scholarly journals Dynamin-mediated Internalization of Caveolae

1998 ◽  
Vol 141 (1) ◽  
pp. 85-99 ◽  
Author(s):  
John R. Henley ◽  
Eugene W.A. Krueger ◽  
Barbara J. Oswald ◽  
Mark A. McNiven
Keyword(s):  
Mammalian Cells ◽  
Immunofluorescence Microscopy ◽  
In Vivo Studies ◽  
Coated Pits ◽  
Toxin B ◽  
Cholera Toxin B ◽  
Double Label ◽  
Biochemical Analyses ◽  
Redundant Functions

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.

scholarly journals Autophagy Deficiency by Atg4B Loss Leads to Metabolomic Alterations in Mice

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 481
Author(s):  
Gemma G. Martínez-García ◽  
Raúl F. Pérez ◽  
Álvaro F. Fernández ◽  
Sylvere Durand ◽  
Guido Kroemer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Mammalian Cells ◽  
Tissue Homeostasis ◽  
In Vivo Studies ◽  
Protective Mechanism ◽  
Cardiac Damage ◽  
Wide Range ◽  
First Time

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b−/− mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

Enhanced xenobiotic transporter expression in normal teleost hepatocytes: response to environmental and chemotherapeutic toxins

2001 ◽  
Vol 204 (2) ◽  
pp. 217-227
Author(s):  
J.A. Albertus ◽  
R.O. Laine
Keyword(s):  
Mammalian Cells ◽  
Cellular Localization ◽  
Fundulus Heteroclitus ◽  
Environmental Pollutants ◽  
Human Tumor ◽  
Aquatic Organisms ◽  
In Vivo Studies ◽  
Multixenobiotic Resistance

Many aquatic organisms are resistant to environmental pollutants, probably because their inherent multi-drug-resistant protein extrusion pump (pgp) can be co-opted to handle man-made pollutants. This mechanism of multixenobiotic resistance is similar to the mechanism of multidrug resistance exhibited in chemotherapy-resistant human tumor cells. In the present study, a variety of techniques were used to characterize this toxin defense system in killifish (Fundulus heteroclitus) hepatocytes. The cellular localization and activity of the putative drug efflux system were evaluated. In addition, in vitro and in vivo studies were used to examine the range of expression of this putative drug transporter in the presence of environmental and chemotherapeutic toxins. The broad range of pgp expression generally observed in transformed mammalian cells was found in normal cells of our teleost model. Our findings suggest that the expression of the pgp gene in the killifish could be an excellent indicator of toxin levels or stressors in the environment.

In vitro and in vivo studies of antimutagenic properties of bioginseng in mammalian cells

1991 ◽  
Vol 111 (5) ◽  
pp. 658-661 ◽  
Author(s):  
N. V. Umnova ◽  
T. L. Michurina ◽  
N. I. Smirnova ◽  
I. V. Aleksandrova ◽  
G. G. Poroshenko
Keyword(s):  
Mammalian Cells ◽  
In Vivo Studies

scholarly journals In vivo co-distribution of fibronectin and actin fibers in granulation tissue: immunofluorescence and electron microscope studies of the fibronexus at the myofibroblast surface.

1984 ◽  
Vol 98 (6) ◽  
pp. 2091-2106 ◽  
Author(s):  
I I Singer ◽  
D W Kawka ◽  
D M Kazazis ◽  
R A Clark
Keyword(s):  
Granulation Tissue ◽  
Wound Repair ◽  
Immunofluorescence Microscopy ◽  
Actin Microfilaments ◽  
Surface Adhesion ◽  
Microfilament Bundles ◽  
Binding Surface ◽  
Double Label ◽  
Fibroblast Adhesion

The fibronexus ( FNX ), a very close transmembrane association of individual extracellular fibronectin fibers and actin microfilaments, was found previously at the substrate-binding surface of fibroblasts in tissue culture (Singer, 1. 1., 1979, Cell, 16:675-685). To determine whether the fibronexus might be involved in fibroblast adhesion during wound healing in vivo, we looked for co-localization of actin and fibronectin in granulation tissue formed within full-thickness guinea pig skin wounds. At 7-9 d, most of the actin fibers were observed to be coincident with congruent fibronectin fibers using double-label immunofluorescence microscopy. These fibronectin and actin fibers were co-localized at the myofibroblast surface surrounding the nucleus, and along attenuated myofibroblast processes which extended deeply into the extracellular matrix. This conspicuous co-distribution of fibronectin and actin fibers prompted us to look for fibronexuses at the myofibroblast surface with electron microscopy. We observed three kinds of FNXs : (a) tandem associations between the termini of individual extracellular fibronectin fibers and actin microfilament bundles at the tips of elongate myofibroblast processes, (b) plaque-like and, (c) track-like FNXs , in which parallel fibronectin and actin fibers were connected by perpendicular transmembranous fibrils. Goniometric studies on the external and internal components of these cross-linking fibrils showed that their membrane-associated ends are probably co-axial. Using immunoelectron microscopy on ultrathin cryosections, we confirmed that the densely staining external portion of these various FNXs does indeed contain fibronectin. The finding that these FNXs appear to connect collagen fibers to intracellular bundles of actin microfilaments is particularly significant. Our studies strongly suggest that the fibronexus is an important in vivo cell surface adhesion site functioning in wound repair, and perhaps within fibronectin-rich tissues during embryogenesis, tumor growth, and inflammation.

scholarly journals WAF1 retards S-phase progression primarily by inhibition of cyclin-dependent kinases.

1997 ◽  
Vol 17 (8) ◽  
pp. 4877-4882 ◽  
Author(s):  
V V Ogryzko ◽  
P Wong ◽  
B H Howard
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
S Phase ◽  
In Vivo Studies ◽  
Nuclear Antigen ◽  
Phase Progression

The p21(WAF1/CIP1/sdi1) gene product (WAF1) inhibits DNA replication in vitro (J. Chen, P. Jackson, M. Kirschner, and A. Dutta, Nature 374:386-388, 1995; S. Waga, G. Hannon, D. Beach, and B. Stillman, Nature 369:574-578, 1994), but in vivo studies on the antiproliferative activity of WAF1 have not resolved G1-phase arrest from potential inhibition of S-phase progression. Here, we demonstrate that elevated WAF1 expression can retard replicative DNA synthesis in vivo. The WAF1-mediated inhibitory effect could be antagonized by cyclin A, cyclin E, or the simian virus 40 small-t antigen with no decrease in the levels of WAF1 protein in transfected cells. Proliferating-cell nuclear antigen (PCNA) overexpression was neither necessary nor sufficient to antagonize WAF1 action. Expression of the N-terminal domain of WAF1, responsible for cyclin-dependent kinase (CDK) interaction, had the same effect as full-length WAF1, while the PCNA binding C terminus exhibited modest activity. We conclude that S-phase progression in mammalian cells is dependent on continuing cyclin and CDK activity and that WAF1 affects S phase primarily through cyclin- and CDK-dependent pathways.

scholarly journals Oregano Phytocomplex Induces Programmed Cell Death in Melanoma Lines via Mitochondria and DNA Damage

Foods ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1486
Author(s):  
Valentina Nanni ◽  
Gabriele Di Marco ◽  
Gianni Sacchetti ◽  
Antonella Canini ◽  
Angelo Gismondi
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Mammalian Cells ◽  
Cell Model ◽  
Scientific Evidence ◽  
Plant Secondary Metabolites ◽  
In Vivo Studies ◽  
Lack Of Information

Plant secondary metabolites possess chemopreventive and antineoplastic properties, but the lack of information about their exact mechanism of action in mammalian cells hinders the translation of these compounds in suitable therapies. In light of this, firstly, Origanum vulgare L. hydroalcoholic extract was chemically characterized by spectrophotometric and chromatographic analyses; then, the molecular bases underlying its antitumor activity on B16-F10 and A375 melanoma cells were investigated. Oregano extract induced oxidative stress and inhibited melanogenesis and tumor cell proliferation, triggering programmed cell death pathways (both apoptosis and necroptosis) through mitochondria and DNA damage. By contrast, oregano extract was safe on healthy tissues, revealing no cytotoxicity and mutagenicity on C2C12 myoblasts, considered as non-tumor proliferating cell model system, and on Salmonella strains, by the Ames test. All these data provide scientific evidence about the potential application of this food plant as an anticancer agent in in vivo studies and clinical trials.

scholarly journals Advances in NAD-Lowering Agents for Cancer Treatment

Nutrients ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1665
Author(s):  
Moustafa S. Ghanem ◽  
Fiammetta Monacelli ◽  
Alessio Nencioni
Keyword(s):  
Mammalian Cells ◽  
In Vivo Studies ◽  
Clinical Activity ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Production Route ◽  
Cellular Events ◽  
Cancer Cell Survival ◽  
Key Enzyme

Nicotinamide adenine dinucleotide (NAD) is an essential redox cofactor, but it also acts as a substrate for NAD-consuming enzymes, regulating cellular events such as DNA repair and gene expression. Since such processes are fundamental to support cancer cell survival and proliferation, sustained NAD production is a hallmark of many types of neoplasms. Depleting intratumor NAD levels, mainly through interference with the NAD-biosynthetic machinery, has emerged as a promising anti-cancer strategy. NAD can be generated from tryptophan or nicotinic acid. In addition, the “salvage pathway” of NAD production, which uses nicotinamide, a byproduct of NAD degradation, as a substrate, is also widely active in mammalian cells and appears to be highly exploited by a subset of human cancers. In fact, research has mainly focused on inhibiting the key enzyme of the latter NAD production route, nicotinamide phosphoribosyltransferase (NAMPT), leading to the identification of numerous inhibitors, including FK866 and CHS-828. Unfortunately, the clinical activity of these agents proved limited, suggesting that the approaches for targeting NAD production in tumors need to be refined. In this contribution, we highlight the recent advancements in this field, including an overview of the NAD-lowering compounds that have been reported so far and the related in vitro and in vivo studies. We also describe the key NAD-producing pathways and their regulation in cancer cells. Finally, we summarize the approaches that have been explored to optimize the therapeutic response to NAMPT inhibitors in cancer.

scholarly journals Temporal analysis of mRNA expression profiles in Orientia infected C3HeB/FeJ mouse

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Chien-Chung Chao ◽  
Ruoting Yang ◽  
Zhiwen Zhang ◽  
Tatyana Belinskaya ◽  
Chye-Teik Chan ◽  
...  
Keyword(s):  
Post Infection ◽  
Mammalian Cells ◽  
Time Course ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
In Vivo Studies ◽  
Coagulation Cascade ◽  
Global Changes

Abstract Background Scrub typhus causes up to 35% mortality if left untreated. One billion people living in the endemic regions are at risk. In spite of its heavy disease burden in some of the most populated areas in the world, there is no vaccine available. Although the disease can be effectively treated by proper antibiotics, timely and accurate diagnosis remains a challenge. Orientia tsutsugamushi infects a variety of mammalian cells in vitro and replicates in the cytoplasm of the infected cells. Microarray analysis has been used extensively to study host-pathogen interactions in in vitro models to understand pathogenesis. However there is a lack of in vivo studies. Results In this study, C3HeB/FeJ (C3H) mice were infected by O. tsutsugamushi via the intraperitoneal route and monitored gene expression at 10 different time points post infection. We observed two distinct types of expression profiles in the genes that we analyzed. There are two valleys (4–18 h and 2–4 days) with low number of differentially expressed genes (DEG) with three peaks with high number of DEG at 2 h, 1-day and 7-day post infection. Further analysis revealed that pathways like complement and coagulation cascade, and blood clotting cascade pathways showed significant global changes throughout entire time course. Real time quantitative Polymerase Chain Reaction (RT-qPCR) confirmed the change of expression for genes involved in complement and coagulation cascade. These results suggested dynamic regulation of the complement and coagulation cascades throughout most of the time post infection while some other specific pathways, such as fatty acid metabolism and tryptophan metabolism, are turned on or off at certain times post infection. Conclusions The findings highlight the complex interconnection among all different biological pathways. It is conceivable that specific pathways such as cell growth control and cell development in the host are affected by Orientia in the initial phase of infection for Orientia to grow intracellularly. Once Orientia is replicating successfully inside the host as infection progresses, the infection could activate pathways involved in cellular immune responses to defend for host cell survival and try to eliminate the pathogen.

scholarly journals Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments.

1985 ◽  
Vol 101 (6) ◽  
pp. 2104-2112 ◽  
Author(s):  
D A Wall ◽  
A L Hubbard
Keyword(s):  
Biochemical Characterization ◽  
Temperature Shift ◽  
Rat Hepatocytes ◽  
Cholesterol Content ◽  
Subcellular Fractionation ◽  
In Vivo Studies ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Early Endosomes

The endocytic compartments of the asialoglycoprotein (ASGP) pathway in rat hepatocytes were studied using a combined morphological and biochemical approach in the isolated perfused liver. Use of electron microscopic tracers and a temperature-shift protocol to synchronize ligand entry confirmed the route of ASGP internalization observed in our previous in vivo studies (1) and established conditions under which we could label the contents of successive compartments in the pathway for subcellular fractionation studies. Three endosomal compartments were demonstrated in which ASGPs appear after they enter the cell via coated pits and vesicles but before they reach their site of degradation in lysosomes. These three compartments could be distinguished by their location within the hepatocyte, by their morphological appearance in situ, and by their density in sucrose gradients. The distributions of ASGP receptors, both accessible and latent (revealed by detergent permeabilization), were also examined and compared with that of ligand during subcellular fractionation. Most accessible ASGP receptors co-distributed with conventional plasma membrane markers. However, hepatocytes contain a substantial intracellular pool of latent ASGP binding sites that exceeds the number of cell surface receptors and whose presence is not dependent on ASGP exposure. The distribution of these latent ASGP receptors on sucrose gradients (detected either immunologically or by binding assays) was coincident with that of ligand sequestered within the early endosome compartments. In addition, both early endosomes and the membrane vesicles containing latent ASGP receptors had high cholesterol content, because both shifted markedly in density upon exposure to digitonin.

A Review of the Genotoxicity of Triallate

2003 ◽  
Vol 22 (3) ◽  
pp. 233-251 ◽  
Author(s):  
Charles E. Healy ◽  
Larry D. Kier ◽  
Fabrice Broeckaert ◽  
Mark A. Martens
Keyword(s):  
Dna Synthesis ◽  
Mammalian Cells ◽  
Mutagenic Activity ◽  
Human Lymphocytes ◽  
In Vivo Studies ◽  
Weight Of Evidence ◽  
Unscheduled Dna Synthesis ◽  
Vitro Assay

Triallate is a selective herbicidal chemical used for control of wild oats in wheat. It has an extensive genotoxicity database that includes a variety of in vitro and in vivo studies. The chemical has produced mixed results in in vitro assay systems. It was genotoxic in bacterial mutation Ames assays, predominantly in Salmonella typhimurium strains TA100 and TA1535 in the presence of S9. Weaker responses have been observed in TA100 and TA1535 in the absence of S9. Mixed results have been observed in strain TA98, whereas no genotoxicity has been observed in strains TA1537 and TA1538. The presence and absence of S9 and its source seem to play a role in the bacterial response to the chemical. There have also been conflicting results in other test systems using other bacterial genera, yeast, and mammalian cells. Chromosome effects assays (sister-chromatid exchange and cytogenetics assays) have produced mixed results with S9 but no genotoxicity without S9. Triallate has not produced any genotoxicity in in vitro DNA damage or unscheduled DNA synthesis assays using EUE cells, human lymphocytes, and rat and mouse hepatocytes. In a series of in vivo genotoxicity assays (cytogenetics, micronucleus, dominant lethal, and unscheduled DNA synthesis), there has been no indication of any adverse genotoxic effect. Metabolism data indicate that the probable explanation for the differences observed between the in vitro studies with S9 and without S9 and between the in vitro and the in vivo studies is the production of a mutagenic intermediate in vitro at high doses of triallate is expected to be at most only transiently present in in vivo studies. The weight of evidence strongly suggests that triallate is not likely to exert mutagenic activity in vivo due to toxicokinetics and metabolic processes leading to detoxification.

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