Polyamine transport system mediates agmatine transport in mammalian cells

2001 ◽  
Vol 281 (1) ◽  
pp. C329-C334 ◽  
Author(s):  
Joseph Satriano ◽  
Masato Isome ◽  
Robert A. Casero ◽  
Scott C. Thomson ◽  
Roland C. Blantz
Keyword(s):  
Transport System ◽  
Mammalian Cells ◽  
Maximal Velocity ◽  
Human Carcinoma ◽  
Mammalian Cell Lines ◽  
Carrier Mediated Transport ◽  
Polyamine Transport ◽  
Polyamine Content ◽  
Polyamine Transporter ◽  
Nih 3T3

Agmatine is a biogenic amine with the capacity to regulate a number of nonreceptor-mediated functions in mammalian cells, including intracellular polyamine content and nitric oxide generation. We observed avid incorporation of agmatine into several mammalian cell lines and herein characterize agmatine transport in mammalian cells. In transformed NIH/3T3 cells, agmatine uptake is energy dependent with a saturable component indicative of carrier-mediated transport. Transport displays an apparent Michaelis-Menten constant of 2.5 μM and a maximal velocity of 280 pmol · min−1 · mg−1 protein and requires a membrane potential across the plasma membrane for uptake. Competition with polyamines, but not cationic molecules that utilize the y+ system transporter, suppresses agmatine uptake. Altering polyamine transporter activity results in parallel changes in polyamine and agmatine uptake. Furthermore, agmatine uptake is abrogated in a polyamine transport-deficient human carcinoma cell line. These lines of evidence demonstrate that agmatine utilizes, and is dependent on, the polyamine transporter for cellular uptake. The fact that this transport system is associated with proliferation could be of consequence to the antiproliferative effects of agmatine.

Polyamine homoeostasis

2009 ◽  
Vol 46 ◽  
pp. 11-24 ◽  
Author(s):  
Lo Persson
Keyword(s):  
Mammalian Cells ◽  
Cell Function ◽  
Protein S ◽  
26S Proteasome ◽  
Adenosylmethionine Decarboxylase ◽  
Specific Effects ◽  
Polyamine Transport ◽  
Polyamine Transporter ◽  
Reading Frames ◽  
Synthesis And Degradation

The polyamines are essential for a variety of functions in the mammalian cell. Although their specific effects have not been fully elucidated, it is clear that the cellular polyamines have to be kept within certain levels for normal cell function. Polyamine homoeostasis in mammalian cells is achieved by a complex network of regulatory mechanisms affecting synthesis and degradation, as well as membrane transport of polyamines. The two key enzymes in the polyamine biosynthetic pathway, ODC (ornithine decarboxylase) and AdoMetDC (S-adenosylmethionine decarboxylase), are strongly regulated by feedback mechanisms at several levels, including transcriptional, translational and post-translational. Some of these mechanisms have been shown to be truly unique and include upstream reading frames and ribosomal frameshifting, as well as ubiquitin-independent proteasomal degradation. SSAT (spermidine/spermine N1-acetyltransferase), which is a crucial enzyme for degradation and efflux of polyamines, is also highly regulated by polyamines. A cellular excess of polyamines rapidly induces SSAT, resulting in increased degradation/efflux of the polyamines. The polyamines appear to induce both transcription and translation of the SSAT mRNA. However, the major part of the polyamine-induced increase in SSAT is caused by a marked stabilization of the enzyme against degradation by the 26S proteasome. In addition, active transport of extracellular polyamines into the cell contributes to cellular polyamine homoeostasis. Depletion of cellular polyamines rapidly induces an increased uptake of exogenous polyamines, whereas an excess of polyamines down-regulates the polyamine transporter(s). However, the protein(s) involved in polyamine transport and the exact mechanisms by which the polyamines regulate the transporter(s) are not yet known.

scholarly journals ATP13A3 is a major component of the enigmatic mammalian polyamine transport system

2020 ◽  
pp. jbc.RA120.013908
Author(s):  
Norin Nabil Hamouda ◽  
Chris Van den Haute ◽  
Roeland Vanhoutte ◽  
Ragna Sannerud ◽  
Mujahid Azfar ◽  
...  
Keyword(s):  
Transport System ◽  
Mammalian Cells ◽  
Polyamine Biosynthesis ◽  
Polyamine Transport System ◽  
Polyamine Transport ◽  
Mrna And Protein Expression ◽  
Transport Inhibitors ◽  
Pulmonary Arterial ◽  
Polyamine Uptake ◽  
P Type

Polyamines, such as putrescine, spermidine and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system (PTS) using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor MGBG (methylglyoxal bis- (guanylhydrazone)), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in wild-type cells induced a CHO-MG phenotype, demonstrating a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3 as a major component of the mammalian PTS that confers sensitivity to MGBG and that has been previously genetically linked with pulmonary arterial hypertension.

scholarly journals Induction of apoptosis in human leukaemic cells by IPENSpm, a novel polyamine analogue and anti-metabolite

2002 ◽  
Vol 367 (1) ◽  
pp. 307-312 ◽  
Author(s):  
Alison V. FRASER ◽  
Patrick M. WOSTER ◽  
Heather M. WALLACE
Keyword(s):  
Polyamine Metabolism ◽  
Decarboxylase Activity ◽  
Dependent Manner ◽  
Polyamine Transport ◽  
Polyamine Content ◽  
Leukaemic Cells ◽  
Polyamine Analogue ◽  
Transport Inhibitors ◽  
Polyamine Transporter ◽  
Induced Apoptosis

Human promyelogenous leukaemic cells (HL-60) were treated with novel spermine analogue, (S)-N1-(2-methyl-1-butyl)-N11-ethyl-4,8-diazaundecane (IPENSpm), and the effects on growth and intracellular polyamine metabolism were measured. IPENSpm was cytotoxic to these cells at concentrations greater than 2.5μM. It induced apoptosis in a caspase-dependent manner and its toxicity profile was comparable with etoposide, a well-known anti-tumour agent and inducer of apoptosis. IPENSpm decreased intracellular polyamine content as a result of changes in ornithine decarboxylase activity and increases in spermidine/spermine N1-acetyltransferase and polyamine export. Analysis showed spermine and spermidine as the major intracellular polyamines, while putrescine and acetyl-polyamines were the main export compounds. IPENSpm used the polyamine transporter system for uptake and its accumulation in cells was prevented by polyamine transport inhibitors. IPENSpm can be classified as a polyamine anti-metabolite and it may be a promising new lead compound in terms of treatment of some human cancers.

scholarly journals Effect of a Bis-Benzyl Polyamine Analogue onPneumocystis carinii

2000 ◽  
Vol 44 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Salim Merali ◽  
Muhamed Sarić ◽  
Kevin Chin ◽  
Allen B. Clarkson
Keyword(s):  
Mammalian Cells ◽  
Pneumocystis Carinii ◽  
Specific Treatment ◽  
Polyamine Metabolism ◽  
Biosynthetic Enzyme ◽  
Polyamine Biosynthesis ◽  
Polyamine Analogues ◽  
Polyamine Content ◽  
Polyamine Analogue ◽  
Polyamine Transporter

ABSTRACT Pneumocystis carinii is the causative agent of P. carinii pneumonia (PCP), an opportunistic infection associated with AIDS and other immunosuppressed conditions. Although polyamine metabolism of this fungus has been shown to be a chemotherapeutic target, this metabolism has not been thoroughly investigated. Reported here is the effect of one polyamine analogue,N,N′-bis{3-[(phenylmethyl)amino]propyl}-1,7-diaminoheptane (BBS), on P. carinii. BBS inhibits the growth of P. carinii in culture, but at concentrations higher than those required to inhibit the growth of other pathogens. However, BBS is at least as active in an animal model of PCP as in other models of diseases studied. BBS causes some reduction in P. cariniipolyamine content and polyamine biosynthetic enzyme activities, but the effect is less than that observed with other pathogens and very much less than the effect of the polyamine biosynthesis inhibitordl-α-difluoromethylornithine. BBS enters P. carinii cells via a polyamine transporter, unlike all other cells that have been studied. P. carinii cells do not remove the benzyl groups of BBS, as is reported for mammalian cells. The most likely mode of action is displacement of natural polyamines. Overall, the activity of BBS provides further evidence that polyamines and polyamine metabolism are rational targets for the development of drugs to treat PCP. Because the details of BBS-P. cariniiinteraction differ from those of other cells studied, polyamine analogues may provide a highly specific treatment for PCP.

Evaluation of the nucleopolyhedrovirus of Anticarsia gemmatalis as a vector for gene therapy in mammals

2020 ◽  
Vol 20 ◽  
Author(s):  
Cintia N. Parsza ◽  
Diego L. Mengual Gómez ◽  
Jorge Alejandro Simonin ◽  
Mariano Nicolás Belaich ◽  
Pablo Daniel Ghiringhelli
Keyword(s):  
Gene Therapy ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Autographa Californica ◽  
Anticarsia Gemmatalis ◽  
Agricultural Pests ◽  
Mammalian Cell Lines ◽  
Delivery Vector ◽  
Wide Range ◽  
Insect Pathogens

Background: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV) has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals, because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. Objective/Method: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. Results: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. Conclusion: Consequently, this insect pathogen is proposed as an alternative of non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

Cytotoxicity Study of Textile Fabrics Impregnated With CuO Nanoparticles in Mammalian Cells

2017 ◽  
Vol 36 (6) ◽  
pp. 478-484 ◽  
Author(s):  
Gagandeep Singh ◽  
James Beddow ◽  
Christopher Mee ◽  
Lidia Maryniak ◽  
Eadaoin M. Joyce ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Dermal Fibroblast ◽  
Copper Oxide Nanoparticles ◽  
Growth Media ◽  
Indirect Contact ◽  
Cuo Nps ◽  
Mammalian Cell Lines ◽  
Textile Fabrics ◽  
Hdf Cells

Copper and copper compounds have multifunctional properties (antibacterial, antiviral, and antifungal) with promising applications. Copper in its nanoparticle (Cu NPs) forms has been widely used in various industrial and commercial applications. In the current research, the cytotoxic effects of textile fabrics impregnated with copper oxide nanoparticles (CuO NPs) were studied in mammalian cell lines. CuO NPs were impregnated onto textile substrates using 2 different techniques: the sonochemical generation and impregnation of NPs from metal complexes ( insitu) and a “throwing the stones” technology using commercially prepared CuO NPs. The cytotoxicity of these 2 textile fabric types was assayed on human dermal fibroblast (HDF) cells and human hepatocellular carcinoma cells (HepG2) and was evaluated by indirect contact using an MTT assay. The impregnated fabrics were not exposed to the cells, rather their leachates were used to test cytotoxicity. The fabrics were soaked into the growth media for up to 7 days, and the leachates from day 1 and day 7 were incubated with the cell lines for 24 hours prior to the testing. The discharge or leaching from antimicrobial nanomaterials into the surroundings and surface waters is posing a serious environmental threat, which needs to be addressed. Hence, with regard to product safety, it is a good approach to study the fabric leachates rather than the intact material. The results showed that CuO NPs are not toxic to HDF cells. However, cytotoxicity was seen in HepG2 cells with cell viability decreasing by 20% to 25% for all the fabrics after 24 hours.

scholarly journals ADP-Ribosylation of Actin by the Clostridium botulinum C2 Toxin in Mammalian Cells Results in Delayed Caspase-Dependent Apoptotic Cell Death

2008 ◽  
Vol 76 (10) ◽  
pp. 4600-4608 ◽  
Author(s):  
Karin Heine ◽  
Sascha Pust ◽  
Stefanie Enzenmüller ◽  
Holger Barth
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Clostridium Botulinum ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Adp Ribosylation ◽  
Mammalian Cell Lines ◽  
Adp Ribosyltransferase ◽  
C2 Toxin

ABSTRACT The binary C2 toxin from Clostridium botulinum mono-ADP-ribosylates G-actin in the cytosol of eukaryotic cells. This modification leads to depolymerization of actin filaments accompanied by cell rounding within 3 h of incubation but does not immediately induce cell death. Here we investigated the long-term responses of mammalian cell lines (HeLa and Vero) following C2 toxin treatment. Cells stayed round even though the toxin was removed from the medium after its internalization into the cells. No unmodified actin reappeared in the C2 toxin-treated cells within 48 h. Despite actin being completely ADP-ribosylated after about 7 h, no obvious decrease in the overall amount of actin was observed for at least 48 h. Therefore, ADP-ribosylation was not a signal for an accelerated degradation of actin in the tested cell lines. C2 toxin treatment resulted in delayed apoptotic cell death that became detectable about 15 to 24 h after toxin application in a portion of the cells. Poly(ADP)-ribosyltransferase 1 (PARP-1) was cleaved in C2 toxin-treated cells, an indication of caspase 3 activation and a hallmark of apoptosis. Furthermore, specific caspase inhibitors prevented C2 toxin-induced apoptosis, implying that caspases 8 and 9 were activated in C2 toxin-treated cells. C2I, the ADP-ribosyltransferase component of the C2 toxin, remained active in the cytosol for at least 48 h, and no extensive degradation of C2I was observed. From our data, we conclude that the long-lived nature of C2I in the host cell cytosol was essential for the nonreversible cytotoxic effect of C2 toxin, resulting in delayed apoptosis of the tested mammalian cells.

scholarly journals Phosphatidylethanol stimulates the plasma-membrane calcium pump from human erythrocytes

1996 ◽  
Vol 317 (3) ◽  
pp. 933-938 ◽  
Author(s):  
Meylin SUJU ◽  
Marbelly DAVILA ◽  
German POLEO ◽  
Roberto DOCAMPO ◽  
Gustavo BENAIM
Keyword(s):  
Plasma Membrane ◽  
Phospholipase D ◽  
Mammalian Cells ◽  
Human Erythrocytes ◽  
Maximal Velocity ◽  
Ethanol Intoxication ◽  
Solubilized Enzyme ◽  
Acidic Phospholipids ◽  
Additive Combination ◽  
Stimulation Of

Phosphatidylethanol is formed by ‘transphosphatidylation’ of phospholipids with ethanol catalysed by phospholipase D and can be accumulated in the plasma membrane of mammalian cells after treatment of animals with ethanol. In the present work we show that phosphatidylalcohols, such as phosphatidylethanol and phosphatidylbutanol, produced a twofold stimulation of the Ca2+-ATPase activity of human erythrocytes. This stimulation occurs with the purified, solubilized enzyme as well as with ghost preparations, where the enzyme is in its natural lipidic environment and is different to that obtained with other acidic phospholipids such as phosphatidylserine. Addition of either phosphatidylserine, phosphatidylethanol or phosphatidylbutanol to the purified Ca2+-ATPase, or to ghosts preparations, increased the affinity of the enzyme for Ca2+ and the maximal velocity of the reaction as compared with controls in the absence of acidic phospholipids. However, in contrast with what occurs with phosphatidylserine, simultaneous addition of phosphatidylalcohols and calmodulin increased the affinity of the enzyme for Ca2+ to a greater extent than each added separately. When ethanol was added to either the purified erythrocyte Ca2+-ATPase or to erythrocyte-ghost preparations in the presence of acidic phospholipids, an additive effect was observed. There was an increase in the affinity for Ca2+ and in the maximal velocity of the reaction, well above the values obtained with ethanol or with the acidic phospholipids tested separately. These findings could have pharmacological importance. It is conceivable that the decrease in the intracellular Ca2+ concentration that has been reported in erythrocytes as a result of ethanol intoxication could be due to the stimulation of the Ca2+-ATPase by the accumulated phosphatidylethanol, to a direct effect of ethanol on the enzyme or to an additive combination of both.

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