scholarly journals Assembly of Retrovirus Capsid-Nucleocapsid Proteins in the Presence of Membranes or RNA

2000 ◽  
Vol 74 (16) ◽  
pp. 7431-7441 ◽  
Author(s):  
Guy Zuber ◽  
Jason McDermott ◽  
Sonya Karanjia ◽  
Weiyi Zhao ◽  
Michael F. Schmid ◽  
...  
Keyword(s):  
Proteolytic Processing ◽  
Nucleocapsid Proteins ◽  
Gag Proteins ◽  
Virus Particles ◽  
His Tag ◽  
Different Types ◽  
Membrane Bound ◽  
Immature Virus ◽  
Local Protein

ABSTRACT Retrovirus Gag precursor (PrGag) proteins direct the assembly of roughly spherical immature virus particles, while after proteolytic processing events, the Gag capsid (CA) and nucleocapsid (NC) domains condense on viral RNAs to form mature retrovirus core structures. To investigate the process of retroviral morphogenesis, we examined the properties of histidine-tagged (His-tagged) Moloney murine leukemia (M-MuLV) capsid plus nucleocapsid (CANC) (His-MoCANC) proteins in vitro. The His-MoCANC proteins bound RNA, possessed nucleic acid-annealing activities, and assembled into strand, circle (or sphere), and tube forms in the presence of RNA. Image analysis of electron micrographs revealed that tubes were formed by cage-like lattices of CANC proteins surrounding at least two different types of protein-free cage holes. By virtue of a His tag association with nickel-chelating lipids, His-MoCANC proteins also assembled into planar sheets on lipid monolayers, mimicking the membrane-associated immature PrGag protein forms. Membrane-bound His-MoCANC proteins organized into two-dimensional (2D) cage-like lattices that were closely related to the tube forms, and in the presence of both nickel-chelating lipids and RNAs, 2D lattice forms appeared similar to lattices assembled in the absence of RNA. Our observations are consistent with a M-MuLV morphogenesis model in which proteolytic processing of membrane-bound Gag proteins permits CA and NC domains to rearrange from an immature spherical structure to a condensed mature form while maintaining local protein-protein contacts.

scholarly journals Spectrum of antiviral activity of 4-aminopyrimidine N-oxides against a broad panel of tick-borne encephalitis virus strains

2020 ◽  
Vol 28 ◽  
pp. 204020662094346
Author(s):  
Evgenia V Dueva ◽  
Ksenia K Tuchynskaya ◽  
Liubov I Kozlovskaya ◽  
Dmitry I Osolodkin ◽  
Kseniya N Sedenkova ◽  
...  
Keyword(s):  
Encephalitis Virus ◽  
Northern Eurasia ◽  
Virus Particles ◽  
E Protein ◽  
Tick Borne Encephalitis ◽  
Tbev Strain ◽  
Amino Phenol ◽  
Tick Borne Encephalitis Virus ◽  
Immature Virus

Tick-borne encephalitis is an important human arbovirus neuroinfection spread across the Northern Eurasia. Inhibitors of tick-borne encephalitis virus (TBEV) strain Absettarov, presumably targeting E protein n-octyl-β-d-glucoside (β-OG) pocket, were reported earlier. In this work, these inhibitors were tested in vitro against seven strains representing three main TBEV subtypes. The most potent compound, 2-[(2-methyl-1-oxido-5,6,7,8-tetrahydroquinazolin-4-yl)amino]-phenol, showed EC50 values lower than 22 µM against all the tested strains. Nevertheless, EC50 values for virus samples of certain strains demonstrated a substantial variation, which appeared to be consistent with the presence of E protein not only in infectious virions, but also in non-infectious and immature virus particles, protein aggregates, and membrane complexes.

scholarly journals CdtC-Induced Processing of Membrane-Bound CdtA Is a Crucial Step inAggregatibacter actinomycetemcomitansCytolethal Distending Toxin Holotoxin Formation

2017 ◽  
Vol 86 (3) ◽  
Author(s):  
Keiko Tsuruda ◽  
Oranart Matangkasombut ◽  
Masaru Ohara ◽  
Motoyuki Sugai
Keyword(s):  
Mammalian Cells ◽  
Proteolytic Processing ◽  
Content Type ◽  
Protein Toxin ◽  
Sequential Event ◽  
Membrane Bound ◽  
Processed Form ◽  
Rapid Processing

ABSTRACTAggregatibacter actinomycetemcomitansis an oral pathogen causing periodontal disease and bacterial endocarditis. It produces cytolethal distending toxin (CDT) that could damage mammalian cells and tissues. CDT is a tripartite protein toxin composed of CdtA, CdtB, and CdtC. We have previously indicated that CdtA is a lipoprotein and that the proteolytic processing of CdtA is important for biogenesis and secretion of CDT holotoxin. Here, we established anin vitroprocessing assay of CdtA and investigated the interactions of CdtA with other Cdt subunits. This assay demonstrated that incubation of membrane-bound CdtA (MCdtA), CdtB, and CdtC immediately generated a processed form of CdtA (CdtA′), which is recovered from the soluble fraction. In contrast, incubation of soluble membrane-unbound CdtA with CdtB and CdtC did not yield any CdtA′. Furthermore, incubation of CdtC with MCdtA was enough to induce rapid processing of MCdtA, whereas CdtB alone was unable to induce the processing. Coimmunoprecipitation demonstrated that CdtA′ and CdtC formed a complex. Furthermore, subsequent addition of CdtB to this reaction mixture resulted in complete CDT holotoxin complex. The cytolethal distending activity assay demonstrated that CDT complex containing CdtA′ showed far stronger cytotoxicity than that containing CdtA. Collectively, our data suggest that CDT holotoxin formationin vivois a sequential event: interaction of MCdtA and CdtC induces proteolytic processing of MCdtA, and the released CdtA′ forms a complex with CdtC. Subsequent binding of CdtB to the CdtA′/CdtC complex results in CDT holotoxin formation.

scholarly journals Ultrastructure of hyphal cells of the dermatophyte Trichophyton tonsurans

2020 ◽  
Author(s):  
Amaliya Stepanova ◽  
G. Sybren de Hoog ◽  
Nataliya Vasilyeva ◽  
Konstantin Raznatovskiy ◽  
Galina Chilina
Keyword(s):  
Aerial Mycelium ◽  
Vegetative Mycelium ◽  
Limited Data ◽  
Trichophyton Tonsurans ◽  
Transmission Electron ◽  
Different Types ◽  
Membrane Bound ◽  
First Time ◽  
Mitochondrial Reticulum

Background and Purpose: Trichophyton tonsurans is a widely distributed anthropophilic dermatophyte causing different diseases of skin. In the literature limited data are available about the morphogenesis of vegetative mycelium of T. tonsurans and related anthropophilic dermatophytes. The aim of present study was to describe ultrastructural patterns of development, cellular organellography and septal pore apparatus structure of in vitro growing vegetative mycelium of T. tonsurans. Materials and Methods: Trichophyton tonsurans strain RCPFF 214/898 was grown on solid Czapek’s Agar (CzA) at 28ºС. For investigation of colonies morphology we used methods of light-, scanning and transmission electron microscopy (SEM and TEM). Results: Differences in morphogenesis of aerial and substrate hyphae were revealed. Mitochondrial reticulum and fibrosinous bodies were shown in T. tonsurans for the first time. The septal pore apparatus in hyphal cells of was comprised Woronin bodies and septal pore plugs. Woronin bodies (0.18 μm), located with 1‒4 near the pore, were spherical, membrane-bound, and had a homogeneous, electron-dense content. The cells of aerial and submerged hyphal cells of T. tonsurans contain two nuclei. Conclusion: Mature cells of substrate hyphae appeared more active than comparable cells in the aerial mycelium. During the maturation process, the differences in number and morphology of mitochondria, number of vacuoles, and in the synthesis of different types of storage substances were revealed. Presence of “mitochondrial reticulum” and variable types of storage substances in submerged hyphal cells suggested higher levels of metabolic activity compared to aerial mycelium.

scholarly journals Inhibition of intracellular proteolytic processing of soluble proproteins by an engineered α2-macroglobulin containing a furin recognition sequence in the bait region

1997 ◽  
Vol 326 (2) ◽  
pp. 507-514 ◽  
Author(s):  
Luc VAN ROMPAEY ◽  
Torik AYOUBI ◽  
Wim VAN DE VEN ◽  
Peter MARYNEN
Keyword(s):  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
Proteolytic Processing ◽  
Soluble Form ◽  
Recognition Sequence ◽  
Bait Region ◽  
Α2 Macroglobulin ◽  
Ester Formation ◽  
Membrane Bound

The bait region of the general protease inhibitor α2-macroglobulin (α2M) was mutated by introducing a recognition sequence of furin. This did not interfere with folding, S-ester formation or tetramerization of the mutant recombinant α2M (rα2M). Mutant rα2M inhibited furin in vitro, by a similar mechanism to that used by plasma α2M to inhibit high-molecular-mass proteases. The mutant α2M was intracellularly active in COS-1 cells in inhibiting the endogenous processing of the soluble substrates for furin (von Willebrand factor, transforming growth factor β1 and a soluble form of the envelope glycoprotein gp160 from HIV-1) but not the membrane-bound form of gp160. The intracellular activity of mutant α2M strongly indicated that α2M attains its native conformation, and thus that the unusual internal S-ester is formed, before α2M passes through the cleavage compartment(s). Our results show for the first time that modulation of the bait region of α2M allows the creation of an inhibitor against membrane-bound proteases. It can be expected that the use of α2M-bait mutants will become important as a technique for the study of various proteolytic processes and for the identification of the proteases involved.

scholarly journals A Glutaredoxin, Encoded by the G4L Gene of Vaccinia Virus, Is Essential for Virion Morphogenesis

2000 ◽  
Vol 74 (19) ◽  
pp. 9175-9183 ◽  
Author(s):  
Christine L. White ◽  
Andrea S. Weisberg ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Virus Replication ◽  
Proteolytic Processing ◽  
Rnase Protection ◽  
Virus Particles ◽  
Late Protein ◽  
Virion Morphogenesis ◽  
Core Components

ABSTRACT Vaccinia virus encodes two glutaredoxins, O2L and G4L, both of which exhibit thioltransferase and dehydroascorbate reductase activities in vitro. Although O2L was previously found to be dispensable for virus replication, we now show that G4L is necessary for virion morphogenesis. RNase protection and Western blotting assays indicated that G4L was expressed at late times after infection and was incorporated into mature virus particles. Attempts to isolate a mutant virus with a deleted G4L gene were unsuccessful, suggesting that the protein was required for virus replication. This interpretation was confirmed by the construction and characterization of a conditional lethal recombinant virus with an inducible copy of the G4L gene replacing the original one. Expression of G4L was proportional to the concentration of inducer, and the amount of glutaredoxin could be varied from barely detectable to greater than normal amounts of protein. Immunogold labeling revealed that the induced G4L protein was associated with immature and mature virions and adjacent cytoplasmic depots. In the absence of inducer, the production of infectious virus was severely inhibited, though viral late protein synthesis appeared unaffected except for decreased maturation-dependent proteolytic processing of certain core components. Electron microscopy of cells infected under nonpermissive conditions revealed an accumulation of crescent membranes on the periphery of electron-dense globular masses but few mature particles. We concluded that the two glutaredoxin homologs encoded by vaccinia virus have different functions and that G4L has a role in virion morphogenesis, perhaps by acting as a redox protein.

scholarly journals Conversion of Membrane-bound Fas(CD95) Ligand to Its Soluble Form Is Associated with Downregulation of Its Proapoptotic Activity and Loss of Liver Toxicity

1998 ◽  
Vol 187 (8) ◽  
pp. 1205-1213 ◽  
Author(s):  
Pascal Schneider ◽  
Nils Holler ◽  
Jean-Luc Bodmer ◽  
Michael Hahne ◽  
Karl Frei ◽  
...  
Keyword(s):  
Fas Ligand ◽  
Liver Toxicity ◽  
Proteolytic Processing ◽  
Soluble Form ◽  
Cross Linking ◽  
Trail Receptors ◽  
Membrane Bound ◽  
Apoptotic Activity

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-α undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-α) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.

Ultrastructural Investigations of the Contractile Filaments of Amoebae

1974 ◽  
Vol 32 ◽  
pp. 188-189
Author(s):  
P.L. Moore
Keyword(s):  
Cytoplasmic Streaming ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Amoeboid Movement ◽  
Different Types ◽  
Chemical Conditions ◽  
Complete Interpretation

Previous freeze fracture results on the intact giant, amoeba Chaos carolinensis indicated the presence of a fibrillar arrangement of filaments within the cytoplasm. A complete interpretation of the three dimensional ultrastructure of these structures, and their possible role in amoeboid movement was not possible, since comparable results could not be obtained with conventional fixation of intact amoebae. Progress in interpreting the freeze fracture images of amoebae required a more thorough understanding of the different types of filaments present in amoebae, and of the ways in which they could be organized while remaining functional.The recent development of a calcium sensitive, demembranated, amoeboid model of Chaos carolinensis has made it possible to achieve a better understanding of such functional arrangements of amoeboid filaments. In these models the motility of demembranated cytoplasm can be controlled in vitro, and the chemical conditions necessary for contractility, and cytoplasmic streaming can be investigated. It is clear from these studies that “fibrils” exist in amoeboid models, and that they are capable of contracting along their length under conditions similar to those which cause contraction in vertebrate muscles.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Recent Advances in Curcumin Nanocarriers for the Treatment of Different Types of Cancer with Special Emphasis on In Vitro Cytotoxicity and Cellular Uptake Studies

2020 ◽  
Vol 10 (5) ◽  
pp. 577-590
Author(s):  
Jai B. Sharma ◽  
Shailendra Bhatt ◽  
Asmita Sharma ◽  
Manish Kumar
Keyword(s):  
Cancer Cells ◽  
Cellular Uptake ◽  
Anticancer Agents ◽  
Targeted Delivery ◽  
In Vitro Cytotoxicity ◽  
Curcumin Nanoparticles ◽  
Cytotoxicity Studies ◽  
Delivery Technique ◽  
Different Types

Background: The potential use of nanocarriers is being explored rapidly for the targeted delivery of anticancer agents. Curcumin is a natural polyphenolic compound obtained from rhizomes of turmeric, belongs to family Zingiberaceae. It possesses chemopreventive and chemotherapeutic activity with low toxicity in almost all types of cancer. The low solubility and bioavailability of curcumin make it unable to use for the clinical purpose. The necessity of an effective strategy to overcome the limitations of curcumin is responsible for the development of its nanocarriers. Objective: This study is aimed to review the role of curcumin nanocarriers for the treatment of cancer with special emphasis on cellular uptake and in vitro cytotoxicity studies. In addition to this, the effect of various ligand conjugated curcumin nanoparticles on different types of cancer was also studied. Methods: A systematic review was conducted by extensively surfing the PubMed, science direct and other portals to get the latest update on recent development in nanocarriers of curcumin. Results: The current data from recent studies showed that nanocarriers of curcumin resulted in the targeted delivery, higher efficacy, enhanced bioavailability and lower toxicity. The curcumin nanoparticles showed significant inhibitory effects on cancer cells as compared to free curcumin. Conclusion: It can be concluded that bioavailability of curcumin and its cytotoxic effect to cancer cells can be enhanced by the development of curcumin based nanocarriers and it was found to be a potential drug delivery technique for the treatment of cancer.

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