scholarly journals Topography of connexin32 in rat liver gap junctions. Evidence for an intramolecular disulphide linkage connecting the two extracellular peptide loops

1991 ◽  
Vol 100 (3) ◽  
pp. 567-578 ◽  
Author(s):  
S. Rahman ◽  
W.H. Evans
Keyword(s):  
Gap Junctions ◽  
Cell Communication ◽  
Amino Acid Sequences ◽  
Transmembrane Domains ◽  
Two Dimensional ◽  
Dimensional Model ◽  
Intracellular Loop ◽  
Extracellular Loops ◽  
Disulphide Bonds ◽  
Peptide Antibodies

A range of anti-peptide antibodies directed towards selected amino acid sequences of connexin32 was prepared and characterised. The site-directed antibodies that identified connexin32 were used to study by immunolocalization and by proteolytic treatment of intact and split gap junctions the arrangement of the protein in the membrane. These studies reinforce models of connexin topography in which the polypeptide traverses the junctional membrane four times, with the amino and carboxyl termini cytoplasmically located. The four transmembrane domains were shown to be linked by two extracellular loops with a single intracellular loop connecting the second and third transmembrane domains. Evidence is presented to show that the two extracellular domains of connexin32, which are important for intercellular adhesion and the insulated bridging of the extracellular space by channels allowing cell-cell communication across the gap junction, are connected by disulphide bond(s). The studies lead to a more detailed two-dimensional model of connexin32 in the membrane, incorporating the favoured theoretical arrangement of disulphide bonds at the extracellular domain of connexin32.

scholarly journals Bovine mRNAs in Cow's Milk Exosomes Are Bioavailable and Translated into Peptides in Non-bovine Systems (P06-030-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Di Wu ◽  
Jiang Shu ◽  
Camila Braga Pereira ◽  
Juan Cui ◽  
Jiri Adamec ◽  
...  
Keyword(s):  
Bovine Milk ◽  
Cell Communication ◽  
Mrna Translation ◽  
Amino Acid Sequences ◽  
Food Allergies ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Funding Sources ◽  
Rabbit Reticulocyte Lysate System ◽  
Gates Foundation

Abstract Objectives Exosomes are natural nanoparticles that participate in cell-to-cell communication by transferring regulatory molecules such as microRNAs from donor cells to recipient cells. Previously, we have demonstrated that exosomes and microRNAs are not exclusively obtained from endogenous synthesis but may also be absorbed from milk. We assessed whether 1) exosomes contain mRNAs (i.e., RNAs other than microRNAs), 2) mRNAs are bioavailable in mice, and 3) bovine mRNAs are translated into peptides. Methods For mRNA analysis, exosomes were isolated from bovine milk using differential ultracentrifugation, and RNA cargos were analyzed by RNA-sequencing. For bioavailability analysis, a synthetic fragment of fluorophore (IRDye)-labeled bovine CSN3 mRNA was transfected into bovine milk exosomes, which were administered to Balb/c mice by oral gavage. Tissues distribution of CSN3 mRNA was assessed 24 h after gavage by using a LiCor Odyssey CLx imager. For analysis of mRNA translation, mRNA from bovine milk exosomes were translated using the rabbit reticulocyte lysate system and BODIPY-labeled lysine. Peptides were separated by two-dimensional gel electrophoresis and fluorescence was visualized using a Typhoon FLA 7000 scanner. Statistical analysis, NA. Results We detected > 3600 bovine mRNAs in exosomes. Most mRNAs were truncated; 107 mRNAs contained their natural ATG start codons. Thirteen of these mRNAs, including CSN3, encoded bovine proteins and peptides with amino acid sequences distinct from those in human and murine orthologs (Table 1). Mice absorbed CSN3 mRNA encapsulated in milk exosomes, which accumulated primarily in the liver (Fig. 1). Nine bovine peptide spots were detected by two-dimensional electrophoresis (Fig. 2). Conclusions Bovine milk exosomes contain mRNAs which are bioavailable and translated into peptides in non-bovine systems. We speculate that food mRNAs might play a role in food allergies and immune tolerance. Future studies: Identification of peptides by LC/MS-MS is in progress. We will assess the relevance of mRNA translation for allergies and tolerance in animal models. Funding Sources NIFA, NIH, Gates Foundation, PureTech, Inc. and USDA Hatch and Multistate. J.Z. is a consultant for PureTech. Supporting Tables, Images and/or Graphs

scholarly journals Intracellular and extracellular loops of LRRC8 are essential for volume-regulated anion channel function

2018 ◽  
Vol 150 (7) ◽  
pp. 1003-1015 ◽  
Author(s):  
Toshiki Yamada ◽  
Kevin Strange
Keyword(s):  
Anion Channel ◽  
Transmembrane Domains ◽  
Channel Structure ◽  
Cell Swelling ◽  
Voltage Sensitivity ◽  
Intracellular Loop ◽  
Heteromeric Channel ◽  
Extracellular Loops ◽  
And Function ◽  
Essential Subunit

The volume-regulated anion channel (VRAC) is expressed ubiquitously in vertebrate cells and mediates swelling-induced release of Cl− and organic solutes. Recent studies by several laboratories have demonstrated conclusively that VRAC is encoded by members of the leucine-rich repeat containing 8 (Lrrc8) gene family, which comprises five members, termed Lrrc8a–e. Numerous observations indicate that VRAC is a heteromeric channel comprising the essential subunit LRRC8A and one or more of the other LRRC8 paralogs. Here we demonstrate that the intracellular loop (IL) connecting transmembrane domains 2 and 3 of LRRC8A and the first extracellular loop (EL1) connecting transmembrane domains 1 and 2 of LRRC8C, LRRC8D, or LRRC8E are both essential for VRAC activity. We generate homomeric VRACs by replacing EL1 of LRRC8A with that of LRRC8C and demonstrate normal regulation by cell swelling and shrinkage. We also observe normal volume-dependent regulation in VRAC homomers in which the IL of LRRC8C, LRRC8D, or LRRC8E is replaced with the LRRC8A IL. A 25–amino acid sequence unique to the LRRC8A IL is sufficient to generate homomeric VRAC activity when inserted into the corresponding region of LRRC8C and LRRC8E. LRRC8 chimeras containing these partial LRRC8A IL sequences exhibit altered anion permeability, rectification, and voltage sensitivity, suggesting that the LRRC8A IL plays a role in VRAC pore structure and function. Our studies provide important new insights into the structure/function roles of the LRRC8 EL1 and IL. Homomeric LRRC8 channels will simplify future studies aimed at understanding channel structure and the longstanding and vexing problem of how VRAC is regulated by cell volume changes.

Gap junctions in the prothoracic glands of the tobacco hornworm, Manduca sexta

1992 ◽  
Vol 50 (1) ◽  
pp. 706-707
Author(s):  
Ji-da Dai ◽  
M. Joseph Costello ◽  
Lawrence I. Gilbert
Keyword(s):  
Gap Junctions ◽  
Small Molecules ◽  
Manduca Sexta ◽  
Freeze Fracture ◽  
Cell Communication ◽  
Cellular Level ◽  
Thin Sections ◽  
Prothoracic Glands ◽  
Polyhydroxylated Steroids ◽  
Stimulation Of

Insect molting and metamorphosis are elicited by a class of polyhydroxylated steroids, ecdysteroids, that originate in the prothoracic glands (PGs). Prothoracicotropic hormone stimulation of steroidogenesis by the PGs at the cellular level involves both calcium and cAMP. Cell-to-cell communication mediated by gap junctions may play a key role in regulating signal transduction by controlling the transmission of small molecules and ions between adjacent cells. This is the first report of gap junctions in the PGs, the evidence obtained by means of SEM, thin sections and freeze-fracture replicas.

Commitment to pro- versus counter-attitudinal behavior and the dynamics of social representations

2002 ◽  
Vol 61 (1) ◽  
pp. 34-44 ◽  
Author(s):  
Eric Tafani ◽  
Lionel Souchet
Keyword(s):  
Higher Education ◽  
Social Representations ◽  
Cognitive Restructuring ◽  
Social Representation ◽  
Two Dimensional ◽  
Dimensional Model ◽  
Social Actions ◽  
The Social ◽  
Two Dimensional Model ◽  
Students Attitudes

This research uses the counter-attitudinal essay paradigm ( Janis & King, 1954 ) to test the effects of social actions on social representations. Thus, students wrote either a pro- or a counter-attitudinal essay on Higher Education. Three forms of counter-attitudinal essays were manipulated countering respectively a) students’ attitudes towards higher education; b) peripheral beliefs or c) central beliefs associated with this representation object. After writing the essay, students expressed their attitudes towards higher education and evaluated different beliefs associated with it. The structural status of these beliefs was also assessed by a “calling into question” test ( Flament, 1994a ). Results show that behavior challenging either an attitude or peripheral beliefs induces a rationalization process, giving rise to minor modifications of the representational field. These modifications are only on the social evaluative dimension of the social representation. On the other hand, when the behavior challenges central beliefs, the same rationalization process induces a cognitive restructuring of the representational field, i.e., a structural change in the representation. These results and their implications for the experimental study of representational dynamics are discussed with regard to the two-dimensional model of social representations ( Moliner, 1994 ) and rationalization theory ( Beauvois & Joule, 1996 ).

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