scholarly journals Sentan: A Novel Specific Component of the Apical Structure of Vertebrate Motile Cilia

2008 ◽  
Vol 19 (12) ◽  
pp. 5338-5346 ◽  
Author(s):  
Akiharu Kubo ◽  
Akiko Yuba-Kubo ◽  
Sachiko Tsukita ◽  
Shoichiro Tsukita ◽  
Masayuki Amagai
Keyword(s):  
Cell Membrane ◽  
Polyclonal Antibodies ◽  
Distal Portion ◽  
Lipid Binding ◽  
Molecular Component ◽  
Ovum Transport ◽  
Membrane Protrusions ◽  
Motile Cilia ◽  
Molecular Components

Human respiratory and oviductal cilia have specific apical structures characterized by a narrowed distal portion and a ciliary crown. These structures are conserved among vertebrates that have air respiration systems; however, the molecular components of these structures have not been defined, and their functions are unknown. To identify the molecular component(s) of the cilia apical structure, we screened EST libraries to identify gene(s) that are exclusively expressed in ciliated tissues, are transcriptionally up-regulated during in vitro ciliogenesis, and are not expressed in testis (because sperm flagella have no such apical structures). One of the identified gene products, named sentan, was localized to the distal tip region of motile cilia. Using anti-sentan polyclonal antibodies and electron microscopy, sentan was shown to localize exclusively to the bridging structure between the cell membrane and peripheral singlet microtubules, which specifically exists in the narrowed distal portion of cilia. Exogenously expressed sentan showed affinity for the membrane protrusions, and a protein–lipid binding assay revealed that sentan bound to phosphatidylserine. These findings suggest that sentan is the first molecular component of the ciliary tip to bridge the cell membrane and peripheral singlet microtubules, making the distal portion of the cilia narrow and stiff to allow for better airway clearance or ovum transport.

Light Chain LC and TAT-EGFP-HCS of Botulinum Toxin Expression and Biological Function in vitro and in vivo

2019 ◽  
Vol 16 (3) ◽  
pp. 175-180
Author(s):  
Fengjin Hao ◽  
Yueqin Feng ◽  
Yifu Guan
Keyword(s):  
Biological Activity ◽  
Botulinum Toxin ◽  
Cell Membrane ◽  
Western Blot ◽  
Biological Function ◽  
C6 Cells ◽  
Green Fluorescence ◽  
Bv2 Cells

Objective: To verify whether the botulinum toxin heavy chain HCS has specific neuronal targeting function and to confirm whether TAT-EGFP-LC has hydrolyzable SNAP-25 and has transmembrane biological activity. Methods: We constructed the pET-28a-TAT-EGFP-HCS/LC plasmid. After the plasmid is expressed and purified, we co-cultured it with nerve cells or tumors. In addition, we used Western-Blot to identify whether protein LC and TAT-EGFP-LC can digest the protein SNAP-25. Results: Fluorescence imaging showed that PC12, BV2, C6 and HeLa cells all showed green fluorescence, and TAT-EGFP-HCS had the strongest fluorescence. Moreover, TAT-EGFP-LC can hydrolyze intracellular SNAP-25 in PC12 cells, C6 cells, BV2 cells and HeLa, whereas LC alone cannot. In addition, the in vivo protein TAT-EGFP-HCS can penetrate the blood-brain barrier and enter mouse brain tissue. Conclusion: TAT-EGFP-HSC expressed in vitro has neural guidance function and can carry large proteins across the cell membrane without influencing the biological activity.

scholarly journals Hexapod Assassins’ Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 819
Author(s):  
Nicolai Rügen ◽  
Timothy P. Jenkins ◽  
Natalie Wielsch ◽  
Heiko Vogel ◽  
Benjamin-Florian Hempel ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Galleria Mellonella ◽  
Systemic Reactions ◽  
Venom Composition ◽  
Assassin Bug ◽  
Molecular Components ◽  
First Time ◽  
Tissue Digestion

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.

scholarly journals DnaA, the Initiator of Escherichia coliChromosomal Replication, Is Located at the Cell Membrane

2000 ◽  
Vol 182 (9) ◽  
pp. 2604-2610 ◽  
Author(s):  
Gillian Newman ◽  
Elliott Crooke
Keyword(s):  
Cell Membrane ◽  
Spatial Organization ◽  
Active Form ◽  
Chromosomal Replication ◽  
E Coli ◽  
Dnaa Protein ◽  
Section Electron ◽  
Acidic Phospholipids

ABSTRACT Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.

scholarly journals Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom

Toxins ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 254
Author(s):  
Shelby S. Szteiter ◽  
Ilse N. Diego ◽  
Jonathan Ortegon ◽  
Eliana Salinas ◽  
Abcde Cirilo ◽  
...  
Keyword(s):  
Snake Venom ◽  
Polyclonal Antibody ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Snake Envenomation ◽  
Disintegrin Domain ◽  
The Common ◽  
Neutralization Ability ◽  
New Strategies

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

scholarly journals Influences of advanced glycosylation end products on the inner blood–retinal barrier in a co-culture cell model in vitro

2020 ◽  
Vol 15 (1) ◽  
pp. 619-628
Author(s):  
Chen Yuan ◽  
Ya Mo ◽  
Jie Yang ◽  
Mei Zhang ◽  
Xuejun Xie
Keyword(s):  
Electrical Resistance ◽  
Cell Model ◽  
Polyclonal Antibodies ◽  
Time Dependent ◽  
Dependent Manner ◽  
Blood Retinal Barrier ◽  
Advanced Glycosylation End Products ◽  
End Products ◽  
Advanced Glycosylation

AbstractAdvanced glycosylation end products (AGEs) are harmful factors that can damage the inner blood–retinal barrier (iBRB). Rat retinal microvascular endothelial cells (RMECs) were isolated and cultured, and identified by anti-CD31 and von Willebrand factor polyclonal antibodies. Similarly, rat retinal Müller glial cells (RMGCs) were identified by H&E staining and with antibodies of glial fibrillary acidic protein and glutamine synthetase. The transepithelial electrical resistance (TEER) value was measured with a Millicell electrical resistance system to observe the leakage of the barrier. Transwell cell plates for co-culturing RMECs with RMGCs were used to construct an iBRB model, which was then tested with the addition of AGEs at final concentrations of 50 and 100 mg/L for 24, 48, and 72 h. AGEs in the in vitro iBRB model constructed by RMEC and RMGC co-culture led to the imbalance of the vascular endothelial growth factor (VEGF) and pigment epithelial derivative factor (PEDF), and the permeability of the RMEC layer increased because the TEER decreased in a dose- and time-dependent manner. AGEs increased VEGF but lowered PEDF in a dose- and time-dependent manner. The intervention with AGEs led to the change of the transendothelial resistance of the RMEC layer likely caused by the increased ratio of VEGF/PEDF.

scholarly journals Intracellular host cell membrane remodelling induced by SARS‐CoV‐2 infection in vitro

2021 ◽  
Author(s):  
Lucio Ayres Caldas ◽  
Fabiana Avila Carneiro ◽  
Fabio Luis Monteiro ◽  
Ingrid Augusto ◽  
Luiza Mendonça Higa ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Host Cell ◽  
Host Cell Membrane

scholarly journals The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells.

1988 ◽  
Vol 107 (2) ◽  
pp. 635-641 ◽  
Author(s):  
J L Salisbury ◽  
A T Baron ◽  
M A Sanders
Keyword(s):  
Nuclear Envelope ◽  
Polyclonal Antibodies ◽  
Flagellar Apparatus ◽  
Immunogold Labeling ◽  
Basal Bodies ◽  
Flagellar Roots ◽  
Cell Biol ◽  
Immunofluorescence Studies ◽  
Descending Fibers

Monoclonal and polyclonal antibodies raised against algal centrin, a protein of algal striated flagellar roots, were used to characterize the occurrence and distribution of this protein in interphase and mitotic Chlamydomonas cells. Chlamydomonas centrin, as identified by Western immunoblot procedures, is a low molecular (20,000-Mr) acidic protein. Immunofluorescence and immunogold labeling demonstrates that centrin is a component of the distal fiber. In addition, centrin-based flagellar roots link the flagellar apparatus to the nucleus. Two major descending fibers extend from the basal bodies toward the nucleus; each descending fiber branches several times giving rise to 8-16 fimbria which surround and embrace the nucleus. Immunogold labeling indicates that these fimbria are juxtaposed to the outer nuclear envelope. Earlier studies have demonstrated that the centrin-based linkage between the flagellar apparatus and the nucleus is contractile, both in vitro and in living Chlamydomonas cells (Wright, R. L., J. Salisbury, and J. Jarvik. 1985. J. Cell Biol. 101:1903-1912; Salisbury, J. L., M. A. Sanders, and L. Harpst. 1987. J. Cell Biol. 105:1799-1805). Immunofluorescence studies show dramatic changes in distribution of the centrin-based system during mitosis that include a transient contraction at preprophase; division, separation, and re-extension during prophase; and a second transient contraction at the metaphase/anaphase boundary. These observations suggest a fundamental role for centrin in motile events during mitosis.

Light-dependent hydration of the space surrounding photoreceptors in the cat retina

1994 ◽  
Vol 11 (4) ◽  
pp. 743-752 ◽  
Author(s):  
Jian-Dong Li ◽  
Victor I. Govardovskii ◽  
Roy H. Steinberg
Keyword(s):  
Mathematical Model ◽  
Time Course ◽  
Distal Portion ◽  
Subretinal Space ◽  
Volume Increase ◽  
Physiological Event ◽  
Cat Retina ◽  
Interphotoreceptor Matrix ◽  
Space Marker

AbstractWe have studied the effect of retinal illumination on the concentration of the extracellular space marker tetramethylammonium (TMA+) in the dark-adapted cat retina using double-barreled ion-selective microelectrodes. The retina was loaded with TMA+ by a single intravitreal injection. Retinal illumination produced a slow decrease in , which was maximal in amplitude in the most distal portion of the space surrounding photoreceptors, the subretinal space. The light-evoked decrease in was considerably slower and of a different overall time course than the light-evoked decrease in , also recorded in the subretinal space. decreased to a peak at 38 s after the onset of illumination, then slowly recovered towards the baseline, and transiently increased following the offset of illumination. It resembled the light-evoked decreases previously recorded in the in vitro preparations of frog (Huang & Karwoski, 1990, 1992) and chick (Li et al., 1992, 1994) but was considerably larger in amplitude, 22% compared with 7%. As in frog, where it was first recorded, the light-evoked decrease is considered to originate from a light-evoked increase in the volume of the subretinal space (or subretinal hydration). A mathematical model accounting for diffusion predicted that the volume increase underlying the response was 63% on average and could be as large as 95% and last for minutes. The estimated volume increase was then used to examine its effect on K+ concentration in the subretinal space. We conclude that a light-dependent hydration of the subretinal space represents a significant physiological event in the intact cat eye, which should affect the organization of the interphotoreceptor matrix, and the concentrations of all ions and metabolites located in the subretinal space.

Identification and characterization of a novel Neospora caninum immune mapped protein 1

Parasitology ◽  
2012 ◽  
Vol 139 (8) ◽  
pp. 998-1004 ◽  
Author(s):  
X. CUI ◽  
T. LEI ◽  
D. Y. YANG ◽  
P. HAO ◽  
Q. LIU
Keyword(s):  
Neospora Caninum ◽  
Polyclonal Antibodies ◽  
Functional Protein ◽  
Reading Frame ◽  
Protein Motifs ◽  
Apicomplexan Parasites ◽  
Potential Vaccine ◽  
Palmitoylation Site

SUMMARYImmune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42·9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.

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