Human α-synemin interacts directly with vinculin and metavinculin

2008 ◽  
Vol 409 (3) ◽  
pp. 657-667 ◽  
Author(s):  
Ning Sun ◽  
David R. Critchley ◽  
Denise Paulin ◽  
Zhenlin Li ◽  
Richard M. Robson
Keyword(s):  
Mammalian Cells ◽  
Striated Muscle ◽  
Splice Variants ◽  
Focal Adhesions ◽  
Muscle Cells ◽  
Intermediate Filament Protein ◽  
Tail Domain ◽  
Protein Protein Interaction

Synemin is a very large, unique member of the IF (intermediate filament) protein superfamily. Association of synemin with the major IF proteins, desmin and/or vimentin, within muscle cells forms heteropolymeric IFs. We have previously identified interactions of avian synemin with α-actinin and vinculin. Avian synemin, however, is expressed as only one form, whereas human synemin is expressed as two major splice variants, namely α- and β-synemins. The larger α-synemin contains an additional 312-amino-acid insert (termed SNTIII) located near the end of the long C-terminal tail domain. Whether α- and β-synemins have different cellular functions is unclear. In the present study we show, by in vitro protein–protein interaction assays, that SNTIII interacts directly with both vinculin and metavinculin. Furthermore, SNTIII interacts with vinculin in vivo, and this association is promoted by PtdIns(4,5)P2. SNTIII also specifically co-localizes with vinculin within focal adhesions when transiently expressed in mammalian cells. In contrast, other regions of synemin show distinct localization patterns in comparison with those of SNTIII, without labelling focal adhesions. Our results indicate that α-synemin, but not β-synemin, interacts with both vinculin and metavinculin, thereby linking the heteropolymeric IFs to adhesion-type junctions, such as the costameres located within human striated muscle cells.

scholarly journals Effects of Trichinella spiralis and its excretory/secretory products on autophagy of host muscle cells in vivo and in vitro

2021 ◽  
Vol 15 (2) ◽  
pp. e0009040
Author(s):  
Xiaoxiang Hu ◽  
Xiaolei Liu ◽  
Xue Bai ◽  
Li Yang ◽  
Jing Ding ◽  
...  
Keyword(s):  
Striated Muscle ◽  
Cell Transformation ◽  
Trichinella Spiralis ◽  
Muscle Cells ◽  
Microbial Infection ◽  
Related Factors ◽  
Parasitic Helminths ◽  
Secretory Products

Trichinella spiralis (T. spiralis) is a widely distributed pathogenic microorganism that causes trichinellosis, a disease that has the potential of causing severe harm to their host. Numerous studies have demonstrated that autophagy can be triggered by microbial infection, such as bacteria, viruses, protozoa, and parasitic helminths. However, it’s still unknown whether autophagy can facilitate host resistance to T. spiralis infection. The present study examined the role of autophagy in striated muscle cell transformation following infection with T. spiralis in BALB/c mice. Transmission electron microscopy (TEM) was used to detect the production of the host diaphragm autophagosome after T. spiralis infection, and changes in the protein and transcriptional levels of autophagic marker proteins were also detected. The significance of autophagy in T. spiralis infection, namely inhibition of T. spiralis growth, was preliminarily evaluated by conducting in vivo experiments using autophagy inhibitors. Besides, we studied the effect of excretory-secretory products (ES) of T. spiralis on autophagy of C2C12 myoblasts. The changes in protein and gene expression levels in autophagy-related pathways in vitro and in vivo were measured as further evidence. The results showed that T. spiralis infection induced autophagy in the host muscle cells. Meanwhile, ES inhibited autophagy of myoblasts in vitro, but this did not affect the cell viability. The upregulation and downregulation of autophagy-related factors in skeletal muscle cells may indicate an adaptive mechanism providing a comfortable niche for the parasite.

scholarly journals The N and C Termini of the Splice Variants of the Human Mitogen-Activated Protein Kinase-Interacting Kinase Mnk2 Determine Activity and Localization

2003 ◽  
Vol 23 (16) ◽  
pp. 5692-5705 ◽  
Author(s):  
Gert C. Scheper ◽  
Josep L. Parra ◽  
Mary Wilson ◽  
Barbara van Kollenburg ◽  
Alfred C. O. Vertegaal ◽  
...  
Keyword(s):  
Map Kinase ◽  
Binding Site ◽  
Mammalian Cells ◽  
Map Kinases ◽  
Splice Variants ◽  
Mitogen Activated Protein Kinase ◽  
Initiation Factor ◽  
Mitogen Activated Protein

ABSTRACT The cap-binding eukaryotic initiation factor eIF4E is phosphorylated by the mitogen-activated protein (MAP) kinase-interacting kinases (Mnk's). Three forms of the Mnk's exist in human cells: Mnk1, Mnk2a, and Mnk2b. These last two are derived from the same gene by alternative splicing and differ only at their C termini. While Mnk2a contains a MAP kinase-binding site in this region, Mnk2b lacks such a sequence and is much less readily activated by MAP kinases in vitro. Expression of Mnk2b in mammalian cells leads to increased phosphorylation of eIF4E, showing that it acts as an eIF4E kinase in vivo. While Mnk2a is cytoplasmic, a substantial amount of Mnk2b is found in the nucleus. Both enzymes contain a stretch of basic residues in their N termini that plays a role in binding to eIF4G and functions as a nuclear localization signal. Binding of eIF4G or nuclear import appears to be regulated by the C terminus of Mnk2a. Furthermore, the MAP kinase-binding site of Mnk2a regulates nuclear entry. Within the nucleus, Mnk2b and certain variants of Mnk2a that are present in the nucleus colocalize with the promyelocytic leukemia protein PML, which also binds to eIF4E.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Cytological changes induced by platinum-thymine in sarcoma-180 cells: Electron microscopy study

1990 ◽  
Vol 48 (3) ◽  
pp. 290-291
Author(s):  
Gustav Ofosu
Keyword(s):  
Mammalian Cells ◽  
Antitumor Agent ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Limiting Factor ◽  
Sarcoma 180 ◽  
Electron Microscopic ◽  
Cytological Changes

Platinum-thymine has been found to be a potent antitumor agent, which is quite soluble in water, and lack nephrotoxicity as the dose-limiting factor. The drug has been shown to interact with DNA and inhibits DNA, RNA and protein synthesis in mammalian cells in vitro. This investigation was undertaken to elucidate the cytotoxic effects of piatinum-thymine on sarcoma-180 cells in vitro ultrastructurally, Sarcoma-180 tumor bearing mice were treated with intraperitoneal injection of platinum-thymine 40mg/kg. A concentration of 60μg/ml dose of platinum-thymine was used in in vitro experiments. Treatments were at varying time intervals of 3, 7 and 21 days for in vivo experiments, and 30, 60 and 120 min., 6, 12, and 24th in vitro. Controls were not treated with platinum-thymine.Electron microscopic analyses of the treated cells in vivo and in vitro showed drastic cytotoxic effect.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica

2020 ◽  
Vol 27 (5) ◽  
pp. 432-446
Author(s):  
Akiko Yamamoto ◽  
Ken-ichiro Matsunaga ◽  
Toyoaki Anai ◽  
Hitoshi Kawano ◽  
Toshihisa Ueda ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Immunohistochemical Analysis ◽  
Protein Characterization ◽  
Intermediate Filament Protein ◽  
Tail Domain ◽  
Animal Cells ◽  
Dugesia Japonica ◽  
Function Relationship

Background: Intermediate Filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. Objective: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. Method: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. Results: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. Conclusions: Together with data from other histological studies, our results suggest that Djf- 1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress.

The Role of nitro (NO2-), chloro (Cl), and fluoro (F) Substitution in the Design of Antileishmanial and Antichagasic Compounds

2020 ◽  
Vol 21 ◽  
Author(s):  
Boniface Pone ◽  
Ferreira Igne Elizabeth
Keyword(s):  
Chagas Disease ◽  
Mammalian Cells ◽  
Neglected Tropical Diseases ◽  
New Drugs ◽  
Pharmacokinetic Studies ◽  
Scientific Publications ◽  
Antitrypanosomal Activity ◽  
Chemical Groups

: Neglected tropical diseases (NTDs) are responsible for over 500,000 deaths annually and are characterized by multiple disabilities. Leishmaniasis and Chagas disease are among the most severe NTDs, and are caused by the Leishmania sp, and Trypanosoma cruzi, respectively. Glucantime, pentamidine and miltefosine are commonly used to treat leishmaniasis, whereas nifurtimox, benznidazole are current treatments for Chagas disease. However, these treatments are associated with drug resistance, and severe side effects. Hence, the development of synthetic products, especially those containing N02, F, or Cl, which chemical groups are known to improve the biological activity. The present work summarizes the information on the antileishmanial and antitrypanosomal activity of nitro-, chloro-, and fluoro-synthetic derivatives. Scientific publications referring to halogenated derivatives in relation to antileishmanial and antitrypanosomal activities were hand searched in databases such as SciFinder, Wiley, Science Direct, PubMed, ACS, Springer, Scielo, and so on. According to the literature information, more than 90 compounds were predicted as lead molecules with reference to their IC50/EC50 values in in vitro studies. It is worth to mention that only active compounds with known cytotoxic effects against mammalian cells were considered in the present study. The observed activity was attributed to the presence of nitro-, fluoro- and chloro-groups in the compound backbone. All in all, nitro and h0alogenated derivatives are active antileishmanial and antitrypanosomal compounds and can serve as baseline for the development of new drugs against leishmaniasis and Chagas disease. However, efforts on in vitro and in vivo toxicity studies of the active synthetic compounds is still needed. Pharmacokinetic studies, and the mechanism of action of the promising compounds need to be explored. The use of new catalysts and chemical transformation can afford unexplored halogenated compounds with improved antileishmanial and antitrypanosomal activity.

Discovery of 3-Cinnamamido-N-Substituted Benzamides as Potential Antimalarial Agents

2020 ◽  
Vol 16 ◽  
Author(s):  
Haicheng Liu ◽  
Yushi Futamura ◽  
Honghai Wu ◽  
Aki Ishiyama ◽  
Taotao Zhang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Antimalarial Activity ◽  
In Vitro Assays ◽  
Compound Library ◽  
Antimalarial Agents ◽  
Phenotypic Screen ◽  
Ic50 Values ◽  
Substituted Benzamides

Background: Malaria is one of the most devastating parasitic diseases, yet the discovery of antimalarial agents remains profoundly challenging. Very few new antimalarials have been developed in the past 50 years, while the emergence of drug-resistance continues to appear. Objective: This study focuses on the discovery, design, synthesis, and antimalarial evaluation of 3-cinnamamido-N-substituted benzamides. Method: In this study, a screening of our compound library was carried out against the multidrug-sensitive Plasmodium falciparum 3D7 strain. Derivatives of the hit were designed, synthesized and tested against P. falciparum 3D7 and the in vivo antimalarial activity of the most active compounds was evaluated using the method of Peters’ 4-day suppressive test. Results: The retrieved hit compound 1 containing a 3-cinnamamido-N-substituted benzamide skeleton showed moderate antimalarial activity (IC50 = 1.20 µM) for the first time. A series of derivatives were then synthesized through a simple four-step workflow, and half of them exhibited slightly better antimalarial effect than the precursor 1 during the subsequent in vitro assays. Additionally, compounds 11, 23, 30 and 31 displayed potent activity with IC50 values of approximately 0.1 µM, and weak cytotoxicity against mammalian cells. However, in vivo antimalarial activity is not effective which might be ascribed to the poor solubility of these compounds. Conclusion: In this study, phenotypic screen of our compound library resulted in the first report of 3-cinnamamide framework with antimalarial activity and 40 derivatives were then designed and synthesized. Subsequent structure-activity studies showed that compounds 11, 23, 30 and 31 exhibited the most potent and selective activity against P. falciparum 3D7 strain with IC50 values around 0.1 µM. Our work herein sets another example of phenotypic screen-based drug discovery, leading to potentially promising candidates of novel antimalarial agents once given further optimization.

scholarly journals In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

2021 ◽  
Vol 7 (6) ◽  
pp. 439
Author(s):  
Tecla Ciociola ◽  
Walter Magliani ◽  
Tiziano De Simone ◽  
Thelma A. Pertinhez ◽  
Stefania Conti ◽  
...  
Keyword(s):  
In Silico ◽  
Mammalian Cells ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Consensus Sequence ◽  
In Silico Analysis ◽  
Significant Activity ◽  
Synthetic Antibody

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

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