scholarly journals Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level.

1996 ◽  
Vol 132 (3) ◽  
pp. 345-357 ◽  
Author(s):  
X M Wang ◽  
J G Peloquin ◽  
Y Zhai ◽  
J C Bulinski ◽  
G G Borisy
Keyword(s):  
Posttranslational Modifications ◽  
Glutathione Transferase ◽  
Cultured Cells ◽  
Human Fibroblasts ◽  
Cellular Level ◽  
Vitro Assay ◽  
Normal Functions ◽  
Projection Domain

Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT-dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system.

scholarly journals Rho5p Is Involved in Mediating the Osmotic Stress Response in Saccharomyces cerevisiae, and Its Activity Is Regulated via Msi1p and Npr1p by Phosphorylation and Ubiquitination

2008 ◽  
Vol 7 (9) ◽  
pp. 1441-1449 ◽  
Author(s):  
Robert B. Annan ◽  
Cunle Wu ◽  
Daniel D. Waller ◽  
Malcolm Whiteway ◽  
David Y. Thomas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Osmotic Stress ◽  
Posttranslational Modifications ◽  
Bound State ◽  
Small Gtpases ◽  
Rho Proteins ◽  
Vitro Assay ◽  
Regulatory Circuit

ABSTRACT Small GTPases of the Rho family act as molecular switches, and modulation of the GTP-bound state of Rho proteins is a well-characterized means of regulating their signaling activity in vivo. In contrast, the regulation of Rho-type GTPases by posttranslational modifications is poorly understood. Here, we present evidence of the control of the Saccharomyces cerevisiae Rho-type GTPase Rho5p by phosphorylation and ubiquitination. Rho5p binds to Ste50p, and the expression of the activated RHO5(Q91H) allele in an Δste50 strain is lethal under conditions of osmotic stress. An overexpression screen identified RGD2 and MSI1 as being high-copy suppressors of the osmotic sensitivity of this lethality. Rgd2p had been identified as being a possible Rho5p GTPase-activating protein based on an in vitro assay; this result supports its function as a regulator of Rho5p activity in vivo. MSI1 was previously identified as being a suppressor of hyperactive Ras/cyclic AMP signaling, where it antagonizes Npr1p kinase activity and promotes ubiquitination. Here, we show that Msi1p also acts via Npr1p to suppress activated Rho5p signaling. Rho5p is ubiquitinated, and its expression is lethal in a strain that is compromised for proteasome activity. These data identify Rho5p as being a target of Msi1p/Npr1p regulation and describe a regulatory circuit involving phosphorylation and ubiquitination.

scholarly journals Iron in cytosolic ferritin can be recycled through lysosomal degradation in human fibroblasts

1998 ◽  
Vol 336 (1) ◽  
pp. 201-205 ◽  
Author(s):  
Derek C. RADISKY ◽  
Jerry KAPLAN
Keyword(s):  
Human Fibroblasts ◽  
Dependent Manner ◽  
Lysosomal Degradation ◽  
Temperature Dependent ◽  
Intracellular Iron ◽  
Vitro Assay ◽  
Cellular Iron ◽  
Horse Spleen Ferritin

Examination of the mechanism of intracellular iron recovery from lysosomally-degraded ferritin in vivo has been complicated by the continuous flux of cellular iron through ferritin molecules. Here we incubated human fibroblasts with cationic ferritin, a derivative of horse spleen ferritin, as a technique for delivering immunologically distinct ferritin molecules directly to lysosomes. Using this method, we found increased endogenous ferritin levels after the cellular degradation of cationic ferritin, demonstrating that cells can utilize lysosomal ferritin to produce increased cytosolic ferritin levels. Further, using an in vitro assay, we showed that isolated lysosomes degrade endogenous ferritin in a time- and temperature-dependent manner. These results are consistent with a model in which cytosolic ferritin is taken into the lysosomes and degraded. The solubilized iron from the ferric core could then be transported across the lysosomal membrane back into the cytosol.

Substrate specificities and activities of AZAP family Arf GAPs in vivo

2008 ◽  
Vol 294 (1) ◽  
pp. C263-C270 ◽  
Author(s):  
Ellen J. Cuthbert ◽  
Kathryn K. Davis ◽  
James E. Casanova
Keyword(s):  
Posttranslational Modifications ◽  
Pleckstrin Homology Domain ◽  
Nucleotide Exchange ◽  
Systematic Analysis ◽  
Vitro Assay ◽  
Substrate Specificities ◽  
Guanine Nucleotide Exchange ◽  
Subcellular Compartmentalization

The ADP-ribosylation factor (Arf) GTPases are important regulators of vesicular transport in eukaryotic cells. Like other GTPases, the Arfs require guanine nucleotide exchange factors to facilitate GTP loading and GTPase-activating proteins (GAPs) to promote GTP hydrolysis. Whereas there are only six mammalian Arfs, the human genome encodes over 20 proteins containing Arf GAP domains. A subset of these, referred to as AZAPs (Randazzo PA, Hirsch DS. Cell Signal 16: 401–413, 2004), are characterized by the presence of at least one NH2-terminal pleckstrin homology domain and two or more ankyrin repeats following the GAP domain. The substrate specificities of these proteins have been previously characterized by using in vitro assay systems. However, a limitation of such assays is that they may not accurately represent intracellular conditions, including posttranslational modifications, or subcellular compartmentalization. Here we present a systematic analysis of the GAP activity of seven AZAPs in vivo, using an assay for measurement of cellular Arf-GTP (Santy LC, Casanova JE. J Cell Biol 154: 599–610, 2001). In agreement with previous in vitro results, we found that ACAP1 and ACAP2 have robust, constitutive Arf6 GAP activity in vivo, with little activity toward Arf1. In contrast, although ARAP1 was initially reported to be an Arf1 GAP, we found that it acts primarily on Arf6 in vivo. Moreover, this activity appears to be regulated through a mechanism involving the NH2-terminal sterile-α motif. AGAP1 is unique among the AZAPs in its specificity for Arf1, and this activity is dependent on its NH2-terminal GTPase-like domain. Finally, we found that expression of AGAP1 induces a surprising reciprocal activation of Arf6, which suggests that regulatory cross talk exists among Arf isoforms.

scholarly journals An in vitro assay and artificial intelligence approach to determine rate constants of nanomaterial-cell interactions

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Edward Price ◽  
Andre J. Gesquiere
Keyword(s):  
Artificial Intelligence ◽  
Exposure Dose ◽  
Cellular Level ◽  
Screening Tools ◽  
Biological Interactions ◽  
Vitro Assay ◽  
Cellular Environment ◽  
Cell Simulation

Abstract In vitro assays and simulation technologies are powerful methodologies that can inform scientists of nanomaterial (NM) distribution and fate in humans or pre-clinical species. For small molecules, less animal data is often needed because there are a multitude of in vitro screening tools and simulation-based approaches to quantify uptake and deliver data that makes extrapolation to in vivo studies feasible. Small molecule simulations work because these materials often diffuse quickly and partition after reaching equilibrium shortly after dosing, but this cannot be applied to NMs. NMs interact with cells through energy dependent pathways, often taking hours or days to become fully internalized within the cellular environment. In vitro screening tools must capture these phenomena so that cell simulations built on mechanism-based models can deliver relationships between exposure dose and mechanistic biology, that is biology representative of fundamental processes involved in NM transport by cells (e.g. membrane adsorption and subsequent internalization). Here, we developed, validated, and applied the FORECAST method, a combination of a calibrated fluorescence assay (CF) with an artificial intelligence-based cell simulation to quantify rates descriptive of the time-dependent mechanistic biological interactions between NMs and individual cells. This work is expected to provide a means of extrapolation to pre-clinical or human biodistribution with cellular level resolution for NMs starting only from in vitro data.

scholarly journals Human Embryonic Stem Cell-Derived Neurons Are Highly Permissive for Varicella-Zoster Virus Lytic Infection

2017 ◽  
Vol 92 (1) ◽  
Author(s):  
Tomohiko Sadaoka ◽  
Cindi L. Schwartz ◽  
Labchan Rajbhandari ◽  
Arun Venkatesan ◽  
Jeffrey I. Cohen
Keyword(s):  
Varicella Zoster Virus ◽  
Cultured Cells ◽  
Human Fibroblasts ◽  
Embryonic Stem ◽  
Vaccine Production ◽  
Varicella Zoster ◽  
Viral Particles ◽  
Human Neurons

ABSTRACTVaricella-zoster virus (VZV) is highly cell associated when grown in culture and has a much higher (4,000- to 20,000-fold increased) particle-to-PFU ratioin vitrothan herpes simplex virus (HSV). In contrast, VZV is highly infectiousin vivoby airborne transmission. Neurons are major targets for VZVin vivo; in neurons, the virus can establish latency and reactivate to produce infectious virus. Using neurons derived from human embryonic stem cells (hESC) and cell-free wild-type (WT) VZV, we demonstrated that neurons are nearly 100 times more permissive for WT VZV infection than very-early-passage human embryonic lung cells or MRC-5 diploid human fibroblasts, the cells used for vaccine production or virus isolation. The peak titers achieved after infection were ∼10-fold higher in human neurons than in MRC-5 cells, and the viral genome copy number-to-PFU ratio for VZV in human neurons was 500, compared with 50,000 for MRC-5 cells. Thus, VZV may not necessarily have a higher particle-to-PFU ratio than other herpesviruses; instead, the cells previously used to propagate virusin vitromay have been suboptimal. Furthermore, based on electron microscopy, neurons infected with VZV produced fewer defective or incomplete viral particles than MRC-5 cells. Our data suggest that neurons derived from hESC may have advantages compared to other cells for studies of VZV pathogenesis, for obtaining stocks of virus with high titers, and for isolating VZV from clinical specimens.IMPORTANCEVaricella-zoster virus (VZV) causes chickenpox and shingles. Cell-free VZV has been difficult to obtain, both forin vitrostudies and for vaccine production. While numerous cells lines have been tested for their ability to produce high titers of VZV, the number of total virus particles relative to the number of viral particles that can form plaques in culture has been reported to be extremely high relative to that in other viruses. We show that VZV grows to much higher titers in human neurons than in other cell typesin vitroand that the number of total virus genomes relative to the number of viral particles that can form plaques in culture is much lower in human neurons than other cultured cells. These findings indicate that human neurons may be useful for studying VZVin vitro, for growing preparations of virus with high titers, and for isolating the virus from human samples.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

Exploiting Anti-Inflammation Effects of Flavonoids in Chronic Inflammatory Diseases

2020 ◽  
Vol 26 (22) ◽  
pp. 2610-2619 ◽  
Author(s):  
Tarique Hussain ◽  
Ghulam Murtaza ◽  
Huansheng Yang ◽  
Muhammad S. Kalhoro ◽  
Dildar H. Kalhoro
Keyword(s):  
Oxidative Stress ◽  
Chronic Diseases ◽  
Inflammatory Diseases ◽  
Therapeutic Option ◽  
Cellular Level ◽  
Antiinflammatory Drugs ◽  
Suitable Alternative ◽  
Chronic Inflammatory Diseases

Background: Inflammation is a complex response of the host defense system to different internal and external stimuli. It is believed that persistent inflammation may lead to chronic inflammatory diseases such as, inflammatory bowel disease, neurological and cardiovascular diseases. Oxidative stress is the main factor responsible for the augmentation of inflammation via various molecular pathways. Therefore, alleviating oxidative stress is effective a therapeutic option against chronic inflammatory diseases. Methods: This review article extends the knowledge of the regulatory mechanisms of flavonoids targeting inflammatory pathways in chronic diseases, which would be the best approach for the development of suitable therapeutic agents against chronic diseases. Results: Since the inflammatory response is initiated by numerous signaling molecules like NF-κB, MAPK, and Arachidonic acid pathways, their encountering function can be evaluated with the activation of Nrf2 pathway, a promising approach to inhibit/prevent chronic inflammatory diseases by flavonoids. Over the last few decades, flavonoids drew much attention as a potent alternative therapeutic agent. Recent clinical evidence has shown significant impacts of flavonoids on chronic diseases in different in-vivo and in-vitro models. Conclusion: Flavonoid compounds can interact with chronic inflammatory diseases at the cellular level and modulate the response of protein pathways. A promising approach is needed to overlook suitable alternative compounds providing more therapeutic efficacy and exerting fewer side effects than commercially available antiinflammatory drugs.

scholarly journals TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection

2021 ◽  
Author(s):  
Hongli Zhou ◽  
Minyu Zhou ◽  
Yue Hu ◽  
Yanin Limpanon ◽  
Yubin Ma ◽  
...  
Keyword(s):  
Cell Death ◽  
Enrichment Analysis ◽  
Angiostrongylus Cantonensis ◽  
Gene Set Enrichment Analysis ◽  
Caspase 8 ◽  
Cns Injury ◽  
Vitro Assay ◽  
Tnf Α

AbstractAngiostrongylus cantonensis (AC) can cause severe eosinophilic meningitis or encephalitis in non-permissive hosts accompanied by apoptosis and necroptosis of brain cells. However, the explicit underlying molecular basis of apoptosis and necroptosis upon AC infection has not yet been elucidated. To determine the specific pathways of apoptosis and necroptosis upon AC infection, gene set enrichment analysis (GSEA) and protein–protein interaction (PPI) analysis for gene expression microarray (accession number: GSE159486) of mouse brain infected by AC revealed that TNF-α likely played a central role in the apoptosis and necroptosis in the context of AC infection, which was further confirmed via an in vivo rescue assay after treating with TNF-α inhibitor. The signalling axes involved in apoptosis and necroptosis were investigated via immunoprecipitation and immunoblotting. Immunofluorescence was used to identify the specific cells that underwent apoptosis or necroptosis. The results showed that TNF-α induced apoptosis of astrocytes through the RIP1/FADD/Caspase-8 axis and induced necroptosis of neurons by the RIP3/MLKL signalling pathway. In addition, in vitro assay revealed that TNF-α secretion by microglia increased upon LSA stimulation and caused necroptosis of neurons. The present study provided the first evidence that TNF-α was secreted by microglia stimulated by AC infection, which caused cell death via parallel pathways of astrocyte apoptosis (mediated by the RIP1/FADD/caspase-8 axis) and neuron necroptosis (driven by the RIP3/MLKL complex). Our research comprehensively elucidated the mechanism of cell death after AC infection and provided new insight into targeting TNF-α signalling as a therapeutic strategy for CNS injury.

scholarly journals E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peng Gao ◽  
Xianwei Ma ◽  
Ming Yuan ◽  
Yulan Yi ◽  
Guoke Liu ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Type I Interferon ◽  
Zinc Fingers ◽  
E3 Ligase ◽  
Type I ◽  
E3 Ligases ◽  
Protein Posttranslational Modifications ◽  
Antiviral Innate Immunity

AbstractUbiquitination is one of the most prevalent protein posttranslational modifications. Here, we show that E3 ligase Nedd4l positively regulates antiviral immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3. Deficiency of Nedd4l significantly impairs type I interferon and proinflammatory cytokine production induced by virus infection both in vitro and in vivo. Nedd4l deficiency inhibits virus-induced ubiquitination of TRAF3, the binding between TRAF3 and TBK1, and subsequent phosphorylation of TBK1 and IRF3. Nedd4l directly interacts with TRAF3 and catalyzes K29-linked ubiquitination of Cys56 and Cys124, two cysteines that constitute zinc fingers, resulting in enhanced association between TRAF3 and E3 ligases, cIAP1/2 and HECTD3, and also increased K48/K63-linked ubiquitination of TRAF3. Mutation of Cys56 and Cys124 diminishes Nedd4l-catalyzed K29-linked ubiquitination, but enhances association between TRAF3 and the E3 ligases, supporting Nedd4l promotes type I interferon production in response to virus by catalyzing ubiquitination of the cysteines in TRAF3.

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