scholarly journals Metalloproteinases secretion from cultured cells: Differentiation by detergents

2018 ◽  
Vol 72 ◽  
pp. 1132-1137
Author(s):  
Rafał Seredyński ◽  
Katarzyna Hotowy ◽  
Elżbieta Czapińska ◽  
Nicole Nowak ◽  
Ewa Terlecka ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cultured Cells ◽  
Intracellular Localization ◽  
Active Form ◽  
Dermal Fibroblasts ◽  
Fluorimetric Assay ◽  
Enzyme Distribution ◽  
A Minor ◽  
Triton X ◽  
In Cells

Aim: Matrix metalloproteinases, particularly MMP-2 and MMP-9, are the active factors influencing the structure and properties of the extracellular matrix. This effect is enhanced in metastasis. Hence, it is necessary to enrich the knowledge of the relation between the accumulation and distribution of the enzymes in cells and the intensity of their secretion. Material/Methods: The study was conducted on three cell lines of dermal origin: normal line of human dermal fibroblasts (NHDF) and two melanoma lines (BM and B16F10). The results were obtained with substrate zymography, immunofluorescence microscopy and detergent-complemented fluorimetric assay. Results: All the studied cell lines revealed relatively rich content of MMP-2 (latent and active) with random intracellular localization. Nevertheless, the enzyme secretion was differentiated in various types of cells. The most intensive proMMP-2 secretion was obtained for NHDF, relatively lower for BM, and the lowest for B16F10 line. Zymographic detection of the active form of MMP-2 was restricted to NHDF and BM cell lines. On the other hand, differentiating usage of detergents Brij 35P and Triton X-100 evidenced an active fraction of MMP-2 present in cells of all studied lines. Triton X-100-generated lysis of cells of high MMP-2 secretion (NHDF, BM) revealed the presence of intracellular inhibitors. Conclusions: From the obtained results it can be concluded that the active form of MMP-2 is localized pericellularly in studied cells, being a minor fraction of a total amount of cellular MMP-2. The rest of the enzyme content is located deeper in cell cytoplasm in a latent form. The activity of the pericellular fraction of the enzyme can be measured with detergent-complemented fluorimetric assay, constituting a potentially useful tool for evaluating the enzyme distribution.

scholarly journals Murine Gammaherpesvirus (MHV-68) Transforms Cultured Cells in vitro

Intervirology ◽  
2015 ◽  
Vol 58 (2) ◽  
pp. 69-72 ◽  
Author(s):  
Veronika Mrázová ◽  
Tatiana Betáková ◽  
Marcela Kúdelová ◽  
Miroslava Šupolíková ◽  
Veronika Lachová ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cultured Cells ◽  
Virus Antigen ◽  
Immunofluorescence Assay ◽  
Dermal Fibroblasts ◽  
Murine Gammaherpesvirus ◽  
Transformed Cell ◽  
Transformed Cell Lines ◽  
Nih 3T3

Human dermal fibroblasts and mouse NIH/3T3 cells acquired the transformed phenotype (‘criss-cross' pattern of growth) after infection with ultraviolet-irradiated murine gammaherpesvirus (MuHV-4 strain 68; MHV-68). These cells with changed phenotype could be serially cultured for 5-6 passages (35-40 days), and then they entered into crisis and most of them died. In a small number of cultures, however, foci of newly transformed cells appeared from which two stable cell lines were derived. After 6-9 cell culture passages of the MHV-68 transformed cell lines, MHV-68 DNA and virus antigen could be detected by PCR and immunofluorescence assay along with the disappearance of actin bundles, indicating that both transformed cell lines might be oncogenic.

scholarly journals Human Cytomegalovirus UL36 Protein Is Dispensable for Viral Replication in Cultured Cells

1999 ◽  
Vol 73 (9) ◽  
pp. 7126-7131 ◽  
Author(s):  
Catherine E. Patterson ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Half Life ◽  
Cultured Cells ◽  
Intracellular Localization ◽  
State Level ◽  
Specific Variation ◽  
Infected Cells ◽  
Ad169 Strain ◽  
Expression Plasmids ◽  
In Cells

ABSTRACT Consistent with earlier analyses of human cytomegalovirus UL36 mRNA, we find that the UL36 protein is present throughout infection. In fact, it is delivered to the infected cell as a constituent of the virion. Curiously, much less UL36 protein accumulated in cells infected with the AD169 strain of human cytomegalovirus than in cells infected with the Towne or Toledo strain, and localization of the protein in cells infected with AD169 is strikingly different from that in cell infected with the Towne or Toledo strain. The variation in steady-state level of the proteins results from different stabilities of the proteins. The UL36 proteins from the three viral strains differ by several amino acid substitutions. However, this variability is not responsible for the different half-lives because the AD169 and Towne proteins, which exhibit very different half-lives within infected cells, exhibit the same half-life when introduced into uninfected cells by transfection with expression plasmids. We demonstrate that the UL36 protein is nonessential for growth in cultured cells, and we propose that the ability of the virus to replicate in the absence of UL36 function likely explains the striking strain-specific variation in the half-life and intracellular localization of the protein.

scholarly journals Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages.

1981 ◽  
Vol 90 (3) ◽  
pp. 665-669 ◽  
Author(s):  
B Poole ◽  
S Ohkuma
Keyword(s):  
Fluorescence Probe ◽  
Spectral Characteristics ◽  
Active Form ◽  
Peritoneal Macrophages ◽  
Weak Bases ◽  
Wide Range ◽  
A Minor ◽  
Energy Dependent ◽  
Mouse Peritoneal Macrophages ◽  
In Cells

The spectral characteristics of dextran, labeled with fluorescein, depend upon pH. We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions. The pH of the medium has no effect on the intralysosomal pH. Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH. However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form. Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH. This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A. The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.

scholarly journals The actin nucleation factor JMY is a negative regulator of neuritogenesis

2011 ◽  
Vol 22 (23) ◽  
pp. 4563-4574 ◽  
Author(s):  
Elif Nur Firat-Karalar ◽  
Peter P. Hsiue ◽  
Matthew D. Welch
Keyword(s):  
Cell Lines ◽  
Regulatory Protein ◽  
Neuronal Cell ◽  
Full Length ◽  
Negative Regulator ◽  
Neurite Formation ◽  
A Minor ◽  
And Function ◽  
In Cells

Junction-mediating and regulatory protein (JMY) is a p53 cofactor that was recently shown to nucleate actin assembly by a hybrid mechanism involving tandem actin monomer binding and Arp2/3 complex activation. However, the regulation and function of JMY remain largely uncharacterized. We examined the activity of JMY in vitro and in cells, its subcellular distribution, and its function in fibroblast and neuronal cell lines. We demonstrated that recombinant full-length JMY and its isolated WASP homology 2 domain, connector, and acidic region (WWWCA) have potent actin-nucleating and Arp2/3-activating abilities in vitro. In contrast, the activity of full-length JMY, but not the isolated WWWCA domain, is suppressed in cells. The WWWCA domain is sufficient to promote actin-based bead motility in cytoplasmic extracts, and this activity depends on its ability to activate the Arp2/3 complex. JMY is expressed at high levels in brain tissue, and in various cell lines JMY is predominantly cytoplasmic, with a minor fraction in the nucleus. Of interest, silencing JMY expression in neuronal cells results in a significant enhancement of the ability of these cells to form neurites, suggesting that JMY functions to suppress neurite formation. This function of JMY requires its actin-nucleating activity. These findings highlight a previously unrecognized function for JMY as a modulator of neuritogenesis.

scholarly journals Gene Therapeutic Approach for Inhibiting Hepatitis C Virus Replication Using a Recombinant Protein That Controls Interferon Expression

2010 ◽  
Vol 54 (12) ◽  
pp. 5048-5056 ◽  
Author(s):  
Chul Hyun Joo ◽  
Uk Lee ◽  
Young Ran Nam ◽  
Jae U. Jung ◽  
Heuiran Lee ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cultured Cells ◽  
Viral Vector ◽  
Wide Spectrum ◽  
Intracellular Localization ◽  
Treatment Modalities ◽  
In Cells

ABSTRACT The hepatitis C virus (HCV) is a continuing threat to public health. The systemic administration of interferon alpha with ribavirin is the only currently approved treatment. However, this treatment is associated with a wide spectrum of systemic side effects that limits its effectiveness; thus, there is an urgent need for new treatment modalities. In this study, we describe a novel anti-HCV strategy employing a recombinant transcription factor that we have engineered in such a way that NS3/4a viral protease controls its intracellular localization, thereby restoring interferon secretion specifically in cells infected with HCV. Proof-of-concept experiments validated the strategy, showing that the recombinant transcription factor was triggered to stimulate interferon promoter by NS3/4A and remained inactive in cells without NS3/4a. Using an adenovirus-associated viral vector delivery system, we found that the recombinant transcription factor inhibited HCV replication effectively in vitro in cultured cells.

scholarly journals The interaction of ATP11C-b with ezrin contributes to its polarized localization

2021 ◽  
Author(s):  
Hiroki Inoue ◽  
Hiroyuki Takatsu ◽  
Asuka Hamamoto ◽  
Masahiro Takayama ◽  
Riki Nakabuchi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Lines ◽  
Splice Variant ◽  
Active Form ◽  
Motile Cell ◽  
Erm Proteins ◽  
Cytoplasmic Region ◽  
Motile Cells ◽  
Exogenous Expression ◽  
In Cells

ATP11C, a member of the P4-ATPase family, translocates phosphatidylserine and phosphatidylethanolamine at the plasma membrane. We previously revealed that its C-terminal splice variant ATP11C-b exhibits polarized localization in motile cell lines, such as MDA-MB-231 and BaF3. In the present study, we found that the C-terminal cytoplasmic region of ATP11C-b interacts specifically with ezrin. Notably, the LLxY motif in the ATP11C-b C-terminal region is crucial for its interaction with ezrin as well as its polarized localization on the plasma membrane. A constitutively active, C-terminal phosphomimetic mutant of ezrin was colocalized with ATP11C-b in polarized motile cells. ATP11C-b was partially mislocalized in cells depleted of ezrin alone, and exhibited greater mislocalization in cells simultaneously depleted of family members, ezrin, radixin, and moesin (ERM), suggesting that ERM proteins, particularly ezrin, contribute to the polarized localization of ATP11C-b. Further, Atp11c knockout resulted in C-terminally phosphorylated ERM proteins mislocalization, which was restored by exogenous expression of ATP11C-b but not ATP11C-a. These observations together indicate that the polarized localizations of ATP11C-b and the active form of ezrin to the plasma membrane are interdependently stabilized.

Absence of temporal ordering of apoptotic features in heat-shock treated leukemia and lymphoma cell lines

1996 ◽  
Vol 54 ◽  
pp. 758-759
Author(s):  
D. W. Fairbain ◽  
M.D. Standing ◽  
K.L. O'Neill
Keyword(s):  
Heat Shock ◽  
Cell Lines ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Resistant Cell ◽  
Membrane Blebbing ◽  
Late Loss ◽  
Induced Apoptosis ◽  
Dying Cells ◽  
In Cells

Apoptosis is a genetically defined response to physiological stimuli that results in cellular suicide. Features common to apoptotic cells include chromatin condensation, oligonucleosomal DNA fragmentation, membrane blebbing, nuclear destruction, and late loss of ability to exclude vital dyes. These characteristics contrast markedly from pathological necrosis, in which membrane integrity loss is demonstrated early, and other features of apoptosis, which allow a non-inflammatory removal of dead and dying cells, are absent. Using heat shock-induced apoptosis as a model for examining stress response in cells, we undertook to categorize a variety of human leukemias and lymphomas with regard to their response to heat shock. We were also interested in determining whether a common temporal order was followed in cells dying by apoptosis. In addition, based on our previous results, we investigated whether increasing heat load resulted in increased apoptosis, with particular interest in relatively resistant cell lines, or whether the mode of death changed from apoptosis to necrosis.

Umatilla Virus Association with Fibrils and Tubules in Cultured Cells

1980 ◽  
Vol 38 ◽  
pp. 476-477
Author(s):  
Neil M. Foster ◽  
Ruth D. Breckon
Keyword(s):  
Bluetongue Virus ◽  
Linear Array ◽  
Cultured Cells ◽  
Intimate Association ◽  
Infected Cells ◽  
Dark Staining ◽  
In Cells

Macrotubules have been described1 in cells infected with Umatilla virus (UMAV), an orbivirus for which bluetongue virus (BTV) is the protype. Macrotubules, often in linear array, were observed in the cytoplasm and in intimate association with viroplasms of infected cells. Macrotubules had outside and inside diameters of 20 and 15 nm and many had dark-staining centers with diameters similar to the interiors of the tubules. UMAV was 60 nm and the RNA core was 30 nm in diameter. This report describes the association of UMAV with macrotubules and two types of microtubules.

Direct protein delivery into intact plant cells using polyhistidine peptides

2021 ◽  
Author(s):  
Yoshino Tanaka ◽  
Yoshihiko Nanasato ◽  
Kousei Omura ◽  
Keita Endoh ◽  
Tsuyoshi Kawano ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fusion Proteins ◽  
Protein Delivery ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Plant Cells ◽  
Adenosine Monophosphate ◽  
Cell Penetrating Peptides ◽  
Intracellular Space ◽  
Intracellular Fluorescence

Abstract Polyhistidine peptides (PHPs), sequences comprising only histidine residues (>His8), are effective cell-penetrating peptides for plant cells. Using PHP-fusion proteins, we aimed to deliver proteins into cultured plant cells from Nicotiana tabacum, Oryza sativa, and Cryptomeria japonica. Co-cultivation of cultured cells with fusion proteins combining maltose-binding protein (MBP), red fluorescent protein (RFP), and various PHPs (MBP-RFP-His8–His20) in one polypeptide showed the cellular uptake of fusion proteins in all plant cell lines. Maximum intracellular fluorescence was shown in MBP-RFP-His20. Further, adenylate cyclase (CyaA), a synthase of cyclic adenosine monophosphate (cAMP) activated by cytosolic calmodulin, was used as a reporter for protein delivery in living cells. A fusion protein combining MBP, RFP, CyaA, and His20 (MBP-RFP-CyaA-His20) was delivered into plant cells and increased intracellular fluorescence and cAMP production in all cell lines. The present study demonstrates that PHPs are effective carriers of proteins into the intracellular space of various cultured plant cells.

scholarly journals 40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David J. Young ◽  
Sezen Meydan ◽  
Nicholas R. Guydosh
Keyword(s):  
Protein Synthesis ◽  
Neurological Disease ◽  
Ribosome Profiling ◽  
Minor Role ◽  
Stop Codons ◽  
Genes Encoding ◽  
Ribosome Footprinting ◽  
A Minor ◽  
And Control ◽  
In Cells

AbstractThe recycling of ribosomes at stop codons for use in further rounds of translation is critical for efficient protein synthesis. Removal of the 60S subunit is catalyzed by the ATPase Rli1 (ABCE1) while removal of the 40S is thought to require Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR). However, it remains unclear how these Tma proteins cause 40S removal and control reinitiation of downstream translation. Here we used a 40S ribosome footprinting strategy to directly observe intermediate steps of ribosome recycling in cells. Deletion of the genes encoding these Tma proteins resulted in broad accumulation of unrecycled 40S subunits at stop codons, directly establishing their role in 40S recycling. Furthermore, the Tma20/Tma22 heterodimer was responsible for a majority of 40S recycling events while Tma64 played a minor role. Introduction of an autism-associated mutation into TMA22 resulted in a loss of 40S recycling activity, linking ribosome recycling and neurological disease.

Sign in / Sign up

Export Citation Format

Share Document