5. We also observed and evaluated the times from the second cleavage to each embryonic developing stage by TLM. We investigated embryonic distribution of DNA, Nanog protein, and Connexin 43 protein with immunofluorescent staining. In the comparison of E4.5 with E5.5, inhibition of gap junction intercellular communication (GJIC) delayed embryonic blastocyst formation. The times from the second cleavage to blastocyst formation were significantly extended in the GJi-treated embryos (control vs with oleamide, 2224 ± 179 min vs 2354 ± 278 min, p = 0.013). Morphological differences were traced in control versus GJi-treated embryos until the hatching stage. Oleamide induced frequent severe collapses of expanded blastocysts (77.4 % versus 26.3 %, p = 0.0001) and aberrant ICM divisions connected to sticky strands (74.3 % versus 5.3 %, p = 0.0001). Immunofluorescent staining indicated Nanog-positive cells were distributed in each divided ICM. GJIC plays an important role in blastocyst formation, collapses of expanded blastocysts, and the ICM construction in mouse embryos.

Membrane shape modulates transmembrane protein distribution.

Science.gov (United States)

Aimon, Sophie; Callan-Jones, Andrew; Berthaud, Alice; Pinot, Mathieu; Toombes, Gilman E S; Bassereau, Patricia

2014-01-27

Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Efect of intercellular extracts from banana inoculated leaves with Mycosphaerella fijiensis Morelet, on chloroplast electronic transport of Grande naine (AAA cv.

Directory of Open Access Journals (Sweden)

Michel Leiva-Mora

2003-01-01

Full Text Available Some foliar pathogens colonize intercellular spaces of damage tissues during infection process, mediated by toxins production and diffusion to kill adjacent healthy cells. Due to the absence of reliable bioassays, the physiologic effects of several phytotoxins are still ignored on cellular membranous systems of the affected cells. In the present work it was extracted the intercellular content from not inoculated and inoculated banana leaves with different Mycosphaerella fijiensis strains. Their effects on chloroplasts of Grande naine cv were evaluated by the absorbance evolution (595 nm of Hill reactive (DCPIP, mixture with 810 ì l of chloroplasts suspension and 99 ì l of the intercellular contents. The electronic exchange on chloroplasts suspension was inhibited by intercellular contents of inoculated leaves. The intercellular contents from leaves inoculated with I1 (high virulence strain had a major inhibiter effect respect to leaves inoculates with G1 strain (low virulence, showing a correspondence between the inhibiter effect of intercellular contents and the affection levels of affected tissues. The procedures used in this work will let to make studies concerned with Mycosphaerella fijiensis-Musa spp interactions and the future breeding programs. Key words: banana breeding, black Sigatoka, host pathogen interaction, physiological bioassays

Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes.

Science.gov (United States)

Abounit, Saïda; Bousset, Luc; Loria, Frida; Zhu, Seng; de Chaumont, Fabrice; Pieri, Laura; Olivo-Marin, Jean-Christophe; Melki, Ronald; Zurzolo, Chiara

2016-10-04

Synucleinopathies such as Parkinson's disease are characterized by the pathological deposition of misfolded α-synuclein aggregates into inclusions throughout the central and peripheral nervous system. Mounting evidence suggests that intercellular propagation of α-synuclein aggregates may contribute to the neuropathology; however, the mechanism by which spread occurs is not fully understood. By using quantitative fluorescence microscopy with co-cultured neurons, here we show that α-synuclein fibrils efficiently transfer from donor to acceptor cells through tunneling nanotubes (TNTs) inside lysosomal vesicles. Following transfer through TNTs, α-synuclein fibrils are able to seed soluble α-synuclein aggregation in the cytosol of acceptor cells. We propose that donor cells overloaded with α-synuclein aggregates in lysosomes dispose of this material by hijacking TNT-mediated intercellular trafficking. Our findings thus reveal a possible novel role of TNTs and lysosomes in the progression of synucleinopathies. © 2016 The Authors.

[Effects of Chinese herbal compound for supplementing qi and activating blood circulation on actin, Cx43 expressions and gap junctional intercellular communication functions of myocardial cells in patients with Coxsackie virus B 3 viral myocarditis].

Science.gov (United States)

Zhang, Ming-xue; He, Wei; Gu, Ping

2010-08-01

To observe the effect of Chinese herbal compound for supplementing qi and activating blood circulation (CHC) on the gap junctional intercellular communication (GJIC) function of myocardial cells in patients with Coxsackie virus B 3 (CVB3) viral myocarditis. Expressions of actin and connexin43 (Cx43) in myocardial cells of patients arranged in three groups (the normal control group, the viral infected group and the CHC treated group) were detected by immunohistochemical method; the fluorescence photobleaching recovery rate of cells was detected by laser scanning confocal microscope. As compared with the viral infected group, the expressions of actin and Cx43 were increased and the GJIC function was improved in the CHC treated group. CHC could antagonize viral injury on skeleton protein, and repair the structure of gap junction channel to improve the GJIC function of myocardial cells after being attacked by CVB3.

A percolation-like model for simulating inter-cellular diffusion in the context of bystander signalling in tumour

International Nuclear Information System (INIS)

Moulton, C.R.; Fleming, A.J.; Ebert, M.A.

2011-01-01

Full text: Despite ongoing active research, the role of the radiation bystander effect in modifying local tissue response to an ionising radiation dose remains unclear. The present study aims to provide new insight by simulating the diffusion-mediated inter-cellular communication processes in 2D and 3D cell-like structures to calculate likely signal ranges in the diffusion limited case. Random walks of individual signalling molecules were tracked between cells with inclusion of molecule-receptor interactions. The resulting diffusion anomaly is a function of cell density, signal uptake probability and the spatial arrangement of cells local to the signal origin. Uptake probability effects dominate percolation effects in disordered media. Diffu sion through 2D structures is more conducive to anomalous diffusion than diffusion through 3D structures. Values for time-dependent diffusion constants and permeability are derived for typical simulation parameters. Even at low signal uptake probabilities the communication range is restricted to a mean value of less than 100 foun owing to complete signal uptake by 600 s. This should be considered in light of the potential influence of signal relaying, flo dynamics or vasculature-mediated signalling.

Different domains are critical for oligomerization compatibility of different connexins

Science.gov (United States)

MARTÍNEZ, Agustín D.; MARIPILLÁN, Jaime; ACUÑA, Rodrigo; MINOGUE, Peter J.; BERTHOUD, Viviana M.; BEYER, Eric C.

2011-01-01

Oligomerization of connexins is a critical step in gap junction channel formation. Some members of the connexin family can oligomerize with other members and form functional heteromeric hemichannels [e.g. Cx43 (connexin 43) and Cx45], but others are incompatible (e.g. Cx43 and Cx26). To find connexin domains important for oligomerization, we constructed chimaeras between Cx43 and Cx26 and studied their ability to oligomerize with wild-type Cx43, Cx45 or Cx26. HeLa cells co-expressing Cx43, Cx45 or Cx26 and individual chimaeric constructs were analysed for interactions between the chimaeras and the wild-type connexins using cell biological (subcellular localization by immunofluorescence), functional (intercellular diffusion of microinjected Lucifer yellow) and biochemical (sedimentation velocity through sucrose gradients) assays. All of the chimaeras containing the third transmembrane domain of Cx43 interacted with wild-type Cx43 on the basis of co-localization, dominant-negative inhibition of intercellular communication, and altered sedimentation velocity. The same chimaeras also interacted with co-expressed Cx45. In contrast, immunofluorescence and intracellular diffusion of tracer suggested that other domains influenced oligomerization compatibility when chimaeras were co-expressed with Cx26. Taken together, these results suggest that amino acids in the third transmembrane domain are critical for oligomerization with Cx43 and Cx45. However, motifs in different domains may determine oligomerization compatibility in members of different connexin subfamilies. PMID:21348854

Exosomes: mediators of communication in eukaryotes

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María A Lopez-Verrilli

2013-01-01

Full Text Available In addition to the established mechanisms of intercellular signaling, a new way of communication has gained much attention in the last decade: communication mediated by exosomes. Exosomes are nanovesicles (with a diameter of 40-120 nm secreted into the extracellular space by the multivesicular endosome after its outer membrane fuses with the plasma membrane. Once released, exosomes modulate the response of the recipient cells that recognize them. This indicates that exosomes operate in a specific manner and participate in the regulation of the target cell. Remarkably, exosomes occur from unicellular organisms to mammals, suggesting an evolutionarily conserved mechanism of communication. In this review we describe the cascade of exosome formation, intracellular traffic, secretion, and internalization by recipient cells, and review their most relevant effects. We also highlight important steps that are still poorly understood.

Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells.

Science.gov (United States)

Babica, Pavel; Zurabian, Rimma; Kumar, Esha R; Chopra, Rajus; Mianecki, Maxwell J; Park, Joon-Suk; Jaša, Libor; Trosko, James E; Upham, Brad L

2016-09-01

Methoxychlor (MXC) and vinclozolin (VIN) are well-recognized endocrine disrupting chemicals known to alter epigenetic regulations and transgenerational inheritance; however, non-endocrine disruption endpoints are also important. Thus, we determined the effects of MXC and VIN on the dysregulation of gap junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells. Both chemicals induced a rapid dysregulation of GJIC at non-cytotoxic doses, with 30 min EC50 values for GJIC inhibition being 10 µM for MXC and 126 µM for VIN. MXC inhibited GJIC for at least 24 h, while VIN effects were transient and GJIC recovered after 4 h. VIN induced rapid hyperphosphorylation and internalization of gap junction protein connexin43, and both chemicals also activated MAPK ERK1/2 and p38. Effects on GJIC were not prevented by MEK1/2 inhibitor, but by an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), resveratrol, and in the case of VIN, also, by a p38 inhibitor. Estrogen (ER) and androgen receptor (AR) modulators (estradiol, ICI 182,780, HPTE, testosterone, flutamide, VIN M2) did not attenuate MXC or VIN effects on GJIC. Our data also indicate that the effects were elicited by the parental compounds of MXC and VIN. Our study provides new evidence that MXC and VIN dysregulate GJIC via mechanisms involving rapid activation of PC-PLC occurring independently of ER- or AR-dependent genomic signaling. Such alterations of rapid intercellular and intracellular signaling events involved in regulations of gene expression, tissue development, function and homeostasis, could also contribute to transgenerational epigenetic effects of endocrine disruptors. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Proteins Play Important Role in Intercellular Adhesion Affecting on Fruit Textural Quality

DEFF Research Database (Denmark)

Bahadur Adhikari, Khem; Shomer, Ilan

2012-01-01

Fruit textural quality is becoming a major quality parameter for export, postharvest preservation, handling and processing. The main determinant of textural quality is intercellular adhesion (ICA) as attributed by the cell wall (CW) and its components. The importance of CW protein in ICA strength......Fruit textural quality is becoming a major quality parameter for export, postharvest preservation, handling and processing. The main determinant of textural quality is intercellular adhesion (ICA) as attributed by the cell wall (CW) and its components. The importance of CW protein in ICA...... strengthening was exempli ed in Medjoul date (Phoenix dactylifera L.) fruit, as a model. Fruit mesocarp sensitively responded to culture environment which was assayed in vitro at pH 3.5( pKa) in presence of organic acid molecules. The max penetration force, as a measure of ICA strength, of p...

Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication

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Eva Skiöldebrand

2018-01-01

Full Text Available Chondrocytes are effectively involved in the pathophysiological processes of inflammation in joints. They form cellular processes in the superficial layer of the articular cartilage and form gap junction coupled syncytium to facilitate cell-to-cell communication. However, very little is known about their physiological cellular identity and communication. The aim with the present work is to evaluate the physiological behavior after stimulation with the inflammatory inducers interleukin-1β and lipopolysaccharide. The cytoskeleton integrity and intracellular Ca2+ release were assessed as indicators of inflammatory state. Cytoskeleton integrity was analyzed through cartilage oligomeric matrix protein and actin labeling with an Alexa 488-conjugated phalloidin probe. Ca2+ responses were assessed through the Ca2+ sensitive fluorophore Fura-2/AM. Western blot analyses of several inflammatory markers were performed. The results show reorganization of the actin filaments. Glutamate, 5-hydoxytryptamine, and ATP evoked intracellular Ca2+ release changed from single peaks to oscillations after inflammatory induction in the chondrocytes. The expression of toll-like receptor 4, the glutamate transporters GLAST and GLT-1, and the matrix metalloproteinase-13 increased. This work demonstrates that chondrocytes are a key part in conditions that lead to inflammation in the cartilage. The inflammatory inducers modulate the cytoskeleton, the Ca2+ signaling, and several inflammatory parameters. In conclusion, our data show that the cellular responses to inflammatory insults from healthy and inflammatory chondrocytes resemble those previously observed in astrocyte and cardiac fibroblasts networks.

Stomatal responses to flooding of the intercellular air spaces suggest a vapor-phase signal between the mesophyll and the guard cells.

Science.gov (United States)

Sibbernsen, Erik; Mott, Keith A

2010-07-01

Flooding the intercellular air spaces of leaves with water was shown to cause rapid closure of stomata in Tradescantia pallida, Lactuca serriola, Helianthus annuus, and Oenothera caespitosa. The response occurred when water was injected into the intercellular spaces, vacuum infiltrated into the intercellular spaces, or forced into the intercellular spaces by pressurizing the xylem. Injecting 50 mm KCl or silicone oil into the intercellular spaces also caused stomata to close, but the response was slower than with distilled water. Epidermis-mesophyll grafts for T. pallida were created by placing the epidermis of one leaf onto the exposed mesophyll of another leaf. Stomata in these grafts opened under light but closed rapidly when water was allowed to wick between epidermis and the mesophyll. When epidermis-mesophyll grafts were constructed with a thin hydrophobic filter between the mesophyll and epidermis stomata responded normally to light and CO(2). These data, when taken together, suggest that the effect of water on stomata is caused partly by dilution of K(+) in the guard cell and partly by the existence of a vapor-phase signal that originates in the mesophyll and causes stomata to open in the light.

Regulation of the Host Antiviral State by Intercellular Communications

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Sonia Assil

2015-08-01

Full Text Available Viruses usually induce a profound remodeling of host cells, including the usurpation of host machinery to support their replication and production of virions to invade new cells. Nonetheless, recognition of viruses by the host often triggers innate immune signaling, preventing viral spread and modulating the function of immune cells. It conventionally occurs through production of antiviral factors and cytokines by infected cells. Virtually all viruses have evolved mechanisms to blunt such responses. Importantly, it is becoming increasingly recognized that infected cells also transmit signals to regulate innate immunity in uninfected neighboring cells. These alternative pathways are notably mediated by vesicular secretion of various virus- and host-derived products (miRNAs, RNAs, and proteins and non-infectious viral particles. In this review, we focus on these newly-described modes of cell-to-cell communications and their impact on neighboring cell functions. The reception of these signals can have anti- and pro-viral impacts, as well as more complex effects in the host such as oncogenesis and inflammation. Therefore, these “broadcasting” functions, which might be tuned by an arms race involving selective evolution driven by either the host or the virus, constitute novel and original regulations of viral infection, either highly localized or systemic.

The predominant mechanism of intercellular calcium wave propagation changes during long-term culture of human osteoblast-like cells

DEFF Research Database (Denmark)

Henriksen, Zanne; Hiken, Jeffrey F; Steinberg, Thomas H

2006-01-01

cells still responded to addition of ATP, but P2Y desensitization did not inhibit ICW propagation. Our data indicate that the relative role of P2Y-mediated and gap junction-mediated ICW changes during osteoblast differentiation in vitro. In less differentiated cells, P2Y-mediated ICW predominate......Intercellular calcium waves (ICW) are calcium transients that spread from cell to cell in response to different stimuli. We previously demonstrated that human osteoblast-like cells in culture propagate ICW in response to mechanical stimulation by two mechanisms. One mechanism involves autocrine...... activation of P2Y receptors, and the other requires gap junctional communication. In the current work we ask whether long-term culture of osteoblast-like cells affects the propagation of ICW by these two mechanisms. Human osteoblast-like cells were isolated from bone marrow. Mechanically induced ICW were...

Intercellular Resistance to BRAF Inhibition Can Be Mediated by Extracellular Vesicle–Associated PDGFRβ

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Laura J. Vella

2017-11-01

Full Text Available Treatment of BRAF mutant melanoma with kinase inhibitors has been associated with rapid tumor regression; however, this clinical benefit is short-lived, and most patients relapse. A number of studies suggest that the extracellular environment promotes BRAF inhibitor resistance and tumor progression. Extracellular vesicles, such as exosomes, are functional mediators in the extracellular environment. They are small vesicles known to carry a concentrated group of functional cargo and serve as intercellular communicators not only locally but also systemically. Increasingly, it is reported that extracellular vesicles facilitate the development of drug resistance in cancer; however, their role in BRAF inhibitor resistance in melanoma is unclear. Here we investigated if extracellular vesicles from BRAF inhibitor–resistant melanoma could influence drug sensitivity in recipient melanoma cells. We demonstrate that the resistance driver, PDGFRβ, can be transferred to recipient melanoma cells via extracellular vesicles, resulting in a dose-dependent activation of PI3K/AKT signaling and escape from MAPK pathway BRAF inhibition. These data suggest that the BRAF inhibitor–sensitive phenotype of metastatic melanoma can be altered by delivery of PDGFRβ by extracellular vesicles derived from neighboring drug-resistant melanoma cells.

HYS-32, a novel analogue of combretastatin A-4, enhances connexin43 expression and gap junction intercellular communication in rat astrocytes.

Science.gov (United States)

Lin, Pei-Chun; Shen, Chien-Chang; Liao, Chih-Kai; Jow, Guey-Mei; Chiu, Chi-Ting; Chung, Tun-Hui; Wu, Jiahn-Chun

2013-05-01

HYS-32 [4-(3,4-dimethoxyphenyl)-3-(naphthalen-2-yl)-2(5H)-furanone] is a new analogue of the anti-tumor compound combretastatin A-4 containing a cis-stilbene moiety. In this study, we investigated its effects on Cx43 gap junction intercellular communication (GJIC) and the signaling pathway involved in rat primary astrocytes. Western blot analyses showed that HYS-32 dose- and time-dependently upregulated Cx43 expression. A confocal microscopic study and scrape-loading/dye transfer analyses demonstrated that HYS-32 (5μM) induced microtubule coiling, accumulation of Cx43 in gap junction plaques, and increased GJIC in astrocytes. The HYS-32-induced microtubule coiling and Cx43 accumulation in gap junction plaques was reversed when HYS-32 was removed. Treatment of astrocytes with cycloheximide resulted in time-dependent degradation of by co-treatment with HYS-32 by increasing the half-life of Cx43. Co-treatment with HYS-32 also prevented the LPS-induced downregulation of Cx43 and inhibition of GJIC in astrocytes. HYS-32 induced activation of PKC, ERK, and JNK, and co-treatment with the PKC inhibitor Go6976 or the ERK inhibitor PD98059, but not the JNK inhibitor SP600125, prevented the HYS-32-induced increase in Cx43 expression and GJIC. Go6976 suppressed the HYS-32-induced PKC phosphorylation and increase in phospho-ERK levels, while PD98059 did not prevent the HYS-32-induced increase in phospho-PKC levels, suggesting that PKC is an upstream effector of ERK. In conclusion, our results show that HYS-32 increases the half-life of Cx43 and enhances Cx43 expression and GJIC in astrocytes via a PKC-ERK signaling cascade. These novel biological effects of HYS-32 on astrocyte gap junctions support its potential for therapeutic use as a protective agent for the central nervous system. Copyright © 2013 Elsevier Ltd. All rights reserved.

Transformed hairy roots of Discaria trinervis: a valuable tool for studying actinorhizal symbiosis in the context of intercellular infection.

Science.gov (United States)

Imanishi, Leandro; Vayssières, Alice; Franche, Claudine; Bogusz, Didier; Wall, Luis; Svistoonoff, Sergio

2011-11-01

Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized. Intercellular infection has been described in Discaria trinervis, an actinorhizal plant belonging to the Rosales order. To decipher the molecular mechanisms underlying intercellular infection with Frankia bacteria, we set up an efficient genetic transformation protocol for D. trinervis based on Agrobacterium rhizogenes. We showed that composite plants with transgenic roots expressing green fluorescent protein can be specifically and efficiently nodulated by Frankia strain BCU110501. Nitrogen fixation rates and feedback inhibition of nodule formation by nitrogen were similar in control and composite plants. In order to challenge the transformation system, the MtEnod11 promoter, a gene from Medicago truncatula widely used as a marker for early infection-related symbiotic events in model legumes, was introduced in D. trinervis. MtEnod11::GUS expression was related to infection zones in root cortex and in the parenchyma of the developing nodule. The ability to study intercellular infection with molecular tools opens new avenues for understanding the evolution of the infection process in nitrogen-fixing root nodule symbioses.

Shaping intercellular channels of plasmodesmata: the structure-to-function missing link.

Science.gov (United States)

Nicolas, William J; Grison, Magali S; Bayer, Emmanuelle M

2017-12-18

Plasmodesmata (PD) are a hallmark of the plant kingdom and a cornerstone of plant biology and physiology, forming the conduits for the cell-to-cell transfer of proteins, RNA and various metabolites, including hormones. They connect the cytosols and endomembranes of cells, which allows enhanced cell-to-cell communication and synchronization. Because of their unique position as intercellular gateways, they are at the frontline of plant defence and signalling and constitute the battleground for virus replication and spreading. The membranous organization of PD is remarkable, where a tightly furled strand of endoplasmic reticulum comes into close apposition with the plasma membrane, the two connected by spoke-like elements. The role of these structural features is, to date, still not completely understood. Recent data on PD seem to point in an unexpected direction, establishing a close parallel between PD and membrane contact sites and defining plasmodesmal membranes as microdomains. However, the implications of this new viewpoint are not fully understood. Aided by available phylogenetic data, this review attempts to reassess the function of the different elements comprising the PD and the relevance of membrane lipid composition and biophysics in defining specialized microdomains of PD, critical for their function. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Biochar and microbial signaling: production conditions determine effects on microbial communication

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Masiello, Caroline A.; Chen, Ye; Gao, Xiaodong; Liu, Shirley; Cheng, Hsiao-Ying; Bennett, Matthew R.; Rudgers, Jennifer A.; Wagner, Daniel S.; Zygourakis, Kyriacos; Silberg, Jonathan J.

2013-01-01

Charcoal has a long soil residence time, which has resulted in its production and use as a carbon sequestration technique (biochar). A range of biological effects can be triggered by soil biochar that can positively and negatively influence carbon storage, such as changing the decomposition rate of organic matter and altering plant biomass production. Sorption of cellular signals has been hypothesized to underlie some of these effects, but it remains unknown whether the binding of biochemical signals occurs, and if so, on time scales relevant to microbial growth and communication. We examined biochar sorption of N-3-oxo-dodecanoyl-L-homoserine lactone, an acyl-homoserine lactone (AHL) intercellular signaling molecule used by many gram-negative soil microbes to regulate gene expression. We show that wood biochars disrupt communication within a growing multicellular system that is made up of sender cells that synthesize AHL and receiver cells that express green fluorescent protein in response to an AHL signal. However, biochar inhibition of AHL-mediated cell-cell communication varied, with the biochar prepared at 700°C (surface area of 301 m2/g) inhibiting cellular communication 10-fold more than an equivalent mass of biochar prepared at 300°C (surface area of 3 m2/g). These findings provide the first direct evidence that biochars elicit a range of effects on gene expression dependent on intercellular signaling, implicating the method of biochar preparation as a parameter that could be tuned to regulate microbial-dependent soil processes, like nitrogen fixation and pest attack of root crops. PMID:24066613

Biochar and microbial signaling: production conditions determine effects on microbial communication.

Science.gov (United States)

Masiello, Caroline A; Chen, Ye; Gao, Xiaodong; Liu, Shirley; Cheng, Hsiao-Ying; Bennett, Matthew R; Rudgers, Jennifer A; Wagner, Daniel S; Zygourakis, Kyriacos; Silberg, Jonathan J

2013-10-15

Charcoal has a long soil residence time, which has resulted in its production and use as a carbon sequestration technique (biochar). A range of biological effects can be triggered by soil biochar that can positively and negatively influence carbon storage, such as changing the decomposition rate of organic matter and altering plant biomass production. Sorption of cellular signals has been hypothesized to underlie some of these effects, but it remains unknown whether the binding of biochemical signals occurs, and if so, on time scales relevant to microbial growth and communication. We examined biochar sorption of N-3-oxo-dodecanoyl-L-homoserine lactone, an acyl-homoserine lactone (AHL) intercellular signaling molecule used by many gram-negative soil microbes to regulate gene expression. We show that wood biochars disrupt communication within a growing multicellular system that is made up of sender cells that synthesize AHL and receiver cells that express green fluorescent protein in response to an AHL signal. However, biochar inhibition of AHL-mediated cell-cell communication varied, with the biochar prepared at 700 °C (surface area of 301 m(2)/g) inhibiting cellular communication 10-fold more than an equivalent mass of biochar prepared at 300 °C (surface area of 3 m(2)/g). These findings provide the first direct evidence that biochars elicit a range of effects on gene expression dependent on intercellular signaling, implicating the method of biochar preparation as a parameter that could be tuned to regulate microbial-dependent soil processes, like nitrogen fixation and pest attack of root crops.

[Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members].

Science.gov (United States)

Goncharuk, M V; Shul'ga, A A; Ermoliuk, Ia S; Tkach, E N; Goncharuk, S A; Pustovalova, Iu E; Mineev, K S; Bocharov, É V; Maslennikov, I V; Arsen'ev, A S; Kirpichnikov, M P

2011-01-01

A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.

Costimulation of N-methyl-d-aspartate and muscarinic neuronal receptors modulates gap junctional communication in striatal astrocytes

OpenAIRE

Rouach, N.; Tencé, M.; Glowinski, J.; Giaume, C.

2002-01-01

Cocultures of neurons and astrocytes from the rat striatum were used to determine whether the stimulation of neuronal receptors could affect the level of intercellular communication mediated by gap junctions in astrocytes. The costimulation of N-methyl-D-asparte (NMDA) and muscarinic receptors led to a prominent reduction of astrocyte gap junctional communication (GJC) in coculture. This treatment was not effective in astrocyte cultures, these cells being devoid of NMDA receptors. Both types ...

Stomatal Responses to Flooding of the Intercellular Air Spaces Suggest a Vapor-Phase Signal Between the Mesophyll and the Guard Cells1[OA

Science.gov (United States)

Sibbernsen, Erik; Mott, Keith A.

2010-01-01

Flooding the intercellular air spaces of leaves with water was shown to cause rapid closure of stomata in Tradescantia pallida, Lactuca serriola, Helianthus annuus, and Oenothera caespitosa. The response occurred when water was injected into the intercellular spaces, vacuum infiltrated into the intercellular spaces, or forced into the intercellular spaces by pressurizing the xylem. Injecting 50 mm KCl or silicone oil into the intercellular spaces also caused stomata to close, but the response was slower than with distilled water. Epidermis-mesophyll grafts for T. pallida were created by placing the epidermis of one leaf onto the exposed mesophyll of another leaf. Stomata in these grafts opened under light but closed rapidly when water was allowed to wick between epidermis and the mesophyll. When epidermis-mesophyll grafts were constructed with a thin hydrophobic filter between the mesophyll and epidermis stomata responded normally to light and CO2. These data, when taken together, suggest that the effect of water on stomata is caused partly by dilution of K+ in the guard cell and partly by the existence of a vapor-phase signal that originates in the mesophyll and causes stomata to open in the light. PMID:20472750

Radiation-induced perturbation of cell-to-cell signalling and communication

International Nuclear Information System (INIS)

Mariotti, L.; Facoetti, A.; Bertolotti, A.; Ranza, E.; Alloni, D.; Ottolenghi, A.

2011-01-01

The investigation of the bystander phenomena (i.e. the induction of damage in cells not directly traversed by radiation) is strictly related to the study of the mechanisms of intercellular communication and of the perturbative effects of radiation. A new possible way to try to solve the bystander puzzle is through a 'systems radiation biology' approach with the total integration of experimental and theoretical activities. In particular, this contribution will focus on: (1) 'ad hoc' experiments designed to quantify key parameters involved in intercellular signalling (focusing, as a pilot study, on release, decay and internalization of interleukin-6 molecules, their modulation by radiation, and possible differences between in vivo/in vitro behaviour); (2) the implementation and the development of two different modelling approaches: a stochastic model (based on a Monte Carlo code) that takes account of the local mechanisms of release and internalization of signalling molecules (e.g. cytokines) and an analytical model where signal molecules are treated as a population and their temporal behaviour is described by differential equations. This approach provided instruments to investigate the complex phenomena of signal transmission and the role of cell communication to guarantee (maintain) the robustness of the in vitro experimental systems against the effects of perturbations. (authors)

The soluble loop BC region guides, but not dictates, the assembly of the transmembrane cytochrome b6.

Directory of Open Access Journals (Sweden)

Lydia Tome-Stangl

Full Text Available Studying folding and assembly of naturally occurring α-helical transmembrane proteins can inspire the design of membrane proteins with defined functions. Thus far, most studies have focused on the role of membrane-integrated protein regions. However, to fully understand folding pathways and stabilization of α-helical membrane proteins, it is vital to also include the role of soluble loops. We have analyzed the impact of interhelical loops on folding, assembly and stability of the heme-containing four-helix bundle transmembrane protein cytochrome b6 that is involved in charge transfer across biomembranes. Cytochrome b6 consists of two transmembrane helical hairpins that sandwich two heme molecules. Our analyses strongly suggest that the loop connecting the helical hairpins is not crucial for positioning the two protein "halves" for proper folding and assembly of the holo-protein. Furthermore, proteolytic removal of any of the remaining two loops, which connect the two transmembrane helices of a hairpin structure, appears to also not crucially effect folding and assembly. Overall, the transmembrane four-helix bundle appears to be mainly stabilized via interhelical interactions in the transmembrane regions, while the soluble loop regions guide assembly and stabilize the holo-protein. The results of this study might steer future strategies aiming at designing heme-binding four-helix bundle structures, involved in transmembrane charge transfer reactions.

Urinary Exosomes: The Potential for Biomarker Utility, Intercellular Signaling and Therapeutics in Urological Malignancy.

Science.gov (United States)

Franzen, Carrie A; Blackwell, Robert H; Foreman, Kimberly E; Kuo, Paul C; Flanigan, Robert C; Gupta, Gopal N

2016-05-01

Exosomes are small secreted vesicles that contain proteins, mRNA and miRNA with the potential to alter signaling pathways in recipient cells. While exosome research has flourished, few publications have specifically considered the role of genitourinary cancer shed exosomes in urine, their implication in disease progression and their usefulness as noninvasive biomarkers. In this review we examined the current literature on the role of exosomes in intercellular communication and as biomarkers, and their potential as delivery vehicles for therapeutic applications in bladder, prostate and renal cancer. We searched PubMed® and Google® with the key words prostate cancer, bladder cancer, kidney cancer, exosomes, microvesicles and urine. Relevant articles, including original research studies and reviews, were selected based on contents. A review of this literature was generated. Cancer exosomes can be isolated from urine using various techniques. Cancer cells have been found to secrete more exosomes than normal cells. These exosomes have a role in cellular communication by interacting with and depositing their cargo in target cells. Bladder, prostate and renal cancer exosomes have been shown to enhance migration, invasion and angiogenesis. These exosomes have also been shown to increase proliferation, confer drug resistance and promote immune evasion. Urinary exosomes can be isolated from bladder, kidney and prostate cancer. They serve as a potential reservoir for biomarker identification. Exosomes also have potential for therapeutics as siRNA or pharmacological agents can be loaded into exosomes. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

Science.gov (United States)

Wang, Hong; Brautigan, David L

2006-11-01

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

Correlation of Aquaporins and Transmembrane Solute Transporters Revealed by Genome-Wide Analysis in Developing Maize Leaf

Directory of Open Access Journals (Sweden)

Xun Yue

2012-01-01

Full Text Available Aquaporins are multifunctional membrane channels that facilitate the transmembrane transport of water and solutes. When transmembrane mineral nutrient transporters exhibit the same expression patterns as aquaporins under diverse temporal and physiological conditions, there is a greater probability that they interact. In this study, genome-wide temporal profiling of transcripts analysis and coexpression network-based approaches are used to examine the significant specificity correlation of aquaporins and transmembrane solute transporters in developing maize leaf. The results indicate that specific maize aquaporins are related to specific transmembrane solute transporters. The analysis demonstrates a systems-level correlation between aquaporins, nutrient transporters, and the homeostasis of mineral nutrients in developing maize leaf. Our results provide a resource for further studies into the physiological function of these aquaporins.

A rapid co-culture stamping device for studying intercellular communication

Science.gov (United States)

Hassanzadeh-Barforoushi, Amin; Shemesh, Jonathan; Farbehi, Nona; Asadnia, Mohsen; Yeoh, Guan Heng; Harvey, Richard P.; Nordon, Robert E.; Warkiani, Majid Ebrahimi

2016-10-01

Regulation of tissue development and repair depends on communication between neighbouring cells. Recent advances in cell micro-contact printing and microfluidics have facilitated the in-vitro study of homotypic and heterotypic cell-cell interaction. Nonetheless, these techniques are still complicated to perform and as a result, are seldom used by biologists. We report here development of a temporarily sealed microfluidic stamping device which utilizes a novel valve design for patterning two adherent cell lines with well-defined interlacing configurations to study cell-cell interactions. We demonstrate post-stamping cell viability of >95%, the stamping of multiple adherent cell types, and the ability to control the seeded cell density. We also show viability, proliferation and migration of cultured cells, enabling analysis of co-culture boundary conditions on cell fate. We also developed an in-vitro model of endothelial and cardiac stem cell interactions, which are thought to regulate coronary repair after myocardial injury. The stamp is fabricated using microfabrication techniques, is operated with a lab pipettor and uses very low reagent volumes of 20 μl with cell injection efficiency of >70%. This easy-to-use device provides a general strategy for micro-patterning of multiple cell types and will be important for studying cell-cell interactions in a multitude of applications.

Investigation of intercellular salicylic acid accumulation during compatible and incompatible Arabidopsis-pseudomonas syringae interactions using a fast neutron-generated mutant allele of EDS5 identified by genetic mapping and whole-genome sequencing.

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Jessie L Carviel

Full Text Available A whole-genome sequencing technique developed to identify fast neutron-induced deletion mutations revealed that iap1-1 is a new allele of EDS5 (eds5-5. RPS2-AvrRpt2-initiated effector-triggered immunity (ETI was compromised in iap1-1/eds5-5 with respect to in planta bacterial levels and the hypersensitive response, while intra- and intercellular free salicylic acid (SA accumulation was greatly reduced, suggesting that SA contributes as both an intracellular signaling molecule and an antimicrobial agent in the intercellular space during ETI. During the compatible interaction between wild-type Col-0 and virulent Pseudomonas syringae pv. tomato (Pst, little intercellular free SA accumulated, which led to the hypothesis that Pst suppresses intercellular SA accumulation. When Col-0 was inoculated with a coronatine-deficient strain of Pst, high levels of intercellular SA accumulation were observed, suggesting that Pst suppresses intercellular SA accumulation using its phytotoxin coronatine. This work suggests that accumulation of SA in the intercellular space is an important component of basal/PAMP-triggered immunity as well as ETI to pathogens that colonize the intercellular space.

Using Information Theory to Assess the Communicative Capacity of Circulating MicroRNA

OpenAIRE

Finn, Nnenna A.; Searles, Charles D.

2013-01-01

The discovery of extracellular microRNAs (miRNAs) and their transport modalities (i.e. microparticles, exosomes, proteins and lipoproteins) has sparked theories regarding their role in intercellular communication. Here, we assessed the information transfer capacity of different miRNA transport modalities in human serum by utilizing basic principles of information theory. Zipf Statistics were calculated for each of the miRNA transport modalities identified in human serum. Our analyses revealed...

Disruption of gap junctional intercellular communication by antibiotic gentamicin is associated with aberrant localization of occludin, N-cadherin, connexin 43, and vimentin in SerW3 Sertoli cells in vitro.

Science.gov (United States)

Bekheet, Souad H M; Stahlmann, Ralf

2009-09-01

Spermatogenesis is a very complex process by which male germ cells differentiate into mature spermatozoa. The sophisticated communication network that controls spermatogenesis can be derailed so that dysfunction of one cell type propagates to all types as a cascade. This accounts for the particular vulnerability of the testis to environmental factors such as drugs and xenobiotics. Sertoli cells play an important role in protecting developing germ cells by forming a physiological barrier, limiting exposure to potentially toxic substrates, or conversely, facilitating uptake of xenobiotics within the testis. In this study, cells from the rat Sertoli line (SerW3) were incubated for 3, 6 and 9 subsequent days in serum free DMEM (SFDM) composed of DMEM supplemented with three different concentrations of antibiotic gentamicin (10, 30, and 100 μg). The effect of the three different concentrations of this antibiotic was determined on Sertoli cell-cell interaction through impaired expression of their constitutive tight junction proteins as early targets for different toxicants in vitro by immunochemistry analysis. The Sertoli SerW3 cell line illustrated the cytotoxicity of GS, as the intercellular junction proteins such as occludin, N-cadherin, connexin 43, and vimentin were delocalized from the membrane to the cytoplasmic compartment during exposure to the antibiotic. This study underlines the potential deleterious effects of the routine use of antibiotics during continuous cell culture.

Involvement of a putative intercellular signal-recognizing G protein-coupled receptor in the engulfment of Salmonella by the protozoan Tetrahymena

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P.N. Agbedanu

2013-07-01

Full Text Available In an effort to investigate the molecular basis of protozoa engulfment-mediated hypervirulence of Salmonella in cattle, we evaluated protozoan G protein-coupled receptors (GPCRs as transducers of Salmonella engulfment by the model protozoan Tetrahymena. Our laboratory previously demonstrated that non-pathogenic protozoa (including Tetrahymena engulf Salmonella and then exacerbate its virulence in cattle, but the mechanistic details of the phenomenon are not fully understood. GPCRs were investigated since these receptors facilitate phagocytosis of particulates by Tetrahymena, and a GPCR apparently modulates bacterial engulfment for the pathogenic protozoan Entamoeba histolytica. A database search identified three putative Tetrahymena GPCRs, based on sequence homologies and predicted transmembrane domains, that were the focus of this study. Salmonella engulfment by Tetrahymena was assessed in the presence of suramin, a non-specific GPCR inhibitor. Salmonella engulfment was also assessed in Tetrahymena in which expression of putative GPCRs was knocked-down using RNAi. A candidate GPCR was then expressed in a heterologous yeast expression system for further characterization. Our results revealed that Tetrahymena were less efficient at engulfing Salmonella in the presence of suramin. Engulfment was reduced concordantly with a reduction in the density of protozoa. RNAi-based studies revealed that knock-down of one the Tetrahymena GPCRs caused diminished engulfment of Salmonella. Tetrahymena lysates activated this receptor in the heterologous expression system. These data demonstrate that the Tetrahymena receptor is a putative GPCR that facilitates bacterial engulfment by Tetrahymena. Activation of the putative GPCR seemed to be related to protozoan cell density, suggesting that its cognate ligand is an intercellular signaling molecule.

Detection of cancerous kidney tissue areas by means of infrared spectroscopy of intercellular fluid

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Urboniene, V.; Jankevicius, F.; Zelvys, A.; Steiner, G.; Sablinskas, V.

2014-03-01

In this work the infrared absorption spectra of intercellular fluid of normal and tumor kidney tissue were recorded and analyzed. The samples were prepared by stamping freshly resected tissue onto a CaF2 substrate. FT-IR spectra obtained from intracellular fluid of tumor tissue exhibit stronger absorption bands in the spectral region from 1000-1200 cm-1 and around 1750 cm-1 than those obtained from normal tissue. It is likely the spectra of extracellular matrix of kidney tumor tissue with large increases in the intensities of these bands represent a higher concentration of fatty acids and glycerol. Amide I and amide II bands are stronger in spectra of normal tissue indicating a higher level of proteins. The results demonstrate that FT-IR spectroscopy of intercellular fluids is a novel approach for a quick diagnosis during surgical resection, which can improve the therapy of kidney tumors.

Exosomal Heat Shock Proteins as New Players in Tumour Cell-to-cell Communication

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Claudia Campanella

2014-06-01

Full Text Available Exosomes have recently been proposed as novel elements in the study of intercellular communication in normal and pathological conditions. The biomolecular composition of exosomes reflects the specialized functions of the original cells. Heat shock proteins (Hsps are a group of chaperone proteins with diverse biological roles. In recent years, many studies have focused on the extracellular roles played by Hsps that appear to be involved in cancer development and immune system stimulation. Hsps localized on the surface of exosomes, secreted by normal and tumour cells, could be key players in intercellular cross-talk, particularly during the course of different diseases, such as cancer. Exosomal Hsps offer significant opportunities for clinical applications, including their use as potential novel biomarkers for the diagnoses or prognoses of different diseases, or for therapeutic applications and drug delivery.

Exosomal Heat Shock Proteins as New Players in Tumour Cell-to-Cell Communication

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Claudia Campanella

2014-06-01

Full Text Available Exosomes have recently been proposed as novel elements in the study of intercellular communication in normal and pathological conditions. The biomolecular composition of exosomes reflects the specialized functions of the original cells. Heat shock proteins (Hsps are a group of chaperone proteins with diverse biological roles. In recent years, many studies have focused on the extracellular roles played by Hsps that appear to be involved in cancer development and immune system stimulation. Hsps localized on the surface of exosomes, secreted by normal and tumour cells, could be key players in intercellular cross-talk, particularly during the course of different diseases, such as cancer. Exosomal Hsps offer significant opportunities for clinical applications, including their use as potential novel biomarkers for the diagnoses or prognoses of different diseases, or for therapeutic applications and drug delivery.

Expression of genes encoding multi-transmembrane proteins in specific primate taste cell populations.

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Bryan D Moyer

Full Text Available BACKGROUND: Using fungiform (FG and circumvallate (CV taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed. METHODOLOGY/PRINCIPAL FINDINGS: Using double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive, sour cells (PKD2L1-positive, as well as other taste cell populations. Transmembrane protein 44 (TMEM44, a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1, a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1, a calcium-binding transmembrane protein; and anoctamin 7 (ANO7, a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B, a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release. CONCLUSIONS/SIGNIFICANCE: Identification of genes encoding multi-transmembrane domain proteins

Regulation Involved in Colonization of Intercellular Spaces of Host Plants in Ralstonia solanacearum

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Yasufumi Hikichi

2017-06-01

Full Text Available A soil-borne bacterium Ralstonia solanacearum invading plant roots first colonizes the intercellular spaces of the root, and eventually enters xylem vessels, where it replicates at high levels leading to wilting symptoms. After invasion into intercellular spaces, R. solanacearum strain OE1-1 attaches to host cells and expression of the hrp genes encoding components of the type III secretion system (T3SS. OE1-1 then constructs T3SS and secrets effectors into host cells, inducing expression of the host gene encoding phosphatidic acid phosphatase. This leads to suppressing plant innate immunity. Then, OE1-1 grows on host cells, inducing quorum sensing (QS. The QS contributes to regulation of OE1-1 colonization of intercellular spaces including mushroom-type biofilm formation on host cells, leading to its virulence. R. solanacearum strains AW1 and K60 produce methyl 3-hydroxypalmitate (3-OH PAME as a QS signal. The methyltransferase PhcB synthesizes 3-OH PAME. When 3-OH PAME reaches a threshold level, it increases the ability of the histidine kinase PhcS to phosphorylate the response regulator PhcR. This results in elevated levels of functional PhcA, the global virulence regulator. On the other hand, strains OE1-1 and GMI1000 produce methyl 3-hydroxymyristate (3-OH MAME as a QS signal. Among R. solanacearum strains, the deduced PhcB and PhcS amino acid sequences are related to the production of QS signals. R. solanacearum produces aryl-furanone secondary metabolites, ralfuranones, which are extracellularly secreted and required for its virulence, dependent on the QS. Interestingly, ralfuranones affect the QS feedback loop. Taken together, integrated signaling via ralfuranones influences the QS, contributing to pathogen virulence.

PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection.

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Marshall, Karen E; Hughson, Andrew; Vascellari, Sarah; Priola, Suzette A; Sakudo, Akikazu; Onodera, Takashi; Baron, Gerald S

2017-01-15

Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrP C ) influences PrP C misfolding into the disease-associated isoform, PrP res , as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrP C away from rafts. Previous studies showed that nonraft transmembrane PrP C variants resist conversion to PrP res when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrP C and GPI-anchored PrP res in distinct membrane environments. Thus, it remained unclear whether transmembrane PrP C might convert to PrP res if seeded by an exogenous source of PrP res not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrP C To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrP C supported persistent PrP res propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrP C is required for persistent PrP res propagation, implicating raft microdomains as a location for conversion. Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrP C

Functional Peptidomics: Combining Discovery-Based Mass Spectrometry and Neurophysiology to Explore Communication of Phase-Resetting Cues in the Rat Suprachiasmatic Nucleus

Science.gov (United States)

Atkins, Norman, Jr.

2009-01-01

Intercellular signaling is vital to communication within neuronal circuits. The suprachiasmatic nucleus (SCN), the master circadian clock of mammals, contains a dense collection of neurons that align their intrinsic rhythmicity with environmental stimulus and physiological state. While peptide physiology has been demonstrated as a contributor to…

The status of intercellular junctions in established lens epithelial cell lines.

Science.gov (United States)

Dave, Alpana; Craig, Jamie E; Sharma, Shiwani

2012-01-01

Cataract is the major cause of vision-related disability worldwide. Mutations in the crystallin genes are the most common known cause of inherited congenital cataract. Mutations in the genes associated with intercellular contacts, such as Nance-Horan Syndrome (NHS) and Ephrin type A receptor-2 (EPHA2), are other recognized causes of congenital cataract. The EPHA2 gene has been also associated with age-related cataract, suggesting that intercellular junctions are important in not only lens development, but also in maintaining lens transparency. The purpose of this study was to analyze the expression and localization of the key cell junction and cytoskeletal proteins, and of NHS and EPHA2, in established lens epithelial cell lines to determine their suitability as model epithelial systems for the functional investigation of genes involved in intercellular contacts and implicated in cataract. The expression and subcellular localization of occludin and zona occludens protein-1 (ZO-1), which are associated with tight junctions; E-cadherin, which is associated with adherence junctions; and the cytoskeletal actin were analyzed in monolayers of a human lens epithelial cell line (SRA 01/04) and a mouse lens epithelial cell line (αTN4). In addition, the expression and subcellular localization of the NHS and EPHA2 proteins were analyzed in these cell lines. Protein or mRNA expression was respectively determined by western blotting or reverse transcription-polymerase chain reaction (RT-PCR), and localization was determined by immunofluorescence labeling. Human SRA 01/04 and mouse αTN4 lens epithelial cells expressed either the proteins of interest or their encoding mRNA. Occludin, ZO-1, and NHS proteins localized to the cellular periphery, whereas E-cadherin, actin, and EPHA2 localized in the cytoplasm in these cell lines. The human SRA 01/04 and mouse αTN4 lens epithelial cells express the key junctional proteins. The localization patterns of these proteins suggest that

Amino-terminal domain of classic cadherins determines the specificity of the adhesive interactions

DEFF Research Database (Denmark)

Klingelhöfer, Jörg; Troyanovsky, R B; Laur, O Y

2000-01-01

Classic cadherins are transmembrane receptors involved in cell type-specific calcium-dependent intercellular adhesion. The specificity of adhesion is mediated by homophilic interactions between cadherins extending from opposing cell surfaces. In addition, classic cadherins can self-associate form......Classic cadherins are transmembrane receptors involved in cell type-specific calcium-dependent intercellular adhesion. The specificity of adhesion is mediated by homophilic interactions between cadherins extending from opposing cell surfaces. In addition, classic cadherins can self....... To study lateral and adhesive intercadherin interactions, we examined interactions between two classic cadherins, E- and P-cadherins, in epithelial A-431 cells co-producing both proteins. We showed that these cells exhibited heterocomplexes consisting of laterally assembled E- and P....... The specificity of adhesive interaction was localized to the amino-terminal (EC1) domain of both cadherins. Thus, EC1 domain of classic cadherins exposes two determinants responsible for nonspecific lateral and cadherin type-specific adhesive dimerization....

Cloning and characterization of SCART1, a novel scavenger receptor cysteine-rich type I transmembrane molecule

DEFF Research Database (Denmark)

Holm, Dorte; Fink, Dorte Rosenbek; Grønlund, Jørn

2009-01-01

We have cloned and characterized a novel murine transmembrane molecule, mSCART1 belonging to the scavenger receptor cysteine-rich superfamily. The cDNA encodes a polypeptide chain of 989 amino acids, organized as a type I transmembrane protein that contains eight extracellular SRCR domains followed...

First principles design of a core bioenergetic transmembrane electron-transfer protein

Energy Technology Data Exchange (ETDEWEB)

Goparaju, Geetha; Fry, Bryan A.; Chobot, Sarah E.; Wiedman, Gregory; Moser, Christopher C.; Leslie Dutton, P.; Discher, Bohdana M.

2016-05-01

Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

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Cinzia Ambrosi

Full Text Available Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26 that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P. Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels

Intercellular K⁺ accumulation depolarizes Type I vestibular hair cells and their associated afferent nerve calyx.

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Contini, D; Zampini, V; Tavazzani, E; Magistretti, J; Russo, G; Prigioni, I; Masetto, S

2012-12-27

Mammalian vestibular organs contain two types of sensory receptors, named Type I and Type II hair cells. While Type II hair cells are contacted by several small afferent nerve terminals, the basolateral surface of Type I hair cells is almost entirely enveloped by a single large afferent nerve terminal, called calyx. Moreover Type I, but not Type II hair cells, express a low-voltage-activated outward K(+) current, I(K,L), which is responsible for their much lower input resistance (Rm) at rest as compared to Type II hair cells. The functional meaning of I(K,L) and associated calyx is still enigmatic. By combining the patch-clamp whole-cell technique with the mouse whole crista preparation, we have recorded the current- and voltage responses of in situ hair cells. Outward K(+) current activation resulted in K(+) accumulation around Type I hair cells, since it induced a rightward shift of the K(+) reversal potential the magnitude of which depended on the amplitude and duration of K(+) current flow. Since this phenomenon was never observed for Type II hair cells, we ascribed it to the presence of a residual calyx limiting K(+) efflux from the synaptic cleft. Intercellular K(+) accumulation added a slow (τ>100ms) depolarizing component to the cell voltage response. In a few cases we were able to record from the calyx and found evidence for intercellular K(+) accumulation as well. The resulting depolarization could trigger a discharge of action potentials in the afferent nerve fiber. Present results support a model where pre- and postsynaptic depolarization produced by intercellular K(+) accumulation cooperates with neurotransmitter exocytosis in sustaining afferent transmission arising from Type I hair cells. While vesicular transmission together with the low Rm of Type I hair cells appears best suited for signaling fast head movements, depolarization produced by intercellular K(+) accumulation could enhance signal transmission during slow head movements. Copyright �

Insight of Transmembrane Processes of Self-Assembling Nanotubes Based on a Cyclic Peptide Using Coarse Grained Molecular Dynamics Simulation.

Science.gov (United States)

Fu, Yankai; Yan, Tingxuan; Xu, Xia

2017-09-28

Transmembrane self-assembling cyclic peptide (SCP) nanotubes are promising candidates for delivering specific molecules through cell membranes. The detailed mechanisms behind the transmembrane processes, as well as stabilization factors of transmembrane structures, are difficult to elucidate through experiments. In this study, the effects of peptide sequence and oligomeric state on the transmembrane capabilities of SCP nanotubes and the perturbation of embedded SCP nanotubes acting on the membrane were investigated based on coarse grained molecular dynamics simulation. The simulation results reveal that hydrophilic SCP oligomers result in the elevation of the energy barrier while the oligomerization of hydrophobic SCPs causes the reduction of the energy barrier, further leading to membrane insertion. Once SCP nanotubes are embedded, membrane properties such as density, thickness, ordering state and lateral mobility are adjusted along the radial direction. This study provides insight into the transmembrane strategy of SCP nanotubes and sheds light on designing novel transport systems.

Compression induced intercellular shaping for some geometric cellular lattices

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Adonai Gimenez Calbo

2001-03-01

Full Text Available The wall perimeter fraction, which contact neighboring cells, was named compression ratio (alpha. A zero compression ratio indicates maximum intercellular (air volume (vG, v/v and neglectable contact among cells, while alpha=1 indicates complete adherence between neighboring cells and no vG in the lattice. The maximum intercellular air volume (beta, v/v, when alpha=0, was 0.593 for triangular, 0.2146 for square and 0,0931 for hexagonal lattices. The equation alpha=1- (vG/beta½ was derived to relate alpha, beta and vG in the studied lattices. The relation (P S=p/alpha between cell turgor (P S and the tissue aggregating pressure (p, defined as the compression to keep in place a layer of cells, was demonstrated using the compression ratio concept. Intercellular deformations of Ipomea batatas L. roots obtained with pressure chamber were used to test alpha, vG, p and P S as a function of compression. Volumetric and transversal elastic extensibilities and the lamella media tearing forces were obtained and alpha constancy was considered as a criteria of cellular shape stability.A fração do perímetro da parede celular em contato com células vizinha foi denominada razão de compressão (alfa. Razão de compressão zero indica volume intercelular (vG, v/v máximo e contato neglível entre as células, enquanto alfa=1 ocorre quando há completa aderência com as células vizinhas (vG=0. O volume (gasoso intercelular máximo (beta, v/v, quando alfa=0, foi 0,593, 0,2146 e 0,0931 para látices triangulares, quadradas e hexagonais. A equação derivada para relacionar alfa, beta and vG nas látices estudadas foi alfa=1- (vG/beta½. A razão de compressão foi em seguida empregada para estabelecer a relação P S=p/alfa entre a pressão de turgescência (P S e a pressão de agregação (p, definida com a compressão para manter uma camada de células no seu lugar. As deformações intercelulares de batata-doce obtidas com procedimentos de c

Markedly diminished epidermal keratinocyte expression of intercellular adhesion molecule-1 (ICAM-1) in Sezary syndrome

Energy Technology Data Exchange (ETDEWEB)

Nickoloff, B.J.; Griffiths, E.M.; Baadsgaard, O.; Voorhees, J.J.; Hanson, C.A.; Cooper, K.D. (Univ. of Michigan Medical Center, Ann Arbor (USA))

1989-04-21

In mucosis fungoides the malignant T cells express lymphocyte function-associated antigen-1, which allows them to bind to epidermal keratinocytes expressing the gamma interferon-inducible intercellular adhesion molecule-1. In this report, a patient with leukemic-stage mucosis fungoides (Sezary syndrome) had widespread erythematous dermal infiltrates containing malignant T cells, but without any epidermotropism. The authors discovered that the T cells expressed normal amounts of functional lymphocyte function-associated antigen-1, but the keratinocytes did not express significant levels of intercellular adhesion molecule-1, which was probably due to the inability of the malignant T cells to produce gamma interferon. These results support the concept that the inability of malignant T cells to enter the epidermis may contribute to emergence of more clinically aggressive T-cell clones that are no longer confined to the skin, but infiltrate the blood, lymph nodes, and viscera, as is seen in Sezary syndrome.

15N and 31P solid-state NMR study of transmembrane domain alignment of M2 protein of influenza A virus in hydrated cylindrical lipid bilayers confined to anodic aluminum oxide nanopores.

Science.gov (United States)

Chekmenev, Eduard Y; Hu, Jun; Gor'kov, Peter L; Brey, William W; Cross, Timothy A; Ruuge, Andres; Smirnov, Alex I

2005-04-01

This communication reports the first example of a high resolution solid-state 15N 2D PISEMA NMR spectrum of a transmembrane peptide aligned using hydrated cylindrical lipid bilayers formed inside nanoporous anodic aluminum oxide (AAO) substrates. The transmembrane domain SSDPLVVA(A-15N)SIIGILHLILWILDRL of M2 protein from influenza A virus was reconstituted in hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers that were macroscopically aligned by a conventional micro slide glass support or by the AAO nanoporous substrate. 15N and 31P NMR spectra demonstrate that both the phospholipids and the protein transmembrane domain are uniformly aligned in the nanopores. Importantly, nanoporous AAO substrates may offer several advantages for membrane protein alignment in solid-state NMR studies compared to conventional methods. Specifically, higher thermal conductivity of aluminum oxide is expected to suppress thermal gradients associated with inhomogeneous radio frequency heating. Another important advantage of the nanoporous AAO substrate is its excellent accessibility to the bilayer surface for exposure to solute molecules. Such high accessibility achieved through the substrate nanochannel network could facilitate a wide range of structure-function studies of membrane proteins by solid-state NMR.

Activation gating kinetics of GIRK channels are mediated by cytoplasmic residues adjacent to transmembrane domains.

Science.gov (United States)

Sadja, Rona; Reuveny, Eitan

2009-01-01

G-protein-coupled inwardly rectifying potassium channels (GIRK/Kir3.x) are involved in neurotransmission-mediated reduction of excitability. The gating mechanism following G protein activation of these channels likely proceeds from movement of inner transmembrane helices to allow K(+) ions movement through the pore of the channel. There is limited understanding of how the binding of G-protein betagamma subunits to cytoplasmic regions of the channel transduces the signal to the transmembrane regions. In this study, we examined the molecular basis that governs the activation kinetics of these channels, using a chimeric approach. We identified two regions as being important in determining the kinetics of activation. One region is the bottom of the outer transmembrane helix (TM1) and the cytoplasmic domain immediately adjacent (the slide helix); and the second region is the bottom of the inner transmembrane helix (TM2) and the cytoplasmic domain immediately adjacent. Interestingly, both of these regions are sufficient in mediating the kinetics of fast activation gating. This result suggests that there is a cooperative movement of either one of these domains to allow fast and efficient activation gating of GIRK channels.

Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

Science.gov (United States)

Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

2014-07-18

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server

DEFF Research Database (Denmark)

Käll, Lukas; Krogh, Anders; Sonnhammer, Erik L L

2007-01-01

. The method makes an optimal choice between transmembrane segments and signal peptides, and also allows constrained and homology-enriched predictions. We here present a web interface (http://phobius.cgb.ki.se and http://phobius.binf.ku.dk) to access Phobius. Udgivelsesdato: 2007-Jul......When using conventional transmembrane topology and signal peptide predictors, such as TMHMM and SignalP, there is a substantial overlap between these two types of predictions. Applying these methods to five complete proteomes, we found that 30-65% of all predicted signal peptides and 25-35% of all...

Structural Insights into Triglyceride Storage Mediated by Fat Storage-Inducing Transmembrane (FIT) Protein 2

Science.gov (United States)

Gross, David A.; Snapp, Erik L.; Silver, David L.

2010-01-01

Fat storage-Inducing Transmembrane proteins 1 & 2 (FIT1/FITM1 and FIT2/FITM2) belong to a unique family of evolutionarily conserved proteins localized to the endoplasmic reticulum that are involved in triglyceride lipid droplet formation. FIT proteins have been shown to mediate the partitioning of cellular triglyceride into lipid droplets, but not triglyceride biosynthesis. FIT proteins do not share primary sequence homology with known proteins and no structural information is available to inform on the mechanism by which FIT proteins function. Here, we present the experimentally-solved topological models for FIT1 and FIT2 using N-glycosylation site mapping and indirect immunofluorescence techniques. These methods indicate that both proteins have six-transmembrane-domains with both N- and C-termini localized to the cytosol. Utilizing this model for structure-function analysis, we identified and characterized a gain-of-function mutant of FIT2 (FLL(157-9)AAA) in transmembrane domain 4 that markedly augmented the total number and mean size of lipid droplets. Using limited-trypsin proteolysis we determined that the FLL(157-9)AAA mutant has enhanced trypsin cleavage at K86 relative to wild-type FIT2, indicating a conformational change. Taken together, these studies indicate that FIT2 is a 6 transmembrane domain-containing protein whose conformation likely regulates its activity in mediating lipid droplet formation. PMID:20520733

Structural insights into triglyceride storage mediated by fat storage-inducing transmembrane (FIT protein 2.

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David A Gross

2010-05-01

Full Text Available Fat storage-Inducing Transmembrane proteins 1 & 2 (FIT1/FITM1 and FIT2/FITM2 belong to a unique family of evolutionarily conserved proteins localized to the endoplasmic reticulum that are involved in triglyceride lipid droplet formation. FIT proteins have been shown to mediate the partitioning of cellular triglyceride into lipid droplets, but not triglyceride biosynthesis. FIT proteins do not share primary sequence homology with known proteins and no structural information is available to inform on the mechanism by which FIT proteins function. Here, we present the experimentally-solved topological models for FIT1 and FIT2 using N-glycosylation site mapping and indirect immunofluorescence techniques. These methods indicate that both proteins have six-transmembrane-domains with both N- and C-termini localized to the cytosol. Utilizing this model for structure-function analysis, we identified and characterized a gain-of-function mutant of FIT2 (FLL(157-9AAA in transmembrane domain 4 that markedly augmented the total number and mean size of lipid droplets. Using limited-trypsin proteolysis we determined that the FLL(157-9AAA mutant has enhanced trypsin cleavage at K86 relative to wild-type FIT2, indicating a conformational change. Taken together, these studies indicate that FIT2 is a 6 transmembrane domain-containing protein whose conformation likely regulates its activity in mediating lipid droplet formation.

Structure of FGFR3 transmembrane domain dimer: implications for signaling and human pathologies.

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Bocharov, Eduard V; Lesovoy, Dmitry M; Goncharuk, Sergey A; Goncharuk, Marina V; Hristova, Kalina; Arseniev, Alexander S

2013-11-05

Fibroblast growth factor receptor 3 (FGFR3) transduces biochemical signals via lateral dimerization in the plasma membrane, and plays an important role in human development and disease. Eight different pathogenic mutations, implicated in cancers and growth disorders, have been identified in the FGFR3 transmembrane segment. Here, we describe the dimerization of the FGFR3 transmembrane domain in membrane-mimicking DPC/SDS (9/1) micelles. In the solved NMR structure, the two transmembrane helices pack into a symmetric left-handed dimer, with intermolecular stacking interactions occurring in the dimer central region. Some pathogenic mutations fall within the helix-helix interface, whereas others are located within a putative alternative interface. This implies that although the observed dimer structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the membrane is complex. We propose an FGFR3 signaling mechanism that is based on the solved structure, available structures of isolated soluble FGFR domains, and published biochemical and biophysical data. Copyright © 2013 Elsevier Ltd. All rights reserved.

Trans-membrane area asymmetry controls the shape of cellular organelles

NARCIS (Netherlands)

Beznoussenko, Galina V; Pilyugin, Sergei S; Geerts, Willie J C; Kozlov, Michael M; Burger, Koert N J; Luini, Alberto; Derganc, Jure; Mironov, Alexander A

2015-01-01

Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA)) determines the membrane curvature within different sites of the organelle.

Modelling of a transmembrane evaporation module for desalination of seawater

NARCIS (Netherlands)

Guijt, C.M.; Racz, I.G.; van Heuven, Jan Willem; Reith, T.; de Haan, A.B.

1999-01-01

Transmembrane evaporation (often called membrane distillation) carried out in a countercurrent flow module, in which incoming cold seawater is heated by the condensing product water flow, is a promising technology for low-cost seawater desalination. This paper presents a model for preliminary design

The effect of water saturation deficit on the volume of intercellular space in laeves

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J. Czerski

2015-01-01

Full Text Available The volume of intercellular spaces in leaves at various stages of water saturation was determined by method of Czerski (1964, 1968. The investigation were performed with the following plant species: Vicia faba L., Nicotiana tabacum L. var. rustica, Solarium tuberosum L. var. Flisak, Helichrysum bracteatum Wild., Bmssica napus L. var. oleifera, Beta vulgaris L. var. saccharifera.

Transmembrane adaptor molecules: a new category of lymphoid-cell markers

Czech Academy of Sciences Publication Activity Database

Tedoldi, S.; Paterson, J.C.; Hansmann, M.-L.; Natkunam, Y.; Rüdiger, T.; Angelisová, Pavla; Du, M.Q.; Roberton, H.; Roncador, G.; Sanchez, L.; Pozzobon, M.; Masir, N.; Barry, R.; Pileri, S.; Mason, D.Y.; Marafioti, T.; Hořejší, Václav

2006-01-01

Roč. 107, č. 1 (2006), s. 213-221 ISSN 0006-4971 R&D Projects: GA MŠk(CZ) 1M0506 Institutional research plan: CEZ:AV0Z50520514 Keywords : transmembrane adaptors * PAG * LIME Subject RIV: EC - Immunology Impact factor: 10.370, year: 2006

The transmembrane collagen COL-99 guides longitudinally extending axons in C. elegans.

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Taylor, Jesse; Unsoeld, Thomas; Hutter, Harald

2018-06-01

We have identified the transmembrane collagen, COL-99, in a genetic screen for novel genes involved in axon guidance in the nematode C. elegans. COL-99 is similar to transmembrane collagens type XIII, XXIII and XXV in vertebrates. col-99 mutants exhibit guidance defects in axons extending along the major longitudinal axon tracts, most prominently the left ventral nerve cord (VNC). COL-99 is expressed in the hypodermis during the time of axon outgrowth. We provide evidence that a furin cleavage site in COL-99 is essential for function, suggesting that COL-99 is released from the cells producing it. Vertebrate homologs of COL-99 have been shown to be expressed in mammalian nervous systems and linked to various neurological disease but have not been associated with guidance of extending neurons. col-99 acts genetically with the discoidin domain receptors ddr-1 and ddr-2, which are expressed by neurons affected in col-99 mutants. Discoidin domain receptors are activated by collagens in vertebrates. DDR-1 and DDR-2 may function as receptors for COL-99. Our results establish a novel role for a transmembrane collagen in axonal guidance and asymmetry establishment of the VNC. Copyright © 2018 Elsevier Inc. All rights reserved.

‘Gap Junctions and Cancer: Communicating for 50 Years’

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Aasen, Trond; Mesnil, Marc; Naus, Christian C.; Lampe, Paul D.; Laird, Dale W.

2017-01-01

Fifty years ago, tumour cells were found to lack electrical coupling, leading to the hypothesis that loss of direct intercellular communication is commonly associated with cancer onset and progression. Subsequent studies linked this phenomenon to gap junctions composed of connexin proteins. While many studies support the notion that connexins are tumour suppressors, recent evidence suggests that, in some tumour types, they may facilitate specific stages of tumour progression through both junctional and non-junctional signalling pathways. This Timeline article highlights the milestones connecting gap junctions to cancer, and underscores important unanswered questions, controversies and therapeutic opportunities in the field. PMID:27782134

Robust synchronization analysis in nonlinear stochastic cellular networks with time-varying delays, intracellular perturbations and intercellular noise.

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Chen, Po-Wei; Chen, Bor-Sen

2011-08-01

Naturally, a cellular network consisted of a large amount of interacting cells is complex. These cells have to be synchronized in order to emerge their phenomena for some biological purposes. However, the inherently stochastic intra and intercellular interactions are noisy and delayed from biochemical processes. In this study, a robust synchronization scheme is proposed for a nonlinear stochastic time-delay coupled cellular network (TdCCN) in spite of the time-varying process delay and intracellular parameter perturbations. Furthermore, a nonlinear stochastic noise filtering ability is also investigated for this synchronized TdCCN against stochastic intercellular and environmental disturbances. Since it is very difficult to solve a robust synchronization problem with the Hamilton-Jacobi inequality (HJI) matrix, a linear matrix inequality (LMI) is employed to solve this problem via the help of a global linearization method. Through this robust synchronization analysis, we can gain a more systemic insight into not only the robust synchronizability but also the noise filtering ability of TdCCN under time-varying process delays, intracellular perturbations and intercellular disturbances. The measures of robustness and noise filtering ability of a synchronized TdCCN have potential application to the designs of neuron transmitters, on-time mass production of biochemical molecules, and synthetic biology. Finally, a benchmark of robust synchronization design in Escherichia coli repressilators is given to confirm the effectiveness of the proposed methods. Copyright © 2011 Elsevier Inc. All rights reserved.

[Research advances in CKLF-like MARVEL transmembrane domain containing member 5].

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Yuan, Ye-qing; Xiao, Yun-bei; Liu, Zhen-hua; Zhang, Xiao-wei; Xu, Tao; Wang, Xiao-feng

2012-12-01

CKLF-like MARVEL transmembrane domain containing member(CMTM)is a novel generic family firstly reported by Peking University Center for Human Disease Genomics. CMTM5 belongs to this family and has exhibited tumor-inhibiting activities. It can encode proteins approaching to the transmembrane 4 superfamily(TM4SF). CMTM5 is broadly expressed in normal adult and fetal human tissues, but is undetectable or down-regulated in most carcinoma cell lines and tissues. Restoration of CMTM5 may inhibit the proliferation, migration, and invasion of carcinoma cells. Although the exact mechanism of its anti-tumor activity remains unclear, CMTM5 may be involved in various signaling pathways governing the occurrence and development of tumors. CMTM5 may be a new target in the gene therapies for tumors, while further studies on CMTM5 and its anti-tumor mechanisms are warranted.

Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor.

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Vanessa Chantreau

Full Text Available The thyrotropin receptor (TSHR is a G protein-coupled receptor (GPCR that is member of the leucine-rich repeat subfamily (LGR. In the absence of crystal structure, the success of rational design of ligands targeting the receptor internal cavity depends on the quality of the TSHR models built. In this subfamily, transmembrane helices (TM 2 and 5 are characterized by the absence of proline compared to most receptors, raising the question of the structural conformation of these helices. To gain insight into the structural properties of these helices, we carried out bioinformatics and experimental studies. Evolutionary analysis of the LGR family revealed a deletion in TM5 but provided no information on TM2. Wild type residues at positions 2.58, 2.59 or 2.60 in TM2 and/or at position 5.50 in TM5 were substituted to proline. Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin. Only proline substitution at position 2.59 maintained complex glycosylation and high membrane expression, supporting occurrence of a bulged TM2. The TSHR transmembrane domain was modeled by homology with the orexin 2 receptor, using a protocol that forced the deletion of one residue in the TM5 bulge of the template. The stability of the model was assessed by molecular dynamics simulations. TM5 straightened during the equilibration phase and was stable for the remainder of the simulations. Our data support a structural model of the TSHR transmembrane domain with a bulged TM2 and a straight TM5 that is specific of glycoprotein hormone receptors.

CHANGES OF INTERCELLULAR COOPERATION IN PERIPHERAL BLOOD IN TREATED PATIENTS WITH CARDIOLOGIC DISEASES

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L. N. Korichkina

2009-01-01

Full Text Available Aim. To study changes of intercellular cooperation in peripheral blood induced by treatment in patients with arterial hypertension (HT, ischemic heart disease (IHD and chronic heart failure (CHF.Material and methods. 610 patients were involved into the study, including 250 patients with HT of stages I-III (50 untreated patients, 150 patients with IHD and 210 patients with CHF of stages I-III. All patients were treated except 50 hypertensive ones. 80 healthy patients (40 men, 40 women were included into control group. Blood smears of patients were evaluated (Romanovsky's stain. A number of leukocyte, autorosettes and autorosettes with erythrocyte lysis was calculated. The cellular association consisting of a neutrophil, monocyte or eosinocyte with 3 or more erythrocytes skintight to their surface defined as autorosettes. Erythrocytes number and hemoglobin level determined in peripheral blood.Results. Single autorosettes in peripheral blood were observed in patients of control group and in untreated patients with HT. Treated patients with HT, IHD and CHF had increased number of autorossets and autorosettes with erythrocytes lysis. This phenomenon resulted in reduction of erythrocytes number and hemoglobin level in peripheral blood.Conclusion. Treated patients with cardiologic diseases had changes in intercellular cooperation. It should be considered at intensive and long term therapy.

Proteomic and Functional Analyses of the Virion Transmembrane Proteome of Cyprinid Herpesvirus 3.

Science.gov (United States)

Vancsok, Catherine; Peñaranda, M Michelle D; Raj, V Stalin; Leroy, Baptiste; Jazowiecka-Rakus, Joanna; Boutier, Maxime; Gao, Yuan; Wilkie, Gavin S; Suárez, Nicolás M; Wattiez, Ruddy; Gillet, Laurent; Davison, Andrew J; Vanderplasschen, Alain F C

2017-11-01

Virion transmembrane proteins (VTPs) mediate key functions in the herpesvirus infectious cycle. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses. The present study was devoted to CyHV-3 VTPs. Using mass spectrometry approaches, we identified 16 VTPs of the CyHV-3 FL strain. Mutagenesis experiments demonstrated that eight of these proteins are essential for viral growth in vitro (open reading frame 32 [ORF32], ORF59, ORF81, ORF83, ORF99, ORF106, ORF115, and ORF131), and eight are nonessential (ORF25, ORF64, ORF65, ORF108, ORF132, ORF136, ORF148, and ORF149). Among the nonessential proteins, deletion of ORF25, ORF132, ORF136, ORF148, or ORF149 affects viral replication in vitro , and deletion of ORF25, ORF64, ORF108, ORF132, or ORF149 impacts plaque size. Lack of ORF148 or ORF25 causes attenuation in vivo to a minor or major extent, respectively. The safety and efficacy of a virus lacking ORF25 were compared to those of a previously described vaccine candidate deleted for ORF56 and ORF57 (Δ56-57). Using quantitative PCR, we demonstrated that the ORF25 deleted virus infects fish through skin infection and then spreads to internal organs as reported previously for the wild-type parental virus and the Δ56-57 virus. However, compared to the parental wild-type virus, the replication of the ORF25-deleted virus was reduced in intensity and duration to levels similar to those observed for the Δ56-57 virus. Vaccination of fish with a virus lacking ORF25 was safe but had low efficacy at the doses tested. This characterization of the virion transmembrane proteome of CyHV-3 provides a firm basis for further research on alloherpesvirus VTPs. IMPORTANCE Virion transmembrane proteins play key roles in the biology of herpesviruses. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses and the causative agent of major economic losses in common and koi carp worldwide. In this study of the virion transmembrane proteome of CyHV-3, the

Transmembrane protein diffusion in gel-supported dual-leaflet membranes.

Science.gov (United States)

Wang, Chih-Ying; Hill, Reghan J

2014-11-18

Tools to measure transmembrane-protein diffusion in lipid bilayer membranes have advanced in recent decades, providing a need for predictive theoretical models that account for interleaflet leaflet friction on tracer mobility. Here we address the fully three-dimensional flows driven by a (nonprotruding) transmembrane protein embedded in a dual-leaflet membrane that is supported above and below by soft porous supports (e.g., hydrogel or extracellular matrix), each of which has a prescribed permeability and solvent viscosity. For asymmetric configurations, i.e., supports with contrasting permeability, as realized for cells in contact with hydrogel scaffolds or culture media, the diffusion coefficient can reflect interleaflet friction. Reasonable approximations, for sufficiently large tracers on low-permeability supports, are furnished by a recent phenomenological theory from the literature. Interpreting literature data, albeit for hard-supported membranes, provides a theoretical basis for the phenomenological Stokes drag law as well as strengthening assertions that nonhydrodynamic interactions are important in supported bilayer systems, possibly leading to overestimates of the membrane/leaflet viscosity. Our theory provides a theoretical foundation for future experimental studies of tracer diffusion in gel-supported membranes.

Effect of flow rate and temperature on transmembrane blood pressure drop in an extracorporeal artificial lung.

Science.gov (United States)

Park, M; Costa, E L V; Maciel, A T; Barbosa, E V S; Hirota, A S; Schettino, G de P; Azevedo, L C P

2014-11-01

Transmembrane pressure drop reflects the resistance of an artificial lung system to blood transit. Decreased resistance (low transmembrane pressure drop) enhances blood flow through the oxygenator, thereby, enhancing gas exchange efficiency. This study is part of a previous one where we observed the behaviour and the modulation of blood pressure drop during the passage of blood through artificial lung membranes. Before and after the induction of multi-organ dysfunction, the animals were instrumented and analysed for venous-venous extracorporeal membrane oxygenation, using a pre-defined sequence of blood flows. Blood flow and revolutions per minute (RPM) of the centrifugal pump varied in a linear fashion. At a blood flow of 5.5 L/min, pre- and post-pump blood pressures reached -120 and 450 mmHg, respectively. Transmembrane pressures showed a significant spread, particularly at blood flows above 2 L/min; over the entire range of blood flow rates, there was a positive association of pressure drop with blood flow (0.005 mmHg/mL/minute of blood flow) and a negative association of pressure drop with temperature (-4.828 mmHg/(°Celsius). These associations were similar when blood flows of below and above 2000 mL/minute were examined. During its passage through the extracorporeal system, blood is exposed to pressure variations from -120 to 450 mmHg. At high blood flows (above 2 L/min), the drop in transmembrane pressure becomes unpredictable and highly variable. Over the entire range of blood flows investigated (0-5500 mL/min), the drop in transmembrane pressure was positively associated with blood flow and negatively associated with body temperature. © The Author(s) 2014.

Evolution of vertebrate interferon inducible transmembrane proteins

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Hickford Danielle

2012-04-01

Full Text Available Abstract Background Interferon inducible transmembrane proteins (IFITMs have diverse roles, including the control of cell proliferation, promotion of homotypic cell adhesion, protection against viral infection, promotion of bone matrix maturation and mineralisation, and mediating germ cell development. Most IFITMs have been well characterised in human and mouse but little published data exists for other animals. This study characterised IFITMs in two distantly related marsupial species, the Australian tammar wallaby and the South American grey short-tailed opossum, and analysed the phylogeny of the IFITM family in vertebrates. Results Five IFITM paralogues were identified in both the tammar and opossum. As in eutherians, most marsupial IFITM genes exist within a cluster, contain two exons and encode proteins with two transmembrane domains. Only two IFITM genes, IFITM5 and IFITM10, have orthologues in both marsupials and eutherians. IFITM5 arose in bony fish and IFITM10 in tetrapods. The bone-specific expression of IFITM5 appears to be restricted to therian mammals, suggesting that its specialised role in bone production is a recent adaptation specific to mammals. IFITM10 is the most highly conserved IFITM, sharing at least 85% amino acid identity between birds, reptiles and mammals and suggesting an important role for this presently uncharacterised protein. Conclusions Like eutherians, marsupials also have multiple IFITM genes that exist in a gene cluster. The differing expression patterns for many of the paralogues, together with poor sequence conservation between species, suggests that IFITM genes have acquired many different roles during vertebrate evolution.

Conformational constraining of inactive and active States of a seven transmembrane receptor by metal ion site engineering in the extracellular end of transmembrane segment V

DEFF Research Database (Denmark)

Rosenkilde, Mette M; David, Ralf; Oerlecke, Ilka

2006-01-01

The extracellular part of transmembrane segment V (TM-V) is expected to be involved in the activation process of 7TM receptors, but its role is far from clear. Here, we study the highly constitutively active CXC-chemokine receptor encoded by human herpesvirus 8 (ORF74-HHV8), in which a metal ion ...

Structural organization of intercellular channels II. Amino terminal domain of the connexins: sequence, functional roles, and structure.

Science.gov (United States)

Beyer, Eric C; Lipkind, Gregory M; Kyle, John W; Berthoud, Viviana M

2012-08-01

The amino terminal domain (NT) of the connexins consists of their first 22-23 amino acids. Site-directed mutagenesis studies have demonstrated that NT amino acids are determinants of gap junction channel properties including unitary conductance, permeability/selectivity, and gating in response to transjunctional voltage. The importance of this region has also been emphasized by the identification of multiple disease-associated connexin mutants affecting amino acid residues in the NT region. The first part of the NT is α-helical. The structure of the Cx26 gap junction channel shows that the NT α-helix localizes within the channel, and lines the wall of the pore. Interactions of the amino acid residues in the NT with those in the transmembrane helices may be critical for holding the channel open. The predicted sites of these interactions and the applicability of the Cx26 structure to the NT of other connexins are considered. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics. Copyright © 2011. Published by Elsevier B.V.

Ochratoxim A alters cell adhesion and gap junction intercellular communication in MDCK cells

International Nuclear Information System (INIS)

Mally, Angela; Decker, Martina; Bekteshi, Michaela; Dekant, Wolfgang

2006-01-01

Ochratoxin A (OTA) is one of the most potent renal carcinogens studied to date, but the mechanism of tumor formation by ochratoxin A remains largely unknown. Cell adhesion and cell-cell communication participate in the regulation of signaling pathways involved in cell proliferation and growth control and it is therefore not surprising that modulation of cell-cell signaling has been implicated in cancer development. Several nephrotoxicants and renal carcinogens have been shown to alter cell-cell signaling by interference with gap junction intercell communication (GJIC) and/or cell adhesion, and the aim of this study was to determine if disruption of cell-cell interactions occurs in kidney epithelial cells in response to OTA treatment. MDCK cells were treated with OTA (0-50 μM) for up to 24 h and gap junction function was analyzed using the scrape-load/dye transfer assay. In addition, expression and intracellular localization of Cx43, E-cadherin and β-catenin were determined by immunoblot and immunofluorescence analysis. A clear decrease in the distance of dye transfer was evident following treatment with OTA at concentrations/incubation times which did not affect cell viability. Consistent with the functional inhibition of GJIC, treatment with OTA resulted in a dose-dependent decrease in Cx43 expression. In contrast to Cx43, OTA did not alter total amount of the adherens junction proteins E-cadherin and β-catenin. Moreover, Western blot analysis of Triton X-100 soluble and insoluble protein fractions did not indicate translocation of cell adhesion molecules from the membrane to the cytoplasm. However, a ∼78 kDa fragment of β-catenin was detected in the detergent soluble fraction, indicating proteolytic cleavage of β-catenin. Immunofluorescence analysis also revealed changes in the pattern of both β-catenin and E-cadherin labeling, suggesting that OTA may alter cell-adhesion. Taken together, these data support the hypothesis that disruption of cell

Exosome-Mediated Genetic Information Transfer, a Missing Piece of Osteoblast-Osteoclast Communication Puzzle.

Science.gov (United States)

Yin, Pengbin; Lv, Houchen; Li, Yi; Deng, Yuan; Zhang, Licheng; Tang, Peifu

2017-01-01

The skeletal system functions and maintains itself based on communication between cells of diverse origins, especially between osteoblasts (OBs) and osteoclasts (OCs), accounting for bone formation and resorption, respectively. Previously, protein-level information exchange has been the research focus, and this has been discussed in detail. The regulative effects of microRNAs (miRNAs) on OB and OC ignite the question as to whether genetic information could be transferred between bone cells. Exosomes, extracellular membrane vesicles 30-100 nm in diameter, have recently been demonstrated to transfer functional proteins, mRNAs, and miRNAs, and serve as mediators of intercellular communication. By reviewing the distinguishing features of exosomes, a hypothesis was formulated and evaluated in this article that exosome-mediated genetic information transfer may represent a novel strategy for OB-OC communication. The exosomes may coordinately regulate these two cells under certain physiological conditions by transferring genetic information. Further research in exosome-shuttered miRNAs in OB-OC communication may add a missing piece to the bone cells communication "puzzle."

Structure and function of the cystic fibrosis transmembrane conductance regulator

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M.M. Morales

1999-08-01

Full Text Available Cystic fibrosis (CF is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR. Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel. Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients. The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP. The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs, and a cytoplasmic domain. In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Cl- channels (ORCCs.

Treatment with the gap junction modifier rotigaptide (ZP123) reduces infarct size in rats with chronic myocardial infarction

DEFF Research Database (Denmark)

Haugan, Ketil; Marcussen, Niels; Kjølbye, Anne Louise

2006-01-01

Treatment with non-selective drugs (eg, long-chain alcohols, halothane) that reduce gap junction intercellular communication (GJIC) is associated with reduced infarct size after myocardial infarction (MI). Therefore, it has been suggested that gap junction intercellular communication stimulating ...

Role of the vaccinia virus O3 protein in cell entry can be fulfilled by its Sequence flexible transmembrane domain

Energy Technology Data Exchange (ETDEWEB)

Satheshkumar, P.S.; Chavre, James; Moss, Bernard, E-mail: bmoss@nih.gov

2013-09-15

The vaccinia virus O3 protein, a component of the entry–fusion complex, is encoded by all chordopoxviruses. We constructed truncation mutants and demonstrated that the transmembrane domain, which comprises two-thirds of this 35 amino acid protein, is necessary and sufficient for interaction with the entry–fusion complex and function in cell entry. Nevertheless, neither single amino acid substitutions nor alanine scanning mutagenesis revealed essential amino acids within the transmembrane domain. Moreover, replication-competent mutant viruses were generated by randomization of 10 amino acids of the transmembrane domain. Of eight unique viruses, two contained only two amino acids in common with wild type and the remainder contained one or none within the randomized sequence. Although these mutant viruses formed normal size plaques, the entry–fusion complex did not co-purify with the mutant O3 proteins suggesting a less stable interaction. Thus, despite low specific sequence requirements, the transmembrane domain is sufficient for function in entry. - Highlights: • The 35 amino acid O3 protein is required for efficient vaccinia virus entry. • The transmembrane domain of O3 is necessary and sufficient for entry. • Mutagenesis demonstrated extreme sequence flexibility compatible with function.

Characterization of the GXXXG motif in the first transmembrane segment of Japanese encephalitis virus precursor membrane (prM protein

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Wu Suh-Chin

2010-05-01

Full Text Available Abstract The interaction between prM and E proteins in flavivirus-infected cells is a major driving force for the assembly of flavivirus particles. We used site-directed mutagenesis to study the potential role of the transmembrane domains of the prM proteins of Japanese encephalitis virus (JEV in prM-E heterodimerization as well as subviral particle formation. Alanine insertion scanning mutagenesis within the GXXXG motif in the first transmembrane segment of JEV prM protein affected the prM-E heterodimerization; its specificity was confirmed by replacing the two glycines of the GXXXG motif with alanine, leucine and valine. The GXXXG motif was found to be conserved in the JEV serocomplex viruses but not other flavivirus groups. These mutants with alanine inserted in the two prM transmembrane segments all impaired subviral particle formation in cell cultures. The prM transmembrane domains of JEV may play importation roles in prM-E heterodimerization and viral particle assembly.

System and methods for predicting transmembrane domains in membrane proteins and mining the genome for recognizing G-protein coupled receptors

Science.gov (United States)

Trabanino, Rene J; Vaidehi, Nagarajan; Hall, Spencer E; Goddard, William A; Floriano, Wely

2013-02-05

The invention provides computer-implemented methods and apparatus implementing a hierarchical protocol using multiscale molecular dynamics and molecular modeling methods to predict the presence of transmembrane regions in proteins, such as G-Protein Coupled Receptors (GPCR), and protein structural models generated according to the protocol. The protocol features a coarse grain sampling method, such as hydrophobicity analysis, to provide a fast and accurate procedure for predicting transmembrane regions. Methods and apparatus of the invention are useful to screen protein or polynucleotide databases for encoded proteins with transmembrane regions, such as GPCRs.

Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes

DEFF Research Database (Denmark)

Sikder, K. U.; Stone, K. A.; Kumar, P. B. S.

2014-01-01

We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that mic......We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find...... that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells. (C) 2014 AIP Publishing LLC....

Modeling the Structure of SARS 3a Transmembrane Protein Using a ...

Indian Academy of Sciences (India)

Modeling the structure of SARS 3a Transmembrane protein using a ... for the implicit membrane molecular dynamics (MD) simulations. ... The coordinates during the simulation were saved every 500 steps, and were used for analysis. ... the pair list for calculation of nonbonded interactions being updated after every 10 steps.

Molecular cloning and tissue-specific expression analysis of mouse spinesin, a type II transmembrane serine protease 5

International Nuclear Information System (INIS)

Watanabe, Yoshihisa; Okui, Akira; Mitsui, Shinichi; Kawarabuki, Kentaro; Yamaguchi, Tatsuyuki; Uemura, Hidetoshi; Yamaguchi, Nozomi

2004-01-01

We have previously reported novel serine proteases isolated from cDNA libraries of the human and mouse central nervous system (CNS) by PCR using degenerate oligodeoxyribonucleotide primers designed on the basis of the serine protease motifs, AAHC and DSGGP. Here we report a newly isolated serine protease from the mouse CNS. This protease is homologous (77.9% identical) to human spinesin type II transmembrane serine protease 5. Mouse spinesin (m-spinesin) is also composed of (from the N-terminus) a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger-receptor-like domain, and a serine protease domain, as is h-spinesin. We also isolated type 1, type 2, and type 3 variant cDNAs of m-spinesin. Full-length spinesin (type 4) and type 3 contain all the domains, whereas type 1 and type 2 variants lack the cytoplasmic, transmembrane, and scavenger-receptor-like domains. Subcellular localization of the variant forms was analyzed using enhanced green fluorescent protein (EGFP) fusion proteins. EGFP-type 4 fusion protein was predominantly localized to the ER, Golgi apparatus, and plasma membrane, whereas EGFP-type 1 was localized to the cytoplasm, reflecting differential classification of m-spinesin variants into transmembrane and cytoplasmic types. We analyzed the distribution of m-spinesin variants in mouse tissues, using RT-PCR with variant-specific primer sets. Interestingly, transmembrane-type spinesin, types 3 and 4, was specifically expressed in the spinal cord, whereas cytoplasmic type, type 1, was expressed in multiple tissues, including the cerebrum and cerebellum. Therefore, m-spinesin variants may have distinct biological functions arising from organ-specific variant expression

Increasing the endogenous NO level causes catalase inactivation and reactivation of intercellular apoptosis signaling specifically in tumor cells.

Science.gov (United States)

Bauer, Georg

2015-12-01

Tumor cells generate extracellular superoxide anions and are protected against intercellular apoptosis-inducing HOCl- and NO/peroxynitrite signaling through the expression of membrane-associated catalase. This enzyme decomposes H2O2 and thus prevents HOCl synthesis. It efficiently interferes with NO/peroxynitrite signaling through oxidation of NO and decomposition of peroxynitrite. The regulatory potential of catalase at the crosspoint of ROS and RNS chemical biology, as well as its high local concentration on the outside of the cell membrane of tumor cells, establish tight control of intercellular signaling and thus prevent tumor cell apoptosis. Therefore, inhibition of catalase or its inactivation by singlet oxygen reactivate intercellular apoptosis-inducing signaling. Nitric oxide and peroxynitrite are connected with catalase in multiple and meaningful ways, as (i) NO can be oxidated by compound I of catalase, (ii) NO can reversibly inhibit catalase, (iii) peroxynitrite can be decomposed by catalase and (iv) the interaction between peroxynitrite and H2O2 leads to the generation of singlet oxygen that inactivates catalase. Therefore, modulation of the concentration of free NO through addition of arginine, inhibition of arginase, induction of NOS expression or inhibition of NO dioxygenase triggers an autoamplificatory biochemical cascade that is based on initial formation of singlet oxygen, amplification of superoxide anion/H2O2 and NO generation through singlet oxygen dependent stimulation of the FAS receptor and caspase-8. Finally, singlet oxygen is generated at sufficiently high concentration to inactivate protective catalase and to reactivate intercellular apoptosis-inducing ROS signaling. This regulatory network allows to establish several pathways for synergistic interactions, like the combination of modulators of NO metabolism with enhancers of superoxide anion generation, modulators of NO metabolism that act at different targets and between modulators of

Increasing the endogenous NO level causes catalase inactivation and reactivation of intercellular apoptosis signaling specifically in tumor cells

Science.gov (United States)

Bauer, Georg

2015-01-01

Tumor cells generate extracellular superoxide anions and are protected against intercellular apoptosis-inducing HOCl- and NO/peroxynitrite signaling through the expression of membrane-associated catalase. This enzyme decomposes H2O2 and thus prevents HOCl synthesis. It efficiently interferes with NO/peroxynitrite signaling through oxidation of NO and decomposition of peroxynitrite. The regulatory potential of catalase at the crosspoint of ROS and RNS chemical biology, as well as its high local concentration on the outside of the cell membrane of tumor cells, establish tight control of intercellular signaling and thus prevent tumor cell apoptosis. Therefore, inhibition of catalase or its inactivation by singlet oxygen reactivate intercellular apoptosis-inducing signaling. Nitric oxide and peroxynitrite are connected with catalase in multiple and meaningful ways, as (i) NO can be oxidated by compound I of catalase, (ii) NO can reversibly inhibit catalase, (iii) peroxynitrite can be decomposed by catalase and (iv) the interaction between peroxynitrite and H2O2 leads to the generation of singlet oxygen that inactivates catalase. Therefore, modulation of the concentration of free NO through addition of arginine, inhibition of arginase, induction of NOS expression or inhibition of NO dioxygenase triggers an autoamplificatory biochemical cascade that is based on initial formation of singlet oxygen, amplification of superoxide anion/H2O2 and NO generation through singlet oxygen dependent stimulation of the FAS receptor and caspase-8. Finally, singlet oxygen is generated at sufficiently high concentration to inactivate protective catalase and to reactivate intercellular apoptosis-inducing ROS signaling. This regulatory network allows to establish several pathways for synergistic interactions, like the combination of modulators of NO metabolism with enhancers of superoxide anion generation, modulators of NO metabolism that act at different targets and between modulators of

Transmembrane Inhibitor of RICTOR/mTORC2 in Hematopoietic Progenitors

Directory of Open Access Journals (Sweden)

Dongjun Lee

2014-11-01

Full Text Available Central to cellular proliferative, survival, and metabolic responses is the serine/threonine kinase mTOR, which is activated in many human cancers. mTOR is present in distinct complexes that are either modulated by AKT (mTORC1 or are upstream and regulatory of it (mTORC2. Governance of mTORC2 activity is poorly understood. Here, we report a transmembrane molecule in hematopoietic progenitor cells that physically interacts with and inhibits RICTOR, an essential component of mTORC2. Upstream of mTORC2 (UT2 negatively regulates mTORC2 enzymatic activity, reducing AKTS473, PKCα, and NDRG1 phosphorylation and increasing FOXO transcriptional activity in an mTORC2-dependent manner. Modulating UT2 levels altered animal survival in a T cell acute lymphoid leukemia (T-ALL model that is known to be mTORC2 sensitive. These studies identify an inhibitory component upstream of mTORC2 in hematopoietic cells that can reduce mortality from NOTCH-induced T-ALL. A transmembrane inhibitor of mTORC2 may provide an attractive target to affect this critical cell regulatory pathway.

TMDIM: an improved algorithm for the structure prediction of transmembrane domains of bitopic dimers

Science.gov (United States)

Cao, Han; Ng, Marcus C. K.; Jusoh, Siti Azma; Tai, Hio Kuan; Siu, Shirley W. I.

2017-09-01

α-Helical transmembrane proteins are the most important drug targets in rational drug development. However, solving the experimental structures of these proteins remains difficult, therefore computational methods to accurately and efficiently predict the structures are in great demand. We present an improved structure prediction method TMDIM based on Park et al. (Proteins 57:577-585, 2004) for predicting bitopic transmembrane protein dimers. Three major algorithmic improvements are introduction of the packing type classification, the multiple-condition decoy filtering, and the cluster-based candidate selection. In a test of predicting nine known bitopic dimers, approximately 78% of our predictions achieved a successful fit (RMSD PHP, MySQL and Apache, with all major browsers supported.

Destruction of the hepatocyte junction by intercellular invasion of Leptospira causes jaundice in a hamster model of Weil's disease.

Science.gov (United States)

Miyahara, Satoshi; Saito, Mitsumasa; Kanemaru, Takaaki; Villanueva, Sharon Y A M; Gloriani, Nina G; Yoshida, Shin-ichi

2014-08-01

Weil's disease, the most severe form of leptospirosis, is characterized by jaundice, haemorrhage and renal failure. The mechanisms of jaundice caused by pathogenic Leptospira remain unclear. We therefore aimed to elucidate the mechanisms by integrating histopathological changes with serum biochemical abnormalities during the development of jaundice in a hamster model of Weil's disease. In this work, we obtained three-dimensional images of infected hamster livers using scanning electron microscope together with freeze-cracking and cross-cutting methods for sample preparation. The images displayed the corkscrew-shaped bacteria, which infiltrated the Disse's space, migrated between hepatocytes, detached the intercellular junctions and disrupted the bile canaliculi. Destruction of bile canaliculi coincided with the elevation of conjugated bilirubin, aspartate transaminase and alkaline phosphatase levels in serum, whereas serum alanine transaminase and γ-glutamyl transpeptidase levels increased slightly, but not significantly. We also found in ex vivo experiments that pathogenic, but not non-pathogenic leptospires, tend to adhere to the perijunctional region of hepatocyte couplets isolated from hamsters and initiate invasion of the intercellular junction within 1 h after co-incubation. Our results suggest that pathogenic leptospires invade the intercellular junctions of host hepatocytes, and this invasion contributes in the disruption of the junction. Subsequently, bile leaks from bile canaliculi and jaundice occurs immediately. Our findings revealed not only a novel pathogenicity of leptospires, but also a novel mechanism of jaundice induced by bacterial infection. © 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.

Biophysical Aspects of Transmembrane Signaling

CERN Document Server

Damjanovich, Sandor

2005-01-01

Transmembrane signaling is one of the most significant cell biological events in the life and death of cells in general and lymphocytes in particular. Until recently biochemists and biophysicists were not accustomed to thinking of these processes from the side of a high number of complex biochemical events and an equally high number of physical changes at molecular and cellular levels at the same time. Both types of researchers were convinced that their findings are the most decisive, having higher importance than the findings of the other scientist population. Both casts were wrong. Life, even at cellular level, has a number of interacting physical and biochemical mechanisms, which finally build up the creation of an "excited" cell that will respond to particular signals from the outer or inner world. This book handles both aspects of the signalling events, and in some cases tries to unify our concepts and help understand the signals that govern the life and death of our cells. Not only the understanding, bu...

Cancer Research Advance in CKLF-like MARVEL Transmembrane Domain Containing Member Family (Review).

Science.gov (United States)

Lu, Jia; Wu, Qian-Qian; Zhou, Ya-Bo; Zhang, Kai-Hua; Pang, Bing-Xin; Li, Liang; Sun, Nan; Wang, Heng-Shu; Zhang, Song; Li, Wen-Jian; Zheng, Wei; Liu, Wei

2016-01-01

CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of genes first reported at international level by Peking University Human Disease Gene Research Center. The gene products are between chemokines and the transmembrane-4 superfamily. Loaceted in several human chromosomes, CMTMs, which are unregulated in kinds of tumors, are potential tumor suppressor genes consisting of CKLF and CMTM1 to CMTM8. CMTMs play important roles in immune, male reproductive and hematopoietic systems. Also, it has been approved that CMTM family has strong connection with diseases of autoimmunity, haematopoietic system and haematopoietic system. The in-depth study in recent years found the close relation between CMTMs and umorigenesis, tumor development and metastasis. CMTM family has a significant clinical value in diagnosis and treatment to the diseases linking to tumor and immune system.

Potential microRNA-mediated oncogenic intercellular communication revealed by pan-cancer analysis

Science.gov (United States)

Li, Yue; Zhang, Zhaolei

2014-11-01

Carcinogenesis consists of oncogenesis and metastasis, and intriguingly microRNAs (miRNAs) are involved in both processes. Although aberrant miRNA activities are prevalent in diverse tumor types, the exact mechanisms for how they regulate cancerous processes are not always clear. To this end, we performed a large-scale pan-cancer analysis via a novel probabilistic approach to infer recurrent miRNA-target interactions implicated in 12 cancer types using data from The Cancer Genome Atlas. We discovered ~20,000 recurrent miRNA regulations, which are enriched for cancer-related miRNAs/genes. Notably, miRNA 200 family (miR-200/141/429) is among the most prominent miRNA regulators, which is known to be involved in metastasis. Importantly, the recurrent miRNA regulatory network is not only enriched for cancer pathways but also for extracellular matrix (ECM) organization and ECM-receptor interactions. The results suggest an intriguing cancer mechanism involving miRNA-mediated cell-to-cell communication, which possibly involves delivery of tumorigenic miRNA messengers to adjacent cells via exosomes. Finally, survival analysis revealed 414 recurrent-prognostic associations, where both gene and miRNA involved in each interaction conferred significant prognostic power in one or more cancer types. Together, our comprehensive pan-cancer analysis provided not only biological insights into metastasis but also brought to bear the clinical relevance of the proposed recurrent miRNA-gene associations.

Relative transmembrane segment rearrangements during BK channel activation resolved by structurally assigned fluorophore–quencher pairing

Science.gov (United States)

Pantazis, Antonios

2012-01-01

Voltage-activated proteins can sense, and respond to, changes in the electric field pervading the cell membrane by virtue of a transmembrane helix bundle, the voltage-sensing domain (VSD). Canonical VSDs consist of four transmembrane helices (S1–S4) of which S4 is considered a principal component because it possesses charged residues immersed in the electric field. Membrane depolarization compels the charges, and by extension S4, to rearrange with respect to the field. The VSD of large-conductance voltage- and Ca-activated K+ (BK) channels exhibits two salient inconsistencies from the canonical VSD model: (1) the BK channel VSD possesses an additional nonconserved transmembrane helix (S0); and (2) it exhibits a “decentralized” distribution of voltage-sensing charges, in helices S2 and S3, in addition to S4. Considering these unique features, the voltage-dependent rearrangements of the BK VSD could differ significantly from the standard model of VSD operation. To understand the mode of operation of this unique VSD, we have optically tracked the relative motions of the BK VSD transmembrane helices during activation, by manipulating the quenching environment of site-directed fluorescent labels with native and introduced Trp residues. Having previously reported that S0 and S4 diverge during activation, in this work we demonstrate that S4 also diverges from S1 and S2, whereas S2, compelled by its voltage-sensing charged residues, moves closer to S1. This information contributes spatial constraints for understanding the BK channel voltage-sensing process, revealing the structural rearrangements in a non-canonical VSD. PMID:22802360

Intercellular deposits of basement membrane material in active human pituitary adenomas detected by immunostaining for laminin and electron microscopy

DEFF Research Database (Denmark)

Holck, S; Wewer, U M; Albrechtsen, R

1986-01-01

and one patient with Cushing's syndrome). Concurrently, at the ultrastructural level, bunches of basement membrane-like material intermingled between the adenoma cells were demonstrated in seven of these ten active adenomas. Furthermore, secretory granules were entrapped occasionally in this intercellular...

Quantitative measurements of intercellular adhesion between a macrophage and cancer cells using a cup-attached AFM chip.

Science.gov (United States)

Kim, Hyonchol; Yamagishi, Ayana; Imaizumi, Miku; Onomura, Yui; Nagasaki, Akira; Miyagi, Yohei; Okada, Tomoko; Nakamura, Chikashi

2017-07-01

Intercellular adhesion between a macrophage and cancer cells was quantitatively measured using atomic force microscopy (AFM). Cup-shaped metal hemispheres were fabricated using polystyrene particles as a template, and a cup was attached to the apex of the AFM cantilever. The cup-attached AFM chip (cup-chip) approached a murine macrophage cell (J774.2), the cell was captured on the inner concave of the cup, and picked up by withdrawing the cup-chip from the substrate. The cell-attached chip was advanced towards a murine breast cancer cell (FP10SC2), and intercellular adhesion between the two cells was quantitatively measured. To compare cell adhesion strength, the work required to separate two adhered cells (separation work) was used as a parameter. Separation work was almost 2-fold larger between a J774.2 cell and FP10SC2 cell than between J774.2 cell and three additional different cancer cells (4T1E, MAT-LyLu, and U-2OS), two FP10SC2 cells, or two J774.2 cells. FP10SC2 was established from 4T1E as a highly metastatic cell line, indicates separation work increased as the malignancy of cancer cells became higher. One possible explanation of the strong adhesion of macrophages to cancer cells observed in this study is that the measurement condition mimicked the microenvironment of tumor-associated macrophages (TAMs) in vivo, and J774.2 cells strongly expressed CD204, which is a marker of TAMs. The results of the present study, which were obtained by measuring cell adhesion strength quantitatively, indicate that the fabricated cup-chip is a useful tool for measuring intercellular adhesion easily and quantitatively. Copyright © 2017 Elsevier B.V. All rights reserved.

Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase

DEFF Research Database (Denmark)

Desai, D M; Sap, J; Schlessinger, J

1993-01-01

CD45, a transmembrane protein tyrosine phosphatase (PTPase), is required for TCR signaling. Multiple CD45 isoforms, differing in the extracellular domain, are expressed in a tissue- and activation-specific manner, suggesting an important function for this domain. We report that a chimeric protein...... that ligand-mediated regulation of receptor-PTPases may have mechanistic similarities with receptor tyrosine kinases....

Electrochemical platform for the detection of transmembrane proteins reconstituted into liposomes

Czech Academy of Sciences Publication Activity Database

Vacek, J.; Zatloukalová, M.; Geletičová, J.; Kubala, M.; Modriansky, M.; Fekete, Ladislav; Mašek, J.; Hubatka, F.; Turánek, J.

2016-01-01

Roč. 88, č. 8 (2016), s. 4548-4556 ISSN 0003-2700 R&D Projects: GA MŠk LO1409; GA MŠk LM2015088 Institutional support: RVO:68378271 Keywords : detection * transmembrane proteins * liposomes * electrochemistry Subject RIV: BM - Solid Matter Physics ; Magnetism OBOR OECD: Condensed matter physics (including formerly solid state physics, supercond.) Impact factor: 6.320, year: 2016

Substrate-modulated unwinding of transmembrane helices in the NSS transporter LeuT.

Science.gov (United States)

Merkle, Patrick S; Gotfryd, Kamil; Cuendet, Michel A; Leth-Espensen, Katrine Z; Gether, Ulrik; Loland, Claus J; Rand, Kasper D

2018-05-01

LeuT, a prokaryotic member of the neurotransmitter:sodium symporter (NSS) family, is an established structural model for mammalian NSS counterparts. We investigate the substrate translocation mechanism of LeuT by measuring the solution-phase structural dynamics of the transporter in distinct functional states by hydrogen/deuterium exchange mass spectrometry (HDX-MS). Our HDX-MS data pinpoint LeuT segments involved in substrate transport and reveal for the first time a comprehensive and detailed view of the dynamics associated with transition of the transporter between outward- and inward-facing configurations in a Na + - and K + -dependent manner. The results suggest that partial unwinding of transmembrane helices 1/5/6/7 drives LeuT from a substrate-bound, outward-facing occluded conformation toward an inward-facing open state. These hitherto unknown, large-scale conformational changes in functionally important transmembrane segments, observed for LeuT in detergent-solubilized form and when embedded in a native-like phospholipid bilayer, could be of physiological relevance for the translocation process.

Crystal structure of the Haemophilus influenzae Hap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation

Science.gov (United States)

Meng, Guoyu; Spahich, Nicole; Kenjale, Roma; Waksman, Gabriel; St Geme, Joseph W

2011-01-01

Bacterial biofilms are complex microbial communities that are common in nature and are being recognized increasingly as an important determinant of bacterial virulence. However, the structural determinants of bacterial aggregation and eventual biofilm formation have been poorly defined. In Gram-negative bacteria, a major subgroup of extracellular proteins called self-associating autotransporters (SAATs) can mediate cell–cell adhesion and facilitate biofilm formation. In this study, we used the Haemophilus influenzae Hap autotransporter as a prototype SAAT to understand how bacteria associate with each other. The crystal structure of the H. influenzae HapS passenger domain (harbouring the SAAT domain) was determined to 2.2 Å by X-ray crystallography, revealing an unprecedented intercellular oligomerization mechanism for cell–cell interaction. The C-terminal SAAT domain folds into a triangular-prism-like structure that can mediate Hap–Hap dimerization and higher degrees of multimerization through its F1–F2 edge and F2 face. The intercellular multimerization can give rise to massive buried surfaces that are required for overcoming the repulsive force between cells, leading to bacterial cell–cell interaction and formation of complex microcolonies. PMID:21841773

Cystic Fibrosis Transmembrane Conductance Regulator Potentiation as a Therapeutic Strategy for Pulmonary Edema: A Proof-of-Concept Study in Pigs.

Science.gov (United States)

Li, Xiaopeng; Vargas Buonfiglio, Luis G; Adam, Ryan J; Stoltz, David A; Zabner, Joseph; Comellas, Alejandro P

2017-12-01

To determine the feasibility of using a cystic fibrosis transmembrane conductance regulator potentiator, ivacaftor (VX-770/Kalydeco, Vertex Pharmaceuticals, Boston, MA), as a therapeutic strategy for treating pulmonary edema. Prospective laboratory animal investigation. Animal research laboratory. Newborn and 3 days to 1 week old pigs. Hydrostatic pulmonary edema was induced in pigs by acute volume overload. Ivacaftor was nebulized into the lung immediately after volume overload. Grams of water per grams of dry lung tissue were determined in the lungs harvested 1 hour after volume overload. Ivacaftor significantly improved alveolar liquid clearance in isolated pig lung lobes ex vivo and reduced edema in a volume overload in vivo pig model of hydrostatic pulmonary edema. To model hydrostatic pressure-induced edema in vitro, we developed a method of applied pressure to the basolateral surface of alveolar epithelia. Elevated hydrostatic pressure resulted in decreased cystic fibrosis transmembrane conductance regulator activity and liquid absorption, an effect which was partially reversed by cystic fibrosis transmembrane conductance regulator potentiation with ivacaftor. Cystic fibrosis transmembrane conductance regulator potentiation by ivacaftor is a novel therapeutic approach for pulmonary edema.

Visfatin Reduces Gap Junction Mediated Cell-to-Cell Communication in Proximal Tubule-Derived Epithelial Cells

Directory of Open Access Journals (Sweden)

Claire E. Hills

2013-11-01

Full Text Available Background/Aims: In the current study we examined if the adipocytokine, visfatin, alters connexin-mediated intercellular communication in proximal tubule-derived epithelial cells. Methods: The effects of visfatin (10-200ng/mL on cell viability and cytotoxicity in HK2-cells were assessed by MTT, crystal violet and lactate dehydrogenase assays. Western blot analysis was used to confirm expression of Cx26, Cx40 and Cx43. The effect of visfatin (10-200ng/mL on TGF-β1 secretion was confirmed by ELISA, and the effects of both TGF-β1 (2-10ng/mL and visfatin (10-200ng/mL on connexin expression were assessed by western blot. Functional intercellular communication was determined using transfer of Lucifer Yellow and paired-whole cell patch clamp electrophysiology. Results: In low glucose (5mM, visfatin (10-200ng/mL did not affect membrane integrity, cytotoxicity or cell viability at 48hrs, but did evoke a concentration-dependent reduction in Cx26 and Cx43 expression. The expression of Cx40 was unaffected. At 48hrs, visfatin (10-200ng/mL increased the secretion of TGF-β1 and the visfatin-evoked changes in connexin expression were mimicked by exogenous application of the pro-fibrotic cytokine (2-10ng/ml. Visfatin reduced dye transfer between coupled cells and decreased functional conductance, with levels falling by 63% as compared to control. Although input resistance was increased following visfatin treatment by 166%, the change was not significant as compared to control. The effects of visfatin on Cx-expression and cell-coupling were blocked in the presence of a TGF-β1 specific neutralizing antibody. Conclusions: The adipocytokine visfatin selectively evoked a non-toxic reduction in connexin expression in HK2-cells. The loss in gap-junction associated proteins was mirrored by a loss in functional conductance between coupled cells. Visfatin increased TGF-β secretion and the pattern of change for connexins expression was mimicked by exogenous

Inhibition of connexin43 gap junction channels by the endocrine disruptor ioxynil

International Nuclear Information System (INIS)

Leithe, Edward; Kjenseth, Ane; Bruun, Jarle; Sirnes, Solveig; Rivedal, Edgar

2010-01-01

Gap junctions are intercellular plasma membrane domains containing channels that mediate transport of ions, metabolites and small signaling molecules between adjacent cells. Gap junctions play important roles in a variety of cellular processes, including regulation of cell growth and differentiation, maintenance of tissue homeostasis and embryogenesis. The constituents of gap junction channels are a family of trans-membrane proteins called connexins, of which the best-studied is connexin43. Connexin43 functions as a tumor suppressor protein in various tissue types and is frequently dysregulated in human cancers. The pesticide ioxynil has previously been shown to act as an endocrine disrupting chemical and has multiple effects on the thyroid axis. Furthermore, both ioxynil and its derivative ioxynil octanoate have been reported to induce tumors in animal bioassays. However, the molecular mechanisms underlying the possible tumorigenic effects of these compounds are unknown. In the present study we show that ioxynil and ioxynil octanoate are strong inhibitors of connexin43 gap junction channels. Both compounds induced rapid loss of connexin43 gap junctions at the plasma membrane and increased connexin43 degradation. Ioxynil octanoate, but not ioxynil, was found to be a strong activator of ERK1/2. The compounds also had different effects on the phosphorylation status of connexin43. Taken together, the data show that ioxynil and ioxynil octanoate are potent inhibitors of intercellular communication via gap junctions.

Gap junction mediated intercellular metabolite transfer in the cochlea is compromised in connexin30 null mice.

Directory of Open Access Journals (Sweden)

Qing Chang

Full Text Available Connexin26 (Cx26 and connexin30 (Cx30 are two major protein subunits that co-assemble to form gap junctions (GJs in the cochlea. Mutations in either one of them are the major cause of non-syndromic prelingual deafness in humans. Because the mechanisms of cochlear pathogenesis caused by Cx mutations are unclear, we investigated effects of Cx30 null mutation on GJ-mediated ionic and metabolic coupling in the cochlea of mice. A novel flattened cochlear preparation was used to directly assess intercellular coupling in the sensory epithelium of the cochlea. Double-electrode patch clamp recordings revealed that the absence of Cx30 did not significantly change GJ conductance among the cochlear supporting cells. The preserved electrical coupling is consistent with immunolabeling data showing extensive Cx26 GJs in the cochlea of the mutant mice. In contrast, dye diffusion assays showed that the rate and extent of intercellular transfer of multiple fluorescent dyes (including a non-metabolizable D-glucose analogue, 2-NBDG among cochlear supporting cells were severely reduced in Cx30 null mice. Since the sensory epithelium in the cochlea is an avascular organ, GJ-facilitated intercellular transfer of nutrient and signaling molecules may play essential roles in cellular homeostasis. To test this possibility, NBDG was used as a tracer to study the contribution of GJs in transporting glucose into the cochlear sensory epithelium when delivered systemically. NBDG uptake in cochlear supporting cells was significantly reduced in Cx30 null mice. The decrease was also observed with GJ blockers or glucose competition, supporting the specificity of our tests. These data indicate that GJs facilitate efficient uptake of glucose in the supporting cells. This study provides the first direct experimental evidence showing that the transfer of metabolically-important molecules in cochlear supporting cells is dependent on the normal function of GJs, thereby suggesting a

In vitro early changes in intercellular junctions by treatment with a chemical carcinogen.

Science.gov (United States)

Tachikawa, T; Kohno, Y; Matsui, Y; Yoshiki, S

1986-06-01

To examine early intercellular junction changes caused by treatment with 9,10-dimethyl-1,2-benzanthracene (DMBA), rat lingual epithelium was cultivated in isolation and observed by electrophysiological, freeze-fracture and whole-mount electron microscopy. Electrophysiological measurements showed a transient decrease in membrane potential of -10.2 mV 6 h after the treatment. It returned to almost the same level as that of the control group 1 day later. Six hours after treatment, input resistance decreased rapidly to 5.3 M omega but increased to 18.0 M omega 12 h after treatment. Transient reduction of input resistance and membrane potential occurred prior to the decrease in the coupling ratio 6 h after treatment with DMBA. In freeze-fracture replicas, the number of gap junctions decreased by approximately 45% of the control value 6 h after treatment with DMBA. At 12 h and thereafter, the number and area of gap junctions subsequently decreased by 60-80% of the control value. Alterations in the number and area of desmosomes were similar to those of the gap junctions. The formation of epithelial cytoskeletons, partially devoid of the 2-4 and 5-8 nm filaments was also observed. A decrease in the density of filament networks beneath the plasma membranes was especially apparent. Treatment with a carcinogen brought about morphological cellular changes as early as 6 h after treatment, and such early changes might trigger metabolic cellular abnormalities. Affected cells appear to move away from normal cells in a process of repeated destruction and revision of intercellular junctions, and cytoskeletons.

Early vertebrate origin and diversification of small transmembrane regulators of cellular ion transport.

Science.gov (United States)

Pirkmajer, Sergej; Kirchner, Henriette; Lundell, Leonidas S; Zelenin, Pavel V; Zierath, Juleen R; Makarova, Kira S; Wolf, Yuri I; Chibalin, Alexander V

2017-07-15

Small transmembrane proteins such as FXYDs, which interact with Na + ,K + -ATPase, and the micropeptides that interact with sarco/endoplasmic reticulum Ca 2+ -ATPase play fundamental roles in regulation of ion transport in vertebrates. Uncertain evolutionary origins and phylogenetic relationships among these regulators of ion transport have led to inconsistencies in their classification across vertebrate species, thus hampering comparative studies of their functions. We discovered the first FXYD homologue in sea lamprey, a basal jawless vertebrate, which suggests small transmembrane regulators of ion transport emerged early in the vertebrate lineage. We also identified 13 gene subfamilies of FXYDs and propose a revised, phylogeny-based FXYD classification that is consistent across vertebrate species. These findings provide an improved framework for investigating physiological and pathophysiological functions of small transmembrane regulators of ion transport. Small transmembrane proteins are important for regulation of cellular ion transport. The most prominent among these are members of the FXYD family (FXYD1-12), which regulate Na + ,K + -ATPase, and phospholamban, sarcolipin, myoregulin and DWORF, which regulate the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA). FXYDs and regulators of SERCA are present in fishes, as well as terrestrial vertebrates; however, their evolutionary origins and phylogenetic relationships are obscure, thus hampering comparative physiological studies. Here we discovered that sea lamprey (Petromyzon marinus), a representative of extant jawless vertebrates (Cyclostomata), expresses an FXYD homologue, which strongly suggests that FXYDs predate the emergence of fishes and other jawed vertebrates (Gnathostomata). Using a combination of sequence-based phylogenetic analysis and conservation of local chromosome context, we determined that FXYDs markedly diversified in the lineages leading to cartilaginous fishes (Chondrichthyes) and bony

MutHTP: Mutations in Human Transmembrane Proteins.

Science.gov (United States)

A, Kulandaisamy; S, Binny Priya; R, Sakthivel; Tarnovskaya, Svetlana; Bizin, Ilya; Hönigschmid, Peter; Frishman, Dmitrij; Gromiha, M Michael

2018-02-01

We have developed a novel database, MutHTP, which contains information on 183395 disease-associated and 17827 neutral mutations in human transmembrane proteins. For each mutation site MutHTP provides a description of its location with respect to the membrane protein topology, structural environment (if available) and functional features. Comprehensive visualization, search, display and download options are available. The database is publicly available at http://www.iitm.ac.in/bioinfo/MutHTP/. The website is implemented using HTML, PHP and javascript and supports recent versions of all major browsers, such as Firefox, Chrome and Opera. gromiha@iitm.ac.in. Supplementary data are available at Bioinformatics online. © The Author (2018). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Promiscuous Seven Transmembrane Receptors Sensing L-α-amino Acids

DEFF Research Database (Denmark)

Smajilovic, Sanela; Wellendorph, Petrine; Bräuner-Osborne, Hans

2014-01-01

A number of nutrient sensing seven trans-membrane (7TM) receptors have been identified and characterized over the past few years. While the sensing mechanisms to carbohydrates and free fatty acids are well understood, the molecular basis of amino acid sensing has recently come to the limelight....... The present review describes the current status of promiscuous L-α-amino acid sensors, the calcium sensing receptor (CaSR), the GPRC6A receptor, the T1R1/T1R3 receptor and also their molecular pharmacology, expression pattern and physiological significance....

Radial distribution of dilated intercellular spaces of the esophageal squamous epithelium in patients with reflux disease exhibiting discrete endoscopic lesions

NARCIS (Netherlands)

Vieth, M; Fiocca, R; Haringsma, J; Delarive, J; Wiesel, PH; Tam, W; Tytgat, GNJ; Dent, J; Edebo, A; Lundell, L; Stolte, M

2004-01-01

Introduction: Dilatation of intercellular spaces of the esophageal squamous epithelium has been suggested as a marker of early acid reflux-induced damage. This change is a potentially useful addition to histomorphological changes that represent so called minimal endoscopic lesions. We have assessed

Vitamin A transport and the transmembrane pore in the cell-surface receptor for plasma retinol binding protein.

Directory of Open Access Journals (Sweden)

Ming Zhong

Full Text Available Vitamin A and its derivatives (retinoids play diverse and crucial functions from embryogenesis to adulthood and are used as therapeutic agents in human medicine for eye and skin diseases, infections and cancer. Plasma retinol binding protein (RBP is the principal and specific vitamin A carrier in the blood and binds vitamin A at 1:1 ratio. STRA6 is the high-affinity membrane receptor for RBP and mediates cellular vitamin A uptake. STRA6 null mice have severely depleted vitamin A reserves for vision and consequently have vision loss, even under vitamin A sufficient conditions. STRA6 null humans have a wide range of severe pathological phenotypes in many organs including the eye, brain, heart and lung. Known membrane transport mechanisms involve transmembrane pores that regulate the transport of the substrate (e.g., the gating of ion channels. STRA6 represents a new type of membrane receptor. How this receptor interacts with its transport substrate vitamin A and the functions of its nine transmembrane domains are still completely unknown. These questions are critical to understanding the molecular basis of STRA6's activities and its regulation. We employ acute chemical modification to introduce chemical side chains to STRA6 in a site-specific manner. We found that modifications with specific chemicals at specific positions in or near the transmembrane domains of this receptor can almost completely suppress its vitamin A transport activity. These experiments provide the first evidence for the existence of a transmembrane pore, analogous to the pore of ion channels, for this new type of cell-surface receptor.

Regulation of germ cell development by intercellular signaling in the mammalian ovarian follicle.

Science.gov (United States)

Clarke, Hugh J

2018-01-01

Prior to ovulation, the mammalian oocyte undergoes a process of differentiation within the ovarian follicle that confers on it the ability to give rise to an embryo. Differentiation comprises two phases-growth, during which the oocyte increases more than 100-fold in volume as it accumulates macromolecules and organelles that will sustain early embryogenesis; and meiotic maturation, during which the oocyte executes the first meiotic division and prepares for the second division. Entry of an oocyte into the growth phase appears to be triggered when the adjacent granulosa cells produce specific growth factors. As the oocyte grows, it elaborates a thick extracellular coat termed the zona pellucida. Nonetheless, cytoplasmic extensions of the adjacent granulosa cells, termed transzonal projections (TZPs), enable them to maintain contact-dependent communication with the oocyte. Through gap junctions located where the TZP tips meet the oocyte membrane, they provide the oocyte with products that sustain its metabolic activity and signals that regulate its differentiation. Conversely, the oocyte secretes diffusible growth factors that regulate proliferation and differentiation of the granulosa cells. Gap junction-permeable products of the granulosa cells prevent precocious initiation of meiotic maturation, and the gap junctions also enable oocyte maturation to begin in response to hormonal signals received by the granulosa cells. Development of the oocyte or the somatic compartment may also be regulated by extracellular vesicles newly identified in follicular fluid and at TZP tips, which could mediate intercellular transfer of macromolecules. Oocyte differentiation thus depends on continuous signaling interactions with the somatic cells of the follicle. WIREs Dev Biol 2018, 7:e294. doi: 10.1002/wdev.294 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Signaling Pathways > Cell Fate Signaling Early Embryonic

Novel approaches to mitigating bacterial biofilm formation and intercellular communication

Science.gov (United States)

Kasper, Stephen H.

Long thought of as solitary single-cell organisms, it is now widely accepted that bacteria can act and cooperate as social organisms. Phenomena such as biofilm formation and quorum sensing (QS) are two intimately intertwined cooperative behaviors that significantly contribute to the pathogenesis of many bacteria. Biofilms are surface associated communities of bacteria encased in a secreted extracellular matrix, which provides several advantages over an individualized lifestyle, such as increased protection from antimicrobial agents as well as enhanced opportunity for the exchange of genetic material. Bacterial QS is a system of population-based communication through the production, sensing, and response to chemical signals, often controlling the expression of diverse virulence factors (e.g. toxins, proteases). Biofilm formation and QS are cooperative processes that are often leveraged as bacteria coordinate infection processes, and can therefore be novel targets for anti-infective treatments that differ from conventional antibiotic treatment. Our lab has previously identified a novel class of small molecules that inhibit biofilm formation and disrupt QS by the pathogenic bacterium Pseudomonas aeruginosa. These organosulfur-based compounds are either natural products or related derivatives of the tropical plant Petiveria alliacea. Because oral biofilm (e.g. dental plaque) is a major conduit of oral and systemic disease, and is also a site for horizontal transfer for genes encoding antibiotic resistance, there exists a need for novel strategies for inhibiting oral biofilm development. Therefore, a small library (˜50 compounds) of structural derivatives was developed and screened for their ability to inhibit biofilm formation by multiple orally associated bacteria. The screening effort uncovered several related compounds that inhibited oral biofilm development. To determine how natural product-based organosulfur compounds could be inducing QS inhibitory effects, an

Generation and Nuclear Translocation of Sumoylated Transmembrane Fragment of Cell Adhesion Molecule L1

Science.gov (United States)

Lutz, David; Wolters-Eisfeld, Gerrit; Joshi, Gunjan; Djogo, Nevena; Jakovcevski, Igor; Schachner, Melitta; Kleene, Ralf

2012-01-01

The functions of the cell adhesion molecule L1 in the developing and adult nervous system are triggered by homophilic and heterophilic interactions that stimulate signal transductions that activate cellular responses. Here, we show that stimulation of signaling by function-triggering L1 antibodies or L1-Fc leads to serine protease-dependent cleavage of full-length L1 at the plasma membrane and generation of a sumoylated transmembrane 70-kDa fragment comprising the intracellular and transmembrane domains and part of the extracellular domain. The 70-kDa transmembrane fragment is transported from the plasma membrane to a late endosomal compartment, released from endosomal membranes into the cytoplasm, and transferred from there into the nucleus by a pathway that depends on importin and chromatin-modifying protein 1. Mutation of the sumoylation site at Lys1172 or of the nuclear localization signal at Lys1147 abolished L1-stimulated generation or nuclear import of the 70-kDa fragment, respectively. Nuclear import of the 70-kDa fragment may activate cellular responses in parallel or in association with phosphorylation-dependent signaling pathways. Alterations in the levels of the 70-kDa fragment during development and in the adult after spinal cord injury or in a mouse model of Alzheimer disease suggest that this fragment is functionally implicated in development, regeneration, neurodegeneration, tumorigenesis, and possibly synaptic plasticity in the mature nervous system. PMID:22431726

Specificity of transmembrane protein palmitoylation in yeast.

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Ayelén González Montoro

Full Text Available Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs, characterized by the presence of a conserved 50- aminoacids domain called "Asp-His-His-Cys- Cysteine Rich Domain" (DHHC-CRD. There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether.Here we investigate the specificity of transmembrane protein palmitoylation in S. cerevisiae, which is carried out predominantly by two PATs, Swf1 and Pfa4. We show that palmitoylation of transmembrane substrates requires dedicated PATs, since other yeast PATs are mostly unable to perform Swf1 or Pfa4 functions, even when overexpressed. Furthermore, we find that Swf1 is highly specific for its substrates, as it is unable to substitute for other PATs. To identify where Swf1 specificity lies, we carried out a bioinformatics survey to identify amino acids responsible for the determination of specificity or Specificity Determination Positions (SDPs and showed experimentally, that mutation of the two best SDP candidates, A145 and K148, results in complete and partial loss of function, respectively. These residues are located within the conserved catalytic DHHC domain suggesting that it could also be involved in the determination of specificity. Finally, we show that modifying the position of the cysteines in Tlg1, a Swf1 substrate, results in lack of palmitoylation, as

Role of α and β Transmembrane Domains in Integrin Clustering

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Amir Shamloo

2015-11-01

Full Text Available Integrins are transmembrane proteins playing a crucial role in the mechanical signal transduction from the outside to the inside of a cell, and vice versa. Nevertheless, this signal transduction could not be implemented by a single protein. Rather, in order for integrins to be able to participate in signal transduction, they need to be activated and produce clusters first. As integrins consist of α- and β-subunits that are separate in the active state, studying both subunits separately is of a great importance, for, in the active state, the distance between α- and β-subunits is long enough that they do not influence one another significantly. Thus, this study aims to investigate the tendency of transmembrane domains of integrins to form homodimers. We used both Steered and MARTINI Coarse-grained molecular dynamics method to perform our simulations, mainly because of a better resolution and computational feasibility that each of these methods could provide to us. Using the Steered molecular dynamics method for α- and β-subunits, we found that the localized lipid packing prevented them from clustering. Nonetheless, the lipid packing phenomenon was found to be an artifact after investigating this process using a coarse grained (CG model. Exploiting the coarse-grained molecular dynamics simulations, we found that α- and β-subunits tend to form a stable homo-dimer.

NTAL (non-T cell activation linker):a transmembrane adaptor protein involved in immunoreceptor signaling

Czech Academy of Sciences Publication Activity Database

Brdička, Tomáš; Imrich, Martin; Angelisová, Pavla; Brdičková, Naděžda; Horváth, Ondřej; Špička, Jiří; Hilgert, Ivan; Lusková, Petra; Dráber, Petr; Novák, P.; Engels, N.; Wienands, J.; Simeoni, L.; Osterreicher, J.; Aguado, E.; Malissen, M.; Schraven, B.; Hořejší, Václav

2002-01-01

Roč. 196, č. 12 (2002), s. 16180-16185 ISSN 0022-1007 R&D Projects: GA MŠk LN00A026 Keywords : NTAL * transmembrane adaptor * immunoreceptor signaling Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 15.838, year: 2002

Comparative characteristic of transmembrane currents and caffeine-induced responses of intact and irradiated small intestine smooth muscle cells

International Nuclear Information System (INIS)

Stepanov, Yu.V.; Gordienko, D.V.; Preobrazhenskaya, T.D.; Stepanova, L.I.; Vojtsitskij, V.M.

1994-01-01

A comparative investigation of transmembrane ion currents and caffeine-induced responses of single smooth muscle cells isolated from the circular layer of rat small intestine was curried out by the method of 'patch-clamp'. No reliable difference in potential-dependent and amplitude-kinetic characteristics of transmembrane ion currents in cells of intact and irradiated with dose of 3 Gy rats was revealed. In cells of irradiated animals external application of caffeine (4 mM) was not accompanied by strong quick-inactivated transient Ca 2+ -dependent potassium current as in control

Transmembrane helical interactions in the CFTR channel pore.

Directory of Open Access Journals (Sweden)

Jhuma Das

2017-06-01

Full Text Available Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR gene affect CFTR protein biogenesis or its function as a chloride channel, resulting in dysregulation of epithelial fluid transport in the lung, pancreas and other organs in cystic fibrosis (CF. Development of pharmaceutical strategies to treat CF requires understanding of the mechanisms underlying channel function. However, incomplete 3D structural information on the unique ABC ion channel, CFTR, hinders elucidation of its functional mechanism and correction of cystic fibrosis causing mutants. Several CFTR homology models have been developed using bacterial ABC transporters as templates but these have low sequence similarity to CFTR and are not ion channels. Here, we refine an earlier model in an outward (OWF and develop an inward (IWF facing model employing an integrated experimental-molecular dynamics simulation (200 ns approach. Our IWF structure agrees well with a recently solved cryo-EM structure of a CFTR IWF state. We utilize cysteine cross-linking to verify positions and orientations of residues within trans-membrane helices (TMHs of the OWF conformation and to reconstruct a physiologically relevant pore structure. Comparison of pore profiles of the two conformations reveal a radius sufficient to permit passage of hydrated Cl- ions in the OWF but not the IWF model. To identify structural determinants that distinguish the two conformations and possible rearrangements of TMHs within them responsible for channel gating, we perform cross-linking by bifunctional reagents of multiple predicted pairs of cysteines in TMH 6 and 12 and 6 and 9. To determine whether the effects of cross-linking on gating observed are the result of switching of the channel from open to close state, we also treat the same residue pairs with monofunctional reagents in separate experiments. Both types of reagents prevent ion currents indicating that pore blockage is primarily responsible.

Resolving the biophysics of axon transmembrane polarization in a single closed-form description

Energy Technology Data Exchange (ETDEWEB)

Melendy, Robert F., E-mail: rfmelendy@liberty.edu [School of Engineering and Computational Sciences, Liberty University, Lynchburg, Virginia 24515 (United States)

2015-12-28

When a depolarizing event occurs across a cell membrane there is a remarkable change in its electrical properties. A complete depolarization event produces a considerably rapid increase in voltage that propagates longitudinally along the axon and is accompanied by changes in axial conductance. A dynamically changing magnetic field is associated with the passage of the action potential down the axon. Over 75 years of research has gone into the quantification of this phenomenon. To date, no unified model exist that resolves transmembrane polarization in a closed-form description. Here, a simple but formative description of propagated signaling phenomena in the membrane of an axon is presented in closed-form. The focus is on using both biophysics and mathematical methods for elucidating the fundamental mechanisms governing transmembrane polarization. The results presented demonstrate how to resolve electromagnetic and thermodynamic factors that govern transmembrane potential. Computational results are supported by well-established quantitative descriptions of propagated signaling phenomena in the membrane of an axon. The findings demonstrate how intracellular conductance, the thermodynamics of magnetization, and current modulation function together in generating an action potential in a unified closed-form description. The work presented in this paper provides compelling evidence that three basic factors contribute to the propagated signaling in the membrane of an axon. It is anticipated this work will compel those in biophysics, physical biology, and in the computational neurosciences to probe deeper into the classical and quantum features of membrane magnetization and signaling. It is hoped that subsequent investigations of this sort will be advanced by the computational features of this model without having to resort to numerical methods of analysis.

Effects of alpha-tocopherol on superoxide production and plasma intercellular adhesion molecule-1 and antibodies to oxidized LDL in chronic smokers

NARCIS (Netherlands)

Tits, van L.J.; Waart, de F.; Hak-Lemmers, H.L.M.; Heijst, P.; Graaf, de J.; Demacker, P.N.; Stalenhoef, A.F.

2001-01-01

Antioxidants have been postulated to exert beneficial effects in atherosclerosis. Atherosclerosis is associated with raised plasma levels of soluble intercellular adhesion molecule-1 (sICAM-1) and autoantibodies against oxidized low-density lipoprotein (oxLDL). It is not known whether antioxidants

Understanding intercellular communication in the brain: Identified ...

Indian Academy of Sciences (India)

The transmission of information between nerve cells in the brain takes place at specialized sites of ... period of 10 days, and the giant chromosomes of larval salivary glands. ... extensive use of Drosophila in order to assay the effects of the genetic changes in ... the muscle responds only to a small percentage of the stimuli.

Intercellular distribution of mutations induced in oopcytes of Drosophila melanogaster by chemical and physical mutagens

International Nuclear Information System (INIS)

Traut, H.

1979-01-01

When females of Drosophila melanogaster are treated with chemical or physical mutagens, not only in one but also in both of the two homologous X chromosomes of a given oocyte, a recessive sex-linked lethal mutation may be induced. A method is described that discriminates between such single and double mutations. A theory is developed to show how a comparison betweeen the expected and the observer frequency of double mutations yields an indication of the intercellular distribution (random or nonrandom) of recessive lethal mutations induced by mutagenic agents in oocytes and, consequently, of the distribution (homogenous or nonhomogeneous) of those agents. Three agents were tested: FUdR (12.5, 50.0 and 81.0 μg/ml), mitomycin C (130.0 μg/ml) and x rays (2000 R, 150 kV). After FUdR feeding, no increase in the mutation frequency usually observed in D. melanogaster without mutagenic treatment was obtained (u = 0.13%, namely three single mutations among 2332 chromosomes tested). After mitomycin C feeding 104 single and three double mutations were obtained. All of the 50 mutations observed after x irradiation were single mutations. The results obtained in the mitomycin C and radiation experiments favor the assumption of a random intercellular distribution of recessive lethal mutations induced by these two agents in oocytes of D. melanogaster. Reasons are discussed why for other types of mutagenic agents nonrandom distributions may be observed with our technique

Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments

Czech Academy of Sciences Publication Activity Database

Vít, O.; Man, Petr; Kádek, Alan; Hausner, Jiří; Sklenář, A.; Harant, K.; Novák, Petr; Scigelová, M.; Wofferndin, G.; Petrák, J.

2016-01-01

Roč. 146, SI (2016), s. 15-22 ISSN 1874-3919 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Integral membrane proteins * CNBr * Transmembrane Subject RIV: CE - Biochemistry Impact factor: 3.914, year: 2016

Impact of axial velocity and transmembrane pressure (TMP) on ARP filter performance

Energy Technology Data Exchange (ETDEWEB)

Poirier, M. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Burket, P. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

2016-02-29

The Savannah River Site (SRS) is currently treating radioactive liquid waste with the Actinide Removal Process (ARP) and the Modular Caustic Side Solvent Extraction Unit (MCU). Recently, the low filter flux through the ARP of approximately 5 gallons per minute has limited the rate at which radioactive liquid waste can be treated. Salt Batch 6 had a lower processing rate and required frequent filter cleaning. Savannah River Remediation (SRR) has a desire to understand the causes of the low filter flux and to increase ARP/MCU throughput. One potential method for increasing filter flux is to adjust the axial velocity and transmembrane pressure (TMP). SRR requested SRNL to conduct bench-scale filter tests to evaluate the effects of axial velocity and transmembrane pressure on crossflow filter flux. The objective of the testing was to determine whether increasing the axial velocity at the ARP could produce a significant increase in filter flux. The authors conducted the tests by preparing slurries containing 6.6 M sodium Salt Batch 6 supernate and 2.5 g MST/L, processing the slurry through a bench-scale crossflow filter unit at varying axial velocity and TMP, and measuring filter flux as a function of time.

A transmembrane polar interaction is involved in the functional regulation of integrin alpha L beta 2.

Science.gov (United States)

Vararattanavech, Ardcharaporn; Chng, Choon-Peng; Parthasarathy, Krupakar; Tang, Xiao-Yan; Torres, Jaume; Tan, Suet-Mien

2010-05-14

Integrins are heterodimeric transmembrane (TM) receptors formed by noncovalent associations of alpha and beta subunits. Each subunit contains a single alpha-helical TM domain. Inside-out activation of an integrin involves the separation of its cytoplasmic tails, leading to disruption of alphabeta TM packing. The leukocyte integrin alpha L beta 2 is required for leukocyte adhesion, migration, proliferation, cytotoxic function, and antigen presentation. In this study, we show by mutagenesis experiments that the packing of alpha L beta 2 TMs is consistent with that of the integrin alpha IIb beta 3 TMs. However, molecular dynamics simulations of alpha L beta 2 TMs in lipids predicted a polar interaction involving the side chains of alpha L Ser1071 and beta2 Thr686 in the outer-membrane association clasp (OMC). This is supported by carbonyl vibrational shifts observed in isotope-labeled alpha L beta 2 TM peptides that were incorporated into lipid bilayers. Molecular dynamics studies simulating the separation of alpha L beta 2 tails showed the presence of polar interaction during the initial perturbation of the inner-membrane association clasp. When the TMs underwent further separation, the polar interaction was disrupted. OMC polar interaction is important in regulating the functions of beta2 integrins because mutations that disrupt the OMC polar interaction generated constitutively activated alpha L beta 2, alpha M beta 2, and alpha X beta 2 in 293T transfectants. We also show that the expression of mutant beta2 Thr686Gly in beta2-deficient T cells rescued cell adhesion to intercellular adhesion molecule 1, but the cells showed overt elongated morphologies in response to chemokine stromal-cell-derived factor 1 alpha treatment as compared to wild-type beta2-expressing cells. These two TM polar residues are totally conserved in other members of the beta2 integrins in humans and across different species. Our results provide an example of the stabilizing effect of polar

Detection of the intercellular adhesion gene cluster (ica in clinical Staphylococcus aureus isolates

Directory of Open Access Journals (Sweden)

Namvar, Amirmorteza Ebrahimzadeh

2013-04-01

Full Text Available [english] is a major hospital and community pathogen having the aptitude to cause a wide variety of infections in men. The ability of microorganisms to produce biofilm facilitates them to withstand the host immune response and is recognized as one factor contributing to chronic or persistent infections. It was demonstrated that the -encoded genes lead to the biosynthesis of polysaccharide intercellular adhesion (PIA molecules, and may be involved in the accumulation phase of biofilm formation. Different studies have shown the decisive role of the gene as virulence factors in staphylococcal infections. This study was carried out to demonstrate the relationship between gene and production of slime layer in strains. Sixty strains were isolated from patients. The isolates were identified morphologically and biochemically following standard laboratory methods. After identification, the staphylococcal isolates were maintained in trypticase soy broth (TSB, to which 15% glycerol was added, and stored at –20°C. Slime formation and biofilm assay was monitored. A PCR assay was developed to identify the presence of (intercellular adhesion gene gene in all isolates. Thirty-nine slime producing colonies with CRA plates (65% formed black colors, the remaining 21 isolates were pink (35%. In the quantitative biofilm assay 35 (58% produced biofilm while 25 (42% isolates did not exhibit this property. All isolates were positive for detection of gene by PCR method. The interaction of and in the investigated isolates may be important in slime layer formation and biofilm phenomena.We propose PCR detection of the gene locus as a rapid and effective method to be used for discrimination between potentially virulent and nonvirulent isolates, with implications for therapeutic and preventive measures pertainin to the management of colonized indwelling catheters.

Intercellular contact: its influence on the Dsub(q) of mammalian cell survival curves

International Nuclear Information System (INIS)

Durand, R.E.; Sutherland, R.M.

1975-01-01

Cell survival in tissues exposed to a given dose of ionizing radiation is usually greater than that of similar cells grown individually in vitro, despite the fact that the radiosensitivities (D 0 ) are virtually identical under the two conditions. An analogous increase in cell survival is observed when Chinese hamster V79-171 cells are grown in suspension culture and irradiated as multicell spheroids. Unfortunately, the information gained from the survival curves so obtained is limited by the inhomogeneity of the cell population with respect to both degree of contact and cell cycle position. The latter can be studied using synchronized small spheroids. The ratio of Dsub(q) of spheroid cells to Dsub(q) of single cells increased as the cells progressed through the cell cycle, from a minimum of 1.3 for G 1 phase cells to a maximum of 2.2 for late S-phase cells. The enhanced survival, or 'contact effect', developed slowly as the spheroids grew, after an initial latent period of about one generation cycle of the cells. A second effect of intercellular contact on mammalian cell survival has also been observed. When cells are assayed under conditions in which intercellular contact is maintained, the net cellular survival is increased further. This effect is different from the usual repair of potentially lethal damage, in that it occurs much more slowly and results in modification of the survival-curve shoulder. Not all cell types tested have shown enhanced survival when grown as spheroids. Several MNNG-induced mutants of the Chinese hamster V79-171 line have been isolated and sublines which do and do not show the contact effect are now available. These may permit study of the mechanism(s) of contact effects. (author)

SCIMP, a transmembrane adaptor protein involved in major histocompatibility complex class II signaling

Czech Academy of Sciences Publication Activity Database

Dráber, Peter; Vonková, Ivana; Štěpánek, Ondřej; Hrdinka, Matouš; Kucová, Markéta; Skopcová, Tereza; Otáhal, Pavel; Angelisová, Pavla; Hořejší, Václav; Yeung, M.; Weiss, A.; Brdička, Tomáš

2011-01-01

Roč. 31, č. 22 (2011), s. 4550-4562 ISSN 0270-7306 R&D Projects: GA MŠk 1M0506; GA ČR GEMEM/09/E011 Institutional research plan: CEZ:AV0Z50520514 Keywords : SCIMP * transmembrane adaptor protein * MHC II Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.527, year: 2011

Expression and regulation of transmembrane transporters in healthy intestine and gastrointestinal diseases

OpenAIRE

Hruz, Petr

2006-01-01

Transmembrane transporters mediate energy dependent or independent translocation of drugs, potentially toxic compounds, and of various endogenous substrates such as bile acids and bilirubin across membranes. In this thesis the focus is on two classes of transporters, the ATPbinding cassette (ABC) transporters, which mediate ATP dependent transport and the solute carriers (SLC) which use electrochemical gradients for their transport. The transporters are expressed on membranes o...

Exosome-Based Cell-Cell Communication in the Tumor Microenvironment

Directory of Open Access Journals (Sweden)

Joana Maia

2018-02-01

Full Text Available Tumors are not isolated entities, but complex systemic networks involving cell-cell communication between transformed and non-transformed cells. The milieu created by tumor-associated cells may either support or halt tumor progression. In addition to cell-cell contact, cells communicate through secreted factors via a highly complex system involving characteristics such as ligand concentration, receptor expression and integration of diverse signaling pathways. Of these, extracellular vesicles, such as exosomes, are emerging as novel cell-cell communication mediators in physiological and pathological scenarios. Exosomes, membrane vesicles of endocytic origin released by all cells (both healthy and diseased, ranging in size from 30 to 150 nm, transport all the main biomolecules, including lipids, proteins, DNAs, messenger RNAs and microRNA, and perform intercellular transfer of components, locally and systemically. By acting not only in tumor cells, but also in tumor-associated cells such as fibroblasts, endothelium, leukocytes and progenitor cells, tumor- and non-tumor cells-derived exosomes have emerged as new players in tumor growth and invasion, tumor-associated angiogenesis, tissue inflammation and immunologic remodeling. In addition, due to their property of carrying molecules from their cell of origin to the peripheral circulation, exosomes have been increasingly studied as sources of tumor biomarkers in liquid biopsies. Here we review the current literature on the participation of exosomes in the communication between tumor and tumor-associated cells, highlighting the role of this process in the setup of tumor microenvironments that modulate tumor initiation and metastasis.

In-vitro investigation of out-of-field cell survival following the delivery of conformal, intensity-modulated radiation therapy (IMRT) and volumetric modulated arc therapy (VMAT) plans

International Nuclear Information System (INIS)

McGarry, Conor K; Hounsell, Alan R; Butterworth, Karl T; Trainor, Colman; McMahon, Stephen J; O'Sullivan, Joe M; Prise, Kevin M

2012-01-01

The aim of this work is to determine the out-of-field survival of cells irradiated with either the primary field or scattered radiation in the presence and absence of intercellular communication following delivery of conformal, IMRT and VMAT treatment plans. Single beam, conformal, IMRT and VMAT plans were created to deliver 3 Gy to half the area of a T80 flask containing either DU-145 or AGO-1522 cells allowing intercellular communication between the in- and out-of-field cell populations. The same plans were delivered to a similar custom made phantom used to hold two T25 culture flasks, one flask in-field and one out-of-field to allow comparison of cell survival responses when intercellular communication is physically inhibited. Plans were created for the delivery of 8 Gy to the more radio-resistant DU-145 cells only in the presence and absence of intercellular communication. Cell survival was determined by clonogenic assay. In both cell lines, the out-of-field survival was not statistically different between delivery techniques for either cell line or dose. There was however, a statistically significant difference between survival out-of-field when intercellular communication was intact (single T80 culture flask) or inhibited (multiple T25 culture flasks) to in-field for all plans. No statistically significant difference was observed in-field with or without cellular communication to out-of-field for all plans. These data demonstrate out-of-field effects as important determinants of cell survival following exposure to modulated irradiation fields when cellular communication between differentially irradiated cell populations is present. This data is further evidence that refinement of existing radiobiological models to include indirect cell killing effects is required. (paper)

Molecular dynamics study of the solvation of an alpha-helical transmembrane peptide by DMSO

NARCIS (Netherlands)

Duarte, A.M.; Mierlo, van C.P.M.; Hemminga, M.A.

2008-01-01

10-ns molecular dynamics study of the solvation of a hydrophobic transmembrane helical peptide in dimethyl sulfoxide (DMSO) is presented. The objective is to analyze how this aprotic polar solvent is able to solvate three groups of amino acid residues (i.e., polar, apolar, and charged) that are

Molecular pharmacological phenotyping of EBI2. An orphan seven-transmembrane receptor with constitutive activity

DEFF Research Database (Denmark)

Rosenkilde, Mette M; Benned-Jensen, Tau; Holst, Peter J

2006-01-01

Epstein-Barr virus (EBV)-induced receptor 2 (EBI2) is an orphan seven-transmembrane (7TM) receptor originally identified as the most up-regulated gene (>200-fold) in EBV-infected cells. Here we show that EBI2 signals with constitutive activity through Galpha(i) as determined by a receptor...

The nectin-1α transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

International Nuclear Information System (INIS)

Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J.

2005-01-01

Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1α involved in cell fusion, we measured the ability of nectin-1α/nectin-2α chimeras, nectin-1α/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1α to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1α cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1α and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1α interaction in fusion

Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

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Adam Bajinting

2017-06-01

Full Text Available Fibroblast growth factor receptors (FGFRs are a family of receptor tyrosine kinases containing three domains: an extracellular receptor domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. FGFRs are activated by fibroblast growth factors (FGFs as part of complex signal transduction cascades regulating angiogenesis, skeletal formation, cell differentiation, proliferation, cell survival, and cancer. We have developed the first recombinant expression system in E. coli to produce a construct of human FGFR2 containing its transmembrane and extracellular receptor domains. We demonstrate that the expressed construct is functional in binding heparin and dimerizing. Size exclusion chromatography demonstrates that the purified FGFR2 does not form a complex with FGF1 or adopts an inactive dimer conformation. Progress towards the successful recombinant production of intact FGFRs will facilitate further biochemical experiments and structure determination that will provide insight into how extracellular FGF binding activates intracellular kinase activity.

Inhibition of luminescence and virulence in the black tiger prawn (Penaeus monodon) pathogen Vibrio harveyi by intercellular signal antagonists.

Science.gov (United States)

Manefield, M; Harris, L; Rice, S A; de Nys, R; Kjelleberg, S

2000-05-01

Expression of luminescence in the Penaeus monodon pathogen Vibrio harveyi is regulated by an intercellular quorum sensing mechanism involving the synthesis and detection of two signaling molecules, one of which is N-hydroxy butanoyl-L-homoserine lactone and the other of which is uncharacterized. Indirect evidence has suggested that virulence, associated with a toxic extracellular protein, and luminescence in V. harveyi are coregulated. In this study the effects of an acylated homoserine lactone antagonist produced by the marine alga Delisea pulchra on luminescence and toxin production in a virulent strain of V. harveyi were analyzed. Luminescence and toxin production were both inhibited by the signal antagonist at concentrations that had no impact on growth. Toxin production was found to be prematurely induced in V. harveyi cultures incubated in a 10% conditioned medium. Additionally, a significant reduction in the toxicity of concentrated supernatant extracts from V. harveyi cultures incubated in the presence of the signal antagonist, as measured by in vivo toxicity assays in mice and prawns, was observed. These results suggest that intercellular signaling antagonists have potential utility in the control of V. harveyi prawn infections.

Inhibition of Luminescence and Virulence in the Black Tiger Prawn (Penaeus monodon) Pathogen Vibrio harveyi by Intercellular Signal Antagonists

Science.gov (United States)

Manefield, Michael; Harris, Lachlan; Rice, Scott A.; de Nys, Rocky; Kjelleberg, Staffan

2000-01-01

Expression of luminescence in the Penaeus monodon pathogen Vibrio harveyi is regulated by an intercellular quorum sensing mechanism involving the synthesis and detection of two signaling molecules, one of which is N-hydroxy butanoyl-l-homoserine lactone and the other of which is uncharacterized. Indirect evidence has suggested that virulence, associated with a toxic extracellular protein, and luminescence in V. harveyi are coregulated. In this study the effects of an acylated homoserine lactone antagonist produced by the marine alga Delisea pulchra on luminescence and toxin production in a virulent strain of V. harveyi were analyzed. Luminescence and toxin production were both inhibited by the signal antagonist at concentrations that had no impact on growth. Toxin production was found to be prematurely induced in V. harveyi cultures incubated in a 10% conditioned medium. Additionally, a significant reduction in the toxicity of concentrated supernatant extracts from V. harveyi cultures incubated in the presence of the signal antagonist, as measured by in vivo toxicity assays in mice and prawns, was observed. These results suggest that intercellular signaling antagonists have potential utility in the control of V. harveyi prawn infections. PMID:10788385

Intercellular signaling pathways active during and after growth and differentiation of the lumbar vertebral growth plate.

Science.gov (United States)

Dahia, Chitra Lekha; Mahoney, Eric J; Durrani, Atiq A; Wylie, Christopher

2011-06-15

Vertebral growth plates at different postnatal ages were assessed for active intercellular signaling pathways. To generate a spatial and temporal map of the major signaling pathways active in the postnatal mouse lumbar vertebral growth plate. The growth of all long bones is known to occur by cartilaginous growth plates. The growth plate is composed of layers of chondrocyets that actively proliferate, differentiate, die and, are replaced by bone. The role of major cell signaling pathways has been suggested for regulation of the fetal long bones. But not much is known about the molecular or cellular signals that control the postnatal vertebral growth plate and hence postnatal vertebral bone growth. Understanding such molecular mechanisms will help design therapeutic treatments for vertebral growth disorders such as scoliosis. Antibodies against activated downstream intermediates were used to identify cells in the growth plate responding to BMP, TGFβ, and FGF in cryosections of lumbar vertebrae from different postnatal age mice to identify the zones that were responding to these signals. Reporter mice were used to identify the chondrocytes responding to hedgehog (Ihh), and Wnt signaling. We present a spatial/temporal map of these signaling pathways during growth, and differentiation of the mouse lumbar vertebral growth plate. During growth and differentiation of the vertebral growth plate, its different components respond at different times to different intercellular signaling ligands. Response to most of these signals is dramatically downregulated at the end of vertebral growth.

Intercellular adhesion molecule-1 blockade attenuates inflammatory response and improves microvascular perfusion in rat pancreas grafts.

Science.gov (United States)

Preissler, Gerhard; Eichhorn, Martin; Waldner, Helmut; Winter, Hauke; Kleespies, Axel; Massberg, Steffen

2012-10-01

After pancreas transplantation (PTx), early capillary malperfusion and leukocyte recruitment indicate the manifestation of severe ischemia/reperfusion injury (IRI). Oscillatory blood-flow redistribution (intermittent capillary perfusion, IP), leading to an overall decrease in erythrocyte flux, precedes complete microvascular perfusion failure with persistent blood flow cessation. We addressed the role of intercellular adhesion molecule-1 (ICAM-1) for leukocyte-endothelial interactions (LEIs) after PTx and evaluated the contribution of IP and malperfusion. Pancreas transplantation was performed in rats after 18-hour preservation, receiving either isotype-matched IgG or monoclonal anti-ICAM-1 antibodies (10 mg/kg intravenously) once before reperfusion. Leukocyte-endothelial interaction, IP, erythrocyte flux, and functional capillary density, respectively, were examined in vivo during 2-hour reperfusion. Nontransplanted animals served as controls. Tissue samples were analyzed by histomorphometry. In grafts of IgG-treated animals, IP was encountered already at an early stage after reperfusion and steadily increased over 2 hours, whereas erythrocyte flux declined continuously. In contrast, inhibition of ICAM-1 significantly improved erythrocyte flux and delayed IP appearance by 2 hours. Further, anti-ICAM-1 significantly reduced LEI and leukocyte tissue infiltration when compared to IgG; edema development was less pronounced in response to anti-ICAM-1 monoclonal antibody. Intercellular adhesion molecule-1 blockade significantly attenuates IRI via immediate reduction of LEI and concomitant improvement of capillary perfusion patterns, emphasizing its central role during IRI in PTx.

The transmembrane region is responsible for targeting of adaptor protein LAX into "heavy rafts''

Czech Academy of Sciences Publication Activity Database

Hrdinka, Matouš; Otáhal, Pavel; Hořejší, Václav

2012-01-01

Roč. 7, č. 5 (2012), e36330 E-ISSN 1932-6203 R&D Projects: GA ČR GEMEM/09/E011; GA MŠk 1M0506 Institutional research plan: CEZ:AV0Z50520514 Keywords : LAX * transmembrane domain * DRM Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.730, year: 2012

Artificial Diels–Alderase based on the transmembrane protein FhuA

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Hassan Osseili

2016-06-01

Full Text Available Copper(I and copper(II complexes were covalently linked to an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA ΔCVFtev. Copper(I was incorporated using an N-heterocyclic carbene (NHC ligand equipped with a maleimide group on the side arm at the imidazole nitrogen. Copper(II was attached by coordination to a terpyridyl ligand. The spacer length was varied in the back of the ligand framework. These biohybrid catalysts were shown to be active in the Diels–Alder reaction of a chalcone derivative with cyclopentadiene to preferentially give the endo product.

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

Science.gov (United States)

Alvares, Stacy M.; Dunn, Clarence A.; Brown, Tod A.; Wayner, Elizabeth E.; Carter, William G.

2008-01-01

Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and Detergent-Resistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals- possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior. PMID:18269919

Transmembrane adaptor proteins in the high-affinity IgE receptor signaling

Czech Academy of Sciences Publication Activity Database

Dráber, Petr; Hálová, Ivana; Levi-Schaffer, F.; Dráberová, Lubica

2012-01-01

Roč. 2, 11.1. (2012), s. 95 ISSN 1664-3224 R&D Projects: GA MŠk 1M0506; GA ČR GA301/09/1826; GA ČR GAP302/10/1759; GA AV ČR KAN200520701 Grant - others:AV ČR(CZ) M200520901 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : IgE receptor * LAT/LAT1 * LAX * NTAL/Lab/LAT2 * PAG/Cbp * mast cells * plasma membrane * transmembrane adaptor proteins Subject RIV: EB - Genetics ; Molecular Biology

The role of extracellular vesicles in phenotypic cancer transformation:

OpenAIRE

Kralj-Iglič, Veronika; Ogorevc, Eva; Veranič, Peter

2013-01-01

Background. Cancer has traditionally been considered as a disease resulting from gene mutations. New findings in biology are challenging gene-centered explanations of cancer progression and redirecting them to the non-genetic origins of tumorigenicity. It has become clear that intercellular communication plays a crucial role in cancer progression. Among the most intriguing ways of intercellular communication is that via extracellular vesicles (EVs). EVs are membrane structures released from v...

Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

OpenAIRE

Kuzma-Kuzniarska, Maria; Yapp, Clarence; Pearson-Jones, Thomas W.; Jones, Andrew K.; Hulley, Philippa A.

2014-01-01

Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows qu...

Transmembrane amyloid-related proteins in CSF as potential biomarkers for Alzheimer’s disease

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Inmaculada eLopez-Font

2015-06-01

Full Text Available In the continuing search for new cerebrospinal fluid (CSF biomarkers for Alzheimer’s disease (AD, reasonable candidates are the secretase enzymes involved in the processing of the amyloid precursor protein (APP, as well as the large proteolytic cleavage fragments sAPPα and sAPPβ. The enzymatic activities of some of these secretases, such as BACE1 and TACE, have been investigated as potential AD biomarkers, and it has been assumed that these activities present in human CSF result from the soluble truncated forms of the membrane-bound enzymes. However, we and others recently identified soluble forms of BACE1 and APP in CSF containing the intracellular domains, as well as the multi-pass transmembrane presenilin-1 (PS1 and other subunits of γ-secretase. We also review recent findings that suggest that most of these soluble transmembrane proteins could display self-association properties based on hydrophobic and/or ionic interactions leading to the formation of heteromeric complexes. The oligomerization state of these potential new biomarkers needs to be taken into consideration for assessing their real potential as CSF biomarkers for AD by adequate molecular tools.

Transmembrane signal transduction by peptide hormones via family B G protein-coupled receptors

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Kelly J Culhane

2015-11-01

Full Text Available Although family B G protein-coupled receptors (GPCRs contain only 15 members, they play key roles in transmembrane signal transduction of hormones. Family B GPCRs are drug targets for developing therapeutics for diseases ranging from metabolic to neurological disorders. Despite their importance, the molecular mechanism of activation of family B GPCRs remains largely unexplored due to the challenges in expression and purification of functional receptors to the quantity for biophysical characterization. Currently, there is no crystal structure available of a full-length family B GPCR. However, structures of key domains, including the extracellular ligand binding regions and seven-helical transmembrane regions, have been solved by X-ray crystallography and NMR, providing insights into the mechanisms of ligand recognition and selectivity, and helical arrangements within the cell membrane. Moreover, biophysical and biochemical methods have been used to explore functions, key residues for signaling, and the kinetics and dynamics of signaling processes. This review summarizes the current knowledge of the signal transduction mechanism of family B GPCRs at the molecular level and comments on the challenges and outlook for mechanistic studies of family B GPCRs.

Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation.

Science.gov (United States)

Liu, Han-Yuan; Chen, Wei-Liang; Ober, Christopher K; Daniel, Susan

2018-01-23

Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

Intrinsic potential of cell membranes: opposite effects of lipid transmembrane asymmetry and asymmetric salt ion distribution

DEFF Research Database (Denmark)

Gurtovenko, Andrey A; Vattulainen, Ilpo

2009-01-01

Using atomic-scale molecular dynamics simulations, we consider the intrinsic cell membrane potential that is found to originate from a subtle interplay between lipid transmembrane asymmetry and the asymmetric distribution of monovalent salt ions on the two sides of the cell membrane. It turns out......Cl saline solution and the PE leaflet is exposed to KCl, the outcome is that the effects of asymmetric lipid and salt ion distributions essentially cancel one another almost completely. Overall, our study highlights the complex nature of the intrinsic potential of cell membranes under physiological...... that both the asymmetric distribution of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids across a membrane and the asymmetric distribution of NaCl and KCl induce nonzero drops in the transmembrane potential. However, these potential drops are opposite in sign. As the PC leaflet faces a Na...

Integrative Modeling of Electrical Properties of Pacemaker Cardiac Cells

Science.gov (United States)

Grigoriev, M.; Babich, L.

2016-06-01

This work represents modeling of electrical properties of pacemaker (sinus) cardiac cells. Special attention is paid to electrical potential arising from transmembrane current of Na+, K+ and Ca2+ ions. This potential is calculated using the NaCaX model. In this respect, molar concentration of ions in the intercellular space which is calculated on the basis of the GENTEX model is essential. Combined use of two different models allows referring this approach to integrative modeling.

Improving cardiac gap junction communication as a new antiarrhythmic mechanism: the action of antiarrhythmic peptides.

Science.gov (United States)

Dhein, Stefan; Hagen, Anja; Jozwiak, Joanna; Dietze, Anna; Garbade, Jens; Barten, Markus; Kostelka, Martin; Mohr, Friedrich-Wilhelm

2010-03-01

Co-ordinated electrical activation of the heart is maintained by intercellular coupling of cardiomyocytes via gap junctional channels located in the intercalated disks. These channels consist of two hexameric hemichannels, docked to each other, provided by either of the adjacent cells. Thus, a complete gap junction channel is made from 12 protein subunits, the connexins. While 21 isoforms of connexins are presently known, cardiomyocytes typically are coupled by Cx43 (most abundant), Cx40 or Cx45. Some years ago, antiarrhythmic peptides were discovered and synthesised, which were shown to increase macroscopic gap junction conductance (electrical coupling) and enhance dye transfer (metabolic coupling). The lead substance of these peptides is AAP10 (H-Gly-Ala-Gly-Hyp-Pro-Tyr-CONH(2)), a peptide with a horseshoe-like spatial structure as became evident from two-dimensional nuclear magnetic resonance studies. A stable D: -amino-acid derivative of AAP10, rotigaptide, as well as a non-peptide analogue, gap-134, has been developed in recent years. Antiarrhythmic peptides act on Cx43 and Cx45 gap junctions but not on Cx40 channels. AAP10 has been shown to enhance intercellular communication in rat, rabbit and human cardiomyocytes. Antiarrhythmic peptides are effective against ventricular tachyarrhythmias, such as late ischaemic (type IB) ventricular fibrillation, CaCl(2) or aconitine-induced arrhythmia. Interestingly, the effect of antiarrhythmic peptides is higher in partially uncoupled cells and was shown to be related to maintained Cx43 phosphorylation, while arrhythmogenic conditions like ischaemia result in Cx43 dephosphorylation and intercellular decoupling. It is still a matter of debate whether these drugs also act against atrial fibrillation. The present review outlines the development of this group of peptides and derivatives, their mode of action and molecular mechanisms, and discusses their possible therapeutic potential.

[The Functional Role of Exosomes in Cancer Biology and Their Potential as Biomarkers and Therapeutic Targets of Cancer].

Science.gov (United States)

Naito, Yutaka; Yoshioka, Yusuke; Ochiya, Takahiro

2015-06-01

Intercellular communication plays an important role in the regulation of various cellular events. In particular, cancer cells and the surrounding cells communicate with each other, and this intercellular communication triggers cancer initiation and progression through the secretion of molecules, including growth factors and cytokines. Recent advances in cancer biology have indicated that small membrane vesicles, termed exosomes, also serve as regulatory agents in intercellular communications. Exosomes contain functional cellular components, including proteins and microRNAs (miRNAs), and they transfer these components to recipient cells. This exosome-mediated intercellular communication leads to increased growth, invasion, and metastasis of cancer. Thus, researchers regard exosomes as important cues to understanding the molecular mechanisms of cancer biology. Indeed, several lines of evidence have demonstrated that exosomes can explain multiple aspects of cancer biology. In addition, increasing evidence suggests that exosomes and their specific molecules are also attractive for use as biomarkers and therapeutic targets in cancer. Recent reports showed the efficacy of a novel diagnosis by detecting component molecules of cancer-derived exosomes, including miRNAs and membrane proteins. Furthermore, clinical trials that test the application of exosomes for cancer therapy have already been reported. From these points of view, we will summarize experimental data that support the role of exosomes in cancer progression and the potential of exosomes for use in novel diagnostic and therapeutic approaches for cancer.

Analysis of Light-Induced Transmembrane Ion Gradients and Membrane Potential in Photosystem I Proteoliposomes

International Nuclear Information System (INIS)

Pennisi, Cristian P.; Greenbaum, Elias; Yoshida, Ken

2010-01-01

Photosystem I (PSI) complexes can support a light-driven electrochemical gradient for protons, which is the driving force for energy-conserving reactions across biological membranes. In this work, a computational model that enables a quantitative description of the light-induced proton gradients across the membrane of PSI proteoliposomes is presented. Using a set of electrodiffusion equations, a compartmental model of a vesicle suspended in aqueous medium was studied. The light-mediated proton movement was modeled as a single proton pumping step with backpressure of the electric potential. The model fits determinations of pH obtained from PSI proteoliposomes illuminated in the presence of mediators of cyclic electron transport. The model also allows analysis of the proton gradients in relation to the transmembrane ion fluxes and electric potential. Sensitivity analysis enabled a determination of the parameters that have greater influence on steady-state levels and onset/decay rates of transmembrane pH and electric potential. This model could be used as a tool for optimizing PSI proteoliposomes for photo-electrochemical applications.

Assessing the utility of photoswitchable fluorescent proteins for tracking intercellular protein movement in the Arabidopsis root.

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Shuang Wu

Full Text Available One way in which cells communicate is through the direct transfer of proteins. In plants, many of these proteins are transcription factors, which are made by one cell type and traffic into another. In order to understand how this movement occurs and its role in development, we would like to track this movement in live, intact plants in real-time. Here we examine the utility of the photoconvertible proteins, Dendra2 and (to a lesser extent EosFP as tags for studying intracellular and intercellular protein movement in the Arabidopsis root. To this end, we made fusions between Dendra2 and six mobile transcription factors. Our results show that Dendra2 is an effective tool for studying protein movement between plant cells. Interestingly, we found that Dendra2 could not simply be swapped into existing constructs that had originally contained GFP. Most of the fusions made in this way failed to produce a fluorescent fusion. In addition we found that the optimal settings for photoconversion of Dendra2 in stably transformed roots were different from what has been published for photoconversion in transient assays in plants or in animal cells. By modifying the confocal setting, we were able to photoconvert Dendra2 in all cell layers in the root. However the efficiency of photoconversion was affected by the position of the cell layer within the root, with more internal tissues requiring more energy. By examining the Dendra2 fusions, we confirmed the mobility of the SHORT-ROOT (SHR and CAPRICE (CPC transcription factors between cells and we further discovered that SHR movement in stele and CPC movement in the epidermis are non-directional.

Soluble intercellular adhesion molecule 1 and flow-mediated dilatation are related to the estimated risk of coronary heart disease independently from each other

NARCIS (Netherlands)

Witte, D.R.; Broekmans, W.M.R.; Kardinaal, A.F.M.; Klöpping-Ketelaars, I.A.A.; Poppel, G. van; Bots, M.L.; Kluft, C.; Princen, J.M.G.

2003-01-01

Background: Flow mediated dilatation (FMD) of the brachial artery and soluble intercellular adhesion molecule 1 (sICAM-1) are measures of distinct functions of the endothelium, reflecting nitric oxide (NO)-mediated and pro-inflammatory status, respectively. The comparative value of the two measures

CD44: molecular interactions, signalling and functions in the nervous system.

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Grzegorz Marek Wilczynski

2015-05-01

Full Text Available CD44 is the major surface hyaluronan receptor implicated in intercellular and cell-matrix adhesion, cell migration and signalling. It is a transmembrane, highly glycosylated protein with several isoforms resulting from alternative gene splicing. The CD44 molecule consists of several domains serving different functions: the N-terminal extracellular domain, the stem region, the transmembrane domain and the C-terminal tail. In the nervous system, CD44 expression occurs in both glial and neuronal cells. The role of CD44 in the physiology and pathology of the nervous system is not entirely understood, however, there exists evidence suggesting it might be involved in the axon guidance, cytoplasmic Ca2+ clearance, dendritic arborization, synaptic transmission, epileptogenesis, oligodendrocyte and astrocyte differentiation, post-traumatic brain repair and brain tumour development.

Coordinated movement of cytoplasmic and transmembrane domains of RyR1 upon gating.

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Montserrat Samsó

2009-04-01

Full Text Available Ryanodine receptor type 1 (RyR1 produces spatially and temporally defined Ca2+ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how the channel gates are unknown. We used EGTA or neuroactive PCB 95 to stabilize the full closed or open states of RyR1. Single-channel measurements in the presence of FKBP12 indicate that PCB 95 inverts the thermodynamic stability of RyR1 and locks it in a long-lived open state whose unitary current is indistinguishable from the native open state. We analyzed two datasets of 15,625 and 18,527 frozen-hydrated RyR1-FKBP12 particles in the closed and open conformations, respectively, by cryo-electron microscopy. Their corresponding three-dimensional structures at 10.2 A resolution refine the structure surrounding the ion pathway previously identified in the closed conformation: two right-handed bundles emerging from the putative ion gate (the cytoplasmic "inner branches" and the transmembrane "inner helices". Furthermore, six of the identifiable transmembrane segments of RyR1 have similar organization to those of the mammalian Kv1.2 potassium channel. Upon gating, the distal cytoplasmic domains move towards the transmembrane domain while the central cytoplasmic domains move away from it, and also away from the 4-fold axis. Along the ion pathway, precise relocation of the inner helices and inner branches results in an approximately 4 A diameter increase of the ion gate. Whereas the inner helices of the K+ channels and of the RyR1 channel cross-correlate best with their corresponding open/closed states, the cytoplasmic inner branches, which are not observed in the K+ channels, appear to have at least as important a role as the inner helices for RyR1 gating. We propose a theoretical model whereby the inner helices, the inner branches, and the h1 densities together create an efficient novel gating mechanism for channel opening by relaxing two right

Intact transmembrane isoforms of the neural cell adhesion molecule are released from the plasma membrane

DEFF Research Database (Denmark)

Olsen, M; Krog, L; Edvardsen, K

1993-01-01

. By density-gradient centrifugation it was shown that shed transmembrane NCAM-B was present in fractions of high, as well as low, density, indicating that a fraction of the shed NCAM is associated with minor plasma membrane fragments. Finally, it was shown that isolated soluble NCAM inhibited cell binding...

Different mechanisms of modulation of gap junction communication by non-genotoxic carcinogens in rat liver in vivo

International Nuclear Information System (INIS)

Cowles, C.; Mally, A.; Chipman, J.K.

2007-01-01

This is a comparative study of the mechanisms by which three different rodent non-genotoxic carcinogens modulate connexin-mediated gap junction intercellular communication in male rat liver in vivo. In the case of the peroxisome proliferating agent Wy-14,643, a non-hepatotoxic dose of 50 mg/kg led to a marked loss of inter-hepatocyte dye transfer associated with a loss of both Cx32 and Cx26 protein expression. In contrast, p,p'-dichlorodiphenyltrichloroethane (DDT) at a non-hepatotoxic dose (25 mg/kg) was not found to alter Cx32 or Cx26 expression or to produce a measurable Cx32 serine phosphorylation but did give a small, significant reduction of cell communication. Carbon tetrachloride (CCl 4 ) did not affect cell communication (despite a small significant reduction of Cx32 content) at a non-hepatotoxic dose. Both loss of communication and Cx32 expression was observed only at a dose that caused hepatocyte toxicity as evidenced by increased serum alanine aminotransferase activity. Overall, the findings emphasise that loss of gap junctional communication in vivo can contribute to carcinogenesis by non-genotoxic carcinogens through different primary mechanism. In contrast to Wy-14,643 and DDT, the results with CCl 4 are consistent with a requirement for hepatotoxicity in its carcinogenic action

Simulations of Skin Barrier Function: Free Energies of Hydrophobic and Hydrophilic Transmembrane Pores in Ceramide Bilayers

NARCIS (Netherlands)

Notman, Rebecca; Anwar, Jamshed; Briels, Willem J.; Noro, Massimo G.; den Otter, Wouter K.

2008-01-01

Transmembrane pore formation is central to many biological processes such as ion transport, cell fusion, and viral infection. Furthermore, pore formation in the ceramide bilayers of the stratum corneum may be an important mechanism by which penetration enhancers such as dimethylsulfoxide (DMSO)

Influence of Ionizing Radiation on Stromal-Epithelial Intercellular Communication in Esophageal Carcinogenesis

Science.gov (United States)

Patel, Zarana S.; Kalabis, Jiri; Rustgi, Anil K.; Cucinotta, Francis A.; Huff, Janice L.

2010-01-01

Esophageal cancer is the 6th leading cause of cancer death worldwide. Its development is associated with a variety of risk factors including tobacco use, heavy alcohol consumption, human papilloma virus infection, and certain dietary factors such as trace mineral and vitamin deficiencies. An association with ionizing radiation exposure is revealed by the high excess relative risk for squamous cell carcinoma of the esophagus observed in the survivors of the atomic bomb detonations in Japan. It is also seen as a secondary malignancy in patients who received radiotherapy for breast and thoracic cancers; additionally, patients with head/neck and oral squamous cell cancers are at increased risk for metachronous esophageal squamous cell cancers. This malignancy is rapidly fatal, mainly because it remains asymptomatic until late, advanced stages when the disease is rarely curable. The stromal microenvironment plays an essential role in the maintenance and modulation of normal epithelial cell growth and differentiation and cross talk between the epithelial and stromal compartments can influence many aspects of malignant progression, including tumor cell proliferation, migration, invasion and recruitment of new blood vessels. To test the hypothesis that radiation exposure plays a role in esophageal carcinogenesis via non-targeted mechanisms involving stromal-epithelial cell communication, we are studying radiation effects on hTERT-immortalized human esophageal epithelial cells and genetic variants grown in co-culture with human esophageal stromal fibroblasts (Okawa et al., Genes & Dev. 2007. 21: 2788-2803). We examined how radiation treatment of stromal fibroblasts affected epithelial migration and invasion, behaviors associated with cancer promotion and progression. Chemotactic and haptotactic migration of epithelial cells stimulated by conditioned media from irradiated fibroblasts was measured using assays conducted in Transwell cell culture chambers. Our results using

Effect of ionizing radiation on transmembrane potential of Streptococcus

International Nuclear Information System (INIS)

Fomenko, B.S.; Akoev, I.G.

1979-01-01

Treatment of Streptococcus faecalis with ionizing radiation at doses of 5 to 100 krad is shown to reduce the energy-dependent accumulation of dibenzyldimethylammonium (DDA + ) by the cell. Since transmembrane potential is the moving force of DDA + transport across the membrane, the decrease in DDA + accumulation is suggested to be due to potential reduction. This radiation effect was not due to inactivation of the potential-generating mechanism; thus, the ATPase activity and glycolytic activity of the irradiated cells were higher than in the control. At the same time, the membranes exhibited an increased permeability for K + and protons, which is probably due to structural rearrangements in the membranes after irradiation. It is suggested that the potential reduction results from the increase in proton permeability of membranes

Indomethacin induced gastropathy in CD18, intercellular adhesion molecule 1, or P-selectin deficient mice

Science.gov (United States)

Morise, Z; Granger, D; Fuseler, J; Anderson, D; Grisham, M

1999-01-01

BACKGROUND—Neutrophil-endothelial cell interactions are thought to play a critical role in the pathophysiology of non-steroidal anti-inflammatory drug (NSAID) induced gastropathy.
AIMS—To optimise a mouse model of NSAID induced gastropathy and to evaluate the importance of adhesion molecules using adhesion molecule deficient mice.
METHODS—Gastropathy was induced in C57BL/6 mice or their adhesion molecule deficient counterparts via oral administration of indomethacin (20 mg/kg). Lesion scores, mucosal permeability, and histopathology were used to assess gastric mucosal injury.
RESULTS—Intragastric administration of indomethacin induced linear haemorrhagic mucosal lesions, primarily in the corpus of the stomach that were first observed at six hours. These lesions continued to develop over the next six hours with maximal lesion scores and mucosal permeabilities at 12 hours. When indomethacin was administered to mice deficient in CD18, intercellular adhesion molecule 1 (ICAM-1), or P-selectin, there were significant decreases in lesion scores compared with their C57BL/6 controls. In addition, mucosal permeabilities were found to be significantly lower in CD18 or ICAM-1 deficient mice observed at 12 hours.
CONCLUSION—Certain leucocyte and endothelial cell adhesion molecules are important determinants for full expression of indomethacin induced gastropathy. It is proposed that this modification of the mouse model may be useful for the investigation of other pathophysiological mechanisms of NSAID induced gastropathy.


Keywords: indomethacin; gastropathy; cyclooxygenase; intercellular adhesion molecule; VCAM; vascular cell adhesion molecule; P-selectin PMID:10486359

Intercellular adhesion molecules (ICAMs) and spermatogenesis

Science.gov (United States)

Xiao, Xiang; Mruk, Dolores D.; Cheng, C. Yan

2013-01-01

BACKGROUND During the seminiferous epithelial cycle, restructuring takes places at the Sertoli–Sertoli and Sertoli–germ cell interface to accommodate spermatogonia/spermatogonial stem cell renewal via mitosis, cell cycle progression and meiosis, spermiogenesis and spermiation since developing germ cells, in particular spermatids, move ‘up and down’ the seminiferous epithelium. Furthermore, preleptotene spermatocytes differentiated from type B spermatogonia residing at the basal compartment must traverse the blood–testis barrier (BTB) to enter the adluminal compartment to prepare for meiosis at Stage VIII of the epithelial cycle, a process also accompanied by the release of sperm at spermiation. These cellular events that take place at the opposite ends of the epithelium are co-ordinated by a functional axis designated the apical ectoplasmic specialization (ES)—BTB—basement membrane. However, the regulatory molecules that co-ordinate cellular events in this axis are not known. METHODS Literature was searched at http://www.pubmed.org and http://scholar.google.com to identify published findings regarding intercellular adhesion molecules (ICAMs) and the regulation of this axis. RESULTS Members of the ICAM family, namely ICAM-1 and ICAM-2, and the biologically active soluble ICAM-1 (sICAM-1) are the likely regulatory molecules that co-ordinate these events. sICAM-1 and ICAM-1 have antagonistic effects on the Sertoli cell tight junction-permeability barrier, involved in Sertoli cell BTB restructuring, whereas ICAM-2 is restricted to the apical ES, regulating spermatid adhesion during the epithelial cycle. Studies in other epithelia/endothelia on the role of the ICAM family in regulating cell movement are discussed and this information has been evaluated and integrated into studies of these proteins in the testis to create a hypothetical model, depicting how ICAMs regulate junction restructuring events during spermatogenesis. CONCLUSIONS ICAMs are crucial

Rescue of Notch signaling in cells incapable of GDP-L-fucose synthesis by gap junction transfer of GDP-L-fucose in Drosophila.

Science.gov (United States)

Ayukawa, Tomonori; Matsumoto, Kenjiroo; Ishikawa, Hiroyuki O; Ishio, Akira; Yamakawa, Tomoko; Aoyama, Naoki; Suzuki, Takuya; Matsuno, Kenji

2012-09-18

Notch (N) is a transmembrane receptor that mediates cell-cell interactions to determine many cell-fate decisions. N contains EGF-like repeats, many of which have an O-fucose glycan modification that regulates N-ligand binding. This modification requires GDP-L-fucose as a donor of fucose. The GDP-L-fucose biosynthetic pathways are well understood, including the de novo pathway, which depends on GDP-mannose 4,6 dehydratase (Gmd) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (Gmer). However, the potential for intercellularly supplied GDP-L-fucose and the molecular basis of such transportation have not been explored in depth. To address these points, we studied the genetic effects of mutating Gmd and Gmer on fucose modifications in Drosophila. We found that these mutants functioned cell-nonautonomously, and that GDP-L-fucose was supplied intercellularly through gap junctions composed of Innexin-2. GDP-L-fucose was not supplied through body fluids from different isolated organs, indicating that the intercellular distribution of GDP-L-fucose is restricted within a given organ. Moreover, the gap junction-mediated supply of GDP-L-fucose was sufficient to support the fucosylation of N-glycans and the O-fucosylation of the N EGF-like repeats. Our results indicate that intercellular delivery is a metabolic pathway for nucleotide sugars in live animals under certain circumstances.

What Can We Learn about Cholesterol's Transmembrane Distribution Based on Cholesterol-Induced Changes in Membrane Dipole Potential?

Czech Academy of Sciences Publication Activity Database

Falkovich, S. G.; Martinez-Seara, Hector; Nesterenko, A. M.; Vattulainen, I.; Gurtovenko, A. A.

2016-01-01

Roč. 7, č. 22 (2016), s. 4585-4590 ISSN 1948-7185 Institutional support: RVO:61388963 Keywords : membrane * cholesterol * membrane asymmetry * membrane dipole potential * transmembrane distribution Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 9.353, year: 2016

Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: Application to the nicotinic acetylcholine receptor

International Nuclear Information System (INIS)

Perez-Ramirez, B.; Martinez-Carrion, M.

1989-01-01

A novel procedure has been developed to specifically label the cytoplasmic domains of transmembrane proteins with the aldehyde pyridoxal 5-phosphate (PLP). Torpedo californica acetylcholine receptor (AcChR) vesicles were loaded with [ 3 H]pyridoxine 5-phosphate ([ 3 H]PNP) and pyridoxine-5-phosphate oxidase, followed by intravesicular enzymatic oxidation of [ 3 H]PNP at 37 degree C in the presence of externally added cytochrome c as a scavenger of possible leaking PLP product. The four receptor subunits were labeled whether the reaction was carried out on the internal surface or separately designed to mark the external one. On the other hand, the relative pyridoxylation of the subunits differed in both cases, reflecting differences in accessible lysyl residues in each side of the membrane. Even though there are no large differences in the total lysine content among the subunits and there are two copies of the α-subunit, internal surface labeling by PLP was greatest for the highest molecular weight (δ) subunit, reinforcing the concept that the four receptor subunits are transmembranous and may protrude into the cytoplasmic face in a fashion that is proportional to their subunit molecular weight. Yet, the labeling data do not fit well to any of the models proposed for AcChR subunit folding. The method described can be used for selective labeling of the cytoplasmic domains of transmembrane proteins in sealed membrane vesicles

Nonpolar interactions between trans-membrane helical EGF peptide and phosphatidylcholines, sphingomyelins and cholesterol. Molecular dynamics simulation studies

NARCIS (Netherlands)

Róg, T.; Murzyn, K.; Karttunen, M.E.J.; Pasenkiewicz-Gierula, M.

2008-01-01

A molecular dynamics simulation study of four lipid bilayers with inserted trans-membrane helical fragment of epithelial growth factor (EGF) receptor (EGF peptide) was performed. The lipid bilayers differ in their lipid composition and consist of (i) unsaturated phosphatidylcholine

Low rate doses effects of gamma radiation on glycoproteins of transmembrane junctions in fibroblasts

International Nuclear Information System (INIS)

Bringas, J.E.; Caceres, J.L.

1996-01-01

Glycoproteins of trans-membrane junctions are molecules that help to bind cells with the extracellular matrix. Integrins are the most important trans-membrane molecules among others. The damage of gamma radiation on those proteins could be an important early event that causes membrane abnormalities which may lead to cell malfunction and cancer induced by radiation due to cell dissociation. Randomized blocks with 3 repetitions of mouse embryo fibroblast cultures, were irradiated with Cobalt-60 gamma rays, during 20 days. Biological damage to glycoproteins and integrins was evaluated by cellular growth and fibroblast proliferative capacity. Integrins damage was studied by isolation by column immunoaffinity chromatography migrated on SDS-Page under reducing and non reducing conditions, and inhibition of integrins extracellular matrix adhesion by monoclonal antibodies effect. The dose/rate (0.05 Gy/day-0.2 Gy/day) of gamma given to cells did not show damage evidence on glycoproteins and integrins. If damage happened, it was repaired by cells very soon, was delayed by continuous cellular division or by glycoproteins characteristic of being multiple extracellular ligatures. Bio effects became more evident with an irradiation time greater than 20 days or a high dose/rate. (authors). 6 refs

Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes

Science.gov (United States)

Sikder, Md. Kabir Uddin; Stone, Kyle A.; Kumar, P. B. Sunil; Laradji, Mohamed

2014-01-01

We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells. PMID:25106608

Role of ATP binding and hydrolysis in the gating of the cystic fibrosis transmembrane conductance regulator

Directory of Open Access Journals (Sweden)

Taras Gout

2012-01-01

Full Text Available The CFTR gene is unique within the ATP-binding cassette (ABC protein family, predominantly of transporters, by coding a chloride channel. The gating mechanism of ABC proteins has been characterized by the ATP Switch model in terms cycles of dimer formation and dissociation linked to ATP binding and hydrolysis, respectively. It would be of interest to assess the extent that Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, a functional channel, fits the ATP Switch model for ABC transporters. Additional transporter mechanisms, namely those of Pgp and HlyB, are discussed for perspective. Literature search of databases selected key references in comparing and contrasting the gating mechanism. CFTR is a functional chloride channel facilitating transmembrane anion flow down electrochemical gradients. A dysfunctional CFTR protein results in cystic fibrosis, a fatal pleiotropic disease currently managed symptomatically. Understanding the gating mechanism will help target drug development aimed at alleviating and curing the disease.

Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

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Zhenyong Wu

2017-10-01

Full Text Available Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs. Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores

Herpes simplex virus glycoprotein D relocates nectin-1 from intercellular contacts.

Science.gov (United States)

Bhargava, Arjun K; Rothlauf, Paul W; Krummenacher, Claude

2016-12-01

Herpes simplex virus (HSV) uses the cell adhesion molecule nectin-1 as a receptor to enter neurons and epithelial cells. The viral glycoprotein D (gD) is used as a non-canonical ligand for nectin-1. The gD binding site on nectin-1 overlaps with a functional adhesive site involved in nectin-nectin homophilic trans-interaction. Consequently, when nectin-1 is engaged with a cellular ligand at cell junctions, the gD binding site is occupied. Here we report that HSV gD is able to disrupt intercellular homophilic trans-interaction of nectin-1 and induce a rapid redistribution of nectin-1 from cell junctions. This movement does not require the receptor's interaction with the actin-binding adaptor afadin. Interaction of nectin-1 with afadin is also dispensable for virion surfing along nectin-1-rich filopodia. Cells seeded on gD-coated surfaces also fail to accumulate nectin-1 at cell contact. These data indicate that HSV gD affects nectin-1 locally through direct interaction and more globally through signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

Ischemic preconditioning protects against gap junctional uncoupling in cardiac myofibroblasts.

Science.gov (United States)

Sundset, Rune; Cooper, Marie; Mikalsen, Svein-Ole; Ytrehus, Kirsti

2004-01-01

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The purpose of this study was to investigate the role of gap junction communication in simulated preconditioning in cultured neonatal rat cardiac myofibroblasts. Gap junctional intercellular communication was assessed by Lucifer yellow dye transfer. Preconditioning preserved intercellular coupling after prolonged ischemia. An initial reduction in coupling in response to the preconditioning stimulus was also observed. This may protect neighboring cells from damaging substances produced during subsequent regional ischemia in vivo, and may preserve gap junctional communication required for enhanced functional recovery during subsequent reperfusion.

Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.

Science.gov (United States)

El Hiani, Yassine; Linsdell, Paul

2015-06-19

As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Gap Junction Intercellular Communication in Bone Marrow Failure

Science.gov (United States)

2012-10-01

traits are responsible for the differences in the severity of these diseases and their relation to skeletal malformations in BMF syndromes. Using a...mesenchymal transition (19-29), and are crucial in the establishment of electrical synapses in the central nervous system, heart , immune system and...developmental defective osteogenesis resulting in craniofacial and skeletal anomalies and associated with heart defects and neurological defects. ODDD

Na,K-ATPase regulates intercellular communication in the vascular wall via cSrc kinase dependent connexin43 phosphorylation

DEFF Research Database (Denmark)

Hangaard, Lise; Bouzinova, Elena; Stæhr, Christian Albeck

2017-01-01

Communication between vascular smooth muscle cells (VSMCs) is dependent on gap junctions and is regulated by the Na-K-ATPase. The Na-K-ATPase is therefore important for synchronized VSMC oscillatory activity, i.e., vasomotion. The signaling between the Na-K-ATPase and gap junctions is unknown. We...... coupling in rat mesenteric small arteries in vitro. Phosphorylation of cSrc kinase and connexin43 (Cx43) were semiquantified by Western blotting. Micromole concentration of ouabain reduced the amplitude of norepinephrine-induced vasomotion and desynchronized Ca2+ transients in VSMC in the arterial wall...

The Single Transmembrane Segment of Minimal Sensor DesK Senses Temperature via a Membrane-Thickness Caliper.

Science.gov (United States)

Inda, Maria E; Oliveira, Rafael G; de Mendoza, Diego; Cybulski, Larisa E

2016-11-01

Thermosensors detect temperature changes and trigger cellular responses crucial for survival at different temperatures. The thermosensor DesK is a transmembrane (TM) histidine kinase which detects a decrease in temperature through its TM segments (TMS). Here, we address a key issue: how a physical stimulus such as temperature can be converted into a cellular response. We show that the thickness of Bacillus lipid membranes varies with temperature and that such variations can be detected by DesK with great precision. On the basis of genetic studies and measurements of in vitro activity of a DesK construct with a single TMS (minimal sensor DesK [MS-DesK]), reconstituted in liposomes, we propose an interplay mechanism directed by a conserved dyad, phenylalanine 8-lysine 10. This dyad is critical to anchor the only transmembrane segment of the MS-DesK construct to the extracellular water-lipid interphase and is required for the transmembrane segment of MS-DesK to function as a caliper for precise measurement of membrane thickness. The data suggest that positively charged lysine 10, which is located in the hydrophobic core of the membrane but is close to the water-lipid interface, pulls the transmembrane region toward the water phase to localize its charge at the interface. Nevertheless, the hydrophobic residue phenylalanine 8, located at the N-terminal extreme of the TMS, has a strong tendency to remain in the lipid phase, impairing access of lysine 10 to the water phase. The outcome of this interplay is a fine-tuned sensitivity to membrane thickness that elicits conformational changes that favor different signaling states of the protein. The ability to sense and respond to extracellular signals is essential for cell survival. One example is the cellular response to temperature variation. How do cells "sense" temperature changes? It has been proposed that the bacterial thermosensor DesK acts as a molecular caliper measuring membrane thickness variations that would occur

A portable lipid bilayer system for environmental sensing with a transmembrane protein.

Directory of Open Access Journals (Sweden)

Ryuji Kawano

Full Text Available This paper describes a portable measurement system for current signals of an ion channel that is composed of a planar lipid bilayer. A stable and reproducible lipid bilayer is formed in outdoor environments by using a droplet contact method with a micropipette. Using this system, we demonstrated that the single-channel recording of a transmembrane protein (alpha-hemolysin was achieved in the field at a high-altitude (∼3623 m. This system would be broadly applicable for obtaining environmental measurements using membrane proteins as a highly sensitive sensor.

Structure, function and physiological consequences of virally encoded chemokine seven transmembrane receptors

DEFF Research Database (Denmark)

Rosenkilde, M M; Smit, M J; Waldhoer, M

2008-01-01

A number of human and animal herpes viruses encode G-protein coupled receptors with seven transmembrane (7TM) segments-most of which are clearly related to human chemokine receptors. It appears, that these receptors are used by the virus for immune evasion, cellular transformation, tissue targeting...... pathogenesis is still poorly understood. Here we focus on the current knowledge of structure, function and trafficking patterns of virally encoded chemokine receptors and further address the putative roles of these receptors in virus survival and host -cell and/or -immune system modulation. Finally, we...

The viral transmembrane superfamily: possible divergence of Arenavirus and Filovirus glycoproteins from a common RNA virus ancestor

Directory of Open Access Journals (Sweden)

Buchmeier Michael J

2001-02-01

Full Text Available Abstract Background Recent studies of viral entry proteins from influenza, measles, human immunodeficiency virus, type 1 (HIV-1, and Ebola virus have shown, first with molecular modeling, and then X-ray crystallographic or other biophysical studies, that these disparate viruses share a coiled-coil type of entry protein. Results Structural models of the transmembrane glycoproteins (GP-2 of the Arenaviruses, lymphochoriomeningitis virus (LCMV and Lassa fever virus, are presented, based on consistent structural propensities despite variation in the amino acid sequence. The principal features of the model, a hydrophobic amino terminus, and two antiparallel helices separated by a glycosylated, antigenic apex, are common to a number of otherwise disparate families of enveloped RNA viruses. Within the first amphipathic helix, demonstrable by circular dichroism of a peptide fragment, there is a highly conserved heptad repeat pattern proposed to mediate multimerization by coiled-coil interactions. The amino terminal 18 amino acids are 28% identical and 50% highly similar to the corresponding region of Ebola, a member of the Filovirus family. Within the second, charged helix just prior to membrane insertion there is also high similarity over the central 18 amino acids in corresponding regions of Lassa and Ebola, which may be further related to the similar region of HIV-1 defining a potent antiviral peptide analogue. Conclusions These findings indicate a common pattern of structure and function among viral transmembrane fusion proteins from a number of virus families. Such a pattern may define a viral transmembrane superfamily that evolved from a common precursor eons ago.

Cellular communication through light.

Directory of Open Access Journals (Sweden)

Daniel Fels

Full Text Available Information transfer is a fundamental of life. A few studies have reported that cells use photons (from an endogenous source as information carriers. This study finds that cells can have an influence on other cells even when separated with a glass barrier, thereby disabling molecule diffusion through the cell-containing medium. As there is still very little known about the potential of photons for intercellular communication this study is designed to test for non-molecule-based triggering of two fundamental properties of life: cell division and energy uptake. The study was performed with a cellular organism, the ciliate Paramecium caudatum. Mutual exposure of cell populations occurred under conditions of darkness and separation with cuvettes (vials allowing photon but not molecule transfer. The cell populations were separated either with glass allowing photon transmission from 340 nm to longer waves, or quartz being transmittable from 150 nm, i.e. from UV-light to longer waves. Even through glass, the cells affected cell division and energy uptake in neighboring cell populations. Depending on the cuvette material and the number of cells involved, these effects were positive or negative. Also, while paired populations with lower growth rates grew uncorrelated, growth of the better growing populations was correlated. As there were significant differences when separating the populations with glass or quartz, it is suggested that the cell populations use two (or more frequencies for cellular information transfer, which influences at least energy uptake, cell division rate and growth correlation. Altogether the study strongly supports a cellular communication system, which is different from a molecule-receptor-based system and hints that photon-triggering is a fine tuning principle in cell chemistry.

The Role of Water Distribution Controlled by Transmembrane Potentials in the Cytochrome c-Cardiolipin Interaction: Revealing from Surface-Enhanced Infrared Absorption Spectroscopy.

Science.gov (United States)

Zeng, Li; Wu, Lie; Liu, Li; Jiang, Xiue

2017-11-02

The interaction of cytochrome c (cyt c) with cardiolipin (CL) plays a crucial role in apoptotic functions, however, the changes of the transmembrane potential in governing the protein behavior at the membrane-water interface have not been studied due to the difficulties in simultaneously monitoring the interaction and regulating the electric field. Herein, surface-enhanced infrared absorption (SEIRA) spectroelectrochemistry is employed to study the mechanism of how the transmembrane potentials control the interaction of cyt c with CL membranes by regulating the electrode potentials of an Au film. When the transmembrane potential decreases, the water content at the interface of the membranes can be increased to slow down protein adsorption through decreasing the hydrogen-bond and hydrophobic interactions, but regulates the redox behavior of CL-bound cyt c through a possible water-facilitated proton-coupled electron transfer process. Our results suggest that the potential drop-induced restructure of the CL conformation and the hydration state could modify the structure and function of CL-bound cyt c on the lipid membrane. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

What Can We Learn about Cholesterol's Transmembrane Distribution Based on Cholesterol-Induced Changes in Membrane Dipole Potential?

DEFF Research Database (Denmark)

Falkovich, Stanislav G.; Martinez-Seara, Hector; Nesterenko, Alexey M.

2016-01-01

Cholesterol is abundant in the plasma membranes of animal cells and is known to regulate a variety of membrane properties. Despite decades of research, the transmembrane distribution of cholesterol is still a matter of debate. Here we consider this outstanding issue through atomistic simulations ...

Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins.

Energy Technology Data Exchange (ETDEWEB)

Davis, Ryan W. (University of New Mexico, Albuquerque, NM); Brozik, James A. (University of New Mexico, Albuquerque, NM); Brozik, Susan Marie; Cox, Jason M. (University of New Mexico, Albuquerque, NM); Lopez, Gabriel P. (University of New Mexico, Albuquerque, NM); Barrick, Todd A. (University of New Mexico, Albuquerque, NM); Flores, Adrean (University of New Mexico, Albuquerque, NM)

2007-03-01

The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.

Cell orientation and regulation of cell–cell communication in human mesenchymal stem cells on different patterns of electrospun fibers

International Nuclear Information System (INIS)

Chang, Jui-Chih; Fujita, Satoshi; Tonami, Hiroyuki; Iwata, Hiroo; Kato, Koichi; Hsu, Shan-hui

2013-01-01

Cell behavior can be manipulated by the topography of the culture surface. In this study, we examined the intercellular communication and osteogenic differentiation of mesenchymal stem cells (MSCs) grown on electrospun fibers with different orientations and densities. Human bone marrow-derived MSCs (hMSCs) were seeded on poly(ε-caprolactone) (PCL) electrospun scaffolds composed of aligned (1D) or cross-aligned (2D) fibers (1.0–1.2 µm diameter) with high, medium, or low fiber densities. It was found that cells preferred to adhere onto electrospun PCL fibers rather than on the flat substrate. The immunofluorescence staining showed that the expression of vinculin, a focal adhesion protein, was limited to the periphery and the two extremities of aligned cells on the edge of the fibers. Electron microscopy showed that cells extended their lamellipodia across the adjacent fibers and proliferated along the direction of fibers. Cells grown on 1D fibrous scaffolds at all fiber densities had an obvious alignment. On 2D fibers, a higher degree of cell alignment was observed at the higher fiber density. On 1D scaffolds, the gap junction intercellular communication (GJIC) quantified by the lucifer yellow dye transfer assay was significantly promoted in the aligned cells in the direction parallel to the fibers but was abolished in the direction perpendicular to the fibers. The expression of osteogenic marker genes (RUNX2, ALP, and OCN) was significantly enhanced in seven days by culture on 1D but not 2D fibers. It was thus proposed that the promoted osteogenic differentiation of hMSCs may be associated with the fiber-guided and directional induction of GJIC. (paper)

Transmembrane Domain Single-Nucleotide Polymorphisms Impair Expression and Transport Activity of ABC Transporter ABCG2

NARCIS (Netherlands)

Sjostedt, N.; Heuvel, J.J.M.W. van den; Koenderink, J.B.; Kidron, H.

2017-01-01

PURPOSE: To study the function and expression of nine naturally occurring single-nucleotide polymorphisms (G406R, F431L, S441N, P480L, F489L, M515R, L525R, A528T and T542A) that are predicted to reside in the transmembrane regions of the ABC transporter ABCG2. METHODS: The transport activity of the

Intercellular production of tamavidin 1, a biotin-binding protein from Tamogitake mushroom, confers resistance to the blast fungus Magnaporthe oryzae in transgenic rice.

Science.gov (United States)

Takakura, Yoshimitsu; Oka, Naomi; Suzuki, Junko; Tsukamoto, Hiroshi; Ishida, Yuji

2012-05-01

The blast fungus Magnaporthe oryzae, one of the most devastating rice pathogens in the world, shows biotin-dependent growth. We have developed a strategy for creating disease resistance to M. oryzae whereby intercellular production of tamavidin 1, a biotin-binding protein from Pleurotus cornucopiae occurs in transgenic rice plants. The gene that encodes tamavidin 1, fused to the sequence for a secretion signal peptide derived from rice chitinase gene, was connected to the Cauliflower mosaic virus 35S promoter, and the resultant construct was introduced into rice. The tamavidin 1 was accumulated at levels of 0.1-0.2% of total soluble leaf proteins in the transgenic rice and it was localized in the intercellular space of rice leaves. The tamavidin 1 purified from the transgenic rice was active, it bound to biotin and inhibited in vitro growth of M. oryzae by causing biotin deficiency. The transgenic rice plants showed a significant resistance to M. oryzae. This study shows the possibility of a new strategy to engineer disease resistance in higher plants by taking advantage of a pathogen's auxotrophy.

Agonists and inverse agonists for the herpesvirus 8-encoded constitutively active seven-transmembrane oncogene product, ORF-74

DEFF Research Database (Denmark)

Rosenkilde, M M; Kledal, T N; Bräuner-Osborne, Hans

1999-01-01

A number of CXC chemokines competed with similar, nanomolar affinity against 125I-interleukin-8 (IL-8) binding to ORF-74, a constitutively active seven-transmembrane receptor encoded by human herpesvirus 8. However, in competition against 125I-labeled growth-related oncogene (GRO)-alpha, the ORF-74...

Different transport behaviors of NH4 (+) and NH3 in transmembrane cyclic peptide nanotubes.

Science.gov (United States)

Zhang, Mingming; Fan, Jianfen; Xu, Jian; Weng, Peipei; Lin, Huifang

2016-10-01

Two water-filled transmembrane cyclic peptide nanotubes (CPNTs) of 8×cyclo-(WL)n=4,5/POPE were chosen to investigate the dependences of the transport properties of the positive NH4 (+) and neutral NH3 on the channel radius. Molecular dynamic simulations revealed that molecular charge, size, ability to form H-bonds and channel radius all significantly influence the behaviors of NH4 (+) and NH3 in a CPNT. Higher electrostatic interactions, more H-bonds, and water-bridges were found in the NH4 (+) system, resulting in NH4 (+) meeting higher energy barriers, while NH3 can enter, exit and permeate the channels effortlessly. This work sheds a first light on the differences between the mechanisms of NH4 (+) and NH3 moving in a CPNT at an atomic level. Graphical Abstract Snapshot of the simulation system of NH4 (+)_octa-CPNT with an NH4 (+) initially positioned at one mouth of the tube, PMF profiles for single NH4 (+) ion and NH3 molecule moving through water-filled transmembrane CPNTs of 8×cyclo-(WL)n=4,5/POPE and sketch graphs of the possible H-bond forms of NH3 and NH4 (+) with the neighboring water.

Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

Science.gov (United States)

Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

2017-11-09

Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

Can membrane-bound carotenoid pigment zeaxanthin carry out a transmembrane proton transfer?

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Kupisz, Kamila; Sujak, Agnieszka; Patyra, Magdalena; Trebacz, Kazimierz; Gruszecki, Wiesław I

2008-10-01

Polar carotenoid pigment zeaxanthin (beta,beta-carotene-3,3'-diol) incorporated into planar lipid membranes formed with diphytanoyl phosphatidylcholine increases the specific electric resistance of the membrane from ca. 4 to 13 x 10(7) Omega cm2 (at 5 mol% zeaxanthin with respect to lipid). Such an observation is consistent with the well known effect of polar carotenoids in decreasing fluidity and structural stabilization of lipid bilayers. Zeaxanthin incorporated into the lipid membrane at 1 mol% has very small effect on the overall membrane resistance but facilitates equilibration of the transmembrane proton gradient, as demonstrated with the application of the H+-sensitive antimony electrodes. Relatively low changes in the electrical potential suggest that the equilibration process may be associated with a symport/antiport activity or with a transmembrane transfer of the molecules of acid. UV-Vis linear dichroism analysis of multibilayer formed with the same lipid-carotenoid system shows that the transition dipole moment of the pigment molecules forms a mean angle of 21 degrees with respect to the axis normal to the plane of the membrane. This means that zeaxanthin spans the membrane and tends to have its two hydroxyl groups anchored in the opposite polar zones of the membrane. Detailed FTIR analysis of beta-carotene and zeaxanthin indicates that the polyene chain of carotenoids is able to form weak hydrogen bonds with water molecules. Possible molecular mechanisms responsible for proton transport by polyenes are discussed, including direct involvement of the polyene chain in proton transfer and indirect effect of the pigment on physical properties of the membrane.

Induction of IL-1 during hemodialysis: Transmembrane passage of intact endotoxins (LPS)

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Laude-Sharp, M.; Caroff, M.; Simard, L.; Pusineri, C.; Kazatchkine, M.D.; Haeffner-Cavaillon, N. (INSERM U 28, Hopital Broussais, Paris (France))

1990-12-01

Circulating monocytes of patients undergoing chronic hemodialysis are triggered to produce interleukin-1 (IL-1) in vivo. Intradialytic induction of IL-1 is associated with complement activation in patients dialyzed with first-use cellulose membranes. Chronic stimulation of IL-1 production occurs because of an yet unidentified mechanism in patients dialyzed with high permeability membranes. The present study demonstrates that intact bacterial lipopolysaccharide (LPS) molecules may cross cuprophan, AN69 and polysulfone membranes under in vitro conditions simulating in vivo hemodialysis. The experiments used purified LPS from Neisseria meningitidis and LPS from Pseudomonas testosteroni, a bacterial strain grown out from a clinically used dialysate. LPS were purified to homogeneity and radiolabeled. Transmembrane passage of 3H-labeled LPS was observed within the first five minutes of dialysis. A total of 0.1 to 1% of 3H-labeled LPS were recovered in the dialysate compartment after one hour of dialysis. High amounts of LPS, representing 40 to 70% of the amount originally present in the dialysate, were absorbed onto high permeability membranes. Low amounts of LPS were absorbed onto cuprophan membranes. The amount of LPS absorbed decreased with the concentration of LPS in the dialysate. LPS recovered from the blood compartment exhibited the same molecular weight as that used to contaminate the dialysate. Biochemically detectable transmembrane passage of LPS was not associated with that of material detectable using the limulus amebocyte lysate (LAL) assay. An IL-1-inducing activity was, however, detected in the blood compartment upon dialysis with high permeability membranes, as previously found by others with cuprophan membranes.

Induction of IL-1 during hemodialysis: Transmembrane passage of intact endotoxins (LPS)

International Nuclear Information System (INIS)

Laude-Sharp, M.; Caroff, M.; Simard, L.; Pusineri, C.; Kazatchkine, M.D.; Haeffner-Cavaillon, N.

1990-01-01

Circulating monocytes of patients undergoing chronic hemodialysis are triggered to produce interleukin-1 (IL-1) in vivo. Intradialytic induction of IL-1 is associated with complement activation in patients dialyzed with first-use cellulose membranes. Chronic stimulation of IL-1 production occurs because of an yet unidentified mechanism in patients dialyzed with high permeability membranes. The present study demonstrates that intact bacterial lipopolysaccharide (LPS) molecules may cross cuprophan, AN69 and polysulfone membranes under in vitro conditions simulating in vivo hemodialysis. The experiments used purified LPS from Neisseria meningitidis and LPS from Pseudomonas testosteroni, a bacterial strain grown out from a clinically used dialysate. LPS were purified to homogeneity and radiolabeled. Transmembrane passage of 3H-labeled LPS was observed within the first five minutes of dialysis. A total of 0.1 to 1% of 3H-labeled LPS were recovered in the dialysate compartment after one hour of dialysis. High amounts of LPS, representing 40 to 70% of the amount originally present in the dialysate, were absorbed onto high permeability membranes. Low amounts of LPS were absorbed onto cuprophan membranes. The amount of LPS absorbed decreased with the concentration of LPS in the dialysate. LPS recovered from the blood compartment exhibited the same molecular weight as that used to contaminate the dialysate. Biochemically detectable transmembrane passage of LPS was not associated with that of material detectable using the limulus amebocyte lysate (LAL) assay. An IL-1-inducing activity was, however, detected in the blood compartment upon dialysis with high permeability membranes, as previously found by others with cuprophan membranes

Interleukin-4 and interleukin-13 compromise the sinonasal epithelial barrier and perturb intercellular junction protein expression.

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Wise, Sarah K; Laury, Adrienne M; Katz, Elizabeth H; Den Beste, Kyle A; Parkos, Charles A; Nusrat, Asma

2014-05-01

Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a "leaky" epithelial barrier phenotype. We hypothesize that T helper 2 (Th2) cytokines interleukin (IL)-4 and IL-13 modulate epithelial junction proteins, thereby contributing to the leaky epithelial barrier. Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n = 6) and 68.6% (n = 8) of baseline, respectively. Tight junction protein junctional adhesion molecule-A (JAM-A) expression decreased 42.2% with IL-4 exposure (n = 9) and 37.5% with IL-13 exposure (n = 9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n = 9) and 32.9% with IL-13 exposure (n = 9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or zonula occludens-1 (ZO-1) with IL-4 or IL-13 exposure. Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity. © 2014 ARS-AAOA, LLC.

IL-4 and IL-13 Compromise the Sinonasal Epithelial Barrier and Perturb Intercellular Junction Protein Expression

Science.gov (United States)

Wise, Sarah K.; Laury, Adrienne M.; Katz, Elizabeth H.; Den Beste, Kyle A.; Parkos, Charles A.; Nusrat, Asma

2014-01-01

Introduction Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a “leaky” epithelial barrier phenotype. We hypothesize that Th2 cytokines IL-4 and IL-13 modulate epithelial junction proteins thereby contributing to the leaky epithelial barrier. Methods Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. Results IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n=6) and 68.6% (n=8) of baseline, respectively. Tight junction protein JAM-A expression decreased 42.2% with IL-4 exposure (n=9) and 37.5% with IL-13 exposure (n=9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n=9) and 32.9% with IL-13 exposure (n=9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or ZO-1 with IL-4 or IL-13 exposure. Conclusion Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity. PMID:24510479

Functional relevance of aromatic residues in the first transmembrane domain of P2X receptors

Czech Academy of Sciences Publication Activity Database

Jindřichová, Marie; Vávra, Vojtěch; Obšil, Tomáš; Stojilkovic, S. S.; Zemková, Hana

2009-01-01

Roč. 109, č. 3 (2009), s. 923-934 ISSN 0022-3042 R&D Projects: GA MŠk(CZ) LC554; GA AV ČR(CZ) IAA5011408; GA AV ČR(CZ) IAA500110702; GA AV ČR(CZ) IAA500110910 Institutional research plan: CEZ:AV0Z50110509 Keywords : purinergic receptors * gating * transmembrane domain Subject RIV: FH - Neuro logy Impact factor: 3.999, year: 2009

Point mutations in the transmembrane region of the clic1 ion channel selectively modify its biophysical properties.

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Stefania Averaimo

Full Text Available Chloride intracellular Channel 1 (CLIC1 is a metamorphic protein that changes from a soluble cytoplasmic protein into a transmembrane protein. Once inserted into membranes, CLIC1 multimerises and is able to form chloride selective ion channels. Whilst CLIC1 behaves as an ion channel both in cells and in artificial lipid bilayers, its structure in the soluble form has led to some uncertainty as to whether it really is an ion channel protein. CLIC1 has a single putative transmembrane region that contains only two charged residues: arginine 29 (Arg29 and lysine 37 (Lys37. As charged residues are likely to have a key role in ion channel function, we hypothesized that mutating them to neutral alanine to generate K37A and R29A CLIC1 would alter the electrophysiological characteristics of CLIC1. By using three different electrophysiological approaches: i single channel Tip-Dip in artificial bilayers using soluble recombinant CLIC1, ii cell-attached and iii whole-cell patch clamp recordings in transiently transfected HEK cells, we determined that the K37A mutation altered the single-channel conductance while the R29A mutation affected the single-channel open probability in response to variation in membrane potential. Our results show that mutation of the two charged amino acids (K37 and R29 in the putative transmembrane region of CLIC1 alters the biophysical properties of the ion channel in both artificial bilayers and cells. Hence these charged residues are directly involved in regulating its ion channel activity. This strongly suggests that, despite its unusual structure, CLIC1 itself is able to form a chloride ion channel.

Differential expression of a novel seven transmembrane domain protein in epididymal fat from aged and diabetic mice.

Science.gov (United States)

Yang, H; Egan, J M; Rodgers, B D; Bernier, M; Montrose-Rafizadeh, C

1999-06-01

To identify novel seven transmembrane domain proteins from 3T3-L1 adipocytes, we used PCR to amplify 3T3-L1 adipocyte complementary DNA (cDNA) with primers homologous to the N- and C-termini of pancreatic glucagon-like peptide-1 (GLP-1) receptor. We screened a cDNA library prepared from fully differentiated 3T3-L1 adipocytes using a 500-bp cDNA PCR product probe. Herein describes the isolation and characterization of a 1.6-kb cDNA clone that encodes a novel 298-amino acid protein that we termed TPRA40 (transmembrane domain protein of 40 kDa regulated in adipocytes). TPRA40 has seven putative transmembrane domains and shows little homology with the known GLP-1 receptor or with other G protein-coupled receptors. The levels of TPRA40 mRNA and protein were higher in 3T3-L1 adipocytes than in 3T3-L1 fibroblasts. TPRA40 is present in a number of mouse and human tissues. Interestingly, TPRA40 mRNA levels were significantly increased by 2- to 3-fold in epididymal fat of 24-month-old mice vs. young controls as well as in db/db and ob/ob mice vs. nondiabetic control littermates. No difference in TPRA40 mRNA levels was observed in brain, heart, skeletal muscle, liver, or kidney. Furthermore, no difference in TPRA40 expression was detected in brown fat of ob/ob mice when compared with age-matched controls. Taken together, these data suggest that TPRA40 represents a novel membrane-associated protein whose expression in white adipose tissue is altered with aging and type 2 diabetes.

Crystallization and preliminary X-ray diffraction analysis of P30, the transmembrane domain of pertactin, an autotransporter from Bordetella pertussis

International Nuclear Information System (INIS)

Zhu, Yanshi; Black, Isobel; Roszak, Aleksander W.; Isaacs, Neil W.

2007-01-01

P30, the transmembrane C-terminal domain of pertactin from B. pertussis has been crystallized after refolding in vitro. Preliminary X-ray crystallographic data are reported. P30, the 32 kDa transmembrane C-terminal domain of pertactin from Bordetella pertussis, is supposed to form a β-barrel inserted into the outer membrane for the translocation of the passenger domain. P30 was cloned and expressed in inclusion bodies in Escherichia coli. After refolding and purification, the protein was crystallized using the sitting-drop vapour-diffusion method at 292 K. The crystals diffract to a resolution limit of 3.5 Å using synchrotron radiation and belong to the hexagonal space group P6 1 22, with unit-cell parameters a = b = 123.27, c = 134.43 Å

Transmembrane molecular transport during versus after extremely large, nanosecond electric pulses.

Science.gov (United States)

Smith, Kyle C; Weaver, James C

2011-08-19

Recently there has been intense and growing interest in the non-thermal biological effects of nanosecond electric pulses, particularly apoptosis induction. These effects have been hypothesized to result from the widespread creation of small, lipidic pores in the plasma and organelle membranes of cells (supra-electroporation) and, more specifically, ionic and molecular transport through these pores. Here we show that transport occurs overwhelmingly after pulsing. First, we show that the electrical drift distance for typical charged solutes during nanosecond pulses (up to 100 ns), even those with very large magnitudes (up to 10 MV/m), ranges from only a fraction of the membrane thickness (5 nm) to several membrane thicknesses. This is much smaller than the diameter of a typical cell (∼16 μm), which implies that molecular drift transport during nanosecond pulses is necessarily minimal. This implication is not dependent on assumptions about pore density or the molecular flux through pores. Second, we show that molecular transport resulting from post-pulse diffusion through minimum-size pores is orders of magnitude larger than electrical drift-driven transport during nanosecond pulses. While field-assisted charge entry and the magnitude of flux favor transport during nanosecond pulses, these effects are too small to overcome the orders of magnitude more time available for post-pulse transport. Therefore, the basic conclusion that essentially all transmembrane molecular transport occurs post-pulse holds across the plausible range of relevant parameters. Our analysis shows that a primary direct consequence of nanosecond electric pulses is the creation (or maintenance) of large populations of small pores in cell membranes that govern post-pulse transmembrane transport of small ions and molecules. Copyright © 2011 Elsevier Inc. All rights reserved.

Effect of intercellular adhesion molecule 1 expression in radiation otitis media murine model

International Nuclear Information System (INIS)

Wang Shengzi; Cheng Qingfang; Lu Shenbin; Liu Jianping; Wang Shuyi

2003-01-01

Objective: To characterize the dose- and time-dependent changes in intercellular adhesion molecule 1 (ICAM-1) expression and the role of this molecule as a mediator of middle ear inflammation induced by radiation. Methods: Radiation-induced otitis media animal models were established by using guinea pigs after 60 Co irradiation with 3 Gy/fraction per day, 5 times per week to a total dose of 15, 30, 45 Gy. The expression of ICAM-1 was studied by SP immunohistochemistry with the relation between radiation dose and infiltration of leukocytes investigated. Results: ICAM-1 was not expressed in the normal epithelium of the middle ear mucosa. Mucosal epithelium strongly expressed ICAM-1 after having been administered with 45 Gy of irradiation showing a significant correlation between the expression of ICAM-1 and the infiltration of leukocytes. Conclusions: Irradiation increases the expression of ICAM-1 in the middle ear mucosa. ICAM-1 may be related to the inflammation in the middle ear after irradiation

Transmembrane START domain proteins: in silico identification, characterization and expression analysis under stress conditions in chickpea (Cicer arietinum L.).

Science.gov (United States)

Satheesh, Viswanathan; Chidambaranathan, Parameswaran; Jagannadham, Prasanth Tejkumar; Kumar, Vajinder; Jain, Pradeep K; Chinnusamy, Viswanathan; Bhat, Shripad R; Srinivasan, R

2016-01-01

Steroidogenic acute regulatory related transfer (StART) proteins that are involved in transport of lipid molecules, play a myriad of functions in insects, mammals and plants. These proteins consist of a modular START domain of approximately 200 amino acids which binds and transfers the lipids. In the present study we have performed a genome-wide search for all START domain proteins in chickpea. The search identified 36 chickpea genes belonging to the START domain family. Through a phylogenetic tree reconstructed with Arabidopsis, rice, chickpea, and soybean START proteins, we were able to identify four transmembrane START (TM-START) proteins in chickpea. These four proteins are homologous to the highly conserved mammalian phosphatidylcholine transfer proteins. Multiple sequence alignment of all the transmembrane containing START proteins from Arabidopsis, rice, chickpea, and soybean revealed that the amino acid residues to which phosphatidylcholine binds in mammals, is also conserved in all these plant species, implying an important functional role and a very similar mode of action of all these proteins across dicots and monocots. This study characterizes a few of the not so well studied transmembrane START superfamily genes that may be involved in stress signaling. Expression analysis in various tissues showed that these genes are predominantly expressed in flowers and roots of chickpea. Three of the chickpea TM-START genes showed induced expression in response to drought, salt, wound and heat stress, suggesting their role in stress response.

Three-dimensional structures of the mammalian multidrug resistance P-glycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding.

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Rosenberg, Mark F; Kamis, Alhaji Bukar; Callaghan, Richard; Higgins, Christopher F; Ford, Robert C

2003-03-07

P-glycoprotein is an ATP-binding cassette transporter that is associated with multidrug resistance and the failure of chemotherapy in human patients. We have previously shown, based on two-dimensional projection maps, that P-glycoprotein undergoes conformational changes upon binding of nucleotide to the intracellular nucleotide binding domains. Here we present the three-dimensional structures of P-glycoprotein in the presence and absence of nucleotide, at a resolution limit of approximately 2 nm, determined by electron crystallography of negatively stained crystals. The data reveal a major reorganization of the transmembrane domains throughout the entire depth of the membrane upon binding of nucleotide. In the absence of nucleotide, the two transmembrane domains form a single barrel 5-6 nm in diameter and about 5 nm deep with a central pore that is open to the extracellular surface and spans much of the membrane depth. Upon binding nucleotide, the transmembrane domains reorganize into three compact domains that are each 2-3 nm in diameter and 5-6 nm deep. This reorganization opens the central pore along its length in a manner that could allow access of hydrophobic drugs (transport substrates) directly from the lipid bilayer to the central pore of the transporter.

TDP-43 or FUS-induced misfolded human wild-type SOD1 can propagate intercellularly in a prion-like fashion.

Science.gov (United States)

Pokrishevsky, Edward; Grad, Leslie I; Cashman, Neil R

2016-03-01

Amyotrophic lateral sclerosis (ALS), which appears to spread through the neuroaxis in a spatiotemporally restricted manner, is linked to heritable mutations in genes encoding SOD1, TDP-43, FUS, C9ORF72, or can occur sporadically without recognized genetic mutations. Misfolded human wild-type (HuWt) SOD1 has been detected in both familial and sporadic ALS patients, despite mutations in SOD1 accounting for only 2% of total cases. We previously showed that accumulation of pathological TDP-43 or FUS coexist with misfolded HuWtSOD1 in patient motor neurons, and can trigger its misfolding in cultured cells. Here, we used immunocytochemistry and immunoprecipitation to demonstrate that TDP-43 or FUS-induced misfolded HuWtSOD1 can propagate from cell-to-cell via conditioned media, and seed cytotoxic misfolding of endogenous HuWtSOD1 in the recipient cells in a prion-like fashion. Knockdown of SOD1 using siRNA in recipient cells, or incubation of conditioned media with misfolded SOD1-specific antibodies, inhibits intercellular transmission, indicating that HuWtSOD1 is an obligate seed and substrate of propagated misfolding. In this system, intercellular spread of SOD1 misfolding is not accompanied by transmission of TDP-43 or FUS pathology. Our findings argue that pathological TDP-43 and FUS may exert motor neuron pathology in ALS through the initiation of propagated misfolding of SOD1.

[Application of Brownian dynamics to the description of transmembrane ion flow as exemplified by the chloride channel of glycine receptor].

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Boronovskiĭ, S E; Nartsissov, Ia R

2009-01-01

Using the Brownian dynamics of the movement of hydrated ion in a viscous water solution, a mathematical model has been built, which describes the transport of charged particles through a single protein pore in a lipid membrane. The dependences of transmembrane ion currents on ion concentrations in solution have been obtained. It was shown that, if the geometry of a membrane pore is identical to that of the inner part of the glycine receptor channel and there is no ion selectivity, then the values of both chloride and sodium currents are not greater than 0.5 pA at the physiological concentrations of these ions. If local charge heterogeneity caused by charged amino acid residues of transmembrane protein segments is included into the model calculations, the chloride current increases to about 3.7 pA, which exceeds more than seven times the value for sodium ions under the conditions of the complex channel geometry in the range of physiological concentrations of ions in the solution. The model takes changes in the density of charge distribution both inside the channel and near the protein surface into account. The alteration of pore geometry can be also considered as a parameter at the researcher's option. Thus, the model appears as an effective tool for the description of transmembrane currents for other types of membrane channels.

Ligand Modulation of the Epstein-Barr Virus-induced Seven-transmembrane Receptor EBI2

DEFF Research Database (Denmark)

Benned-Jensen, Tau; Smethurst, Christopher; Holst, Peter Johannes

2011-01-01

The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we...... with similar potency. Overexpression of EBI2 profoundly potentiated antibody-stimulated ex vivo proliferation of murine B cells compared with WT cells, whereas this was equivalently reduced for EBI2-deficient B cells. Inhibition of EBI2 constitutive activity suppressed the proliferation in all cases...

C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication.

Science.gov (United States)

Wang, Juan; Silva, Malan; Haas, Leonard A; Morsci, Natalia S; Nguyen, Ken C Q; Hall, David H; Barr, Maureen M

2014-03-03

Cells release extracellular vesicles (ECVs) that play important roles in intercellular communication and may mediate a broad range of physiological and pathological processes. Many fundamental aspects of ECV biogenesis and signaling have yet to be determined, with ECV detection being a challenge and obstacle due to the small size (100 nm) of the ECVs. We developed an in vivo system to visualize the dynamic release of GFP-labeled ECVs. We show here that specific Caenorhabdidits elegans ciliated sensory neurons shed and release ECVs containing GFP-tagged polycystins LOV-1 and PKD-2. These ECVs are also abundant in the lumen surrounding the cilium. Electron tomography and genetic analysis indicate that ECV biogenesis occurs via budding from the plasma membrane at the ciliary base and not via fusion of multivesicular bodies. Intraflagellar transport and kinesin-3 KLP-6 are required for environmental release of PKD-2::GFP-containing ECVs. ECVs isolated from wild-type animals induce male tail-chasing behavior, while ECVs isolated from klp-6 animals and lacking PKD-2::GFP do not. We conclude that environmentally released ECVs play a role in animal communication and mating-related behaviors. Copyright © 2014 Elsevier Ltd. All rights reserved.

Monolayer freeze-fracture autoradiography: quantitative analysis of the transmembrane distribution of radioiodinated concanavalin A

International Nuclear Information System (INIS)

Fisher, K.A.

1982-01-01

The technique of monolayer freeze-fracture autoradiography (MONOFARG) has been developed and the principles, quantitation, and application of the method are described. Cell monolayers attached to polylysine-treated glass were freeze-fractured, shadowed, and coated with dry, Parlodion-supported Ilford L4 photographic emulsion at room temperature. Quantitative aspects of MONOFARG were examined using radioiodinated test systems. Background was routinely -4 grains/μm 2 /day, the highest overall efficiency was between 25% and 45%, and grain density and efficiency were dependent on radiation dose for iodine-125 and D-19 development. Corrected grain densities were linearly proportional to iodine-125 concentration. The method was applied to an examination of the transmembrane distribution of radioiodinated and fluoresceinated concanavalin A ( 125 I-FITC-Con-A). Human erythrocytes were labeled, column-purified, freeze-dried or freeze-fractured, autoradiographed, and examined by electron microscopy. The number of silver grains per square micrometer of unsplit single membrane was essentially identical to that of split extracellular membrane halves. These data demonstrate that 125 I-FITC-Con-A partitions exclusively with the extracellular half of the membrane upon freeze-fracturing and can be used as a quantitative marker for the fraction of extracellular split membrane halves. This method should be able to provide new information about certain transmembrane properties of biological membrane molecules and probes, as well as about the process of freeze-fracture per se

Crystallization and preliminary X-ray diffraction analysis of P30, the transmembrane domain of pertactin, an autotransporter from Bordetella pertussis

Energy Technology Data Exchange (ETDEWEB)

Zhu, Yanshi; Black, Isobel; Roszak, Aleksander W.; Isaacs, Neil W., E-mail: n.isaacs@chem.gla.ac.uk [Department of Chemistry and WestChem, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom)

2007-07-01

P30, the transmembrane C-terminal domain of pertactin from B. pertussis has been crystallized after refolding in vitro. Preliminary X-ray crystallographic data are reported. P30, the 32 kDa transmembrane C-terminal domain of pertactin from Bordetella pertussis, is supposed to form a β-barrel inserted into the outer membrane for the translocation of the passenger domain. P30 was cloned and expressed in inclusion bodies in Escherichia coli. After refolding and purification, the protein was crystallized using the sitting-drop vapour-diffusion method at 292 K. The crystals diffract to a resolution limit of 3.5 Å using synchrotron radiation and belong to the hexagonal space group P6{sub 1}22, with unit-cell parameters a = b = 123.27, c = 134.43 Å.

Signaling through intercellular adhesion molecule 1 (ICAM-1) in a B cell lymphoma line

DEFF Research Database (Denmark)

Holland, J; Owens, T

1997-01-01

Intercellular adhesion molecule 1 (ICAM-1) (CD54) is an adhesion molecule of the immunoglobulin superfamily. The interaction between ICAM-1 on B lymphocytes and leukocyte function-associated antigen 1 on T cells plays a major role in several aspects of the immune response, including T-dependent B...... cell activation. While it was originally believed that ICAM-1 played a purely adhesive role, recent evidence suggests that it can itself transduce biochemical signals. We demonstrate that cross-linking of ICAM-1 results in the up-regulation of class II major histocompatibility complex, and we...... investigate the biochemical mechanism for the signaling role of ICAM-1. We show that cross-linking of ICAM-1 on the B lymphoma line A20 induces an increase in tyrosine phosphorylation of several cellular proteins, including the Src family kinase p53/p56(lyn). In vitro kinase assays showed that Lyn kinase...

Deorphanizing the human transmembrane genome: A landscape of uncharacterized membrane proteins.

Science.gov (United States)

Babcock, Joseph J; Li, Min

2014-01-01

The sequencing of the human genome has fueled the last decade of work to functionally characterize genome content. An important subset of genes encodes membrane proteins, which are the targets of many drugs. They reside in lipid bilayers, restricting their endogenous activity to a relatively specialized biochemical environment. Without a reference phenotype, the application of systematic screens to profile candidate membrane proteins is not immediately possible. Bioinformatics has begun to show its effectiveness in focusing the functional characterization of orphan proteins of a particular functional class, such as channels or receptors. Here we discuss integration of experimental and bioinformatics approaches for characterizing the orphan membrane proteome. By analyzing the human genome, a landscape reference for the human transmembrane genome is provided.

Role of Intercellular Adhesion Molecule-1 in Radiation-Induced Brain Injury

International Nuclear Information System (INIS)

Wu, K.-L.; Tu Ba; Li Yuqing; Wong, C. Shun

2010-01-01

Purpose: To determine the role of intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of brain injury after irradiation (IR). Methods and Materials: We assessed the expression of ICAM-1 in mouse brain after cranial IR and determined the histopathologic and behavioral changes in mice that were either wildtype (+/+) or knockout (-/-) of the ICAM-1 gene after IR. Results: There was an early dose-dependent increase in ICAM-1 mRNA and protein expression after IR. Increased ICAM-1 immunoreactivity was observed in endothelia and glia of ICAM-1+/+ mice up to 8 months after IR. ICAM-1-/- mice showed no expression. ICAM-1+/+ and ICAM-1-/- mice showed similar vascular abnormalities at 2 months after 10-17 Gy, and there was evidence for demyelination and inhibition of hippocampal neurogenesis at 8 months after 10 Gy. After 10 Gy, irradiated ICAM-1+/+ and ICAM-1-/- mice showed similar behavioral changes at 2-6 months in open field, light-dark chamber, and T-maze compared with age-matched genotype controls. Conclusion: There is early and late upregulation of ICAM-1 in the vasculature and glia of mouse brain after IR. ICAM-1, however, does not have a causative role in the histopathologic injury and behavioral dysfunction after moderate single doses of cranial IR.

Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions

International Nuclear Information System (INIS)

Yokawa, Satoru; Suzuki, Takahiro; Inouye, Satoshi; Inoh, Yoshikazu; Suzuki, Ryo; Kanamori, Takao; Furuno, Tadahide; Hirashima, Naohide

2017-01-01

We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase from the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion. - Highlights: • The fused protein of proglucagon to Gaussia luciferase was used as a reporter. • The fusion protein was highly expressed using a preferred human-codon optimized gene. • Glucagon secretion stimulated by depolarization was determined by luminescence. • Glucagon secretion in α cells was visualized by bioluminescence imaging. • Glucagon secretion sites were localized in the intercellular contact regions.

Defining the transmembrane helix of M2 protein from influenza A by molecular dynamics simulations in a lipid bilayer

NARCIS (Netherlands)

Forrest, LR; Tieleman, DP; Sansom, MSP

Integral membrane proteins containing at least one transmembrane (TM) alpha-helix are believed to account for between 20% and 30% of most genomes. There are several algorithms that accurately predict the number and position of TM helices within,a membrane protein sequence. However, these methods

Self-Assembling Organic Nanopores as Synthetic Transmembrane Channels with Tunable Functions

Science.gov (United States)

Wei, Xiaoxi

A long-standing goal in the area of supramolecular self-assembly involves the development of synthetic ion/water channels capable of mimicking the mass-transport characteristics of biological channels and pores. Few examples of artificial transmembrane channels with large lumen, high conductivity and selectivity are known. A review of pronounced biological transmembrane protein channels and some representative synthetic models have been provided in Chapter 1, followed by our discovery and initial investigation of shape-persistent oligoamide and phenylene ethynylene macrocycles as synthetic ion/water channels. In Chapter 2, the systematic structural modification of oligoamide macrocycles 1, the so-called first-generation of these shape-persistent macrocycles, has led to third-generation macrocycles 3. The third generation was found to exhibit unprecedented, strong intermolecular association in both the solid state and solution via multiple techniques including X-ray diffraction (XRD), SEM, and 1H NMR. Fluorescence spectroscopy paired with dynamic light scattering (DLS) revealed that macrocycles 3 can assemble into a singly dispersed nanotubular structure in solution. The resultant self-assembling pores consisting of 3 were examined by HPTS-LUVs assays and BLM studies (Chapter 3) and found to form cation-selective (PK+/PCl- = 69:1) transmembrane ion channels with large conductance (200 ˜ 2000 pS for alkali cations) and high stability with open times reaching to 103 seconds. Tuning the aggregation state of macrocycles by choosing an appropriate polar solvent mixture (i.e., 3:1, THF:DMF, v/v) and concentration led to the formation of ion channels with well-defined square top behavior. A parallel study using DLS to examine the size of aggregates was used in conjunction with channel activity assays (LUVs/BLM) to reveal the effects of the aggregation state on channel activity. Empirical evidence now clearly indicates that a preassembled state, perhaps that of a

PheVI:09 (Phe6.44) as a sliding microswitch in seven-transmembrane (7TM) G protein-coupled receptor activation

DEFF Research Database (Denmark)

Valentin-Hansen, Louise; Holst, Birgitte; Frimurer, Thomas M

2012-01-01

In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational......-V into a tight pocket generated by five hydrophobic residues protruding from TM-III and TM-V. Of these, the residue in position III:16 (3.40) (often an Ile or Val) appears to function as a barrier or gate for the transition between inactive and active conformation. Mutational analysis showed that PheVI:09...... an aromatic microswitch that stabilizes the active, outward tilted conformation of TM-VI relative to TM-III by sliding into a tight hydrophobic pocket between TM-III and TM-V and that the hydrophobic residue in position III:16 constitutes a gate for this transition....

Rapid construction of mechanically- confined multi- cellular structures using dendrimeric intercellular linker.

Science.gov (United States)

Mo, Xuejun; Li, Qiushi; Yi Lui, Lena Wai; Zheng, Baixue; Kang, Chiang Huen; Nugraha, Bramasta; Yue, Zhilian; Jia, Rui Rui; Fu, Hong Xia; Choudhury, Deepak; Arooz, Talha; Yan, Jie; Lim, Chwee Teck; Shen, Shali; Hong Tan, Choon; Yu, Hanry

2010-10-01

Tissue constructs that mimic the in vivo cell-cell and cell-matrix interactions are especially useful for applications involving the cell- dense and matrix- poor internal organs. Rapid and precise arrangement of cells into functional tissue constructs remains a challenge in tissue engineering. We demonstrate rapid assembly of C3A cells into multi- cell structures using a dendrimeric intercellular linker. The linker is composed of oleyl- polyethylene glycol (PEG) derivatives conjugated to a 16 arms- polypropylenimine hexadecaamine (DAB) dendrimer. The positively charged multivalent dendrimer concentrates the linker onto the negatively charged cell surface to facilitate efficient insertion of the hydrophobic oleyl groups into the cellular membrane. Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular structures within minutes. The cells exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and other functions in the constructs. We constructed defined multi- cellular structures such as rings, sheets or branching rods that can serve as potential tissue building blocks to be further assembled into complex 3D tissue constructs for biomedical applications. 2010 Elsevier Ltd. All rights reserved.

Localization of the transmembrane proteoglycan syndecan-4 and its regulatory kinases in costameres of rat cardiomyocytes: a deconvolution microscopic study

DEFF Research Database (Denmark)

VanWinkle, W Barry; Snuggs, Mark B; De Hostos, Eugenio L

2002-01-01

Syndecan-4 (syn-4), a transmembrane heparan sulfate-containing proteoglycan, is unique among the four members of the syndecan family in its specific cellular localization to complex cytoskeletal adhesion sites, i.e., focal adhesions. During early phenotypic redifferentiation of neonatal cardiomyo...

Identification and functional comparison of seven-transmembrane G-protein-coupled BILF1 receptors in recently discovered nonhuman primate lymphocryptoviruses

DEFF Research Database (Denmark)

Spiess, Katja; Fares, Suzan; Sparre-Ulrich, Alexander H

2015-01-01

Coevolution of herpesviruses with their respective host has resulted in a delicate balance between virus-encoded immune evasion mechanisms and host antiviral immunity. BILF1 encoded by human Epstein-Barr virus (EBV) is a 7-transmembrane (7TM) G-protein-coupled receptor (GPCR) with multiple immuno...

Using information theory to assess the communicative capacity of circulating microRNA.

Science.gov (United States)

Finn, Nnenna A; Searles, Charles D

2013-10-11

The discovery of extracellular microRNAs (miRNAs) and their transport modalities (i.e., microparticles, exosomes, proteins and lipoproteins) has sparked theories regarding their role in intercellular communication. Here, we assessed the information transfer capacity of different miRNA transport modalities in human serum by utilizing basic principles of information theory. Zipf Statistics were calculated for each of the miRNA transport modalities identified in human serum. Our analyses revealed that miRNA-mediated information transfer is redundant, as evidenced by negative Zipf's Statistics with magnitudes greater than one. In healthy subjects, the potential communicative capacity of miRNA in complex with circulating proteins was significantly lower than that of miRNA encapsulated in circulating microparticles and exosomes. Moreover, the presence of coronary heart disease significantly lowered the communicative capacity of all circulating miRNA transport modalities. To assess the internal organization of circulating miRNA signals, Shannon's zero- and first-order entropies were calculated. Microparticles (MPs) exhibited the lowest Shannon entropic slope, indicating a relatively high capacity for information transfer. Furthermore, compared to the other miRNA transport modalities, MPs appeared to be the most efficient at transferring miRNA to cultured endothelial cells. Taken together, these findings suggest that although all transport modalities have the capacity for miRNA-based information transfer, MPs may be the simplest and most robust way to achieve miRNA-based signal transduction in sera. This study presents a novel method for analyzing the quantitative capacity of miRNA-mediated information transfer while providing insight into the communicative characteristics of distinct circulating miRNA transport modalities. Published by Elsevier Inc.

Exploiting hydrophobicity for efficient production of transmembrane helices for structure determination by NMR spectroscopy

DEFF Research Database (Denmark)

Bugge, Katrine Østergaard; Steinocher, Helena; Brooks, Andrew J.

2015-01-01

-labeled protein. In this work, we have exploited the hydrophobic nature of membrane proteins to develop a simple and efficient production scheme for isotope-labeled single-pass transmembrane domains (TMDs) with or without intrinsically disordered regions. We have evaluated the applicability and limitations...... of the strategy using seven membrane protein variants that differ in their overall hydrophobicity and length and show a recovery for suitable variants of >70%. The developed production scheme is cost-efficient and easy to implement and has the potential to facilitate an increase in the number of structures...

Connexin Communication Compartments and Wound Repair in Epithelial Tissue.

Science.gov (United States)

Chanson, Marc; Watanabe, Masakatsu; O'Shaughnessy, Erin M; Zoso, Alice; Martin, Patricia E

2018-05-03

Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems.

Pentameric ligand-gated ion channels exhibit distinct transmembrane domain archetypes for folding/expression and function.

Science.gov (United States)

Therien, J P Daniel; Baenziger, John E

2017-03-27

Although transmembrane helix-helix interactions must be strong enough to drive folding, they must still permit the inter-helix movements associated with conformational change. Interactions between the outermost M4 and adjacent M1 and M3 α-helices of pentameric ligand-gated ion channels have been implicated in folding and function. Here, we evaluate the role of different physical interactions at this interface in the function of two prokaryotic homologs, GLIC and ELIC. Strikingly, disruption of most interactions in GLIC lead to either a reduction or a complete loss of expression and/or function, while analogous disruptions in ELIC often lead to gains in function. Structural comparisons suggest that GLIC and ELIC represent distinct transmembrane domain archetypes. One archetype, exemplified by GLIC, the glycine and GABA receptors and the glutamate activated chloride channel, has extensive aromatic contacts that govern M4-M1/M3 interactions and that are essential for expression and function. The other archetype, exemplified by ELIC and both the nicotinic acetylcholine and serotonin receptors, has relatively few aromatic contacts that are detrimental to function. These archetypes likely have evolved different mechanisms to balance the need for strong M4 "binding" to M1/M3 to promote folding/expression, and the need for weaker interactions that allow for greater conformational flexibility.

Impact of biofilm accumulation on transmembrane and feed channel pressure drop: Effects of crossflow velocity, feed spacer and biodegradable nutrient

KAUST Repository

Dreszer, C.; Flemming, H. C.; Zwijnenburg, A.; Kruithof, J. C.; Vrouwenvelder, Johannes S.

2014-01-01

. As biodegradable nutrient, acetate was dosed to the feed water (1.0 and 0.25mgL-1 carbon) to enhance biofilm accumulation in the monitors. The studies showed that biofilm formation caused an increased transmembrane resistance and feed channel pressure drop

A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER

Directory of Open Access Journals (Sweden)

Haga Christopher L

2007-09-01

Full Text Available Abstract Background In mouse the cytokine interleukin-7 (IL-7 is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER. The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR, a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules.

Eavesdropping on altered cell-to-cell signaling in cancer by secretome profiling.

Science.gov (United States)

Klinke, David J

2016-01-01

In the past decade, cumulative clinical experiences with molecular targeted therapies and immunotherapies for cancer have promoted a shift in our conceptual understanding of cancer. This view shifted from viewing solid tumors as a homogeneous mass of malignant cells to viewing tumors as heterogeneous structures that are dynamically shaped by intercellular interactions among the variety of stromal, immune, and malignant cells present within the tumor microenvironment. As in any dynamic system, identifying how cells communicate to maintain homeostasis and how this communication is altered during oncogenesis are key hurdles for developing therapies to restore normal tissue homeostasis. Here, I discuss tissues as dynamic systems, using the mammary gland as an example, and the evolutionary concepts applied to oncogenesis. Drawing from these concepts, I present 2 competing hypotheses for how intercellular communication might be altered during oncogenesis. As an initial test of these competing hypotheses, a recent secretome comparison between normal human mammary and HER2+ breast cancer cell lines suggested that the particular proteins secreted by the malignant cells reflect a convergent evolutionary path associated with oncogenesis in a specific anatomical niche, despite arising in different individuals. Overall, this study illustrates the emerging power of secretome proteomics to probe, in an unbiased way, how intercellular communication changes during oncogenesis.

Identification of eight novel mutations in a collaborative analysis of a part of the second transmembrane domain of the CFTR gene

Energy Technology Data Exchange (ETDEWEB)

Mercier, B.; Audrezet, M.P.; Guillermit, H.; Quere, I.; Verlingue, C.; Ferec, C. (CDTS, Brest (France)); Lissens, W.; Bonduelle, M.; Liebaers, I. (University Hospital VUB, Brussels (Belgium)); Novelli, G.; Sangiuolo, F.; Dallapiccola, B. (IRCCS, Rotondo (Italy)); Kalaydjieva, L. (Inst. of Obstetrics, Sofia (Bulgaria)); Arce, M. De; Cashman, S. (Trinity College, Dublin (Ireland)); Kapranov, N. (NRC of medical Genetics, Moscow (Russian Federation)); Canki Klain, N. (Tozd Univerzitetna Ginekoloska Klinika, Ljubljana (Yugoslavia)); Lenoir, G. (Hopital des Enfants Malades Necker, Paris (France)); Chauveau, P. (Centre Hospitalier General, Le Havre (France)); Lanaerts, C. (Centre Hospitalier Regional et Universitaire, Amiens (France)); Rault, G. (Centre Helio-Marin, Roscoff (France))

1993-04-01

Cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible, when mutated, for cystic fibrosis (CF), spans over 230 kb on the long arm of chromosome 7 and is composed of 27 exons. The most common mutation responsible for CF worldwide is the deletion of a phenylalanine amino acid at codon 508 in the first nucleotide-binding fold and accounts for approximately 70% of CF chromosomes studied. More than 250 other mutations have been reported through the CF Genetic Analysis Consortium. The majority of the mutations previously described lie in the two nucleotide-binding folds. To explore exhaustively other regions of the gene, particularly exons coding for transmembrane domains, the authors have initiated a collaborative study between different laboratories to screen 369 non-[Delta]F508 CF chromosomes of seven ethnic European populations (Belgian, French, Breton, Irish, Italian, Yugoslavian, Russian). Among these chromosomes carrying an unidentified mutation, 63 were from Brittany, 50 of various French origin, 45 of Irish origin, 56 of Italian origin, 41 of Belgian origin, 2 of Turkish origin, 38 of Yugoslavian origin, 22 of Russian origin, and 52 of Bulgarian origin. Diagnostic criteria for CF included at least one positive sweat test and pulmonary disease with or without pancreatic disease. Using a denaturing gradient gel electrophoresis (DGGE) assay, they have identified eight novel mutations in exon 17b coding for part of the second transmembrane domain of the CFTR and they describe them in this report. 8 refs., 1 fig., 1 tab.

Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins.

Science.gov (United States)

Gaines, J C; Acebes, S; Virrueta, A; Butler, M; Regan, L; O'Hern, C S

2018-05-01

We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. © 2018 Wiley Periodicals, Inc.

Incorporation of transmembrane hydroxide transport into the chemiosmotic theory.

Science.gov (United States)

de Grey, A D

1999-10-01

A cornerstone of textbook bioenergetics is that oxidative ATP synthesis in mitochondria requires, in normal conditions of internal and external pH, a potential difference (delta psi) of well over 100 mV between the aqueous compartments that the energy-transducing membrane separates. Measurements of delta psi inferred from diffusion of membrane-permeant ions confirm this, but those using microelectrodes consistently find no such delta psi--a result ostensibly irreconcilable with the chemiosmotic theory. Transmembrane hydroxide transport necessarily accompanies mitochondrial ATP synthesis, due to the action of several carrier proteins; this nullifies some of the proton transport by the respiratory chain. Here, it is proposed that these carriers' structure causes the path of this "lost" proton flow to include a component perpendicular to the membrane but within the aqueous phases, so maintaining a steady-state proton-motive force between the water at each membrane surface and in the adjacent bulk medium. The conflicting measurements of delta psi are shown to be consistent with the response of this system to its chemical environment.

Transmembrane peptides as sensors of the membrane physical state

Science.gov (United States)

Piotto, Stefano; Di Biasi, Luigi; Sessa, Lucia; Concilio, Simona

2018-05-01

Cell membranes are commonly considered fundamental structures having multiple roles such as confinement, storage of lipids, sustain and control of membrane proteins. In spite of their importance, many aspects remain unclear. The number of lipid types is orders of magnitude larger than the number of amino acids, and this compositional complexity is not clearly embedded in any membrane model. A diffused hypothesis is that the large lipid palette permits to recruit and organize specific proteins controlling the formation of specialized lipid domains and the lateral pressure profile of the bilayer. Unfortunately, a satisfactory knowledge of lipid abundance remains utopian because of the technical difficulties in isolating definite membrane regions. More importantly, a theoretical framework where to fit the lipidomic data is still missing. In this work, we wish to utilize the amino acid sequence and frequency of the membrane proteins as bioinformatics sensors of cell bilayers. The use of an alignment-free method to find a correlation between the sequences of transmembrane portion of membrane proteins with the membrane physical state suggested a new approach for the discovery of antimicrobial peptides.

A monocyte chemotaxis inhibiting factor in serum of HIV infected men shares epitopes with the HIV transmembrane protein gp41

NARCIS (Netherlands)

Tas, M.; Drexhage, H. A.; Goudsmit, J.

1988-01-01

This report describes that gp41, the transmembranous envelope protein of HIV, is able to inhibit monocyte chemotaxis (measured as FMLP-induced polarization). To study the presence of such immunosuppressive HIV env proteins in the circulation of HIV-infected men, fractions were prepared from serum

Selective elimination of high constitutive activity or chemokine binding in the human herpesvirus 8 encoded seven transmembrane oncogene ORF74

DEFF Research Database (Denmark)

Rosenkilde, M M; Kledal, T N; Holst, Peter Johannes

2000-01-01

Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines, e.g. growth-related oncogene-alpha, and inhibited by angiostatic chemokines e.g. interferon-gamma-inducible protein. Transgenic mice...

Dilated intercellular space diameter as marker of reflux-related mucosal injury in children with chronic cough and gastro-oesophageal reflux disease.

Science.gov (United States)

Borrelli, O; Mancini, V; Thapar, N; Ribolsi, M; Emerenziani, S; de'Angelis, G; Bizzarri, B; Lindley, K J; Cicala, M

2014-04-01

The diagnostic corroboration of the relationship between gastro-oesophageal reflux disease (GERD) and chronic cough remains challenging. To compare oesophageal mucosal intercellular space diameter (ISD) in children with GERD, children with gastro-oesophageal reflux (GER)-related cough (GrC) and a control group, and to explore the relationship between baseline impedance levels and dilated ISD in children with GER-related cough. Forty children with GERD, 15 children with GrC and 12 controls prospectively underwent oesophagogastroduodenoscopy (EGD) with oesophageal biopsies taken 2-3 cm above squamocolumnar junction. ISD were quantified using transmission electron microscopy. Impedance-pH monitoring with evaluation of baseline impedance in the most distal impedance channel was performed in both patient groups. A significant difference in mean ISD values was found between GrC patients (0.9 ± 0.2 μm) and controls (0.5 ± 0.2 μm, P reflux parameter. Finally, there was no correlation between ISD and distal baseline impedance values (r:-0.35; NS). In children with reflux-related cough, dilated intercellular space diameter appears to be an objective and useful marker of oesophageal mucosal injury regardless of acid exposure, and its evaluation should be considered for those patients where the diagnosis is uncertain. In children with reflux-related cough, baseline impedance levels have no role in identifying reflux-induced oesophageal mucosal ultrastructural changes. © 2014 John Wiley & Sons Ltd.

Vectorial signalling mechanism required for cell-cell communication during sporulation in Bacillus subtilis.

Science.gov (United States)

Diez, Veronica; Schujman, Gustavo E; Gueiros-Filho, Frederico J; de Mendoza, Diego

2012-01-01

Spore formation in Bacillus subtilis takes place in a sporangium consisting of two chambers, the forespore and the mother cell, which are linked by pathways of cell-cell communication. One pathway, which couples the proteolytic activation of the mother cell transcription factor σ(E) to the action of a forespore synthesized signal molecule, SpoIIR, has remained enigmatic. Signalling by SpoIIR requires the protein to be exported to the intermembrane space between forespore and mother cell, where it will interact with and activate the integral membrane protease SpoIIGA. Here we show that SpoIIR signal activity as well as the cleavage of its N-terminal extension is strictly dependent on the prespore fatty acid biosynthetic machinery. We also report that a conserved threonine residue (T27) in SpoIIR is required for processing, suggesting that signalling of SpoIIR is dependent on fatty acid synthesis probably because of acylation of T27. In addition, SpoIIR localization in the forespore septal membrane depends on the presence of SpoIIGA. The orchestration of σ(E) activation in the intercellular space by an acylated signal protein provides a new paradigm to ensure local transmission of a weak signal across the bilayer to control cell-cell communication during development. © 2011 Blackwell Publishing Ltd.

Transfected HEK293 Cells Expressing Functional Recombinant Intercellular Adhesion Molecule 1 (ICAM-1) - A Receptor Associated with Severe Plasmodium falciparum Malaria

DEFF Research Database (Denmark)

Bengtsson, Anja; Joergensen, Louise; Barbati, Zachary R

2013-01-01

Intercellular adhesion molecule 1 (ICAM-1) is a membrane-bound glycoprotein expressed on endothelial cells and cells of the immune system. Human ICAM-1 mediates adhesion and migration of leucocytes, and is implicated in inflammatory pathologies, autoimmune diseases and in many cancer processes....... Additionally, ICAM-1 acts as receptor for pathogens like human rhinovirus and Plasmodium falciparum malaria parasites. A group of related P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains, the DBLβ, mediates ICAM-1 binding of P. falciparum-infected erythrocytes. This ICAM‑1-binding phenotype has...

Coarse Grained Molecular Dynamics Simulations of Transmembrane Protein-Lipid Systems

Directory of Open Access Journals (Sweden)

Peter Spijker

2010-06-01

Full Text Available Many biological cellular processes occur at the micro- or millisecond time scale. With traditional all-atom molecular modeling techniques it is difficult to investigate the dynamics of long time scales or large systems, such as protein aggregation or activation. Coarse graining (CG can be used to reduce the number of degrees of freedom in such a system, and reduce the computational complexity. In this paper the first version of a coarse grained model for transmembrane proteins is presented. This model differs from other coarse grained protein models due to the introduction of a novel angle potential as well as a hydrogen bonding potential. These new potentials are used to stabilize the backbone. The model has been validated by investigating the adaptation of the hydrophobic mismatch induced by the insertion of WALP-peptides into a lipid membrane, showing that the first step in the adaptation is an increase in the membrane thickness, followed by a tilting of the peptide.

Soluble intercellular adhesion molecule-1 (sICAM-1) and soluble interleukin-2 receptors (sIL-2R) in scleroderma skin

DEFF Research Database (Denmark)

Søndergaard, Klaus; Deleuran, Mette; Heickendorff, Lene

1998-01-01

In order to investigate whether soluble intercellular adhesion molecule-1 (sICAM-1) and soluble interleukin-2 receptors (sIL-2R) were present in scleroderma skin, and to compare their levels to concentrations measured in plasma and clinical parameters, we examined suction blister fluid and plasma...... from 13 patients with systemic sclerosis and 11 healthy volunteers. Suction blisters and biopsies were from the transition zone between normal skin and scleroderma, and uninvolved abdominal skin. The levels of sICAM-1 and sIL-2R were significantly increased in both plasma and suction blister fluid from...

Interspecies communication between plant and mouse gut host cells through edible plant derived exosome-like nanoparticles.

Science.gov (United States)

Mu, Jingyao; Zhuang, Xiaoying; Wang, Qilong; Jiang, Hong; Deng, Zhong-Bin; Wang, Baomei; Zhang, Lifeng; Kakar, Sham; Jun, Yan; Miller, Donald; Zhang, Huang-Ge

2014-07-01

Exosomes, small vesicles participating in intercellular communication, have been extensively studied recently; however, the role of edible plant derived exosomes in interspecies communication has not been investigated. Here, we investigate the biological effects of edible plant derived exosome-like nanoparticles (EPDENs) on mammalian cells. In this study, exosome-like nanoparticles from four edible plants were isolated and characterized. We show that these EPDENs contain proteins, lipids, and microRNA. EPDENs are taken up by intestinal macrophages and stem cells. The results generated from EPDEN-transfected macrophages indicate that ginger EPDENs preferentially induce the expression of the antioxidation gene, heme oxygenase-1 and the anti-inflammatory cytokine, IL-10; whereas grapefruit, ginger, and carrot EPDENs promote activation of nuclear factor like (erythroid-derived 2). Furthermore, analysis of the intestines of canonical Wnt-reporter mice, i.e. B6.Cg-Tg(BAT-lacZ)3Picc/J mice, revealed that the numbers of β-galactosidase(+) (β-Gal) intestinal crypts are increased, suggesting that EPDEN treatment of mice leads to Wnt-mediated activation of the TCF4 transcription machinery in the crypts. The data suggest a role for EPDEN-mediated interspecies communication by inducing expression of genes for anti-inflammation cytokines, antioxidation, and activation of Wnt signaling, which are crucial for maintaining intestinal homeostasis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modulation of vesicular catecholamine release from rat PC12 cells

NARCIS (Netherlands)

Westerink, R.H.S.

2002-01-01

Intercellular communication is of vital importance for the nervous system, since the nervous system is the main coordinating system in animals. Nerve cell communication is initiated by the release of chemical messengers, neurotransmitters, from the presynaptic nerve cell. The neurotransmitters, such

Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR).

Science.gov (United States)

Rosenberg, Mark F; Kamis, Alhaji Bukar; Aleksandrov, Luba A; Ford, Robert C; Riordan, John R

2004-09-10

The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that is mutated in patients suffering from cystic fibrosis. Here we report the purification and first crystallization of wild-type human CFTR. Functional characterization of the material showed it to be highly active. Electron crystallography of negatively stained two-dimensional crystals of CFTR has revealed the overall architecture of this channel for two different conformational states. These show a strong structural homology to two conformational states of another eukaryotic ATP-binding cassette transporter, P-glycoprotein. In contrast to P-glycoprotein, however, both conformational states can be observed in the presence of a nucleotide, which may be related to the role of CFTR as an ion channel rather than a transporter. The hypothesis that the two conformations could represent the "open" and "closed" states of the channel is considered.

Simulations of Skin Barrier Function: Free Energies of Hydrophobic and Hydrophilic Transmembrane Pores in Ceramide Bilayers

OpenAIRE

Notman, Rebecca; Anwar, Jamshed; Briels, W. J.; Noro, Massimo G.; den Otter, Wouter K.

2008-01-01

Transmembrane pore formation is central to many biological processes such as ion transport, cell fusion, and viral infection. Furthermore, pore formation in the ceramide bilayers of the stratum corneum may be an important mechanism by which penetration enhancers such as dimethylsulfoxide (DMSO) weaken the barrier function of the skin. We have used the potential of mean constraint force (PMCF) method to calculate the free energy of pore formation in ceramide bilayers in both the innate gel pha...

Requirement of transmembrane domain for CD154 association to lipid rafts and subsequent biological events.

Directory of Open Access Journals (Sweden)

Nadir Benslimane

Full Text Available Interaction of CD40 with CD154 leads to recruitment of both molecules into lipid rafts, resulting in bi-directional cell activation. The precise mechanism by which CD154 is translocated into lipid rafts and its impact on CD154 signaling remain largely unknown. Our aim is to identify the domain of CD154 facilitating its association to lipid rafts and the impact of such association on signaling events and cytokine production. Thus, we generated Jurkat cell lines expressing truncated CD154 lacking the cytoplasmic domain or chimeric CD154 in which the transmembrane domain was replaced by that of transferrin receptor I, known to be excluded from lipid rafts. Our results show that cell stimulation with soluble CD40 leads to the association of CD154 wild-type and CD154-truncated, but not CD154-chimera, with lipid rafts. This is correlated with failure of CD154-chimera to activate Akt and p38 MAP kinases, known effectors of CD154 signaling. We also found that CD154-chimera lost the ability to promote IL-2 production upon T cell stimulation with anti-CD3/CD28 and soluble CD40. These results demonstrate the implication of the transmembrane domain of CD154 in lipid raft association, and that this association is necessary for CD154-mediated Akt and p38 activation with consequent enhancement of IL-2 production.

Dynamic monitoring of transmembrane potential changes: a study of ion channels using an electrical double layer-gated FET biosensor.

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Pulikkathodi, Anil Kumar; Sarangadharan, Indu; Chen, Yi-Hong; Lee, Geng-Yen; Chyi, Jen-Inn; Lee, Gwo-Bin; Wang, Yu-Lin

2018-03-27

In this research, we have designed, fabricated and characterized an electrical double layer (EDL)-gated AlGaN/GaN high electron mobility transistor (HEMT) biosensor array to study the transmembrane potential changes of cells. The sensor array platform is designed to detect and count circulating tumor cells (CTCs) of colorectal cancer (CRC) and investigate cellular bioelectric signals. Using the EDL FET biosensor platform, cellular responses can be studied in physiological salt concentrations, thereby eliminating complex automation. Upon investigation, we discovered that our sensor response follows the transmembrane potential changes of captured cells. Our whole cell sensor platform can be used to monitor the dynamic changes in the membrane potential of cells. The effects of continuously changing electrolyte ion concentrations and ion channel blocking using cadmium are investigated. This methodology has the potential to be used as an electrophysiological probe for studying ion channel gating and the interaction of biomolecules in cells. The sensor can also be a point-of-care diagnostic tool for rapid screening of diseases.

3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction.

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Johanna L Höög

2016-01-01

Full Text Available Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility.We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum.The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life cycle of this human parasite.

Ligand-Receptor Interaction-Mediated Transmembrane Transport of Dendrimer-like Soft Nanoparticles: Mechanisms and Complicated Diffusive Dynamics.

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Liang, Junshi; Chen, Pengyu; Dong, Bojun; Huang, Zihan; Zhao, Kongyin; Yan, Li-Tang

2016-05-09

Nearly all nanomedical applications of dendrimer-like soft nanoparticles rely on the functionality of attached ligands. Understanding how the ligands interact with the receptors in cell membrane and its further effect on the cellular uptake of dendrimer-like soft nanoparticles is thereby a key issue for their better application in nanomedicine. However, the essential mechanism and detailed kinetics for the ligand-receptor interaction-mediated transmembrane transport of such unconventional nanoparticles remain poorly elucidated. Here, using coarse-grained simulations, we present the very first study of molecular mechanism and kinetics behaviors for the transmembrane transport of dendrimer-like soft nanoparticles conjugated with ligands. A phase diagram of interaction states is constructed through examining ligand densities and membrane tensions that allows us to identify novel endocytosis mechanisms featured by the direct wrapping and the penetration-extraction vesiculation. The results provide an in-depth insight into the diffusivity of receptors and dendrimer in the membrane plane and demonstrate how the ligand density influences receptor diffusion and uptake kinetics. It is interesting to find that the ligand-conjugated dendrimers present superdiffusive behaviors on a membrane, which is revealed to be driven by the random fluctuation dynamics of the membrane. The findings facilitate our understanding of some recent experimental observations and could establish fundamental principles for the future development of such important nanomaterials for widespread nanomedical applications.

Association of Intercellular Adhesion Molecule 1 (ICAM1 with Diabetes and Diabetic Nephropathy

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Harvest F Gu

2013-01-01

Full Text Available Diabetes and diabetic nephropathy are complex diseases affected by genetic and environmental factors. Identification of the susceptibility genes and investigation of their roles may provide useful information for better understanding of the pathogenesis and for developing novel therapeutic approaches. Intercellular adhesion molecule 1 (ICAM1 is a cell surface glycoprotein expressed on endothelial cells and leukocytes in the immune system. The ICAM1 gene is located on chromosome 19p13 within the linkage region of diabetes. In the recent years, accumulating reports have implicated that genetic polymorphisms in the ICAM1 gene are associated with diabetes and diabetic nephropathy. Serum ICAM1 levels in diabetes patients and the icam1 gene expression in kidney tissues of diabetic animals are increased compared to the controls. Therefore, ICAM1 may play a role in the development of diabetes and diabetic nephropathy. In this review, we present genomic structure, variation and regulation of the ICAM1 gene, summarized genetic and biological studies of this gene in diabetes and diabetic nephropathy and discussed about the potential application using ICAM1 as a biomarker and target for prediction and treatment of diabetes and diabetic nephropathy.

Loss of intercellular adhesion leads to differential accumulation of hypericin in bladder cancer

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Lucky, S. Sasidharan; Bhuvaneswari, Ramaswamy; Chin, William W. L.; Lau, Weber K. O.; Olivo, Malini C. D.

2009-06-01

Photodynamic diagnosis (PDD) exploits the photoactive nature of certain compounds, namely photosensitizers, in order to enhance the visual demarcation between normal and neoplastic tissue. Hypericin is one such potent photosensitizer that preferentially accumulate in neoplastic tissue, and fluoresce in the visible spectrum when illuminated with light of an appropriate wavelength. In our study, we investigated the role of E-cadherin in the selective permeation of hypericin in bladder cancer tissues. Clinical studies were done on a series of 43 histologically graded bladder cancer biopsy specimens, obtained from 28 patients who received intravesical instillations with 8μM hypericin solution for at least 2 hours. Immunohistochemical staining was used to assess the expression of E-cadherin, in the cryosectioned tissues. Hypericin uptake was examined by fluorescence microscopy. Immunohistochemical staining showed a clear expression of E-cadherin along the urothelial lining of the normal and pre-malignant tissues. Partial expression of these cell adhesion molecules were still observed in malignant tissues, however there was a loss of expression to variable extends along the urothelium. Thus, loss of intercellular adhesion can be associated with enhanced hypericin permeation through paracellular diffusion.

Intercellular adhesion molecule-1 augments myoblast adhesion and fusion through homophilic trans-interactions.

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Pizza, Francis X; Martin, Ryan A; Springer, Evan M; Leffler, Maxwell S; Woelmer, Bryce R; Recker, Isaac J; Leaman, Douglas W

2017-07-11

The overall objective of the study was to identify mechanisms through which intercellular adhesion molecule-1 (ICAM-1) augments the adhesive and fusogenic properties of myogenic cells. Hypotheses were tested using cultured myoblasts and fibroblasts, which do not constitutively express ICAM-1, and myoblasts and fibroblasts forced to express full length ICAM-1 or a truncated form lacking the cytoplasmic domain of ICAM-1. ICAM-1 mediated myoblast adhesion and fusion were quantified using novel assays and cell mixing experiments. We report that ICAM-1 augments myoblast adhesion to myoblasts and myotubes through homophilic trans-interactions. Such adhesive interactions enhanced levels of active Rac in adherent and fusing myoblasts, as well as triggered lamellipodia, spreading, and fusion of myoblasts through the signaling function of the cytoplasmic domain of ICAM-1. Rac inhibition negated ICAM-1 mediated lamellipodia, spreading, and fusion of myoblasts. The fusogenic property of ICAM-1-ICAM-1 interactions was restricted to myogenic cells, as forced expression of ICAM-1 by fibroblasts did not augment their fusion to ICAM-1+ myoblasts/myotubes. We conclude that ICAM-1 augments myoblast adhesion and fusion through its ability to self-associate and initiate Rac-mediated remodeling of the actin cytoskeleton.

Azorhizobium caulinodans Transmembrane Chemoreceptor TlpA1 Involved in Host Colonization and Nodulation on Roots and Stems

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Wei Liu

2017-07-01

Full Text Available Azorhizobium caulinodans ORS571 is a motile soil bacterium that interacts symbiotically with legume host Sesbania rostrata, forming nitrogen-fixing root and stem nodules. Bacterial chemotaxis plays an important role in establishing this symbiotic relationship. To determine the contribution of chemotaxis to symbiosis in A. caulinodans ORS571-S. rostrata, we characterized the function of TlpA1 (transducer-like protein in A. caulinodans, a chemoreceptor predicted by SMART (Simple Modular Architecture Research Tool, containing two N-terminal transmembrane regions. The tlpA1 gene is located immediately upstream of the unique che gene cluster and is transcriptionally co-oriented. We found that a ΔtlpA1 mutant is severely impaired for chemotaxis to various organic acids, glycerol and proline. Furthermore, biofilm forming ability of the strain carrying the mutation is reduced under certain growth conditions. Interestingly, competitive colonization ability on S. rostrata root surfaces is impaired in the ΔtlpA1 mutant, suggesting that chemotaxis of the A. caulinodans ORS571 contributes to root colonization. We also found that TlpA1 promotes competitive nodulation not only on roots but also on stems of S. rostrata. Taken together, our data strongly suggest that TlpA1 is a transmembrane chemoreceptor involved in A. caulinodans-S. rostrata symbiosis.

Corruption of host seven-transmembrane proteins by pathogenic microbes: a common theme in animals and plants?

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Panstruga, Ralph; Schulze-Lefert, Paul

2003-04-01

Human diseases like AIDS, malaria, and pneumonia are caused by pathogens that corrupt host chemokine G-protein coupled receptors for molecular docking. Comparatively, little is known about plant host factors that are required for pathogenesis and that may serve as receptors for the entry of pathogenic microbes. Here, we review potential analogies between human chemokine receptors and the plant seven-transmembrane MLO protein, a candidate serving a dual role as docking molecule and defence modulator for the phytopathogenic powdery mildew fungus.

The psychostimulant modafinil enhances gap junctional communication in cortical astrocytes.

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Liu, Xinhe; Petit, Jean-Marie; Ezan, Pascal; Gyger, Joël; Magistretti, Pierre; Giaume, Christian

2013-12-01

Sleep-wake cycle is characterized by changes in neuronal network activity. However, for the last decade there is increasing evidence that neuroglial interaction may play a role in the modulation of sleep homeostasis and that astrocytes have a critical impact in this process. Interestingly, astrocytes are organized into communicating networks based on their high expression of connexins, which are the molecular constituents of gap junction channels. Thus, neuroglial interactions should also be considered as the result of the interplay between neuronal and astroglial networks. Here, we investigate the effect of modafinil, a wakefulness-promoting agent, on astrocyte gap junctional communication. We report that in the cortex modafinil injection increases the expression of mRNA and protein of connexin 30 but not those of connexin 43, the other major astroglial connexin. These increases are correlated with an enhancement of intercellular dye coupling in cortical astrocytes, which is abolished when neuronal activity is silenced by tetrodotoxin. Moreover, gamma-hydroxybutyric acid, which at a millimolar concentration induces sleep, has an opposite effect on astroglial gap junctions in an activity-independent manner. These results support the proposition that astroglia may play an important role in complex physiological brain functions, such as sleep regulation, and that neuroglial networking interaction is modified during sleep-wake cycle. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'. Copyright © 2013. Published by Elsevier Ltd.

Regulation of Endothelial Adherens Junctions by Tyrosine Phosphorylation

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Adam, Alejandro Pablo

2015-01-01

Endothelial cells form a semipermeable, regulated barrier that limits the passage of fluid, small molecules, and leukocytes between the bloodstream and the surrounding tissues. The adherens junction, a major mechanism of intercellular adhesion, is comprised of transmembrane cadherins forming homotypic interactions between adjacent cells and associated cytoplasmic catenins linking the cadherins to the cytoskeleton. Inflammatory conditions promote the disassembly of the adherens junction and a loss of intercellular adhesion, creating openings or gaps in the endothelium through which small molecules diffuse and leukocytes transmigrate. Tyrosine kinase signaling has emerged as a central regulator of the inflammatory response, partly through direct phosphorylation and dephosphorylation of the adherens junction components. This review discusses the findings that support and those that argue against a direct effect of cadherin and catenin phosphorylation in the disassembly of the adherens junction. Recent findings indicate a complex interaction between kinases, phosphatases, and the adherens junction components that allow a fine regulation of the endothelial permeability to small molecules, leukocyte migration, and barrier resealing. PMID:26556953

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator

NARCIS (Netherlands)

A. Luciani (Alessandro); V.R. Villella (Valeria Rachela); S. Esposito (Susanna); M. Gavina (Manuela); I. Russo (Ilaria); M. Silano (Marco); S. Guido (Stefano); M. Pettoello-Mantovani (Massimo); R. Carnuccio (Rosa); B.J. Scholte (Bob); A. de Matteis (Antonella); M.C. Maiuri (Maria Chiara); V. Raia (Valeria); A. Luini (Alberto); H.K. Kroemer (Heyo); L. Maiuri (Luigi)

2012-01-01

textabstractChannel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded

Intercellular communication in Helicobacter pylori: luxS is essential for the production of an extracellular signaling molecule.

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Forsyth, M H; Cover, T L

2000-06-01

Individual bacteria of numerous species can communicate and coordinate their actions via the production, release, and detection of extracellular signaling molecules. In this study, we used the Vibrio harveyi luminescence bioassay to determine whether Helicobacter pylori produces such a factor. Cell-free conditioned media from H. pylori strains 60190 and 26695 each induced >100-fold-greater luminescence in V. harveyi than did sterile culture medium. The H. pylori signaling molecule had a molecular mass of 100-fold-greater luminescence in the V. harveyi bioassay than did conditioned medium from either mutant strain. Production of the signaling molecule was restored in an H. pylori luxS null mutant strain by complementation with a single intact copy of luxS placed in a heterologous site on the chromosome. In addition, Escherichia coli DH5alpha produced autoinducer activity following the introduction of an intact copy of luxS from H. pylori. Production of the signaling molecule by H. pylori was growth phase dependent, with maximal production occurring in the mid-exponential phase of growth. Transcription of H. pylori vacA also was growth phase dependent, but this phenomenon was not dependent on luxS activity. These data indicate that H. pylori produces an extracellular signaling molecule related to AI-2 from V. harveyi. We speculate that this signaling molecule may play a role in regulating H. pylori gene expression.

Transmembrane Peptides as Sensors of the Membrane Physical State

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Stefano Piotto

2018-05-01

Full Text Available Cell membranes are commonly considered fundamental structures having multiple roles such as confinement, storage of lipids, sustain and control of membrane proteins. In spite of their importance, many aspects remain unclear. The number of lipid types is orders of magnitude larger than the number of amino acids, and this compositional complexity is not clearly embedded in any membrane model. A diffused hypothesis is that the large lipid palette permits to recruit and organize specific proteins controlling the formation of specialized lipid domains and the lateral pressure profile of the bilayer. Unfortunately, a satisfactory knowledge of lipid abundance remains utopian because of the technical difficulties in isolating definite membrane regions. More importantly, a theoretical framework where to fit the lipidomic data is still missing. In this work, we wish to utilize the amino acid sequence and frequency of the membrane proteins as bioinformatics sensors of cell bilayers. The use of an alignment-free method to find a correlation between the sequences of transmembrane portion of membrane proteins with the membrane physical state (MPS suggested a new approach for the discovery of antimicrobial peptides.

Research Advances in CKLFSF-like MARVEL Transmembrane Domain Containing Member 3.

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Hu, Feng-zhan; Sheng, Zheng-zuo; Qin, Cai-peng; Xu, Tao

2016-06-10

CKLF-like MARVEL transmembrane domain containing member/chemokine-like factor super family member (CKLFSF/CMTM) is a novel tumor suppressor gene. CMTM3 is broadly expressed in normal human tissues and evolutionary conserved,especially in testis,spleen,and some cells of peripheral blood mononuclear cells. However,its expression is undetectable or down-regulated in most carcinoma cell lines and tissues. Restoration of CMTM3 may inhibit the proliferation,migration,and invasion of carcinoma cells. Although the exact mechanism of its anti-tumor activity remains unclear,CKLFSF3/CMTM3 is closely connected with immune system and associated with sex during tumorigenesis. The study advances of CKLFSF3/CMTM3 are elaborated in this review as CMTM3 may be a new target in the gene therapies for tumors,especially genitourinary tumors,while further studies on CMTM3 and its anti-tumor mechanisms are warranted.

The Self-Identity Protein IdsD Is Communicated between Cells in Swarming Proteus mirabilis Colonies.

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Saak, Christina C; Gibbs, Karine A

2016-12-15

Proteus mirabilis is a social bacterium that is capable of self (kin) versus nonself recognition. Swarming colonies of this bacterium expand outward on surfaces to centimeter-scale distances due to the collective motility of individual cells. Colonies of genetically distinct populations remain separate, while those of identical populations merge. Ids proteins are essential for this recognition behavior. Two of these proteins, IdsD and IdsE, encode identity information for each strain. These two proteins bind in vitro in an allele-restrictive manner. IdsD-IdsE binding is correlated with the merging of populations, whereas a lack of binding is correlated with the separation of populations. Key questions remained about the in vivo interactions of IdsD and IdsE, specifically, whether IdsD and IdsE bind within single cells or whether IdsD-IdsE interactions occur across neighboring cells and, if so, which of the two proteins is exchanged. Here we demonstrate that IdsD must originate from another cell to communicate identity and that this nonresident IdsD interacts with IdsE resident in the recipient cell. Furthermore, we show that unbound IdsD in recipient cells does not cause cell death and instead appears to contribute to a restriction in the expansion radius of the swarming colony. We conclude that P. mirabilis communicates IdsD between neighboring cells for nonlethal kin recognition, which suggests that the Ids proteins constitute a type of cell-cell communication. We demonstrate that self (kin) versus nonself recognition in P. mirabilis entails the cell-cell communication of an identity-encoding protein that is exported from one cell and received by another. We further show that this intercellular exchange affects swarm colony expansion in a nonlethal manner, which adds social communication to the list of potential swarm-related regulatory factors. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Monitoring of the Proton Electrochemical Gradient in Reconstituted Vesicles: Quantitative Measurements of Both Transmembrane Potential and Intravesicular pH by Ratiometric Fluorescent Probes

Czech Academy of Sciences Publication Activity Database

Holoubek, A.; Večeř, J.; Sigler, Karel

2007-01-01

Roč. 17, - (2007), s. 201-213 ISSN 1053-0509 Institutional research plan: CEZ:AV0Z50200510 Keywords : transmembrane potential * intracellular ph * oxonol dyes Subject RIV: EE - Microbiology, Virology Impact factor: 2.101, year: 2007

Impact of biofilm accumulation on transmembrane and feed channel pressure drop: Effects of crossflow velocity, feed spacer and biodegradable nutrient

KAUST Repository

Dreszer, C.

2014-03-01

Biofilm formation causes performance loss in spiral-wound membrane systems. In this study a microfiltration membrane was used in experiments to simulate fouling in spiral-wound reverse osmosis (RO) and nanofiltration (NF) membrane modules without the influence of concentration polarization. The resistance of a microfiltration membrane is much lower than the intrinsic biofilm resistance, enabling the detection of biofilm accumulation in an early stage. The impact of biofilm accumulation on the transmembrane (biofilm) resistance and feed channel pressure drop as a function of the crossflow velocity (0.05 and 0.20ms-1) and feed spacer presence was studied in transparent membrane biofouling monitors operated at a permeate flux of 20Lm-2h-1. As biodegradable nutrient, acetate was dosed to the feed water (1.0 and 0.25mgL-1 carbon) to enhance biofilm accumulation in the monitors. The studies showed that biofilm formation caused an increased transmembrane resistance and feed channel pressure drop. The effect was strongest at the highest crossflow velocity (0.2ms-1) and in the presence of a feed spacer. Simulating conditions as currently applied in nanofiltration and reverse osmosis installations (crossflow velocity 0.2ms-1 and standard feed spacer) showed that the impact of biofilm formation on performance, in terms of transmembrane and feed channel pressure drop, was strong. This emphasized the importance of hydrodynamics and feed spacer design. Biomass accumulation was related to the nutrient load (nutrient concentration and linear flow velocity). Reducing the nutrient concentration of the feed water enabled the application of higher crossflow velocities. Pretreatment to remove biodegradable nutrient and removal of biomass from the membrane elements played an important part to prevent or restrict biofouling. © 2013 Elsevier Ltd.

In vivo evidence for a functional role of both tumor necrosis factor (TNF) receptors and transmembrane TNF in experimental hepatitis.

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Küsters, S; Tiegs, G; Alexopoulou, L; Pasparakis, M; Douni, E; Künstle, G; Bluethmann, H; Wendel, A; Pfizenmaier, K; Kollias, G; Grell, M

1997-11-01

The significance of tumor necrosis factor receptor 1 (TNFR1) for TNF function in vivo is well documented, whereas the role of TNFR2 so far remains obscure. In a model of concanavalin A (Con A)-induced, CD4+ T cell-dependent experimental hepatitis in mice, in which TNF is a central mediator of apoptotic and necrotic liver damage, we now provide evidence for an essential in vivo function of TNFR2 in this pathophysiological process. We demonstrate that a cooperation of TNFR1 and TNFR2 is required for hepatotoxicity as mice deficient of either receptor were resistant against Con A. A significant role of TNFR2 for Con A-induced hepatitis is also shown by the enhanced sensitivity of transgenic mice overexpressing the human TNFR2. The ligand for cytotoxic signaling via both TNF receptors is the precursor of soluble TNF, i.e. transmembrane TNF. Indeed, transmembrane TNF is sufficient to mediate hepatic damage, as transgenic mice deficient in wild-type soluble TNF but expressing a mutated nonsecretable form of TNF developed inflammatory liver disease.

Connexin Communication Compartments and Wound Repair in Epithelial Tissue

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Marc Chanson

2018-05-01

Full Text Available Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems.

Regulation of Connexin-Based Channels by Fatty Acids

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Puebla, Carlos; Retamal, Mauricio A.; Acuña, Rodrigo; Sáez, Juan C.

2017-01-01

In this mini-review, we briefly summarize the current knowledge about the effects of fatty acids (FAs) on connexin-based channels, as well as discuss the limited information about the impact FAs may have on pannexins (Panxs). FAs regulate diverse cellular functions, some of which are explained by changes in the activity of channels constituted by connexins (Cxs) or Panxs, which are known to play critical roles in maintaining the functional integrity of diverse organs and tissues. Cxs are transmembrane proteins that oligomerize into hexamers to form hemichannels (HCs), which in turn can assemble into dodecamers to form gap junction channels (GJCs). While GJCs communicate the cytoplasm of contacting cells, HCs serve as pathways for the exchange of ions and small molecules between the intra and extracellular milieu. Panxs, as well as Cx HCs, form channels at the plasma membrane that enable the interchange of molecules between the intra and extracellular spaces. Both Cx- and Panx-based channels are controlled by several post-translational modifications. However, the mechanism of action of FAs on these channels has not been described in detail. It has been shown however that FAs frequently decrease GJC-mediated cell-cell communication. The opposite effect also has been described for HC or Panx-dependent intercellular communication, where, the acute FA effect can be reversed upon washout. Additionally, changes in GJCs mediated by FAs have been associated with post-translational modifications (e.g., phosphorylation), and seem to be directly related to chemical properties of FAs (e.g., length of carbon chain and/or degree of saturation), but this possible link remains poorly understood. PMID:28174541

Sustained inhibition of rat myometrial gap junctions and contractions by lindane

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Grindatti Carmen M

2003-10-01

Full Text Available Abstract Background Gap junctions increase in size and abundance coincident with parturition, forming an intercellular communication network that permits the uterus to develop the forceful, coordinated contractions necessary for delivery of the fetus. Lindane, a pesticide used in the human and veterinary treatment of scabies and lice as well as in agricultural applications, inhibits uterine contractions in vitro, inhibits myometrial gap junctions, and has been associated with prolonged gestation length in rats. The aim of the present study was to investigate whether brief exposures to lindane would elicit sustained inhibition of rat uterine contractile activity and myometrial gap junction intercellular communication. Methods To examine effects on uterine contraction, longitudinal uterine strips isolated from late gestation (day 20 rats were exposed to lindane in muscle baths and monitored for changes in spontaneous phasic contractions during and after exposure to lindane. Lucifer yellow dye transfer between myometrial cells in culture was used to monitor gap junction intercellular communication. Results During a 1-h exposure, 10 micro M and 100 micro M lindane decreased peak force and frequency of uterine contraction but 1 micro M lindane did not. After removal of the exposure buffer, contraction force remained significantly depressed in uterine strips exposed to 100 micro M lindane, returning to less than 50% basal levels 5 h after cessation of lindane exposure. In cultured myometrial myocytes, significant sustained inhibition of Lucifer yellow dye transfer was observed 24 h after lindane exposures as brief as 10 min and as low as 0.1 micro M lindane. Conclusion Brief in vitro exposures to lindane have long-term effects on myometrial functions that are necessary for parturition, inhibiting spontaneous phasic contractions in late gestation rat uterus and gap junction intercellular communication in myometrial cell cultures.

Two novel mutations in the sixth transmembrane segment of the thyrotropin receptor gene causing hyperfunctioning thyroid nodules.

Science.gov (United States)

Gozu, Hulya; Avsar, Melike; Bircan, Rifat; Claus, Maren; Sahin, Serap; Sezgin, Ozlem; Deyneli, Oguzhan; Paschke, Ralf; Cirakoglu, Beyazit; Akalin, Sema

2005-04-01

Autonomously functioning thyroid nodules (AFTNs) can present as hyperfunctioning adenomas or toxic multinodular goiters. In the last decade, a large number of activating mutations have been identified in the thyrotropin receptor (TSHR) gene in autonomously functioning thyroid nodules. Most have been situated close to, or within the sixth transmembrane segment and third intracellular loop of the TSHR where the receptor interacts with the Gs protein. In this study we describe two novel mutations in the sixth transmembrane segment of the TSHR causing hyperfunctioning thyroid nodules. Genomic DNAs were isolated from four hyperfunctioning thyroid nodules, normal tissues and peripheral leukocytes of two patients with toxic multinodular goiter. After amplifying the related regions, TSHR and G(s)alpha genes were analyzed by single-strand conformation polymorphism (SSCP) analysis. The precise localization of the mutations was identified by automatic DNA sequence analysis. Functional studies were done by site-directed mutagenesis and transfection of a mutant construct into COS-7 cells. We identified two novel TSHR mutations in two hyperfunctioning thyroid nodules: Phe631Val in the first patient and Iso630Met in the second patient. Both mutant receptors display an increase in constitutive stimulation of basal cyclic adenosine monophosphate (cAMP) levels compared to the wild-type receptor. This confirms that these mutant receptors cause hyperfunctioning thyroid nodules.

Solid state deuterium nuclear magnetic resonance detection of transmembrane-potential-driven tetraphenylphosphonium redistribution across Giant Unilamellar Vesicle bilayers

International Nuclear Information System (INIS)

Franzin, Carla Maria Mirella

1995-01-01

It has been demonstrated that deuterium nuclear magnetic resonance ( 2 H NMR) of Giant Unilamellar Vesicles (GUVs) consisting of specifically choline-deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), plus 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and cholesterol can be used to monitor the transbilayer redistribution of tetraphenylphosphonium (TPP + ) in response to a transmembrane potential (δψ tm ). The 2 H quadrupolar splittings (δν Q 's) measured reflect the level of TPP + bound at the membrane surface due to the latter's effect on the membrane surface electrostatic potential, ψ s . Results reveal the appearance of two distinct δν Q 's, due to differences in bound TPP + at the inner versus the outer monolayer in response to a δψ tm . The observed values of the δν Q 's agree with theoretical predictions based on a derived mathematical model that takes into account δψ tm , plus ψ s , plus the equilibrium binding of TPP + from solution onto the membrane surface, plus the sensitivity of δν Q to the amount of bound TPP + . This model identifies experimental factors that lead to improvements in spectral resolution. Henceforth, 2 H NMR is a valuable tool for quantifying transmembrane asymmetries of ψ s . (author)

Gap junction modulation by extracellular signaling molecules: the thymus model

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Alves L.A.

2000-01-01

Full Text Available Gap junctions are intercellular channels which connect adjacent cells and allow direct exchange of molecules of low molecular weight between them. Such a communication has been described as fundamental in many systems due to its importance in coordination, proliferation and differentiation. Recently, it has been shown that gap junctional intercellular communication (GJIC can be modulated by several extracellular soluble factors such as classical hormones, neurotransmitters, interleukins, growth factors and some paracrine substances. Herein, we discuss some aspects of the general modulation of GJIC by extracellular messenger molecules and more particularly the regulation of such communication in the thymus gland. Additionally, we discuss recent data concerning the study of different neuropeptides and hormones in the modulation of GJIC in thymic epithelial cells. We also suggest that the thymus may be viewed as a model to study the modulation of gap junction communication by different extracellular messengers involved in non-classical circuits, since this organ is under bidirectional neuroimmunoendocrine control.

Biased and Constitutive Signaling in the CC-Chemokine Receptor CCR5 by manipulating the Interface between Transmembrane Helix 6 and 7

DEFF Research Database (Denmark)

Steen, Anne; Thiele, Stefanie; Guo, Dong

2013-01-01

The equilibrium state of CCR5 is manipulated here toward either activation or inactivation by introduction of single amino acid substitutions in the transmembrane domains (TMs) 6 and 7. Insertion of a steric hindrance mutation in the center of TM7 (G286F in position VII:09/7.42) resulted in biase...

Molecular dynamics simulation of the thermosensitivity of the human connexin 26 hemichannel

Science.gov (United States)

Alizadeh, Hadi; Davoodi, Jamal; Zeilinger, Carsten; Rafii-Tabar, Hashem

2018-01-01

Connexin hemichannels mediate cytoplasm and extracellular milieu communication by exchanging a variety of cytoplasmic molecules and ions. These hemichannels can be regulated by external stimuli such as mechanical stress, applied voltage, pH and temperature changes. Although there are many studies on structures and functions of connexin 26 in contexts of pH, ion concentration and voltage, employing computational methods, no such study has been performed so far involving temperature changes. In this study, using molecular dynamics simulation, we investigate thermosensitivity of the human Connexin 26 hemichannel. Our results show that the channel approaches a structurally closed state at lower temperature compared to higher temperature. This is in fair agreement with experimental results that indicate channel closure at lower temperature. Furthermore, our MD simulation results show that some regions of connexin 26 hemichannel are more sensitive to temperature compared to other regions. Whereas the intercellular half of the channel does not show any considerable response to temperature during the simulation time accessible in this study, the cytoplasmic half approaches a closed structural state at lower temperature compared to the higher temperature. Specifically, our results suggest that the cytoplasmic loop, the cytoplasmic half of the second transmembrane helix, and the N-terminus helix play a dominant role in temperature gating.

Clustering and negative feedback by endocytosis in planar cell polarity signaling is modulated by ubiquitinylation of prickle.

Directory of Open Access Journals (Sweden)

Bomsoo Cho

2015-05-01

Full Text Available The core components of the planar cell polarity (PCP signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. We find that the E3 ubiquitin ligase complex Cullin1(Cul1/SkpA/Supernumerary limbs(Slimb regulates the stability of one of the peripheral membrane components, Prickle (Pk. Excess Pk disrupts PCP feedback and prevents asymmetry. We show that Pk participates in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang and Flamingo (Fmi, and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.

NMR studies of transmembrane electron transport in human erythrocytes

International Nuclear Information System (INIS)

Kennett, E.C.; Bubb, W.A.; Kuchel, P.W.

2002-01-01

Full text: Electron transport systems exist in the plasma membranes of all cells. These systems appear to play a role in cell growth and proliferation, intracellular signalling, hormone responses, apoptotic events, cell defence and perhaps most importantly they enable the cell to respond to changes in the redox state of both the intra- and extracellular environments. Previously, 13 C NMR has been used to study transmembrane electron transport in human erythrocytes, specifically the reduction of extracellular 13 C-ferricyanide. NMR is a particularly useful tool for studying such systems as changes in the metabolic state of the cell can be observed concomitantly with extracellular reductase activity. We investigated the oxidation of extracellular NADH by human erythrocytes using 1 H and 31 P NMR spectroscopy. Recent results for glucose-starved human erythrocytes indicate that, under these conditions, extracellular NADH can be oxidised at the plasma membrane with the electron transfer across the membrane resulting in reduction of intracellular NAD + . The activity is inhibited by known trans-plasma membrane electron transport inhibitors (capsaicin and atebrin) and is unaffected by inhibition of the erythrocyte Band 3 anion transporter. These results suggest that electron import from extracellular NADH allows the cell to re-establish a reducing environment after the normal redox balance is disturbed

Phe783, Thr797, and Asp804 in transmembrane hairpin M5-M6 of Na+,K+-ATPase play a key role in ouabain binding.

Science.gov (United States)

Qiu, Li Yan; Koenderink, Jan B; Swarts, Herman G P; Willems, Peter H G M; De Pont, Jan Joep H H M

2003-11-21

Ouabain is a glycoside that binds to and inhibits the action of Na+,K+-ATPase. Little is known, however, about the specific requirements of the protein surface for glycoside binding. Using chimeras of gastric H+,K+-ATPase and Na+,K+-ATPase, we demonstrated previously that the combined presence of transmembrane hairpins M3-M4 and M5-M6 of Na+,K+-ATPase in a backbone of H+,K+-ATPase (HN34/56) is both required and sufficient for high affinity ouabain binding. Since replacement of transmembrane hairpin M3-M4 by the N terminus up to transmembrane segment 3 (HNN3/56) resulted in a low affinity ouabain binding, hairpin M5-M6 seems to be essential for ouabain binding. To assess which residues of M5-M6 are required for ouabain action, we divided this transmembrane hairpin in seven parts and individually replaced these parts by the corresponding sequences of H+,K+-ATPase in chimera HN34/56. Three of these chimeras failed to bind ouabain following expression in Xenopus laevis oocytes. Altogether, these three chimeras contained 7 amino acids that were specific for Na+,K+-ATPase. Individual replacement of these 7 amino acids by the corresponding amino acids in H+,K+-ATPase revealed a dramatic loss of ouabain binding for F783Y, T797C, and D804E. As a proof of principle, the Na+,K+-ATPase equivalents of these 3 amino acids were introduced in different combinations in chimera HN34. The presence of all 3 amino acids appeared to be required for ouabain action. Docking of ouabain onto a three-dimensional-model of Na+,K+-ATPase suggests that Asp804, in contrast to Phe783 and Thr797, does not actually form part of the ouabain-binding pocket. Most likely, the presence of this amino acid is required for adopting of the proper conformation for ouabain binding.

Transmembrane transporter expression regulated by the glucosylceramide pathway in Cryptococcus neoformans.

Science.gov (United States)

Singh, Arpita; Rella, Antonella; Schwacke, John; Vacchi-Suzzi, Caterina; Luberto, Chiara; Del Poeta, Maurizio

2015-11-16

disrupt transmembrane signaling complex, which in turn contributes to cryptococcal osmotic, pH, ion homeostasis and its pathobiology. Six genes identified from gene expression microarrays by gene set enrichment analysis and validated by RT-PCR, are membrane located and associated with the growth defect at neutral-alkaline pH due to the absence and or presence of a structurally modified GlcCer. They may be involved in the transmembrane signaling network in Cryptococcus neoformans, and therefore the pathobiology of the fungus in these conditions.

Biologically Inspired Intercellular Slot Synchronization

Directory of Open Access Journals (Sweden)

Alexander Tyrrell

2009-01-01

Full Text Available The present article develops a decentralized interbase station slot synchronization algorithm suitable for cellular mobile communication systems. The proposed cellular firefly synchronization (CelFSync algorithm is derived from the theory of pulse-coupled oscillators, common to describe synchronization phenomena in biological systems, such as the spontaneous synchronization of fireflies. In order to maintain synchronization among base stations (BSs, even when there is no direct link between adjacent BSs, some selected user terminals (UTs participate in the network synchronization process. Synchronization emerges by exchanging two distinct synchronization words, one transmitted by BSs and the other by active UTs, without any a priori assumption on the initial timing misalignments of BSs and UTs. In large-scale networks with inter-BS site distances up to a few kilometers, propagation delays severely affect the attainable timing accuracy of CelFSync. We show that by an appropriate combination of CelFSync with the timing advance procedure, which aligns uplink transmission of UTs to arrive simultaneously at the BS, a timing accuracy within a fraction of the inter-BS propagation delay is retained.

Increased Throughput in Ion Channel Drug Development and Exploration by Automation of Electrophysiology

DEFF Research Database (Denmark)

Willumsen, N. J.

2006-01-01

Ion channels constitute macromolecular communication gates that are present in the membranes of all living cells. They are crucial for practically any physiological process, either as chemical or electrical signal transducers or as transmembrane routes for the bulk transport of salts. Not surpris......Ion channels constitute macromolecular communication gates that are present in the membranes of all living cells. They are crucial for practically any physiological process, either as chemical or electrical signal transducers or as transmembrane routes for the bulk transport of salts...

The Size of Activating and Inhibitory Killer Ig-like Receptor Nanoclusters Is Controlled by the Transmembrane Sequence and Affects Signaling

Directory of Open Access Journals (Sweden)

Anna Oszmiana

2016-05-01

Full Text Available Super-resolution microscopy has revealed that immune cell receptors are organized in nanoscale clusters at cell surfaces and immune synapses. However, mechanisms and functions for this nanoscale organization remain unclear. Here, we used super-resolution microscopy to compare the surface organization of paired killer Ig-like receptors (KIR, KIR2DL1 and KIR2DS1, on human primary natural killer cells and cell lines. Activating KIR2DS1 assembled in clusters two-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters is controlled by transmembrane amino acid 233, a lysine in KIR2DS1. Super-resolution microscopy also revealed two ways in which the nanoscale clustering of KIR affects signaling. First, KIR2DS1 and DAP12 nanoclusters are juxtaposed in the resting cell state but coalesce upon receptor ligation. Second, quantitative super-resolution microscopy revealed that phosphorylation of the kinase ZAP-70 or phosphatase SHP-1 is favored in larger KIR nanoclusters. Thus, the size of KIR nanoclusters depends on the transmembrane sequence and affects downstream signaling.

Transmembrane topology of the acetylcholine receptor examined in reconstituted vesicles

International Nuclear Information System (INIS)

McCrea, P.D.

1987-01-01

Each of the five acetylcholine receptor (AChR) subunits, α 2 β-γδ, is believed to have the same number of transmembrane crossing and to share the same general folding pattern. AChR isolated from the electric organ of electric fish is predominantly dimeric. We have used this bridge as a marker for the C-terminus of the δ subunit, and presumably that of the other subunits in addition. The disulfide's accessibility to hydrophilic reductants, principally glutathione (GSH), was tested in a reconstituted vesicle system. The reduction of the δ-δ desulfide, as evidenced by the transition of AChrR dimers to monomers, was quantitatively monitored on velocity sedimentation sucrose gradients. Alternatively, the reduction of δ 2 to δ was followed by employing non-reducing SDS-PAGE. Reductants such as GSH were able to access the bridge in intact right-side-out vesicles. No acceleration of this process was evident when the vesicles were disrupted by freeze-thaw or by detergents. Control experiments which determined the rate of reduction of entrapped diphtheria toxin, or that of 3 H-GSH efflux, demonstrated that intact reconstituted vesicles provide an adequate permeability barrier to GSH access of their intravesicular space

Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia.

Directory of Open Access Journals (Sweden)

Fumio Yoshikawa

Full Text Available BACKGROUND: Phospholipase D (PLD catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x(4-Asp (HKD motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4. METHODOLOGY/PRINCIPAL FINDINGS: PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity. CONCLUSIONS/SIGNIFICANCE: Results showed that PLD4 is a non

Hydrophobic interaction between contiguous residues in the S6 transmembrane segment acts as a stimuli integration node in the BK channel

Science.gov (United States)

Carrasquel-Ursulaez, Willy; Contreras, Gustavo F.; Sepúlveda, Romina V.; Aguayo, Daniel; González-Nilo, Fernando

2015-01-01

Large-conductance Ca2+- and voltage-activated K+ channel (BK) open probability is enhanced by depolarization, increasing Ca2+ concentration, or both. These stimuli activate modular voltage and Ca2+ sensors that are allosterically coupled to channel gating. Here, we report a point mutation of a phenylalanine (F380A) in the S6 transmembrane helix that, in the absence of internal Ca2+, profoundly hinders channel opening while showing only minor effects on the voltage sensor active–resting equilibrium. Interpretation of these results using an allosteric model suggests that the F380A mutation greatly increases the free energy difference between open and closed states and uncouples Ca2+ binding from voltage sensor activation and voltage sensor activation from channel opening. However, the presence of a bulky and more hydrophobic amino acid in the F380 position (F380W) increases the intrinsic open–closed equilibrium, weakening the coupling between both sensors with the pore domain. Based on these functional experiments and molecular dynamics simulations, we propose that F380 interacts with another S6 hydrophobic residue (L377) in contiguous subunits. This pair forms a hydrophobic ring important in determining the open–closed equilibrium and, like an integration node, participates in the communication between sensors and between the sensors and pore. Moreover, because of its effects on open probabilities, the F380A mutant can be used for detailed voltage sensor experiments in the presence of permeant cations. PMID:25548136

Regulation of KV channel voltage-dependent activation by transmembrane β subunits

Directory of Open Access Journals (Sweden)

Xiaohui eSun

2012-04-01

Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

Critical role of gap junction communication, calcium and nitric oxide signaling in bystander responses to focal photodynamic injury.

Science.gov (United States)

Calì, Bianca; Ceolin, Stefano; Ceriani, Federico; Bortolozzi, Mario; Agnellini, Andrielly H R; Zorzi, Veronica; Predonzani, Andrea; Bronte, Vincenzo; Molon, Barbara; Mammano, Fabio

2015-04-30

Ionizing and nonionizing radiation affect not only directly targeted cells but also surrounding "bystander" cells. The underlying mechanisms and therapeutic role of bystander responses remain incompletely defined. Here we show that photosentizer activation in a single cell triggers apoptosis in bystander cancer cells, which are electrically coupled by gap junction channels and support the propagation of a Ca2+ wave initiated in the irradiated cell. The latter also acts as source of nitric oxide (NO) that diffuses to bystander cells, in which NO levels are further increased by a mechanism compatible with Ca(2+)-dependent enzymatic production. We detected similar signals in tumors grown in dorsal skinfold chambers applied to live mice. Pharmacological blockade of connexin channels significantly reduced the extent of apoptosis in bystander cells, consistent with a critical role played by intercellular communication, Ca2+ and NO in the bystander effects triggered by photodynamic therapy.

Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists

DEFF Research Database (Denmark)

Christensen, Gitte L; Kelstrup, Christian D; Lyngsø, Christina

2010-01-01

(q)-dependent and -independent AT(1)R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity......Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The angiotensin II type 1 receptor (AT(1)R) is a prototypical 7TMR...... and quantity of Galpha(q) protein-independent signaling and uncovers novel signaling pathways. We foresee that the amount and diversity of G protein-independent signaling may be more pronounced than previously recognized for other 7TMRs as well. Quantitative mass spectrometry is a promising tool for evaluation...

Synthesis of a designed transmembrane protein by thioether ligation of solubilised segments : Nα-haloacetylated peptides survived resin cleavage using TFA with EDT as scavenger

NARCIS (Netherlands)

Englebretsen, D.R; Choma, C.T.; Robillard, G.T.

1998-01-01

Nα-haloacetylated peptides made by Fmoc solid phase synthesis survived cleavage when EDT was used as a cleavage component. Two segments of a desgned transmembrane protein, one bromoacetylated, the other containing a cysteine, and each bearing a "solubilising tail" peptide, were synthesised by Fmoc

Molecular Simulations of Sequence-Specific Association of Transmembrane Proteins in Lipid Bilayers

Science.gov (United States)

Doxastakis, Manolis; Prakash, Anupam; Janosi, Lorant

2011-03-01

Association of membrane proteins is central in material and information flow across the cellular membranes. Amino-acid sequence and the membrane environment are two critical factors controlling association, however, quantitative knowledge on such contributions is limited. In this work, we study the dimerization of helices in lipid bilayers using extensive parallel Monte Carlo simulations with recently developed algorithms. The dimerization of Glycophorin A is examined employing a coarse-grain model that retains a level of amino-acid specificity, in three different phospholipid bilayers. Association is driven by a balance of protein-protein and lipid-induced interactions with the latter playing a major role at short separations. Following a different approach, the effect of amino-acid sequence is studied using the four transmembrane domains of the epidermal growth factor receptor family in identical lipid environments. Detailed characterization of dimer formation and estimates of the free energy of association reveal that these helices present significant affinity to self-associate with certain dimers forming non-specific interfaces.

Poxvirus-encoded TNF decoy receptors inhibit the biological activity of transmembrane TNF.

Science.gov (United States)

Pontejo, Sergio M; Alejo, Ali; Alcami, Antonio

2015-10-01

Poxviruses encode up to four different soluble TNF receptors, named cytokine response modifier B (CrmB), CrmC, CrmD and CrmE. These proteins mimic the extracellular domain of the cellular TNF receptors to bind and inhibit the activity of TNF and, in some cases, other TNF superfamily ligands. Most of these ligands are released after the enzymic cleavage of a membrane precursor. However, transmembrane TNF (tmTNF) is not only a precursor of soluble TNF but also exerts specific pro-inflammatory and immunological activities. Here, we report that viral TNF receptors bound and inhibited tmTNF and describe some interesting differences in their activity against the soluble cytokine. Thus, CrmE, which does not inhibit mouse soluble TNF, could block murine tmTNF-induced cytotoxicity. We propose that this anti-tmTNF effect should be taken into consideration when assessing the role of viral TNF decoy receptors in the pathogenesis of poxvirus.

cGAS-Mediated Innate Immunity Spreads Intercellularly through HIV-1 Env-Induced Membrane Fusion Sites.

Science.gov (United States)

Xu, Shuting; Ducroux, Aurélie; Ponnurangam, Aparna; Vieyres, Gabrielle; Franz, Sergej; Müsken, Mathias; Zillinger, Thomas; Malassa, Angelina; Ewald, Ellen; Hornung, Veit; Barchet, Winfried; Häussler, Susanne; Pietschmann, Thomas; Goffinet, Christine

2016-10-12

Upon sensing cytoplasmic retroviral DNA in infected cells, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide cGAMP, which activates STING to trigger a type I interferon (IFN) response. We find that membrane fusion-inducing contact between donor cells expressing the HIV envelope (Env) and primary macrophages endogenously expressing the HIV receptor CD4 and coreceptor enable intercellular transfer of cGAMP. This cGAMP exchange results in STING-dependent antiviral IFN responses in target macrophages and protection from HIV infection. Furthermore, under conditions allowing cell-to-cell transmission of HIV-1, infected primary T cells, but not cell-free virions, deliver cGAMP to autologous macrophages through HIV-1 Env and CD4/coreceptor-mediated membrane fusion sites and induce a STING-dependent, but cGAS-independent, IFN response in target cells. Collectively, these findings identify an infection-specific mode of horizontal transfer of cGAMP between primary immune cells that may boost antiviral responses, particularly in infected tissues in which cell-to-cell transmission of virions exceeds cell-free infection. Copyright © 2016 Elsevier Inc. All rights reserved.

Domino-Like Intercellular Delivery of Undecylenic Acid-Conjugated Porous Silicon Nanoparticles for Deep Tumor Penetration.

Science.gov (United States)

Yong, Tuying; Hu, Jun; Zhang, Xiaoqiong; Li, Fuying; Yang, Hao; Gan, Lu; Yang, Xiangliang

2016-10-05

Improving the intratumoral distribution of anticancer agents remains the critical challenge for developing efficient cancer chemotherapy. Luminescent porous silicon nanoparticles (PSiNPs) have attracted considerable attention in the biomedical field especially in drug delivery. Here, we described the lysosomal exocytosis-mediated domino-like intercellular delivery of undecylenic acid-conjugated PSiNPs (UA-PSiNPs) for deep tumor penetration. UA-PSiNPs with significantly improved stability in physiological conditions were internalized into tumor cells by macropinocytosis-, caveolae-, and clathrin-mediated endocytosis and mainly colocalized with Golgi apparatus and lysosomes. Substantial evidence showed that UA-PSiNPs was excreted from cells via lysosomal exocytosis after cellular uptake. The exocytosed UA-PSiNPs induced a domino-like infection of adjacent cancer cells and allowed encapsulated doxorubicin (DOX) to deeply penetrate into both three-dimensional tumor spheroids and in vivo tumors. In addition, DOX-loaded UA-PSiNPs exhibited strong antitumor activity and few side effects in vivo. This study demonstrated that UA-PSiNPs as a drug carrier might be applied for deep tumor penetration, offering a new insight into the design of more efficient delivery systems of anticancer drugs.

Short length transmembrane domains having voluminous exoplasmic halves determine retention of Type II membrane proteins in the Golgi complex

OpenAIRE

Quiroga, Rodrigo; Trenchi, Alejandra; Gonzalez Montoro, Ayelén; Valdez, Javier Esteban; Maccioni, Hugo Jose Fernando

2017-01-01

It is still unclear why some proteins that travel along the secretory pathway are retained in the Golgi complex whereas others make their way to the plasma membrane. Recent bioinformatic analyses on a large number of single-spanning membrane proteins support the hypothesis that specific features of the transmembrane domain (TMD) are relevant to the sorting of these proteins to particular organelles. Here we experimentally test this hypothesis for Golgi and plasma membrane proteins. Using the ...

Overexpression of transmembrane protein 168 in the mouse nucleus accumbens induces anxiety and sensorimotor gating deficit.

Directory of Open Access Journals (Sweden)

Kequan Fu

Full Text Available Transmembrane protein 168 (TMEM168 comprises 697 amino acid residues, including some putative transmembrane domains. It is reported that TMEM168 controls methamphetamine (METH dependence in the nucleus accumbens (NAc of mice. Moreover, a strong link between METH dependence-induced adaptive changes in the brain and mood disorders has been evaluated. In the present study, we investigated the effects of accumbal TMEM168 in a battery of behavioral paradigms. The adeno-associated virus (AAV Tmem168 vector was injected into the NAc of C57BL/6J mice (NAc-TMEM mice. Subsequently, the accumbal TMEM168 mRNA was increased approximately by seven-fold when compared with the NAc-Mock mice (controls. The NAc-TMEM mice reported no change in the locomotor activity, cognitive ability, social interaction, and depression-like behaviors; however, TMEM168 overexpression enhanced anxiety in the elevated-plus maze and light/dark box test. The increased anxiety was reversed by pretreatment with the antianxiety drug diazepam (0.3 mg/kg i.p.. Moreover, the NAc-TMEM mice exhibited decreased prepulse inhibition (PPI in the startle response test, and the induced schizophrenia-like behavior was reversed by pretreatment with the antipsychotic drug risperidone (0.01 mg/kg i.p.. Furthermore, accumbal TMEM168 overexpression decreased the basal levels of extracellular GABA in the NAc and the high K+ (100 mM-stimulated GABA elevation; however, the total contents of GABA in the NAc remained unaffected. These results suggest that the TMEM168-regulated GABAergic neuronal system in the NAc might become a novel target while studying the etiology of anxiety and sensorimotor gating deficits.

Extracellular Vesicles, Tunneling Nanotubes, and Cellular Interplay: Synergies and Missing Links

Science.gov (United States)

Nawaz, Muhammad; Fatima, Farah

2017-01-01

The process of intercellular communication seems to have been a highly conserved evolutionary process. Higher eukaryotes use several means of intercellular communication to address both the changing physiological demands of the body and to fight against diseases. In recent years, there has been an increasing interest in understanding how cell-derived nanovesicles, known as extracellular vesicles (EVs), can function as normal paracrine mediators of intercellular communication, but can also elicit disease progression and may be used for innovative therapies. Over the last decade, a large body of evidence has accumulated to show that cells use cytoplasmic extensions comprising open-ended channels called tunneling nanotubes (TNTs) to connect cells at a long distance and facilitate the exchange of cytoplasmic material. TNTs are a different means of communication to classical gap junctions or cell fusions; since they are characterized by long distance bridging that transfers cytoplasmic organelles and intracellular vesicles between cells and represent the process of heteroplasmy. The role of EVs in cell communication is relatively well-understood, but how TNTs fit into this process is just emerging. The aim of this review is to describe the relationship between TNTs and EVs, and to discuss the synergies between these two crucial processes in the context of normal cellular cross-talk, physiological roles, modulation of immune responses, development of diseases, and their combinatory effects in tissue repair. At the present time this review appears to be the first summary of the implications of the overlapping roles of TNTs and EVs. We believe that a better appreciation of these parallel processes will improve our understanding on how these nanoscale conduits can be utilized as novel tools for targeted therapies. PMID:28770210

Trafficking and function of the cystic fibrosis transmembrane conductance regulator: a complex network of posttranslational modifications

Science.gov (United States)

McClure, Michelle L.; Barnes, Stephen; Brodsky, Jeffrey L.

2016-01-01

Posttranslational modifications add diversity to protein function. Throughout its life cycle, the cystic fibrosis transmembrane conductance regulator (CFTR) undergoes numerous covalent posttranslational modifications (PTMs), including glycosylation, ubiquitination, sumoylation, phosphorylation, and palmitoylation. These modifications regulate key steps during protein biogenesis, such as protein folding, trafficking, stability, function, and association with protein partners and therefore may serve as targets for therapeutic manipulation. More generally, an improved understanding of molecular mechanisms that underlie CFTR PTMs may suggest novel treatment strategies for CF and perhaps other protein conformational diseases. This review provides a comprehensive summary of co- and posttranslational CFTR modifications and their significance with regard to protein biogenesis. PMID:27474090

Population Based Assessment of MHC Class I Antigens Down Regulation as Markers of Increased Risk for Development and Progression of Breast Cancer from Benign Breast Lesions

National Research Council Canada - National Science Library

Worsham, Maria J

2007-01-01

.... The major histocompatibility complex (MHC) class I molecules are found on the cell membrane of all cells in the body and are involved in intercellular communications and in complex interactions with the immune...

Population Based Assessment of MHC Class I Antigens Down Regulation as Markers of Increased Risk for Development and Progression of Breast Cancer from Benign Breast Lesions

National Research Council Canada - National Science Library

Worsham, Maria

2001-01-01

.... The major histocompatibility complex (MHC) class I molecules are found on the cell membrane of all cells in the body and are involved in intercellular communications and in complex interactions with the immune...

Population Based Assessment of MHC Class I Antigens Down Regulation as Markers of Increased Risk for Development and Progression of Breast Cancer from Benign Breast Lesions

National Research Council Canada - National Science Library

Worsham, Maria J; Raju, Usha; Abrams, Judith

2005-01-01

.... The major histocompatibility complex (MHC) class I molecules are found on the cell membrane of all cells in the body and are involved in intercellular communications and in complex interactions with the immune...

Population Based Assessment of MHC Class 1 Antigens Down Regulation as Marker in Increased Risk for Development and Progression of Breast Cancer From Benign Breast Lesions

National Research Council Canada - National Science Library

Worsham, Maria J

2006-01-01

.... The major histocompatibility complex (MHC) class I molecules are found on the cell membrane of all cells in the body and are involved in intercellular communications and in complex interactions with the immune...

Population Based Assessment of MHC Class I Antigens Down Regulation as Markers of Increased Risk for Development and Progression of Breast Cancer From Benign Breast Lesions

National Research Council Canada - National Science Library

Worsham, Maria

2004-01-01

.... The major histocompatibility complex (MHC) class I molecules are found on the cell membrane of all cells in the body and are involved in intercellular communications and in complex interactions with the immune...

Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase.

Directory of Open Access Journals (Sweden)

Steffen Kawelke

Full Text Available Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2 and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2 was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity.

In vitro and in situ intercellular adhesion molecule-1 (ICAM-1) expression by endothelial cells lining a polyester fabric.

Science.gov (United States)

Rémy, M; Valli, N; Brethes, D; Labrugère, C; Porté-Durrieu, M C; Dobrova, N B; Novikova, S P; Gorodkov, A J; Bordenave, L

1999-02-01

In order to improve long-term patency of vascular grafts, the promising concept of endothelial cell seeding is actually under investigation. Our laboratory tested a polyester coated with albumin and chitosan which permits a rapid colonization by human umbilical vein endothelial cells (HUVEC) and it seems relevant to test in vitro the expression of adhesive molecules expressed by cells with regard to the inflammatory process. We studied intercellular adhesion molecule-1 (ICAM-1) expression and focused our work on the determination of ICAM-1 sites expressed per adherent cell lining the biomaterial, thus in situ, in comparison to control HUVEC on plastic wells: the results obtained by binding experiments were correlated to flow cytometry analyses and showed that the polyester does not induce a proinflammatory state and that HUVEC covering the structure are able to respond to a stimulus.

Interactions between charged residues in the transmembrane segments of the voltage-sensing domain in the hERG channel.

Science.gov (United States)

Zhang, M; Liu, J; Jiang, M; Wu, D-M; Sonawane, K; Guy, H R; Tseng, G-N

2005-10-01

Studies on voltage-gated K channels such as Shaker have shown that positive charges in the voltage-sensor (S4) can form salt bridges with negative charges in the surrounding transmembrane segments in a state-dependent manner, and different charge pairings can stabilize the channels in closed or open states. The goal of this study is to identify such charge interactions in the hERG channel. This knowledge can provide constraints on the spatial relationship among transmembrane segments in the channel's voltage-sensing domain, which are necessary for modeling its structure. We first study the effects of reversing S4's positive charges on channel activation. Reversing positive charges at the outer (K525D) and inner (K538D) ends of S4 markedly accelerates hERG activation, whereas reversing the 4 positive charges in between either has no effect or slows activation. We then use the 'mutant cycle analysis' to test whether D456 (outer end of S2) and D411 (inner end of S1) can pair with K525 and K538, respectively. Other positive charges predicted to be able, or unable, to interact with D456 or D411 are also included in the analysis. The results are consistent with predictions based on the distribution of these charged residues, and confirm that there is functional coupling between D456 and K525 and between D411 and K538.

Comparative exploration of hydrogen sulfide and water transmembrane free energy surfaces via orthogonal space tempering free energy sampling.

Science.gov (United States)

Lv, Chao; Aitchison, Erick W; Wu, Dongsheng; Zheng, Lianqing; Cheng, Xiaolin; Yang, Wei

2016-03-05

Hydrogen sulfide (H2 S), a commonly known toxic gas compound, possesses unique chemical features that allow this small solute molecule to quickly diffuse through cell membranes. Taking advantage of the recent orthogonal space tempering (OST) method, we comparatively mapped the transmembrane free energy landscapes of H2 S and its structural analogue, water (H2 O), seeking to decipher the molecular determinants that govern their drastically different permeabilities. As revealed by our OST sampling results, in contrast to the highly polar water solute, hydrogen sulfide is evidently amphipathic, and thus inside membrane is favorably localized at the interfacial region, that is, the interface between the polar head-group and nonpolar acyl chain regions. Because the membrane binding affinity of H2 S is mainly governed by its small hydrophobic moiety and the barrier height inbetween the interfacial region and the membrane center is largely determined by its moderate polarity, the transmembrane free energy barriers to encounter by this toxic molecule are very small. Moreover when H2 S diffuses from the bulk solution to the membrane center, the above two effects nearly cancel each other, so as to lead to a negligible free energy difference. This study not only explains why H2 S can quickly pass through cell membranes but also provides a practical illustration on how to use the OST free energy sampling method to conveniently analyze complex molecular processes. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Regulation of Yersina pestis Virulence by AI-2 Mediated Quorum Sensing

Energy Technology Data Exchange (ETDEWEB)

Segelke, B; Hok, S; Lao, V; Corzett, M; Garcia, E

2010-03-29

The proposed research was motivated by an interest in understanding Y. pestis virulence mechanisms and bacteria cell-cell communication. It is expected that a greater understanding of virulence mechanisms will ultimately lead to biothreat countermeasures and novel therapeutics. Y. pestis is the etiological agent of plague, the most devastating disease in human history. Y. pestis infection has a high mortality rate and a short incubation before mortality. There is no widely available and effective vaccine for Y. pestis and multi-drug resistant strains are emerging. Y. pestis is a recognized biothreat agent based on the wide distribution of the bacteria in research laboratories around the world and on the knowledge that methods exist to produce and aerosolize large amounts of bacteria. We hypothesized that cell-cell communication via signaling molecules, or quorum sensing, by Y. pestis is important for the regulation of virulence factor gene expression during host invasion, though a causative link had never been established. Quorum sensing is a mode of intercellular communication which enables orchestration of gene expression for many bacteria as a function of population density and available evidence suggests there may be a link between quorum sensing and regulation of Y. pesits virulence. Several pathogenic bacteria have been shown to regulate expression of virulence factor genes, including genes encoding type III secretion, via quorum sensing. The Y. pestis genome encodes several cell-cell signaling pathways and the interaction of at least three of these are thought to be involved in one or more modes of host invasion. Furthermore, Y. pestis gene expression array studies carried out at LLNL have established a correlation between expression of known virulence factors and genes involved in processing of the AI-2 quorum sensing signal. This was a basic research project that was intended to provide new insights into bacterial intercellular communication and how it is

Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis

International Nuclear Information System (INIS)

Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T.; Pierre, Philippe; Chadee, Deborah N.; Pizza, Francis X.

2015-01-01

We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast–myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube–myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube–myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. - Highlights: • We examined mechanisms through which skeletal muscle cell expression of ICAM-1 facilitates events of in vitro myogenesis. • Expression of ICAM-1 by cultured myoblasts did not influence their ability to proliferate or differentiate. • Skeletal muscle cell expression of ICAM-1 augmented myoblast fusion, myotube alignment, myotube–myotube fusion, and myotube size. • ICAM-1 augmented myogenic processes through

Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis

Energy Technology Data Exchange (ETDEWEB)

Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T. [Department of Kinesiology, The University of Toledo, Toledo, OH (United States); Pierre, Philippe [Centre d’Immunologie de Marseille-Luminy U2M, Aix-Marseille Université, Marseille (France); INSERM U631, Institut National de la Santé et Recherche Médicale, Marseille (France); CNRS UMR6102, Centre National de la Recherche Scientifique, Marseille (France); Chadee, Deborah N. [Department of Biological Sciences, The University of Toledo, Toledo, OH (United States); Pizza, Francis X., E-mail: Francis.Pizza@utoledo.edu [Department of Kinesiology, The University of Toledo, Toledo, OH (United States)

2015-02-15

We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast–myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube–myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube–myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. - Highlights: • We examined mechanisms through which skeletal muscle cell expression of ICAM-1 facilitates events of in vitro myogenesis. • Expression of ICAM-1 by cultured myoblasts did not influence their ability to proliferate or differentiate. • Skeletal muscle cell expression of ICAM-1 augmented myoblast fusion, myotube alignment, myotube–myotube fusion, and myotube size. • ICAM-1 augmented myogenic processes through

Intercultural Communication Ethics and Communication Competence%Intercultural Communication Ethics and Communication Competence

Institute of Scientific and Technical Information of China (English)

时婷洁

2012-01-01

This paper investigates intercultural communication ethics is a vital element to promote intercultural communication competence. Firstly, it defines the concept of intercultural communication ethics; Secondly, it illustrates the relation between ethics and the key point of intercultural communication competence; and finally addresses how intercultural communication ethics can improve intercultural communication competence.

Stability of Intercellular Exchange of Biochemical Substances Affected by Variability of Environmental Parameters

Science.gov (United States)

Mihailović, Dragutin T.; Budinčević, Mirko; Balaž, Igor; Mihailović, Anja

Communication between cells is realized by exchange of biochemical substances. Due to internal organization of living systems and variability of external parameters, the exchange is heavily influenced by perturbations of various parameters at almost all stages of the process. Since communication is one of essential processes for functioning of living systems it is of interest to investigate conditions for its stability. Using previously developed simplified model of bacterial communication in a form of coupled difference logistic equations we investigate stability of exchange of signaling molecules under variability of internal and external parameters.

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper

NARCIS (Netherlands)

Lener, Thomas; Gimona, Mario; Aigner, Ludwig; Börger, Verena; Buzas, Edit; Camussi, Giovanni; Chaput, Nathalie; Chatterjee, Devasis; Court, Felipe A; Del Portillo, Hernando A; O'Driscoll, Lorraine; Fais, Stefano; Falcon-Perez, Juan M; Felderhoff-Mueser, Ursula; Fraile, Lorenzo; Gho, Yong Song; Görgens, André; Gupta, Ramesh C; Hendrix, An; Hermann, Dirk M; Hill, Andrew F; Hochberg, Fred; Horn, Peter A; de Kleijn, Dominique; Kordelas, Lambros; Kramer, Boris W; Krämer-Albers, Eva-Maria; Laner-Plamberger, Sandra; Laitinen, Saara; Leonardi, Tommaso; Lorenowicz, Magdalena J; Lim, Sai Kiang; Lötvall, Jan; Maguire, Casey A; Marcilla, Antonio; Nazarenko, Irina; Ochiya, Takahiro; Patel, Tushar; Pedersen, Shona; Pocsfalvi, Gabriella; Pluchino, Stefano; Quesenberry, Peter; Reischl, Ilona G; Rivera, Francisco J; Sanzenbacher, Ralf; Schallmoser, Katharina; Slaper-Cortenbach, Ineke; Strunk, Dirk; Tonn, Torsten; Vader, Pieter; van Balkom, Bas W M; Wauben, Marca|info:eu-repo/dai/nl/112675735; Andaloussi, Samir El; Théry, Clotilde; Rohde, Eva; Giebel, Bernd

2015-01-01

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information

17β estradiol regulation of connexin 43-based gap junction and mechanosensitivity through classical estrogen receptor pathway in osteocyte-like MLO-Y4 cells.

KAUST Repository

Ren, Jian; Wang, Xuhui; Wang, Guangchao; Wu, Junhua

2013-01-01

Connexin 43 (Cx43) plays an essential role in osteocyte mechanotransduction. Although estrogen involves in the adaptive responses of bone cells to mechanical loadings, its effects on osteocytic Cx43-based gap junction intercellular communication

Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners.

Science.gov (United States)

Muramatsu, Takashi

2016-05-01

Basigin, also called CD147 or EMMPRIN, is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. Basigin has isoforms; the common form (basigin or basigin-2) has two immunoglobulin domains, and the extended form (basigin-1) has three. Basigin is the receptor for cyclophilins, S100A9 and platelet glycoprotein VI, whereas basigin-1 serves as the receptor for the rod-derived cone viability factor. Basigin tightly associates with monocarboxylate transporters and is essential for their cell surface translocation and activities. In the same membrane plane, basigin also associates with other proteins including GLUT1, CD44 and CD98. The carbohydrate portion of basigin is recognized by lectins, such as galectin-3 and E-selectin. These molecular recognitions form the basis for the role of basigin in the transport of nutrients, migration of inflammatory leukocytes and induction of matrix metalloproteinases. Basigin is important in vision, spermatogenesis and other physiological phenomena, and plays significant roles in the pathogenesis of numerous diseases, including cancer. Basigin is also the receptor for an invasive protein RH5, which is present in malaria parasites. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society.

Effects of transmembrane hydraulic pressure on performance of forward osmosis membranes.

Science.gov (United States)

Coday, Bryan D; Heil, Dean M; Xu, Pei; Cath, Tzahi Y

2013-03-05

Forward osmosis (FO) is an emerging membrane separation process that continues to be tested and implemented in various industrial water and wastewater treatment applications. The growing interests in the technology have prompted laboratories and manufacturers to adopt standard testing methods to ensure accurate comparison of membrane performance under laboratory-controlled conditions; however, standardized methods might not capture specific operating conditions unique to industrial applications. Experiments with cellulose triacetate (CTA) and polyamide thin-film composite (TFC) FO membranes demonstrated that hydraulic transmembrane pressure (TMP), common in industrial operation of FO membrane elements, could affect membrane performance. Experiments were conducted with three FO membranes and with increasing TMP up to a maximum of 50 psi (3.45 bar). The feed solution was a mixture of salts and the draw solution was either a NaCl solution or concentrated seawater at similar osmotic pressure. Results revealed that TMP minimally affected water flux, reverse salt flux (RSF), and solute rejection of the CTA membrane. However, water flux through TFC membranes might slightly increase with increasing TMP, and RSF substantially declines with increasing TMP. It was observed that rejection of feed constituents was influenced by TMP and RSF.

Transfer of vesicles from Schwann cell to axon: a novel mechanism of communication in the peripheral nervous system

Directory of Open Access Journals (Sweden)

María Alejandra eLopez-Verrilli

2012-06-01

Full Text Available Schwann cells (SCs are the glial component of the peripheral nervous system, with essential roles during development and maintenance of axons, as well as during regenerative processes after nerve injury. SCs increase conduction velocities by myelinating axons, regulate synaptic activity at presynaptic nerve terminals and are a source of trophic factors to neurons. Thus, development and maintenance of peripheral nerves are crucially dependent on local signalling between SCs and axons. In addition to the classic mechanisms of intercellular signalling, the possibility of communication through secreted vesicles has been poorly explored to date. Interesting recent findings suggest the occurrence of lateral transfer mediated by vesicles from glial cells to axons that could have important roles in axonal growth and axonal regeneration. Here, we review the role of vesicular transfer from SCs to axons and propose the benefits of this means in supporting neuronal and axonal maintenance and regeneration after nerve damage.

Disruption in connexin-based communication is associated with intracellular Ca²⁺ signal alterations in astrocytes from Niemann-Pick type C mice.

Directory of Open Access Journals (Sweden)

Pablo J Sáez

Full Text Available Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1⁻/⁻ showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1⁻/⁻ hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1⁻/⁻ astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1⁻/⁻ astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.

Disruption in connexin-based communication is associated with intracellular Ca²⁺ signal alterations in astrocytes from Niemann-Pick type C mice.

Science.gov (United States)

Sáez, Pablo J; Orellana, Juan A; Vega-Riveros, Natalia; Figueroa, Vania A; Hernández, Diego E; Castro, Juan F; Klein, Andrés D; Jiang, Jean X; Zanlungo, Silvana; Sáez, Juan C

2013-01-01

Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1⁻/⁻) showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1⁻/⁻ hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1⁻/⁻ astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1⁻/⁻ astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.

Nanosecond pulsed electric fields depolarize transmembrane potential via voltage-gated K+, Ca2+ and TRPM8 channels in U87 glioblastoma cells.

Science.gov (United States)

Burke, Ryan C; Bardet, Sylvia M; Carr, Lynn; Romanenko, Sergii; Arnaud-Cormos, Delia; Leveque, Philippe; O'Connor, Rodney P

2017-10-01

Nanosecond pulsed electric fields (nsPEFs) have a variety of applications in the biomedical and biotechnology industries. Cancer treatment has been at the forefront of investigations thus far as nsPEFs permeabilize cellular and intracellular membranes leading to apoptosis and necrosis. nsPEFs may also influence ion channel gating and have the potential to modulate cell physiology without poration of the membrane. This phenomenon was explored using live cell imaging and a sensitive fluorescent probe of transmembrane voltage in the human glioblastoma cell line, U87 MG, known to express a number of voltage-gated ion channels. The specific ion channels involved in the nsPEF response were screened using a membrane potential imaging approach and a combination of pharmacological antagonists and ion substitutions. It was found that a single 10ns pulsed electric field of 34kV/cm depolarizes the transmembrane potential of cells by acting on specific voltage-sensitive ion channels; namely the voltage and Ca2 + gated BK potassium channel, L- and T-type calcium channels, and the TRPM8 transient receptor potential channel. Copyright © 2017 Elsevier B.V. All rights reserved.

Proteomic insights into intra- and intercellular plant-bacteria symbiotic association during root nodule formation

Directory of Open Access Journals (Sweden)

Afshin eSalavati

2013-02-01

Full Text Available Over the last several decades, there have been a large number of studies done on the all aspects of legumes and bacteria which participate in nitrogen-fixing symbiosis. The analysis of legume-bacteria interaction is not just a matter of numerical complexity in terms of variants of gene products that can arise from a single gene. Bacteria regulate their quorum-sensing genes to enhance their ability to induce conjugation of plasmids and symbiotic islands, and various protein secretion mechanisms; that can stimulate a collection of chain reactions including species-specific combinations of plant-secretion isoflavonoids, complicated calcium signaling pathways and autoregulation of nodulation mechanisms. Quorum-sensing systems are introduced by the intra- and intercellular organization of gene products lead to protein–protein interactions or targeting of proteins to specific cellular structures. In this study, an attempt has been made to review significant contributions related to nodule formation and development and their impacts on cell proteome for better understanding of plant-bacterium interaction mechanism at protein level. This review would not only provide new insights into the plant-bacteria symbiosis response mechanisms but would also highlights the importance of studying changes in protein abundance inside and outside of cells in response to symbiosis. Furthermore, the application to agriculture programe of plant-bacteria interaction will be discussed.

A human/mouse chimeric monoclonal antibody against intercellular adhesion molecule-1 for tumor radioimmunoimaging

International Nuclear Information System (INIS)

Yamamura, Miyuki; Hinoda, Yuji; Sasaki, Shigeru; Tsujisaki, Masayuki; Imai, Kohzoh; Oriuchi, Noboru; Endo, Keigo.

1996-01-01

A mouse-human chimeric antibody for intercellular adhesion molecule-1 (ICAM-1) was established by using heavy chain loss mouse mutant hybridoma and human immunoglobulin expression vector. The HA58 hybridoma secreted anti-ICAM-1 monoclonal antibody (MoAb) (IgG1,κ). The gene of the mouse variable region of heavy chain was amplified and cloned by the polymerase chain reaction technique directly from the HA58 hybridoma RNA. The variable region of heavy chain was joined with an expression vector which contains human γ1 constant gene. The expression vector was transfected into heavy chain loss mutant cells HA58-7, which produced only murine immunoglobulin light chains. The resultant chimeric MoAb HA58, chHA58, retained full-binding reactivity to ICAM-1 compared with murine HA58 parental antibody. The chimeric MoAb chHA58 showed little antibody dependent cell-mediated cytotoxic activity against cultured tumor cells. Biodistribution studies with 99m Tc-labeled chHA58 in nude mice bearing human gastric carcinoma JRST cells, demonstrated that the tumor-blood ratio was 1.55 at 18 h after injection, when the tumors were clearly visible in gamma scintigraphy. These data suggest that chHA58 may be of practical use for radioimmunoimaging of a wide variety of tumors. (author)

Effects of centrifugation on transmembrane water loss from normal and pathologic erythrocytes

International Nuclear Information System (INIS)

Kaperonis, A.A.; Chien, S.

1989-01-01

Plasma 125 I-albumin was used as a marker of extracellular dilution in order to study the effect of high-speed centrifugation on transmembrane water distribution in several types of human red cells, including normal (AA), hemoglobin variants (beta A, AS, SC, beta S, and SS), and those from patients with hereditary spherocytosis. SS and AA erythrocytes were also examined for changes in intracellular hemoglobin concentration of three different density fractions and with increasing duration of spin. The minimum force and duration of centrifugation required to impair water permeability were found to vary with the red cell type, the anticoagulant used (heparin or EDTA), the initial hematocrit of the sample centrifuged, as well as among the individual erythrocyte fractions within the same sample. When subjecting pathologic erythrocytes to high-speed centrifugation, the 125 I-albumin dilution technique can be used to determine whether the centrifugation procedure has led to an artifactual red cell water loss and to correct for this when it does occur. An abnormal membrane susceptibility to mechanical stress was demonstrated in erythrocytes from patients with hereditary spherocytosis and several hemoglobinopathies

Pannexin 1 channels play essential roles in urothelial mechanotransduction and intercellular signaling.

Directory of Open Access Journals (Sweden)

Hiromitsu Negoro

Full Text Available Urothelial cells respond to bladder distension with ATP release, and ATP signaling within the bladder and from the bladder to the CNS is essential for proper bladder function. In other cell types, pannexin 1 (Panx1 channels provide a pathway for mechanically-induced ATP efflux and for ATP-induced ATP release through interaction with P2X7 receptors (P2X7Rs. We report that Panx1 and P2X7R are functionally expressed in the bladder mucosa and in immortalized human urothelial cells (TRT-HU1, and participate in urothelial ATP release and signaling. ATP release from isolated rat bladders induced by distention was reduced by the Panx1 channel blocker mefloquine (MFQ and was blunted in mice lacking Panx1 or P2X7R expression. Hypoosmotic shock induced YoPro dye uptake was inhibited by MFQ and the P2X7R blocker A438079 in TRT-HU1 cells, and was also blunted in primary urothelial cells derived from mice lacking Panx1 or P2X7R expression. Rinsing-induced mechanical stimulation of TRT-HU1 cells triggered ATP release, which was reduced by MFQ and potentiated in low divalent cation solution (LDPBS, a condition known to enhance P2X7R activation. ATP signaling evaluated as intercellular Ca2+ wave radius was significantly larger in LDPBS, reduced by MFQ and by apyrase (ATP scavenger. These findings indicate that Panx1 participates in urothelial mechanotransduction and signaling by providing a direct pathway for mechanically-induced ATP release and by functionally interacting with P2X7Rs.

Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development.

Science.gov (United States)

Sun, Hao; Lagarrigue, Frederic; Gingras, Alexandre R; Fan, Zhichao; Ley, Klaus; Ginsberg, Mark H

2018-04-02

Integrin activation regulates adhesion, extracellular matrix assembly, and cell migration, thereby playing an indispensable role in development and in many pathological processes. A proline mutation in the central integrin β3 transmembrane domain (TMD) creates a flexible kink that uncouples the topology of the inner half of the TMD from the outer half. In this study, using leukocyte integrin α4β7, which enables development of gut-associated lymphoid tissue (GALT), we examined the biological effect of such a proline mutation and report that it impairs agonist-induced talin-mediated activation of integrin α4β7, thereby inhibiting rolling lymphocyte arrest, a key step in transmigration. Furthermore, the α4β7(L721P) mutation blocks lymphocyte homing to and development of the GALT. These studies show that impairing the ability of an integrin β TMD to transmit talin-induced TMD topology inhibits agonist-induced physiological integrin activation and biological function in development. © 2018 Sun et al.

Non-equilibrium dynamics of 2D liquid crystals driven by transmembrane gas flow.

Science.gov (United States)

Seki, Kazuyoshi; Ueda, Ken; Okumura, Yu-ichi; Tabe, Yuka

2011-07-20

Free-standing films composed of several layers of chiral smectic liquid crystals (SmC*) exhibited unidirectional director precession under various vapor transfers across the films. When the transferred vapors were general organic solvents, the precession speed linearly depended on the momentum of the transmembrane vapors, where the proportional constant was independent of the kind of vapor. In contrast, the same SmC* films under water transfer exhibited precession in the opposite direction. As a possible reason for the rotational inversion, we suggest the competition of two origins for the torques, one of which is microscopic and the other macroscopic. Next, we tried to move an external object by making use of the liquid crystal (LC) motion. When a solid or a liquid particle was set on a film under vapor transfer, the particle was rotated in the same direction as the LC molecules. Using home-made laser tweezers, we measured the force transmitted from the film to the particle, which we found to be several pN.

Effects of lead intoxication on intercellular junctions and biochemical alterations of the renal proximal tubule cells.

Science.gov (United States)

Navarro-Moreno, L G; Quintanar-Escorza, M A; González, S; Mondragón, R; Cerbón-Solorzáno, J; Valdés, J; Calderón-Salinas, J V

2009-10-01

Lead intoxication is a worldwide health problem which frequently affects the kidney. In this work, we studied the effects of chronic lead intoxication (500 ppm of Pb in drinking water during seven months) on the structure, function and biochemical properties of rat proximal tubule cells. Lead-exposed animals showed increased lead concentration in kidney, reduction of calcium and amino acids uptake, oxidative damage and glucosuria, proteinuria, hematuria and reduced urinary pH. These biochemical and physiological alterations were related to striking morphological modifications in the structure of tubule epithelial cells and in the morphology of their mitochondria, nuclei, lysosomes, basal and apical membranes. Interestingly, in addition to the nuclei, inclusion bodies were found in the cytoplasm and in mitochondria. The epithelial cell structure modifications included an early loss of the apical microvillae, followed by a decrement of the luminal space and the respective apposition and proximity of apical membranes, resulting in the formation of atypical intercellular contacts and adhesion structures. Similar but less marked alterations were observed in subacute lead intoxication as well. Our work contributes in the understanding of the physiopathology of lead intoxication on the structure of renal tubular epithelial cell-cell contacts in vivo.

Direct and indirect inactivation of tumor cell protective catalase by salicylic acid and anthocyanidins reactivates intercellular ROS signaling and allows for synergistic effects.

Science.gov (United States)

Scheit, Katrin; Bauer, Georg

2015-03-01

Salicylic acid and anthocyanidins are known as plant-derived antioxidants, but also can provoke paradoxically seeming prooxidant effects in vitro. These prooxidant effects are connected to the potential of salicylic acid and anthocyanidins to induce apoptosis selectively in tumor cells in vitro and to inhibit tumor growth in animal models. Several epidemiological studies have shown that salicylic acid and its prodrug acetylsalicylic acid are tumor-preventive for humans. The mechanism of salicylic acid- and anthocyanidin-dependent antitumor effects has remained enigmatic so far. Extracellular apoptosis-inducing reactive oxygen species signaling through the NO/peroxynitrite and the HOCl signaling pathway specifically induces apoptosis in transformed cells. Tumor cells have acquired resistance against intercellular reactive oxygen species signaling through expression of membrane-associated catalase. Here, we show that salicylic acid and anthocyanidins inactivate tumor cell protective catalase and thus reactive apoptosis-inducing intercellular reactive oxygen species signaling of tumor cells and the mitochondrial pathway of apoptosis Salicylic acid inhibits catalase directly through its potential to transform compound I of catalase into the inactive compound II. In contrast, anthocyanidins provoke a complex mechanism for catalase inactivation that is initiated by anthocyanidin-mediated inhibition of NO dioxygenase. This allows the formation of extracellular singlet oxygen through the reaction between H(2)O(2) and peroxynitrite, amplification through a caspase8-dependent step and subsequent singlet oxygen-mediated inactivation of catalase. The combination of salicylic acid and anthocyanidins allows for a remarkable synergistic effect in apoptosis induction. This effect may be potentially useful to elaborate novel therapeutic approaches and crucial for the interpretation of epidemiological results related to the antitumor effects of secondary plant compounds. © The

Effect of transmembrane pressure control on energy efficiency during skim milk concentration by ultrafiltration at 10 and 50°C.

Science.gov (United States)

Méthot-Hains, S; Benoit, S; Bouchard, C; Doyen, A; Bazinet, L; Pouliot, Y

2016-11-01

The efficiency of the ultrafiltration process during skim milk concentration was studied using both dynamic and constant (465 or 672kPa) transmembrane pressure experiments at refrigerated temperature (10°C) and high temperature (50°C). The pilot-scale module was equipped with a 10-kDa polyethersulfone spiral-wound membrane element with a surface area of 2.04m 2 . Permeation flux, resistance-in-series model, mineral and protein rejection, and energy consumption were studied as a function of temperature and transmembrane pressure applied. Higher permeation flux values were systematically obtained at 50°C. Also, a significant temperature effect was found for calcium rejection, which was lower at 10°C compared with 50°C. Total hydraulic resistance and reversible fouling resistance were higher at 50°C than at 10°C. No change in protein rejection was observed, depending on the operating mode studied. Permeation flux, which was higher at 50°C, had lower pumping energy consumption compared with ultrafiltration at the colder temperature. Also, the low ultrafiltration temperature required a higher total energy consumption to reach the 3.6× retentate compared with ultrafiltration at 50°C. Overall, our study shows that the operating parameters and temperature can be optimized using an energy efficiency ratio. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Role of the transmembrane domain of the VanT serine racemase in resistance to vancomycin in Enterococcus gallinarum BM4174.

Science.gov (United States)

Arias, C A; Peña, J; Panesso, D; Reynolds, P

2003-03-01

Enterococcus gallinarum BM4175 (a vancomycin-susceptible derivative of BM4174 obtained by insertional inactivation of vanC-1) was transformed with plasmid constructs pCA10 (containing the genes necessary for resistance, vanC-1-XYc-T), pJP1 (with a fragment lacking the DNA encoding the transmembrane region of VanT, -vanC-1-XYc-T((Delta))(2-322)-) and with plasmids containing fragments encoding either the transmembrane (mvanT(1-322)) or racemase (svanT(323-698)) domains of VanT under the control of a constitutive promoter. Accumulated peptidoglycan precursors were measured in all strains in the presence of L-Ser, D-Ser (50 mM) or in the absence of any growth supplement. Uptake of 0.1 mM L-[(14)C]serine was also determined in BM4174, BM4175 and BM4175/pCA10. Vancomycin resistance was restored in BM4175 transformed with pCA10(C-1-XYc-T), and the profile of peptidoglycan precursors was similar to wild-type E. gallinarum BM4174. Transformation of E. gallinarum BM4175 with plasmid pJP1(vanC-1-XYc-T((Delta))(2-322)) resulted in: (i) vancomycin MICs remaining within susceptible levels (VanT is likely to be involved in the transport of L-Ser, and that in its absence the resistance phenotype is compromised.

Optical and non-optical methods for detection and characterization of microparticles and exosomes

NARCIS (Netherlands)

van der Pol, E.; Hoekstra, A.G.; Sturk, A.; Otto, C.; van Leeuwen, T.G.; Nieuwland, R.

2010-01-01

Microparticles and exosomes are cell-derived microvesicles present in body fluids that play a role in coagulation, inflammation, cellular homeostasis and survival, intercellular communication, and transport. Despite increasing scientific and clinical interest, no standard procedures are available

Exosomes in cancer theranostic: Diamonds in the rough.

Science.gov (United States)

Cordonnier, Marine; Chanteloup, Gaëtan; Isambert, Nicolas; Seigneuric, Renaud; Fumoleau, Pierre; Garrido, Carmen; Gobbo, Jessica

2017-03-04

During the last 10 years, exosomes, which are small vesicles of 50-200 nm diameter of endosomal origin, have aroused a great interest in the scientific and clinical community for their roles in intercellular communication in almost all physiological and pathological processes. Most cells can potentially release these nanovesicles that share with the parent cell a similar lipid bilayer with transmembrane proteins and a panel of enclosed soluble proteins such as heat shock proteins and genetic material, thus acting as potential nanoshuttles of biomarkers. Exosomes surface proteins allow their targeting and capture by recipient cells, while the exosomes' content can modify the physiological state of recipient cells. Tumor derived exosomes by interacting with other cells of the tumor microenvironment modulate tumor progression, angiogenic switch, metastasis, and immune escape. Targeting tumor-derived exosomes might be an interesting approach in cancer therapy. Furthermore, because a key issue to improve cancer patients' outcome relies on earlier cancer diagnosis (metastases, as opposed to the primary tumor, are responsible for most cancer deaths) exosomes have been put forward as promising biomarker candidates for cancer diagnosis and prognosis. This review summarizes the roles of exosomes in cancer and clinical interest, focusing on the importance of exosomal heat shock proteins (HSP). The challenges of clinical translation of HSP-exosomes as therapeutic targets and biomarkers for early cancer detection are also discussed.

Emerging role of cystic fibrosis transmembrane conductance regulator- an epithelial chloride channel in gastrointestinal cancers

Institute of Scientific and Technical Information of China (English)

Yuning Hou; Xiaoqing Guan; Zhe Yang; Chunying Li

2016-01-01

Cystic fibrosis transmembrane conductance regulator(CFTR), a glycoprotein with 1480 amino acids, has been well established as a chloride channel mainly expressed in the epithelial cells of various tissues and organs such as lungs, sweat glands, gastrointestinal system, and reproductive organs. Although defective CFTR leads to cystic fibrosis, a common genetic disorder in the Caucasian population, there is accumulating evidence that suggests a novel role of CFTR in various cancers, especially in gastroenterological cancers, such as pancreatic cancer and colon cancer. In this review, we summarize the emerging findings that link CFTR with various cancers, with focus on the association between CFTR defects and gastrointestinal cancers as well as the underlying mechanisms. Further study of CFTR in cancer biology may help pave a new way for the diagnosis and treatment of gastrointestinal cancers.

Extracellular vesicles: Exosomes, microvesicles, and friends

NARCIS (Netherlands)

Raposo, G.; Stoorvogel, W.|info:eu-repo/dai/nl/074352385

2013-01-01

Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for

Communication without communication

Directory of Open Access Journals (Sweden)

Bratina Boris R.

2017-01-01

Full Text Available This paper analyses the necessary conditions for successful communication. It is well known that post-modernity, described as an era of control, produces only decentralized, imploded subjectivities, who are neither able to question their own being nor to relate one with another in authentic bonds of communication. Today, virtual communication has become an ultimate model of every possible communication whatsoever. The authors, therefore, pose the question of conditions for possibility of subjectivities who would be able and apt for authentic communication, wherein faith, fidelity, truth, and capability of keeping one's word occupy the central place.

Combinatorial solid-phase glycopeptide libraries - Synthesis and application in biomolecular interaction studies and biosensors

NARCIS (Netherlands)

Maljaars, C.E.P.

2006-01-01

Carbohydrates play vital roles in all forms of life. Attached to proteins and lipids they serve as recognition elements in several biological interaction processes, such as intercellular communication, immune response, viral and parasitical infections, signal transduction, and development. Synthetic

Molecular dynamics simulation studies of transmembrane transport of chemical components in Chinese herbs and the function of platycodin D in a biological membrane

Directory of Open Access Journals (Sweden)

Shufang Yang

2017-04-01

Conclusion: The Martini force field was successfully applied to the study of the interaction between herbal compounds and a biological membrane. By combining the dynamics equilibrium morphology, the distribution of drugs inside and outside the biomembrane, and the interaction sites of drugs on the DPPC bilayer, factors influencing transmembrane transport of drugs were elucidated and the function of platycodin D in a biological membrane was reproduced.

Effect of drugs on lipid methylation, receptor-adenylate cyclase coupling and cyclic AMP secretion in Dictyostelium discoideum

NARCIS (Netherlands)

Van Waarde, Aren; Van Haastert, P.J.M.

1986-01-01

Intercellular communication in Dictyostelium discoldeum takes place by means of cyclic AMP-induced cyclic AMP-synthesis and secretion. Since phospholipid methylation has been suggested to play a role in receptor-adenylate cyclase coupling, we examined the effects of transmethylation inhibitors on

Biological properties of extracellular vesicles and their physiological functions

NARCIS (Netherlands)

Yáñez-Mó, María; Siljander, Pia R-M; Andreu, Zoraida; Zavec, Apolonija Bedina; Borràs, Francesc E; Buzas, Edit I; Buzas, Krisztina; Casal, Enriqueta; Cappello, Francesco; Carvalho, Joana; Colás, Eva; Cordeiro-da Silva, Anabela; Fais, Stefano; Falcon-Perez, Juan M; Ghobrial, Irene M; Giebel, Bernd; Gimona, Mario; Graner, Michael; Gursel, Ihsan; Gursel, Mayda; Heegaard, Niels H H; Hendrix, An; Kierulf, Peter; Kokubun, Katsutoshi; Kosanovic, Maja; Kralj-Iglic, Veronika; Krämer-Albers, Eva-Maria; Laitinen, Saara; Lässer, Cecilia; Lener, Thomas; Ligeti, Erzsébet; Linē, Aija; Lipps, Georg; Llorente, Alicia; Lötvall, Jan; Manček-Keber, Mateja; Marcilla, Antonio; Mittelbrunn, Maria; Nazarenko, Irina; Nolte-'t Hoen, Esther N M; Nyman, Tuula A; O'Driscoll, Lorraine; Olivan, Mireia; Oliveira, Carla; Pállinger, Éva; Del Portillo, Hernando A; Reventós, Jaume; Rigau, Marina; Rohde, Eva; Sammar, Marei; Sánchez-Madrid, Francisco; Santarém, N; Schallmoser, Katharina; Ostenfeld, Marie Stampe; Stoorvogel, Willem|info:eu-repo/dai/nl/074352385; Stukelj, Roman; Van der Grein, Susanne G|info:eu-repo/dai/nl/412755211; Vasconcelos, M Helena; Wauben, Marca H M|info:eu-repo/dai/nl/112675735; De Wever, Olivier

2015-01-01

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological

Studying extracellular vesicle transfer by a Cre-loxP method

NARCIS (Netherlands)

Zomer, Anoek; Steenbeek, Sander Christiaan; Maynard, Carrie; van Rheenen, Jacco

Extracellular vesicle (EV) transfer is increasingly recognized as an important mode of intercellular communication by transferring a wide variety of biomolecules between cells. The characterization of in vitro- or ex vivo-isolated EVs has considerably contributed to the understanding of biological

Earthworm's immunity in the nanomaterial world: new room, future challenges

DEFF Research Database (Denmark)

Hayashi, Yuya; Engelmann, Péter

2013-01-01

of the immune responses to nanoparticles with references to other invertebrate species and vertebrate counterparts. We discuss the significant contribution of amoebocytes as nanoparticle scavengers and suggest a possibility of studying inter-cellular communications in coelomocytes. Implications from the leading...

House dust mite induces expression of intercellular adhesion molecule-1 in EoL-1 human eosinophilic leukemic cells.

Science.gov (United States)

Kwon, Byoung Chul; Sohn, Myung Hyun; Kim, Kyung Won; Kim, Eun Soo; Kim, Kyu-Earn; Shin, Myeong Heon

2007-10-01

The house dust mite (HDM) is considered to be the most common indoor allergen associated with bronchial asthma. In this study, we investigated whether crude extract of the HDM Dermatophagoides farinae could activate human eosinophilic leukemic cells (EoL-1) to induce upregulation of cell-surface adhesion molecules. When EoL-1 cells were incubated with D. farinae extract, expression of intercellular adhesion molecule-1 (ICAM-1) significantly increased on the cell surfaces compared to cells incubated with medium alone. In contrast, surface expression of CD11b and CD49d in EoL-1 cells was not affected by D. farinae extract. In addition, pretreatment of cells with NF-kappaB inhibitor (MG-132) or JNK inhibitor (SP600125) significantly inhibited ICAM-1 expression promoted by HDM extract. However, neither p38 MAP kinase inhibitor nor MEK inhibitor prevented HDM-induced ICAM-1 expression in EoL-1 cells. These results suggest that crude extract of D. farinae induces ICAM-1 expression in EoL-1 cells through signaling pathways involving both NF-kappaB and JNK.

The Cucumber leaf spot virus p25 auxiliary replicase protein binds and modifies the endoplasmic reticulum via N-terminal transmembrane domains

Energy Technology Data Exchange (ETDEWEB)

Ghoshal, Kankana [University of British Columbia, Faculty of Land and Food Systems, Vancouver, British Columbia, Canada V6T 1Z4 (Canada); Theilmann, Jane; Reade, Ron; Sanfacon, Helene [Agriculture and Agri-Food Canada Pacific Agri-Food Research Centre, 4200 Hwy 97, Summerland, British Columbia, Canada V0H 1Z0 (Canada); Rochon, D’Ann, E-mail: dann.rochon@agr.gc.ca [University of British Columbia, Faculty of Land and Food Systems, Vancouver, British Columbia, Canada V6T 1Z4 (Canada); Agriculture and Agri-Food Canada Pacific Agri-Food Research Centre, 4200 Hwy 97, Summerland, British Columbia, Canada V0H 1Z0 (Canada)

2014-11-15

Cucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified including the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication. - Highlights: • The CLSV p25 auxiliary replicase targets the endoplasmic reticulum (ER). • Targeting of CLSV p25 is associated with ER restructuring. • Restructuring of the ER occurs during CLSV infection. • CLSV p25 contains 3 predicted transmembrane domains 2 of which are required for ER targeting. • Vesicles derived from the ER may be sites of CLSV replication.

The Cucumber leaf spot virus p25 auxiliary replicase protein binds and modifies the endoplasmic reticulum via N-terminal transmembrane domains

International Nuclear Information System (INIS)

Ghoshal, Kankana; Theilmann, Jane; Reade, Ron; Sanfacon, Helene; Rochon, D’Ann

2014-01-01

Cucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified including the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication. - Highlights: • The CLSV p25 auxiliary replicase targets the endoplasmic reticulum (ER). • Targeting of CLSV p25 is associated with ER restructuring. • Restructuring of the ER occurs during CLSV infection. • CLSV p25 contains 3 predicted transmembrane domains 2 of which are required for ER targeting. • Vesicles derived from the ER may be sites of CLSV replication

Evolutionary and Comparative Genomics to Drive Rational Drug Design, with Particular Focus on Neuropeptide Seven-Transmembrane Receptors.

Science.gov (United States)

Furlong, Michael; Seong, Jae Young

2017-01-01

Seven transmembrane receptors (7TMRs), also known as G protein-coupled receptors, are popular targets of drug development, particularly 7TMR systems that are activated by peptide ligands. Although many pharmaceutical drugs have been discovered via conventional bulk analysis techniques the increasing availability of structural and evolutionary data are facilitating change to rational, targeted drug design. This article discusses the appeal of neuropeptide-7TMR systems as drug targets and provides an overview of concepts in the evolution of vertebrate genomes and gene families. Subsequently, methods that use evolutionary concepts and comparative analysis techniques to aid in gene discovery, gene function identification, and novel drug design are provided along with case study examples.

Structure and Mechanism of Proton Transport Through the Transmembrane Tetrameric M2 Protein Bundle of the Influenza A Virus

Energy Technology Data Exchange (ETDEWEB)

R Acharya; V Carnevale; G Fiorin; B Levine; A Polishchuk; V Balannick; I Samish; R Lamb; L Pinto; et al.

2011-12-31

The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2{sup +} and 3{sup +} with a pK{sub a} near 6. A 1.65 {angstrom} resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.

Decreased in vitro fertility in male rats exposed to fluoride-induced oxidative stress damage and mitochondrial transmembrane potential loss

International Nuclear Information System (INIS)

Izquierdo-Vega, Jeannett A.; Sanchez-Gutierrez, Manuel; Razo, Luz Maria del

2008-01-01

Fluorosis, caused by drinking water contamination with inorganic fluoride, is a public health problem in many areas around the world. The aim of the study was to evaluate the effect of environmentally relevant doses of fluoride on in vitro fertilization (IVF) capacity of spermatozoa, and its relationship to spermatozoa mitochondrial transmembrane potential (ΔΨ m ). Male Wistar rats were administered at 5 mg fluoride/kg body mass/24 h, or deionized water orally for 8 weeks. We evaluated several spermatozoa parameters in treated and untreated rats: i) standard quality analysis, ii) superoxide dismutase (SOD) activity, iii) the generation of superoxide anion (O 2 ·- ), iv) lipid peroxidation concentration, v) ultrastructural analyses of spermatozoa using transmission electron microscopy, vi) ΔΨ m , vii) acrosome reaction, and viii) IVF capability. Spermatozoa from fluoride-treated rats exhibited a significant decrease in SOD activity (∼ 33%), accompanied with a significant increase in the generation of O 2 · (∼ 40%), a significant decrease in ΔΨ m (∼ 33%), and a significant increase in lipid peroxidation concentration (∼ 50%), relative to spermatozoa from the control group. Consistent with this finding, spermatozoa from fluoride-treated rats exhibited altered plasmatic membrane. In addition, the percentage of fluoride-treated spermatozoa capable of undergoing the acrosome reaction was decreased relative to control spermatozoa (34 vs. 55%), while the percentage fluoride-treated spermatozoa capable of oocyte fertilization was also significantly lower than the control group (13 vs. 71%). These observations suggest that subchronic exposure to fluoride causes oxidative stress damage and loss of mitochondrial transmembrane potential, resulting in reduced fertility

Brucella Intracellular Life Relies on the Transmembrane Protein CD98 Heavy Chain.

Science.gov (United States)

Keriel, Anne; Botella, Eric; Estrach, Soline; Bragagnolo, Gabriel; Vergunst, Annette C; Feral, Chloe C; O'Callaghan, David

2015-06-01

Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Effects of centrifugation on transmembrane water loss from normal and pathologic erythrocytes

Energy Technology Data Exchange (ETDEWEB)

Kaperonis, A.A.; Chien, S.

1989-02-01

Plasma /sup 125/I-albumin was used as a marker of extracellular dilution in order to study the effect of high-speed centrifugation on transmembrane water distribution in several types of human red cells, including normal (AA), hemoglobin variants (beta A, AS, SC, beta S, and SS), and those from patients with hereditary spherocytosis. SS and AA erythrocytes were also examined for changes in intracellular hemoglobin concentration of three different density fractions and with increasing duration of spin. The minimum force and duration of centrifugation required to impair water permeability were found to vary with the red cell type, the anticoagulant used (heparin or EDTA), the initial hematocrit of the sample centrifuged, as well as among the individual erythrocyte fractions within the same sample. When subjecting pathologic erythrocytes to high-speed centrifugation, the /sup 125/I-albumin dilution technique can be used to determine whether the centrifugation procedure has led to an artifactual red cell water loss and to correct for this when it does occur. An abnormal membrane susceptibility to mechanical stress was demonstrated in erythrocytes from patients with hereditary spherocytosis and several hemoglobinopathies.

Detection of cancerous biological tissue areas by means of infrared absorption and SERS spectroscopy of intercellular fluid

Science.gov (United States)

Velicka, M.; Urboniene, V.; Ceponkus, J.; Pucetaite, M.; Jankevicius, F.; Sablinskas, V.

2015-08-01

We present a novel approach to the detection of cancerous kidney tissue areas by measuring vibrational spectra (IR absorption or SERS) of intercellular fluid taken from the tissue. The method is based on spectral analysis of cancerous and normal tissue areas in order to find specific spectral markers. The samples were prepared by sliding the kidney tissue over a substrate - surface of diamond ATR crystal in case of IR absorption or calcium fluoride optical window in case of SERS. For producing the SERS signal the dried fluid film was covered by silver nanoparticle colloidal solution. In order to suppress fluorescence background the measurements were performed in the NIR spectral region with the excitation wavelength of 1064 nm. The most significant spectral differences - spectral markers - were found in the region between 400 and 1800 cm-1, where spectral bands related to various vibrations of fatty acids, glycolipids and carbohydrates are located. Spectral markers in the IR and SERS spectra are different and the methods can complement each other. Both of them have potential to be used directly during surgery. Additionally, IR absorption spectroscopy in ATR mode can be combined with waveguide probe what makes this method usable in vivo.

Mobility of the native Bacillus subtilis conjugative plasmid pLS20 is regulated by intercellular signaling.

Science.gov (United States)

Singh, Praveen K; Ramachandran, Gayetri; Ramos-Ruiz, Ricardo; Peiró-Pastor, Ramón; Abia, David; Wu, Ling J; Meijer, Wilfried J J

2013-10-01

Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default "OFF" state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.

Fluorophores, environments, and quantification techniques in the analysis of transmembrane helix interaction using FRET.

Science.gov (United States)

Khadria, Ambalika S; Senes, Alessandro

2015-07-01

Förster resonance energy transfer (FRET) has been widely used as a spectroscopic tool in vitro to study the interactions between transmembrane (TM) helices in detergent and lipid environments. This technique has been instrumental to many studies that have greatly contributed to quantitative understanding of the physical principles that govern helix-helix interactions in the membrane. These studies have also improved our understanding of the biological role of oligomerization in membrane proteins. In this review, we focus on the combinations of fluorophores used, the membrane mimetic environments, and measurement techniques that have been applied to study model systems as well as biological oligomeric complexes in vitro. We highlight the different formalisms used to calculate FRET efficiency and the challenges associated with accurate quantification. The goal is to provide the reader with a comparative summary of the relevant literature for planning and designing FRET experiments aimed at measuring TM helix-helix associations. © 2015 Wiley Periodicals, Inc.

Transmembrane carbonic anhydrase isozymes IX and XII in the female mouse reproductive organs

Directory of Open Access Journals (Sweden)

Tomas Eija

2004-10-01

Full Text Available Abstract Background Carbonic anhydrase (CA classically catalyses the reversible hydration of dissolved CO2 to form bicarbonate ions and protons. The twelve active CA isozymes are thought to regulate a variety of cellular functions including several processes in the reproductive systems. Methods The present study was designed to investigate the expression of transmembrane CAs, CA IX and XII, in the mouse uterus, ovary and placenta. The expression of CA IX and XII was examined by immunoperoxidase staining method and western blotting. CA II and XIII served as positive controls since they are known to be present in the mouse reproductive tract. Results The data of our study indicated that CA XII is expressed in the mouse endometrium. Only very faint signal was observed in the corpus luteum of the ovary and the placenta remained mainly negative. CA IX showed weak reaction in the endometrial epithelium, while it was completely absent in the ovary and placenta. Conclusion The conservation of CA XII expression in both mouse and human endometrium suggests a role for this isozyme in reproductive physiology.

Molecular Dynamics Simulations of Orai Reveal How the Third Transmembrane Segment Contributes to Hydration and Ca2+ Selectivity in Calcium Release-Activated Calcium Channels.

Science.gov (United States)

Alavizargar, Azadeh; Berti, Claudio; Ejtehadi, Mohammad Reza; Furini, Simone

2018-04-26

Calcium release-activated calcium (CRAC) channels open upon depletion of Ca 2+ from the endoplasmic reticulum, and when open, they are permeable to a selective flux of calcium ions. The atomic structure of Orai, the pore domain of CRAC channels, from Drosophila melanogaster has revealed many details about conduction and selectivity in this family of ion channels. However, it is still unclear how residues on the third transmembrane helix can affect the conduction properties of the channel. Here, molecular dynamics and Brownian dynamics simulations were employed to analyze how a conserved glutamate residue on the third transmembrane helix (E262) contributes to selectivity. The comparison between the wild-type and mutated channels revealed a severe impact of the mutation on the hydration pattern of the pore domain and on the dynamics of residues K270, and Brownian dynamics simulations proved that the altered configuration of residues K270 in the mutated channel impairs selectivity to Ca 2+ over Na + . The crevices of water molecules, revealed by molecular dynamics simulations, are perfectly located to contribute to the dynamics of the hydrophobic gate and the basic gate, suggesting a possible role in channel opening and in selectivity function.

Hypercapnia modulates cAMP signalling and cystic fibrosis transmembrane conductance regulator‐dependent anion and fluid secretion in airway epithelia

Science.gov (United States)

Turner, Mark J.; Saint‐Criq, Vinciane; Patel, Waseema; Ibrahim, Salam H.; Verdon, Bernard; Ward, Christopher; Garnett, James P.; Tarran, Robert; Cann, Martin J.

2015-01-01

Key points Raised arterial blood CO2 (hypercapnia) is a feature of many lung diseases.CO2 has been shown to act as a cell signalling molecule in human cells, notably by influencing the levels of cell signalling second messengers: cAMP and Ca2+.Hypercapnia reduced cAMP‐stimulated cystic fibrosis transmembrane conductance regulator‐dependent anion and fluid transport in Calu‐3 cells and primary human airway epithelia but did not affect cAMP‐regulated HCO3 − transport via pendrin or Na+/HCO3 − cotransporters.These results further support the role of CO2 as a cell signalling molecule and suggests CO2‐induced reductions in airway anion and fluid transport may impair innate defence mechanisms of the lungs. Abstract Hypercapnia is clinically defined as an arterial blood partial pressure of CO2 of above 40 mmHg and is a feature of chronic lung disease. In previous studies we have demonstrated that hypercapnia modulates agonist‐stimulated cAMP levels through effects on transmembrane adenylyl cyclase activity. In the airways, cAMP is known to regulate cystic fibrosis transmembrane conductance regulator (CFTR)‐mediated anion and fluid secretion, which contributes to airway surface liquid homeostasis. The aim of the current work was to investigate if hypercapnia could modulate cAMP‐regulated ion and fluid transport in human airway epithelial cells. We found that acute exposure to hypercapnia significantly reduced forskolin‐stimulated elevations in intracellular cAMP as well as both adenosine‐ and forskolin‐stimulated increases in CFTR‐dependent transepithelial short‐circuit current, in polarised cultures of Calu‐3 human airway cells. This CO2‐induced reduction in anion secretion was not due to a decrease in HCO3 − transport given that neither a change in CFTR‐dependent HCO3 − efflux nor Na+/HCO3 − cotransporter‐dependent HCO3 − influx were CO2‐sensitive. Hypercapnia also reduced the volume of forskolin‐stimulated fluid

Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

Science.gov (United States)

Papa, S; Lorusso, M; Izzo, G; Capuano, F

1981-02-15

1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi

Characterization of two types of prostasomes with distinct molecular compositions

NARCIS (Netherlands)

Aalberts, M.|info:eu-repo/dai/nl/313888515

2012-01-01

During the last years it has become evident that many different cell types can communicate with each other through intercellular transfer of extracellular vesicles. Such cell-derived membrane vesicles function in several physiological processes and also in disease. This thesis describes two

Cardiac myocyte-derived follistatin-like 1 prevents renal injury in a subtotal nephrectomy model

NARCIS (Netherlands)

Hayakawa, Satoko; Ohashi, Koji; Shibata, Rei; Kataoka, Yoshiyuki; Miyabe, Megumi; Enomoto, Takashi; Joki, Yusuke; Shimizu, Yuuki; Kambara, Takahiro; Uemura, Yusuke; Yuasa, Daisuke; Ogawa, Hayato; Matsuo, Kazuhiro; Hiramatsu-Ito, Mizuho; van den Hoff, Maurice J. B.; Walsh, Kenneth; Murohara, Toyoaki; Ouchi, Noriyuki

2015-01-01

Heart disease contributes to the progression of CKD. Heart tissue produces a number of secreted proteins, also known as cardiokines, which participate in intercellular and intertissue communication. We recently reported that follistatin-like 1 (Fstl1) functions as a cardiokine with cardioprotective

Repurposing metformin as a quorum sensing inhibitor in ...

African Journals Online (AJOL)

Background: Quorum sensing is a mechanism of intercellular communication that controls the production of virulence factors in Pseudomonas aeruginosa. Inhibition of quorum sensing can disarm the virulence factors without exerting stress on bacterial growth that leads to emergence of antibiotic resistance. Objectives: ...

Gap junctional communication modulates gene transcription by altering the recruitment of Sp1 and Sp3 to connexin-response elements in osteoblast promoters

Science.gov (United States)

Stains, Joseph P.; Lecanda, Fernando; Screen, Joanne; Towler, Dwight A.; Civitelli, Roberto

2003-01-01

Loss-of-function mutations of gap junction proteins, connexins, represent a mechanism of disease in a variety of tissues. We have shown that recessive (gene deletion) or dominant (connexin45 overexpression) disruption of connexin43 function results in osteoblast dysfunction and abnormal expression of osteoblast genes, including down-regulation of osteocalcin transcription. To elucidate the molecular mechanisms of gap junction-sensitive transcriptional regulation, we systematically analyzed the rat osteocalcin promoter for sensitivity to gap junctional intercellular communication. We identified an Sp1/Sp3 containing complex that assembles on a minimal element in the -70 to -57 region of the osteocalcin promoter in a gap junction-dependent manner. This CT-rich connexin-response element is necessary and sufficient to confer gap junction sensitivity to the osteocalcin proximal promoter. Repression of osteocalcin transcription occurs as a result of displacement of the stimulatory Sp1 by the inhibitory Sp3 on the promoter when gap junctional communication is perturbed. Modulation of Sp1/Sp3 recruitment also occurs on the collagen Ialpha1 promoter and translates into gap junction-sensitive transcriptional control of collagen Ialpha1 gene expression. Thus, regulation of Sp1/Sp3 recruitment to the promoter may represent a potential general mechanism for transcriptional control of target genes by signals passing through gap junctions.

Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

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Caballero-Rivera, Daniel; Cruz-Nieves, Omar A; Oyola-Cintrón, Jessica; Torres-Núñez, David A; Otero-Cruz, José D

2011-01-01

The nicotinic acetylcholine receptor (nAChR) is a member of a family of ligand-gated ion channels that mediate diverse physiological functions, including fast synaptic transmission along the peripheral and central nervous systems. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, a high-resolution atomic structure of the nAChR still remains elusive. In this study, we extended the Fourier transform coupled tryptophan scanning mutagenesis (FT-TrpScanM) approach to gain insight into the secondary structure of the δM3 transmembrane domain of the Torpedo californica nAChR, to monitor conformational changes experienced by this domain during channel gating, and to identify which lipid-exposed positions are linked to the regulation of ion channel kinetics. The perturbations produced by periodic tryptophan substitutions along the δM3 transmembrane domain were characterized by two-electrode voltage clamp and 125I-labeled α-bungarotoxin binding assays. The periodicity profiles and Fourier transform spectra of this domain revealed similar helical structures for the closed- and open-channel states. However, changes in the oscillation patterns observed between positions Val-299 and Val-304 during transition between the closed- and open-channel states can be explained by the structural effects caused by the presence of a bending point introduced by a Thr-Gly motif at positions 300–301. The changes in periodicity and localization of residues between the closed-and open-channel states could indicate a structural transition between helix types in this segment of the domain. Overall, the data further demonstrate a functional link between the lipid-exposed transmembrane domain and the nAChR gating machinery. PMID:21785268

Molecular characterization of the gerbil C5a receptor and identification of a transmembrane domain V amino acid that is crucial for small molecule antagonist interaction.

Science.gov (United States)

Waters, Stephen M; Brodbeck, Robbin M; Steflik, Jeremy; Yu, Jianying; Baltazar, Carolyn; Peck, Amy E; Severance, Daniel; Zhang, Lu Yan; Currie, Kevin; Chenard, Bertrand L; Hutchison, Alan J; Maynard, George; Krause, James E

2005-12-09

Anaphylatoxin C5a is a potent inflammatory mediator associated with pathogenesis and progression of several inflammation-associated disorders. Small molecule C5a receptor (C5aR) antagonist development is hampered by species-specific receptor biology and the associated inability to use standard rat and mouse in vivo models. Gerbil is one rodent species reportedly responsive to small molecule C5aR antagonists with human C5aR affinity. We report the identification of the gerbil C5aR cDNA using a degenerate primer PCR cloning strategy. The nucleotide sequence revealed an open reading frame encoding a 347-amino acid protein. The cloned receptor (expressed in Sf9 cells) bound recombinant human C5a with nanomolar affinity. Alignment of the gerbil C5aR sequence with those from other species showed that a Trp residue in transmembrane domain V is the only transmembrane domain amino acid unique to small molecule C5aR antagonist-responsive species (i.e. gerbil, human, and non-human primate). Site-directed mutagenesis was used to generate human and mouse C5aRs with a residue exchange of this Trp residue. Mutation of Trp to Leu in human C5aR completely eliminated small molecule antagonist-receptor interaction. In contrast, mutation of Leu to Trp in mouse C5aR enabled small molecule antagonist-receptor interaction. This crucial Trp residue is located deeper within transmembrane domain V than residues reportedly involved in C5a- and cyclic peptide C5a antagonist-receptor interaction, suggesting a novel interaction site(s) for small molecule antagonists. These data provide insight into the basis for small molecule antagonist species selectivity and further define sites critical for C5aR activation and function.

Microsomal Prostaglandin E Synthase-1 Facilitates an Intercellular Interaction between CD4⁺ T Cells through IL-1β Autocrine Function in Experimental Autoimmune Encephalomyelitis.

Science.gov (United States)

Takemiya, Takako; Takeuchi, Chisen; Kawakami, Marumi

2017-12-19

Microsomal prostaglandin synthetase-1 (mPGES-1) is an inducible terminal enzyme that produces prostaglandin E₂ (PGE₂). In our previous study, we investigated the role of mPGES-1 in the inflammation and demyelination observed in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, using mPGES - 1 -deficient ( mPGES-1 -/- ) and wild-type (wt) mice. We found that mPGES-1 facilitated inflammation, demyelination, and paralysis and was induced in vascular endothelial cells and macrophages and microglia around inflammatory foci. Here, we investigated the role of interleukin-1β (IL-1β) in the intercellular mechanism stimulated by mPGES-1 in EAE spinal cords in the presence of inflammation. We found that the area invaded by CD4-positive (CD4⁺) T cells was extensive, and that PGE₂ receptors EP1-4 were more induced in activated CD4⁺ T cells of wt mice than in those of mPGES - 1 -/- mice. Moreover, IL-1β and IL-1 receptor 1 (IL-1r1) were produced by 65% and 48% of CD4⁺ T cells in wt mice and by 44% and 27% of CD4⁺ T cells in mPGES-1 -/- mice. Furthermore, interleukin-17 (IL-17) was released from the activated CD4⁺ T cells. Therefore, mPGES-1 stimulates an intercellular interaction between CD4⁺ T cells by upregulating the autocrine function of IL-1β in activated CD4⁺ T cells, which release IL-17 to facilitate axonal and myelin damage in EAE mice.

A chemical biology approach to interrogate quorum-sensing regulated behaviors at the molecular and cellular level.

Science.gov (United States)

Lowery, Colin A; Matamouros, Susana; Niessen, Sherry; Zhu, Jie; Scolnick, Jonathan; Lively, Jenny M; Cravatt, Benjamin F; Miller, Samuel I; Kaufmann, Gunnar F; Janda, Kim D

2013-07-25

Small molecule probes have been used extensively to explore biologic systems and elucidate cellular signaling pathways. In this study, we use an inhibitor of bacterial communication to monitor changes in the proteome of Salmonella enterica serovar Typhimurium with the aim of discovering unrecognized processes regulated by AI-2-based quorum-sensing (QS), a mechanism of bacterial intercellular communication that allows for the coordination of gene expression in a cell density-dependent manner. In S. typhimurium, this system regulates the uptake and catabolism of intercellular signals and has been implicated in pathogenesis, including the invasion of host epithelial cells. We demonstrate that our QS antagonist is capable of selectively inhibiting the expression of known QS-regulated proteins in S. typhimurium, thus attesting that QS inhibitors may be used to confirm proposed and elucidate previously unidentified QS pathways without relying on genetic manipulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

How cancer cells dictate their microenvironment: present roles of extracellular vesicles.

Science.gov (United States)

Naito, Yutaka; Yoshioka, Yusuke; Yamamoto, Yusuke; Ochiya, Takahiro

2017-02-01

Intercellular communication plays an important role in cancer initiation and progression through secretory molecules, including growth factors and cytokines. Recent advances have revealed that small membrane vesicles, termed extracellular vesicles (EVs), served as a regulatory agent in the intercellular communication of cancer. EVs enable the transfer of functional molecules, including proteins, mRNA and microRNAs (miRNAs), into recipient cells. Cancer cells utilize EVs to dictate the unique phenotype of surrounding cells, thereby promoting cancer progression. Against such "education" by cancer cells, non-tumoral cells suppress cancer initiation and progression via EVs. Therefore, researchers consider EVs to be important cues to clarify the molecular mechanisms of cancer biology. Understanding the functions of EVs in cancer progression is an important aspect of cancer biology that has not been previously elucidated. In this review, we summarize experimental data that indicate the pivotal roles of EVs in cancer progression.

Endocochlear potential generation is associated with intercellular communication in the stria vascularis: structural analysis in the viable dominant spotting mouse mutant.

Science.gov (United States)

Carlisle, L; Steel, K; Forge, A

1990-11-01

Deafness in the viable dominant spotting mouse mutant is due to a primary defect of the stria vascularis which results in absence of the positive endocochlear potential in scala media. Endocochlear potentials were measured and the structure of stria vascularis of mutants with potentials close to zero was compared with that in normal littermate controls by use of morphometric methods. The stria vascularis was significantly thinner in mutants. Marginal cells were not significantly different from controls in terms of volume density or intramembrane particle density but the network density of tight junctions was significantly reduced in the mutants. A virtual absence of gap junctions between basal cells and marginal or intermediate cells was observed, but intramembrane particle density and junctional complexes between adjacent basal cells were not different from controls. The volume density of basal cells was significantly greater in mutants. Intermediate cells accounted for a significantly smaller volume density of the stria vascularis in mutants and had a lower density of intramembrane particles than controls. Melanocytes were not identified in the stria vascularis of mutants. These results suggest that communication between marginal, intermediate and basal cells might be important to the normal function of the stria vascularis.

Communications device identification methods, communications methods, wireless communications readers, wireless communications systems, and articles of manufacture

Science.gov (United States)

Steele, Kerry D [Kennewick, WA; Anderson, Gordon A [Benton City, WA; Gilbert, Ronald W [Morgan Hill, CA

2011-02-01

Communications device identification methods, communications methods, wireless communications readers, wireless communications systems, and articles of manufacture are described. In one aspect, a communications device identification method includes providing identification information regarding a group of wireless identification devices within a wireless communications range of a reader, using the provided identification information, selecting one of a plurality of different search procedures for identifying unidentified ones of the wireless identification devices within the wireless communications range, and identifying at least some of the unidentified ones of the wireless identification devices using the selected one of the search procedures.

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

Directory of Open Access Journals (Sweden)

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

The transmembrane domain of the p75 neurotrophin receptor stimulates phosphorylation of the TrkB tyrosine kinase receptor.

Science.gov (United States)

Saadipour, Khalil; MacLean, Michael; Pirkle, Sean; Ali, Solav; Lopez-Redondo, Maria-Luisa; Stokes, David L; Chao, Moses V

2017-10-06

The function of protein products generated from intramembraneous cleavage by the γ-secretase complex is not well defined. The γ-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein that results in Aβ, a transmembrane (TM) peptide. Another protein that undergoes very similar γ-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of TrkB (tyrosine kinase receptor B). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time-dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Because this residue is just before the γ-secretase cleavage site, we then investigated whether the p75(αγ) peptide, which is a product of both α- and γ-cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether, our results suggest that TrkB activation by p75(αγ) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimer's disease, and epilepsy. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Pseudo-communication vs Quasi-communication

Directory of Open Access Journals (Sweden)

Елена Константиновна Черничкина

2016-12-01

Full Text Available The article is devoted to the analysis of such specific forms of human interaction as quasi- and pseudo-communication. The authors specify the terms which sometimes are used interchangeably. The aim of the conducted research is to find out and demonstrate existing differences and similarities of these communicative phenomena on the basis of theoretical and empirical analysis of the research material in the Russian and English languages. The authors describe communicative features of these phenomena and consider the reasons for such forms of communication and their increased use at present. The research material is represented fiction extracts, film scripts, jokes, print media, a collection of oral speech records both in Russian and English. The authors make use of the following research methods: definitional analysis (to define the terminology of the research, the method of linguistic observation and introspection (to select the communicative situations, the descriptive-analytical method and the method of comparative analysis (to identify similarities and differences of the target phenomena, and the conversational analysis method (to view productivity and effectiveness of a dialogue, etc. The classification of possible forms of their existence in different discourses is suggested. The authors assume that both pseudo- and quasi-communication are characterized as fictitious forms of human interaction with some noticeable violation of the basic communicative model. Pseudo-communication suffers from the discrepancy of the meaning of a coded and decoded message. The authors put forward the main parameters of scientific classification of it as follows: adequate understanding, intentionality, and the stage of communicative action where the failure takes place. At the same time they stress the necessity to distinguish the cases of pseudo talks from phatic and indirect communication. Quasi-communcation is marked by the lack of a real partner and hence