scholarly journals Requirement for the Mitochondrial Pyruvate Carrier in Mammalian Development Revealed by a Hypomorphic Allelic Series

2016 ◽  
Vol 36 (15) ◽  
pp. 2089-2104 ◽  
Author(s):  
Caitlyn E. Bowman ◽  
Liang Zhao ◽  
Thomas Hartung ◽  
Michael J. Wolfgang
Keyword(s):  
Mammalian Development ◽  
Allelic Series ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Pyruvate Carrier ◽  
Essential Components ◽  
Transcriptional Changes ◽  
Perinatal Lethality ◽  
Necessary And Sufficient ◽  
Dose Dependent ◽  
Specific Deletion

Glucose and oxygen are two of the most important molecules transferred from mother to fetus during eutherian pregnancy, and the metabolic fates of these nutrients converge at the transport and metabolism of pyruvate in mitochondria. Pyruvate enters the mitochondrial matrix through the mitochondrial pyruvate carrier (MPC), a complex in the inner mitochondrial membrane that consists of two essential components, MPC1 and MPC2. Here, we define the requirement for mitochondrial pyruvate metabolism during development with a progressive allelic series of Mpc1 deficiency in mouse. Mpc1 deletion was homozygous lethal in midgestation, but Mpc1 hypomorphs and tissue-specific deletion of Mpc1 presented as early perinatal lethality. The allelic series demonstrated that graded suppression of MPC resulted in dose-dependent metabolic and transcriptional changes. Steady-state metabolomics analysis of brain and liver from Mpc1 hypomorphic embryos identified compensatory changes in amino acid and lipid metabolism. Flux assays in Mpc1-deficient embryonic fibroblasts also reflected these changes, including a dramatic increase in mitochondrial alanine utilization. The mitochondrial alanine transaminase GPT2 was found to be necessary and sufficient for increased alanine flux upon MPC inhibition. These data show that impaired mitochondrial pyruvate transport results in biosynthetic deficiencies that can be mitigated in part by alternative anaplerotic substratesin utero.

Identification and Functional Expression of the Mitochondrial Pyruvate Carrier

Science ◽  
2012 ◽  
Vol 337 (6090) ◽  
pp. 93-96 ◽  
Author(s):  
Sébastien Herzig ◽  
Etienne Raemy ◽  
Sylvie Montessuit ◽  
Jean-Luc Veuthey ◽  
Nicola Zamboni ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Lactococcus Lactis ◽  
Mitochondrial Membrane ◽  
Functional Expression ◽  
Inner Mitochondrial Membrane ◽  
Molecular Identity ◽  
Mitochondrial Pyruvate Carrier ◽  
Essential Components ◽  
Yeast Mutants

The transport of pyruvate, the end product of glycolysis, into mitochondria is an essential process that provides the organelle with a major oxidative fuel. Although the existence of a specific mitochondrial pyruvate carrier (MPC) has been anticipated, its molecular identity remained unknown. We report that MPC is a heterocomplex formed by two members of a family of previously uncharacterized membrane proteins that are conserved from yeast to mammals. Members of the MPC family were found in the inner mitochondrial membrane, and yeast mutants lacking MPC proteins showed severe defects in mitochondrial pyruvate uptake. Coexpression of mouse MPC1 and MPC2 in Lactococcus lactis promoted transport of pyruvate across the membrane. These observations firmly establish these proteins as essential components of the MPC.

Dose dependent transcriptional changes in circulating myeloid cells following chronic ethanol consumption

Alcohol ◽  
2018 ◽  
Vol 66 ◽  
pp. 95 ◽  
Author(s):  
S. Sureshchandra ◽  
A. Jankeel ◽  
C. Stull ◽  
K. Grant ◽  
Ilhem Messaoudi
Keyword(s):  
Myeloid Cells ◽  
Ethanol Consumption ◽  
Chronic Ethanol ◽  
Chronic Ethanol Consumption ◽  
Transcriptional Changes ◽  
Dose Dependent

Cytotoxic effects of cisplatin, cis-dichlorodiammineplatinum(II), on Tetrahymena

1988 ◽  
Vol 90 (4) ◽  
pp. 707-716
Author(s):  
J.R. Nilsson
Keyword(s):  
Mitochondrial Membrane ◽  
Prolonged Treatment ◽  
Dependent Manner ◽  
Inner Mitochondrial Membrane ◽  
Cell Organelles ◽  
Cell Content ◽  
Cytotoxic Effects ◽  
Dense Granules ◽  
High Concentrations ◽  
Dose Dependent

A study was made of the effects of cisplatin, cis-dichlorodiammineplatinum(II) (5–250 mg l-1), on the physiology and fine structure of Tetrahymena. The physiological effects observed were dose-dependent. Endocytosis was inhibited reversibly in all, but late in the high, concentrations. After an initial dose-related increase, due to division of cells most advanced in the cell cycle, proliferation ceased for at least two normal cell generations (6 h) in 50 and 100 mg drug l-1, but for 24 h in 250 mg l-1, after which multiplication was resumed in a dose-dependent manner. Exposure to cisplatin resulted in the appearance of small, refractive granules and platinum (i.e. electron-dense material) accumulated in these granules. Fine structural observations of cells exposed to 250 mg drug l-1 showed nucleolar fusion and appearance initially of lipid droplets, dense granules and autophagosomes. A time-dependent redistribution of cell organelles was revealed by morphometry; in particular, the mitochondria increased in number, but decreased in size. Moreover, after prolonged treatment (24 h) and without cell division, the inner mitochondrial membrane had diminished and the ratio of the inner to the outer mitochondrial membrane was only half of the value for control mitochondria. Concomitantly with this decrease, the cell content of ATP was reduced to a similar extent. The findings indicate a specific action of cisplatin on mitochondria, resembling that induced in Tetrahymena by chloramphenicol and methotrexate.

scholarly journals Independent regulation of vertebral number and vertebral identity by microRNA-196 paralogs

2015 ◽  
Vol 112 (35) ◽  
pp. E4884-E4893 ◽  
Author(s):  
Siew Fen Lisa Wong ◽  
Vikram Agarwal ◽  
Jennifer H. Mansfield ◽  
Nicolas Denans ◽  
Matthew G. Schwartz ◽  
...  
Keyword(s):  
Target Genes ◽  
Genetic Networks ◽  
Molecular Signatures ◽  
Mammalian Development ◽  
Allelic Series ◽  
Vertebral Number ◽  
Full Extent ◽  
Axis Elongation ◽  
Axis Extension ◽  
Additional Role

The Hox genes play a central role in patterning the embryonic anterior-to-posterior axis. An important function of Hox activity in vertebrates is the specification of different vertebral morphologies, with an additional role in axis elongation emerging. The miR-196 family of microRNAs (miRNAs) are predicted to extensively target Hox 3′ UTRs, although the full extent to which miR-196 regulates Hox expression dynamics and influences mammalian development remains to be elucidated. Here we used an extensive allelic series of mouse knockouts to show that the miR-196 family of miRNAs is essential both for properly patterning vertebral identity at different axial levels and for modulating the total number of vertebrae. All three miR-196 paralogs, 196a1, 196a2, and 196b, act redundantly to pattern the midthoracic region, whereas 196a2 and 196b have an additive role in controlling the number of rib-bearing vertebra and positioning of the sacrum. Independent of this, 196a1, 196a2, and 196b act redundantly to constrain total vertebral number. Loss of miR-196 leads to a collective up-regulation of numerous trunk Hox target genes with a concomitant delay in activation of caudal Hox genes, which are proposed to signal the end of axis extension. Additionally, we identified altered molecular signatures associated with the Wnt, Fgf, and Notch/segmentation pathways and demonstrate that miR-196 has the potential to regulate Wnt activity by multiple mechanisms. By feeding into, and thereby integrating, multiple genetic networks controlling vertebral number and identity, miR-196 is a critical player defining axial formulae.

scholarly journals Hair Loss and Defective T- and B-Cell Function in Mice Lacking ORAI1

2008 ◽  
Vol 28 (17) ◽  
pp. 5209-5222 ◽  
Author(s):  
Yousang Gwack ◽  
Sonal Srikanth ◽  
Masatsugu Oh-hora ◽  
Patrick G. Hogan ◽  
Edward D. Lamperti ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Hair Loss ◽  
Cell Function ◽  
Severe Combined Immunodeficiency ◽  
Immunohistochemical Analysis ◽  
Cell Receptor ◽  
Crac Channel ◽  
Perinatal Lethality ◽  
Specific Deletion

ABSTRACT ORAI1 is a pore subunit of the store-operated Ca2+ release-activated Ca2+ (CRAC) channel. To examine the physiological consequences of ORAI1 deficiency, we generated mice with targeted disruption of the Orai1 gene. The results of immunohistochemical analysis showed that ORAI1 is expressed in lymphocytes, skin, and muscle of wild-type mice and is not expressed in Orai1 −/− mice. Orai1 −/− mice with the inbred C57BL/6 background showed perinatal lethality, which was overcome by crossing them to outbred ICR mice. Orai1 −/− mice were small in size, with eyelid irritation and sporadic hair loss resembling the cyclical alopecia observed in mice with keratinocyte-specific deletion of the Cnb1 gene. T and B cells developed normally in Orai1 −/− mice, but B cells showed a substantial decrease in Ca2+ influx and cell proliferation in response to B-cell receptor stimulation. Naïve and differentiated Orai1 −/− T cells showed substantial reductions in store-operated Ca2+ entry, CRAC currents, and cytokine production. These features are consistent with the severe combined immunodeficiency and mild extraimmunological symptoms observed in a patient with a missense mutation in human ORAI1 and distinguish the ORAI1-null mice described here from a previously reported Orai1 gene-trap mutant mouse which may be a hypomorph rather than a true null.

scholarly journals Drosophila Hedgehog can act as a morphogen in the absence of regulated Ci processing

2020 ◽  
Author(s):  
Jamie C. Little ◽  
Elisa Garcia-Garcia ◽  
Amanda Sul ◽  
Daniel Kalderon
Keyword(s):  
Protein Profile ◽  
Gene Activation ◽  
Proteolytic Processing ◽  
Full Length ◽  
Target Cells ◽  
Linear Gradient ◽  
Gli Proteins ◽  
Transcriptional Changes ◽  
Wing Imaginal Discs ◽  
Dose Dependent

SummaryExtracellular Hedgehog (Hh) proteins induce transcriptional changes in target cells by inhibiting the proteolytic processing of full-length Drosophila Ci or mammalian Gli proteins to nuclear transcriptional repressors and by activating the full-length proteins, which are otherwise held inactive by cytoplasmic binding partners and subject to accelerated degradation following activation. We used Ci variants expressed at physiological levels to investigate the contributions of these mechanisms to dose-dependent Hh signaling at the anteroposterior (AP) border of Drosophila wing imaginal discs. Ci variants that cannot be processed supported a normal pattern of graded target gene activation and the development of adults with normal wing morphology when supplemented by constitutive Ci repressor, showing that Hh can signal normally in the absence of regulated processing. The full-length Ci-155 protein profile of these variants revealed a linear gradient of Hh-stimulated degradation, allowing derivation of a spatial profile of inhibition of processing of normal C-155 by Hh. The processing-resistant Ci variants were also significantly activated in the absence of Hh by elimination of Cos2, acting through association with the CORD domain of Ci, or PKA, revealing separate inhibitory roles of these two components in addition to their well-established roles in promoting Ci-155 processing.

scholarly journals Activation State-Dependent Interaction between Gαi and p67phox

2006 ◽  
Vol 26 (13) ◽  
pp. 5190-5200 ◽  
Author(s):  
Caroline Marty ◽  
Tohru Kozasa ◽  
Mark T. Quinn ◽  
Richard D. Ye
Keyword(s):  
Nadph Oxidase ◽  
G Proteins ◽  
Heterotrimeric G Proteins ◽  
Phagocyte Nadph Oxidase ◽  
State Dependent ◽  
Multiple Protein ◽  
Essential Components ◽  
Dose Dependent Decrease ◽  
The Relationship ◽  
Dose Dependent

ABSTRACT The phagocyte NADPH oxidase consists of multiple protein subunits that interact with each other to form a functional superoxide-generating complex. Although the essential components for superoxide production have been well characterized, other proteins potentially involved in the regulation of NADPH oxidase activation remain to be identified. We report here that the Gαi subunit of heterotrimeric G proteins is a novel binding partner for p67phox in transfected HEK293T cells and peripheral blood polymorphonuclear leukocytes. p67phox preferably interacted with inactive Gαi. Expression of p67phox caused a dose-dependent decrease in intracellular cyclic AMP concentration, suggesting altered function of Gαi. We identified a fragment of p67phox, consisting of the PB1 domain and the C-terminal SH3 domain, to be critical for the interaction with Gαi. Because these domains are involved in the interaction with p47phox and p40phox, the relationship between the respective binding events was investigated. Wild-type Gαi, but not its QL mutant, could promote the interaction between p67phox and p47phox. However, the interaction between p67phox and p40phox was not affected by either Gαi form. These results provide the first evidence for an interaction between p67phox and an alpha subunit of heterotrimeric G proteins, suggesting a potential role for Gαi in the regulation or activation of NADPH oxidase.

Abstract 1068: Keratinocyte-specific deletion in mice reveals gene dose-dependent function of SIRT1 in tumorigenesis.

2013 ◽  
Author(s):  
Mei Ming ◽  
Keyoumars Soltani ◽  
Christopher R. Shea ◽  
Xiaoling Li ◽  
Yu-ying He
Keyword(s):  
Gene Dose ◽  
Dependent Function ◽  
Dose Dependent ◽  
Specific Deletion

Accumulation of D-arginine by rat liver mitochondria

1987 ◽  
Vol 65 (12) ◽  
pp. 1057-1063 ◽  
Author(s):  
Rafael Villalobos-Molina ◽  
J. Pablo Pardo ◽  
Alfredo Saavedra-Molina ◽  
Enrique Piña
Keyword(s):  
Membrane Potential ◽  
Rat Liver ◽  
Mitochondrial Respiration ◽  
Mitochondrial Membrane ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Inner Mitochondrial Membrane ◽  
Osmotic Swelling ◽  
Dose Dependent Fashion ◽  
Dose Dependent

The permeability of the inner mitochondrial membrane from rat liver to D-arginine was studied. By using safranin as a probe of the membrane potential, depolarization of energized liver mitochondria occurred in a dose-dependent fashion starting at 3.3 mmol/L of D- or DL-arginine. When ethidium bromide fluorescence was employed, a decrease in the membrane potential due to D- or DL-arginine was observed. A parallel significant change in succinate-induced respiration in rat liver mitochondria was found in response to osmotic swelling in 125 mmol/L of D-arginine salts. L-Arginine, L-glutamine, L-asparagine, L-ornithine, D-ornithine, and L-lysine did not modify the membrane potential at the concentrations tested. D-Arginine was not transformed into citrulline, but 1.0 mmol/L of the D-amino acid inhibited, by 42%, the state 3 of mitochondrial respiration using succinate as substrate. When D-arginine was used in combination with nigericin, a 40% inhibition of mitochondrial respiration in state 3 was recorded with succinate and with glutamate–malate as substrates.

scholarly journals DNA Methyltransferase Is Actively Retained in the Cytoplasm during Early Development

1999 ◽  
Vol 147 (1) ◽  
pp. 25-32 ◽  
Author(s):  
M. Cristina Cardoso ◽  
Heinrich Leonhardt
Keyword(s):  
Early Development ◽  
Nuclear Localization ◽  
Dna Methyltransferase ◽  
Blastocyst Stage ◽  
Mammalian Development ◽  
Nuclear Localization Signals ◽  
Early Embryos ◽  
Necessary And Sufficient ◽  
Early Mammalian Development ◽  
Dna Substrate

The overall DNA methylation level sharply decreases from the zygote to the blastocyst stage despite the presence of high levels of DNA methyltransferase (Dnmt1). Surprisingly, the enzyme is localized in the cytoplasm of early embryos despite the presence of several functional nuclear localization signals. We mapped a region in the NH2-terminal, regulatory domain of Dnmt1 that is necessary and sufficient for cytoplasmic retention during early development. Altogether, our results suggest that Dnmt1 is actively retained in the cytoplasm, which prevents binding to its DNA substrate in the nucleus and thereby contributes to the erasure of gamete-specific epigenetic information during early mammalian development.

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